binding of the complement intermediate c56 to zymosan in acute ...

Clin. exp. Immunol. (1975) 20, 113-124.

BINDING OF THE COMPLEMENTINTERMEDIATE C56 TO ZYMOSAN IN

ACUTE PHASE HUMAN SERA

PATRICIA J. BAKER, L. G. RUBIN, T. F. LINT,B. C. McLEOD AND H. GEWURZ

Department ofImmunology, Rush Medical College, and Department of Microbiology,University of Illinois Medical Center, Chicago, Illinois, U.S.A.

(Received 19 August 1974)

SUMMARY

C56 is known to appear in the fluid phase when zymosan is incubated at 370C withcertain acute phase 'reactor' sera. In the present study, C56 was detected bound tothe zymosan particle prior to its appearance free in solution. In reactor sera C56was formed and released with kinetics similar to that of the generation and decayof a C56-binding site formed when zymosan was incubated with normal serum.Bound and fluid phase C56 was detected only in reactor sera, and was generatedonly by agents known preferentially to activate the properdin pathway. Elution ofC56 from zymosan in hypertonic salt solutions proved to be a convenient step inthe partial purification of large amounts of this haemolytically active bimolecularcomplex.

INTRODUCTION

In their elucidation of the mechanism which accounts for 'reactive lysis', Thompson &Lachmann (1970) observed that the addition of zymosan (Z) or related compounds tocertain acute phase sera resulted in the appearance of persisting C56 complexes, and thatC56 could be eluted from Z (Thompson & Lachmann, 1970; Lachmann & Thompson, 1970).In recent studies of the formation of C56 in acute phase sera, we observed that this inter-mediate could be detected on the Z particles prior to its appearance in the fluid phase. Thepresent study was directed toward defining the characteristics of the C56-Z interaction inreactor sera, and offers elution of C56 from Z as a convenient step in the preparation of thisactive bimolecular complex.

Correspondence: Dr Henry Gewurz, Department of Immunology, 1753 West Congress Parkway, Chicago,Illinois 60612, U.S.A.

H 113

114 Patricia J. Baker et al.

MATERIALS AND METHODSBuffersGVB refers to isotonic veronal-NaCl buffer containing 0-1% gelatin, pH 7 3 (Mayer,

1961). GVB2` refers to GVB with 0- 15 mm Ca2" and I f0 mm Mg2". EDTA-GVB was pre-pared by addition of one part of 0d1 M EDTA pH 7-3 to nine parts of GVB. GGVB2 + wasprepared by addition of equal volumes of GVB and 500 dextrose brought to 0 15 mm Ca2 +

and 1.0 mM Mg2 + (Nelson et al., 1966).

SeraNormal human serum (NHS) and reactor sera were obtained from healthy laboratory

personnel. These sera were categorized by use of reactor screen plates and maintained at- 70'C. Guinea-pig complement (GPC) was obtained from Texas Biologicals, Incorporated,as fresh, frozen, pooled sera and was maintained at -700C until use.

ReagentsZymosan (Z) lot 71C-1370, was purchased from Sigma Chemicals and stored as a 4 mg/ml

suspension in 0-15 M NaCl at 4°C. Agarose was obtained from L'Industrie Biologique,France. Endotoxin (Escherichia coli lipopolysaccharide) lot 3123-10 was obtained fromDifco Laboratories; suspensions containing 20 mg/ml 0 15 M NaCl were heated in a boilingwater bath for 5 min immediately prior to use. Streptokinase-streptodornase Varidase®(SK-SD) was obtained from Lederle as a lyophilized powder and contained 5000 units ofstreptokinase and at least 1250 units of streptodornase/ml. Human immune serum globulin(HGG) was purchased from Armour Pharmaceutical Company and contained 165 + 15 mgof gamma-globulin/ml. C7, C8 and C9 were obtained as lyophilized powders from CordisLaboratories. C56 was prepared from reactor sera as described by Lachmann & Thompson(1970).

ErythrocytesSheep erythrocytes (E) were drawn into acid-citrate-dextrose anticoagulant. The cells

were washed twice in five volumes of 0-15 M NaCl, once in EDTA-GVB, once in GVB2",resuspended to 109/ml in GGVB2+ containing 0-001 M NaN3 and stored at 4°C until use.

Sensitized sheep erythrocytes (EA) were prepared as described (Mayer, 1961). EAClgp4-7huwere obtained from Cordis Laboratories.

Antisera

Rabbit anti-human factor B (C3PA) was obtained from Behring Diagnostics. Goat anti-human C5 was purchased from Meloy Laboratories. Antihuman gamma-globulin and anti-bovine serum albumin were produced in rabbits according to the method of Campbell et al.(1970).

Formation of complement-consuming complexesComplexes of HGG-anti-HGG were formed at equivalence; 1 ml containing 330 pg of

Zymosan-C56 interactions in human sera

HGG was mixed with 1 ml of rabbit antisera, and the precipitate was recovered by centri-fugation, washed thoroughly and resuspended to 2 ml in 0 15 M NaCi. BSA-anti-BSA com-plexes also were formed at equivalence; 6 ml containing 400 pg of BSA/ml were mixed with6 ml of anti-BSA, washed thoroughly and resuspended in 10 ml of 0 15 M NaCi. HGG,16-5 mg/ml, was aggregated by incubation at 630C for 20 min (AHGG).

Zymosan-serum interactionsZ was added to serum (4 mg/ml), and the sample was incubated in a 370C water bath for

1 hr with frequent shaking. The activated serum was recovered by centrifugation at 1000 gfor 3 min at 40C. The Z pellet was washed twice in fifteen volumes of ice-cold distilled water,followed by recentrifugation. C56 was eluted from washed Z pellets by incubation in 0 5M NaCl, at 220C for 15 min and recovered by centrifugation.

ChromatographyWhatman DE-52 was washed and packed in glass columns (2.5 x 13 cm) in starting buffer;

0-01 M sodium phosphate, pH 7 4, containing 4000 (v/v) glycerol. The C56-rich eluate fromZ, and a euglobulin precipitate of activated reactor serum prepared according to Lachmann& Thompson (1970), respectively, were applied to identical DE-52 columns, and the boundproteins were eluted with a 0-01-03 M linear salt gradient. Fractions were analysed for C56by reactor plates and for protein by absorbance at 280 nm.The Z eluate was also chromatographed on a Sephadex G-200 column (3 x 85 cm) after

concentration and dialysis into starting buffer, which contained 0 1 M NaCi, 0-05 M Tris and0*001 M NaN3 at pH 7-2. The flow rate was 12 ml/hr; 3-5-ml fractions were collected, theprotein was monitored by absorption at 280 nm, and C56 activity was assayed on reactorplates.

Haemolytic reactions in gel'Reactor screen plates' were prepared as previously described (Thompson & Rowe, 1968),

containing 109 EA incorporated into 0-7500 agarose in 4 0 ml of GVB2". Normal sera andtest samples were applied to adjacent wells and the plates were incubated for 1 hr at 37°Cand then for 24 hr at 22°C. A test serum was considered 'reactor' if an arc of lysis appearedconcave to the test serum well.

Plates to quantify the reactor state ('reactor plates') were prepared as previously described(Lachmann & Thompson, 1970; McLeod et al., 1974a). In this assay serum was activatedduring a 1-hr incubation with Z (4 mg/ml) at 37°C prior to application to an agarose platecontaining unsensitized E in a 0.7500 gel containing 0 01 ml ofGPC in 4 ml ofEDTA-GVB.This plate quantitatively measured C56 complexes by radial haemolysis.The electrophoretic migration ofC56 in the Z eluate was determined by placing test samples

in wells in a 100 agarose gel containing 2-5 x 108 E/ml of EDTA-GVB. A 25 mA currentwas applied until the Bromphenol Blue marker dye reached the wicks. To detect C56 the

115


plate was overlayed with a 1: 20 dilution ofEDTA for 10 min, drained and incubated at 220 Cin a humidity chamber and lysis recorded at 24 hr.

EC567formationThe stable EC567 intermediate was formed by adding 0 1 ml of C56 to 0 3 ml EDTA-

GVB containing 5 x 106 E and ten units of C7. The mixture was incubated at 370C for 12 min,then centrifuged at 1000 g for 3 min. The cell pellet was washed twice in five volumes ofEDTA-GVB. A dilution of C56 was chosen so that the untreated control would yield70-90O EC567. These cells were lyse dby incubation for 15 min at 370C with 0 5 ml of a1:80 dilution of GPC in EDTA-GVB. The remaining cells were lysed in 1 0 ml distilledH20, and the optical density at 412 nm was determined.

RESULTSAbsorption of C56 to zymosanWhen Z is incubated with 'reactor' serum, stable C56 appears in the fluid phase (Lach-

mann & Thompson, 1970). This can readily be measured when the activated serum (withor without the Z) is added to an agarose gel which contains unsensitized erythrocytes and

FIG. 1. Elution of C56 from Z. Reactor serum was activated with Z at 370C for 1 hr and the

suspension or centrifuged fractions were added to a reactor plate containing E and GPC-EDTAin agarose gel. Lysis is seen upon addition to the wells of the entire suspension (a), the centri-

fuged serum alone (b), the washed Z resuspended in 0-15 m NaCl (c), and a 1.0 m Na~l eluateof the washed Z (d). in washed suspensions (c, d), C56-initiated lysis is seen in the absence ofC567-INH.

C-EDTA (Fig. 1, wells a and b). The ability of C56 to induce a zone lysis is influenced bysuch factors as the presence of an inhibitor of the trimolecular complement complex C567termed C567-INH (McLeod, Baker & Gewurz, 1974b, 1975). In the present experiments, Z,which had been preincubated with reactor serum, was centrifuged and washed to remove non-

adsorbed serum components. Subsequent addition of either the Z suspension, or an eluateof it, to the 'reactor plates' resulted in lysis characteristic of C56 in the absence of C567-INHand any other potentially inhibitory serum factors. The resulting uninterrupted zones of

haemolysis are seen in Fig. 1, wells c and d.

Zymosan-C56 interactions in human sera 117

FIG. 2. Electrophoresis of a Z eluate. (a) The 1 0 M NaCl eluate from Z used to activate a reactorserum was electrophoresed in an agarose gel containing E in EDTA, and C56 activity localizedby haemolysis upon overlaying GPC-EDTA at the termination of the electrophoresis interval.(b) Simultaneous electrophoresis of known C56, purified from a euglobulin precipitate of anactivated reactor serum, is shown for comparison.

The lytic activity eluted from zymosan was characterized in order to verify that it wasactually C56. It was inactivated upon preincubation with C7 or anti-C5, but not by pre-incubation with antisera to C3 or factor B. When electrophoresed in agarose gels containingerythrocytes, it migrated identically to purified C56 (Fig. 2). It was formed and detected inreactor sera but not in normal sera. When the active Z eluate was applied to a DE-52 columnand eluted with a continuous salt gradient, the activity eluted at a relative salt concentrationof 0O 18 M, where C56 is known to elute (Fig. 3). Further, it had a specific activity much greaterthan that seen either in a Z-activated 'reactor' serum or in a euglobulin precipitate of suchserum (Fig. 3a), applied to a similar column. When the Z eluate was applied to a SephadexG-200 column, the activity eluted between the void volume and the marker IgG (Fig. 4)identical to the expected position of C56 derived from the fluid phase of activated reactorserum (Lachmann & Thompson, 1970) and again, with a greater specific activity. It was freeofC8 and C9 as determined by haemolytic assays using EAC1-7, and C567-INH as described(McLeod, Baker & Gewurz, 1974a). We therefore concluded that the activity eluted fromZ was C56, and that elution from Z could be a useful step in its preparation.

Conditions optimalfor elution of C56The optimal conditions for elution or preservation of C56 and Z were determined. Z-C56

complexes were formed at 37°C and, washed at 0°C, as described above, and eluted for 15min at 22°C with NaCl solutions of varying ionic strengths from 0 01 to 03 M. All the de-tectable C56 was found to elute at ionic strengths of 0 4 M or greater, while C56 remainedmaximally attached to Z at ionic strengths of 0-01 M or less. Therefore, subsequent washes


E00O

To

E

C

.V

a

0

E.2CD

F

.0

0

0 2 4 6 8 10 12 14 16 18 20 22 24 26Fraction number

FIG. 3. DE-52 fractionation of proteins eluted from Z. (a) A 1 0 M NaCl eluate of Z used to acti-vate a reactor serum was applied to a 2-7 x 3 cm column equilibrated with 0 01 M Na phosphatebuffer (pH 7 4) containing 40% v/v glycerol, eluted with 0 01-0 3 M NaCl gradient and C56activity was localized. (b) For comparison, a euglobulin precipitation of Z-activated reactorserum was chromatographed on an identical column. (- *) Diameter of reactive lysis zone(mm). (SO--) OD280 nm-

were performed in 0 01 M NaCl, while elutions were performed in 0-5 M NaCl (Fig. 5).The kinetics of the appearance of C56 both in the fluid phase and on the Z particle were

investigated (Fig. 6). C56 was detected on the Z particle before it was detectable free insolution. Bound C56 was detectable by 10 min, and peak C56 concentration on the particlewas reached in 15 min. Thereafter progressive loss of C56 from Z was seen, and by 2 hr itwas no longer detectable bound to the particles. By contrast, C56 was not seen free in solu-tion until 15 min, at which time most of the detectable C56 still was on the Z. It progressivelyaccumulated in the fluid phase where maximum concentrations were reached after 2 hr; nodecay of C56 in the fluid phase was noted during the 6-hr observation period. Thus, the


Vold C56 IgG Albbunin0300

0-250

0-200

C

0-1500 I-07

00500*4e ~....e~e.* *^..........ee-^. ee-*-.o 3

O~~~~~~~~~~~~~~~~~~~ ES

31 39 47 55 63 71 79 87 95 103Fraction number

FIG. 4. Sephadex G-200 fractionation of proteins eluted from Z. A 160 M NaC eluate of Z usedto activate a reactor serum was applied to a Sephadex G-200 column; fractions were collectedand tested for C56 by radial haemolysis and protein was monitored by absorbance at 280 nm.(0) GD280 nm. (0) Diameter of reactive lysis zone (mm).

6-5 _

60~~~~~~~~~~~~~~~~~

0-05°0 * -_.

z ~~~~~~~~~~~~~~~~~~~3 5 O30_-

0 02 04 06 08 10 12 14 16 8 20 30

Concentration of NoC emboyed for erution (M)

FIG. 5. Elution of C56 from zymosan. Zymosan used to activate 'reactor' serum was treated withNaCl solutions of varying ionic strengths, and these eluates (o), as well as eluates of the treatedzymosan resuspended in 0-15 M NaCI (o), were assayed for C56 in reactor plates by radialhaemolytic diffusion.

binding of C56 to Z is of limited duration, and C56 could be optimally eluted after 10-15min incubation at 22°C.


80_

_ 700 / A

760 - A

'b 5-0

40)4-0 A

3zoAI! 9* . 42 3 4 5

Time (hr)

FIG. 6. Generation of C56. Z was incubated with 'reactor' serum at 370C and samples wereremoved at varying times and centrifuged. The Z pellet was washed in 0 01 M NaCI and C56was eluted with 0 5 M NaCl. The C56 in both the serum supernate and the eluate were assayedin reactor plates by radial haemolysis. (e) C56 elutable from zymosan. (A) C56 in serum.

Agents optimalfor formation offluid phase and bound C56Previous investigations ofThompson & Rowe (1968) had indicated that only agents which

now are considered to be preferential activators of the alternative complement; pathway(Gotze & Mtiller-Eberhard, 1970) were effective in generating persisting fluid phase C56 uponinteraction with reactor sera. We reinvestigated this aspect, searching both for fluid phaseand agent-bound C56. A variety of agents in amounts selected to totally deplete haemolyticC activity were preincubated with reactor sera (Table 1). No C56 was detectable either in thefluid phase or on the centrifuged pellet when sheep EA, BSA-anti-BSA, HGG-anti-HGG,AHGG or SK-SD (supernate only) were tested. By contrast, agents (agarose and endotoxiclipopolysaccharide) which, like Z, were preferential activators of the alternative pathway, werecapable of generating C56 in solution and had detectable C56 bound to their surfaces. C56was more efficiently and conveniently eluted from Z than from agarose or LPS. We concludethat generation of C56 in reactor serum is a property of activators of the properdin pathway.

Generation of a C56-binding site(s) on zymosan

C56 did not bind to untreated Z during incubations of 5-60 min at 37°C or 0°C. To testwhether normal serum could produce a C56-binding site on Z, Z and fresh normal serum werepreincubated at 37°C for varying intervals, the Z-serum complex was washed, and partiallypurified C56 was added to these complexes. After incubation for 30 min at 37°C, the mix-tures were further washed in low ionic strength buffer and thereafter examined for thepresence of C56 on the serum-treated Z particles. As shown in Fig. 7, a transitory ability to

bind C56 appeared during the interaction of Z with normal serum. The rate of formationand decay of this site(s) was similar to the kinetics of appearance and disappearance ofelutable C56 from Z in reactor sera. This site(s) was not formed when serum and Z were


preincubated in EDTA or at 0C, nor was if formed in serum heat-inactivated at 560C for30 min. We conclude that the binding site for C56 is generated by interaction of Z withnormal serum if reaction conditions suitable for activation of complement are provided.

TABLE 1. Generation and binding of C56 in 'reactor' serum*

Detectable C56Agent Amount Supernate Agent

Sheep EA 1 x 109 cells 0 0BSA-anti-BSA 110 pug 0 0HGG-anti-HGG 60jpg 0 0AHGG 4100 ug 0 0SK-SD 1000 units of SK 0

250 units of SDAgarose 50,ug + +Zymosan 2000 pg + +Endotoxin 2000 pg + +

* The agent was incubated with 0-2 ml of human reactor serumat 370C for 60 min. In all mixtures, haemolytic complement activitywas completely depleted. The mixtures were centrifuged and boththe supernate and the agent, resuspended in 0-15 M NaCI, wereassayed for C56 activity in a reactor plate.

100 _

o 80 0

i 60

0 40

10 20 30 40 50 60 70 80 90 100 110 120 130 180

Time (min)

FIG. 7. Generation of C56-binding site upon interaction of NHS and Z. NHS was incubatedwith Z at 370C. At varying times samples were removed and the Z recovered and washed oncein 0 01 M NaCl. The serum-treated Z was incubated with purified C56 for 15 min at 220C, centri-fuged, and washed, and bound C56 was eluted with 0-5 M NaCI. The percentage of C56 boundand subsequently eluted was assayed by its ability to form EC567 in the presence of E andC7.


DISCUSSION

Lachmann & Thompson (1970) had eluted stable C56 from Z which had been preincubatedwith human reactor sera. We were surprised to observe that C56 was detectable on Z evenprior to its appearance in the fluid phase, and this proved to be of value in recovering largeamounts of this intermediate. Elution ofC56 from Z resulted in a reagent which upon furtherpurification was found to be free of C7, C8 and C9 and C567-INH. This C56 was suitablefor study of its interaction with components of the complement attack mechanism (Kolbet al., 1972). C56 previously had been separated from Z and cobra venom factor-activatedreactor sera by preincubation and chromatography (Lachmann & Thompson, 1970;Goldman, Ruddy & Austen, 1972), as well as by addition of purified C5 and C6 to EAC-14oxy23 cells (Goldman & Austen, 1974). Goldlust et al. (1971, 1974) had observed unstableC5b,6 to appear in the fluid phase when EAC1423 was incubated with purified guinea-pigC5 and C6; it is not clear whether the more stable C56 reported by others and ourselvesworking with human reactor sera is exclusively a species difference, or also is influenced bythe reactor state or the use of whole serum rather than purified components.

It has been shown (Shin, Pickering & Mayer, 1971) that either guinea-pig C5 (C5b) or a

complex of guinea-pig C5b and C6 (C5b,6) can bind to, and elute from erythrocytes possess-ing bound C3b sites (Goldlust et al., 1971, 1974). Furthermore, activation of the alternativepathway in guinea-pig serum with Z generated a multiunit C5-cleaving enzyme system con-taining at least C3b and properdin factor B (Brade et al., 1973; Nicholson et al., 1974). Thisalternative pathway enzyme system might also be expected to reversibly bind C5b,6 andthus be analogous to the human C56-binding site which we have studied here (Fig. 7). Thekinetics of formation and decay of the C56-binding site generated on Z by human serum issimilar to the generation and decay of the C5-cleaving enzyme in guinea-pig serum (Bradeet al., 1973). Formation of the C56-binding site seemed to require participation of thecomplement system since it was not observed when heated serum was used or when pre-incubations were performed at 0°C or in the presence of EDTA. It is not yet clear whetherit can be formed via both the primary and the alternative complement pathways. The appear-ance of the C56-binding site seemed to correlate with the appearance of C56 bound to Z inreactor sera and the decay of the site seemed to correlate closely with the release of C56 fromZ in reactor sera (Fig. 6).We were unable to demonstrate C56 in reactor sera when complement was consumed by

a variety of agents which chiefly activate the primary complement pathway, including HGG,immune complexes, SK-SD mixtures, and EA; in these instances, no C56 was found in thefluid phase or on the particle surface. By contrast, activators of the alternative pathwayincluding agarose and endotoxin, like Z, resulted in the appearance of C56 detectable on theparticle surface as well as in the fluid phase. It seems that generation of persisting C56 inwhole serum occurs preferentially via activation of the alternative pathway. C56 had been


observed in the fluid phase by others after activation of the primary complement pathway(Goldman & Austen, 1974; Goldlust et al., 1971, 1974), but only when the purified com-plement components C5 and C6 were added to a C5-consuming intermediate. The under-lying basis for this preferential formation of persisting C56 in whole serum by activators ofthe alternative pathway is not yet clear.

Persisting C56 is found on particles and in the fluid phase when activators of thealternative pathway are added to certain (reactor) sera but not upon addition to normal sera.A lowered level of C7 has been reported to be one basis for the preferential appearance ofC56 in acute phase sera (Lachmann & Thompson, 1970); however, C7 levels are normal incertain reactor sera (Schutte et al., 1975), suggesting that additional factors also favour for-mation of persisting C56. The identification and role of such factors in acute inflammatoryreactions, like the role of C56 and C567 complexes themselves, remains to be determined.

ACKNOWLEDGMENTS

Supported by grants from the National Institutes of Health (numbers 5 ROI Al 10784-02),the American Heart Association, the Leukemia Research Foundation and the Fay-HunterTrust. H.G. is Thomas J. Coogan, Sr, Professor and Chairman of Immunology.

REFERENCES

BRADE, V., LEE, G.D., NICHOLSON, A., SHIN, H.S. & MAYER, M.M. (1973) The reaction of zymosan with theproperdin system in normal and C4-deficient guinea pig serum. Demonstration of C3- and C5-cleavingmulti-unit enzymes, both containing Factor B and acceleration of their formation by the classicalcomplement pathway. J. Immunol. 111, 1389.

CAMPBELL, D.H., GARVEY, J.S., CREMER, N.E. & SUSSDORF, D.H. (1970) Methods in Immunology, 2nd edition,p. 159. Benjamin, New York.

GOLDLUST, M.B., SHIN, H.S., HAMMER, C.H. & MAYER, M.M. (1974) Studies of complement complex C5b,6eluted from EAC-6: reaction of C5b,6 with EAC 4b,3b and evidence on the role of C2a and C3b in theactivation of C5. J. Immunol. 113, 998.

GOLDLUST, M.B., SHIN, H.S. & MAYER, M.M. (1971) Activated C5: its stabilization by complexing with C6and the catalytic role of cell-bound C2a in its formation. J. Immunol. 107, 318.

GOLDMAN, J.N. & AUSTEN, K.F. (1974) Reaction mechanism of nascent C567 (reactive lysis). II. Killingof a rough form of Escherichia coli by C567, C8 and C9. J. infect. Dis. 129, 444.

GOLDMAN, J.N., RUDDY, S. & AUSTEN, K.F. (1972) Reaction mechanisms of nascent C567 (reactive lysis).I. Reaction characteristics for production of EC567 and lysis by C8 and C9. J. Immunol. 109, 353.

GOTZE, 0. & MULLER-EBERHARD, H.J. (1970) Lysis of erythrocytes by complement in the absence of anti-body. J. exp. Med. 132, 898.

KOLB, W.P., HAXBY, J.A., ARROYAVE, C.M. & MULLER-EBERHARD, H.J. (1972) Molecular analysis of themembrane attack mechanism of complement. J. exp. Med. 135, 549.

LACHMANN, P.J. & THOMPSON, R.A. (1970) Reactive lysis: the complement-mediated lysis of unsensitizedcells. II. The characterization of activated reactor as C56 and the participation of C8 and C9. J. exp.Med. 131, 643.

124 Patricia J. Baker et al.MAYER, M.M. (1961) Complement and complement fixation. Experimental Immunochemistry (ed. by E. A.

Kabat and M. M. Mayer), p. 113. Charles C. Thomas, Springfield, Illinois.MCLEOD, B., BAKER, P. & GEWURZ, H. (1974a) Studies on the inhibition of C56-initiated lysis (reactive lysis).

I. Description of the phenomenon and methods of assay. Immunology, 26, 1145.MCLEOD, B., BAKER, P. & GEWURZ, H. (1974b) Studies on the inhibition of C56-initiated lysis. II. C567-

INH-an inhibitor of the C567 trimolecular complex of complement. Int. Arch. Allergy, 47, 623.MCLEOD, B., BAKER, P. & GEWURZ, H. (1975) Studies on the inhibition of C56-initiated lysis (reactive lysis).

III. Characterization of the inhibitory activity of C567-INH and its mode of action. Immunology, 28,133.

NELSON, R.A., JR, JENSEN, J., GIGLI, I. & TAMURA, N. (1966) Methods for the separation, purification andmeasurement of nine components of hemolytic complement in guinea pig serum. Immunochemistry, 3,111.

NICHOLSON, A., BRADE, V., LEE, G.O., SHIN, H.S. & MAYER, M.M. (1974) Kinetic studies of the formationof the properdin system enzymes in zymosan: evidence that nascent C3b controls the rate of assembly.J. Immunol. 112, 1115.

SCHUTTE, M., DICAMELLI, R., MURPHY, P., SADOVE, M. & GEWURZ, H. (1975) Effects of anesthesia, surgeryand inflammation upon host defense mechanisms. I. Effects upon the complement system. Int. Arch.Allergy. (In Press.)

SHIN, H.S., PICKERING, R.J. & MAYER, M.M. (1971) The fifth component of the guinea pig complementsystem. III. Dissociation and transfer of C5b, and the probable site of C5b fixation. J. Immunol. 106,480.

THOMPSON, R.A. & LACHMANN, P.J. (1970) Reactive lysis: the complement-mediated lysis of unsensitizedcells. I. The characterization of the indicator factor and it's identification as C7. J. exp. Med. 131, 629.

THOMPSON, R.A. & ROWE, D.S. (1968) Reactive hemolysis: a distinctive form of red cell lysis. Immunology,14, 745.

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