Complement Mol Immunol

Molecular Immunology 38 (2001) 77–131

Abstracts

Complement in human disease�

www.elsevier.com/locate/molimm

1 The role of complement in the regulation of renalfibrogenesis in adriamycin nephropathy

K. Abe, T. Springall, S.H. Sacks, N.S. Sheerin

Department of Nephrology and Transplantation, Guy’sHospital, King’s Collage London, London, UK

Tubulointerstitial fibrosis is the final pathway inmany chronic renal diseases and is characterized bythe accumulation of extracellular matrix proteins inparticular type IV collagen (ColIV). Heat ShockProtein 47 (HSP47) is a collagen-binding protein thatacts as a molecular chaperone during the synthesisand secretion of procollagen. The synthesis of HSP47has been shown to parallel that of collagen in physio-logical and pathophysiological conditions. In addition,it is recognized that complement is activated at thesite of tubulointerstitial fibrosis suggesting a patho-genic role. To investigate whether complement influ-ences the progression of interstitial fibrosis weinduced persistent proteinuria in BALB/c mice by theintravenous injection of adriamycin.

Tubulo-interstitial fibrosis was evident 6 weeks afterdisease induction by light microscopy. Immunohisto-chemistry demonstrated complement (C3) depositionat the site of injury. By reverse transcriptase-poly-merase chain reaction (RT-PCR), HSP47 and ColIVmRNA expression was increased at 6 weeks. HSP47mRNA, by in situ hybridisation, and protein expres-sion were observed in tubular epithelial cells espe-cially in areas of interstitial damage. Distribution ofColIV was similar to that of HSP47. To determinewhether complement activation could influence ex-pression of these pro-fibrotic factors tubular epithelialcells were incubated with sub-lytic concentrations

C5b-9. This resulted in the up-regulation of HSP47and ColIV mRNA.

In summary, C3 deposition is seen at the site oftissue injury co-localizing with increased HSP47 andColIV synthesis. In vitro we have shown that C5b-9increases the level of mRNA for both HSP47 andColIV in tubular epithelial cells. Complement activa-tion in the tubulo-interstitial compartment couldtherefore promote interstitial fibrosis.

2 The enzymatic characterization of the MBL-associ-ated serine proteases MASP-1 and MASP-2

G. Ambrus1, P. Gal1, A. Laich2, R.B. Sim2, P.Zavodszky1

1Institute of Enzymology, Biological Research Center,Hungarian Academy of Sciences, Budapest, Hungary ;2Immunochemistry Unit, Department of Biochemistry,Uni�ersity of Oxford, Oxford, UK

Antibody independent activation of the complementsystem via the lectin pathway is initiated, when man-nan-binding lectin (MBL) binds to carbohydrate moi-eties on the surface of invading pathogens. Serineproteases attached to MBL convey the activation sig-nal by cleaving subsequent complement components.Inconsistent data have been presented on the sub-strate specificity of the MASP proteases and the se-quence of activation is not clearly established. Littleis known about the autoactivation capacity of MASP-1 and the esterolytic activities of the MASPs on syn-thetic substrates has not been described yet. Similarly,the question whether MASP-1 is capable of activatingMASP-2 and vice versa is still left open.

To answer these questions we cloned the C-termi-nal �B catalytic regions of MASP-1 and MASP-2.Both constructs contained the CCP(1)-CCP(2)-SP do-mains and were expressed in Escherichia coli in the

� Abstracts presented at the 8th European Meeting on Complementin Human Disease, 8–12th September 2001, held in Strasbourg, France.Abstracts are printed alphabetically with regards to first author.

PII: S 0 1 6 1 -5890 (01 )00037 -2

Abstracts / Molecular Immunology 38 (2001) 77–13178

form of inclusion bodies. Proteins were renatured andpurified by ion exchange and hydrophobic interactionchromatography. Both �B fragments eluted in theiractivated forms, which was reflected in the cleavage ofthe Arg-Ile activation bond. The restoration of enzymaticactivity were monitored by the cleavage of syntheticsubstrates. MASP-2 showed esterolytic activities com-parable to C1s and exhibited kcat/Km values half as highas typical for C1s. Upon prolonged incubation MASP-1cleaved C3 with low efficiency suggesting that thisreaction has no substantial physiological relevance.

3 IVIG protects against mesenteric ischemia-reperfusioninjury in rats by site-specific scavenging of C3

J. Anderson1,2, T. Shea-Donahue3, S. Rehring1,2, S.Fleming4, G. Tsokos3,4, M. Basta5

1Department of Surgery, Walter Reed Army MedicalCenter, Washington, DC, USA ; 2Department of Surgery,Uniformed Ser�ices Uni�ersity of the Health Sciences,Bethesda, MD, USA ; 3Department of Medicine, Uni-formed Ser�ices Uni�ersity of the Health Sciences,Bethesda, MD, USA ; 4Department of Cellular Injury,Walter Reed Army Institute of Research, Sil�er Spring,MD, USA ; 5National Institutes of Health, Bethesda, MD,USA

Mesenteric ischemia-reperfusion (I/R) injury in rats isan in vivo model relevant for a variety of clinicalconditions, such as hemorrhagic shock, burn, myocardialinfarction and multiple organ failure. Several lines ofevidence imply an important role of complement inmucosal damage triggered by mesenteric ischemia-reper-fusion. On the other hand, therapy with high-doseintravenous immunoglobulin (IVIG) appears to be pro-tective against immune-mediated tissue damage. Whilethe mechanism of this beneficial effect still remainsunclear, it was demonstrated that immunoglobulinmolecules have the capacity to intercept deposition ofC3b and C4b on target cells and, thereby, attenuatesubsequent damage. Here, we present evidence that IVIGpre-treatment in a dose–responsive manner preservednormal intestinal architecture following induction ofishemia-reperfusion events. Immuno-histochemistry ofintestine of rats subjected to I/R injury showed intensestaining for rat C3 and IgG, indicating complementactivation and up-regulation of host IgG; non-intestinalorgans showed no evidence of increased C3 and IgGexpression. Only background staining for C3 and IgGwas observed in controls. Furthermore, in IVIG-treatedanimals human IgG co-localized with rat C3, primarilyin and around intestinal capillaries, suggesting in situcomplex formation and protection from complement-

mediated injury by the C3-scavenging mechanism. Ourstudy provides the first visualization of this mechanismof beneficial effect of IVIG and also strongly suggest thatimmunomodulation with IVIG has a therapeutic poten-tial in conditions related to intestinal ischemia-reperfu-sion.

4 Assembly of C3BbP convertases on a polymer surface

J. Andersson, K. Nilsson Ekdahl, U.R. Nilsson, B.Nilsson

Section of Clinical Immunology, Uni�ersity Hospital ofUppsala, Uppsala, Sweden

Whole blood in contact with a biomaterial surfacetriggers complement activation. On many biomaterialsthe trigger of the activation is the alternative pathway(APW). In order to regulate APW, it is important to gainfurther insight into how the APW C3 convertase isassembled on the surface. In this study, the convertasewas built up of purified components on a polymersurface. The surface was either uncoated or coated withfibrinogen, BSA, or IgG. The binding of proteins to thesurface was monitored using a Quartz Crystal Microbal-ance that assesses the mass bound to the surface. WhenC3, factor B and factor D was sequentially incubatedthree times on a surface coated with C3b, there was anequal mass bound in each cycle. However, when prop-erdin was added with factor B, the bound mass wassignificantly higher and increased each cycle. This wasalso the case on a surface coated with C3 although themass was smaller compared with C3b. Adsorbed C3 alsobound monoclonal antibodies against epitopes of boundC3b and which are not available in native fluid-phase C3.Of the coated surfaces, the BSA and, in particular, theIgG surface promoted assembly of the convertase to ahigher degree than fibrinogen. These findings suggest thatadsorbed C3 might trigger complement activation uponblood–biomaterial contact and confirms that properdinis an important regulator of the assembly of the initialAPW C3 convertases on polymer surfaces.

5 Immune adherence receptors of primates: variation instructure but retention of function

J.P. Atkinson1, V.B. Subramanian1, F. Zhang1, M.E.Medof2

1Washington Uni�ersity School of Medicine, Departmentof Medicine, Di�ision of Rheumatology, St. Louis, MO,USA ; 2Case Western Reser�e, Department of Pathologyand Medicine, Cle�eland, OH, USA

Abstracts / Molecular Immunology 38 (2001) 77–131 79

The immune adherence (IA) phenomenon of pri-mates refers to the binding of C4b/C3b opsonizedimmune complexes to complement receptor (CR1,CD35) of erythrocytes (E), a key step in the physio-logic processing of IC. Expression of CR1 by primateE varies compared with human with respect to mol.wt. (55–130 vs. the 220 kDa), copies per cell, func-tional sites/receptor and mode of membrane an-choring (transmembrane or GPI). These ‘short’ formsare exclusively or predominantly expressed by E andlikely arose in response to environmental pressurefrom Plasmodium falciparum. A site on CR1 whichinteracts with malarial adhesions is located in theCOOH-terminal long homologous repeat D (LHR-D)which is not present in the smaller CR1s of OldWorld primates. Using E from human (hu) and, asthe representative primate species, chimpanzee (ch) E,we assessed the effect of receptor copy number (hu=250/E vs. ch=14 000/E) and number of ligand inter-active sites/receptor (hu=3/CR1 vs. ch=1/CR1) inthe binding of BSA/anti-BSA IC. By Scatchard analy-sis hu CR1 had a 3- to 4-fold higher affinity forC3b-dimers (9.2 vs. 2.4 nM). At optimal conditionsfor each E preparation, IC binding was 15–20%greater by ch E, even though ch CR1 has only onebinding site and a lower affinity. Binding over a widerange of E concentrations demonstrated that 30–40-fold fewer ch than hu E were required for compara-ble IC binding. Employing FITC labeled Fabfragments of anti-CR1 mAb (7G9), immunostainingdemonstrated larger CR1 clusters on ch E than hu-man E. These results suggest that primates expressboth a larger number of and more highly clusteredCR1 on their E in order to compensate for havingfewer binding sites.

6 Enhanced sensitivity of apoptotic cells to comple-ment-mediated lysis is mediated by caspases

G. Attali, Z. Fishelson

Department of Cell Biology and Histology, SacklerSchool of Medicine, Tel A�i� Uni�ersity, Tel A�i�69978, Israel

Apoptotic cells are known to activate complementand to bind complement proteins like C1q and C3b.This is believed to play a role in clearance of apop-totic cells by phagocytes armed with complement re-ceptors. The level of resistance of apoptotic cells tolysis by the complement membrane attack complex(MAC) has not been well studied yet. Human JurkatT lymphoma cells were induced by antibodies directedto Fas (CD95) to undergo apoptosis and their inter-

action with human complement was analyzed. Ourresults clearly indicate that early apoptotic Jurkatcells, long before they undergo through the series ofmorphological apoptotic changes, are more sensitiveto sublytic and lytic MAC doses than control cells.Complement activation by stimulated Jurkat cells wasFactor B-dependent and calcium ion-independent, i.e.activation was mediated mainly by the alternativecomplement pathway. As determined by flow cytome-try, upon complement activation, both C3 and C9were deposited more on the surface of apoptotic cellsthan on control cells. This was accompanied by en-hanced lysis (Chromium-51 release and trypan blueinclusion) of the apoptotic cells. Caspase inhibitorsprevented the increment in cell death of anti-Fastreated cells but did not lower it below the level ofcell death observe in control cells. These findings andothers suggest that, (1) complement activation byapoptotic cells may culminate in MAC formation andcell lysis; (2) Fas-mediated caspase activation en-hances complement activation on Jurkat cells; and (3)complement-mediated lysis of untreated Jurkat cells isinsensitive to caspase inhibitors, i.e. caspases are notinvolved in MAC-mediated cell killing.

7 Complement is activated in congestive heart failureand modulated by intravenous immunoglobulin treat-ment

P. Aukrust1,2, K.T. Lappegard5,6, L. Gullestad4, T.Ueland 2,3, H. Aass4, L. Wikeby2,4, S. Simonsen4, S.S.Frøland1,2, T.E. Mollnes5

1Section of Clinical Immunology and Infectious Dis-eases, Uni�ersity of Oslo, Oslo, Norway ; 2Research In-stitute for Internal Medicine, Uni�ersity of Oslo, Oslo,Norway ; 3Section of Endocrinology, Medical Depart-ment, Uni�ersity of Oslo, Oslo, Norway ; 4Departmentof Cardiology, Rikshospitalet, Uni�ersity of Oslo, Oslo,Norway ; 5Department of Immunology, Nordland Cen-tral Hospital, Bodø ; 6Department of Medicine, Nord-land Central Hospital, Bodø, and Uni�ersity ofTromsø, Tromsø, Norway

There is increasing evidence that the innate immunesystem plays a role in the pathogenesis of congestiveheart failure (CHF). However, the role of comple-ment in CHF remains elusive. We studied comple-ment activation in 39 patients with stable CHF beforeand during intravenous immunoglobulin (IVIG) treat-ment, recently shown to improve left ventricular ejec-tion fraction (LVEF) in these patients (L. Gullestadet al. Circulation 2001; 103: 220–225). Comparedwith healthy controls, CHF patients had markedly el-


evated systemic levels of C1rs-C1inhibitor complexes,C3bBbP, C3bc and TCC, indicating activation of boththe classical, alternative and terminal complement path-ways. Furthermore, analysis of paired coronary sinusand pulmonary artery samples in CHF patients demon-strated local complement activation within the my-ocardium. IVIG substantially increased complementactivation in contrast to placebo both during induction(first week) and maintenance (6 months) therapy. WhileIVIG improved LVEF, the IVIG-induced complementactivation was inversely correlated with LVEF im-provement. Complement inhibition may be a futuretherapeutic approach for CHF. Although IVIG mayprotect tissue from complement damage by deviatingthe activation to the fluid-phase, this increased sys-temic activation may be disadvantageous. Therefore,more specific complement inhibitors should be in-vestigated.

8 Molecular characterization of factor I deficiency in twoBrazilian sisters

G.V. Baracho1, V. Nudelman2, L. Isaac1

1Departamento de Imunologia, Instituto CienciasBiomedicas, Uni�ersidade Sao Paulo, Sao Paulo, Brazil ;2Escola Paulista de Medicina, Uni�ersidade Federal deSao Paulo, Sao Paulo, Brazil

Factor I (fI) is one of the most important regulatoryproteins of the complement system. The complete defi-ciency of this protein is rare and its molecular basis hasbeen elucidated in only two studies (Vyse et al., 1996;Morley et al., 1998). Here, we have studied two Brazil-ian fI-deficient sisters. Southern blotting analysis of theprobands’ fI gene after digestion with either Eco RI orPst I revealed no significant rearrangements, insertionsor deletions. We analyzed the cDNA obtained from theprobands’ LPS-stimulated fibroblasts by reverse tran-scriptase-polymerase chain reaction (RT-PCR). All am-plified products had the same size as those of control’sfibroblast cDNA. However, using primers that spanexons 1–5 in quantitative RT-PCR assays, we observedsignificantly less products for the deficients’ fI cDNA.This could indicate the presence of unstable fI-mRNAmolecules. Sequencing of subcloned proband cDNArevealed a two base pair insertion (AT) between posi-tions 1204 and 1205 in the 11th exon. This insertionwas confirmed on the probands’ genomic DNA and inhalf of the mother’s clones. This mutation creates astop codon 12 bp downstream of the insertion site. Thisexplains the absence of fI protein and may be related tothe instability of its mRNA. (Supported by FAPESPand CNPq).

10 Neuroprotection by C1 inhibitor in a mouse model oftransient and focal brain ischemia

L. Bergamaschini1, M. Barba2, L. Catapano2, C.Storini2, A. De Luigi2, E. Rossi1, A.M. Arabia3, M.G.De Simoni2

1Ospedale Maggiore, Uni�ersity of Milan, Milan, Italy ;2Mario Negri Institute for Pharmacological ResearchMilan, Milan, Italy ; 3Baker Medical Research Institute,Melbourne, Australia

There is evidence that inflammation and tissue de-struction in several diseases of central nervous system isdue to the activation of complement. We evaluated theeffect of C1-INH in a model of focal ischemia obtainedby reversible occlusion of the middle cerebral artery inCD1 mice. The occurrence of ischemia was verified byan electrocorticographic recording. When given at thebeginning of the 2 h ischemic period, C1-INH (15 Uper mouse iv) significantly improved both general (C1-INH, 7.2�0.5; saline, 11.4�1.3) and focal (C1-INH,12�2.3; saline, 22.6�1.1) deficits assessed after 48 hby neurological scales unique to the mouse. At thesame time C1-INH significantly reduced infarct volumecalculated by 2,3,5-triphenyltetrazolium chloride stain-ing on coronal slices and quantified by Analyzing Im-age System (C1-INH, 6.7�1.4%; saline, 17.7�3.9%).Neutral red staining, allowing to define the brain areasinvolved in the ischemic injury, further evidencedstrong neuroprotective effect of C1-INH. GFAP im-munohistochemistry demonstrated that activation ofastrocytes was also markedly reduced by C1-INH.When given at the end of focal ischemia, C1-INH wasstill effective in reducing neurological defects, infarctvolume and inflammatory reaction. Our findings showthe relevance of complement in ischemia/reperfusionbrain injury and that complement therapeutics couldhave a major impact on the quality of life for subjectswith ischemic diseases.

11 Structural requirements for the complement regula-tory activities of C4b-binding protein

Anna M. Blom, Lena Kask, Bjorn Dahlback

Department of Clinical Chemistry, Uni�ersity HospitalMalmo, Lund Uni�ersity, Sweden

C4b-binding protein (C4BP), a disulfide-linked poly-mer of seven identical �-chains and a unique �-chain,regulates the classical pathway of complement. The �-and �-chains are composed of 8 and 3 complementcontrol protein (CCP) domains, respectively. To eluci-


date the structural requirements for the interactionbetween C4b and the �-chain, we created nineteenrecombinant C4BP variants. Six truncated monomericvariants, nine polymeric variants in which individualCCPs were deleted, and finally four variants in whichdouble alanine residues were introduced betweenCCPs were functionally characterized. We found thatall three N-terminal CCP domains contribute to thebinding of C4b and were important for full functionalactivity, CCP2 and CCP3 being the most important.The spatial arrangements of the first CCPs are impor-tant, as introduction of alanine residues between CCP1/2, CCP2/3 and CCP3/4 results in functional impair-ment.

We also found that C4BP is able to serve as acofactor to Factor I in the cleavage of C3b and can,therefore, influence the alternative pathway of com-plement. We found that C4BP requires all four N-ter-minal CCPs of the �-chain, with CCP2 and CCP3being the most important, to act as a cofactor in thecleavage of C3b. Also, a cluster of positively chargedamino acids on the interface between CCP1 andCCP2 is involved in the binding. Compared with theinteraction with C4b, we conclude that binding ofC3b requires larger molecular surface but is based onionic interactions as it is also in case of C4b.

12 Novel synthetic small molecule inhibitor of C1s ex-erts cardioprotective effects in ischemia-reperfusion in-jury in rabbits

Michael Buerke1, Hansjorg Schwertz1, MartinSchmidt2, H. Hillen2, Werner Seitz2, Jurgen Meyer1,Harald Darius1

1II. Department of Medicine, Johannes Gutenberg Uni-�ersity, 55101 Mainz, Germany ; 2BASF Pharmaceuti-cal, Ludwigshafen, Germany

Myocardial ischemia-reperfusion injury can be re-lated to leukocyte activation with subsequent releaseof cytokines and oxygen-derived free radicals. In thisregard, activation of the classical complement systemplays an important role by generation of chemotacticagents, adhesion molecule expression, neutrophil accu-mulation, and direct cytotoxic effects.

In the present study, the cardioprotective effects ofa novel small molecule C1 inhibitor (C1s-INH-248,BASF, Ludwigshafen, Germany) were examined in arabbit model of myocardial ischemia (I) and reperfu-sion (R; i.e. 60 min I+180 min R). In in vitro tests(enzyme activity, liposome lysis, and sheep red celllysis) C1s-INH-248 demonstrated profound inhibitory

potency. C1s-INH-248 (1 mg/kg b.w.) administered 5min prior to reperfusion significantly attenuated my-ocardial injury compared with vehicle treated animals(31.9�2.5% vs. 8�1.6% necrosis/area-at-risk, P�0.01). The reduction of myocardial injury was alsoobserved as diminished plasma creatine kinase activityin C1s-INH-248 treated animals (70.7�6.8 vs. 45.1�3.9 U/g protein after 3 h of reperfusion P�0.05).Further, cardiac myeloperoxidase (MPO) activity (i.e.a marker of PMN accumulation) in the ischemic andnecrotic area was significantly reduced following C1s-INH-248 treatment compared to vehicle treated ani-mals (0.4�0.05 vs. 1.31�0.23 U/100 mg tissue innecrotic area, P�0.01). This antineutrophil effect wasconfirmed by histological analysis of ischemic reper-fused myocardium of C1s-INH-248 treated animals.Further, immunohistochemical analysis of ischemic-reperfused myocardial tissue of vehicle treated demon-strated deposition of C5b-9 on the vascularendothelium as well as on cardiac myocytes. Blockingof the classical complement pathway with the C1s-INH-248 resulted in reduced deposition of C5b-9 oncardiac myocytes and cardiac vessels.

Thus, blocking the classical complement pathwaywith a highly specific and potent inhibitor of the acti-vated C1 complex appears to be an effective mean topreserve ischemic myocardium from injury followingreperfusion. The mechanisms of the cardioprotectiveeffect of C1s-INH-248 appears to be due to blockingof complement activation and reduced neutrophil ac-cumulation resulting in diminished myocardial necro-sis following ischemia and reperfusion.

13 Complement biosynthesis in human trophoblast cells

R. Bulla1, D. Lorenzon1, P. Macor1, D. De Santo2,F. De Seta2, S. Guaschino2, F. Tedesco1

1Department of Physiology and Pathology, Uni�ersityof Trieste, Italy ; 2DSRS, Section of Obstretrics andGynecology, Uni�ersity of Trieste and IRCCS BurloGarofolo, Trieste, Italy

The surface of villi at the fetomaternal interface iscovered by two layers of trophoblast cells, cytotro-phoblast (CTB) lying on the basal membrane and theexternally situated syncytiotrophoblast (STB). Ex-travillous trophoblast (EVT) represents an additionalpopulation migrating to the decidua. Villous tro-phoblast cells express C regulatory proteins as ameans of protection against C attack.

We now show that these cells synthesize C compo-nents. Two trophoblast cell lines were used, the


BeWo cell line, which exhibits morphological character-istics of mature trophoblast, and HTR-8/Svneo cell line,which resembles EVT. Trophoblast cells (TC) purifiedfrom first trimester placental tissue obtained from elec-tive termination of pregnancy were also used. Reversetranscriptase-polymerase chain reaction (RT-PCR)analysis revealed that both cell lines and purified TCexpressed abundant messages for C3 and C4. Withregard to the late C components, the messages for C7and C9 were relatively stronger than those of C8�, C8�and C6, whereas the message for C5 was undetectable.Enzyme linked immunosorbent assay (ELISA) revealedthat BeWo secreted approximately 3 ng per 106 cells per72 h of both C3 and C4, whereas the amount of C4secreted by HTR8 was 5–6 fold higher and that of C3was essentially similar to the amount secreted by BeWo.Interestingly, secretion of C4, but not of C3, wasup-regulated by IFN-� to values of approximately 60 ngper 106 for both cell lines, whereas IL-1� and TNF-�were ineffective. C6 was the only late componentsecreted by the two cell lines in measurable amounts(about 1–3 ng per 106 cells per 72 h) and was notregulated by cytokines.

14 ACE inhibitor ramipril down-regulates C3 gene ex-pression in renal proximal tubular epithelial cells(PTECs)

V. Cappiello, V. Montinaro, G. Castellano, F.P. Schena

Di�ision of Nephrology, Department of Emergency andOrgan Transplantation, Uni�ersity of Bari, Policlinico,Bari, Italy

Proximal tubular epithelial cells (PTECs) are able tosynthesize and release C3, especially under conditions oftubulo-interstitial damage (TID). Chronic TID is associ-ated with development of progressive renalinsufficiency.

ACE inhibitor drugs have been demonstrated to slowdown the progressive decline of renal function. SinceC3, produced locally in renal tissue, may participate inthe pathogenesis of progression of renal damage, wetested the hypothesis that ACE inhibitors may modulateC3 gene expression in PTECs. We studied in culture theeffects of two ACE inhibitors (ramipril, enalapril) andan angiotensin II receptor antagonist losartan (all 1 �mfinal concentration) on a transformed human PTEC line(HK-2) in presence of IL-1� (100 U/ml), an efficaciousstimulus for C3 gene induction. C3 gene expression wasanalyzed by reverse transcription (RT) of RNA and

polymerase chain reaction (PCR). After 12 h of cultureno difference was observed in C3 gene expression inten-sity induced by IL-1� with all drugs tested. At 24 h aspecific down-regulation of C3 was evident only in thepresence of ramipril. At 48 h the effect of the drug wasno more evident and C3 expression was comparable inall conditions. In conclusion, ACE inhibitor ramipril isable to down-regulate C3 gene expression in PTECs.This finding may have relevance in the protective effectof ramipril on TID in chronic nephropathies.

15 The course of lymphoproliferative disorders associatedwith the acquired deficiency of C1-inhibitor

A. Carugati, D. Gioffre, L.C. Zingale, E. Pappalardo,A. Agostoni, M. Cicardi

Internal Medicine Department, Uni�ersity of Milan, Mi-lano, Italy

Acquired angioedema (AAE) is a rare disease withonset in adulthood, due to the non-genetic deficiency ofC1-inhibitor (C1-INH). It is characterized by hyperacti-vation of the classical complement pathway and acceler-ated C1-INH catabolism, resulting in low levels ofC1-INH, C4 and C1q. In 1986 autoantibodies to C1-INH (C1-INH-Abs), causing its functional inactivation,have been identified in serum of patients with AAE.Among the great variety of diseases coexisting withAAE, the association between AAE and lymphoprolif-erative disorders (LD) is the most frequent.

We evaluated the presence of C1-INH-Abs and thesigns of B-cell-line proliferation in 19 patients followedat our Department (median follow-up 9.4 years). Six-teen patients had C1-INH-Abs, 14 had a LD and 1patient had neither C1-INH-Abs nor LD. Eleven pa-tients had a monoclonal component, whose class, ineight cases, matched with that of the C1-INH-Ab, andfor ten of them the monoclonal component appearsbenign after a median follow-up of 6.5 years. Fourpatients developed overt LD (1 CLL, 1 Waldenstromdisease, 2 NHL), all but one after onset of angioedema.Production of C1-INH-Ab could not be shown in any ofthe four neoplastic clones. Two of these four patientshad neither C1-INH-Ab nor monoclonal components.One showed inconstantly C1-INH-Abs and the neoplas-tic clone was immature and not immunoglobulin-secret-ing. The fourth patient had Waldenstrom disease withIgG anti-C1-INH.

Our data confirm the association between AAE andLD, but they do not support the view that the neoplasticclones directly originate from those producing the C1-INH-Ab.


16 E. coli-induced upregulation of granulocyte CR3 con-tributes to oxidative burst and is totally C5a receptordependent

D. Christiansen1, H. Fure1, G. Bergseth1, J.D. Lambris2,T.E. Mollnes1

1Department of Immunology, Nordland Central Hospital,Bodø, and Uni�ersity of Tromsø, Norway ; 2Protein Chem-istry Laboratory, Uni�ersity of Pennsyl�ania, Philadel-phia, USA

Complement plays a major role in many aspects ofinflammation, including granulocyte oxidative burst.Using a novel human whole blood model anticoagulatedwith the specific thrombin inhibitor lepirudin (see ab-stract by Fure H. et al.) we studied the role of comple-ment in Escherichia coli-induced upregulation ofgranulocyte CR3 (CD11b) and the role of CR3 inoxidative burst. The C3 binding peptide Compstatin anda C5a receptor antagonist (C5aRag) were used to inhibitcomplement. CR3 was examined in blood incubated withE. coli (4×106 per ml blood) for 10 min and analysedusing flow cytometry. The effect of CR3 on granulocyteoxidative burst was measured by flow cytometry afterincubation of blood for 10 min with 1×108 E. coli perml blood in the presence or absence of a blockinganti-CD11b antibody. Compstatin and C5aRag indepen-dently inhibited the upregulation of granulocyte CR3dose-dependently and completely, suggesting that CR3upregulation was not only dependent on complementactivation, but solely on the C5aR. Furthermore, gran-ulocyte oxidative burst was reduced by 30% by blockingCR3. We conclude that E. coli-induced upregulation ofgranulocyte CR3 is totally dependent on the C5aR andthat this upregulation enhances E. coli-induced oxidativeburst of granulocytes. The data highlights the C5a/C5aRinteractions as therapeutic target in conditions withdetrimental complement-induced oxidative burst.

17 Bactericidal action of complement in normal cord serumagainst Escherichia coli strains isolated from urine ofchildren with urinary tract infections

A. Cisowska, S. Jankowski

Department of Biology and Medical Parasitology, Medi-cal Uni�ersity, Wroclaw, Poland

Objecti�es: It is known that Escherichia coli rods withK1 capsule are associated with neonatal meningitis,septicaemia and childhood pyelonephritis. The aim of thestudy was to comparison of E. coli strains with K1surface antigen (K1+ ) and rods without this antigen

(K1− ) sensitivity to human normal cord serum (HNCS)and determination of the mechanism of its activityagainst these strains.

Methods: Seventy E. coli strains (35 K1+ and 35K1− ) isolated from urine cultures obtained from chil-dren with urinary tract infections were tested. Sensitivityof the investigated strains to bactericidal activity of 50%HNCS was assessed according to Jankowski S. et al.,(FEMS Immunol. Med. Microbiol., 1996, 13: 59–64).The alternative complement activation mechanism wasblocked thermally (CS50°C/20 min), and the classical oneby addition to the serum of EGTA and MgCl2 suspension(CSMgEGTA).

Results: E. coli (82.8%) K1+ strains were HNCS-re-sistant, whereas the percentage of such rods in the E. coliK1− pool was 62.8%. In further investigations, HNCS-sensitive strains were selected from both groups and theirsensitivity to CS50°C/20 min and CSMgEGTA wasdetermined. On comparison of the bacterial survivalrates, two mechanisms of serum activity were distin-guished in the K1+ and four in the K1− pool. AmongK1+ rods, an important role of the classical mechanismof complement activation was observed (50% strains), aswell as parallel activation of the classical and alternativeone. In the K1− group, the alternative route waspredominant in cytolysis (69.2% of strains). The involve-ment of the classical or alternative pathway, only theclassical one or co-involvement of both in bactericidalserum activity was observed in single cases only.

Conclusions: E. coli K1+ strains are characterizedwith lower sensitivity to HNCS bactericidal effect incomparison with K1− rods. The alternative mechanismof complement activation was important in the serumbactericidal activity against E. coli K1− strains.

18 Production and characterisation of C3d oliogomers fornovel vaccines

V. Cox, P. Rowling, M. Steward, J. Bright, S. Gallagher,P. Kareclas, C. Harding, R. Smith, L. Affleck.

Adprotech Ltd., Chesterford Research Park, SaffronWalden, Essex CB10 1XL, UK

Fusion of tandem arrays of the complement compo-nent C3d to an antigen can enhance humoral responsesby several orders of magnitude when administered as arecombinant fusion protein (Dempsey et al., 1996). Themechanism is thought to involve targeting of antigen byC3d to its receptor (CD21) on B cells and folliculardendritic cells. Multi-copy C3d-antigen fusions can alsobe used in the context of a DNA vaccine resulting insignificant increases in antibody titre and avidity leading


to enhanced protection in an influenza model (Ross etal., 2001). In both studies, the arrays of C3d wereencoded by repeated copies of a gene identical towild-type apart from mutation of the natural thiolestersite. Where identical DNA sequences are duplicated, therisk of homologous recombination or genomic integra-tion exists. In the study of Dempsey et al., this resultedin very low yields of the full length fusion protein. Inthis study, we describe novel C3d genes in which theDNA (but not the encoded protein) sequence is variedand which are resistant to homologous recombinationin concatameric arrays (Dempsey et al., 1996). Stableexpression in eukaryotic hosts was used to producepractical amounts of recombinant human and murineC3d oligomers. The purification and characterisation ofthe oligomers is described together with functional as-says based on the binding of C3d to CD21. Recombi-nant proteins containing multiple copies of C3d showedenhanced affinity for CD21 over monomeric C3d andform the basis for a range of novel vaccines.

19 A new mannan-binding lectin associated serineprotease, MASP-3, and its association with distinct com-plexes of the MBL complement activation pathway

Mads R. Dahl1, Steffen Thiel1, Misao Matsushita2,Teizo Fujita2, Anthony C. Willis3, Tove Christensen1,Thomas Vorup-Jensen1, Jens C. Jensenius1

1Department of Medical Microbiology and Immunology,Uni�ersity of Aarhus, DK 8000, Aarhus, Denmark ;2Department of Biochemistry, Fukushima Medical Uni-�ersity, Fukushima 960-1295, Japan ; 3MRC Immuno-chemistry Unit, Uni�ersity of Oxford, OX1 3QU, UK

The innate immune system, as well as being essentialfor the immediate antimicrobial defence, also subserveimportant roles for establishing clonal immune re-sponses. The third pathway of complement activation,the mannan-binding lectin (MBL) pathway, is part ofthe innate immune defence. The binding of MBL tomicrobial carbohydrates activates the MBL-associatedserine proteases (MASPs), which recruit the comple-ment factors, C4 and C2, to generate the C3 convertaseor directly activates C3. We present a new member ofthe MBL complex, MASP-3, which is generatedthrough alternative splicing of a complex MASP-1/3gene. MASP-3 is phylogenetically highly conserved withthe B chain showing 65 and 95% sequence identity tothe shark and pig homologues, respectively. The desig-nation of MASP-3 as a protease is based on homologyto known MASPs. Different MBL oligomers werefound to have distinct MASP composition and differentbiological activities. MASP-1 and MAp19 and direct

C3-cleaving activity are associated with smalleroligomers whereas MASP-3 is found together withMASP-2 on larger oligomers.

20 Patterns within short consensus repeats define genesof the RCA complex: origin and functions of CCPs

Roger L. Dawkins, Richard A. Davies, JosephF. Williamson, Jemma Berry, Silvana Gaudieri

Centre for Molecular Immunology and Instrumentation,Uni�ersity of Western Australia, Queen ElizabethII Medical Centre, Nedlands, WA, Australia

Complement control proteins (CCPs) contain re-peated protein domains, short consensus repeats(SCRs), which must be relevant to complement activa-tion, coagulation, viral binding, foetal implantation andself–nonself recognition. Although SCRs share somediscontinuous and imperfect motifs, there are manyvariable positions and indels, which make classificationdifficult.

Using domain-by-domain phylogenetic analysis, wehave found that most domains can be classified intoonly 11 groups, designated a, b, c, d, e, f, g, h, i, j, ork and that each particular CCP is characterised by theorder of representatives of these groups. For example,human complement receptor 1 (CR1) has ‘a, j, e, f, b, k,d’ repeated four times followed by ‘c, h’. Most interest-ingly, specific sets of domains or SCRs appear to beassociated with specific functions. The aje set is a featureof several CCPs including CRRY and appears to beassociated with the success or failure of implantationinteralia. Synonymous and nonsynonymous profileswithin the SCR sets appear to reflect different functionsand selection pressures. Examples support the conceptthat CCP sets will prove to be important in evolution ofacquired and innate immunity.

21 Complement factor I is upregulated in rat hepatocytesand HUVEC by interleukin-6 but not by interferon-�,interleukin-1� or tumor necrosis factor-�

T. Demberg, G. Schlaf, O. Gotze

Department of Immunology, Georg-August Uni�ersitatGottingen, Kreuzbergring 57, D-37075 Gottingen,Germany

Complement factor I (FI) is a regulatory serineprotease of the complement system which cleaves threepeptide bonds in the �-chain of C3b and two bonds inthe �-chain of C4b and thus prevents the assembly of


the C3 and C5 convertases. FI is mainly synthesizedin the liver i.e. in hepatocytes (HC). We have investi-gated the proinflammatory cytokines IL-6, IL-1�,TNF-� and IFN-� for their potential role in the regu-lation of FI expression. Of the investigated cytokinesonly IL-6 increased the FI-specific reverse transcrip-tase-polymerase chain reaction (RT-PCR) signal inisolated HC and in the two rat hepatoma-derived celllines FAO and H4IIE. Quantitative competitive RT-PCR showed an IL-6 induced upregulation of FI-spe-cific mRNA by about 10-fold. These data were inaccord with Northern Blot analyses in which the FI-mRNA was upregulated by IL-6 between 5- and 7-fold. IL-6 also increased FI-protein in cell culturesupernatants by about 5-fold as detected by a semi-quantitative immunoblot using a newly generatedmonoclonal antibody specific for rat FI. Also, on theprotein level, the cytokines IL-1�, TNF-� and IFN-�had no regulatory effects. An extrahepatic source ofFI are human umbilical vein endothelial cells (HU-VEC) in which the synthesis of FI is upregulated byIL-6. This is in accord with the upregulation observedin rat HC and two rat hepatoma cell lines (FAO andH4IIE), demonstrating that the regulation of FI issimilar in HUVEC and HC. These results are in con-trast to the previously described IFN-�-mediated up-regulation of FI in HUVEC and suggest, in accordwith other investigations on extrahepatic sources ofFI (e.g. myoblasts) that IFN-� has probably noprominent role in the regulation of FI.

22 Effect of heparin on biological activity ofAlzheimer’s amyloid-� peptide

C. Donarini1, E. Rossi1, A. De Luigi2, M.G. DeSimoni2, A. Agostoni1, L. Bergamaschini1

1Ospedale Maggiore, Uni�ersity of Milan, Milan, Italy ;2Mario Negri Institute for Pharmacol Research, Milan,Italy

Amyloid-� protein (A�) is implicated in the patho-genesis of Alzheimer’s disease because of its neurotox-icity and its ability to trigger local inflammation andneurodegeneration. Compounds that interact with theamino acids of the N-terminal region, can reduce thebiological activity of A�. We evaluated the effect ofheparin on the ability of A�1-42 to activate comple-ment and contact system and to induce neurotoxicity.A�1-42 was incubated with heparin (up to 1/15 mo-lar) for 15 min at room temperature followed by 30min at 37°C in presence of normal plasma as sourceof complement and contact system factors. Heparin

reduced, in a dose-dependent manner, complementand contact system activation, assessed by measuringC4 and high molecular weight kininogen (HK) cleav-age by densitometric analysis of immunostained blot-ting membranes after sodium dodecylsul-phate-polyacrylamide gel electrophoresis (SDS-PAGE). Congo Red staining served to determine theinteraction of A�1-42 with heparin. Heparin reducedsignificantly citotoxicity of A� on PC12 cells assessedby measuring MTT conversion and LDH release.Since heparin acrosses blood–brain barrier, heparintreatment could represent a new therapeutical strategyto reduce progressive neurodegeneration in AD brain.

23 Characterisation of a complete C1s deficiency: thefirst case of a non functional protein

M.-A. Dragon-Durey1, C. Lebbe2, V. Fremeaux-Bacchi1, B. Bienvenu2, J. Blouin1, I. Hammoud1, P.Morel2, M.D. Kazatchkine1, W.H. Fridman1

1Departement d’Immunologie, Hopital EuropeenGeorges Pompidou, Paris, France ; 2Ser�ice de Derma-tologie, Hopital St Louis, Paris, France

We report a case of a 23-year-old Tunisian manwho presented with a lupus erythematosus syndromesince age of 8 years. He presented with predominanceof skin lesions (photosensibility, acrosclerosis, vasculi-tis) and joint involvement and an increased suscepti-bility to infectious diseases. A fluorescent antinuclearantibody was found with a speckled pattern associ-ated with anti-SSA, Sm and RNP antibodies. Thispatient’s complement profile was characterised by theabsence of CH50 activity, C1 functional activity lessthan 10% with normal levels of C2 and C4. The addi-tion of normal plasma or purified C1s to the patient’splasma restored its ability to sustain classical pathwayactivation. C1s and C1r Western-blot analysis usingpolyclonal antibodies showed normal-weight C1s andC1r proteins in his plasma and no additional bandwas revealed. Exon-specific amplification of genomicDNA by polymerase chain reaction followed by directsequence analysis revealed a homozygous nucleotidicsubstitution in the C1s gene exon XII. This substitu-tion leads to the change of the glycine196 to a valinelocated immediately after the active serine proteasesite Ser195. These data suggest that this mutation dis-turbs the functional serine protease activity of C1s.We report for the first time a case of complete C1sdeficiency associated with a non functional protein(C1s type II deficiency).


24 High avidity IgG response against meningococcalcapsular polysaccharide serogroup C in vaccinated comple-ment deficient persons

M. Drogari-Apiranthitou1, E. Luinge1, J. Dankert1, E.J.Kuijper1,2

1Department of Medical Microbiology, Academic MedicalCentre, Uni�ersity of Amsterdam, Amsterdam, TheNetherlands ; 2Department of Medical Microbiology, Uni-�ersity Medical Centre of Leiden, Leiden, The Netherlands

Increase of specific total IgG indicates immune re-sponse to vaccination, but does not guarantee whethervaccination will be effective. To assess protection againstmeningococcal disease the serum bactericidal activity(SBA) assay is a suitable test. However, SBA assay islaborious and difficult to standardize. High avidity IgGantibodies are considered to be more specific and they areexpected to confer protection. Therefore, we studied thedevelopment of high avidity IgG antibodies againstmeningococccal capsular polysaccharide C in 2 C3- and17 late complement component deficient (CD) individu-als vaccinated with a tetravalent meningococcal capsularpolyssaccharide vaccine and revaccinated 7 years later,using a modified enzyme linked immunosorbent assay(ELISA).

The values of high avidity IgG antibodies 6 monthsafter vaccination in the CD persons did not differsignificantly from those in a control group of 16 healthyunrelated individuals. However, when compared with theSBA titres, a correlation was found only in the CD group(r Spearman=0.526, P�0.05). Seven years after vacci-nation the high avidity IgG titres decreased similarly inboth groups. SBA titres were also reduced in bothgroups, but no correlation with high avidity IgG wasfound. Three months after revaccination high avidityantibodies in the CD individuals increased reaching thevalues at 6 months after vaccination, but no correlationwith SBA titres was found.

These results indicate that high avidity meningococcalanticapsular antibodies might not always be bactericidal,therefore the modified ELISA used cannot substitute thefunctional SBA test to assess vaccine efficacy.

25 Complement activation and formation of the comple-ment membrane attack complex (MAC) on serogroup bmeningococci in the presence or absence of bactericidalantibodies

M. Drogari-Apiranthitou1, E.J. Kuijper1,2, N. Dekker1,J. Dankert1

1Department of Medical Microbiology, Academic MedicalCentre, Uni�ersity of Amsterdam, Amsterdam, TheNetherlands ; 2Department of Medical Microbiology, Uni-�ersity Medical Centre of Leiden, Leiden, The Netherlands

The influence of antibodies on complement depositionon serogroup B meningococcal strain H44/76, its variantsdifferent in capsulation and expression of the class 1porin (PorA) and its LPS-deficient isogenic mutantpLAK33 were studied in whole-cell enzyme linked im-munosorbent assays (ELISAs) with opsonized bacteria.Two normal sera, one bactericidal and one non-bacteri-cidal against H44/76 and an a-�-globulinemic serum wereused for sensibilisation of meningococci.

Incubation with the bactericidal serum resulted inhigher membrane attack complex (MAC) deposition onH44/76 and pLAK33 than with the non-bactericidalserum while the amounts of C3b and iC3b did not differsignificantly. C3b, iC3b and MAC on the unencapsulatedv24 variant in the two normal sera were similar comparedto H44/76. The amounts of C3b on v24 and pLAK33 inthe a-�-globulinemic serum were comparable to those inthe two normal sera, indicating IgG- and LPS-indepen-dent complement activation.

The amounts of C3b and MAC on PorA(+ ) orPorA(− ) strains did not differ significantly. Addition ofan anti-PorA chimeric antibody to the non-bactericidalnormal serum resulted in a dose-related increase of thebactericidal activity against H44/76PorA(+ ), but theamounts of C3b, iC3b and MAC on the bacterial surfaceremained unchanged.

In conclusion, complement deposition on meningo-cocci is mainly IgG-independent and is not influenced byPorA or LPS. Addition of bactericidal antibodies in thenon-immune serum albeit promotes lysis, does not influ-ence the amounts of C3b or MAC deposited. Thissupports the hypothesis that proper MAC insertionrather than quantity of MAC formed on the bacter-ial surface is of importance for efficient meningococcallysis.

26 C1 inhibitor biosynthesis to study its intracellularreactivities

C. Drouet1, M. Koenig1, C. Dumestre-Perard1, D.Ponard1, P. Maurel2, M. Tosi3, M.G. Colomb1

1Uni�ersite Joseph Fourier JE2236, F-38042 Grenoble,France ; 2INSERM U128, F-34293 Montpellier, France ;3INSERM EPI9906 F-76000 Rouen, France

C1 inhibitor (C1Inh) is a blood serpin known fordeficiencies associated with angioedema. DNA alter-ations have been typed, but very little is known at theprotein level about eventual intracellular expressionproducts. Our purpose is to set up an experimental basisto analyse the intracellular activities of C1Inh.


(1) C1Inh is secreted by cultured primary hepato-cytes (100 ng/�g DNA/day) and HepG2 (2.6 ng/�gDNA/day), as a 105 kDa glycoprotein; higher MWforms are secreted by HepG2. Pulse-chase experi-ments with HepG2 show that C1Inh is detected as afully glycosylated protein within 30–45 min in lysatesand after 60 min in supernatants.

(2) The serpin reactivity of HepG2 intracellularC1Inh is analysed in cell lysates on exogenous acti-vated C1s. Its reactivity is also tested with HepG2transfected with a Hepatitis C virus construct andexpressing the NS3 serine protease. In this case highMW association forms of C1Inh are detected in thecorresponding lysates, associations which reflect animportant role of C1Inh in pathogen control.

(3) The reactivity of normal and abnormal C1Inh isanalysed in a minigene construct representing the cod-ing and regulatory sequences (Tosi, unpublished) andused to transfect HepG2, with preliminary results asin 1. This cellular system will be developed later toanalyse the intracellular fate of abnormal C1Inh, suchas the interaction with chaperones.

27 Complement C4 monitoring in the follow-up ofchronic hepatitis C treatment

C. Dumestre-Perard1, D. Ponard1, C. Drouet1, V.Leroy2, J.P. Zarski2, M.G. Colomb1

1Laboratoire d’Immunologie, CHU Grenoble, France ;2Departement d’Hepato-Gastroenterologie, CHUGrenoble, France

This study concerns 66 patients with chronic HCVinfection, treated with interferon-� alone or with in-terferon-� + ribavirin and 50 healthy patients ascontrol. Complement blood tests were performed at 0,3, 6, 9 and 12 months of treatment on C1q, C3, C4,MBL, C1s-C1inhibitor complexes, total (TH50) andC4 (C4 H) hemolytic activities; specific C4 was takenas the C4H/C4 protein ratio. Rheumatoid factor (RF)was also titrated.

A significant reduction of TH50 and specific C4,compared with healthy controls were observed beforeonset of the treatment, the other parameters were notaffected and no C1s-C1 inhibitor complexes were de-tected. At the same time, a significant reduction inspecific C4 was observed in relapsers in contrast withsustained responders. These results point to a poten-tial predictive interest of this parameter to monitorthe response to therapy.

Restoration of specific C4 at 6 months was better

in responders compared with non-responders.Complement activation in chronic hepatitis C does

not seem to involve the C1 stage of the classicalpathway. A neg ative correlation between specific C4and RF titers suggests a possible involvement of RFin C4 activation, via the lectin pathway (no C1s-C1inhibitor complexes).

Specific C4 monitoring appears as a valuable basisin the follow-up of chronic hepatitis C treatment, to-gether with the other conventional investigations.

28 Role of complement and neutrophil migration in re-nal ischaemia/reperfusion injury

C.A. Farrar, W. Zhou, S.H.Sacks

Department of Nephrology and Transplantation, Guy’sHospital, London, UK

Ischaemia/reperfusion (I/R) injury occurs uponreperfusion after an extended period of ischaemia.Three components are known to contribute to theinjury process, namely oxidant stress, infiltration ofneutrophils and complement activation. Previouswork in this laboratory employed a model of renalI/R injury in C3-deficient mice that clearly indicatedcomplement as an important mediator of I/R injury.To investigate the role of neutrophils and their inter-action with complement in renal I/R injury, C3-defi-cient and wild type mice were treated with antiP-selectin mAb (which is able to inhibit the migrationof neutrophils), prior to induction of renal ischaemia.Following reperfusion, renal function and morpholog-ical changes were examined.

In this study, we showed that either complement orp-selectin blockade alone is able to reduce renal I/Rinjury in mice. Combined blockade of these two com-ponents, resulted in a further reduction of renal in-jury. Compared with control mAb treatment, antiP-selectin mAb treated WT and C3-deficient miceshowed 14 and 40% reduction of injury, respectively.

Histological and immunochemical analysis demon-strated that renal tubular damage and neutrophilinfiltration were minimized in anti p-selectin mAbtreated mice, but was more pronounced in C3-defi-cient mice.

These data suggest that both complement and neu-trophils play an important role in renal I/R injury.These two components could be potential targets fortherapeutic intervention. Since the effects of comple-ment and selectin blockade are additive they may actby mutually exclusive routes.


29 Concentrations of regulatory complement proteins inBrazilian healthy children and adults

P. Ferreira de Paula1, P.R. Junior2, V.P.L. Ferriani2, V.Nudelman3, L. Isaac1

1Departamento de Imunologia ICB/USP, Brazil ;2Departamento de Pediatria-FMRPUSP-RP, Brazil ;3HIAE-SP, Brazil

Here we established the normal ranges (Healy, 1969)of concentrations of factor H (fH), factor I (fI), prop-erdin and C4 binding protein (C4BP) in healthy Brazil-ian children from 1 month through 12 years and as wellas in healthy adults. Protein concentrations were deter-mined by single radial immunodiffusion. The resultswere expressed, respectively, as lower limit, mean, up-per limit [age range, (n)]. For fH, 271.80, 563.80,1060.97 �g/ml [1 month–1 year, (39)]; 325.10, 689.30,1284.17 �g/ml [1–6 years, (33)]; 223.26, 695.60, 1712.42�g/ml [6–13 years, (31)] and 228.56, 453.80, 829.00�g/ml [adults, (30)]. For fI, 23.76, 57.82, 120.78 �g/ml[1 month–1 year, (39)]; 29.97, 57.93, 103.54 �g/ml [1–6years, (27)]; 34.03, 57.06, 91.00 �g/ml [6–13 years, (25)]and 38.78, 63.91, 100.49 �g/ml [adults, (30)]. For C4BP,21.12, 70.30, 179.04% NHS [1 month–1 year, (43)];32.06, 62.72, 112.88% NHS [1–6 years (36)]; 32.63,74.86, 150.90% NHS [6–13 years (33)] and 37.17, 64.25,104.63% NHS [adults (34)]. For properdin, 7.52, 18.38,38.94 �g/ml [1 month–1 year, (37)]; 13.23, 23.08, 38.01�g/ml [1–6 years (24)]; 11.02, 21.36, 38.10 �g/ml [6–13years (33)] and 13.42, 25.03, 43.95 �g/ml [adults (16)].These reference values are important to clinical andlaboratory investigators of disorders associated withlow levels of these proteins. (Supported by FAPESPand CNPq).

30 Transendothelial migration of polymorphonuclearleukocytes induced in vitro and in vivo by the cytolyti-cally inactive terminal complement complex

F. Fischetti1, M. Pausa2, R. Bulla3, E. Vecile3, F.Bossi3, A. Dobrina3, F. Tedesco3

1Department of Medicine and Neurology, Uni�ersity ofTrieste, Trieste, Italy ; 2DSRS, Section of Obstretricsand Gynecology, Uni�ersity of Trieste and IRCCS BurloGarofolo, Trieste, Trieste, Italy ; 3Department of Physi-ology and Pathology, Uni�ersity of Trieste, Trieste, Italy

In previous studies, we have shown that the cytolyti-cally inactive terminal complement complex (iTCC)binds to endothelial cells (ECs) and stimulates surfaceexpression of adhesion molecules. Data are now pre-

sented indicating that iTCC promotes inflammationacting on the abluminal side of ECs.

Intravital microscopy was used to monitor in vivoleukocyte trafficking across rat mesenteric postcapillaryvenules induced by topical administration of iTCC toileal mesentery. After intravenous infusion of a fluores-cent marker, leukocytes became visible by epifluores-cent transillumination and started rolling within 15 minfrom the administration of iTCC. The cells adheredalmost completely to endothelium after one hour andemigrated out of the vessels in the following 3 h. C5acaused a similar effect, though less marked, whileboiled iTCC was inactive, thus excluding the contribu-tion of contaminating LPS.

iTCC was also effective in stimulating migration ofPMN across EC monolayer incubated for 4 h with thecomplex added to the lower chamber of the transwell,whereas 30 min incubation was sufficient for C5a toinduce passage of PMN. Contaminating C5a was notresponsible for the effect of iTCC because the complexhad no chemotactic activity and contained negligibleamount of C5a. Despite the ability of iTCC to stimu-late the release of IL-8 by ECs, anti-IL-8 antibodiesfailed to inhibit iTCC-induced migration of PMN,which, instead, was partially blocked by anti-PECAMantibodies.

These results suggest that iTCC be included amongthe C activation products that may act at tissue levelpromoting recruitment of leukocytes at inflammatorysites.

31 Expression and characterisation of a membrane-targeted soluble recombinant form of rat Crry for use inanti-complement therapy

D.A. Fraser1, C.L. Harris1, R.J. Mead1, J.R. Bright2,S.P. Gallagher2, R.A. Smith2, B.P. Morgan1

1Complement Biology Group, Department of MedicalBiochemistry, Uni�ersity of Wales College of Medicine,Cardiff, UK ; 2AdProTech Ltd., Royston, Herts., UK

The complement (C) system, when activated in anunregulated way or at an inappropriate site, contributesto pathology in inflammatory diseases. Previous studieshave shown that soluble recombinant regulators of Care effective in animal models and in some humandiseases. However, limitations include cost, rapid clear-ance and unwanted systemic effects. To address someof these limitations, bacterial expression of regulatorshas been optimised and a membrane targeting systeminvolving the addition of a membrane address tag,termed ‘addressin’, to the C regulator developed. Whenadministered directly to sites of inflammation, ad-dressin-tagged human regulators have been shown to be


retained and inhibit C activation locally. To test theefficacy of tagged rat C regulators we expressed atruncated soluble form of Crry in bacteria and com-pared tagged and untagged Crry in vitro and in vivo.A soluble recombinant form of Crry, containing onlythe first four SCR was first expressed in a mammalianexpression system and shown to be functional as afluid phase regulator. To generate the quantities re-quired for addressin tagging and testing in vivo, Crrywas expressed in bacteria, successfully solubilised frominclusion bodies and refolded. Refolded protein wasrecognised by antibodies specific for native proteinand had full C regulatory activity in vitro. Attachmentof a membrane-address tag conferred membrane-bind-ing capacity and greatly increased C regulatory func-tion. Preliminary studies in vivo demonstrate thataddressin-tagged Crry is well tolerated and has anextended half-life when compared with untagged. Thisagent is now being applied to rat models of arthritisand other inflammatory diseases.

32 High prevalence of monoclonal gammopathy in pa-tients presenting with acquired angioedema of type II: aseries of 19 patients

V. Fremeaux-Bacchi1, M.T. Guinnepain2, P. Cacoub 3,M.A. Dragon-Durey1, W.H. Fridman 1, M.D.Kazatchkine1

1Departement d’Immunologie, Hopital EuropeenGeorges Pompidou, Paris, France ; 2Centre d’allergolo-gie clinique, Institut Pasteur, Paris, France ; 3Ser�ice deMedecine Interne, Hopital Pitie-Salpetriere, Paris,France

The syndrome of acquired angioedema type II ischaracterized by the presence of anti-C1INH antibod-ies and of a circulating low molecular weight function-ally inactive form of C1 INH in plasma. This subtypeof C1 inhibitor deficiency will lead to recurrentepisodes of angioedema. We report on the clinical andimmunological features of a series of 19 patients withacquired type II C1 INH deficiency. At the time ofdiagnosis, two thirds of the patients exhibited a mono-clonal immunoglobulin peak in serum that was of thesame isotype as that of the circulating anti-C1 INHantibody. Three of the 12 patients with monoclonalgammopathy developed a malignant lymphoprolifera-tive disease within 4 years after the first symptoms ofangioedema. Our observations established that ac-quired C1 INH deficiency of type I and type II shouldnot be distinguished on the presence or absence oflymphoproliferation. During long-term follow-up, ap-

proximately 30% of monoclonal gammopathy of unde-termined significance (MGUS) patients developmultiple myeloma or a related plasma cells disorder.Since none of the features defining MGUS is uni-formly helpful in predicting the risk for malignantdisease, patients should be followed up on a regularbasis indefinitely. Recognition of the syndrome is im-portant not only for diagnostic purposes but for inves-tigating and treating any associated underlyinglymphoproliferative disorders.

33 Improving the effectiveness of complement in hostdefense, a novel herpes simplex virus (HSV) vaccinestrategy

H.M. Friedman, Y. Chang, M. Jiang, K. Judson, J.Lubinski

Di�ision of Infectious Diseases, Department ofMedicine, Uni�ersity of Pennsyl�ania, Philadelphia, PA,USA

HSV type 1 (HSV-1) encodes two complement inter-fering glycoproteins, gC and gE. gC binds C3b andblocks C5 and P binding to C3b, thereby acting as aninhibitor of the complement cascade, while gE blocksC1q binding to antibodies on the virus. HSV-1 wild-type virus is resistant to antibody-enhanced comple-ment neutralization because of gC and gE; however,HSV-1 mutant virus lacking gC and gE complementinteracting domains is 3–4 log10 more susceptible toantibody-enhanced complement neutralization in vitro,and �1000-fold less virulent that wild-type virus invivo.

We postulate that blocking complement interactingdomains on wild-type HSV should greatly increase theactivity of complement in host defense and improvethe effectiveness of antibodies produced to vaccinessuch as the GlaxoSmithKline glycoprotein gD vaccinenow under development. We examined whether it ispossible to block wild-type HSV-1 complement inter-acting domains on gC. High titers of gC antibodiesare produced during natural HSV-1 infection; how-ever, these antibodies do not block gC–C3b interac-tions. Nevertheless, several monoclonal antibodiesblock this interaction. We used a murine flank modelto examine whether monoclonal antibody to the gCC3b binding domain combined with antibody to theC5/P domain modifies wild-type HSV-1 virulence. An-tibodies markedly reduced virulence in a complement-dependent fashion, since antibodies were effective in


complement intact animals, but not in C3 knockoutmice. We conclude that it is possible to block gCcomplement interacting domains, and hypothesize thatthis approach may greatly improve vaccine efficacy byenhancing complement activity in host defense againstHSV.

34 Membrane attack complex (MAC) on Candidamodulates expression of human polymorphonuclearleukocyte (PMN) surface molecules-further evidence fora MAC receptor on PMNs

A. Fuchs1, C.P. Lell1, C. Lass-Florl1, C. Speth1, H.Stoiber1, M.P. Dierich1, A. Orren2, R. Wurzner1

1Institute of Hygiene and Social Medicine, Uni�ersityof Innsbruck, Innsbruck, Austria ; 2Department of Mi-crobiology, National Uni�ersity of Ireland, Galway,Ireland

Complement is a highly redundant innate immunesystem activated by either of the three pathways. Forimmune defence against fungi, displaying a dense cellwall, activation of phagocytes, mainly by chemotaxis,and opsonisation are considered to be much moreimportant than lysis. Interestingly, both former mech-anisms do not rely on only one component, C5a, butalso C4a and C3a attract phagocytes, whereas opsoni-sation proceeds via C1q or fragments derived fromactivated C3.

Recently, we have shown that polymorphonuclearleukocytes (PMNs) secrete C7 and that this releasewas triggered when PMNs were incubated with Can-dida which had been opsonised with normal humanserum but not when the yeast had been opsonisedwith serum lacking the ability to generate a MAC,i.e. C6 deficient serum.

We now show that membrane attack complex(MAC) on the surface of Candida also represents anadditional trigger for a selective modulation of PMNsurface antigens. While Candida opsonised with nor-mal human serum upregulated CD66b and downregu-lated CD16 on the surface, this CD66b-upregulationwas significantly less pronounced when C6 deficientserum, C7 deficient sera, or C6/C7-depleted serumwere used for the opsonisation whereas the CD16 ex-pression was not significantly altered-C3 depositionwas pronounced and similar for all sera.

These findings corroborate our hypothesis thatMAC on the fungal surface may represent an addi-tional trigger for PMNs, suggesting a new role forthe MAC on target membranes as modulator ofPMN functions locally at the site of inflammationand point towards a MAC-binding protein/receptoron PMNs.

35 Novel anti-factor D monoclonal antibody inhibitscomplement and leukocyte activation in a baboon modelof cardiopulmonary bypass

M. Fung1, A. U� ndar2, H.C. Eichstaedt3, F.J. Clubb,Jr.3, M. Lu1, J.E. Bigley3, W.K. Vaughn3, C.D.Fraser, Jr.2,4

1Tanox, Inc., Houston, TX, USA ; 2Michael E. De-Bakey Department of Surgery, Baylor College ofMedicine, Houston, TX, USA ; 3Cullen Cardio�ascularSurgery Research Laboratories, Texas Heart Institute,Houston, TX, USA ; 4Congenital Heart Surgery Ser-�ice, Texas Children’s Hospital, Houston, TX, USA

Patients undergoing cardiopulmonary bypass (CPB)frequently manifest a generalized systemic inflamma-tion that is associated with complement and leukocyteactivation. We investigated the effects of inhibition ofthe alternative complement pathway on systemic infl-ammation and tissue injury, using a novel anti-factorD monoclonal antibody, Mab 166-32, during hy-pothermic CPB in baboons. Seven adult baboonswere treated with a single injection of Mab 166-32 at5 mg/kg and another seven animals with saline ascontrol. Blood samples were collected before CPB,during CPB and 1, 2, 3, 6, and 18 h after CPB.Assays were performed on the blood chemistry andthe activation of complement and leukocytes. Ascompared with the control animals, Mab-166-32-treated animals showed statistically significant reduc-tion in plasma levels of Bb, C3a and IL-6 and in theexpression of CD11b on neutrophils and monocytes.Plasma levels of CK, LDH, and creatinine were alsosignificantly reduced in the antibody group. In con-clusion, inhibition of complement activation via thealternative pathway by anti-factor D Mab 166-32 sig-nificantly reduces leukocyte activation and myocardialand renal injury in our baboon model of CPB.

36 E. coli-induced oxidative burst is complement depen-dent and can be abolished by different modes of com-plement inhibition

H. Fure1, D. Christiansen1, O.L. Brekke2, G.Bergseth1, K.T. Lappegard3, J.D. Lambris4, M.Fung5, T.E. Mollnes1

1Deptartment of Immunology, Nordland Central Hospi-tal, Bodø, and Uni�ersity of Tromsø, Norway ;2Department of Clinical Chemistry, Nordland CentralHospital, Bodø, and Uni�ersity of Tromsø, Norway ;3Medical Department, Nordland Central Hospital,Bodø, and Uni�ersity of Tromsø, Norway ; 4ProteinChemistry Laboratory, Uni�ersity of Pennsyl�ania,USA ; 5Tanox Inc., Houston, TX, USA


Complement-mediated leukocyte activation inducesoxidative burst with the release of reactive oxygenspecies toxic not only to microbes, but also to the host.We developed a novel human whole blood model usingthe specific thrombin inhibitor lepirudin as anticoagu-lant. In contrast to heparin, lepirudin had no effect oncomplement and was found to be an ideal anticoagu-lant for studying the interactions between various infl-ammatory mediators in whole blood incubated withEscherichia coli. Granulocyte oxidative burst wasquantified by a flow cytometric method based on oxi-dation of dihydrorhodamin 123, and the role of com-plement was investigated by a panel of specificcomplement inhibitors. Anti-C2 reduced oxidativeburst moderately, indicating some activation throughclassical and/or MBL pathway, whereas the effect ofanti-factor D was more pronounced. Combining anti-C2 and anti-factor D virtually abolished oxidativeburst, as did the C3 inhibitor Compstatin. Blocking C5cleavage by anti-C5 revealed that the oxidative burstwas mainly mediated by terminal pathway activation.Finally, a C5a receptor antagonist demonstrated theC5a receptor to be crucial for the induction of oxida-tive burst. In conclusion, E. coli-induced granulocyteoxidative burst is totally complement dependent. Dif-ferent strategies for complement inhibition is described.

37 Local C4 synthesis restores impaired immune re-sponse to T-dependent antigens

M. Gadjeva, A. Verschoor, H. Jezak, M. Caroll

Center for Blood Research, Har�ard Medical School,221 Longwood A�e., Boston, MA 02115, USA

Mice with disrupted C4 locus (C4−/− ) like C4deficient guinea pigs, have an impaired immune re-sponse to T-dependent antigens, which is characterizedby reduced number and size of germinal centers. Wehave generated C4 chimeras with wild type bone mar-row derived from MHC matched littermates to test iflocal synthesis of C4 in the secondary lymphoid com-partments (e.g. spleen and lymph nodes) can restorethe humoral response. C4 chimeras were immunizedwith NP-keyhole limpet hemocyanin (NP-KLH) i.v. orinfected with Herpes Simplex Virus-1 (HSV-1)intradermally.

The engraftment re-established local C4 synthesis inthe spleen and lymph nodes as detected by in situhybridization with C4 RNA probes and immunochisto-chemical analysis. More importantly, the engraftmentrestored the humoral immune response to both NP-KLH and virus. MOMA2+ macrophages were a ma-jor source of C4 synthesis as determined by reverse

transcriptase-polymerase chain reaction (RT-PCR).Regulation of C4 synthesis by macrophages in vivo isunknown. However, results with cell lines implicateinvolvement of IFN� stimulation. Experiments are inprogress to clarify C4 regulation in vivo.

Activation of complement via the classical pathwayleads to antigen tagging with C4/C3 activation frag-ments, which is important for co-receptor signaling onB-lymphocytes and antigen trapping on follicular den-dritic cells. The data is consistent with the hypothesisthat complement activation via the classical pathway isrequired for enhancing the T-dependent response toinert antigens injected i.v. or infectious virus intro-duced intradermally.

38 The murine C1r/C1s gene family cluster

G. Garnier1, A. Circolo2, Y. Xu2 and J.E. Volanakis1,2

1Biomedical Sciences Research Center A. Fleming, Vari,Greece ; 2Di�ision of Clinical Immunology and Rheuma-tology, Uni�ersity of Alabama at Birmingham, Birming-ham, AL, USA

The region of the human chromosome 12 encom-passing the C1r and C1s genes is still incompletelycharacterized. In the mouse, both genes map near thetelomeric end of chromosome 6, but only cDNA se-quences (partially for C1s), are available.

Evidence for duplication of both C1r and C1s genesin the mouse was obtained from Southern blot analysisof genomic DNA. A 120 kb clone containing themurine homologues of the human C1r and C1s genesand two C1r-related genes was isolated from a BAClibrary. As in the humans, the C1r and C1s genes areoriented 3� to 3�, 8 kb apart and have a similar exon/in-tron organization in relation to their protein domains.The ORFs for C1r and C1s have 81 and 77% nucle-otide identity and 80 and 73% amino acid identity,respectively, with their human counterparts. The tran-scriptional initiation sites were identified by cap-specificRACE analysis of the mRNAs. Tissue distribution ofconstitutive and LPS-regulated mRNA expression ofthe two genes is almost identical, suggesting that theyare controlled by similar or shared mechanisms. One ofthe C1r-related genes, encodes a truncated C1r-likeprotein and lies less than 5 kb from the 5� end of theC1r gene in the same orientation. The other C1r-re-lated gene cross-hybridizes with C1r probes and itslocation and orientation within the cluster is understudy. To our knowledge, this is the first evidence ofC1r and C1s genes being part of a larger cluster ofstructurally related genes.


39 A novel murine complement-related gene encoding aC1r-like protein

G. Garnier1, A. Circolo2, J.E. Volanakis1,2

1Biomedical Sciences Research Center A. Fleming,Vari, Greece ; 2Di�ision of Clinical Immunology andRheumatology, Uni�ersity of Alabama at Birmingham,Birmingham, AL, USA

A gene encoding a C1r-like protein (C1r-lp) wasisolated from a murine genomic library. The gene islocated less than 5 kb upstream of the C1r gene, inthe same transcriptional orientation and comparedwith C1r carries a large deletion. It is expressedmainly in the liver and to a lesser extent in othertissues in a pattern similar to that of C1r and C1s.Full-length open reading frame cDNA clones predicta 50.6 kDa protein with 48% overall amino acid iden-tity to C1r. C1r-lp consists of a putative leader pep-tide followed by a CUB module, a truncatedcomplement control protein module, a connecting seg-ment (CS)-like region and a serine protease (SP) do-main sharing 60% residue identity with C1r. Alongwith C1r, C1s, MASP-2 and MASP-3, C1r-lp belongsto the AGY group of serine proteases as it containsan intronless SP domain featuring a valine at position+3 of an AGC-encoded active site serine and lacksthe histidine loop disulfide bridge. The catalytic triadresidues and the cysteines of the CS region and SPdomain are conserved. However, a serine residue ispresent at the bottom of the primary specificitypocket at position 189 (chymotrypsinogen numbering)instead of the typical aspartic of SP with trypsin-likespecificity. Furthermore, C1r-lp lacks the highly con-served activating cleavage site arginine and the con-served sequence at the aminoterminus of active SP.Thus, C1r-lp, although highly homologous to C1r,may lack proteolytic activity, perhaps serving a regu-latory function in the classical pathway.

40 Association of mannose-binding lectin gene variationwith systemic lupus erythematosus and disease severityin a population based cohort

Peter Garred1, Anne Voss2, Hans O. Madsen1, PeterJunker2

1Tissue Typing Laboratory-7631, Department of Clini-cal Immunology, Rigshospitalet, Copenhagen, Den-mark ; 2Department of Medicine, Rheumatology unit,Odense Uni�ersity Hospital, Odense, Denmark

Objecti�e: In hospital-based cohorts low serum lev-els of mannose-binding lectin (MBL) due to MBLvariant alleles have been shown to be a predisposingfactor for systemic lupus erythematosus (SLE) as wellas a risk factor for secondary infections. However,the importance of MBL for SLE in population-basedcohorts is unknown.

Methods: MBL alleles and serum concentrationswere determined in 99 patients with SLE recruitedfrom a representative Danish region. Patients wereclassified according to the 1982 revised ACR criteriaas Definite SLE (D-SLE) (n=77) fulfilling �4 crite-ria and Incomplete SLE (I-SLE) (n=22) with�4 criteria. As controls served 250 healthy volunteers.

Results: MBL variant alleles were observed in48.5% of all SLE patients compared with 37.2% ofthe controls (P=0.07), while the correspondingfigures in D-SLE and I-SLE were 51.9 and 36.4%,respectively (P=0.02 and P�0.99). The time periodfrom the diagnosis of SLE until the D-SLE definitionwas met was shorter in MBL variant allele carriers(P=0.04) and they had higher disease activity (ESRand SLEDAI) in a prospective follow-up period of 2years (P�0.04). MBL variant allele carriers had anincreased risk of acquiring complicating infections(P=0.03, odds ratio=2.8, 95% CI, 1.1–7.1) and theannual number of infections was about two timeshigher (P=0.02). The carriers of MBL variant alleleswere particularly prone to have respiratory infections(P=0.0006).

Conclusion: In SLE patients from a representativearea of Denmark an increased frequency of carriersof MBL variant alleles was found provided that theyfulfilled �4 ACR criteria. Moreover, the presence ofMBL variant alleles was associated with increasedrisk of disease activity and of complicating infections.Thus, MBL deficiency may not predispose to SLE assuch, but is rather a disease modifier associated withdisease severity.

41 Ectosomes expressing DAF, MCP, CR1 and L-se-lectin are released by PMN and bind to monocytic andendothelial cells

O. Gasser, C. Hess, S. Miot, J.A. Schifferli

Department of Research, Uni�ersity Hospital Basel,Basel, Switzerland

Ectosomes are microvesicles released from the cell-surface of activated polymorphonuclear leukocyte(PMN). They are found in vivo, in the circulation ofpatients with sepsis, as well as in extravascular spaces


at the site of local inflammation. Using a membrane-interchelating dye combined with mAb, we analysedthe expression of various proteins by ectosomes. Ec-tosomes were found to express complement-regulatorymolecules (CR1, DAF and MCP), adhesion-molecules(L-selectin, LFA-1/CD11a, Mac-1/CD11b), HLA classI, Fc�RIII and CD66b. By contrast, CD14, Fc�RIIand uPAR were absent, indicating a specific selectionprocess to take place at the time ectosomes areformed. Furthermore, enzymatically active MMP-9and myeloperoxidase (MPO) as well as elastase andPR3 were detected on ectosomes. Taking advantageof MPO deficient PMN it was possible to demon-strate that ectosomes acquire soluble MPO from thefluid-phase. Binding of annexin-V proved thatectosomes expose phosphatidyl-serine in the outerleaflet of the membrane-bilayer. Ectosomes wereshown to bind specifically to a monocytic cell line(THP-1) and to endothelial cells (HUVEC) byfluorescence microscopy and FACScan-analysis.Taken together these data suggest that ectosomes aremultifunctional units, capable of binding to cell-sur-faces. Whether they modify the function of cells re-mains to be studied.

42 A syngeneic rat colorectal carcinoma model to studythe effect of membrane bound complement regulatoryproteins on mAb-immunotherapy

K.A. Gelderman, W. Bruin, P.J.K. Kuppen, G.J.Fleuren, A. Gorter

Department of Pathology, Leiden Uni�ersity MedicalCenter, Leiden, The Netherlands

An effective local inflammatory response on tumorcells can be induced by complement activation. Thelevel of complement activation on these cells is regu-lated by expression of membrane-bound complementregulatory proteins (mCRP). Riethmuller et al. (1998)have shown that 17-1A monoclonal antibody (mAb)-immunotherapy is effective as an adjuvant therapy forcolorectal carcinoma. However, the magnitude of thiseffect may be limited due to overexpression of mCRPon colorectal carcinoma cells. Juhl et al. (1997) haveshown that conjugating cobra venom factor (CVF) to17-1A increased the amount of C3 deposited on thetumor cells. Our aim is to develop an experimentalanimal model to study the inhibitory effect of mCRPon mAb-immunotherapy in vivo. In the presentstudy, we have measured the expression of mCRP on

the rat colorectal carcinoma cell line CC531 and wehave compared the effect of the anti-CC531 MG4mAb with an MG4-CVF conjugate on C3 depositionon and lysis of CC531 cells. We have found thatMG4-CVF was a more efficient activator of the com-plement system than MG4. MG4-CVF was able toactivate the complement system via the classical aswell as the alternative pathway. Furthermore, wehave injected rats bearing syngeneic CC531 tumorswith MG4 isotype switch variants (IgG1 and IgG2a).MG4 was shown to home to the tumor site. There-fore, this syngeneic colorectal model offers an oppor-tunity to study the effect of mCRP on mAb-immuno-therapy in vivo.

43 The effect of gut hypoperfusion on complement andcontact system activation during orthotopic liver trans-plantation in man

G. Gobbo, E. Rossi, C. Donarini, F. Macera, A.Agostoni, L. Bergamaschini

Ospedale Maggiore, IRCCS, Uni�ersity of Milan, Mi-lan, Italy

Complement and contact system activation isthought to play a key role in reperfusion tissue injuryduring orthotopic liver transplantation (OLT). We in-vestigated the role of the grafted liver and the gut inactivation of the above systems in 20 subjects withend stage chronic liver disease, undergoing to OLT.

Methods: Blood samples were collected prior to thestart of surgery (T0) from central venous catheter,from portal vein immediately before declamping (T1),from hepatic vein 10 min after portal vein declamping(T2) and at the end of surgical procedure (T3) fromcentral venous catheter. Complement and contact sys-tem activation was assessed by measuring plasma lev-els of sC5b-9, C3a, and of thrombin–antithrombincomplexes (TAT) and factor XIIa, respectively.

Results: Before (T1) and after reperfusion (T2, oc-casionally T3) plasma levels of C3a, sC5b-9, TATand FXIIa were significantly higher that those at T0.However, the increase of plasma concentration wasmaximum between T0 and T1.

Conclusions: These data confirm that complementand contact system activation occurs during OLT.However, the finding that hypoperfusion of the gut,secondary to clamping of the portal and inferiorcaval veins, plays a major role in such an activationsupports the use of complement inhibitors before andduring gut hypoperfusion.


44 Mast cells mediate the inflammatory response inimmune complex peritonitis independent of TNF-� and KC

J. Godau, T. Heller, A. Kola, A. Klos, J. Kohl

Institute of Medical Microbiology, Hanno�er MedicalSchool, Hanno�er, Germany

Polymorphonuclear leukocyte (PMN) recruitment inthe reverse passive Arthus reaction model of IC peritoni-tis in BALB/c or C57BL/6 mice depends on activationof the C5a receptor or Fc�Rs I and III (Heller et al., J.Immunol. 1999, 162:5657 and 163:985). Activation ofFc�RIII leads to release of TNF-�, which, surprisinglydoes not contribute to peritoneal PMN influx. In con-trast, TNF-� release from mast cells of Kit +/+ mice5 min after Ag/Ab application was reported to be crucialfor PMN migration, and was absent in mast cell deficientKitW/KitW−v mice (Zhang et al., Science 1992,258:1957). We found no TNF-� peak between 2 and 10min in peritoneal lavage fluid of Kit +/+ and KitW/KitW−v mice after onset of IC peritonitis, but the samekinetic as in C57BL/6 or BALB/c mice with a TNF-�peak after 90 min. These data suggest that TNF-� is notcrucial for PMN migration. In agreement with Zhang etal. virtually no PMNs were found in peritoneum of KitW/W−v mice. Treatment of C57BL/6 mice with com-pound 48/80 leading to mast cell ‘depletion’, i.e. degran-ulation of mast cells for several days, did not reducePMN migration, neither did depletion of peritonealmacrophages by clodronate.

Huge amounts of C-X-C chemokine KC were foundin peritoneal lavage of Kit +/+ , Kit W/W−v, andclodronate or compound 48/80 treated C57BL/6 mice 90and 120 min after onset of IC peritonitis and stillremained high after 6 h, which is different to TNF-�?(zero at 6 h). Thus, mast cells but not the macrophagesare critically involved in PMN recruitment by mecha-nisms independent of TNF-� or KC.

45 The glomerulonerhritis clinical variants and the comple-ment system in children

A. Goraynova1, H. Goloshjapova2, I. Popova2

1Medical Academy, Voronezh, Russia ; 2Regional Chil-dren’s Hospital, Voronezh, Russia

It is known the complement decrease is the typicalfeature of two clinical variants of glomerulonephritis(GN) when the arterial hypertension (AH) and hema-turia (H) are presented, nephritic syndrome (NiS) andnephrotic syndrome with hypertension, hematuria(NS+AH+H). The nephrotic syndrome (‘minimalchange disease’) and GN with isolated urinary syndrome

do not have hypocomplementemia. We studied thecomplement level in 268 patients with GN. There were41.3�3.9 CH100 in nephritic syndrome, 46.6�2.7 innephrotic syndrome with hypertension, hematuria,64.5�2.0 in isolated urinary syndrome and 64.3�4.0 innephrotic syndrome. We found out that the most patientsyounger 4 years had GN with NS and the high comlpe-ment level. There are the connection between Streptococ-cal infection and GN. The high level of antistreptococcalantibodies was found in 69% patients with NiS, 38.5%in NS+AH+H, 15% in NS, 60% in IVS. Thus, only twoclinical variants had Streptococcal infection, NiS, IVS.The hypocomplementemia and C1–C5 components de-crease were found in NiS, but there were normal levelsof complement and decrease of C1 and C3 componentsin IVS. None of the clinical variants of GN in patientsof the first 3 years of life did not have hypocomple-mentemia. According to this data, we think that thecomplement is very important in pathogenesis of GN inchildren elder than 4 years when the immune status hasbeen formed.

46 C4 allotypes in IgA deficient patients

A.S. Grumach1, R. Rutz2, M. Kirschfink2

1Laboratory of Medical In�estigation in Allergy andImmunology, Faculty of Medicine, Uni�ersity of SaoPaulo, Sao Paulo, SP, Brazil ; 2Institute of Immunology,Uni�ersity of Heidelberg, Heidelberg, Germany

Introduction: IgA deficiency (IgAD) is the most com-mon primary immunodeficiency. Although IgAD predis-poses to a variety of diseases, asymptomatic individualsare often found in screening the normal population. Theoccurrence of clinical manifestations as recurrent infec-tions, autoimmunity and allergy, has been associatedwith additional IgG subclass deficiencies but only fewsymptomatic IgAD had decreased IgG subclass levels inour population. We, therefore, decided to extend ouranalysis to C4 allotypes in order to possible correlationof the clinical manifestations with an impairment ofcomplement.

Methods: Thirty symptomatic IgAD patients (�7mg/dl) were evaluated for immunoglobulins (nephelome-try), IgG subclass (ELISA) and C4 allotypes serum levels(ELISA). Clinical data were correlated to results from thelaboratorial analysis.

Results: Patients (6/30) had decreased serum C4Alevels (recurrent infections (2); allergy (3); auto immunehepatitis (1); and 3/30 had lower C4B values (allergy (2),juvenile rheumatoid arthritis (1) compared with a normalcontrol group of healthy donors. IgG2 and IgG4 sub-classes were deficient in 2/30 patients and isolated IgG4concentrations were deficient in 8/30. Low serum levelsof C4 allotypes were not associated with a distinct pattern


of clinical manifestations, neither with infections norautoimmune processes.

Conclusion: Clinical symptoms in IgAD patients couldnot be related to partial deficiency of a specific C4allotype, although several patients had reduced serumC4A or C4B levels.

47 Continuous 48-h C1-inhibitor treatment followingreperfusion in patients with acute myocardial infarction

C. Erik Hack, Chris de Zwaan, Machteld Tissing, AppieH. Kleine, Hein J.J. Wellens, Marja P. van Dieijen-Vis-ser, Wim Th. Hermens

CLB, Sanquin Blood Supply Foundation, Amsterdam;Cardio�ascular Research Institute Maastricht,Maastricht, The Netherlands

Background: Local complement activation in ischemicmyocardium contributes to myocardial infrction size.Inhibition of complement activation by C1-inhibitor(C1inh) has been shown to reduce ischemic myocardialinjury in animal models. We studied the effects of C1inhin patients with acute myocardial infarction (AMI) in anopen-study and compared the effects of treatment withthe course of AMI in historical control patients.

Methods: Twenty-two patients with AMI receivedC1inh following reperfusion. Possible interference ofC1inh with thrombolytic therapy was prevented by lateadministration of C1inh, starting 6 h after the onset ofsymptoms. An initial intravenous loading dose of C1inhwas followed by continuous infusion for 48 h, using threeescalating dosage schemes, aiming at increasing plasmaC1inh levels 2- to 3-fold for at least 48 h. Efficacy oftreatment was estimated from C4 activation fragments.Myocardial injury was estimated from the area under theplasma curve (AUC) of the myocardial marker proteinstroponine T (TnT) and creatine kinase isoenzyme MB(CK−MBmass).

Findings: C1inh was well-tolerated, drug-related ad-verse events were not observed. Target plasma levels ofC1inh corresponded to values of 48.2�1.6 ml/kg forC1inh distribution space and 0.020�0.001 per h for theC1inh elimination constant (mean�S.E.M., n=22).Efficacy of C1inh was demonstrated by a significantdose-dependent reduction of C4 activation fragments(P=0.005). In 13 patients who received thrombolytictherapy between 1 and 2 h after onset of symptoms,average AUC was reduced by 36 and 57% (P=0.001) forTnT and CK−MBmass, respectively.

Interpretation: Our findings demonstrate that continu-ous 48-h treatment with C1inh provides safe and effectiveinhibition of complement activation after AMI and maysignificantly reduce myocardial injury, when given inaddition to reperfusion therapy. Our results warrant

double-blind studies on the effects of C1inh therapy inpatients with AMI.

48 Combined heterozygous deficiencies of the classicalcomplement pathway proteins C2 AN C4 (C2*Q0/C4A*Q0 and C2*Q0/C4B*Q0 deficiencies)

D. Hartmann1, V. Fremeaux-Bacchi2, A. Meyer1, B.Uring-Lambert1, M. Kazatchkine2, G. Hauptmann1

1Laboratory of Immunology, Uni�ersity Hospital, Stras-bourg, Paris, France ; 2Laboratory of Immunology andINSERM, U430 Paris, France

HLA haplotypes carrying C2 or C4 null alleles(C2*Q0, C4A*Q0, C4B*Q0) represent up to 20% of theHLA haplotypes in the general Caucasian population. Acombination of a haplotype containing C2*Q0 with ahaplotype containing C4A*Q0 or C4B*Q0 results in acombined heterozygous deficiency of C2 and C4. Thiskind of complement deficiency is not a rare event becauseit occurs in 1 out of 1000 people. We have reportedpreviously on several cases of this kind of deficiency (J.Clin. Immunol., 17: 176–84, 1997). According to ourexperience about 30% of the deficient individuals presentwith SLE or another autoimmune disorder. This defi-ciency represents, therefore, a strong marker of diseaseand, according to recent studies, intervenes directly in thepathogenesis of autoimmune disorders. This deficiency isgenerally not evidenced because it is difficult to beassessed and differentiated from complement consump-tion. However, this kind of deficiency should be sus-pected in patients presenting with a persistentcomplement profile of moderately decreased CH50, lowC2, low normal or low C4 and normal C3 levels.Additional analyses necessary to confirm this kind ofdeficiency include C4 polymorphism determination,molecular biology analyses of the C2 and C4 genes, HLAtyping of the patient and relatives. We shall presentseveral well documented and recently analysed cases ofsuch deficiencies.

49 Molecular basis of hereditary membranoproliferativeglomerulonephritis in factor H-deficient pigs

G.A. Hegasy1,2, T. Manuelian2, K. Hogasen3, J. Jansen3,J. Hellwage4, P.F. Zipfel1,2

1Bernhard Nocht Institute for Tropical Medicine, Ham-burg, Germany2Department of Infection Biology, Hans Knoell Institutefor Natural Products Research, Jena, Germany ; 3Instituteof Immunology, The National Hospital, Uni�ersity of


Oslo, Oslo, Norway ; 4Molecular Immunobiology Group,Hans Knoell Institute for Natural Products Research,Jena, Germany

Recently, we reported a hereditary deficiency of thecomplement-inhibitory protein factor H (FH) in pigs,which led to the development of lethal membranoprolif-erative glomerulonephritis type II in homozygous ani-mals. Here we report the molecular aspects of thisdeficiency. Expression analyses of the proteins inplasma by western blotting showed subtotal levels ofFH in diseased pigs, while expression of additionalmembers of the FH protein family was normal or evenincreased. Protein extract prepared from liver tissueshowed markedly increased FH protein levels in thediseased pigs but not in their healthy littermates. Im-munostaining with �-pig FH antiserum revealed moder-ate staining of the septa and weak reactivity in theparenchyma in healthy pigs, while a strong intracellularstaining was observed in the hepatocytes of the diseasedanimals. Sequence analyses identified two mutations inthe FH gene of diseased animals: One exchange [G1590 C] results in an amino acid change [Val 493 Leu]in SCR 9 and a second exchange [T 3610 G] leads to asubstitution of amino acid [Ile 1166 Arg] in SCR 20.Our data shows that in the diseased animals mutatedfactor H is synthesized but the secretion of the proteinis blocked. This block of factor H secretion is indicativefor the pathophysiological mechanisms of factor Hmutations reported recently in humans with hemolyticuremic syndrome.

50 Association of HIV-1 with erythrocytes in vivo

C. Hess1, T. Klimkait2, L. Schlapbach1, S. Sadallah1, G.Balestra1, C. Schafer3, V. Del Zenero3, M. Battegay4,J.A. Schifferli1

1Department of Research, Uni�ersity Hospital Basel,Basel, Switzerland ; 2Institute of Medical Microbiology,Uni�ersity of Basel, Basel, Switzerland ; 3PCR-Labora-tories, Viollier Institute, Basel, Switzerland ; 4Basel Cen-tre for Clinical HIV-Research, Uni�ersity HospitalBasel, Basel, Switzerland

In vitro, complement proteins deposited on HIV-1/anti-HIV-1 immune complexes (ICs) have been shownto target these complexes to the immune-adherencereceptor (CR1/CD35) on erythrocytes.

By use of a radiolabelled model-IC we established amethod to isolate erythrocyte-associated ICs in vivo.Purity of the erythrocyte-preparations was determinedby flow-cytometry. Leukocytes and erythrocytes

purified from 1 ml of whole blood (wb) of HIV-1-in-fected individuals were tested for their content in HIV-1RNA. Contaminating leukocytes could be excluded asthe source for HIV-RNA in the erythrocyte-prepara-tions. A number of 80 out of the 82 patients (98%)tested positive for HIV-1 RNA in erythrocyte-prepara-tions. Copy-numbers of HIV-1 RNA associated witherythrocytes/1 ml wb ranged from �20 to 82 878.HIV-1 RNA associated with erythrocytes correlatedwith the number of HIV-1 RNA-copies in plasma. Theplasma viral load was below detection limit (�20copies per ml) in 23/82 patients. Within this subgroup,erythrocyte-associated HIV-1 RNA was detected withcopy-numbers ranging from �20 to 82 878/erythro-cytes/1 ml of wb.

Chronic exposure of the study population to above-normal amounts of ICs was reflected by a low CH50 in40% (32 of 80), a below-normal number of CD35/ery-throcyte as well as an inverse correlation of the meannumber of CD35/erythrocytes and HIV-1 plasma-viremia. No difference in the anti-HIV-1 antibody pat-tern and no difference in C3 and C4-levels was foundbetween patients having high as compared with patientshaving low erythrocyte-associated HIV-1 RNA-copy-numbers.

In conclusion, HIV-1 is transported by erythrocytesin vivo. Whether binding of HIV-1 to erythrocytes invivo is complement mediated remains to be defined.

51 Characterisation of cell activation through cyt2 andcyt1 tail of MCP

Stephen Hiscox, Carmen W. van den Berg

Department of Pharmacology, UWCM, Heath Park,Cardiff CF14 4XN, UK

We have previously observed that MCP on U937cells is able to induce cell activation as observed by arelease of calcium from intracellular stores and tyrosinephosphorylation of several intracellular proteins. U937cells express MCP both with the cyt1 and with the cyt2tail. In order to investigate the contribution of eithertail to the cell activation events, we generatedtransmembrane anchored forms of CD59 (which isnormally not expressed on U937 cells) with either thecyt1 or the cyt2 tail. The construct with the cyt2 taildisplayed the strongest signalling capacity both in cal-cium release experiments and in tyrosine phosphoryla-tion. It had been shown previously that the cyt2 tail canbe phosphorylated by src-kinases. Src-kinases reside indetergent resistant microdomains and in order forproteins to become phosphorylated, they have to comeinto close proximity with these kinases. Using detergent


solubilisation followed by sucrose floatation we observed that the cyt1 and cyt2 constructs in their nativestate are easily solubilised by detergents but upon cross-linking become more detergent resistant and associatewith the src-kinase containing lipid microdomains, anobservation which may explain their signalling capacity.

52 Decay acceleration factor (DAF), complement receptor1 (CR1), and factor H dissociate the AP C3 convertase(C3bBb) via sites on the type A domain of Bb

D. Hourcade1, L. Mitchell1, L. Kuttner-Kondo2, M.Krych-Goldberg1, J.P. Atkinson1, M.E. Medof2

1Di�ision of Rheumatology, Department of Medicine,Washington Uni�ersity School of Medicine, St. Louis,MO, USA ; 2Case Western Reser�e, Department ofPathology and Medicine, Cle�eland, OH, USA

The AP C3 convertase, C3bBb(Mg2+), is subject toirreversible dissociation (decay acceleration) by threeproteins, decay accelerating factor (DAF), CR1, andfactor H. We have begun to map the factor B (FB) sitescritical to these interactions. We generated a panel of FBmutations, focussing on the type A domain because itcarries divalent cation and C3b-binding elements. C3bBbcomplexes were assembled with the mutants and sub-jected to decay acceleration. Two critical FB sites wereidentified with a structural model, (1) several mutationscentered at adjacent � helices 4 and 5 (Q335, Y338, S339,D382) caused substantial resistance to DAF and CR1-mediated decay acceleration but not factor H; (2) severalmutations centered at the � 1 helix and adjoining loops(especially D254G) caused resistance to decay accelera-tion mediated by all three regulators and also increasedC3b-binding affinity and C3bBb stability. In the simplestinterpretation of these results, DAF and CR1 directlyinteract with C3bBb at � 4/5; factor H likely interacts atsome other location, possibly on the C3b subunit.Mutations at the C3b/Bb interface interfere with thenormal dissociation of C3b from Bb, whether it isspontaneous or promoted by DAF, CR1, or factor H.

53 C4 � chain peptide interferes with the formation of theclassical pathway C2 convertase

J.M. Inal, J.A. Schifferli

Department of Research, Uni�ersity Hospital Basel, Basel,Switzerland

We previously described a peptide based on theextracellular domain one (ed1) region of Schistosoma

haematobium-trispanning orphan receptor (TOR) show-ing sequence homology to part of the human C4 � chain(C4 residues 225 to 251). The ed1 peptide and a derivativethereof interfere with complement activation by bindingto human C2. Ed1 Peptides and sham peptide were usedin a C2-dependent haemolytic assay using C2-deficientserum and purified C2. Ed1 Was found to give a totalinhibition of haemolytic activity at 0.01 nM. Isenman etal. (J. Immunol. 2000 165: 2518–27), have described anamino-terminal region of the C4 ��-chain important forinteraction of C4b with C2. From amino acid sequencehomology with Sh-TOR, which binds C2, we have beenable to predict a region within the � chain of the humancomplement protein, C4b which could also interact withC2. Compared with sham peptide, which did not inhibitcomplement, peptides based on the region F231–Y251 ofthe C4 �-chain showed a dose-dependent inhibition oflysis in the C2 haemolytic assay, with complete inhibitionat a concentration of 0.1 nM. Since in this assaycomplement inhibition was achieved by pre-incubationof C4 �-chain peptides with C2, and thus presumably byinhibiting the formation of the classical pathway C3convertase (C4b2a), we propose C4 F231–Y251 as anadditional C2-binding site on C4.

54 The complement anaphylatoxins co-stimulate IL-8production by human astrocytes in the presence of IL-1

A. Ischenko1, A.-C. Jauneau2, A. Zhakhov1, M.-T.Shouft2, I. Khajueva1, A. Trofimov1, M. Fontaine2

1Institute of Highly Pure Biopreparations, St. Petersburg,Russia ; 2INSERM U519, Rouen, France

Both the complement and cytokines are involved ininflammatory processes of immune and central nervoussystems. However a little is known about interactionbetween these systems in CNS. The potency of anaphy-latoxins C3a, C5a and C-terminal multiple-associatedpeptides MAP-C3a64–78 and MAP-C5a61–74 to regulateproduction of IL-8 induced by inflammatory cytokinesIL-1� and TNF� in astrocytes were investigated. For thispurpose human fetal astrocytes or glioblastoma cell lineswere incubated for 24 and 48 h with different mixturescontaining various concentrations of IL-1� or TNF�(0.5–1000 pg/ml) and fixed amounts of each of testedcomplement polypeptides. IL-8 enzyme linked immuno-sorbent assays (ELISA) of cell supernantants showedthat all tested anaphylatoxin molecules increased levelsof the IL-1�-mediated IL-8 secretion. At the sametime the anaphylatoxins did not affect IL-8 levelsinduced by TNF�. The highest concentrations of C5a(50 nM) and C3a (500 nM) up-regulated the levels


of IL-8 to 100%. The anaphylatoxins were able tostimulate the release of IL-8 even if IL-1� was used insub-inducible concentration 0.5 pg/ml. In other experi-ments, conducted with lower concentrations of anaphy-latoxins (1–100 nM), their potency to co-stimulate IL-8secretion were shown. The effects of these stimulatorscould be partly inhibited by pertussis toxin (PTX) andby IL-1ra. When using the PTX, only a contribution ofanaphylatoxins to the IL-8 release was inhibited. Sig-nificant inhibitory effect was observed after joint incu-bation of cells with IL-1ra, IL-1� and anaphylatoxins.The expression of IL-8 mRNA in astrocytes was stud-ied with using of anaphylatoxin molecules, and levels ofexpression was analyzed by ribonuclease protection as-say. After 4 h stimulation of astrocytes, the levels ofIL-8 mRNA were elevated by 2–10 times for 10 nM ofnatural C3a, C5a and of the MAP-peptides. Theseresults show that the molecules of activated comple-ment can up-regulate effect of IL-1 for production ofthe potent inflammatory chemokine IL-8 in CNS.

55 Binding of the pneumococcal HIC protein to SCRs8–11 of factor H helps serotype 3 S. Pneumoniae toevade complement attack and opsonophagocytosis

Hanna Jarva1, Robert Janulzcyk2, Jens Hellwage3, Pe-ter Zipfel3, Lars Bjorck2, Seppo Meri1

1Haartman Institute, Uni�ersity of Helsinki, Helsinki,Finland ; 2Uni�ersity of Lund, Lund, Sweden ; 3HansKnoll Institute, Jena, Germany

Streptococcus pneumoniae is an important cause ofupper and lower respiratory tract infections, meningitis,peritonitis, bacterial arthritis and sepsis. Despite antibi-otic therapy it still causes significant morbidity andmortality worldwide. The capsule is the most importantsingle virulence factor but other mechanisms also con-tribute. Serotype 3 pneumococci are known to be resis-tant to phagocytosis. On their surface they express thefactor H-binding inhibitor of complement (Hic) thatbinds complement factor H. In the present study, welocalized the binding site of Hic on factor H andstudied the functional consequences of this interaction.

Using a radioligand assay, surface plasmon reso-nance analysis, a microtiter plate binding assay andrecombinant fragments of factor H the binding site ofpurified Hic was localized to SCRs 8–11 on factor H.Hic thus binds to a novel microbial interaction site onfactor H. The only other ligand known so far for SCRs8–11of factor H is C-reactive protein, that also binds tothe pneumococcal C-polysaccharide.

The functional significance of the Hic-factor H inter-action was studied by a C3b cofactor assay and enzyme

linked immunosorbent assays (ELISA) to measure theformation of iC3b and terminal complement complexes(TCC). Hic-expressing pneumococci bound factor H,which acted as a cofactor for factor I in the cleavage ofC3b. In the ELISA assays, more iC3b and less TCCbecame formed on the surface of serum-incubated Hic-positive than Hic-negative pneumococci.

In conclusion, Hic binds strongly to SCRs 8–11 offactor H. Bound factor H remains functionally activeand stops complement activation at the C3 level. Thus,Hic is one of the factors with which serotype 3 pneumo-cocci avoid opsonophagocytosis and SCRs 8–11 repre-sent a novel functional region on factor H.

56 C3a and C5a anaphylatoxins induce upregulation ofneurotrophin expression by astrocytes

A.-C. Jauneau1, A. Ischenko2, P.Chan1, C. Patte3, A.Chatagner1, H. Vaudry3, M. Fontaine1

1INSERM U519, IFR 23, Faculte de Medecine et dePharmacie, Rouen, France ; 2Institute of Highly PureBiopreparation, St Petersburg, Russia ; 3INSERM U413,IFR 23, Faculte des Sciences, Rouen, France

C3a and C5a anaphylatoxins are two proinflamma-tory peptides generated during complement activationthat act through distinct Gi-protein coupled receptorsnamed C3aR and C5aR, respectively. Previously, ourlaboratory has shown that astrocytes expressed C3aRand C5aR constitutively and were able to produce afunctional complement system. In this study, we exam-ined the effect of anaphylatoxin stimulation on neuro-trophin expression. On human T98 cell line, bysemi-quantitative reverse transcription-polymerasechain reaction (RT-PCR), NGF principally but alsoBDNF and NT-3 mRNAs expression was markedlyincreased after 4 h of stimulation but not CNTF. Thesestudies were also done on rat primary astrocytes bymulti-probe RNase protection assay. We observed thatstimulation of rat astrocytes by anaphylatoxins inducedan increased of NGF mRNA expression but neither forCNTF nor BDNF. These responses were completelyinhibited by pretreating cells with pertussis toxin, whichconfirmed that the signal was mediated via Gi-protein.The NGF response was also investigated at the proteinlevel after stimulation of human T98 cell line by ana-phylatoxins. In comparison to non-stimulated cells, weshowed a weak increase of NGF secretion in superna-tants of stimulated cells.

The anaphylatoxin up-regulation of some neuro-trophin genes suggests that the complement might playa role in neuroprotection.


57 Characterization of the promoter of human procathep-sin-L gene, a proteinase which cleaves human C3, the thirdcomponent of complement and confers high tumorigenicand metastatic properties to human melanoma cells

Didier Jean, Nathalie Guillaume, Raymond Frade

INSERM U354, Centre INSERM, Hopital Saint-An-toine, 75012 Paris, France

We previously demonstrated that overexpression ofprocathepsin-L, a cystein protease which cleaves humanC3 (the third component of complement) in melanomacells increased their tumorigenicity and switched theirphenotype from nonmetastatic to highly metastatic cells.Indeed, enforced expression of procathepsin-L by humanmelanoma cells arms them with the ability to inactivatecomplement-mediated lysis and contributes to tumorgrowth and metastasis (Frade et al., Cancer Res., 1998).In this report we analyzed procathepsin-L mRNA ex-pression in human melanoma and found, using reversetranscription-polymerase chain reaction (RT-PCR), thatprocathepsin-L mRNA level was higher in metastaticthan in non-metastatic melanoma cells. These datasuggested regulation of procathepsin-L at the transcrip-tional level and led us to characterize the promoter ofhuman procathepsin-L. We cloned the 5�-flanking regionof the human procathepsin-L gene using a PCR-basedmethod for DNA walking on uncloned genome DNAand determined the nucleotide sequence of 3.2 kb up-stream from the translation site. Transient transfectionstudies using a luciferase reporter gene demonstrated thatthe 5�-flanking region contained a functional promoter.We identified promoter regions, which are important forprocathepsin-L gene expression and its regulation inhuman melanoma cells. We also analyzed transcriptionfactors, which bind to activating region of the procathep-sin-L promoter and regulate procathepsin-L expression.All these data allowed to clarify the regulation ofprocathepsin-L in highly metastatic human melanomacells.

58 Naturally occurring anti-C3b antibodies, binding to C3convertase precursors, may counteract nephritic factor

Emiliana Jelezarova1, Markus Schlumberger1, Peter J.Spath2, Salima Sadallah3, Jurg A. Schifferli3, MargaritaLopez-Trascasa4, Michael Kirschfink5, Hans U. Lutz1

1Institute of Biochemistry, Swiss Federal Institute ofTechnology, ETH-Zentrum, CH 8092 Zurich, Switzer-land ; 2ZLB Bioplasma AG, Bern, Switzerland ;3Department of Medicine, Uni�ersity Hospital Basel,Basel, Switzerland ; 4National Institute of Health, Madrid,

Spain ; 5Institute of Immunology, Uni�ersity of Heidelberg,Heidelberg, Germany

Patients with membranoproliferative glomeru-lonephritis type II (MPGN II) may have low C3 concen-trations due to C3 nephritic factors (C3NeFs) thatstabilize the C3 convertase and induce C3 consumption.In remission their C3 concentration normalizes andC3NeF activity, as measured by a hemolytic assay is low.Among nine patients from which we purified IgG wefound some in remission whose IgG yet bound three tosix times more to C3 convertase than normal IgG in anenzyme linked immunosorbent assays (ELISA). Anamount of 40–95% of their binding to C3 convertase,however, resulted from binding to C3b. The higher thetiter of anti-C3b, the lower was their C3NeF functionalactivity, as determined by a C3 convertase stabilizationassay (J. Immunol. Meth. 251 (2001) 45–52). IgG fromone patient in remission with 95% binding to C3bdestabilized C3 convertase and gave readings belownormal IgG. Thus, it appears as if remission could occurnot only by the loss of C3NeF, but also by upregulationof naturally occurring antibodies (NAbs) to C3b whichmay counteract C3NeF by binding to C3 convertaseprecursors. To test this hypothesis, IgG preparationsfrom patients were absorbed on immobilized C3. Ab-sorption increased the C3 convertase stabilizing effect by1.5 fold in IgG from a patient with 33% binding to C3b.This suggests that absorption on C3 eliminatedmolecules, which evidently counteracted C3NeF. In onepatient in remission with 95% binding to C3b, IgGabsorption on C3 eliminated the destabilizing effect onC3 convertase. Thus, anti-C3b NAbs may be involved inremission of patients with C3NeF and MPGN II.

59 C3NeF Determined by a solid phase immunoassay inpatients with membranoproliferative glomerulonephritis(MPGN), systemic lupus erythematosus (SLE) and poststreptococcal nephritis (PSGN)

E. Jelezarova1, H.U. Lutz1, M. Schlumberger1, S.Sadallah2, M. Trendelenburg2, M. Kirschfink3, M.Lopez-Trascasa4, P.J. Spath5, J.A. Schifferli2

1Institute of Biochemistry, Swiss Federal Institute ofTechnology, ETH-Zentrum, Zurich, Switzerland ;2Department of Research, Uni�ersity Hospital Basel,Basel, Switzerland ; 3Institute of Immunology, Uni�ersityHeidelberg, Heidelberg, Germany ; 4Unidad de Immunolo-gia, Hospital La Paz, Madrid, Spain ; 5ZLB BioplasmaAG, Bern, Switzerland

We have established in parallel different assays tobetter define C3NeF; two enzyme linked immunosorbent


assays (ELISAs) using as antigens C3bBb or C3b alonefixed onto a solid phase (antigenic NeF), and a functionalC3NeF assay (Jelezarova et al., 2001, J. Immunol. Meth.251, 45–52). C3NeF was considered to be present whenthe binding to C3bBb was significantly higher than toC3b. This antigenic C3NeF assay was tested on acollection of serum samples from patients with membra-noproliferative glomerulonephritis (MPGN) I–III. Pa-tients (9 of 21) had a positive C3NeF and in 20/21 casesour ELISA data corresponded to those obtained by ahemolytic assay. The antigenic C3NeF results obtainedwith serum were confirmed by using the purified IgGfractions in the nine patients with MPGN II. There wasalso a direct correlation (R2=0.89) between the antigenicand the functional C3NeF assay for these IgG fractions.

In 11 newly diagnosed patients with either MPGN IIor lipodystrophy 6/9 sera were C3NeF positive. In fourMPGN patients the C3NeF became negative in a follow-up sample. The antigenic C3NeF assay was negative in18/18 SLE patients, although nine had nephritis. Amongpatients with post streptococcal nephritis (PSGN) 2/18patients had C3NeF (however, the diagnosis was basedonly on serological data). The level of anti-C3b Ab washighly variable from one individual to another in alldiseases.

The antigenic assays defined here will be used toanalyze longitudinally the correlation between C3NeFlevels, anti-C3 antibodies and nephritis and may provideclinicians with a screening test for C3NeF, which mightreplace hemolytic assays.

60 The mechanism of HIV trapping in lymphoid tissue

L. Kacani1, W.M. Prodinger1, G.M. Sprinzl2, H. Stoiber1,S. Dopper1, F. Steindl3, M.P. Dierich1

1Institute of Hygiene and Social Medicine, Ludwig Boltz-man Institute of AIDS Research, Uni�ersity of Innsbruck,Innsbruck, Austria ; 2Department of Otorhinolaryngology,Uni�ersity Hospital, Innsbruck, Austria ; 3Institute ofApplied Microbiology, Uni�ersity of Agriculture, Vienna,Austria

Follicular dendritic cells (FDC) are essential con-stituents of germinal centres (GC) of lymphoid follicles,where they form a three-dimensional (3-D) network andtrap antigens complexed with immunoglobulins andcomplement on their surface. Immunohistochemicalstudies of lymphoid tissue (LT) have established that mostof HIV is produced by CD4+ T lymphocytes and issubsequently stored on the surface of FDC in a comple-ment-dependent manner. Quantitative image analysisdemonstrated that, after transition to the chronic phase

of HIV-1 infection, viral RNA is found to more than 98%in virions bound extracellularly to FDC.

Here, tonsillar tissue from HIV-infected individualswas studied to define the contribution of particularcomplement receptors (CR) to virus trapping in LT.CR2 (CD21) was identified as the main binding sitefor HIV in GC, since antibodies blocking the CR2-C3d/iC3b interaction effectively detached 62–77% ofvirions. Similarly, these antibodies removed sub-stantial amounts of virus from LT of patients whoseplasma viral load had been reduced by highlyactive antiretroviral therapy (HAART) below de-tectionlimit for more than 1 year. Interference with the attach-ment process by targeting CR2 appears feasible and maycontribute to the design of new therapeutic strategies.

This work was supported in part by grants from theAustrian Science Found (P14661-PAT), the 5th FrameWork of the EU (QLK 2-1999-01215), Ludwig-Boltz-mann Institute for AIDS Research and the State of Tyrol.

61 Structural requirements for the polymerization of thecomplement inhibitor C4b-binding protein

Lena Kask, Bjorn Dahlback, Andreas Hillarp, Anna M.Blom

Department of Clinical Chemistry, Uni�ersity Hospital,Lund Uni�ersity, Malmo, Sweden

C4b-binding protein (C4BP) is a plasma protein thatinhibits the classical pathway of the complement system.The most abundant form of C4BP in plasma consists of7 �-chains and one �-chain and both types of subunitscontain complement control protein (CCP) domains.Apart from the CCP domains, the subunits also have acarboxy non-repeat region that is involved in the poly-merization of C4BP. This part is believed to form anamphipathic helix with one polar/charged side and onehydrophobic side. In human, but not in some species, theC-terminal part also contains two cysteine residues whichmakes disulphide bridges. The polymeric structure ofC4BP makes it a more effective regulator of clusteredC3-convertase than monomeric C4BP. Pulse-chase anal-ysis of human kidney cells expressing C4BP showed thatalready after 5 min of chase C4BP is fully assembled.Treatment with Brefeldin A, Endoglycosidase H and lowtemperature showed that the assembly occurs in the ER.The pulse labelled C4BP was transported out of the cellin only 40 min. Furthermore, we constructed and ex-pressed C4BP mutants without the two C-terminal cys-teine residues as well as truncated variants lacking partsof the C-terminal region. Gel filtration analysis showed


that both disulphide bridges and the amphipathic helixare important for the polymerization of C4BP.The mutant without both C-terminal cysteine resi-dues migrated as a polymer during gel filtration inhigh molar salt showing that hydrophobic forces arecrucial for the interactions between the amphipathichelices.

In summary, we found that C4BP assembles inthe ER and both the disulphide bridges and thehydrophobic forces are important for the polymeri-zation.

62 Membrane cofactor protein (CD46) enhances NF-�B-activation during T cell-stimulation

C. Kemper, J.P. Atkinson

Rheumatology Di�ision, Department of Medicine, Wash-ington Uni�ersity School of Medicine, St. Louis, MO,USA

Membrane cofactor protein (MCP; CD46) is a widelyexpressed complement inhibitor. MCP also plays a rolein reproduction and serves as a receptor for multiplepathogens. Cross-linking of MCP on cultured humanmonocyte-macrophages by measles virus, C3b-dimers ormAbs leads to IL-12 down regulation. Further, bindingof Neisseria gonorrhoeae to MCP on human epithelialcells lines caused a calcium flux and cross-linking of MCPon T cells induces cell proliferation. In addition, follow-ing cross-linking with mAbs, one of the two cytoplasmictails of MCP (CYT-2) is tyrosine-phosphorylated by a srckinase (lck) in the Jurkat T cell line. To identify intracel-lular proteins that interact with CYT-2, we performed ayeast two-hybrid screen using a Jurkat cDNA library. AcDNA-fragment of the NF-�B inducing kinase (NIK), acentral molecule in the NF-�B pathway, was identifiedas a positive clone. Cross-linking of MCP with Abs orC3b-dimers alone did not induce NF-�B-activation.However, cross-linking of MCP in the presence ofanti-CD3 Abs or C3b-dimers caused a two-fold enhance-ment of activation of NF-�B as compared with CD3-ac-tivation alone and mAbs to MCP induced proliferationin purified primary CD4+ T cells. These data indicatethat MCP can function as a costimulatory moleculeduring T cell receptor stimulation by enhancing NF-�B-activation. Although we have not yet been able todemonstrate a direct interaction of MCP with NIK,co-transfection of a kinase-dead NIK cDNA fragmentdecreases NF-�B-activation in Jurkat cells. We are alsoanalyzing the impact of MCP-activation on additionaltranscription factors and signalling pathways known tobe involved in T cell-activation.

63 Characterization of complement deposition in relation-ship to regulatory protein expression in normal andAlzheimer’s disease brain

C. Kemper1, J. Price2, J. Longwith1, T. Oglesby1, J.P.Atkinson1

1Department of Medicine, Washington Uni�ersity Schoolof Medicine, St. Louis, MO, USA ; 2Department ofAnatomy and Neurobiology, Washington Uni�ersitySchool of Medicine, St. Louis, MO, USA

Senile plaques and neurofibrillary tangles are hall-marks of Alzheimer’s disease (AD). Senile plaques resultfrom the accumulation of �-amyloid and complementfragments are present in these lesions secondary toclassical pathway (CP) activation by the aggregatedamyloid fibers. Since complement activation on hosttissue is inhibited by the membrane complement regula-tors Membrane cofactor protein (MCP), decay accelerat-ing factor (DAF), CD59 and CR1, we first characterizedtheir expression. In healthy and AD brains, endothelialcells of vessels stained strongly (+ ) for the three widelyexpressed inhibitors and were weakly (+ ) for CR1.DAF, MCP and CD59 were also weakly expressed in theneuropil in normal and AD brain. The four inhibitoryproteins were not detected in association with amyloidplaques, cells surrounding plaques or neuronal tangles.Therefore, a major regulatory process that controlscomplement activation in most tissues is not available tomodulate complement activation in these AD lesions.Next, we analyzed complement deposition in AD brainsof individuals in which the clinical stage of the diseasehad been rigorously defined. In concert with priorstudies, C1q, C3c, C3d, C4c, C4d and C5b-9 werelocalized to diffuse, condensed and cored amyloidplaques. While the magnitude of complement depositioncorrelated with the severity of the AD, complementdeposition in plaques was present at the earliest stagesand even in normal individuals destined to develop AD.C3 and C4 fragments and C5b-9 were also detected onneuronal plaques and tangles, especially in late stages (3and 4) AD. The presence of C4c and C3c in plaquesfurther suggests a failure of adequate regulation ofcomplement deposition in AD.

64 Structure and surface distribution of erythrocyte CR1molecular species in rhesus monkey (Macaca mulatta)

A. Kisserli1, C.D. Muller2, B. Donvito1, A. Gimenez1, N.Godin1, M. Tonye Libyh1, V. Duret1, B. Reveil1, F.Philbert1, T. Tabary1, J.H.M. Cohen1

1Laboratoire d’Immunologie UFR Medecine, Physio-Pathologie Dysimmunitaire Humaine (PPDH), EA3309


Pole Biomolecules IFR 53, URCA 51092 Reims, Cedex,France. [email protected]; 2Pharmacologie etPhysico-Chimie des Interactions Cellulaires et Molecu-laires (UMR 7034 du CNRS), UFR de Sciences Pharma-ceutiques, Uni�ersite Louis Pasteur de Strasbourg, 74route du Rhin, B.P. 24, 67401 Illkirch, Cedex, France

CR1 (CD35, the C3b/C4b receptor) is present aterythrocyte (E) surface in Primates, allowing them to useE for opsonised immunes complexes (OIC) transporta-tion and clearing, contrary to other mammals which useplatelets for that purpose. In man, CR1 is a transmem-brane glycoprotein expressed at low level (200–1000antigenic binding sites per E) but characterised by aunique cluster distribution, enhancing multivalent inter-actions between CR1 and OIC. CR1 is more expressedon monkeys E than on human E. A shorter form of CR1has been described in various monkey species rankingfrom 65 to 165 kDa in Baboon and Rhesus, respectively,and reported to be GPI anchored in Baboon and Chimps.The expression of the ‘human type’ E-CR1 in monkeyshas not been extensively addressed, although reported inGorilla, Orangutan and Gibbon, due to the fact that‘short’ CR1 type accounted for most if not all E-CR1 inthe species studied. CR1 molecular species, surfacedistribution and density have been studied on Rhesusmonkey E by using a set of anti-CR1 monoclonalantibodies, Western-Blotting, flow cytometry and confo-cal microscopy. Rhesus monkeys expressed both atransmembrane ‘human-type’ CR1 like man in the samedensity range and with the same cluster distribution, aswell as a shorter (165 kDa) ‘monkey-type’, GPI-anchoredE-CR1, that was distributed according to a non-clusteredlinear pattern upon optical microscopy. Confocal mi-croscopy analysis revealed micro-granular aspect of thisapparently linear staining. A Rhesus monkey individualtotally deficient in ‘monkey-type’ CR1 who only exhib-ited the ‘human-type’ of CR1 was also observed. Short‘monkey-type’ E CR1 in monkeys is 5–20 fold moreexpressed at E surface than the ‘human-type’. The highlevel of expression of ‘monkey-type’ CR1 on Rhesusmonkey E surface is not due to the lack of ‘human-type’CR1. Respective role of the two CR1 molecular specieson monkey E remains to be investigated.

65 Regulation of the MBL-associated serine proteases(MASPs)

M. Kojima1, K. Hajela1, K. Whaley2, W. Schwaeble3,R.B. Sim1

1MRC Immunochemistry Unit, Department of Biochem-istry, Oxford Uni�ersity, Oxford, UK ; 2Medical School,

Uni�ersity of Kuwait, Kuwait ; 3Department of Microbiol-ogy and Immunology, Leicester Uni�ersity, Leicester, UK

The mannose-binding lectin (MBL)-associated serineproteases (MASPs) are homologous to the complementC1r and C1s proteases. We have studied the regulationof MASP-1 and MASP-2 by protease inhibitors inplasma. We have not yet extended this study to therecently-described MASP-3. Like activated C1r and C1s,MASP-1 and MASP-2 form stable complexes with theserpin, C1-inhibitor (C1inh). Attempts to isolate MASP-1 directly from serum using polyclonal antibody affinityresins reveal the presence of high levels of MASP-1–C1inh complexes in serum. Interaction of C1inh withactivated MASP-1 and MASP-2 bound to MBL-mannansurfaces causes dissociation of the C1inh–protease com-plexes from the mannan, in a way similar to the dissoci-ation of C1r–C1s–C1inh complexes from C1q bound toimmune complexes. Although interaction of �-2-macroglobulin (�2M) with MBL and the MASPs hasbeen reported, �2M does not inhibit interaction ofMASP-1 and MASP-2 with C1inh, suggesting that �2Mis not a significant inhibitor of the MASPs. Direct studieswith purified �2M, however, show that MASP-1, but notMASP-2, does cleave �2M, and MASP-1 can becomecovalently bound to �2M. It appears likely that, as withseveral other plasma proteases, the serpin is the signifi-cant physiological inhibitor, and reacts much faster withMASP-1 than does �2M.

66 The anaphylatoxins C3a and C5a contribute to bacte-rial clearance and survival in a murine model of sepsis

A. Kola1, M. Friedrichsen1, R.S. Ames2, A. Klos1, J.Kohl1

1Institute of Medical Microbiology, Hano�er MedicalSchool, Hano�er, Germany ; 2Glaxo SmithKline, King ofPrussia, USA

We established the caecal ligation and puncture (CLP)model of septic peritonitis in C57BL/6 mice using 21Gneedles, which resulted in a mortality of 70% on day 4.Six hours after CLP, bacterial counts were 5×107 cfu/mlperitoneal-lavage or 2×105 cfu/ml blood. In a peritoneallavage taken 1 h after CLP, 10% of cells were PMN. Thisnumber increased to 65% 6 h after CLP. The phagocy-totic potency of PMN was tested in blood and peritoneallavage by FACS-analysis using opsonized FITC labeledEscherichia coli. Six hours after onset of CLP, 80% ofcirculating PMN were able to phagocyte the bacteria,which is the same percentage as in sham treated animals.Only 20% of peritoneal derived PMNs ingested the


bacteria. Twenty-four hours after CLP, phagocytosis ofcirculating PMN was still 80%, whereas only 7% ofperitoneal PMNs showed phagocytosis.

Treatment of animals with the non-peptidic C3a-an-tagonist SB 290157 significantly decreased intraperi-toneal bacterial counts (6×105 vs. 5×107 cfu/mlperitoneal-lavage, P�0.05) 6 h after CLP. Bacterialcounts in blood were not affected. In addition, thepercentage of PMNs in the peritoneal lavage increasedfrom 65 to 80% (P�0.05), which may account for theimproved bacterial clearance. In addition, the effect ofC5aR blockade on animal survival was tested. Micetreated with a C5aR antagonist still suffered frominfection, but none of the animals died during the first4 days (n=4). These data suggest an important role ofC3a and C5a in bacterial clearance and animal survivalin septic peritonitis.

67 Complement resistance of Borrelia burgdorferi bybinding of complement regulatory proteins FHL-1/re-conectin and factor H

P. Kraiczy1, C. Skerka2, M. Kirschfink3, V. Brade1,P.F. Zipfel2

1Institute of Medical Microbiology, Uni�ersity Hospitalof Frankfurt, Frankfurt/M, Germany ; 2Department ofInfection Biology, Hans-Knoell-Institute for NaturalProducts Research, Jena, Germany ; 3Institute of Im-munology, Uni�ersity of Heidelberg, Heidelberg,Germany

Borrelia burgdorferi sensu stricto (s.s.), B. garinii andB. afzelii are the causative agents of Lyme disease.Organisms of the three genospecies show a strikingheterogeneity according to their susceptibility to com-plement-mediated bacteriolysis. Thus, we investigatedwhether this heterogeneity correlates with the acquisi-tion of the human complement regulators FHL-1/re-conectin and factor H. By analyzing 21 borrelialisolates we show that only serum-resistant B. afzelii andB. burgdorferi s.s. isolates bind specifically FHL-1/re-conectin and factor H, while serum-sensitive B. gariniiisolates display no binding activity. The surface-at-tached complement regulators retain cofactor activityfor factor I-mediated inactivation of C3b. By ligandblotting with FHL-1/reconectin a total of up to threeborrelial proteins termed c� omplement r� egulatora� cquiring s� urface p� roteins (CRASPs) are identified inserum-resistant Borreliae, which are responsible for thesurface attachment of this human immune regulator.By using recombinant deletion mutants the bindingdomains are localized to the C-terminal short consensusrepeats SCRs 5-7 of FHL-1/reconectin. By analyzing

Factor H binding to serum-resistant Borreliae addi-tional proteins could be identified in serum-resistantisolates only, which exclusively bind this human im-mune regulator. In summary, we describe an immuneevasion mechanism of serum-resistant B. burgdorferi, asthese pathogens acquire human complement regulatorsin order to control complement activation on theirsurface and to prevent formation of toxic activationproducts (supported by DFG Br 446/11-1 and Zi 342/5).

68 Structure-guided mutagenesis of a CR1 active siteidentifies a positively charged cluster as a part of theligand binding pocket

M. Krych-Goldberg1, P.N. Barlow2, R. Hauhart1, R.Mallin2, B.O Smith3, J.P. Atkinson1

1Rheumatology Di�ision, Department of Medicine,Washington Uni�ersity, St. Louis, MO, USA ;2Edinburgh Centre for Protein Technology, Uni�ersity ofEdinburgh, Edinburgh, UK ; 3Institute of Cell and Molec-ular Biology, Uni�ersity of Edinburgh, Edinburgh, UK

Each active site of CR1 requires three CCPs forfunction. Previous mutagenesis of site 2, composed ofCCPs 8–10 and duplicated in CCPs 15–17, revealedtwo potential contact points in the middle CCP andsuggested key residues in the last CCP but identified noresidues in the first CCP. We have recently solved thestructure of CCP 15–17 by nuclear magnetic resistance(NMR) and employed this structure to guide furthermutagenesis. Solvent exposed, clustered, positivelycharged amino acid side chains were considered to bethe best candidates for contact points because ourprevious work showed that interaction of CR1 andC3b/C4b occurs through ionic interactions betweenpositively charged residues in CR1 and negativelycharged residues in C3b/C4b. Since two potential con-tact points in CCP 16 (or 9) were known, amino acidsthat occupy the same face of the molecule were consid-ered to be the strongest candidates in CCP 15. Based onthis rationale, K912, K914, and R933 were all changed toE. To test if this cluster cooperates with K1016, a likelycontact point in CCP 16, we constructed an additionalmutant in which these charge-reversals of module 15were combined with a K1016E mutation. Lastly, to testthe role of another likely candidate in CCP 16, K964was altered in addition to the four substitutions above.We found that the mutations in CCP 15 have a pro-found effect on functionality of the active site. This isthe first example of using an experimentally determinedstructure of a complement regulator to rationally guidemutagenesis experiments.


69 The ancient complement system. III. MBL, clusterin,and vitronectin levels in breast milk

S. Kuipers1, M. Harmsen1, K. Takahashi2, H. van Dijk1

1Eijkman-Winkler Institute, Uni�ersity Medical CenterUtrecht, Utrecht, The Netherlands ; 2Department of Pe-diatrics, Massachusetts General Hospital, Boston, MA,USA

Aim of the study: To establish occurrence and levelsof ancient complement components in humancolostrum and mature breast milk.

Background: Complement components C3 and C4have been well studied in colostrum and breast milk.However, little is known about the occurrence andlevels of soluble terminal complement pathway in-hibitors vitronectin (VN) and clusterin (CL), and theleading protein of the lectin pathway, mannose-bindinglectin (MBL).

Materials and methods: Colostrum and breast milkobtained from healthy Dutch women were tested forCL, VN, and MBL levels by MoAb-based, protein-spe-cific, competitive enzyme linked immunosorbent assay(ELISA) procedures.

Results: All colostrum and breast milk samples stud-ied so far, contained substantial amounts of the threeproteins. Surprisingly, MBL was not associated withsecretory IgA, which is mannosylated and abundant inbreast milk.

Discussion: Our data extend data of colleagues whodemonstrated the presence of C3 and C4 in breast milk.The role of ancient complement components in breastmilk with regard to infant protection against inflamma-tory diseases of childhood, e.g. respiratory and gastro-intestinal infections needs further study.

70 The ancient complement system. IV. A novel strategyto quantify mannose-binding lectin (MBL) levels in serumand other body fluids

S. Kuipers1, P.C. Aerts1, M. Harmsen1, K.Takahashi2,J.M. Hament1, H. van Dijk1

1Eijkman-Winkler Institute, Uni�ersity Medical CenterUtrecht, AZU G04.614, NL-3584 CX Utrecht, TheNetherlands ; 2Department of Pediatrics, MassachusettsGeneral Hospital, Boston, MA, USA

Aim of the study: To estimate mannose-binding lectin(MBL) levels in serum and e.g. breast-milk samplesbased on a combination of a functional and a competi-tive immunosorbent assay.

Background: MBL is an ancient complement compo-nent involved in antibody-independent complement ac-tivation and opsonization of microorganisms with ahigh carbohydrate content. In Caucasians, there is quitea high prevalence of MBL deficiency of about 10%. Wedeveloped an analytical strategy to identify deficientsubjects in relatively large study populations (with e.g.recurrent infections).

Materials and methods: A functional assay was de-vised based on the principle of bystander hemolysis;cells of fresh baker’s yeast (Saccharomyces cere�isiae)were used as MBL activators, while serum of knownMBL-deficient subjects was employed as reagent serum;chicken erythrocytes were used as target cells. An ear-lier described, MoAb-based, competitive enzyme linkedimmunosorbent assay (ELISA) was used to quantifylow MBL levels.

Results: The functional assay was used as a firstscreening for samples of supposedly MBL-deficient sub-jects. Deficiencies were confirmed and furtherquantified by competitive MBL ELISA. The resultsobtained with the functional assay correlated well (P�0.0001) with data generated with the immunoenzymeassay.

Discussion: We conclude that the combination offunctional and immunochemic assays as described hereis very useful in defining MBL deficiency in relativelylarge study populations.

71 Hereditary angioedema responding to plasma but notto commercially available C1-inhibitor

P.J. Lachmann1, P.W. Ewan1, R.A. Harrison1, E.P.Wraight2

1MRC Molecular Immunopathology Unit, Cambridge,England, UK ; 2Department of Nuclear Medicine, Adden-brookes Hospital, Cambridge, England, UK

The patient is a British female with typical attacks ofHAE starting from the age of 25 but not diagnosed tillthe age of 40. She has low levels of C1 inhibitor and atypical complement profile. There is no antecedent fam-ily history but her two sons also have C1 inhibitordeficiency.

At the time of presentation she was having attacksinvolving the respiratory tract and attacks of boweledema. She was treated with a combination of stana-zolol and tranexamic acid with partial benefit. On threeoccasions attacks were treated with commercially avail-able C1 inhibitor concentrates (Immuno AG) withoutbenefit. Administration of fresh plasma was, however,successful in bringing the attacks to an end— thoughshe had an anaphylactic reaction to one plasmainfusion.


On laboratory testing the commercial C1-inhibitor hadidentical potency with freshly isolated C1-inhibitor. Toinvestigate the puzzling failure of the commercial in-hibitor to work in vivo a simultaneous double turn-overstudy was carried out, the commercial preparation beinglabelled with 131I and the freshly isolated inhibitor with125I. To our surprise the fractional catabolic rate of thecommercial preparation was much lower, i.e. 0.7% ofintravascular pool per hour compared with 2.0% for thefreshly isolated inhibitor.

We suggest that the method of rendering the commer-cial preparation virus free by heating it in the presenceof sugars gives the molecule a shell that hinders itstransport into the extracellular space. This finding alsoreinforces the evidence that the pathogenetic events inHAE take place outside blood vessels.

72 Cross-talk between the human complement classical andalternative pathways: evidence for a C4bBb ‘hybrid’ C3convertase

A. Laich, R.B. Sim

MRC Immunochemistry Unit, Department of Biochem-istry, Uni�ersity of Oxford, Oxford, UK

The complement system is activated via three or moredifferent pathways currently named the classical, thealternative and lectin pathways. There are several reportsthat components of different pathways can cross-interactwith each other, for example, C1r and C1s can interactwith mannose-binding lectin (MBL). Classical pathwayactivity has been reported in homozygous C2-deficientindividuals. Based on this observation, we investigatedcross-interaction between alternative (AP) and classical(CP) pathways.

A crucial step in activating the complement system isthe breakdown and turnover of C3, which is activated bythe C3-convertases of both the CP and AP (C4b2a andC3bBb, respectively). C3b and C4b are homologous asare C2 and FB. C2 was previously not known to interactwith C3b, nor FB with C4b.

Purified human C4b, C2, C3(H2O) and factor B wereused to look into formation of ‘hybrid’ C3 convertases.No interaction between C3b and C2 was found bySurface Plasmon Resonance (BIAcore®) whereas C4b–FB interaction was clearly detected. We were further ableto demonstrate that C4b supports the cleavage of factorB by factor D. Finally, cleavage of 125I-C3 by C4bBb wasevaluated and gave strong evidence that the ‘hybrid’convertase C4bBb can cleave and activate C3 in vitro.This activity is very low, but is consistent with some ofthe ‘C2-bypass’ observations of others.

73 Studies on the relationship between complement andleukocyte activation in blood circulated in uncoated andheparin coated PVC tubing

K.T. Lappegard1,2, G. Bergseth1, J.D. Lambris3, J.Riesenfeld4, T.E. Mollnes1

1Department of Immunology, Nordland Central Hospital,Bodø, Norway ; 2Department of Medicine, Nordland Cen-tral Hospital, Bodø and Uni�ersity of Tromsø, Tromsø,Norway ; 3Department of Pathology, Uni�ersity of Penn-syl�ania, PA, USA ; 4Carmeda AB, Stockholm, Sweden

Surface coating with heparin effectively inhibits theactivation of complement and leukocytes in blood ex-posed to artificial materials. We investigated to whatextent the complement inhibitory effect of end-pointattached heparin (CBAS) is responsible for the inhibitionof leukocyte activation in an in vitro model of extracor-poreal circulation. Blood was circulated in CBAS coatedpolyvinyl chloride (PVC) tubing, and in uncoated PVCtubing with or without the specific C3 inhibitor Comp-statin. The expression of CD11b (CR3) and formationof platelet-leukocyte conjugates were examined for gran-ulocytes and monocytes separately using flow cytometry.The substantial increase in granulocyte CD11b observedfor uncoated PVC was reduced by approximately 50%when Compstatin was added in doses that completelyblocked complement activation. Heparin coating, whichalso effectively inhibited complement activation, com-pletely prevented granulocyte CD11b expression. Mono-cyte CD11b expression was less complement dependent,whereas the heparin coating prevented CD11b expres-sion on monocytes as efficiently as on granulocytes. Theheparin coating completely abolished the PVC inducedformation of granulocyte- and monocyte-platelet conju-gates. Compstatin partly inhibited this conjugate forma-tion and the effect was more pronounced forgranulocytes than for monocytes. Thus, the leukocytecompatibility of this heparin coating is only partiallyexplained by its complement inhibitory properties.

74 Low molecular weight dextran sulfate acts as an‘endothelial cell-protectant’ and prevents complement acti-vation as well as ischemia/reperfusion injury in vitro

Thomas Laumonier1, Sonja Grunenfelder1, ElenaKorchagina2, Nicolai Bovin2, Paul Mohacsi1, RobertRieben1

1Heart Transplantation Laboratory, Cardiology, Uni�er-sity Hospital, 3010 Bern, Switzerland ; 2Shemyakin andO�chinniko� Institute of Bioorganic Chemistry, Moscow,Russia


Low molecular weight dextran sulfate (sulfatedpolyglucose, DXS) is known to block complement acti-vation and regulate the coagulation cascade by potenti-ation of C1-inhibitor. The effect of DXS and a similarsubstance (Fucoidan) were tested in different in vitromodels of pig-to-human xenotransplantation and is-chemia/reperfusion (I/R) injury.

A FACS assay with human endothelial cells (EC) orpig cells (PK15 as well as pig EC) was developed toanalyze complement deposition and cell survival afterI/R injury. Another experimental model using rat aortawas used to confirm these results in a more complextissue system.

Complement deposition time-dependently increasedon human EC after incubation on ice (‘ischemia’) fol-lowed by ‘reperfusion’ with human serum, but wasdose-dependently inhibited by DXS or fucoidan(IC50=0.7 mg/ml). In an in vitro xenotransplantationmodel using non ischemic PK15, DXS (0.2 mg/ml) wasable to inhibit C3c deposition when added during incu-bation with human serum (mean fluorescence (MF)=16) but was not effective when added prior toincubation (MF=50). In fact, fluorescence-labeledDXS preferentially bound to pig epithelial cells (PK15)that were pre-incubated with fresh human serum (dam-aged) as compared with untreated PK15. In addition,we observed specific DXS staining on ischemic (3 hincubation on ice) rat endothelium, but not on controlswithout cold storage.

Our results indicate that DXS is able to bind to‘injured’ endothelium and to protect it from I/R injury.We speculate that the negatively charged nature ofthese substances might play a role and allow them toact as a ‘repair glue’ to replace heparan sulfate proteo-glycans on the EC surface that are released uponactivation of the cells. Further investigations using anin vivo transplantation model are currently under wayto test the ability of DXS/Fucoidan to protect a donororgan from I/R injury and xenografts from hyperacuterejection.

75 Reduced FC�II receptor but normal complement re-ceptor in CPLA2-deficient PLB-985 like cells

R. Levy, A. Konforty, I. Hazan-Halevy, Z. Hazan-Ei-tan, R. Dana

Infectious Diseases Laboratory, Department of ClinicalBiochemistry, Faculty of Health Sciences, Ben-GurionUni�ersity of the Nege� and Soroka Medical Center,Beer She�a, Israel

We have previously established a model of cytosolicphospholipase A2 (cPLA2)-deficient differentiated PLB-

985 cells (PLB-D cells) and demonstrated thatcPLA2-generated arachidonic acid (AA) is essential forNADPH oxidase activation. In the present study weused this model to determine the physiological role ofcPLA2 in the regulation of Fc� receptors (Fc�R) andcomplement receptors (CR1 and CR3). Parent PLBcells and PLB-D cells were differentiated toward thegranulocyte or the monocyte lineage using 1.25%dimethyl sulfoxide (DMSO) or 5×10−8 M 1,25 dihy-droxyvitamin D3 (1,25(OH)2D3), respectively. Similarexpression of the four receptors studied (Fc�RI,Fc�RII, CR1 and CR3) was detected in parent PLBcells and in PLB-D cells differentiated toward the gran-ulocyte lineage. When differentiation was induced tothe monocyte lineage reduced expression of Fc�RIIwas detected in differentiated PLB-D cells, while theother three receptors were expressed normally. Theaddition of exogenous AA (10 �M) each day during 4days of differentiation induced by 1,25(OH)2D3 causedelevations in Fc�RII expression. The results suggestthat cPLA2-generated AA participates in regulating ofFc�RII expression during differentiation with1,25(OH)2D3 to the monocytic-like phenotype.

76 Activation of tyrosine kinase PYK2 in neutrophil-likecells stimulated by opsonized zymosan

R. Levy, Z. Shmelzer, I. Hazan-Halevy

Infectious Disease Laboratory, Department of ClinicalBiochemistry, Faculty of Health, Soroka Medical Centerand Ben Gurion Uni�ersity, Beer She�a, Israel

Neutrophils bind to opsonized microorganisms viaspecific receptors for IgG antibodies (Fc�R) and forthe complement protein C3b receptor (CR) which trig-ger phagocytosis and the oxidative processes. Experi-ments done in our laboratory show that the signaltransduction pathway induced by serum opsonized zy-mosan (OZ), is dependent on tyrosine phosphorylationupstream to MAP kinase. OZ activates two MAP ki-nase isoforms, p38 and ERK1/2. Both p38 and ERKare required to induce complete activation of cPLA2

generating arachidonic acid, which was shown to beessential for the stimulation of NADPH oxidase. Inaddition, stimulation of phagocytic cells by OZ inducesan elevation in intracellular calcium ion concentration.Proline rich tyrosine kinase 2 (PYK2) was recentlyfound in neuronal cells. It is activated by a variety ofextra cellular signals, which elevate intracellular cal-cium concentration. PYK2 undergoes tyrosine phos-phorylation for its activation. PYK2 was shown totrigger the activation of JNK, p38 and ERK in differ-ent systems. The aim of the present research was to


study the involvement of the tyrosine kinase PYK2 inthe signal transduction pathways initiated by OZ inphagocytes. The levels of PYK2 in PLB-985 cells in-creased during 4 days of differentiation reaching itshighest level on the fourth day. The Anti PYK2 rec-ognized two bands, suggesting that the PLB cells ex-press two PYK2 isoenzymes. The tyrosinephosphorylation of PYK2 was detected 10 s afterstimulation, peaked 3 min later, and totally decreasedat 30 min. Inactivated opsonized zymosan (iOZ) andOZ caused a similar tyrosine phosphorylation ofPYK2, indicating that the activation of PYK2 is me-diated by synergism between Fc�RIIA and Fc�RIIIB,while C3b is not involved in the activation of PYK2.

77 A patient with complete C4 deficiency and membra-nous nephropathy: response to intravenousimmunoglobulin

K. Lhotta1, P. Eder2, H.J. Rumpelt3, G. Mayer1, R.Wurzner4

1Department of Clinical Nephrology, Innsbruck Uni�er-sity Hospital, Innsbruck, Austria ; 2Bruneck Hospital,Italy ; 3Institute of Pathology, Heilbronn Hospital,Heilbronn, Germany ; 4Institute of Hygiene, InnsbruckUni�ersity, Innsbruck, Austria

A 16-year-old boy with complete C4 deficiency re-ported previously to suffer from mesangioproliferativeglomerulonephritis developed heavy proteinuria andthe nephrotic syndrome following a skin infection.High dose steroids proved ineffective and renal func-tion started to deteriorate. A renal biopsy nowshowed membranous nephropathy stage I in additionto mesangial proliferation. On electron microscopynumerous subepithelial irregular electron-dense de-posits, often the size of humps, were visible. Treat-ment with high dose intravenous immunoglobulin wasstarted. The patient received 1 g/kg BW every 4weeks for a total number of ten courses. During thattreatment renal function normalized and proteinuriadecreased from 10 to 0.9 g/day. Terminal complementcomplex concentrations in plasma and urine were ele-vated and did not change significantly during treat-ment. A repeat renal biopsy showed membranousnephropathy stage III without tubulointerstitialchanges. This is the first report of membranousnephropathy in a patient with complete C4 deficiency.High dose intravenous immunoglobulin seems to be apromising, well tolerated treatment for patients withcomplete C4 deficiency and glomerulonephritis.

78 Requirement for decay accelerating factor (DAF) inprotecting the kidney physiologically

F. Lin1, S.N. Emancipator1, D.J. Salant2, M.E.Medof1

1Institute of Pathology, Case Western Reser�e Uni�er-sity, Cle�eland, OH, USA ; 2Department of Medicine,Boston Uni�ersity Medical Center, Boston, MA, USA

Decay accelerating factor (DAF) is one of severalregulators that function to protect self cells from de-position of autologous C3b on their surfaces. Its rela-tive importance in vivo, however, is incompletelyunderstood. As one approach to address this issue,we induced nephrotoxic serum (NTS) nephritis inwild type mice and Daf 1 gene-floxed mice devoid ofrenal DAF expression. In this experimental condition,NTS IgG is administered in a low enough dose (0.5mg i.v.) in which glomerular injury is entirely comple-ment-dependent and after 18 h, renal injury is as-sessed by proteinuria, and by histological andimmunohistochemical analysis of kidneys. Fifteen nor-mal and 15 DAF-deficient mice were studied. Baselinealbuminuria in the DAF− mice was 115.9�41.4 �g/mg creatinine as compared with 85.7�32.3 �g/mgcreatinine in DAF+ littermates (P=0.075). Followingadministration of NTS IgG, albuminuria increased to2001.7�688.7 �g/mg creatinine as compared with799.7�340.5 �g/mg creatinine in the controls (P=0.0003). Histological analyses of kidneys showedgreater cellularity in the DAF− animals as comparedwith the wild type controls. Immunohistochemicalstaining showed equivalent deposition of IgG in theDAF− and DAF+ animals. This contrasted withmassive deposition of C3b in the former and none inthe latter. The results clearly show that DAF is essen-tial physiologically in protecting glomeruli against au-tologous complement attack, which is classicalpathway-mediated.

79 Lupus erythematosus in patients with genetically de-termined type I C2 deficiency

D. Lipsker1, C. Gilliot1, B. Uring-Lambert2, A.Meyer2, J. Goetz2, E. Grosshans1, G. Hauptmann2

1Department of Dermatology, Uni�ersity Hospital,Strasbourg, France ; 2Laboratory of Immunology, Uni-�ersity Hospital, Strasbourg, France

The purpose of this study was to evaluate clinicaland immunological characteristics of lupus erythe-


matosus in patients with genetically determined type I C2deficiency. This is a retrospective study including allpatients with type I C2 deficiency diagnosed between1997 and 1998 at the Immunology Laboratory of theStrasbourg University Hospital. The typical 28 bp dele-tion was detected by polymerase chain reaction (PCR)analysis and patient charts of 70 subjects with C2deficiency were reviewed. Eleven patients had lupuserythematosus and among them, five had a complete C2deficiency and six had a partial C2 deficiency. Eightpatients had systemic lupus erythematosus, two hadsubacute cutaneous lupus erythematosus and one patienthad disseminated discoid lupus erythematosus. Photo-sensitivity was present in 73% of the patients and 64%tested positive for anti-Ro(SSA) antibodies. Renal in-volvement, which required immunosuppressive therapy,was present in 54% of the patients. Patients (90%) werefound to be positive for antinuclear antibodies and 54%of the patients were tested positive for anti-dsDNAantibodies. Analysis of the polymorphism of the fourthcomponent of complement performed in 82% of thepatients revealed that the C2-deficient gene was associ-ated with the C4A4 and C4B2 allotypes as previouslydemonstrated. Most patients with lupus erythematosusassociated with C2 type I deficiency presented withphotosensitivity and this is probably related to thepresence of anti-Ro(SSA) autoantibodies. The maininformation obtained from this study on the clinicalpoint of view is that the prognosis for those patients isnot better than that for patients with lupus erythemato-sus in general.

80 Immunohistochemical analysis of human and rat C3areceptor expression by the use of monoclonal antibodies

A.E. Luhmann1, A. Fayyazi2, O. Scheel1, O. Gotze1, J.Zwirner1

1Department of Immunology, Georg-August-Uni�ersity,Gottingen, Germany ; 2Department of Pathology, Georg-August-Uni�ersity, Gottingen, Germany

The anaphylatoxin C3a mediates pro- as well asantiinflammatory activities on binding to its receptor onleukocytes. Recently, distinct C3a receptor (C3aR) ex-pression has also been demonstrated in vascular andbronchial smooth muscle and endothelial cells as well asin bronchial and alveolar epithelial cells. To reexaminethe tissue distribution of C3aR, monoclonal antibodies(mAbs) against the human (mAbs hC3aRZ3 andhC3aRZ8) and rat (mAb rC3aRZ1) C3aR were raisedwhich recognized epitopes on their large extracellularloop. Antibodies hC3aRZ8 and rC3aRZ1 detected C3aRexpression in alveolar and interstitial macrophages but

not in epithelial, endothelial or smooth muscle cells oncryostat sections of the normal human and rat lung. Also,in the normal human and rat liver, kidney and intestine,C3aR expression was only noted in cells belonging to themononuclear phagocyte system. In contrast, mAbhC3aRZ3 against the human C3aR not only stainedmonocytes/macrophages and their derivatives (Kupffercells and Hofbauer cells) but also different types ofsmooth muscle cells in all organs tested including pla-centa and umbilical cord. Our results demonstrate thatcells of the mononuclear phagocyte system abundantlyexpress C3aR whereas its expression in normal epithelialcells is absent or below the detection limit of our sensitivestaining method. The evidence for an ubiquitous expres-sion of C3aR in normal human smooth muscle cells isbased on reactivity of only one of our mAbs and maypossibly be explained by its binding to a cross-reactingepitope.

81 Structural studies on decay accelerating factor andother complement regulators

P. Lukacik, P. Williams, S. Lea

Laboratory of Molecular Biophysics, Department of Bio-chemistry, Uni�ersity of Oxford, South Parks Road,Oxford OXI 3QU

To date our understanding of human complementregulation at the molecular level has been limited toknowledge of the 3D structure of CD59 and structuresof pathogen-derived regulators of human complement,whilst our understanding of the vast majority of shortconsensus repeat (SCR) domain containing regulatorshas been informed by studies of isolated SCR domainsand pairs of domains. We can build reasonably accuratemodels of individual SCRs and this combined with ourassumption that domain linkages were flexible suggestedthat we could build models to assist understanding of thebiology of the multiple SCR domain containing comple-ment regulators. Recent structures have shown that thisassumption of flexibility does not hold for all SCR-SCRjunctions. Since function is spread over multiple SCRsdetermination of the structures of multiple SCRs will berequired to fully understand how the structure of theprotein relates to its biology.

Our group is using X-ray crystallography and surfaceplasmon-resonance to extend our understanding of com-plement regulation via biophysical studies on a varietyof SCR domain containing complement regulators. Todate we have focused on decay accelerating factor (DAF)and have grown crystals of a variety of constructsconsisting of differing numbers of the 4 SCR domains.We have deterimined the structure of a C-terminalfragment of DAF consisting of SCRs 3 and 4 at 1.7A� .


This structure reveals the most detailed view of SCRdomains to date and shows the orientation of domains3 and 4 to be unusually fixed and linear. The implicationsof this structure for DAF function in complementregulation, CD97 and pathogen binding will be discussedalong with more recent results from crystals of all 4 DAFSCR domains and crystals of the rat complement regu-lator CRRY and active fragments of human CR1.

82 Characterization of mutated factor H in serum ofpatients suffering from atypical form of hemolytic uremicsyndrome (HUS)

T. Manuelian1, J. Caprioli2, M. Noris2, G. Remuzzi, P.F.Zipfel1

1Department of Infection Biology, Hans Knoell Institutefor Natural Products Research, Jena, Germany ; 2MarioNegri Institute for Pharmacological Research, Bergamo,Italy

Hemolytic uremic syndrome (HUS) is a disease char-acterised by microangiopathic hemolytic anemia, throm-bocytopenia and acute renal failure. Recent genetic datareveal a clear association of complement regulator factorH with this fatal disease. Clustering of identified muta-tions in the C-terminus, particularly within SCR 20,reveal a hot spot central for the pathogenesis of thedisease. In order to show the role of the mutated factorH protein, we analysed plasma factor H of HUS patientswith the identified Arg 1210 Cys mutation. Western Blotanalysis shows unusual mobility of the mutated protein,which has an apparent mobility of 170 kDa, as comparedwith the 150 kDa form present in plasma of healthyindividuals. A functional difference of these proteins interms of heparin binding and interaction shows the firsttime a functional difference of the mutated protein. Thisfinding may explain how factor H mutations affectprotein function and how mutated regulatory proteinsallow local endothelial damage.

83 High levels of human complement receptor 2 (CR2,CD21) expression in Cr2−/− mice results in a markedreduction in peripheral B cell numbers

Kevin J. Marchbank1, Matthew Gibson2, B. PaulMorgan1, V. Michael Holers2

1Complement Biology Group, Department of MedicalBiochemistry, U.W.C.M., Heath Park, Cardiff, UK ;2Department of Medicine and Immunology, U.C.H.S.C.,4200 East 9th A�enue, Den�er, CO, USA

We have recently shown that low level expression ofhuman complement receptor 2 (CR2, CD21) in Cr2−/−

mice restores humoral immune function. As part of ourongoing investigation into the role of CR2 in the immuneresponse, we have generated mice with increasingamounts of human CR2 (hCR2) expressed on their Bcells. This was achieved using an immunoglobulinlambda mini-gene containing hCR2 cDNA. The lambdahCR2 transgene was injected into fertilized mouse ovausing standard techniques. Southern blot and polymerasechain reaction (PCR) analysis of tail DNA from theresulting mice revealed the presence of seven genotypepositive mice. Analysis of peripheral blood leukocyteswas carried out by flow cytometry and demonstrated thattransgenic mice had been generated either with high,medium or low expression of hCR2 on their B cells.Examination of various tissues from these mice usingreverse transcription (RT)-PCR revealed that hCR2 wasexpressed exclusively in the B cell compartment. LambdaCR2 positive mice with the highest levels of hCR2expression had a marked reduction in peripheral B cellnumbers when compared with littermate controls. Micewith medium or low levels of hCR2 expression hadessentially normal B cell numbers. Analysis of the spleensand the mesenteric lymph nodes of these mice confirmeda reduction in B cell numbers. Analysis of bone marrowB cells from these mice revealed a decrease in mature Bcells (IgD positive fraction) whilst immature B cellsappeared normal. This defect has remained consistentduring backcross onto the mouse CR2 knock-out back-ground (currently at F5). We are investigating themechanism of B cell loss in these high hCR2 expressingmice in order to gain a greater understanding of thisintriguing finding.

84 Heterogeneity of C1q autoantibodies in patients withlow concentrations of circulating C1q

U. Martensson1, G. Sturfelt2, A.G. Sjoholm1

1Institute of Laboratory Medicine, Section of MIG, LundUni�ersity, Lund, Sweden ; 2Department of Rheumatology,Lund Uni�ersity Hospital, Lund, Sweden

Autoantibodies reacting with the collagen-like regionof C1q have been reported in many diseases. We foundC1q antibodies with enzyme linked immunosorbentassays (ELISA) in 69 consecutive patients with lowconcentrations of circulating C1q. Further investigationof the sera by immunoblot (IB) analyses with reducedC1q showed staining of one or two of the C1q-chains in29% of the sera. The most common pattern was reactivityagainst the B and C chains of the molecule. Isoelectricfocusing of sera followed by detection of C1q-specific


antibodies showed polyclonal patterns both in IB-pos-itive sera and in IB-negative sera. C1q antibodieswere mainly of the IgG1 and/or IgG2 subclasses. Thissubclass distribution was found both in the IB-posi-tive and the IB-negative groups. Patient records werereviewed. With few exceptions the patients had sys-temic lupus erythematosis (SLE) or the hypocomple-mentemic urticarial vasculitis syndrome (HUVS). Themajority of the SLE patients were IB-negative. TheIB-positive pattern with reactivity to B and C chainswas typical for patients with HUVS and was found inall HUVS patients with WHO class III or IVglomerulonephritis. Antibodies to dsDNA werepresent in 79% of the SLE patients, but only in oneHUVS patient (5%).

The findings indicate that the heterogeneity of C1qantibodies has diagnostic importance. The possibilitymay be considered that the differences also havefunctional implications.

85 CR1 on erythrocytes of Brazilian systemic lupuserythematosus patients: expression and ability to bindimmune complexes opsonized with complement fromnormal human serum

C.M. Marzocchi-Machado1,2, C.M.O.S. Alves1,A.E.C.S. Azzolini1, A.C. Morselli Polizello1, Y.M.Lucisano-Valim1

1Department of Physics and Chemistry, Faculty ofPharmaceutical Sciences of Ribeirao Preto, Uni�ersi-dade de Sao Paulo, Ribeirao Preto, SP, Brazil ;2Department of Parasitology, Microbiology and Im-munology, Faculty of Medicine of Ribeirao Preto, Uni-�ersidade de Sao Paulo, Ribeirao Preto, SP, Brazil

Hypocomplementemia and low expression of CR1on erythrocytes (E) of patients with systemic lupuserythematosus (SLE) are associated with defectiveclearance of circulating immune complexes (IC) andso they may have pathogenic significance. Here, weinvestigated whether the reduced CR1/E in SLE pa-tients per se might affect the binding of IC to ery-throcyte CR1. First we analysed the expression ofCR1 on E of active (n=30) and inactive (n=34)SLE patients using a FITC-conjugated mouse anti-CR1 monoclonal antibody E11 and flow cytometry.Both groups of patients had a significant reducedCR1/E expression (P�0.001, Kruskal–Wallis andDunn’s tests) compared with healthy controls (n=40). It was also observed that the number of E bear-ing CR1 was reduced in both groups of SLE patientsstudied. Second we determined the functional activityof CR1/E by measuring the binding to E of FITC-

BSA/rabbit anti-bovine serum albumin (BSA) com-plexes, formed at equivalence, which were opsonizedwith complement from normal human serum (NHS).As background control the IC were incubated withheat-inactivated serum and the binding assay wasevaluated by flow cytometry. We did not find differ-ences in the ability of E to bind IC/NHS between thepatients and control groups. These results show thatan effective coating of the complexes with comple-ment are sufficient to ligate them to CR1 preferen-tially than the normal level of receptor expression.Considering the involvement of low levels of comple-ment and CR1/E expression on complex processing,these data support the hypothesis that the serumcomplement level has a major role in the im-munopathogenesis of SLE.

Supported by FAPESP 96/09626-2.

86 The yeast Candida albicans binds alternative path-way complement regulators factor H and FHL-1

T. Meri1,2, A. Hartmann1, D. Lenk 1, R. Eck1, R.Wurzner3, J. Hellwage4, S. Meri2, P.F. Zipfel1

1Department of Infection Biology, Hans Knoell Insti-tute for Natural Products Research, Jena, Germany ;2Haartman Institute, Department of Bacteriology andImmunology, Uni�ersity of Helsinki, Helsinki, Finland ;3Institute for Hygiene, Uni�ersity of Innsbruck, Inns-bruck, Austria ; 4Molecular Immunobiology Group,Hans Knoell Institute for Natural Products Research,Jena, Germany

Candida albicans is the most common causativeagent for fungal systemic infections in immunocom-promised patients and also causes mucocutaneous in-fections in various patient groups. This pathogenicfungus activates both the alternative and classicalpathways of the complement system, however, the es-cape mechanisms to the destructive activation prod-ucts are not known. In this study we have analyzedbinding of complement alternative pathway regulatorsfactor H, which consists of 20 short consensus repeat(SCR) units and of the alternatively spliced productof the same gene, FHL-1 (SCRs 1–7) to C. albicans.Binding of both factor H and FHL-1 to the surfaceof C. albicans was detected by western blotting afterincubation in human serum and elution of boundproteins. This binding was further confirmed by en-zyme linked immunosorbent assays (ELISA) and im-munofluorescence staining analyses. Absorption assayswith recombinant forms of factor H and FHL-1 con-sisting of SCRs 1–7, 8–20 or 15–20 showed bindingparticularly of the N-terminal SCRs 1–7. FACS-


analyses confirmed the binding results. Although thebinding site seems located within the region commonto factor H and FHL-1, the FACS data show a pre-ferred binding of the factor H protein. These datashow the presence of Factor H and FHL-1 bindingproteins on the surface of the yeast C. albicans. Fur-ther analyses will indicate whether these are related tothe previously reported yeast complement receptorsexpressed on normal cells and their hyphal forms.

87 Polymorphism of the fourth component of comple-ment in groups of children with recurrent infections andantibody deficiency

A. Metin1, O� . Sanal1, F. Ersoy1, I. Tezcan1, A.I.Berkel1, C. Irkec2

1Immunology Unit, Hacettepe Uni�ersity ChildrensHospital, Ankara, Turkey ; 2Neurology Department,Gazi Uni�ersity Faculty of Medicine, Ankara, Turkey

C4 allotypes and disease associations have been thesubject of many studies. However, whether the C4deficiency is causative or simply serve as a marker fora linked disease susceptibility gene is unknown. Inthis study, the analysis of polymorphism in the fourthcomponent of complement (C4) was performed onethylenediaminetetraacetic acid (EDTA)-plasma byhigh-voltage agarose gel electrophoresis followed byimmunofixation. C4B allotypes were further detectedby western blots with monoclonal antibody 1228 (antiC4B/Ch1 reactivity). The frequencies of C4A andC4B alleles were determined in 12 children with anti-body deficiency (8 CVID, 2 sIgA deficiency, 2 specificantibody deficiency; group I) and 27 children withoutimmunodeficiency but recurrent infections (group II).The results of polymorphism from the above groupswere compared with those observed in 142 randomlyselected Turkish individuals without history of recur-rent infection or autoimmune disease (control group).In group I one patient had heterozygous C4B nullallele and another patient had homozygous C4A nullallele among CVID patients. One patient with specificantibody deficiency had heterozygous C4A null allele.In group II, there were three C4A-C4B double het-erozygous null, one C4A heterozygous null and fourC4B heterozygous null alleles. In the control group28 C4B heterozygous and one homozygous null alle-les and seven C4A-C4B double heterozygous null and21 C4A heterozygous null alleles were observed.

C4 polymorphism should be further studied in pa-tients with increased susceptibility to infection in or-der to detect its role as a contributory factor.

88 Identification of point mutations in the SCR do-mains of the factor B gene in two caucasian families

Meyer1, I. Jahn1, J. Goetz1, B. Uring-Lambert1, M.M.Tongio2, P. Perrier3, G. Hauptmann1

1Institute of Immunology, CHU Strasbourg, Stras-bourg, France ; 2Etablissement de Transfusion Sanguine,CHU Strasbourg, Strasbourg, France ; 3Etablissementde Transfusion Sanguine, Nancy, France

Factor B (BF) deficiency has so far only be de-scribed in the heterozygous state but the correspond-ing gene defect(s) have never been described. DNAsequencing of the BF gene was performed in mem-bers of two Caucasian families where a genetic abnor-mality of factor B has previously been reported; onefamily with the segregation of a hemolytically inactivevariant (BF*F055) and another family with the segre-gation of a null allele (BF*Q0). Genomic DNA wasamplified with primers constructed according to thehuman BF sequence (GenBank accession number AF019413). The amplification products were sequencedon an ABI PRISM 310 Genetic Analyser. TheBFF*055 variant was found within the HLA –A*2,B*51, C2*C, BF*F055, C4A*3, C4B*1, DRB1*01,DQB1*05 haplotype. A point mutation was evidencedin three family members at position 1186 of the cod-ing sequence, replacing a cytosine by a thymine,which results in the substitution of an arginine by acysteine within the short consensus repeat (SCR3) do-main at amino-acid position 157. The BF*Q0 variantwas found within the HLA-A1, B57, C2*C, BF*Q0,C4A*6, C4B*1, DRB1*07, DQB1*03 haplotype. Apoint mutation was found in four family members atposition 307 of the coding sequence, replacing a cy-tosine by a thymine which results in the substitutionof an arginine by a cysteine within the SCR1 domainat amino-acid position 49. These findings representthe first report on the molecular basis of genetic ab-normalities detected within the BF gene.

89 No evidence for proteolytic cleavage of CR1 onerythrocytes of normal individuals and factor I deficientpatients

S. Miot1, J. Marfurt2, E. Lach-Trifilieff3, C. Gonza-lez-Rubio4, M. Lopez-Trascasa 4, S. Sadallah1, J.A.Schifferli1

1Department of Research, Uni�ersity Hospital Basel,Basel, Switzerland ; 2Swiss Tropical Institute, Basel,Switzerland ; 3No�artis, Orsham, England, UK ; 4Unidadde Immunologia, Hospital Uni�ersitario La Paz,Madrid, Spain


During the physiological in vivo ageing of erythro-cytes (E), the number of CR1 per cell decreases byapproximately 2/3 in 30 days. The CR1 loss is enhancedin various diseases such as SLE, AIDS and particularlyin factor I deficiency. This loss could be due to prote-olytic cleavage leaving remnant CR1 stumps on the E.A rabbit polyclonal Ab raised against the intracellulardomain of CR1 so called tail recognised specificallyCR1 of E and urinary vesicles (UV) on Western blots.No CR1 stumps could be detected on E from healthydonors and three patients with factor I deficiency.Using the same Ab in a sandwich enzyme linked im-munosorbent assays (ELISA), the signal provided byCR1 was specifically blocked as expected by the tailpeptide, but not by E membranes immunodepleted ofwhole CR1. Thus normal E and E from factor Ideficient patients did not express CR1 stumps. Mi-crovesicles enriched in CR1 and decay acceleratingfactor (DAF) are released from E aged in vitro by ATPdepletion, leading to the same loss of both molecules onE. When comparing reticulocytes and E, CR1 andDAF were lost similarly in 15 normal individuals,suggesting that vesiculation may be at the origin ofCR1 loss. However, the very high rate of CR1 loss infactor I deficiency contrasted with a normal loss ofDAF. Thus in these patients the reduction of CR1 wasspecific, not due to physiological ageing and not relatedto simple proteolytic cleavage at the cell surface.

90 Complement C5 inhibition attenuates inducible nitricoxide synthase (iNOS) following intestinal ischemia-reperfusion

Michael C. Montalto, Kiochiro Wada, Jessica R. Lopesda Rosa, Gregory L. Stahl

Center for Experimental Therapeutics and ReperfusionInjury, Department of Anesthesia, Brigham and Women’sHospital, Har�ard Medical School, Boston, MA, USA

The terminal complement cascade plays an importantrole in mediating tissue injury following gastrointestinalischemia and reperfusion (GI/R). Recent studies sug-gest that inducible nitric oxide synthase (iNOS) may bea potent inflammatory mediator during GI/R. How-ever, the role of complement in regulating iNOS duringischemia-reperfusion is unknown. This study sought todetermine the effects of complement inhibition oniNOS mRNA and protein levels in the rat model ofGI/R. Intestinal ischemia was induced in rats by clamp-ing the superior mesenteric artery for 90 min followedby 60 min of reperfusion. An anti-C5 monoclonal anti-body (18A) (20 mg/kg) or vehicle was administered 60min prior to ischemia. Semi-quantitative reverse tran-

scription-polymerase chain reaction (RT-PCR) revealeda significant (P�0.05) increase of inducible nitric oxidesynthase (iNOS) mRNA (2.6�0.76) compared withsham 0.44�0.12) in the intestine following GI/R. In-testinal iNOS mRNA was significantly (P�0.05 com-pared with vehicle) attenuated by18A treatment (0.76�0.23). Immunohistochemistrydemonstrated that iNOS protein levels were also in-creased by GI/R and were attenuated by 18A treat-ment. Further, staining for nitrotyrosine showed that18A reduced protein nitrosylation in the reperfusedintestine. Superoxide dismutase (Cu/Zn) mRNA levelswere unaffected by GI/R or anti-C5 treatment. Thesedata suggest that activation of the late complementcascade results in elevated levels of iNOS and, further,that inhibition of complement may afford protection byattenuating the nitrosylation of proteins in intestinaltissue.

91 CD40–CD40L interaction induces C3 production inrenal proximal tubular epithelial cells (PTECs)

V. Montinaro, G. Castellano, V. Cappiello, P. Pon-trelli, M. Ursi, L. Gesualdo, F.P. Schena

Di�ision of Nephrology, Department of Emergency andOrgan Transplantation, Uni�ersity of Bari, Policlinico,Bari, Italy

Proximal tubular epithelial cells (PTECs) are cellularmediators of the development of renal tubulo-intersti-tial damage (TID). Among other pro-inflammatory products, PTECs are able to synthesizeC3, which may contribute to TID. PTEC expressCD40, a costimulatory molecule. CD40 ligand(CD40L), expressed by infiltrating lymphocytes, mayrepresent a potent mechanism of cellular activation inTID by CD40 cross-linking on PTECs. We evaluatedthe effect of CD40–CD40L interaction on C3 geneexpression and secretion in human PTECs, comparedwith other proinflammatory mediators. Primary humanPTEC were grown in culture and stimulated in vitro for72 h with IL-1� (100 U/ml), IFN-� (500 U/ml) and/orCD40L, expressed by transfected and irradiated murinefibroblasts (L cell+ ). Negative control were represent-ed by untrasfected L cells (L cell−).Production of C3 was evaluated by northern blot andenzyme linked immunosorbent assays (ELISA). Afternormalization versus GAPDH, in co-culture with L cellC3 gene expression was up-regulated by 50–200% com-pared with basal in different PTEC lines. Similar find-ings were obtained with IL-1�. The association ofIL-1+CD40L did not induce synergistic or additiveeffect. IFN-� provoked a synergistic increase of C3


gene expression when associated to CD40L (twice theadditive% increase by CD40L and IFN-�). ELISA ex-pressed as OD ratio confirmed that CD40L stimula-tion induced a secretion of C3 in supernatant. Inconclusion, cellular immune mechanisms (CD40–CD40L interaction) may induce C3 gene expressionand synthesis by PTECs.

92 Direct binding of C1q to apoptotic cells and cellblebs induces complement activation

A.J. Nauta1, M.R. Daha1, O. Tijsma1, R.Nieuwland2, C.E. Hack3, A. Roos1

1Department of Nephrology, Leiden Uni�ersity MedicalCenter, Leiden, The Netherlands ; 2Department of Clin-ical Chemistry, Leiden Uni�ersity Medical Center, Lei-den, The Netherlands ; 3CLB, Sanquin Blood SupplyFoundation, Amsterdam, The Netherlands

Deficiency of early components of the classicalpathway of complement, particularly C1q, predisposesto the development of systemic lupus erythematosus.Several studies have suggested an association betweenthe classical complement pathway and the clearanceof apoptotic cells. C1q deficient mice develop a lupus-like renal disease, which is associated with the pres-ence of multiple apoptotic bodies in the kidney. Inthe present study, we examine the direct binding ofC1q to apoptotic cells and isolated cell blebs. Jurkatcells were rendered apoptotic by treatment withetoposide and binding of highly purified C1q to cellsand isolated cell-derived blebs was studied by flowcytometry. We demonstrate that C1q binds directly toapoptotic Jurkat cells, mainly in a late stage of theapoptotic process, and to blebs derived from theseapoptotic cells. Binding of C1q to apoptotic cells oc-curs via the globular heads of C1q and induces acti-vation of the classical complement pathway, as shownby surface deposition of C4 and C3. C1q bindingstudies on erythrocytes with membrane-exposed phos-pholipids, as well as inhibition studies with AnnexinV suggest that the ligand of C1q on apoptotic cells isdifferent from phosphatidylserine. In addition, for thefirst time, we demonstrate that surface-bound C1qwas present on a subpopulation of microparticles iso-lated from human plasma. Taken together, these ob-servations demonstrate that C1q binds directly toapoptotic cells and blebs derived therefrom and sup-port a role for C1q, possibly in concert with C4 andC3, in the clearance of apoptotic cells and blebs bythe phagocytic system.

93 Sublytic C5b-9 induces translational activation inhuman aortic endothelial cells. Role of p70 S6 kinase

Florin Niculescu, Sorana Hila, Matthew Fosbrink,Lucian Soane, Horea Rus

Uni�ersity of Maryland, School of Medicine, Balti-more, MD 21201, USA

Proliferation of vascular endothelial cells (EC) con-tributes to intimal hyperplasia during atherogenesis,but the factors regulating their proliferation are notwell known. In the present study, we report that sub-lytic C5b-9 assembly induced proliferation of differen-tiated human aortic EC in culture. We alsoinvestigated if C5b-9 cell cycle induction is mediatedthrough activation of phosphatidylinositol 3-kinase(PI 3-kinase)-p70 S6 kinase signaling pathway. PI 3-kinase and p70 S6 kinase were rapidly activated uponstimulation by C5b-9, but not by C5b6. Rapamycininhibits p70 S6 kinase activation by C5b-9. Pretreat-ment with LY294002 and pertussis toxin inhibit p70S6 kinase activation by C5b-9, suggesting the involve-ment of Gi protein-PI 3-kinase in the activation ofp70 S6 kinase. A complex between ERK1 and p70 S6kinase was documented through immunoprecipitationand PD98059 significantly decreased p70 S6 kinaseactivity. C5b-9 induced DNA synthesis was abolishedby pretreatment with inhibitors of Gi protein, PI 3-ki-nase and rapamycin. Cell cycle reentry induced byC5b-9, as shown by gene array analysis occurredthrough activation of cdk4, cdk2, cyclin E and in-creased Rb protein and PCNA levels. Activation ofthese genes and c-myc was inhibited by pretreatmentwith rapamycin. These data indicated that transla-tional activation is an essential step in C5b-9 inducedaortic EC proliferation.

94 Serum from patients with recurrent HUS and TTPinduces endothelial damage and thrombus formation:role of complement

M. Noris1, F. Casiraghi1, M.P. Ruiz Torres1, J.Caprioli1, M. Galbusera1, G. Remuzzi1,2

1Mario Negri Institute, Italy ; 2Ospedali Riuniti diBergamo, Italy

Hemolytic uremic syndrome (HUS) and thromboticthrombocytopenic purpura (TTP) are diseases of mi-croangiopathic hemolytic anemia and thrombocytope-nia with signs of renal and brain involvement.Activation of the alternative pathway of complementmay play a role in inducing microangiopathic dam-


age, however direct experimental evidence is lacking.Here, we evaluated whether serum from patients withrecurrent HUS/TTP (n=8, studied both during the acutephase and at remission) induced cytotoxicity and pro-mote thrombus formation in human microvascular en-dothelial cells (HMEC-1) and whether these effects weremediated by complement-activation products. Cytotoxi-city was evaluated by measuring, after 4 h incubation, thepercent of specific 51Cr release over controls (HMEC-1exposed to serum from healthy subjects, n=16). Throm-bus formation was assessed on HMEC-1 pre-exposed toserum (4 h) from patients or from controls and thenperfused (3.5 min) at 60 dyn/cm2 with control wholeblood. The area occupied by thrombi was measured.Results are as follows:

C3 in serumSerum Serum induced-

thrombi areacytotoxicity (mg/dl)

(mm2 103)(% 51Cr-release)

Acute 31.58�7.49§3.98�0.580 71.1�5.1§0

0.63�0.35*+sCR-1

3.38�2.83*heat-inactivated 1.61�0.91*

16.87�2.281.34�0.48 102.6�6.3Remission

10.70�1.60Controls 96.0�3.1

Data are mean�S.E.; 0, P�0.05 versus remission; *,P�0.05 versus acute; §, P�0.05 versus ctrs.

Cytotoxicity and thrombus formation induced byserum from patients with acute HUS/TTP were signifi-cantly prevented by blocking complement by heat inac-tivation or by addition of human soluble complementreceptor 1 (sCR1, 150 �g/ml). Reduced serum levels ofC3 (table) and glomerular and small vessels C3 depositsin renal biopsies were found in patients with acuteHUS/TTP, while serum C4 levels were normal, confirm-ing in vivo the activation of the alternative pathway ofcomplement. These results provide evidence that comple-ment is a potent mediator of endothelial damage andthrombus formation in HUS/TTP and hopefully offernew perspectives for the therapy of this life-threateningdisease.

95 Multi-drug resistant ovarian carcinoma cells developincreased complement resistance

K. Odening1, K. Jurianz1, Z. Fishelson2, M. Kirschfink1

1Institute of Immunology, Uni�ersity of Heidelberg,Heidelberg, Germany ; 2Department of Cell Biology andHistology, Sackler School of Medicine, Tel A�i�, Israel

Multi-drug resistance of human tumor cells is consid-ered a major problem in cancer chemotherapy. Resis-

tance of tumor cells to various chemotherapeutic agentsis often caused by overexpression of P-glycoprotein(P-gp), a gene product of the multi-drug resistance gene1 (MDR1). There are contradictory data on a possiblecorrelation between multi-drug resistance of tumor cellsand their susceptibility to complement-mediated lysis.

In the present study, we investigated the sensitivity ofthe human ovarian carcinoma cell line OAW42, of itsP-gp positive drug resistant variants, OAW-Tax (taxolresistant) and OAW-Dox (doxorubicin resistant), and ofthe P-gp negative revertant lines, OAW-Tax-rev andOAW-Dox-rev, to complement-mediated cytotoxicity.Both drug resistant lines, OAW-Tax and OAW-Dox,were more resistant to complement-mediated lysis whencompared with the parental line OAW42. Quantitativeflow cytometry analysis of membrane complement regu-latory proteins revealed that OAW-Tax and OAW-Doxcells up-regulated the expression of CD59�CD46�CD55 in comparison to OAW42 cells. With loss of P-gp,revertant tumor cells, OAW-Tax-rev and OAW-Dox-rev,became increasingly complement susceptible, goingalong with a down-regulation of the expression of CD55and CD46, but not of CD59, to the level of the parentalline. Neutralization of mCRP by specific antibodiesincreased complement-mediated tumor cell killing. Infirst experiments, blocking P-glycoprotein function bythe monoclonal anti-P-gp antibody MRK16, or byverapamil, a Ca2+-channel-antagonist, had no signifi-cant effect on complement susceptibility.

96 The role of C9 in complement-mediated lipopolysaccha-ride release from Escherichia coli J5

A. Orren1, A.M. O’Hara1, A.P. Moran1, B.P. Morgan2,R. Wurzner3

1Department of Microbiology, National Uni�ersity ofIreland, Galway, Ireland ; 2Department of Medical Bio-chemistry, Uni�ersity of Wales, Wales, UK ; 3Institute ofHygiene and Sozialmedizin, Leopold Franzens Uni�ersity,Innsbruck, Austria

Escherichia coli J5 (J5) strains selectively incorporateexogenous galactose into lipopolysaccharides (LPS) andthis property was used to label the LPS of livingorganisms with tritium (H3-LPS). J5(2877) is a substrainof J5 that expresses both incomplete Rc and complete R3LPS core types, and also O side-chain when grown in thepresence of galactose, whereas J5(UK) predominatelyexpresses R3 core-type and never expresses O side-chain.Incubation of labelled organisms with 50% normalhuman serum (NHS) led to the release of between 64 and96% of incorporated radioactivity by 30 min, and therewas no statistical


difference between the strains. Negligible release oc-curred with heat inactivated serum or C6, C7 or C9deficient sera.

C9 deficient serum (C9D; Hobart et al. Clin. Exp.Immunol. 106, 500 (1997)), obtained from an Englishpatient, showed total absence of C9 functional activ-ity; however, it retained moderate bactericidal activityagainst J5 (2877). The C9D serum was reconstitutedwith immuno-affinity purified C9 to 60 �g/ml (normalC9 concentration) and used in the LPS release assay.Reconstitution of C9D serum led to a significant(P�0.0015) increase in H3-LPS release to 2.4- and2.9-fold times that in C9D after 30 and 60 min, re-spectively; however, the level of H3-LPS release pro-duced by NHS was not achieved. Also subtotal C9deficient serum (C9SD) (0.12 �g/ml) produced moreH3-LPS release than did reconstituted C9D serum de-spite the lower C9 concentration. Thus there are ad-ditional factors such as aggregation of purified C9, orpossibly properties of the C9D serum itself, whichlimit the efficiency C9 reconstitution.

97 Quantitative evaluation of C1 inhibitor mRNA inperipheral blood mononuclear cells of patients withhereditary angioedema

E. Pappalardo, M.A. Terlizzi, A. Carugati, L.C. Zin-gale, A. Agostoni, M. Cicardi

Dpt Medicina Interna IRCCS Ospedale Maggiore, Mi-lan Uni�ersity, Milan, Italy

C1-Inh levels in HAE, a heterozygous defect, arelargely below the expected 50% of normal. Whetherthis is due to defective synthesis or increased con-sumption is not defined. To answer this question, weset up a quantitative method for measurement of C1-Inh mRNA expression in peripheral blood mononu-clear cells (PBMC).

We studied 28 patients with HAE type I (14 menand 14 women, median age 32, range 5–73) and 26controls (nine men and 17 women, median age 44,range 18–72). Total RNA was extracted by RNAzolB kit (Celbio) from PBMC. Specific mRNAs for C1-Inh and for glyceraldehyde 3 phosphate dehydroge-nase (Gapdh), as an endogenous control, wereretrotranscribed into cDNA and quantitated by realtime polymerase chain reaction (PCR) on TaqManapparatus (Applied Biosystems (AB)). Standardcurves for each transcript were obtained by serial di-lution of stock solution of specific cDNAs cloned intoa pCR 2.1 vectors. C1-inh mRNA copy number wereexpressed as percentage of the copy number ofGapdh mRNA present in the same sample inter-assay

C.V. was �12% for C1-inh and Gapdh, whereas in-tra-assay C.V. was �1%. Sensitivity was around tencopies for C1-Inh and Gapdh. Specificity was pro-vided by primers and probes spanning among exonsseparated by large intron.

C1-Inh mRNA in HAE patients was 54% that ofnormal subjects, 0.47 (range 0.03–1.74) and 0.87(range 0.15–3.03) respectively, P=0.004. Our dataindicate that C1-Inh expression in cells from HAEpatients is very close to 50% of that of normalsubjects. Thus, C1-Inh plasma levels below 50% inHAE patients are probably due to increased con-sumption.

98 B cell autoreactivity in C4 deficient mice

Elahna Paul1,2, Olga O. Pozdnyakova1, Michael C.Carroll1

1Center for Blood Research, Boston, MA, USA ;2Children’s Hospital Nephrology Di�ision, Boston, MA,USA

Deficiency in the human classical complement sys-tem is a major risk factor for lupus-like autoimmunedisease. Genetic deficiencies of components C1q orC4, in particular, are associated with a high incidenceof systemic lupus erythematosus (SLE). In this pre-sentation, we show that mice deficient in complementcomponent C4 (C4−/− ) are autoreactive, similar toC1q−/− mice and also to complement deficient hu-mans. These knock-out animals have evidence of im-paired B cell tolerance and display early features ofimmune complex disease. At 6 months of age, anti-dsDNA antibodies of the IgM isotype are signifi-cantly elevated in sera of C4−/− mice comparedwith complement sufficient controls (C+ ; P=0.04).Subsequent development of IgG autoantibodies by 9months of age (P=0.004) may implicate a T-cell de-pendent process, and by 12 months, C4−/− femaleshave IgM, IgG and C1q immune complexes depositedin renal glomeruli. Although C4−/− mice at 3months of age have no elevation of serum autoanti-bodies, their splenic B cells can nonetheless be trig-gered by bacterial LPS to secrete IgM anti-dsDNAantibodies in vitro. This apparent skewing of the Bcell repertoire suggests that initial seroconversion toautoreactivity is not simply a matter of impaired au-toantibody clearance in the absence of C4, but is afunction of complement dependent B cell regulation.Importantly, much like C4 deficient humans who de-velop autoimmune disease, the autoreactive phenotype


of C4−/− mice is evident across a mixture of ge-netic backgrounds.

99 gC1qR/p33 expression in human coronary arteryatherosclerotic lesions

E.I.B. Peerschke1, J.O. Minta2, A. Gotlieb2, B.Ghebrehiwet3

1Department of Pathology, Weill Medical College,Cornell Uni�ersity, NY, USA ; 2Department of Pathol-ogy, Uni�ersity of Toronto, Toronto, Canada ;3Department of Medicine, State Uni�ersity of NewYork, Stony Brook, NY, USA

gC1q-R/p33 is a multifunctional and multicompart-mental cellular protein which is postulated to play arole in inflammation, thrombosis, and atherosclerosis.The present studies were undertaken to examinegC1q-R expression in atherosclerosis. Specimens ofhuman coronary arteries, obtained at endarterectomyor autopsy, were fixed in 4% buffered para-formalde-hyde or histochoice and embedded in paraffin. Serialcross-sections were placed on microscope slides, de-paraffinized in xylene, dehydrated in alcohol, andtreated with 3% H2O2 to block endogenous perox-idase activity. gC1qR expression was examined by im-munohistochemistry using the avidin–biotin compleximmunoperoxidase activity technique with biotinylatedmonoclonal anti gC1qR antibody (60.11) or a combi-nation of polyclonal anti gC1q-R and biotinylatedsecondary antibody. Sections were counterstained withhematoxylin/eosin, and examined. Control immunos-taining was performed by replacing primary antibodywith phosphate buffer saline (PBS), non-immuneserum or polyclonal rabbit anti-ovalbumin. The re-sults show specific gC1qR staining in and aroundatherosclerotic lesions. Foam cells in necrotic areas inthe deeper intima, and the vessel walls in the adventi-tia showed the strongest gC1qR staining. Moreover,the necrotic center of calcified atherosclerotic plaquesstained strongly for gC1qR. gC1qR staining of en-dothelial cells was noted also. In contrast, uninvolvedvascular sites with mononuclear cell infiltrates in thefirst few layers of the intima, demonstrated only spo-radic and weak gC1qR staining. These results providestrong evidence for upregulated gC1q-R expression inatherosclerotic lesions, and lend to the hypothesis thatduring plaque rupture, exposure of blood to gC1qRpresent in atherosclerotic lesions may contribute tolocal inflammation via activation of coagulation andkinin cascades.

100 FHL-1 and C4BP bind to M18 group A strepto-coccal cells through different surface molecules

David Perez-Caballero1, Pilar Sanchez-Corral1, Sebas-tian Alberti3, Michael Wessels4, Santiago Rodrıguezde Cordoba1,2

1Departamento de Inmunologıa, Centro de In�estiga-ciones Biologicas, Consejo Superior de In�estigacionesCientıficas, Velazquez 144, 28006-Madrid, Spain ;2Unidad de Patologıa Molecular, Fundacion JimenezDıaz, A�. Reyes Catolicos 2, 28040-Madrid, Spain ;3Unidad de In�estigacion, Hospital Son Dureta, AndreaDoria 55, Palma de Mallorca, Spain ; 4Channing Labo-ratory, Boston, MA 02115, USA

Group A Streptococcus pyogenes, the most frequentbacterial cause of suppurative infections in humans,expresses on the cell surface proteins with capacity tobind the human complement regulators factor H,FHL-1 and C4BP. This has been interpreted as amechanism developed by this pathogen to avoid thecomplement system of the host and to decreasephagocytosis by macrophages and polymorphonuclearcells. The binding of these regulators to the strepto-coccal surfaces has been analyzed using strains carry-ing different M serotypes (M4, M5, M6 and M22).Based on these studies it has been assumed that thethree regulators bind to different sites in same strep-tococcal M protein. We have previously shown thatS. pyogenes strains of the M18 serotype have bindingcapacity for factor H, FHL-1 and C4BP. To evaluatethe contribution of the M protein to the interactionsbetween M18 strains and the three regulators, wehave performed binding assays in a M18 strain (87–282) and in a mutant of this strain carrying a disrup-tion of the emm18 gene (282KZ). We have alsoincluded in these experiments the acapsular variantsof the 87–282 and 282KZ strains, TX72 and TX74,respectively. Our conclusions are, (1) the capsule doesnot affect the binding of complement regulators tothe surface of S. pyogenes ; (2) factor H and FHL-1do not bind to the mutant strains 282KZ and TX74,indicating that this interaction requires expression ofthe M18 protein; (3) expression of the M18 protein isnot required for C4BP binding, suggesting that otherstreptococcal surface proteins in the 87–282 strainmediate the interaction with C4BP.

101 Membranoproliferative glomerulonephritis in factorH-deficient mice

M.C. Pickering1, J. Warren1, A.E. Bygrave1, J. Moss2,R.B. Sim3, H.T. Cook2, M.J. Walport1, M. Botto1


1Rheumatology Section, Imperial College School ofMedicine, London, UK ; 2Department of Histopathology,Hammersmith Hospital, London, UK ; 3MRC Immuno-chemistry Unit, Oxford, UK

Complement factor H (FH) is the major fluid-phaseinhibitory protein of the alternative pathway C3 conver-tase. FH deficiency in humans and pigs is associated withmembranoproliferative glomerulonephritis (MPGN). Totest the hypothesis that uncontrolled C3 activation maycause the development of MPGN we have generatedFH-deficient mice by gene targeting. FH−/− mice hadno detectable FH on western blotting, whilst het-erozygous mice (FH+/− ) had approximately 50%normal levels. FH−/− mice had markedly reduced C3levels, the majority below the limit of detection usingrocket immuno-electrophoresis (�30 mg/l). C3 levelswere also significantly reduced in FH+/− mice(342.3�90.7 mg/l) compared with wild-type controls(476�104.9 mg/l). Linear deposition of glomerular C3was detected in FH−/− mice from the first week of life,in the absence of IgG deposition. At 2 months electron-dense, sub-endothelial deposits were present. By 6months MPGN was apparent with mesangial hypercellu-larity and thickening of peripheral capillary loops. Theeffect of FH deficiency on experimental glomeru-lonephritis was investigated using the accelerated serumnephrotoxic nephritis (NTN) model. Compared withcontrols, FH−/− mice developed an accelerated dis-ease with proteinuria, haematuria and hypoalbu-minaemia at day 3 after injection of nephrotoxic serum.To test whether this phenotype is dependent on uncon-trolled C3 activation, FH−/− mice were crossed withfactor B (Bf)-deficient animals. FH Bf-deficient mice hadnormal C3 levels, did not develop glomerular C3 depo-sition or MPGN and do not demonstrate increaseddisease susceptibility in NTN. These findings indicatethat FH-deficient mice develop secondary C3 deficiencyand MPGN and that this is dependent on C3 activation.

102 Abrogation of acute rejection in the absence of locallysynthesized complement C3

J.R. Pratt, S.A. Basheer, S.H. Sacks

Department of Nephrology and Transplantation, KingsCollege, Uni�ersity of London, London, UK

We investigated the role of renal tubule epithelial cell(TEC) C3 synthesis in renal transplantation using C3knockout C57BL/6 mice. We stimulated normal C57BL/6 (H2b) TEC in vitro with IFN�, and detected increasedC3 mRNA and protein synthesis compared to unstimu-lated TEC, and found C3 deposited on the TEC surface.

IFN� treated C3 sufficient or C3 deficient TEC were usedas stimulators to splenocytes taken from B10Br (H2k)recipients of BL/6 renal allografts at day 14 post trans-plant, when graft histology showed severe acute rejec-tion. By IL-2 enzyme linked immunosorbent assay(ELISA) of supernatants from these co-cultures we founda reduction in allostimulation (P�0.001) if the TECwere deficient in the synthesis of C3 (i.e. from a C3knockout BL/6 mouse). By three-colour FACS we alsofound in these day 14 allograft recipients that approxi-mately 25% of spleen CD4 cells were CR1/2 positivecompared with �5% in normal animals, and that 90%of such CD4+CR1/2+ cells produced IL-2. In survivalstudies B10Br recipients of C3 sufficient BL/6 renalallografts survived for a mean of 12.5 days (n=10) whenBUN levels reached �100 mmol/l (normal BUN �10mmol/l). In comparison B10Br recipients of C3 deficientBL/6 grafts did not succumb to acute rejection andsurvived for �100 days (n=8) in the absence of anyimmunosuppression, but with gradually deterioratingrenal function. These data suggest that allostimulation isenhanced if the transplanted organ is capable of C3synthesis that may interact with C3 receptor bearing CD4cells, putatively of Th1 type, known to be important indriving allorejection.

103 Application of a novel targeted complement regulatorto renal transplant ischaemia/reperfusion injury

J.R. Pratt1, M.E. Jones1, I. Dodd2, R.A.G. Smith2, S.H.Sacks1,

1Department of Nephrology and Transplantation, KingsCollege, Uni�ersity of London, London, UK ; 2AdProTechplc, Herts, UK

In order to examine the role of complement in trans-plant ischaemia/reperfusion injury, we have developed anovel strategy to apply a targeted complement regulatorto donor organs. Using a linear arrangement of mem-brane-binding units, termed sequential membrane ad-dressins (SMAs), that in concert allow strong binding tocell membranes, we have targeted an active fragment ofhuman complement receptor type 1 (CD35; a regulatorof complement activation) to donor kidney tissue. Weused this construct, APT070, in rat renal isografts as amodel of transplant ischaemia/reperfusion injury. Donorkidneys were perfused either with Marshalls preservationsolution containing APT070 (n=16), or Marshalls alone(n=16), and exposed to 30 mins cold ischaemia and 60mins warm ischaemia. Recipients were analysed over 20weeks. In the first week post transplant we found reduceddeposition of C3 and C5b-9 in APT070 perfused kidneys,and reduced tubular necrosis, vascular damage and


neutrophil infiltration-indicative of complement activa-tion. Analysis of renal function detected improvementsover the first week (P=0.0004), and over 20 weeks(P= �0.0001) in the treated group compared to con-trols. At 20 weeks, treated isografts exhibited reducedchronic tubular atrophy, oedema and arteriosclerosiscompared with controls. Various studies suggest thatacute inhibition of innate immune mechanisms such asthe complement system, can produce significant benefitin transplantation. The novel construct used here mayfind a role in clinical transplantation and in other diseasesas a therapeutic strategy, for targeting complementinhibition to specific sites of inflammation.

104 Blocking complement receptor 2 on follicular dendriticcells

W.M. Prodinger, W. Sterlacci, S. Fritz, M. Wurm, L.Kacani

Institute for Hygiene and Social Medicine, Uni�ersity ofInnsbruck, Innsbruck, Austria

Follicular dendritic cells (FDC) in germinal centresexpress all complement receptors (CR), CR1 and CR2 inhigh amounts. Binding of C-coated substrates to germi-nal centres on frozen tonsil sections was used to test towhich extent mAB FE8, previously shown to block CR2,can inhibit the FDC interaction with C.

Fluorescent microagarose beads were coated eitherwith iC3b or C3d. mABs against CR (2.5 �g/ml) wereadded to sections before or after incubation with beadsand bound beads were counted on 125 �m GC-areas onconsecutive sections. Alternatively, fluoresceinated heat-aggregated IgG coated with C3 fragments (HAGG-C)was used as C-coated substrate.

Binding of iC3b-beads to GC was not inhibited by FE8or anti-CR3, whereas CR1-blocking mABS 1B4 and 3D9reduced binding to 48 (�6)% and 59 (�12)% of thecontrol, respectively, and to 6 (�2)% together with FE8.As expected, FE8 reduced binding of C3d-beads to 14(�10)%, whereas anti-CR1 had no effect alone, but didso in combination with FE8 (7�4%). Furthermore, FE8(�anti-CR1) could clearly reduce, but not fully abrogatebinding of HAGG-C to FDC. Neither blockade of CR3nor of Fc�RII reduced HAGG-C binding (neither aloneor in combinations).

We conclude that the binding of C-coated substratesto FDC could be inhibited by anti-CR2 together withanti-CR1 mABs to a substantial extent, but not com-pletely. The implications for the removal of infectiousagents or (auto-)antigens from FDC will be discussed.

(Supported by the Austrian Science Funds, grantF-202).

105 A stop codon in exon 13 causes the complete lack ofhuman complement component C3 deficiency

E.S. Reis, G.V. Baracho, A.S. Lima, L. Isaac,

Departamento de Imunologia, ICB, Uni�ersidade de SaoPaulo, Sao Paulo, Brazil

C3 deficiency is a rare autosomic recessive inheriteddisease and the study of its molecular basis for thisdeficiency has been identified in only six individuals. Ineach case, different causes were responsible for the lackof this protein in the patient’s serum. Here, we identifythe mutations responsible for a new C3 deficiency foundin a Brazilian family.

Total RNA was purified from LPS-stimulated fibrob-lasts and the C3 cDNA was obtained by reverse tran-scriptase-polymerase chain reaction (RT-PCR). Whenwe compared the products from the proband’s and thecontrol’s C3 cDNA no significant size differences werefound. However, the proband produced �10-fold lessC3 mRNA than the normal control. Sequencing studiesrevealed two polymorphic regions, a silent mutation atnucleotide 972 and a mutation at position 1001 in thecDNA, which causes proline to be replaced by leucine.

More importantly we also found a stop codon in exon13 caused by a G to A substitution at position 1716. Thismutation was confirmed in the proband’s (homozygoticfor this mutation) and maternal (heterozygotic) genomicDNA. This indicates that the presence of this stop codonis responsible for the lack of C3 in this proband. We havenot yet determined the connection, if any, between thepoint mutation and the low yield of C3 mRNA. (Sup-ported by FAPESP and CNPq).

106 Hereditary angioedema by deficiency of C1 inhibitor:presence of five new mutations at exon eight

O. Roche, A. Blanch, G. Fontan, M. Lopez-Trascasa

Immunology Unit, Hospital Uni�ersitario ‘La Paz’, Paseode la Castellana 261, 28046 Madrid, Spain

We have started a project with the aim to characterizethe mutations in Spanish patients affected by hereditaryangioedema (HAE). For that, we collected DNA andRNA samples from patients of different regions of thecountry. For the molecular studies, we started with theexon eight screening, and we amplified this exon with twoprimers described by Verpy et al. 1996 (Am. J. Hum.Genet. 59, 308–319). The sequencing reaction was devel-oped with these two primers and with a more internalprimer described by Bissler et al. 1997 (Proc. Assoc. Am.Physicians 109, 164–173).


As the result of the analysis of mutations at exon eightin samples of 45 unrelated patients from 45 pedigrees, wefound mutations in 13 of them. Nine out these patientsshowed point mutations and the rest, frameshift muta-tions.

Two of the point mutations were not previouslyreported. One of these, is a missense mutation inducinga change at the Val451 by Gly. The second pointmutation was the change of stop codon by Arg and theresultant protein could be as long as 563 aminoacidsinstead of 479 (normal protein).

Furthermore, in four patients we found three unre-ported frameshift mutations (one deletion and two inser-tions) two of them located at an imperfect repeat orquasipalindrome.

We are developing SSCP and sequencing the rest of theexons in order to characterize the mutations in the restof the Spanish families affected by HAE.

107 Peptide-induced inhibition of the classical pathway ofcomplement activation at the level of C1q

A. Roos1, A.J. Nauta1, D. Broers1, M.C. Faber-Krol1,J.W. Drijfhout2, M.R. Daha1

1Department of Nephrology, Leiden Uni�ersity MedicalCenter, Leiden, The Netherlands ; 2Department of Im-munohematology and Bloodbank, Leiden Uni�ersity Med-ical Center, Leiden, The Netherlands

In conditions such as immune complex diseases andhyperacute xenograft rejection, undesired activation ofthe complement system is a major contributing factor inpathogenesis. We aim on prevention of complement-me-diated damage by specific inhibition of the classicalcomplement pathway, thus not affecting the anti-micro-bial functions of complement system via the alternativepathway and the MBL pathway. Therefore, 42 peptidespreviously selected from phage-displayed peptide li-braries on basis of C1q binding (Lauvrak et al., Biol.Chem. 1997) were synthesized and examined for theirability to inhibit the function of C1q. From 7 peptidesthat showed inhibition of C1q hemolytic activity but noinhibition of the alternative complement pathway, onepeptide (2J) was selected and further studied. Peptide 2Jinhibited the hemolytic activity of C1q from human,chimpanzee, rhesus monkey, rat and mouse origin, allwith a similar dose–response relationship (IC50 2–6�M). Binding of C1q to peptide 2J involved the globularhead domain of C1q. In line with this interaction, peptide2J dose-dependently inhibited the binding of C1q to IgGand blocked activation of C4 and C3 and formation ofC5b-9 induced by immobilized IgM, as assessed byenzyme linked immunosorbent assay (ELISA). Further-more, the peptide strongly inhibited the deposition of C4

and C3 on pig cells induced by their exposure to humanserum. We conclude that this peptide is a promising toolfor the development of a therapeutic inhibitor of theearliest step of the classical complement pathway, i.e. thebinding of C1q to its target.

108 Structure– function relationships between smallpoxinhibitor of complement enzymes (SPICE) and vacciniacomplement control protein (VCP)

A.M. Rosengard, Y. Liu, R.A. Jimenez

Department of Pathology and Laboratory Medicine, Uni-�ersity of Pennsyl�ania, Philadelphia, PA, USA

The genome of the most virulent orthopox virus,variola, encodes a putative complement regulatoryprotein (CRP), composed of four SCRs, with a sequencediffering from its vaccinia virus homologue, VCP, byonly 12 amino acids or 6%. Our laboratory has expressedand functionally characterized the variola CRP, referredto as SPICE. We have previously demonstrated thatSPICE functions more like human CR1 (hCR1) withregard to species-specificity and is more effective atinhibiting human complement when compared with VCPin a CH50 assay. Here, we report a comparison of FactorI-mediated cleavage of C3b and C4b in the fluid phaseusing VCP and SPICE as cofactors. Unlike VCP, whichserves as a cofactor for the proteolysis of C3b toiC3b1/iC3b2, SPICE promotes proteolysis of iC3b2 toC3c, similar to the activity of CR1, but is 12 times fasterthan VCP. All three CRPs function as cofactors toinactivate C4b to iC4b, yet SPICE is 4-fold faster thanVCP. To map the binding sites of VCP and SPICE, wegenerated anti-VCP/SPICE monoclonal antibodies(mAbs). We identified unique mAbs that interfere withC3b or C4b degradation suggesting that the differentactivities of SPICE and VCP may be due to overlappingbinding sites that all include SCR4. These studies suggestthat SPICE is a biological mimic of hCR1 and thischaracteristic may be important in viral pathogenesis.Further structure– function studies of SPICE and VCPare likely to elucidate the structural basis for the func-tional diversity and species-specificity among SCR-con-taining proteins.

109 Towards a CD59-based therapeutic: GPI anchorsubstitution with a membrane-targeting address

P.J.E. Rowling1, J. White1, L. Mansfield1, M. Steward1,J.R. Bright1, P.J. Lachmann2, G.P. Smith1, R.A.G.Smith1


1AdProTech, Chesterford Research Park, SaffronWalden, Essex CB10 1XL, UK ; 2Department of ClinicalVeterinary Medicine, Uni�ersity of Cambridge, Mading-ley Road, Cambridge, UK

There is considerable interest in developing CD59 asa therapeutic protein to protect cells against terminalpathway-mediated damage in a number of diseases (e.g.PNH, transplantation and multiple sclerosis). CD59 isnormally expressed on cells as a GPI-anchored protein.Soluble forms of CD59 that lack a GPI-anchor havebeen expressed but show diminished cytoprotective ac-tivity. We investigated whether the activity of recombi-nant soluble CD59 expressed from a bacterial hostcould be enhanced by the addition of a cell membranetargeting peptide. CD59 with an N-terminal hexahis-tidine tag and C-terminal cysteine was expressed asinclusion bodies in Escherichia coli, refolded andpurified. A membrane-targeting peptide (MSWP-1) wasthen coupled to CD59 in a site-specific manner throughthe C-terminal cysteine. The activity of recombinantforms of CD59 and membrane-binding derivatives werecompared with native (GPI-anchored) CD59 using areactive lysis assay. Identical activities were observedfor the membrane-targeted derivatives of recombinantCD59 and the native (GPI-anchored) protein. Theseproteins were 350-fold more potent than the soluble(anchorless) forms of CD59. These data suggest a routeto the manufacture of pharmaceutically viable forms ofCD59 that involves high level expression of the proteinin E. coli followed by site-specific modification withsynthetic membrane-targeting peptides.

110 Assessment of the oxidative burst and the receptorsCR1, CR3, FC�RII, FC�RIII in leucocytes from Graves’disease patients

E.M.S. Russo-Carbolante1, A.C.M. Polizello2, A.E.C.S.Azzolini2, Y.M. Lucisano-Valim2

1Department of Biochemistry and Immunology, Facultyof Medicine, Uni�ersity of Sao Paulo at Ribeirao Preto,SP, Brazil ; 2Department of Physics and Chemistry, Fac-ulty of Pharmaceutical Sciences, Uni�ersity of SaoPaulo at Ribeirao Preto, SP, Brazil. E-mail:[email protected]

Graves’ disease (GD) is a disease of unknown aetiol-ogy. Several abnormalities of the immune system havebeen described either within the thyroid gland or in theperipheral blood. Our studies were performed on pe-ripheral blood.

This study included patients in hyperthyroid statuswith or without treatment and patients in normal thy-roid status during treatment or without treatment (re-mission phase). Control subjects were sex and agematched healthy individuals.

Oxidative burst of neutrophils from patients andcontrols were assessed by luminol and lucigenin depen-dent chemiluminescense assays. Neutrophils from eachindividual were stimulated with IC (ova/IgG anti-ova),IC opsonized with complement from NHS or serumfrom GD patients. The results showed a statisticallysignificant enhanced oxidative burst of PMN from GDpatients.

Whole blood samples were incubated with FITC orPE marked antibodies against CD11b, CD35, Fc�RIIand Fc�RIII. Analyses were made by flow cytometry.Receptors of neutrophils and lymphocytes were mea-sured. In our studies, the density of the tested receptorson patients’ cells are similar to those found on controls’cells.

It is possible that the oxidative burst is elevatedbecause of a deficient anti oxidant enzymatic system.The greater respiratory burst found in patients’ PMN isneither dependent of an enhanced opsonizing activity ofcomplement system nor of an up regulation of CR orFcR in neutrophils. There are no differences betweenT1/2 values of controls and patients serum comple-ment. Some authors suggested that a decreased CD11bexpression could be a GD marker. Ours results do notsupport this hypothesis.

111 Autoantibodies against complement receptor 1(CD35)

S. Sadallah1, C. Hess1, M. Trendelenburg1, C. Vedeler2,M. Lopez-Trascasa3, J.A. Schifferli1

1Department of Research, Uni�ersity Hospital Basel,Basel, Switzerland ; 2Department of Neurology, Uni�er-sity of Bergen, Bergen, Norway ; 3Unidad de Immunolo-gia, Hospital La Paz, Madrid, Spain

In previous work, we have shown that specific an-tiCR1 autoAb were found in normal blood donors(BD; 3%) and in SLE patients (25%). Here, we furthercharacterized antiCR1 autoAb and compared them toalloAb against CR1 (McCoy).

Purified IgG from the plasma of antiCR1 positiveBD reacted in rCR1 enzyme linked immunosorbentassay (ELISA) and also with rCR1 in Western blot. Inaddition, the passage of positive plasma on a rCR1affinity chromatography column induced the depletionof the antiCR1 autoAb, while the fractions recovered


by acid elution contained the specific binding activityagainst CR1. Alloreactive anti-McCoy Ab detected byELISA were blocked by the addition of an excess ofrCR1 or erythrocytes, whereas the BD antiCR1 autoAbwere not.

Increased frequency of antiCR1 autoAb was found inliver cirrhosis 15/41 (36%), HIV 23/76 (30%), patientswith anticardiolipin autoAb 4/21 (19%), multiple sclero-sis 7/50 (14%), myeloma 8/56 (14%), but not in acutepoststreptococcal glomerulonephritis 1/32 (3%), com-pared with normals (3%). There was no correlationbetween the presence of antiC1q and antiCR1 autoAb.In human immuno deficiency virus (HIV) patients, thepresence of antiCR1 autoAb did not correlate with alow CR1 number on erythrocytes or a low level ofsCR1 in plasma.

Our results suggest that (i) antiCR1 autoAb arepresent in various diseases, (ii) these autoAb are notdirectly involved in the loss of CR1 on erythrocytes,(iii) by contrast to alloAb, antiCR1 autoAb should notinterfere with rCR1 administered to patients.

112 Functional characterization of Factor H in atypicalhemolytic uremic syndrome

Pilar Sanchez-Corral1, David Perez-Caballero1, Caro-lina Gonzalez-Rubio3, Margarita Lopez-Trascasa3,Santiago Rodrıguez de Cordoba1,2

1Departamento de Inmunologıa, Centro de In�estiga-ciones Biologicas, Consejo Superior de In�estigacionesCientıficas, Velazquez 144, 28006-Madrid, Spain ;2Unidad de Patologıa Molecular, Fundacion JimenezDıaz, A�. Reyes Catolicos 2, 28040-Madrid, Spain ;3Unidad de Inmunologıa, Hospital Uni�ersitario La Paz,Paseo de la Castellana 261, 28046-Madrid, Spain

Hemolytic-uremic syndrome (HUS) is a microvascu-lature disorder leading to microangiopathic hemolyticanemia, thrombocytopenia and acute renal failure.Most cases of HUS are associated with epidemics ofdiarrhea caused by verocytotoxin-producing bacteria,but atypical cases of HUS not associated to diarrhea(aHUS) do also occur. Although most aHUS patientspresent normal complement profiles and their plasmalevels of Factor H are within the normal range, involve-ment of Factor H in aHUS has been recently demon-strated. Three different studies, including the workfrom our laboratory, have identified mutations in thefactor H gene (HF1) in 10–30% of aHUS patients.Most of these mutations are missense mutations that,with no exception, alter amino acid residues in SCR16-20, suggesting that a specific dysfunction in the protec-tion of cellular surfaces by Factor H is a major

pathogenic condition underlying aHUS. As many as 11different missense mutations (T956M, Q1058E,D1119G, W1183L, T1184R, L1189R, S1191L, V1197A,R1210C, R1215G, R1215Q) have been identified thusfar in aHUS patients from different countries. To deter-mine the functional consequences of the HF1 mutationsfound in aHUS we have purified the Factor H from thesera of different patients. We have also expressed re-combinant Factor H molecules carrying the T956M,W1183, L1189R or V1197A HF1 mutations. Analysisof the C3b-binding and Factor I-mediated cofactoractivities show differences between the Factor Hmolecules associated with aHUS and the moleculesfrom normal healthy individuals.

113 Functional analysis of beneficial autoantibodies tothe precursor and pathological autoantibodies to the as-sembled C3 convertase

Markus Schlumberger1, Emiliana Jelezarova1, Peter J.Spath2, Salima Sadallah3, Jurg A. Schifferli3, Hans U.Lutz1

1Institute of Biochemistry, Swiss Federal Institute ofTechnology, ETH-Zentrum, CH 8092 Zurich, Switzer-land ; 2ZLB Bioplasma AG, Bern, Switzerland ;3Department of Medicine, Uni�ersity Hospital Basel,Basel, Switzerland

C3 nephritic factors (C3NeFs) are pathological IgGautoantibodies found in membranoproliferativeglomerulonephritis (MPGN). These autoantibodies sta-bilize the C3 convertase and induce C3b deposition tokidney glomeruli. The functional properties of C3NeFswere analyzed in a solid phase assay by determining theextent by which purified IgG from patients’ serumstabilized a C3 convertase assembled on either C3b orC3b2–IgG complexes. C3 convertase activity was deter-mined by the ability to activate labeled C3. NascentC3b was captured via co-immobilized dipeptide con-taining a tyrosine residue. The dose response of C3NeFfunctional activity was linear from 0 to 400 �g/ml oftotal IgG from MPGN II patients (J. Immunol. Meth-ods 251 (2001) 45–52). Naturally occurring IgG anti-C3/C3b antibodies (anti-C3 NAbs) are present inpooled human IgG†for intravenous application (IVIG).IVIG (Lutz et al. Blood 88 (1996) 184–193) and anti-C3 NAbs attenuate complement amplification by stim-ulating inactivation of properdin-stabilized C3b2–IgGcomplexes which are powerful precursors of C3 conver-tase (Jelezarova et al., Biochem. J. 349 (2000) 217–223).These complexes are the major C3 convertase precursorin blood (Jelezarova, Lutz, Mol. Immunol. 36 (1999)837–842). Since they possess dimeric C3b they allow


anti-C3 NAbs to bind bivalently. Anti-C3 NAbs wereassayed by their ability to reverse the stabilizing effectof properdin and stimulate the Factor H and I depen-dent inactivation of the C3b2–IgG complexes. Anti-C3NAb activity was measured after incubation by assem-bly of C3 convertase on the residual precursors. Prelim-inary results indicate that anti-C3 NAbs affinitypurified from IVIG had a destabilizing effect on C3convertase precursors.

114 Accelerated Fas-mediated apoptosis of monocytesand macrophages from patients with systemic lupus ery-thematosus impairs uptake of iC3b-opsonized apoptoticcells by human monocyte-derived macrophages

Ygal Shoshan, Elias Toubi, Dror Mevorach

The Laboratory for Cellular and Molecular Immunology,Department of Medicine, Hadassah Hospital and theHebrew Uni�ersity, Jerusalem, Israel

Impaired handling of apoptotic cells has been sug-gested as an important factor in the development ofsystemic lupus erythematosus (SLE). We recentlyshowed a role for the complement system in the re-moval of apoptotic cells. We studied the in vitro func-tion of macrophages, from 40 patients with SLE andtheir matched controls, in the removal of heterologousapoptotic cells opsonized by iC3b. Phagocytosis/bind-ing index of apoptotic cells opsonized by iC3b wassignificantly lower in patients with SLE and averaged71%�37 of that of healthy individuals (P�0.002) and69%�35 of patients with rheumatoid arthritis (P�0.007). SLE patients had increased apoptosis of bothfreshly isolated monocytes (P�0.001) and maturingmacrophages (P�0.04) that lead to decreased densityof monocyte-derived macrophages. Adding soluble Fas-receptor inhibited apoptosis indicating Fas-mediatedapoptosis. As demonstrated in healthy controls, de-creased density of macrophages, by itself, caused signifi-cant decreased uptake of apoptotic cells to theremaining macrophages. Maintaining normal density inSLE patients either by an increased initial density orusing soluble Fas restored the clearance capacity of theindividual macrophages in the majority of patients.Conclusion: Impaired in vitro uptake of iC3b opsonizedapoptotic thymocytes, demonstrated in SLE patients,was mainly due to accelerated apoptosis of the mono-cytes/macrophages. Accelerated apoptosis of phago-cytes is a novel mechanism of impairment of binding orphagocytosis of apoptotic cells that not only reducesthe number of professional phagocytes but also altersthe function of the remaining macrophages.

115 Preclinical and clinical progression of a membrane-targeted complement regulator therapeutic

R.A.G. Smith1, I. Dodd1, R.G. Oldroyd1, J. Harry1, C.Clarke2, P. Rolan2, L. Dawes3

1Adprotech Ltd., Chesterford Research Park, LittleChesterford, Saffron Walden, Essex CB10 1XL, UK ;2Mede�al Ltd., Skelton House, Lloyd Street North,Manchester M15 6SH, UK ; 3Department of Rheumatol-ogy, Uni�ersity Hospital of Wales, Heath Park, CardiffCF14 4XW, UK

Unregulated complement activation occurs in a num-ber of disease states and contributes to the underlyingpathology. Derivatives of natural regulators of thecomplement pathways, for example soluble complementreceptor Type I (sCR1; sCD35), have been demon-strated to ameliorate disease in a range of animalmodels. We have constructed an Escherichia coli-ex-pressed minimised version of CR1 and greatly im-proved its biological efficacy by a site-directed chemicalmodification using a membrane-targeting moiety; thismodified molecule is termed APT070. The core proteinis expressed at high yield and can be purified andmodified with high efficiency. Cell-based haemolyticassays carried out in the presence of serum in vitrodemonstrate that APT070 is at least 100-fold morepotent than the parent unmodified protein. APT070 hasbeen shown to be active in two animal models ofrheumatoid arthritis and to be retained in the synovialspace following intra-articular injection. A stable,lyophilised formulation has been developed and pre-clinical toxicology, pharmacokinetic and biodistribu-tion studies in appropriate animal species have beencompleted successfully. The compound has now enteredan ascending-dose Phase I study in human volunteers.Preliminary data indicate that APT070 is well-tolerated.A Phase II study is planned of the safety and effects ofsingle increasing intra-articular doses of APT070 onclinical inflammation and complement system markersin the knee joints of patients with established rheuma-toid arthritis.

116 C5b-9 complement complex protects oligodendro-cytes from apoptosis by regulating BAD and BCL-2through phosphatidylinositol 3-kinase/AKT and ERKpathways

Lucian Soane, Florin Niculescu, Horea Rus, Moon L.Shin

School of Medicine, Uni�ersity of Maryland, Baltimore,MD 21201, USA


Differentiation and apoptosis of oligodendrocyte(OLG) progenitor cells are concomitantly induced bydeprivation of serum growth factors. We showed thatthis is associated with a rapid decline of BCL-2 mRNAand caspase-3 dependent cell death. Caspase-3 activa-tion and cell death are inhibited by sublytic C5b-9assembly. In this study, we studied the role of mito-chondria and the regulation of mitochondrial pathwayof apoptosis by C5b-9 in OLG. In serum-free medium,apoptosis progression is accompanied with decreasedphosphatidylinositol 3-kinase (PI-3K) and Akt activity,cytochrome-c release, and activation of caspase-9. Allof these activities were reversed by C5b-9. Investigationof signal transduction pathways activated by C5b-9 andinvolved in OLG survival revealed the requirement ofPI-3K. The PI-3K-dependent phosphorylation of BADat Ser112 and Ser136 resulted in dissociation of BADfrom the BAD/BCL–XL complex. The PI-3K inhibitorLY294002 was able to reverse the rescue of OLG fromcell death by C5b-9. In addition, ERK, activated byC5b-9 in many cell types including OLG, also causedphosphorylation of mitochondrial BCL-2. The mito-chondrial pathway of apoptosis of OLG is, therefore,modulated by C5b-9 through regulating assembly ofanti-apoptotic BCL-2/BCL–XL with pro-apoptoticproteins such as BAD. This mechanism may be in-volved in OLG survival and remyelination in inflamma-tory demyelinating disorders such as multiple sclerosis.

118 Towards a human C3d-based vaccine for malaria

M. Steward1, V. Cox1, R. Oldroyd1, S. Gallagher1, P.Kareclas1, C. Ragnauth1, J. Bright1, L. Miller2, A.Holder3, S. Ogun3, T. Ross4, R. Smith4

1Adprotech Ltd., Chesterford Research Park, SaffronWalden, Essex CB10 1XL, UK ; 2National Institute ofHealth, 31 Center Dri�e MSC 2137, Bethesda, MD20892-2137, USA ; 3Di�ision of Parasitology, NationalInstitute for Medical Research, The Ridgeway, Mill Hill,London NW 7 1AA, UK ; 4Department of Microbiologyand Immunology, School of Medicine, East CarolinaUni�ersity, Green�ille, NC 27858, USA

The complement component C3d when fused intandem arrays to an antigen can enhance the humoralresponses to the antigen by several orders of magnitudeand reduce the threshold dose of antigen which canelicit an immune response (Dempsey et al., 1996. Sci-ence, 271, 348–350). This has significant implicationsfor the development of effective and economic vaccines.This study describes the construction of human malariavaccine candidates, which encode three copies of hu-man C3d fused to an antigen known as MSP1.19.

MSP1.19 is derived from a merozoite surface antigen ofPlasmodium falciparum and has been identified as avaccine candidate for malaria, but only poor immuneresponses were observed in a recent human trial (Keitelet al., 2000. Vaccine 18, 531–539). To test the vaccinein a mouse model, the murine equivalent of the humanvaccine was produced, using murine C3d and the equiv-alent antigen from the mouse malaria Plasmodiumyoelii. Mice were immunized with antigen-C3d fusionscontaining two or three copies of C3d, administered asa recombinant fusion protein or encoded within a DNAvaccine. Two copies of C3d fused to the antigen as afusion protein were sufficient to produce high titrespecific antibodies and a protective response in theabsence of adjuvant, where control antigen producedno response. No auto-antibodies to the C3d componentwere detected and the immunogens resulted in no localor systemic adverse effects. The responses to DNAvaccines containing the same fusions will be presented.

119 Role of complement in pseudoallergic reactions toliposomal and micellar formulations of intravenous drugs

J. Szebeni1, L. Baranyi1, Y. Barenholz2, Y. Talmon3, D.Danino3, S. Savay1, P. Laverman4, J.M. Metselaar5, G.Storm5, M. Basta6, R. Bunger7, C.R. Alving1

1Department of Membrane Biochemistry, Walter ReedArmy Institute of Research, Sil�er Spring, USA ;2Laboratory of Membrane and Liposome Research, He-brew Uni�ersity, Jerusalem, Israel ; 3Department ofChemical Engineering, Technion-Israel Institute of Tech-nology, Haifa, Israel ; 4Department of Nuclear Medicine,Uni�ersity Medical Center Nijmegen, Nijmegen, TheNetherlands ; 5Utrecht Institute for Pharmaceutical Sci-ence, Utrecht Uni�ersity, Utrecht, The Netherlands ;6Epilepsy Research Branch, National Institute of Neuro-logical Disorders and Stroke, Bethesda, MD, USA ;7Department of Physiology, Uniformed Ser�ices Uni�er-sity of Health Sciences, Bethesda, MD, USA

Conjugation of polyethyleneglycol (PEG) to lipo-somes has been used to increase the circulation time ofliposomal drugs and diagnostic agents, such as liposo-mal doxorubicin (Doxil®) and 99m-Tc-HYNIC PEGliposomes. Likewise, micellar solvent systems, such asCremophor EL (CrEL), have been widely used for theintravenous delivery of water insoluble drugs, such asthe anticancer agent paclitaxel (Taxol). Both liposomaldrugs and Taxol can cause severe immediate hypersen-sitivity reactions (HSRs) that cannot be explained withIgE-mediated (Type I) allergy. Here, we present evi-dence for that both agents are potent complement (C)activators in human and animal serum, and that lipo-somes cause dramatic hemodynamic changes in pigs via


C activation, which mimic some of the human symp-toms. The HSR to liposomes in pigs and C activationby Taxol in vitro were inhibited by soluble C receptor1 (sCR1) and intravenous immunoglobulin (IVIG). Inthe case of liposomes, C activation was found to bedue, at least in part, to the binding of natural antibod-ies to the vesicles, whereas C activation by Taxol wasrelated to particle formation in plasma from CrELmicelles and/or crystalline drug molecules. Our datasuggest that C activation explains the HSRs to pegy-lated liposomal drugs as well as to Taxol, and that pigsprovide a sensitive model to study these reactions. Cactivation-related pseudoallergy (CARPA) may repre-sent a subcategory of Type I HSRs that can be pre-vented or treated with C inhibitors. References, Szebeniet al., Circulation 1999;99 2302; Am. J. Physiol.2000;279:H1319; J. Liposome Res. 2000;10:347; Intern.Immunopharm., 200;1:721.

120 �-Amyloid fibrils bind C1q through its globularregions and activate C1 under physiological conditions

P. Tacnet1, M. Galvan2, S. Chevallier1, A.J. Tenner2,G.J. Arlaud1

1Laboratoire d’Enzymologie Moleculaire, Institut de Bi-ologie Structurale, 41 rue Jules Horowitz, 38027 Greno-ble Cedex 1, France ; 2Department of Molecular Biologyand Biochemistry, Uni�ersity of California, Ir�ine, CA,USA

Previous studies based on the use of serum as asource of C have shown that fibrils of �-amyloid pep-tides that accumulate in the brain of patients withAlzheimer’s disease have the ability to bind C1q and toactivate the classical C pathway. The objective of thepresent work was to test the ability of fibrils of peptideA�1-42 to trigger direct activation of the C1 complex,and to carry out further investigations on the site(s) ofC1q involved in the interaction with A�1-42. Using C1reconstituted from purified C1q, C1r and C1s, it wasshown that A�1-42 fibrils trigger direct C1 activation,both in the absence of C1 inhibitor and at C1 inhibitor,C1 ratios up to 8:0, i.e. under conditions consistentwith the physiological context in serum. The truncatedpeptide A�12-42 and the double mutant (D7N, E11Q)of A�1-42 did not yield C1 activation, providing furtherevidence that the C1 binding site of �-amyloid fibrils islocated in the acidic N-terminal 1–11 region of theA�1-42 peptide. Binding studies performed using asolid-phase assay provided strong evidence that C1qinteracts with A�1-42 fibrils through its C-terminalglobular regions. These findings were confirmed using aC4 consumption assay. In contrast to previous studies

which had used a different experimental design, nosignificant involvement of the C1q collagen-like domainwas detected. These observations provide direct evi-dence of the ability of �-amyloid fibrils to triggeractivation of the classical C pathway, and further sup-port the hypothesis that C activation may be a compo-nent of the pathogenesis of Alzheimer’s disease.

121 Mechanism of induction of complement susceptibilityof erythrocytes induced by spider and bacterialsphingomyelinases

Denise V. Tambourgi1, Marcelo de Sousa da Silva1,Stephen J. Billington2, Rute M.G. de Andrade1,Fabio C. Magnoli1, J. Glenn Songer2, Carmen W. vanden Berg3

1Laboratorio de Imunoquımica, Instituto Butantan, SaoPaulo, Brazil ; 2Department of Veterinary Science andMicrobiology, Uni�ersity of Arizona, Tucson, AZ, USA ;3Department of Pharmacology, Therapeutics and Toxi-cology, College of Medicine, Uni�ersity of Wales,Cardiff, UK

We have recently shown that the sphingomyelinase Dtoxins P1 and P2 from the spider Loxosceles intermediainduces complement (C)-dependent lysis of autologouserythrocytes (E) by induction of cleavage of cell surfaceglycophorins (GPs) through activation of an endoge-nous metalloproteinase facilitating the activation of thealternative pathway (AP) of C. Phospholipase D (PLD)from Corynebacterium pseudotuberculosis shows somedegree of homology with the spider sphingomyelinasesand can induce similar clinical symptoms to that ob-served after spider envenomation. The aim of this studywas to investigate if the bacterial PLD induced haemol-ysis of human E was C-dependent and if cleavage ofGPs occurred. We show here that haemolysis of bothPLD and P1 treated human E was C dependent. Anal-ysis of the C-pathways involved showed that PLD, incontrast to P1, did not induce susceptibility to lysis bythe AP. Furthermore, only P1 was able to inducecleavage of GPs. However, both PLD and P1 treatmentof human E induced susceptibility to activation of theclassical pathway (CP) of C. Investigation of the CPinitiating components indicated that the pentraxins,serum amyloid P- and C-reactive protein, bound to thecells. Polyclonal antisera to the pentraxins inhibitedC-mediated lysis, suggesting a role for pentraxins ininitiation of C-activation. The binding of the pentraxinsmay involve exposure of phosphatidyl-serine (PS), as aconsequence of loss of membrane asymmetry sincebinding of annexin V-FITC to the toxin treated cellswas observed. Exposure of PS has been shown to resultin activation of the C-system.


122 Complement activation by phospholipids (PL)

L.A. Tan, R.B. Sim

MRC Immunochemistry Unit, Department of Biochem-istry, Uni�ersity of Oxford, South Parks Road, OxfordOX1 3QU, UK

C1q is a major recognition protein of the innateimmune system. In the absence of antibodies, C1q bindsdirectly to a variety of substances and particles includingbacteria, viruses, polyanions, subcellular organelles andlarge effects on the binding of C1q. The classical pathwayin whole serum was activated by PA, CL, PS, PG, PI andPE coated on microtitre plates. Multilamellar vesicleswere made with 25% PC, 45% cholesterol and 30% PL(PA, CL, PS, PG, PI or PE). Strong binding of C1q wasobserved only to CL vesicles (at 150 mM NaCl and pH7.4), and binding of IGs appeared to be negligible.However, at lower salt strength (in DGVB+ + ), theclassical pathway was activated by the anionic PLs PA,CL, PS, PG and PI but not by PE.

123 Impact of exposure to high glucose conditions on C3expression by human peritoneal mesothelial cells

Sydney Tang, Catherine X.R. Chen, Susan Yung, JosephC.K. Leung, Daniel T.M. Chan, Kar Neng Lai

Department of Medicine, Uni�ersity of Hong Kong, QueenMary Hospital, Hong Kong SAR, Peoples Republic ofChina

Accumulating evidence indicates that complement isactivated in the peritoneal cavity during chronic peri-toneal dialysis using hypertonic dextrose solutions. Hu-man peritoneal mesothelial cells (HPMC) have beenshown to produce complement factors and regulatoryproteins in vitro. Complement could, therefore, play arole in the pathogenesis of peritoneal injury and fibrosisrelated to long term exposure of the peritoneum todialysis solutions, which contain high glucose content.To investigate this, we examined whether glucose mightaffect complement synthesis in HPMC.

HPMC were obtained from omental tissues of con-sented patients undergoing elective abdominal surgery.The cells were isolated, characterized, and grown inmedium 199. Confluent, growth-arrested HPMC wereexposed to medium containing physiological (5 mmol/l),and supraphysiological (30 and 75 mmol/l) concentra-tions of glucose for 48 h. Cells were lysed, reverse-tran-scribed and subjected to polymerase chain reaction(PCR) analysis of C3 mRNA expression using sequence-

specific cDNA primers. Culture supernatants were col-lected and assayed for C3 protein by sandwich enzymelinked immunosorbent assay (ELISA).

Constitutive C3 gene expression and protein secretionby HPMC was unaffected after exposure to glucose indoses resembling those of commercially available dialysissolutions.

Acute exposure to high glucose levels does not regulatecomplement synthesis in HPMC. Further studies areneeded to clarify the role of complement in dialysis-re-lated peritoneal fibrosis.

124 APT070 a novel membrane targeted C3 convertaseinhibitor, attenuates islet damage in an in vitro model ofhyperacute pancreatic islet xenograft rejection

T. Titus1, L. Badet1, P. Mc Shane1, R.A.G. Smith2,D.W.R. Gray1

1Nuffield Department of Surgery, John Radcliffe Hospital,Uni�ersity of Oxford, Oxford OX3 9DU, UK ; 2AdprotechLtd., Chesterford Research Park, Little Chesterford, Saf-fron Walden, Essex CB10 1XL, UK

Complement activation plays a key role in hyperacutexenograft rejection and therefore efficient complementinhibitors are of great interest. In islet transplantation,hyperacute rejection of xenogeneic islets occurs only inprimate recipients. We tested a novel membrane-targetedmodification of a CR1 fragment (APT070) for its abilityto abrogate damage to islets in an in vitro model ofhyperacute rejection. The efficacy of APT070 on the earlyaccelerated destruction of mouse islets was assessed bylabeling C57Bl6 mouse islets with APT070 under opti-mised conditions, incubating with human blood andassay of proinsulin levels in serum. The effect of addi-tional complement inhibition by human CD46 transgenicexpression was assessed by using CD46 transgenic mouseislets (C57Bl6 background) in this system. APT070reduced destruction of C57Bl6 islets in human blood(relative lysis, 21% for APT070 labelled islets vs. 52% forunlabelled islets). The presence of human CD46 astransgene on the mouse islet decreased relative lysisfurther to 8.5%. APT070 protected against complementattack by binding to target cells under a range ofconditions and provided protection in this model, whichwas additive to that mediated by transgenesis with CD46.APT070 and related agents appear to be promisingtherapeutic agents for providing localised protectionagainst complement mediated destruction without sys-temic effects in islet xenotransplantation, and in vivoexperiments to confirm these findings are justified.


125 Hereditary human complement C3 deficiency due toreduced levels of C3 mRNA

A.G. Ulbrich1, M.P.C. Florido1, V. Nudelman2, E.S.Reis2, G.V. Baracho2, L. Isaac1

1Departamento de Imunologia, Instituto de CienciasBiomedicas, Uni�ersidade de Sao Paulo, Sao Paulo,Brazil ; 2Ambulatorio de Pediatria e Imunologia, EscolaPaulista de Medicina, Uni�ersidade Federal de SaoPaulo, Sao Paulo, Brazil

The aim of this work was to study an 8-year-oldboy (L.A.S.) of consanguineous parents, who pre-sented with recurrent bacterial infections, vasculitisand extremely low levels of serum C3 (0.15 �g/ml).Their parents had four other children who died priorto the age of 6 months due to unspecified infectiousand a cardiopathy. The classical and alternative path-way hemolytic activities and the generation of op-sonins and chemotactic factors derived from theactivation of the complement system were markedlyaffected in the probands’s serum. In vitro addition ofpurified C3 restored the classical pathway-dependenthemolytic activity of his serum. Autoradiographs ofthe proband’s LPS-stimulated and 35S-labelled fibrob-last supernatants after sodium dodecyl sulphate-poly-acrylamide gel electrophoresis (SDS-PAGE) revealedno C3 �- or �-chains while his mother presentedchains with normal sizes. The amount of C3 mRNAsynthesized by the proband’s fibroblasts, as evaluatedby reverse transcriptase-polymerase chain reaction(RT-PCR) assays, was greatly reduced. No great in-sertions or deletions could be observed in theproband’s gene by Southern Blotting analysis. Thesedata suggest that a diminished production or stabilityof this mRNA may be the cause for this deficiency(supported by FAPESP and CNPq).

126 The role of complement in the pathogenesis of infl-ammatory bowel disease (IBD)

Lilian Varga1, Laszlo Bene2, Agota Kovacs2, PalHorvath1, Henriette Farkas3, George Fust3, KataMiklos1

1National Institute of Haematology and Immunology,Budapest, Hungary ; 2Peterfy S. Hospital, Budapest,Hungary ; 3Semmelweis Uni�ersity, Budapest, Hungary

Endoscopy with biopsy is a routine examinationmethod in inflammatory bowel disease (IBD) diagno-sis. Our aim to find a specific immunological assay

indicating the patients’ actual clinical status, andstudy the role of complement system in the pathogen-esis of IBD.

Sera of 225 IBD patients — 140 with Crohn’s dis-ease (CD) and 85 with ulcerative colitis (UC) — werescreened for the level of C3, C4 and C1 esterase in-hibitor (C1-inh). Clinical events (duration of the dis-ease, extraintestinal manifestations, disease activity,colon state with CD diagnosis) were compared withlaboratory parameters.

Increased C1-inh level significantly associated withactivity of CD and UC (P=0.001), and with occur-rence of extraintestinal manifestations (P=0.03).Measurements of C3, C4, C1-inh in a serum samplesimultaneously may have a differential diagnosticvalue. Elevated levels of these parameters were foundin CD (P=0.002). Novel observation that autoanti-bodies against C1q was detected in 45/207 (22%) andanti-C1-inh in 11/205 cases of patients. Our resultssuggest that complement measurements can be usefulin the management of IBD.

This work was supported by OTKA (T032661).

127 Differential expression of murine MBL-A andMBL-C in B cells, dendritic cells and macrophages byimmunregulatory and proinflammatory cytokines

S. Wagner, W. Walter, M. Loos

Institute of Medical Microbiology and Hygiene, Johan-nes Gutenberg-Uni�ersity, Mainz, Germany

Mannan-binding lectin (MBL) is a serum C-typelectin of the collectin family, which has been charac-terized in different species and has been found in twoforms (MBL-A and MBL-C) in rodent and in OldWorld monkeys. Although the liver has been de-scribed to be the site of MBL-A and MBL-C synthe-sis, less is known about their differential expressionand functional relevance in antigen presenting cells(APCs).

In order to gain more insight as to whether MBL-Aand MBL-C are differentially expressed in APCs weanalyzed the regulation of MBL-A and MBL-C genesin freshly isolated murine CD11c+ dentritic cells(DCs), CD19+ B cells and CD11b+ macrophages(M�) upon stimulation with a panel of immunregula-tory and pro-inflammatory cytokines, including IL-4,IL-10, vIL-10, TGF�2, GM-CSF, IFN�. MBL-A andMBL-C appear to be differentially expressed as deter-mined by ratio polymerase chain reaction (PCR) andWestern blot analyses.


In detail, constitutive predominant MBL-C mRNAexpression was observed in DCs, B cells and peritonealM�, independently whether they were stimulated. Incomparison, MBL-A gene expression is induced byIL-4, GM-CSF and IFN� in splenic B cells whereasMBL-A is expressed in unstimulated, GM-CSF andIFN� treated splenic DCs. In addition, peritoneal M�exhibit inducible MBL-A expression by IL-4, GM-CSFand TGF�2/IFN� stimulation.

In conclusion, these results demonstrate that MBL-Aand MBL-C are differentially expressed in APCs andsuggest that alternative MBL isoforms may participatein a different way in host defense against invadingpathogens by the innate immune system.

(Supported by DFG, SFB 490, Project C3).

128 Characterization of human mannan-binding lectin(HU RMBL) expressed in CHO, COS-7, HEP G2 andlepitopterian insect cells

S. Wagner, E. Dorr, W. Walter, F. Petry, M. Loos

Institute of Medical Microbiology and Hygiene, Johan-nes Gutenberg-Uni�ersity, Mainz, Germany

Human mannan-binding lectin (MBL), a C-typelectin of the collectin family, is associated in serum withtwo different forms of serine esterases (MBL-associatedserine esterases, MASP), MASP1 and MASP2. Afterbinding to carbohydrate residues e.g. on the surface ofvarious microorganisms MBL activates antibody —independently the classical pathway by cleavage of C4.Since the serum concentration of huMBL is very low,ranging between 0 and 5 �g/ml, we therefore expressedhuMBL in CHO, COS-7, HepG2 and lepitopterianinsect cells and compared it to huMBL purified fromnormal human serum.

The highest level of huMBL expression was detectedin insect cells, followed by COS-7, CHO and HepG2cells as determined by a mannan–enzyme linked im-munosorbent assay (ELISA) system. All recombinantMBLs were able to mediate the activation of the classi-cal complement pathway in comparable concentrations.

Sucrose gradient ultracentrifugation experiments re-vealed that in contrast to huMBL, purified from nor-mal human serum, recombinant MBLs showed asmaller oligomeric structure. Since rhuMBL expressedin CHO, COS-7, HepG2 and lepitopterian insect cells isfunctionally active similar to huMBL it will be a helpfultool to further analyse the role of MBL in innateimmunity.

(Supported by DFG, SFB 490, Project C3).

129 In vitro xenotransplantation model for the analysis ofnovel, pathway-specific complement inhibitors

Alexander Walpen, Paul Mohacsi, Robert Rieben

Heart Transplantation Laboratory, Cardiology, SwissCardio�ascular Center, Uni�ersity Hospital, Bern,Switzerland

Background: We are studying the problem of anti-body dependent, complement (C) mediated hyperacuterejection in xenotransplantation models. Grafts fromDAF/CD59 transgenic animals who should serve asorgan donors still face the problem of acute vascularrejection in which the complement system plays animportant role. We asked the questions which C-path-ways are involved in graft rejection and/or lysis of cellsand how we can specifically inhibit them.

Methods: Erythrocyte lysis tests were used in whichthe involvement of the antibodies against the majorxenograft epitope (anti-Gal) as well as the classical(CP), alternative (AP) and lectin (LP) pathways werestudied by means of immunoabsorption (IA), specifi-cally C-deficient human sera (NHS), and C-inhibitors.

Results: NHS depleted of anti-Gal by IA showed areversible decrease of hemolysis compared with NHS.Experiments with factor B depleted NHS showed areduction of hemolysis of about 90%. A similar effectwas observed for C1q- and C2-depleted NHS (−45,−65%, respectively). Low molecular dextran sulfateand Pentaglobin® (IgM enriched IVIg preparation)added to NHS also reduced hemolysis by dose-depen-dently inhibiting the CP. In addition, dextran sulfateinhibited the AP, on which Pentaglobin® had no effect.

Discussion: The AP as well as anti-Gal antibodiesplay an important role in rabbit erythrocyte lysis. Theresults with the C2 and C1q depleted sera indicate aninvolvement of the CP. The latter may be even moreimportant in PK15 lysis as IVIg preparations are ableto inhibit human serum cytotoxicity towards PK15cells, but cannot block the AP. These results are cur-rently being validated by FACS analysis of C deposi-tion on different cells.

130 Schistosoma japonicum activates complement via allthree major pathways

W. Wang1,2, A. Ruppel2, J.L. Li3, J. Jensenius4, M.Kirschfink2

1Institute of Immunology, Uni�ersity of Heidelberg,Heidelberg, Germany ; 2Department of Tropical Hygiene,Uni�ersity of Heidelberg, Heidelberg, Germany ;3Department of Parasitology, Tongji Medical College,


Huazhong Uni�ersity, Wuhan, People’s Republic ofChina ; 4Institute of Microbiology, Uni�ersity ofAarhus, Aarhus, Denmark

Infections with Schistosoma japonicum constituteone of the major parasitic diseases in China. We andothers have previously demonstrated that the parasiteSchisto-soma mansoni, prevalent in Africa, is able toactivate complement in vitro and in vivo. In thisstudy, we investigated in a comprehensive analysis thepossible interaction of complement with S. japonicumin vitro. Direct binding of mannan-binding lectin(MBL) and C1q on schistosomula (Sla) was verifiedby immunofluorescent assays incubating Sla with hu-man serum as well as with purified MBL or C1q,respectively. Activation of all major pathways, theclassical, the MBL as well as the alternative pathwayby Sla of S. japonicum was verified by incubation ofthe parasites in vitro with human sera deficient inFactor B, C1q, or MBL. Binding of C4, C3 and C5b-9 to the parasite surface was observed in all threedeficient sera, although to a different degree, and wassignificantly augmented upon reconstitution withpurified factor B, C1q or MBL, respectively. Classicalpathway activation was further increased upon prein-cubation of the parasites with sera from schistosomia-sis patients or with S. japonicum-infected rabbitserum.

These data provide the first evidence that schistoso-mula of S. japonicum activate human complement viathe classical, the alternative as well as the MBL-pathway.

131 Improving decay-accelerating factor (DAF, CD55)by substituting a GPI-anchor with a membrane-target-ing address

J. White1, N. Giddings1, D. Esser1, K. Thurston1, M.Steward1, J.R. Bright1, B.P. Morgan2, G.P. Smith1,R.A.G Smith1

1AdProTech, Chesterford Research Park, SaffronWalden, Essex CB10 1XL, UK ; 2Department of Medi-cal Biochemistry, Uni�ersity of Wales College ofMedicine, Heath Park, Cardiff CF4 4XN, UK

Decay-accelerating factor (DAF, CD55) is a GPI-anchored glycoprotein that accelerates the decay ofC3 and C5 convertases. Because inappropriate com-plement activation results in tissue damage in manydiseases there is significant interest in developing aform of DAF suitable for clinical use. We have de-vised a strategy to produce a recombinant form of

DAF that is targeted to the cell membrane. A recom-binant gene that encoded the four short consensusrepeat domains of DAF was modified to introduce aunique cysteine at the C-terminus of the encodedpolypeptide. The resulting construct was expressed inEscherichia coli, with and without a hexahistidine tag,refolded and purified. After selective activation of theC-terminal thiol, these proteins were converted tomembrane-targeted derivatives by reaction with amyristoylated peptide, MSWP1. The resulting com-pounds possessed complement inhibitory activity inboth classical and alternative pathway assays. The ap-parent specific activity of DAF-MSWP1 was 50-foldgreater than for the native (GPI-anchored) DAF, and500-fold greater than soluble (anchorless) forms ofDAF. These data demonstrate that the activity ofGPI-anchored proteins can be improved by substitu-tion of the GPI anchor by post-translational modifi-cation with synthetic membrane-targeting peptidesand further suggest that these derivatives may haveclinical utility.

132 Deletion of the gene encoding CD59 in mice in-creases disease severity in a murine model of rheuma-toid arthritis

A.S. Williams1, D.S. Holt2, P.J. Richards1, B.D.Williams1, B.P. Morgan2

1Department of Rheumatology, Uni�ersity of WalesCollege of Medicine, Cardiff, UK ; 2Complement Biol-ogy Group, Department of Medical Biochemistry, Uni-�ersity of Wales College of Medicine, Cardiff, UK

Rheumatoid arthritis (RA) is a chronic joint diseaseof unknown etiology. The stimulus that triggers andmaintains joint inflammation is unidentified, althoughevidence suggests that complement (C) is involved.Here we address the role in arthritis of C regulationin the terminal pathway using mice rendered deficientby gene deletion in the membrane regulator CD59.Monoarticular murine antigen-induced arthritis(mAIA), a recognised model of RA, was induced inage-matched wild-type (CD59+/+) and deficient(CD59−/−) mice. Disease progression was monitoredover a 21 day timecourse by comparing diameters ofthe arthritic and contralateral normal knee joints(knee swelling). Histological progression of diseasewas assessed in animals sacrificed at intervals. Kneeswelling was significantly greater (P�0.0001) inCD59−/− mice when compared with CD59+/+ con-trols 1 day after arthritis induction and remainedgreater through day 7 post-induction (P�0.02). Byday 21, no significant swelling was apparent in either


group. However, histological assessment of arthriticknee joint sections 21 days after arthritis inductiondemonstrated that joint pathology (cartilage destruc-tion, bone erosion and cellular infiltration) was sig-nificantly worse (P�0.05) in CD59−/− mice(arthritis index=6.41�1.8 vs. 2.75�1.5 in controls).These data demonstrate that C regulation in the ter-minal pathway restricts development of arthritis inthe mAIA model and that deficiency of CD59 in thejoint is associated with increased disease severity inthis model. The findings support results obtainedfrom antibody neutralisation of CD59 in rat jointsand raise the possibility that inhibition of the termi-nal pathway might be an effective therapy in inflam-matory arthritidies in man.

133 Deletion of the gene encoding C3 decreases diseaseseverity in a murine model of rheumatoid arthritis

A.S. Williams1, R.J. Mead2, P.J. Richards1, M.C.Carroll3, B.P. Morgan2

1Department of Rheumatology, Uni�ersity of WalesCollege of Medicine, Cardiff, UK ; 2Complement Biol-ogy Group, Department of Medical Biochemistry, Uni-�ersity of Wales College of Medicine, Cardiff, UK ;3Department of Pathology, Har�ard Medical School,Boston, MA 02165, USA

Rheumatoid arthritis (RA) is a chronic joint diseaseof unknown etiology. There is evidence to supportthe idea that activation of complement (C) within therheumatoid joint is important. Here we address therole of complement in arthritis using mice rendereddeficient by gene deletion in the key component of allactivation pathways, C3. Monoarticular antigen-in-duced arthritis (mAIA), a recognised model of RA,was induced in age-matched wild-type (C57Bl/6(j);C3+/+) and C3 deficient (C3−/−) mice. Disease pro-gression was monitored over a 14 day timecourse bycomparing diameters of the arthritic and contralateralnormal knee joints (knee swelling). Knee swelling wassignificantly reduced (P�0.05) in C3−/− mice whencompared with C3+/+ controls 2 days after arthritisinduction and remained lower through day 14 post-induction (P�0.02). Histological assessment ofarthritic knee joint sections confirmed that jointpathology (cartilage destruction, bone erosion and cel-lular infiltration) was much reduced in C3−/− micewhen compared with controls. These data demon-strate unequivocally that complement activation isnecessary for the development of arthritis in themAIA model. This finding supports results obtained

from our earlier studies in rat joints demonstratingefficacy of C3 convertase regulators (sCR1 orAPT070) in AIA and strengthen the conviction thatlocal or systemic inhibition of C activation will beeffective in treating inflammatory arthritidies in man.

134 Genetic diversity of complement components C4Aand C4B and correlations of C4 gene dosages with C4haemolytic activities and protein levels in a central Eu-ropean population

Y. Yang1, E.K. Chung1, B. Zhou1, C.A. Blanchong1,C.Y. Yu1, M. Kovacs2, I. Karadi2, G. Fust2, L.Varga3

1Children’s Research Institute and College of Medicineand Public Health, The Ohio State Uni�ersity, Colum-bus, OH 43205, USA ; 2Third Department of Medicine,Semmelweis Medical Uni�ersity, Budapest, Hungary ;3National Institute of Haematology and Immunology,Budapest, Hungary

The genetics of human complement component C4is remarkably complex, with unusually frequent varia-tions in the number and size of C4A and C4B genes,and polymorphisms in biochemical properties andserum levels of C4A and C4B proteins. We havestudied the diversity of complement C4A and C4Bfrom 125 healthy individuals from Budapest, Hun-gary. For each sample, C4A and C4B phenotypeswere determined by immunofixation experiments, C4haemolytic activity by effective molecule titration, andtotal C4 protein levels by single radial immunodiffu-sion. The number and size of the C4A and C4Bgenes present were elucidated by specific and defini-tive PshAI and TaqI RFLPs using probes of the RP-C4-CYP21-TNX (RCCX) modules. The frequency ofindividuals with 2, 3, 4 and 5 complement C4 genesin diploid genomes are 1.6, 21.6, 64.8 and 12%, re-spectively. Altogether 481 C4A and C4B genes arepresent in the 250 haplotypes. Among these C4 genes78.6% have the endogenous retrovirus HERV-K(C4)incorporated into intron nine of the long C4 genes;21.4% of C4 genes are the short version without thisendogenous retrovirus. The C4 protein levels andhaemolytic activities showed a statistically significantcorrelation with the C4 gene number. In addition,plasma samples from individuals with short C4 genesor combinations of long and short C4 genes haverelatively higher C4 protein levels than those withlong C4 genes only. The results of this study helpestablish the foundation of human C4 genetics, andprovide the reference for epidemiological studies forthe associations of C4 with diseases such as coronaryheart disease and systemic lupus erythematosus.


135 Complement receptor c3 (CR3): a public transducerof innate immunity signals in macrophages

Eitan Yefenof

The Lautenberg Center for General and Tumor Immunol-ogy, Hadassah Medical Center, Hebrew Uni�ersity,P.O.B. 12272, Jerusalem 91120, Israel

CR3 (CD11b/CD18) is expressed on a variety ofimmune cells and interacts with multiple ligands. Itsaffinity to some of its ligands can be modified byactivation of other membrane proteins, such as selectinsand receptors for chemokines and cytokines. CR3 canalso transmit signals emanating at GPI-anchoredproteins such as Fc�RIII, CD14 and urokinase plasmino-gen activator receptor (uPAR). Accordingly, CR3 isconsidered to be a ‘public transducer’.

In an attempt to clarify how CR3 of macrophagesfunctions, we searched for CR3 associated molecules. Bycoimmunoprecipitation, we have identified a 16 kDaprotein with a PI of 5.1 (P16/5.1) that is not covalentlyassociated with CR3. Microsequencing of P16/5.1 indi-cated exclusive homology to the �-galactoside bindinglectin galectin-1. Antibodies specific to galectin-1 reactedwith P16/5.1 and its association with CR3 was specificallyinhibited by lactose, which binds with high affinity togalectin-1. Double color immunofluorescense stainingrevealed the expression of galectin-1 on the macrophagesurface and its co-localization with CR3. We proposemodels describing the possible role of galectin-1 in theCR3 complex. (1) Association of galectin-1 with �-galac-toside residues on the extra-cellular domain of CR3 maymodify the binding affinity of the receptor to its ligand,or be involved in its signal transduction. (2) Homo-dimeric galectin-1 possessing a bivalent sugar binding sitemay act as an extra-cellular adapter molecule thatcrosslinks CR3 and other receptors. These possibilitiesare not mutually exclusive and can clarify the mode bywhich CR3 functions.

136 Anti-C5 monoclonal antibody attenuates intestinalischemia–reperfusion injury in mice

Hui Zhao, Gregory L. Stahl

Department of Anesthesia, Brigham and Women’s Hospi-tal, Center for Experimental Therapeutics and ReperfusionInjury, Har�ard Medical School, Boston, MA, USA

Previous studies have shown that the terminal comple-ment cascade plays an important role in mediating localand remote tissue injury following gastrointestinal is-

chemia and reperfusion (GI/R) in the rat. However, therole of C5a/C5b-9 following GI/R injury in mice has notbeen evaluated. In this study, murine monoclonal anti-body against C5, which blocks the generation of C5a andC5b-9, was given to mice undergoing GI/R. Intestinalischemia was induced by clamping the superior mesen-teric artery for 20 or 30 min and then reperfused for 1or 3 h. Anti-C5 monoclonal antibody (BB5.1; 40 mg/kg)or isotype control antibody was administered 5 min priorto ischemia. Anti-C5 treated mice demonstrated signifi-cantly less loss of intestinal lactate dehydrogenase (LDH)and significantly less accumulation of myeloperoxidase(MPO) activity (i.e. PMN infiltration) in the intestine andlung. Irreversible intestinal tissue injury (i.e. LDH loss)induced by 30 min ischemia and 1 h of reperfusion wasobserved. However, anti-C5 demonstrated significantprotection against PMN (i.e. MPO activity) infiltrationinto the lung following this prolonged period of ischemia.These data suggest that the late complement componentsplay an important role in lung injury following GI/R inmice. Further, local intestinal injury, if reversible, can beattenuated by anti-C5 treatment in mice. These data alsodemonstrated the potential clinical implication anti-C5treatment during gastrointestinal ischemia and reperfu-sion and its action on remote organs (e.g. lung).

137 Human tumor cells secrete complement regulatorymolecules Factor H and FHL-1

S. Ziegler1, K. Jurianz1, T. Manuelian2, P. Zipfel2, M.Kirschfink1

1Institute of Immunology, Uni�ersity of Heidelberg, Ger-many ; 2Hans Knoll Institute for Natural Product Re-search, Jena, Germany

Factor H and Factor H-like protein 1 (fHL-1) asimportant regulators of the alternative pathway acceler-ate the decay of the alternative pathway convertase andexhibit co-factor activity for Factor I.

In the present study, we investigated the secretion offH/fHL-1 by tumor cells of different origin, such aserythroleukemia (K562), ovarian carcinoma (MT, GG,GP, SKOV-3) and mammary carcinoma (BT474, T47D).K562, MT and SKOV-3 tumor cells constitutivelysecreted fH and fHL-1, whereas in culture supernatantsof GG, GP, BT474 and T47D no fH could be detectedby enzyme linked immunosorbent assay (ELISA). Re-verse transcriptase-polymerase chain reaction (RT-PCR)analysis revealed expression of mRNA for fH and fHL-1in K562 and MT cells, which was confirmed on proteinlevel by Western blot. Treatment of MT cells with IFN�,IL-1� or TGF� and of SKOV-3 cells with IFN� or IL-6increased secretion of fH/fHL-1 up to five times. IFN�


and TGF� induced production of fH/fHL-1 in theovarian carcinoma cell line GP. Secreted fH/fHL-1 wascapable to bind to immobilized C3b and acceler-ateddecay of alternative pathway convertase as measured ina haemolytic inhibition assay, thus indicating functionalactivity of the tumor cell derived regulators. Preliminary

data indicate that fH/fHL-1 is also expressed on themembrane of various tumor cell lines, as revealed byFACS analysis using polyclonal anti-fH antibodies.

Secreted functional active complement inhibitors fHand fHL-1 by tumor cells represent another mechanismto resist complement-mediated cytotoxicity.

Complement Mol Immunol

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