Introduction

Rheumatoid arthritis (RA) is a common, debilitating disease.The development of effective therapies for RA has been hin-dered by inadequate understanding of disease pathogenesis.Although RA is a systemic disease, synovial joints are themain targets of inflammation. There are two main patho-logical features in RA: inflammation of the synovial com-partment and destruction of cartilage and bone. Synovitis ischaracterized by synovial lining cell (SLC) hyperplasia, production of pro-inflammatory cytokines including TNF-aand IL-1 by synovial cells, angiogenesis and a CD4+

T-lymphocyte-predominant mononuclear cell infiltration ofthe synovium.

Electron microscopy revealed that two major cell popula-tions constitute the normal synovial lining layer.1 Type-ASLC are macrophage-like and are thought to be derived frommonocyte precursors in the bone marrow (BM).2 Type-B SLCare fibroblast-like, of mesenchymal origin and specialized forprotein synthesis and secretion.3 However, recent data suggest that the synovial cell phenotype may be morecomplex.4–6 In RA, hyperplasia of the SLC layer results fromincreased numbers of both type-A and type-B SLC,7 togetherwith invasion of blood-borne inflammatory cells. Jointdestruction is mediated by hyperplastic synovial membrane-derived tissue called ‘pannus’, which forms at the interface ofthe synovium and cartilage.8 Pannus is predominantly composed of fibroblast-like SLC.9,10 Rheumatoid arthritissynovial fibroblasts co-engrafted with normal human carti-lage in severe combined immunodeficient (SCID) mice

invade cartilage,11 but normal dermal fibroblasts and non-RASLC do not, suggesting that the invasive phenotype is specific to RA synovial lining fibroblasts.11,12

The question of which cellular actions and interactions are most important in RA remains controversial, but mostevidence favours T-lymphocyte-dependent pathways.13

However, there is increasing support for the concept that T-lymphocyte-independent pathways may contribute signifi-cantly to RA. In one scenario, SLC are ‘pseudo-transformed’in RA by an as yet unknown mechanism. Subsequently, RASLC proliferate and/or fail to undergo normal apoptosis, havealtered cell surface adhesion molecule expression andproduce matrix-degrading enzymes to facilitate invasion intocartilage.13

This report describes the characterization of a novel mAbdesignated 1D5 that was raised against RA-derived synoviallining cells in order to better understand the dramatic func-tional changes these cells undergo in RA.

Materials and Methods

Tissues

Human tissues were obtained from the Royal Children’s and RoyalMelbourne Hospitals according to procedures approved by localinstitutional committees. Synovium was obtained from patientsundergoing joint replacement surgery. Other tissues includingthymus and tonsils were removed from children undergoing cardiacsurgery or tonsillectomy, respectively. Leukaemic bone marrow(BM) samples were obtained from BM biopsy specimens from individuals with acute myeloblastic and chronic lymphoblasticleukaemias (AML and CLL, respectively).

Cell culture

Human lymphoid cell lines were obtained from the Walter and ElizaHall Institute of Medical Research (WEHI) Hybridoma Cell Bank

Immunology and Cell Biology (2001) 79, 419–428

Research Article

Characterization of a human synovial cell antigen: VCAM-1 andinflammatory arthritis

RA CARTER, 1 K O’DONNELL, 1 S SACHTHEP, 2 F CICUTTINI , 3 AW BOYD 4 andIP WICKS 1,2

1Reid Rheumatology Laboratory, Autoimmunity and Transplantation Division, Walter and Eliza Hall Institute ofMedical Research, 2Rheumatology Unit, Royal Melbourne Hospital, Parkville, 3Monash Medical School, Prahran,Victoria and 4Queensland Institute of Medical Research, Brisbane, Queensland, Australia

Summary The contribution of synovial cells to the pathogenesis of rheumatoid arthritis (RA) is only partlyunderstood. Monoclonal antibody (mAb) 1D5 is one of very few mAb ever raised against RA synovial cells in orderto study the biology of these cells. Studies on the expression pattern and structural features of the 1D5 Ag suggestthat 1D5 recognizes human vascular cell adhesion molecule-1 (VCAM-1), which is an intercellular adhesion molecule. Vascular cell adhesion molecule-1 may be involved in a number of crucial intercellular interactions in RA.

Key words: rheumatoid arthritis, synovial cells, vascular cell adhesion molecule-1.

Correspondence: Prof. I Wicks, Autoimmunity and Transplanta-tion Division, Walter and Eliza Hall Institute of Medical Research,Post Office, Royal Melbourne Hospital, Parkville, Victoria 3050,Australia. Email: wicks@wehi.edu.au

Received 15 January 2001; accepted 4 April 2001.

(Melbourne, Vic., Australia) or purchased from the American TypeCulture Collection (ATCC; Rockville, MD, USA). Cell lines werecultured in RPMI 1640 (Invitrogen Australia, Melbourne, Vic., Australia) media supplemented with 100 U/mL penicillin (Com-monwealth Serum Laboratories (CSL), Parkville, Vic., Australia),100 mg/L streptomycin (CSL) and 10% v/v heat-inactivated FCS(HI-FCS) (Flow Laboratories, North Ryde, NSW, Australia) unlessotherwise stated.

Chinese hamster ovary (CHO) cells transfected with a constructcontaining cDNA encoding full-length human vascular cell adhesionmolecule-1 (VCAM-1)14 (VCAM-CHO) were kindly provided by Dr J Gamble (Hanson Cancer Centre, Adelaide, SA, Australia).Chinese hamster ovary cells were cultured in HAM-F12 media (GibcoBRL) supplemented with 100 U/mL penicillin (CSL),100 mg/L streptomycin (CSL) and 10% v/v HI-FCS (Flow Labora-tories). VCAM-CHO were cultured in CHO media further supple-mented with 2 mmol/L geneticin (GibcoBRL).

Human umbilical vein endothelial cells (HUVEC) were kindlyprovided by Dr L Schofield (WEHI, Melbourne, Vic., Australia).HUVEC (passage number 2–8) were grown in tissue culture flasksor plates (Becton Dickinson, Franklin Lakes, NJ, USA) precoatedwith 0.1% w/v gelatin solution. The HUVEC culture media com-prised Media 199 (M199; GibcoBRL) supplemented with antibiotics(100 U/mL penicillin, 100 mg/L streptomycin), 20% v/v HI-FCS,50 µg/mL bovine neural tissue endothelial cell growth supplement(ECGS; Sigma, St Louis, MO, USA) and 20 mmol/L HEPES (GibcoBRL), pH 7.2.

Antibodies, cytokines and other reagents

Monoclonal antibodies were used as dilutions of purified IgG fromascites fluid or miniPERM Bioreactor culture supernatants. All antibodies (Ab) were titrated before use to determine appropriatedilutions for immunoperoxidase and immunofluorescence staining.

Production of monoclonal antibody 1D5 The 1D5 hybridoma wasgenerated following fusion of hypoxanthine/aminopterin/thymidine(HAT)-sensitive murine Sp2/NS-1 myeloma cells with splenocytesfrom immunized Balb/c mice.15 Mice were immunized intra-peritoneally with single cell suspensions grown from synovial tissueexplants obtained from an individual with RA. Hybridomas produc-ing mAb reactive with frozen sections of RA synovial tissues wereidentified. The mAb 1D5 strongly labelled RA synovial tissue andwas chosen for further characterization. The isotype of mAb 1D5(IgG1k) was determined using a Serotec MMT RC1 isotyping kit(Serotec, Oxford, UK).

Other monoclonal antibodies Other mAb used included VIII-6G10(mouse antimacaque VCAM-1 hybridoma, shown to cross-react withhuman tissue;16 ATCC), 1H4 (mouse antihuman intercellular adhesion molecule (ICAM-1) hybridoma, WEHI), 2.06 (mouse anti-human MHC class II, WEHI), EMBII-8 (macrophage marker; mouseantihuman CD68; DAKO Corporation, Carpinteria, CA, USA) andV9 (fibroblast marker; mouse antihuman vimentin; DAKO Corpora-tion). Purified mouse Ig (Sigma) or irrelevant isotype-matched mAbwere used as controls. For immunofluorescence staining, FITC-conjugated sheep-antimouse IgG (Silenus, Melbourne, Vic., Aust-ralia) was used as the secondary reagent.

Cytokines Recombinant human (rhu) TNF-a, rhu IFN-g, rhu IL-1band LPS were purchased from Genentech Inc. (San Francisco, CA,USA), Genzyme Diagnostics (Cambridge, MA, USA), Amgen(Thousand Oaks, CA, USA) and Sigma, respectively.

Production and purification of monoclonal antibodies

1D5, VIII-6G10, 1H4 and M/K-2.7 hybridomas were cultured at high density in miniPERM bioreactor apparatus (Heraeus Instru-ments, South Plainfield, NJ, USA). Monoclonal antibodies werepurified from the supernatants by protein A-Sepharose or protein G-Sepharose affinity chromotography. 1D5 and 1H4 mAb were purified from mouse ascites fluid by protein A-Sepharose or proteinG-Sepharose affinity chromotography.

Cytokine activation of cultured HUVEC

The HUVEC were grown to subconfluency in HUVEC media ingelatin-coated 12- or 24-well plates (Becton Dickinson). Cells werestimulated for 6–72 h with various cytokines and were recovered bytrypsin-EDTA (GibcoBRL) treatment for flow cytometry.

Immunofluorescence staining and flow cytometry

Cells were labelled with primary mAb specific for cell surface molecules or isotype-matched control Ab. After incubation for20 min at 4°C, cells were washed and centrifuged, then labelled for20 min with FITC-conjugated secondary Ab reagents. Cells werewashed, resuspended in fixative (2.5% v/v formaldehyde solution,2% w/v glucose, 0.02% v/v sodium azide in human tonicity (HT)-PBS) and analysed by flow cytometry (FACScan; Becton Dickinson). Data were acquired and analysed using CellQuest(Becton Dickinson) or WEASEL (Dr F Battye, WEHI) software.

Immunohistochemistry

Single-colour immunohistochemistry was performed on frozen tissuesections using an avidin–biotin-based immunoperoxidase technique(DAKO Labelled Streptavidin–Biotin (LSAB)2 Horseradish Peroxi-dase (HRP) System, DAKO). To avoid non-specific mAb binding, Fcreceptors were blocked with 20% v/v normal sheep serum (Serotec).Slides were incubated with primary mAb for 2 h at room temperatureor overnight at 4°C. Sections were washed and incubated withbiotinylated rabbit antimouse IgG and avidin-biotinylated peroxidasecomplexes according to the manufacturer’s instructions (DAKO).Staining was developed with 3,3¢-diaminobenzidinetetrahydro-chloride (DAB)/H

2O

2(Sigma) as the chromogen substrate and

counterstained with haematoxylin. Sections incubated with isotype-matched irrelevant mAb were processed as outlined above.

Preparation of cell suspensions from human tissues

Freshly removed tonsils were dissected and pressed through a nylonmesh sieve. To further disperse remaining clumps of tissue, the suspension was drawn up and expelled several times through a 5 mLsyringe fitted with a 19-gauge needle. The suspension was washedand resuspended in HT-RPMI/10% v/v FCS. Cell viability wasassessed by trypan blue (Sigma) exclusion and was always > 95%.

Rheumatoid arthritis synovium samples were dissected anddigested using an enzyme cocktail containing 2.4 mg/mL dispase II(Roche Diagnostics Australia, Melbourne, Vic., Australia), 1 mg/mLcollagenase Type II (Sigma) and 100 mg/mL DNase I (Roche Diag-nostics). A single-cell suspension was obtained by washing througha nylon mesh sieve.

Preparation of cell and tissue lysates

Cell lines or cell suspensions prepared from human tissues wereincubated in lysis buffer (25 mmol/L Tris-HCl pH 7.5, 150 mmol/L

RA Carter et al.420

NaCl, 1% w/v CHAPS with protease inhibitors – 10 µmol/L E-64(Roche Diagnostics), 100 µmol/L leupeptin (Sigma), 10 mmol/LEDTA (Sigma), 1 µmol/L pepstatin A (Auspep, Parkville, Vic., Australia), 1 mmol/L phenylmethylsulfonyl fluoride (PMSF, RocheDiagnostics), 10 mmol/L 10-phenanthroline (Sigma)) on ice for30 min. Lysates were homogenized in a dounce homogenizer andcentrifuged to remove debris. Synovial fluid samples were incubatedwith 100 U bovine hyaluronidase (Sigma) for 60 min at 37°C. Theprotein concentrations of the cell or tissue lysates were estimatedusing the procedure of Bradford et al.17

Immunoprecipitation

Chinese hamster ovary or VCAM-CHO cell lysates were preclearedwith protein G-Sepharose beads (Pharmacia Corporation, Peapack,NJ, USA) for 2 h at 4°C to remove proteins non-specifically bindingto the beads. Antigen–antibody complexes were precipitated fromthe cell lysates by incubation at 4°C overnight with protein G-Sepharose that had been coupled to either VIII-6G10 or 1D5. Afterthree washes in HT-PBS-0.5% (v/v) Triton X-100 (Sigma), pelletswere resuspended in Laemmli sample buffer,18 boiled for 5 min andwere then subjected to SDS-PAGE and western blot analysis.

SDS-PAGE and western blot analysis

Equivalent protein concentrations of cell and tissue lysates were subjected to SDS-PAGE under both reducing and non-reducing conditions.18 Western blot analysis was performed using previouslydescribed methods.19

Results

Cellular distribution of the 1D5 antigen

1D5 was chosen for further characterization because itstrongly stained synovial cells in sections of RA tissue. Flowcytometric and immunohistochemistry studies were per-formed using mAb 1D5 to determine the cellular distribution

of the 1D5 Ag. A panel of primary cells and tumour cell lineswas examined by flow cytometry for surface expression ofthe 1D5 antigen (Table 1). Cell lines of lymphoid, myelo-monocytic and erythroid origin as well as samples of normaland leukaemic peripheral blood and bone marrow were negative for 1D5 expression. Monoclonal antibody 1D5 wasalso unreactive with human hepatoma and melanoma celllines. Of the samples tested, only the SV40-transformed bonemarrow stromal cell line, 197/17 and single-cell suspensionsprepared from human tonsil, thymus and RA synoviumstained positively. Flow cytometric analysis revealed that 1D5stained a subset of primary RA synovial cells (Fig. 1). Theseresults are consistent with 1D5 recognizing a cell surfacemolecule expressed by some, but not all, adherent cells fromorganized tissues and not from circulating cells.

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Table 1 Human cell lines and tissues tested for expression of 1D5 antigen*

Cell type Cell line Expression as assessed by flow cytometric analysisT lymphocyte HSB2, Jurkat, JM, Molt4, HPB-ALL –Pre-B lymphocyte Lila, LK63 –B lymphocyte Ramos, Raji, U266, Nalm, REH –Myelomonocytic U937, HL60 –Erythroid K562 –Stem cells DU528 –Bone marrow stroma 197/17 +Hepatoma HepG2 –Melanoma M96, C32 –

TissuesRA synovium +Tonsil +Thymus +Bone marrow –AML peripheral blood –AML BM –CLL peripheral blood –CLL BM –

*Cell lines or single-cell suspensions prepared from tissues were stained with isotype-matched control mAb or 1D5 and analysed by flowcytometry. AML, acute myeloblastic leukaemia; BM, bone marrow; CLL, chronic lymphoblastic leukaemia; RA, rheumatoid arthritis.

Figure 1 Expression of the 1D5 Ag on rheumatoid arthritis(RA) synovial cells. Primary RA synovial cells were stained withisotype-matched control mAb ( ) or 1D5 ( ) and analysed by flow cytometry.

Tissue distribution of the 1D5 antigen

To examine the tissue distribution of the 1D5 Ag in situ,immunohistochemistry studies were performed on frozensections from various human tissues. In RA synovium(Fig. 2, panel b), 1D5 Ag expression was predominantlyobserved in the synovial lining layer.

In tonsil tissue, strong 1D5 Ag staining was observed in structures resembling lymphoid follicles (Fig. 2, panel c).In the thymus, 1D5 Ag expression (Fig. 2, panel d) wasobserved as discrete, strongly staining regions, scatteredthroughout the thymic cortex and medulla. Breast carcinomatissue was negative for 1D5 expression (Fig. 2, panel a).

Molecular weight of the 1D5 antigen

Total cellular lysates were prepared from RA synovial tissueand tonsil samples. Tissue lysates were analysed by reducingand non-reducing SDS-PAGE and western blot analysis toestimate the MW of the 1D5 Ag. Figure 3 shows that mAb1D5 detected a major band migrating with a MW of approxi-mately 100 kDa in both RA synovial and tonsil lysates. Asingle band of similar size was detected when lysates wereseparated under reducing conditions.

Induction of 1D5 antigen expression by inflammatorymediators

In view of the MW and tissue distribution of the Ag, we rea-soned that 1D5 may recognize an adhesion molecule (AM).

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Figure 2 Monoclonal antibody 1D5 stains rheumatoid arthritis (RA) synovium, thymus and tonsil. Frozen sections of (a) breast carcinoma, (b) RA synovium, (c) tonsil and (d) thymus were stained by indirect immunoperoxidase with 1D5. Bound mAb was detectedby rabbit antimouse Ig, followed by staining with labelled streptavidin–biotin/horseradish peroxidase (HRP) amplification reagent andDAB. Magnification: ´100.

Figure 3 Monoclonal antibody 1D5 detects a major bandmigrating with a MW of 100 kDa in both rheumatoid arthritis(RA) synovial and tonsil lysates. Five micrograms of humantonsil (lane T) or RA synovium (lane S) lysate was analysed bySDS-PAGE and transferred to a nitrocellulose membrane. Themembrane was probed with a 1:1000 dilution of 1D5. BoundmAb were detected with a sheep antimouse Ig-horseradish peroxidase (HRP) conjugated secondary Ab and enhanced chemiluminescence. The position of molecular weight markers(kD) is shown.

Many AM are expressed by endothelial cells and can beupregulated by cytokines. To determine whether the 1D5 Agis inducible on cytokine-stimulated endothelium, a series ofexperiments were performed using HUVEC. The expressionpattern of the 1D5 Ag was compared with those of ICAM-1and VCAM-1, as detected by the mAb 1H4 and VIII-6G10,respectively. In all instances, 1D5 and VIII-6G10 stainingwas indistinguishable (VIII-6G10 data not shown). Flowcytometric analysis of unstimulated HUVEC revealed nodetectable expression of the 1D5 Ag or VCAM-1 (Fig. 4a). Incontrast, resting HUVEC expressed moderate levels ofICAM-1. Stimulation of HUVEC with 10 ng/mL TNF-a for48 h induced expression of both the 1D5 Ag and VCAM-1,whereas ICAM-1 expression increased above basal levels.Similarly, 1D5 Ag, VCAM-1 and increased ICAM-1 expression

were seen following 48 h incubation of cells with LPS. Treatment of HUVEC with IFN-g increased ICAM-1 expres-sion, but did not induce expression of either the 1D5 Ag orVCAM-1.

To determine the MW of the 1D5 Ag expressed by TNF-a-stimulated HUVEC, lysates were analysed by SDS-PAGE and western blot analysis. 1D5 detected a single bandthat migrated with a molecular weight of approximately100 kDa (Fig. 4b) in TNF-a-stimulated HUVEC, but not inun-stimulated HUVEC.

Kinetics of 1D5 expression following TNF-a stimulation

The kinetics of 1D5 Ag expression on HUVEC were com-pared to those of ICAM-1 and VCAM-1 as determined byflow cytometry. HUVEC were stimulated with 10 ng/mL rhuTNF-a and expression of AM and the 1D5 Ag were examinedat 6, 12, 24, 48 and 72 h after the addition of cytokine(Fig. 5). In all instances, 1D5 Ag and VCAM-1 (as detectedby mAb VIII-6G10) expression was indistinguishable. 1D5Ag/VCAM-1 expression was not detected on unstimulatedHUVEC, whereas ICAM-1 was constitutively expressed atmoderate levels. Induction of the 1D5 Ag was identical tothat of VCAM-1, with maximal expression occurringbetween 24 and 48 h after cytokine stimulation. In contrast,ICAM-1 expression continued to increase throughout thetime-course (Fig. 5).

Recognition of VCAM-transfected CHO cells, but notCHO cells, by 1D5

The above data suggest that 1D5 recognizes human VCAM-1.To confirm this hypothesis, the mAb was used to stain CHOcells stably transfected with VCAM-1 (VCAM-CHO) forflow cytometric and western blot analysis. As a positivecontrol, cells were stained in parallel with VIII-6G10 (anti-human VCAM-1). In all instances, 1D5 and VIII-6G10 stain-ing was indistinguishable (VIII-6G10 data not shown).

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Figure 4 Expression of the 1D5 Ag is inducible on cytokine-stimulated endothelium. (a) Human umbilical vein endothelialcells (HUVEC) stimulated with either 10 ng/mL recombinanthuman (rhu) TNF-a, LPS or rhu IFN-g for 48 h were stained withisotype-matched control mAb, 1D5, VIII-6G10 (antihuman vascular cell adhesion molecule-1 (VCAM-1) mAb) or 1H4(antihuman intercellular adhesion molecule-1 (ICAM-1) mAb)and analysed by flow cytometry. Expression of the 1D5 Ag was compared to VCAM-1 and ICAM-1. As expression of the 1D5 Agand VCAM-1 (as detected by VIII-6G10) was indistinguishable,only 1D5 Ag profiles are shown. (b) Monoclonal antibody 1D5detects a major band migrating with a MW of 100 kDa in lysateprepared from rhu TNF-a-stimulated HUVEC. Five microgramsof lysate from unstimulated HUVEC (–TNF-a) or HUVEC stim-ulated with 10 ng/mL rhu TNF-a for 48 h (+TNF-a) was sepa-rated on SDS-PAGE and transferred to a nitrocellulosemembrane. The membrane was probed with a 1:1000 dilution of 1D5. Bound mAb were detected with a sheep antimouse Ig-horseradish peroxidase (HRP) conjugated secondary antibodyand enhanced chemiluminescence. The position of molecularweight markers (kD) is shown.

Flow cytometric analysis revealed that 1D5 stronglystained VCAM-CHO, but did not react with untransfectedCHO cells (Fig. 6a). When VCAM-CHO cell lysates wereanalysed by non-reducing SDS-PAGE and western blotanalysis (Fig. 6b), both VIII-6G10 and 1D5 detected singlebands of approximately 100 kDa. Neither mAb reacted withnon-transfected CHO cell lysates by western blot. Attempts todirectly immunoprecipitate the 1D5 antigen from VCAM-CHO cell lysates were unsuccessful. However, by westernblot analysis, mAb 1D5 recognized a major protein band ofapproximately 100 kDa from VCAM-CHO cell lysates,which had been immunoprecipitated with mAb VIII-6G10(Fig. 7, lane A). The lower MW band probably representspartial protein breakdown, but may also be due to differentialglycosylation of VCAM-1 in CHO cells. No reactive proteinbands were detected from non-transfected CHO cell lysatesimmunoprecipitated with mAb VIII-6G10 (Fig. 7, lane B).Together, these results provide strong evidence that 1D5 rec-ognizes human VCAM-1.

Soluble 1D5 antigen is found in RA synovial fluid andVCAM-CHO supernatant

Synovial fluid (SF) samples from individuals with kneetrauma, osteoarthritis (OA) or RA were analysed by SDS-PAGE and western blot analysis. Figure 8 shows that mAb1D5 detected a major band migrating with a MW of approxi-mately 100 kDa in RA SF samples under reducing condi-tions. Less intense bands of similar size were detected innormal or OA SF. Similar results were obtained when lysateswere separated under non-reducing conditions (data notshown). Interestingly, supernatants from CHO cells stablytransfected with VCAM-1 (VCAM-CHO) also containedsoluble VCAM-1 (Fig. 7).

Discussion

This report describes the characterization of a novel mAbdesignated 1D5, which is one of very few mAb ever raised

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Figure 5 Induction of the 1D5 Ag on cytokine-stimulated endothelium. Human umbilical vein endothelial cells stimulated with10 ng/mL recombinant human (rhu) TNF-a for periods of 0–72 h were stained with isotype-matched control mAb, 1D5, VIII-6G10 (anti-human vascular cell adhesion molecule-1 (VCAM-1) mAb) or 1H4 (antihuman intercellular adhesion molecule-1 (ICAM-1) mAb) andsecondary antibodies and analysed by flow cytometry. Expression of the 1D5 Ag was compared to VCAM-1 and ICAM-1. As expressionof the 1D5 Ag and VCAM-1 was indistinguishable, only 1D5 Ag profiles are shown.

against RA SLC. Our studies on the expression pattern andstructural features of the 1D5 Ag show that it is a cell surfacemolecule of approximately 100 kDa, which is expressed inorganized tissues rather than on circulating cells. It is alsoinducible on endothelial cells in response to TNF-a and LPS,but not IFN-g.

The expression of a number of AM is elevated on RA synovium including ICAM-1 and VCAM-1.20 The MW ofthese AM21 are comparable to the 1D5 Ag. However, in con-trast to the 1D5 Ag, ICAM-1 is broadly distributed.21 TheMW and pattern of expression of the 1D5 Ag is most similarto VCAM-1; under both reducing and non-reducing SDS-PAGE, the MW of VCAM-1 is approximately 100 kDa.14

Moreover, like VCAM-1,21 1D5 Ag expression is not detectedon circulating cells.

Support for the specificity of mAb 1D5 for VCAM-1came from our studies comparing endothelial cell expressionof the 1D5 Ag, VCAM-1 and ICAM-1. Cytokine-stimulatedHUVEC were stained with a panel of mAb that included1H4, VIII-6G10 and 1D5 and analysed by flow cytometry.1H4 and VIII-6G10 staining was consistent with previousreports of ICAM-1 and VCAM-1 expression by cytokine-stimulated HUVEC.22 Moreover, the staining profiles of 1D5were identical to those of VIII-6G10. Similarly, the inductionkinetics of 1D5 Ag expression on TNF-a-stimulated HUVECwere comparable to those of VCAM-1, as detected by VIII-6G10; peak expression of both molecules occurred between24 and 48 h after stimulation. In contrast, ICAM-1 expres-sion (as detected by 1H4) continued to increase throughoutthe time-course.

Vascular cell adhesion molecule-1 is not expressed onresting vascular endothelium, but is rapidly induced inresponse to a number of inflammatory stimuli, includingTNF-a and IL-1.14 Under these conditions, VCAM-1 is animportant mediator of leucocyte recruitment to sites ofinflammation. Inflammation-associated VCAM-1 expressionhas been detected on large- and small-vessel endotheliumfrom many tissues, including RA synovium.23,24 Vascular celladhesion molecule-1 is also constitutively expressed onseveral other non-vascular cell types including type B SLC,25

bone marrow-derived stromal cells,26 thymic macrophages,27

thymic epithelial cells28 and follicular dendritic cells intonsils.29 The distribution of 1D5 staining in RA synovium,tonsil and thymus are consistent with these observations.

Gene knockout studies show that VCAM-1-mediated celladhesion is essential for efficient placentation and normalcardiac development.30,31 Vascular cell adhesion molecule-1mediates haemopoietic progenitor cell adhesion to BM

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Figure 6 The specificity of mAb 1D5 for human vascular celladhesion molecule-1 (VCAM-1). (a) Untransfected Chinesehamster ovary (CHO) cells or VCAM-1-transfected CHO cells(VCAM-CHO) were stained with isotype-matched control mAb,1D5 or VIII-6G10 (antihuman VCAM-1 mAb) and analysed byflow cytometry. Expression of the 1D5 Ag was compared toVCAM-1. (b) Five micrograms of untransfected CHO or VCAM-CHO cell lysate was analysed in duplicate by SDS-PAGE andtransferred to a nitrocellulose membrane. The membrane wasdivided and probed with a 1:1000 dilution of either VIII-6G10(left panel) or 1D5 (right panel). Bound mAb were detected witha sheep antimouse Ig-HRP conjugated secondary Ab andenhanced chemiluminescence. The position of molecular weightmarkers (kD) is shown.

Figure 7 Isolation of vascular cell adhesion molecule-1(VCAM-1) by immunoprecipitation from VCAM-CHO cells.VCAM-CHO (lane A) or CHO (lane B) cell lysates immuno-precipitated with VIII-6G10 were analysed by SDS-PAGE andtransferred to a nitrocellulose membrane. The membrane wasprobed with a 1:1000 dilution of 1D5. Bound mAb were detectedwith a sheep antimouse Ig-horseradish peroxidase conjugatedsecondary antibody and enhanced chemiluminescence. The posi-tion of molecular weight markers (kD) is shown.

stroma26 and VCAM-1+ thymic stromal cells interact withdeveloping thymocytes.28 Vascular cell adhesion molecule-1may also be involved in T-lymphocyte activation. Studiesusing immobilized anti-TCR or CD3 mAb and solubleVCAM-1 (sVCAM-1) demonstrated VCAM-1 could pro-vide costimulatory signals for CD4+ T lymphocytes.32,33

Monoclonal antibodies against VCAM-1 reduced mixedlymphocyte responses in vitro34,35 and the incidence andseverity of graft-versus-host disease in vivo.36 Tonsillar follicular dendritic cells (FDC) and germinal centre B lym-phocytes interact through VCAM-1 and ICAM-1 adhesionpathways.37

Vascular cell adhesion molecule-1 is strongly expressedon several cell types in RA synovium including type B SLC,25 follicular dendritic cells in lymphoid follicles38,39 andendothelial cells.24 There are multiple potential roles forVCAM-1 in the pathogenesis of RA. Because adhesion ofinfiltrating leucocytes to vascular endothelium is the firststep in the recruitment of these cells to the synovium, celladhesion molecule mediated interactions are crucial to theinitiation and maintenance of the inflammatory response.Vascular cell adhesion molecule-1 may facilitate leucocyterecruitment and retention within the inflamed synovial com-partment.23,25 In vitro, peripheral blood T lymphocytes bind to type B SLC through a VCAM-1-dependent pathway.23,40

In vitro studies demonstrated that RA synovial stromal celllines promote B lymphocyte survival through a VCAM-1-dependent induction of the anti-apoptotic molecule Bcl-x

L.41

Vascular cell adhesion molecule-1 may also facilitate syn-ovial attachment to cartilage and bone11.

Elevated levels of sVCAM-1 and other adhesion moleculeswere found in serum in RA, antiphospholipid syndrome andin the cerebrospinal fluid of patients with SLE and multiplesclerosis.20 Elevated serum sVCAM-1 correlated stronglywith disease activity in lupus nephritis.20 Soluble VCAM-1 isangiogenic in the rat cornea, which is used as an in vivomodel of angiogenesis. In addition, RA SF induced angio-

genesis in this system and was blocked by mAb inhibition toVCAM-1.42 Soluble VCAM-1 may mediate chemotaxis ofmonocytes43 and T lymphocytes44 in RA SF. The mechanismsinvolved in the generation of sVCAM-1 are unknown, but areconsistent with proteolytic cleavage at a site close to the pointof membrane insertion.45 In HUVEC, IL-1 can induce shed-ding of sVCAM-1 and sICAM-1 from the cell surface.45 Ourfinding of sVCAM-1 in non-inflammatory as well as inflam-matory joint fluid may suggest a role for sVCAM-1 in therecruitment of monocytes to become type A SLC.

In summary, this report details the characterization of theAg recognized by a novel mAb designated 1D5, which wasraised against RA synovial cells. The expression pattern andstructural features of the 1D5 Ag suggest that it recognizeshuman VCAM-1. In membrane-bound form, VCAM-1 is anintercellular adhesion molecule that mediates high affinityadhesion of circulating leucocytes to activated endothelium.Much less is known about the soluble form of VCAM-1,which may be a physiological antagonist of VCAM-1, butmay also have direct effects. The function of VCAM-1outside the vascular system is less well understood. Thepathogenesis of RA is complex, but it is clear that AM includ-ing VCAM-1 may be important in the initiation and perpetu-ation of disease. Potential roles that VCAM-1 may play in inflammatory joint disease include the recruitment and retention of leucocytes in the inflamed synovium,23,25 angio-genesis,42 invasion of pannus11 and T- and B-lymphocyte activation and survival within the synovial compartment.41,46

Vascular cell adhesion molecule-1 may, therefore, represent anovel therapeutic target in RA.

Acknowledgements

This work was supported by the Reid Charitable Trusts. Wethank A D’Amico and L Dougherty for technical assistance,S Mihajlovic for histology and D Advani, P Maltezos and B Mesiti for help with figures.

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Figure 8 Soluble 1D5 Ag isdetected in synovial fluid (SF).Five micrograms of lysate pre-pared from SF samples from indi-viduals with knee trauma (lane N),osteoarthritis (OA) (lane OA) orRA (lanes labelled RA) were separated on SDS-PAGE andtransferred to a nitrocellulosemembrane. Five micrograms ofVCAM-CHO or CHO cell super-natants were also included as positive and negative controls,respectively. The membrane wasprobed with a 1:1000 dilution of1D5. Bound mAb were detectedwith a sheep antimouse Ig-horse-radish peroxidase conjugated secondary Ab and enhancedchemiluminescence. The positionof molecular weight markers (kD)is shown.

References

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