Scand. J. Immunol. 17, 463-470, 1983

Regulation of the Binding of C3-Coated Particlesto Human Lymphocytes by Human ComplementComponent H*

M. F O N T A I N E . M. DA VEAU & D. GI LBERT

INSERM U-7«. Bols-Guillaume. France

Fontaine. M., Davcau, M. & Gilbert, D, Regulation of the Bindina of C3-Coateil ParticlesIO Human Lymphocytes by Human Complement Component H, Scand. J. Immunol. 17,463^70, 1983.

Human complement component H was found to modify greatly the binding of C3-coatedparticles to lymphocytes. We used an e.xperimentai model in which lymphocytes weremixed successively with various amounts of H and C3b-coatcd erythrocytcs. At least threemechanisms were postulated to interpret the phetiomenon: (i) release of endogenous 1by lymphtxrytes triggered by H through specific binding sites, (ii) cleavage of iC3b by I,promoted by complenKnt receptor type one, and (iii) inhibition of immune adherence byH. Such qualitative and quantitative changes in C3-coated particle recognition by thebinding sites mi^t mediate important functions of lymphocytes.

M. Fontaine. INSERM U-78, 543, Chemin de la Bretique. 762M) Bois-Guillaume, France

Human complement component H is a ^-globulin [18] that controls the activation ofthealternative pathway. It blocks the access of B toC3b, dislodges Bb from the complex C3b.Bb,and modulates C3b attack by I [2. 17. 20. 29.30].

Lambris et al. [13] found that peripheralblood lymphocytes {PBL) had membranereceptors for H. The binding of H to PBLinduced the release of endogenous I that couldcleave cell-bound C3b into iC3b. So, cell-bound iC3b bound to cell complement receptor(CR) type two (CR2) and type three (CR3) butnot to CR type one (CRI). More recently,Schmitt ('/ ol. [24] suggested the presence ofspecific sites on complement receptor-positive(CR') cells to which soluble and cell-btJund Hand cell-bound iC3b could bind.

In the present study we examined the role ofH in the interactions between PBL and cell-bound C3b. Our results argue for an H doseeffect on the binding of opsonized particles tolymphocytes that leads to qualitative and quanti-

tative changes in C3-coated particle recognitionby the binding sites.

MATERIALS AND METHODS

Celh. Human peripheral blood lymphocytes(HPBL). B lymphocytes free of montvytes, and Tlymphocytes were prepared us described [H], Mono-cyte contamination was asse.ssed by peroxidasestaining.

Complement-coated erythrocytes were preparedfrom sheep erythrocytes (E) sensitized by rabbit anti-sheep haemolysis and then incubated for 5 min at37'C with an R.I reagent (6) to make F\ CT-Q cells.EA Ci?2 cells were washed three limes with coldde^ttrosc gclaiin barbital (Veronal") bulfer (DGVB).The cetls were then incubated with puriftcd C3 for5 min at 37 C to make F.A CT?3Tb cells. The cellswere washed as described ahovc and resuspended inDGVB at a coneentration of 10* cells/ml andstored at 0 C.

CR as.stiy.\. To determine the binding of C-coatederythrocytcs to lymphocytes, 5 x lO** lyniphixryteswere mixed with 10" C-coatetl particles. The reactionmixture was adjusted to 0.2 ml with RPMI. Theincubation was maintained for 30 min at 37'C with

* Nometiclature of the alternative activating pathway of complement: WHO Report, Hur, J. Immtinol,I1.66S.(I981).

0300-9475/83/0500-0463 S02.00 .r 1983 Blackwell Scientific Publications 46.3

464 M. Fontaine, M. Daveau & D. Gilbert

constant and gentle stirring and then counted forrosette forinatiiin. Cells that bimnd three or morecrythrcxrytes were counteil as rosettes.

For preincubation of lymphocytes. 5x10* cellswere mixed with biilTcr alone or wilh variousamounts of \\ during 30 min at M C in a fma!volutiic nf UK) /.I. Cells were then used in CR assayswithotit being washed, a described above. Forinhibition experiments, immune sera (that is,anii-l) were added before the rosetting assay andincuhatcd further 30 min at 37 C. Cells were notwashed before rt>setting expcrinwnts.

Immune adherence was performed as described[111, u-iing human O" erythroc\tcs (10" cells/ml) inmierotitre plates. For inhibition experiments, thetest was done on opalin plates with dill'ercnt concen-trations of human erythroeytes. Sheep erythroeyteswere coated with complement ci>mponent H hymeans of CrCb at a density equivalent to thatobtained by active deposition of H on EA C1423cells. The density of H on erythrtx'ytcs was estimatedby an agglutination assay using an anti-H antibody.

Binding of fluid-phase H to lymphocytes wasvisualized by immunofluorescence using rabbit anti-H untibodicsand sheep fluoixsceinatcd antibodies toanti-rabbit IgG (Institut i\)steur).

5 X IO'* HPBL were incubated (30 min. 37 C) withvarious amounts of H in a final volume of 200/iIRPMI. Controls were set up without H, Cells werewashed thiee times in RPM! and then reacted wilhanti-H antihodies. After three washings in barbital(VcronalHbutTered saline (VBS). they were incubatedwith the (luoresccinated aniihody. Lymphocytes werewashed twice in VBS. airnlried, and mounted undergiyccrol for observation under a Zeiss microscopeequipped with epifluorescence.

Complement components and antisera. Highlypurified C3 was prepared from fresh normal humanserum by Rivanol and euglohulin precipitation [27]followed by an atT'tnity chromatography on aSepharose anti-'impiuities" column [5]. This stepmade pnssihle the complete depletion of H and 05from C.T preparations.

H was prepared from the eugiobulin fraction offresh normal human serum hy ion-exchange chroma-lography on DEAE-Sephaeel (Pharmacia) followedby a gel liltration on Sephacry! .S-300 (Pharmacia)(RipiK'he ft al.. submitted for publicaiion).

The purity of C3 and H was as.sesscd by immuno-electrophoresis. immunodiffusion. and haemagglu-tination. using the following antisera; anti-wholehuman scrum. anti-C3d (Organon Tcknika). anti-1(Kent Lahoratt>ries), anti-C3c. anti-H (prepared inour laboratory). anti-C4bp (Dr Colomh. Grenoble),and by haemolytic assays of C3 and C"5.

The homogeneity of C3 and H preparations waschecked by sodium dodecyl sulphate polyacrylamidegel electrophoresis (SDS-PAGE) [28].

The presence of I in preparations was alsocheeked hy inhibition o( immune adherence.

Radinlahelting of O . Highly purified C3 waslabelled with Na '-'''I hy the chloramitie-T method[I0|. The speciHc activity WHS I /iCi/mg C3 withoutalteration of the haemolytic fictivity of C3.

EA CR'2 cells were allowed to react with ^"I-C3

as described above. EA CTO5 eelK were mixed withPBL stimulated by H or not. Rusettes were counted,harvested, counted for radioactivity, and analysedby SnS PAGE [28]. The pellets were incubated with2% SI>S. 4 M tirea. and 5",, |8-mercaptoethanol for5 min at 90 C and applied onto 7.5",, acrylamidegels. After electrophoresis, gels were sliced into2-mm sections and counted for radiLiactivity in agamma counter. Gels containing purified C3 andmoleeular weight markers (low molecular weightmarkers from Pharmacia) were stained with Coo-ma.ssie brilliant blue R-250 and desiaincil.

RESULTS

// preparations

The M preparations used in these studies wereconcentrated to 0.9 mg/ml. giving in the roset-ting assay a final concentration equivalent to theserum concentration of this protein. At theseconcentrations. H appeared in SDS-PAGE as asingle polypeptide chain of I5O.(KX) daltons(Fig. I), but by gel filtration experiments onSephacryl S-300 cquilibtatcd with O.LS M NaCl,0.02 M IiDTA. and 0.05 M <-aminocaproic acid.

Ftc;. 1. Demonstration of the homogeneity of H andC3 preparations by SDS PAGK. Panel A: Poly-acrylamide (4-.10",,) gradient gel. purilied H (withoutmcrcaptocthiniol). Panel B: 7.5",', polyacrylamidegels with reduced samples (5";, mercaptoethanoi).

H; 2 = purilied C3.

Interactions H/Lymphocytes 465

pH 7.2, a 300.000 molecular weight was found,indicating a dimeric structure of H at thiscoticentration. These preparations were free ofI as detertnined by Ouchterlony analysis withanti-I. The preparation did not abolish theimmune adherence phenomenon (see below),and factor H remained bound to EA C1423 ceilsafter an overnight incubation.

Rosetting assays

The influence of R3 and C3 concentrationswas studied in detail to elitiiinate the influence ofC4 and to obtain maximutn rosette formationwith C3-coated particles. In our experiments, nobinding of EA Ci4 cells to lymphocytes wasobserved when there were two SA C14 sites percell. Eig. 2 shows the dose dependence of EAC1423 rosette formation on the concentration ofC3 used to coat EA CT42 cells. We chose a C3concentration that gave 1(X),000 molecules ofC3 deposited per cell (determined with - I C3).With tiative HPBL, EA CT423 alone gave anaverage rosette formation of 20% (M = 19.6,a=±3.7) .

Influetice of H concetttratians on the EA CJ423rosette formation

Fig. 3 shows clearly the influence of H on EAC1423 rosette formation wiih PBL whenlymphocytes were pre-incubated with variousconcentrations of factor H. At serum concen-tration. H considerably enhanced the number ofrosettes that reached 30% (M = 30, a= ±8.8).

5 10INPUT OF C3 EACI42

Etn. 2. Dependence of the rosette formation be-tween human peripheral blood lymphocytes and EAC1423b on the amount of C3 offered per EA

0,25 0,5H CONCENTRiTION « IQ- mg/ml

EiCi. 3. Binding of EA C1423b to human peripheralblood lymphocytes preincubated with variousconcentrations of H.

whereas weak concentrations considerably de-creased the EA CR23 rosette formation until8% (M = 8.5, a= ± 2.4). Eor very weak concen-trations, the number of rosettes reached theplateau value of EA Cf423 rosettes with PBL.The phenomenon was specific f"or B lympho-cytes. This phenomenon was quite reproducible,since we obtained the same variation of rosetteformation over 30 experiments.

Presence of a receptor specific for factor H

Increased rosette formation of EA CI423cells with PBL In the presence of high concen-trations of factor H could be due to a depositionofJ4 on EA C142'3 cells and binding of EAC1423 H cells onto a receptor specific for H.This possibility was studied by direct binding ofsoluble factor H and visualization by an indirectimmunolluorescence assay and by rosetting ofsheep erythrocytes eoated with H by means ofCrCht. At serum concentration both methodsfailed to demonstrate a binding of factoi H onPBL. Only 5",; of cells were positive by bothrnethods. But by increasing the concentration ofH ( X 6), a strong fluorescence could be observedon B lymphocytes. Seventy to 80% of PBL werestrongly .stained. Despite the presence of Hreceptor, it appeared from these experiments thatincreased rosette formation was probably notdue to a binding of EA C1423 cells through abinding by factor H to its specific receptor.

Evidettce for release of ettdogenotis factor Idttritig incuhatiott of PBL with H

Lambris et al. [13] recently demonstrated the

466 M. Fontaine, A/. Daveau & D. Gilbert

release of endogenous factor I from lympho-cytes after stimulation by H. To investigate theparticipation of factor 1 in our experiments, wechose two concentrations of factor H (finalconcentration, 0.45 and 0.025 mg/tnl) for thepreincubation of lymphocytes before the roset-ting assay.

Under these conditions the number of rosettesobserved was 30 /„ and 8 " „ , respectively,whereas without H. 20% of total lymphocytesrosetted with EA CWH (Fig. 4). If anti-Iantibodies were added to the incubationmixture simultaneously with factor H. novariation of the number of EA CI423 rosetteswas observed. These results clearly indicatedthe participation of factor I during the tosettingassay for high or weak concenttations o\' H. Thephenomenon wa,s also abrogated by addition ofEDTA or NaN;i duting the ineubation of PBLwith H. These results ruled out a possibleparticipation of exogenous factor ! in thesephenotnena.

30

20

10

J30

20

10

30 •

e 20

0 0 025 0.5

H CONCENTRATION mq/ml

FIG. 4. EfFecl of inhibition of H on EA CRl lbbinding to lymphocytes by anti-I aniibodies.

To demonstrate a conversion of C3b by Iduring the rosetting procedure, EA CI423 cellswere prepared with '-•' I C3. After the rosettingassay, EA C1423 cells were collected, washed,and solubilized in 2% SDS. 4 M urea, and5% /3-mercaptoethanol. und analysed by acryla-mide SDS (Fig. 5). It api'wared that /3-chain waslabelled to a lesser extent than a-chain was. Innative C3 (not shown), radioactivity present inj8<hain represented only 25 "„ ofthe total radio-

30

zo

10

5 10 15 I'OGEL SLICE NUMBER

FtG. 5. SDS-PAGE paitems of cell-bound C3b afterincubation with lymphocytes preincubated with (A)buffer: (B) H 0.5mg,ml; (C) H 0.025 mg/ml.'•''•l-Iabellcd C3, CS, and ovalbumin were used asinternal markers. Positions of > and /? chains, C3,and ovalbumin are indicated by arrows. Cellularinlermediaies were harvesietl afler incubation wiihlymphocytes, washed, counted, and solubilized inSDS-PAGE sample bulTer. Afier electropboresisgets were sliced into 2-mm sections and counted forradioactiviiy. Results are expressed for each sliceas the percentage ofthe total radioactivity present inihe gel. Gel containing ' •"'I-C3 was sliced and theradioactivity counted. Gels containing C3 andovalbumin were slained and destained,

activity incorporated in C3. The positions ofnative y. and ^ chains are indicated by arrows inFig. 5. Al l the radioactivity behind the x-chainposition was assumed to be complexed z chain.

Interactions Ht Lymphocytes 467

The same repartition of radioactivity wasobserved for cell-bound C3b (panel A) but«' chain of C3b migrated more slowly ihan« chain in native C3. indicating thai x' chain hadbound covalently to a protein species of mem-brane sheep erythrocyte. When EA C14T3 cellswere ineubated with lymphoeytes prestimulatedwith H at serum concentration. SDS-PAGEanalysis of cell-bound C5b indicated a stronglymarked modification of C3b. No radioactivitywas found in the gel behind the 3t-cliain position,but an increase of radioactivity was found in the^-chain region, representing 46";, of the totalradioactivity (panel B).

For low concentrations of H (0.025 mg/ml),the radioaciiviiy profile observed was identicalto ihat of native cell-bound CMb (panel C). Butafter harvesting of cellular intermediates at theend of the rosetting assay, a marked decreaseof total radioactivity was observed (50%).indicating a release of C3 fragments in the fluidphase. For serum concentrations of H, theradioactivity present on the cells after the roset-ting assay was quite constant.

Inhibition ol' the immune adherence phenomenon

Control experiments aimed at checking thepurity of H preparation revealed that thispreparation was not able to abrogate immuneadherence, whereas it could do so when theCR' cell concentration was decreased. Toobserve the agglutination, a technique onopalin plates was used, which enabled us to

study the kinetics of (he inhibition. Table 1shows (hat H could abrogate immune adherence,but only transiently. The inhibition was moreobvious when the concentration of CRlreceptor was decreased.

D I S C U S S I O N

The influence of complement componenl H onthe binding of C3-coated particles lo lympho-cytes was first obser\ed by Okuda & Tachibana[19]. The way this component interferes in therosetting assay of cell-bound C.lb and lympho-cytes seems complex and tiuiliipic. Theseauthors suggested that H induced a eonfor-mational change of cell-bound ( 3 b that wasrecognized by a new type of receptors on Rajicells. Lambris et al. [13] reported that H couldtrigger lymphocyte via a specific reeeptor forthis protein and induce the release of endo-genous factor I that could cleave C3b inloiC3b. Therefore, the newly formed eellularintermediates bound to CR2 and CR3 recep-tors. Using a dilTerent experimental approachincluding EDTA in all experiments. Schmitt('/ (//. [24] demonstrated that V.A. C1^23 cellscould bind to lymphocytes in the presence of Hthrough the receptors of complement compo-nent H. The release of I was then blocked byEDTA [13].

In this study, we followed an experimentalprocedure as close as possible to an in vivoevent: EDTA was not used, and cells were not

I. Inhibition of immune adherence by H

(>• erythrocyteconcentration

108

IO«

10*

Reaclion(imes(min)

136

136I36

0.5

001

OO

OO

OO

H

0.1

002001

OO

O

concentration

0.025 0

224

02

OO

O

(mg,ml)

.005 0

444

1i2

001

.001

444

2222•>

2

0

444222

to K

l K

l

One volume of EA Cl^I^h cells (IC cells/ml) was mixed wiili one volume ofO' erythro-eytes antl one volume of hulTcr or H solution onto an opiilin plate. Agglutination was moni-tored by continuou.s ohservalioti under a niLignKyijig glass. Zero indicates no aggtulinalinn:(he strength of agglutination was marked from I to 4.

468 .\/. Faniaine, M. Daveau & D. Gilbert

washed after stimulalion of lymphocytes by H.To rule out the participation of exogenouscontaminaling complement components (that is.I. C5), we used \ery highly puriiied componentsC} and H. As described by others [13]. Happeared heterogeneous and ubie to aggregateeasily. In ihe present work. H appeared as asingle polypeptide chain of 150.000 daltons indenaturing medium that elulcd on gel filtrationwith an apparent molecular weight of 300,000.suggesting a dimeric structure for this proiein inphysiological conditions.

The prelreatmcnt of lymphocytes with Hbefurc the rosetting assay had conspicuouseffects on the binding of C 3-coatcd particles tolymphocytes. These effects were dose-dependent(Fig. 3). To understand how cell-bound C3bbinds lo lymphocytes in the presence of H. weinvestigated the structure of cell-bound C3bafter the roseiiing assay. Three mechanisms atleast mighi occur in these phenomena: li)participation of lymphtx-ytc endogenous factor1; tii) inhibition of the imnuine adherencephenomenon by H at weak concentrations ofC R l ; (iii) binding of cell-bound C3b to lympho-cytes through II receptors in the presence ofIhis componenl.

In our experiments, the third mechanismseemed unlikely despite the presence of Hreceptors on B lymphocytes. This receptor wasvisualized only if H was very concentrated(6 mg/ml). hut at this *.onccniration H certainlyaggregated. Sheep eryihrucytcs coaled with H hymeans of CiCi:i failed to exhibit it>settcs withHPBL (< 5%) even ai u density identical to thatobtained with EA CI423h-H intermediate.Nevertheless, the possibiliiy thai H has dilTerentstructures on EA CI423 cells and on CrCLisheep erylhrocytes could not be ruled out. C3are dept^ itcd in large clusters around the EACT45 sites: the fixation oi' H on Ihese cellstherefore certainly leads to high-density Hregions on the cells, whereas with H CrCla-coated sheep erythrocytes, H is evenly distri-buted on the cell membrane.

The release of lymphocyte-endogenous I wasquite obvious if lymphocytes were preincubatedwith H. Whaiever the H conceniration was, thephenomenon was completely abrogated byaddition of anti-l antibodies in the mediumbefore the rosetting assay. This inhibition wasquite specific. For the scrum concentration ofH. the electrophorelic puttern of cell-bound

'^*l C3b afler the rosetling assay clearlydemonstrated the conversion of C3b into iC3b.The conversion was visualized by the cleavage ofthe x' chain of t'3b. giving a polyi'wptide chainof 6S,000 daltons [ I . 7, 15], which appeared inthe gel near the posiiion of fi chain. «' chain ofC3b is covalently linked to compt»nents of sheeperylhrocytc membrane [14]. A cleavage of the3f' chain is therefore observed by the shifting ofihe radioactivity below the pt>sition of i' chain.

For serum concentrations of factor H, iherewas no release of radioactivity in the lluid phase.Inversely, at the H conccntraiion giving mini-mum rosetie formation, a large release of radio-activity in the Huid phase was ob.served. Thiswas certainly due to Ihe cleavage of iC3b intoC'3d,g [12] by I in the presence of CRl, since thisconversion was not observed in the absence oflymphtKyies. Recently, ditTereni authors showedthai I is able lo cleave iC3b inio C3c and C3d.g[16,. 21]. This conversion required the presenceofCRI( [2 I ] ; Medof. M. E.. Ida, K. & Nussenz-weig. V., J. t'.v/i. Med.. in press, see Ref. 21). .^ithai concentration, the conversion of C'3b intoiC3b was not complete, since Ihe profile of radio-activity of the remaining cellular intermediatewas similar to that of cell-bound C3b. On amolecular basis, C3d,g represented nearly 20%of the total radioactivity, and in view of thecovalent association of C3d with erythrocytemembrane that led to a dispersion in size ofcomplexed C3d species, no representalive bandofC'3d was visualized on the gel.

The elTect of II on EA Cr423b binding toiymplwKytes was not due to exogenous factor I,since EDTA and NaN.1 completely inhibited thephenomenon. These reactants are known toblock the release of endogenous I [13].

H was able lo inhibil the binding of C3b toCRl, as demonstrated by our experiments withinhibition of immune adherence using humaneryihrocyles. This inhibition was observed onlyif tbe concentration of CRl was considerablydecreased (under 10" cells CRI /ml ) ; othervv'iseihe phenomenon was very weak and transient,and it disappeared with prolonged incubationlime. In the roselting assay, Ihc conceniration ofCRl ' cells was in the range of the humanerythrocyte concentration for which immuneadherence could be abrogated by H. A similarobservation was made by Ciaither ct al. [7],

The CRl molecule was tirsl isolated byFearon [4J, who demonstrated that this protein

was able to regulate the alternative pathwayactivation as well as H did. These data suggestedthat CRI and H bound toC3bat the same site oron very close ones on C3b and certainlyexhibited rather identical strticttires [3]. So itwas not surprising that H could inhibit immuneadherence under particular conditions.

Therefore, at H serum concentration, lympho-cytes releaM:d into the fluid phase a sufficientamount of I that cleaved C3b into iC3b. Thecomplete conversion led to a total and rapidincrease of rosette formation. At that concen-Iration H cornpeted with CRI and thereforeinhibited the cleavage of iC3b into C3c andC3d,g. Rosetting tests were carried out alphysiological ionic strength. Under theseconditions H did not induce iC 3b cleavage by I[21]. iC3b could exhibit two dilferent structures—iClbi (75 K + 68 K T 4 6 K) and iC3b:; (75 K+ 68 K+43 K) [9. 26]. Under our experimentalcotidltions C3b was certainly degraded to theiC3b:; structure [21], so we visualized the CRIand CR2 receptors [23]. A 30",. rosette for-mation is in agreement with previous workusing cell-bound iC3b [25].

At a lower concentration of factor H (0.025mg/ml). lymphocytes released I also, bul H didnot interfere with CRI that could promotecleavage of iC3b into C3c and C3d,g thatremained bound to the cells. C3d,g-coatedcells bound to CR2 and CR3 receptors [23]. An8",, rosette formation for cell-bound C3d,g wasin agreement with previous work on CR3 [22],indicating average values of 9",, CR2 lympho-cytes and 3.5% CR3' lymphocytes. Forweaker concentrations of H, all the phenomenadescribed above disappeared slowly, and theoriginal number of rosette formation of EACl'42lB was obtained. Such guaiitaiive andquantitative changes in C3-coated particlerecognition by the binding sites may mediateimportant lymphocyte functions.

ACKNOWLEDGMENTS

The authors thank Dr Cotomb and Dr Villiersfor providing anti-C4bp and Mrs A. Chaubeand Mrs M. M. Fontaine for help in preparingthe manuscript. This work was supported by agram of INSERM (C. R. L. 82.10.40) atid by theUniversity of Rouen,

Interactions HI Lymphocytes 469

REFERENCES

1 Carlo, J.R., Ruddy, S., Studcr. E.J. & Conrad,I>.H. Complement receptor binding of C3b-iroated cells treated with CMb inactivutor J3iH-globulin and trypsin. J. Immunol. 123.52.1,1979.

2 Conrad, D,M., Carlo. J.R. & RmUly, S. Quanti-tutivc analysis of binding und influence olalternative pathway components on binding.J.e.xp. Mcd. 147. 1792, 1978.

y Dierich. M.P.. Ehlen, T. & Mussel, M.H.Description of a single, specilic and sensitive icsifor the detection of detergent solubili/ed C3breceptor. Immiinobiol. 140. 241, 1981.

4 Fearon, D.T. Regulation of the anipliticationCVConvcrta.sf of hurnan complement hy aninhihitory protein isolated from huniati erythro-cyte membrane. Prov. naiti. .•Uiiil. Sci. lb, 5S67.1979.

5 Fontaine. M. & Rivat, C. A study of the break-down of the ihird component of htiman comple-ment (C3). .Ann. Itnmunol. (Inst. PasU-ur) I30C,.149. 1979.

6 Fontaine. M.. Joisel. F. & Dumouchel. L.Preparation of an R3 reagent (serum depleted ofthe third component of human complement (C3))by immunoadsorptioii. Application to thehemolytic assay of human C.V J. linnttmol. Meth.33, 145. 19X0.

7 Gaither. T.A.. Hammer, CH, & Frank. M.M.Stuilie-i of thi: molecular mechanisms of C3binuciivation und asimpiificd assay of iH and theC.lb inactivator (C3b INAl. J. Immunol. 123,1195.1979.

8 Gilbert. D.. Daveau. M. & Fontaine, M.DitTcrential binding of IgG subclasses to cn^yme-treiited human lymphocytes. Scand. J. Immunol.14, 21. 1981.

9 Miirrisoii. R.A. & Lachmann, P.J. The physio-logical breakdown o\' ihc third cnmponciit ofhunuu) coinplcnieni. Mol. Itttmunol. 17, 9, I9K0.

10 Hunter. W.M. Radioimmunoassay 17. I. 17. 35,m: Weir, D.M. (cd.) Hamllwok of ExperimvntalImmunology. Vol. I. 2nd edition, HIackweltScientific Publications. Oxford. 1973.

11 Uchmann, PJ., Hobart, M.J. & Aston. W.P.Complement technology 5. 1. 5. 17. in: Weir.D.M. (cd.) Handlxtok of fLxperimetual Immuno-logy. Vol. I, 2nd edition, Blackwell ScientificPublications, Oxford, 1973.

12 Lychmami, P.J.. Pangburn, M.K. & Oldroyd,R.G. Breakdown of C3 after cumplcnicnt acti-vation. Idcntitiealion of a new fragment, C3g.using monoclonal antibodies. J. e.vp. Med. 156,205, 1982.

13 Lambris. J.D., Dobson, N.J. & Ross. G.D.Release of endogenous C3b inactivator fromlymphocytes in response to triggering membranereccptoni for ^iH-globuIin. J. exp, Med. 152,1625. 1980.

14 Law, S.K. & Levine. R.P. Interaction betweenthe ihird complement protein and cell surfacemacromolecules. Proc. num. Acad. Set. 74, 2701.1977.

31

470 M. Fontaine., M. Daveau <t D. Gilbert

15 Law, S.K.. Fearon. D.T. & Levinc, R.I*. Actionof the C.lb-inactivator on cell bound d b . J.Immunol. 122.759. 1979.

16 Medicus, R.G. & Arnaout, M.A. Release orC3cfrom bound C3bi by C.lb inaetivalor. Mol.Immunol. 19, 1.186, 1982.

17 Nagaki, K., Iida, K., Okubti, M. & Inai, S.Reaction mechanisms of fii H-globulin. Ini..'\rchs Allergy Appl. Imtmin. 57, 221. 1978.

18 Nilsson. U.R. & MuUer-Ebcrhard, H.J. Isolationof /Jif-globulin from human serum and itscharacterization as the lifth component ofcomplement. J. cxp. Mcd. 122. 277. 1965.

19 Okuda. T. & Tachihana, T. Complement recep-tors on Raji cx'lls. The presence of a new type ofCi recepior. Immtint>logy4\, 159, 1980.

20 Pangburn, M. & Mullcr-ElKrhiinl, H.J. Comple-mcni C.I convertase; cell surface restriction of^ iH control and generation of restriction onneuraminidase treated cells. Proc. ttatn. .Acad.5f/. 75, 2416, 1978.

21 Ross, G.D., Lambris, J.D., Cain, J.A. &Newman, S.L. Generation of three differentfragments of bound C.I with puriiied factor 1 orserum. I requirements for factor H vs CRI co-factor activity. J. Immunol. 129. 2051, 1982.

22 Ross, G.D. & Lambris, J.I). Identitication of aC3bi specific nwmbrane complement receptorthat is expretised on lymphocytes, monocytes,neutrophils and erythrocytcs. J. e.xp. Mat. 155.96. 1982.

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Received 4 October 1982Reeeived in revised form 4 January 1983