Immunology 1994 82 516-521

Plasma clearance of the human C5a anaphylatoxin by binding to

leucocyte C5a receptors

M. OPPERMANN & 0. GOTZE Department of Immunology, University of Gottingen, Gottingen, Germany

SUMMARY

The C5a anaphylatoxin is a potent complement-derived mediator of inflammation withchemotactic activity. In this study the possible role of specific high-affinity binding sites for C5aon peripheral blood leucocytes for the removal ofC5a from human blood plasma was investigated.The addition of purified granulocytes or mononuclear cells to complement-activated plasmaresulted in the rapid and dose-dependent removal of up to 80% of plasma C5a, as determined byELISA. The specific role of leucocyte C5a receptors (C5aR) in the plasma clearance of C5a was

demonstrated by the inhibition of C5a uptake by the preincubation of cells with the C5aR-specificmonoclonal antibody S5/1. Furthermore, U937 cells which had been induced by db-cAMP toexpress C5aR, but not undifferentiated U937 cells, were capable of removing CSa from plasma.The inhibition of C5aR internalization by monensin did not affect C5a uptake by leucocytes. Theco-incubation with leucocytes had no effect on the plasma clearance of complement activationproducts C3a or terminal complement complex (TCC), as determined by this in vitro assay. Thebinding of the CSa anaphylatoxin to cellular receptors represents an effective control mechanismthat protects the organism from systemic effects of this potent phlogistic mediator.

INTRODUCTION

The complement-derived anaphylatoxins C5a and C5a(desArg)are potent mediators of inflammation which contribute sig-nificantly, by their various biological functions, to this localizedand protective response.' These effects are elicited via high-affinity binding to cell-surface receptors (C5aR) and subse-quent activation of G protein-coupled signal transductionpathways. The anaphylatoxins C5a/C5a(desArg), which aregenerated at inflammatory sites increase the permeability ofsmall blood vessels, augment the adherence of circulatingphagocytic leucocytes to the vascular endothelium, and promotethe directional migration of these cells towards increasingconcentrations of C5a. In addition to directional migration,phagocytes also respond to these chemotactic peptides byreleasing secretory constituents such as lysosomal enzymes andreactive oxygen metabolites. By targeting effector cells to aninflammatory site, C5a and C5a(desArg) help to focus the

Received 8 February 1994; revised 26 March 1994; accepted 31March 1994.

Abbreviations: AU, arbitrary unit; C5aR, receptor for the C5aanaphylatoxin; CVF, cobra venom factor; db-cAMP, dibutyrylic cyclicAMP; EDTA, ethylenediamine tetraacetic acid; mAb, monoclonalantibody; MERGETPA, DL-mercaptomethyl-3-guanidino-ethylthio-propanoic acid; p-APMSF, (p-amidinophenyl)methanesulphonylfluoride; PBMC, peripheral blood mononuclear cells; PMN, poly-morphonuclear leucocyte; TCC, terminal complement complex.

Correspondence: Dr M. Oppermann, Howard Hughes MedicalInstitute, Duke University Medical Center, P.O. Box 3821, DurhamNC 27710, U.S.A.

defensive response to injurious agents and to thereby limitdamage to the host tissue which would result from excessiveand undirected inflammation.

While C5a locally acts as a potent pro-inflammatory agent,its systemic effects have to be carefully balanced by efficientcontrol mechanisms. C5a biological activities are controlled onat least three levels. Firstly, on a cellular level, homologousdesensitization attenuates excessive activation of cells uponrepeated challenge with C5a. Agonist-induced phosphorylationof the C5aR may be critical for the functional alteration ofthe receptor during desensitization.2 Secondly, serum con-tains anaphylatoxin-inhibiting activity which apparently isidentical with the serum carboxypeptidase N.3 This enzymerapidly removes arginyl residues from the carboxyterminus ofhuman C5a and thereby converts this anaphylatoxin into itsdesArg form. Nevertheless, C5a(desArg) still expresses signifi-cant chemotactic and other biological activities.4 Human C5a isexceptionally sensitive to the action of serum carboxypep-tidases. This control mechanism may therefore not be effectivein other species where C5a biological activity has been shownnot to be significantly affected by carboxypeptidases. Finally, ithas been shown in rabbits that CSa biological activity and 1251Ilabelled C5a is rapidly cleared from the circulation under in vivoconditions.5'6 As the half-life ofC5a in neutropenic animals wassignificantly prolonged, a hypothesis was put forward thathigh-affinity binding of C5a and C5a(desArg) to cellularreceptors on neutrophils accounts for the rapid elimination ofC5-derived anaphylatoxins from plasma. Neutrophilic granu-locytes express C5aR with a Kd of 2 nm and an estimated5 x 104_- .1 x 105 binding sites/cell.7 However, the specific role

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Clearance of CSafrom plasma

of leucocyte CSa receptors in the removal of CSa from plasmacould not be directly addressed in these studies.

We have recently produced and characterized monoclonalantibodies (mAb) that are directed against the human C5aRand block C5a binding to its receptor.8 These reagents now

allowed us to demonstrate a role for the leucocyte C5aR in therapid uptake and removal of C5a from human plasma.

MATERIALS AND METHODS

Reagents, antibodiesDibutyryl cyclic AMP and cobra venom factor (CVF) fromNaja naja kaouthia were purchased from Sigma (Deisenhofen,Germany). Monensin, p-APMSF and MERGETPA were fromCalbiochem (Frankfurt, Germany). Fluorescein isothiocyanate-labelled DAKO-CD15 was from Dako (Hamburg, Germany).The mAb S5/1 (mouse IgG2a/K) was generated following theimmunization of mice with a synthetic peptide which cor-

responds to the receptor's amino terminal domain.8 This mAbrecognizes the CSaR on myeloid cells from peripheral bloodand competitively inhibits C5a binding to this receptor.

Quantification of complement activation productsPlasma concentrations of the complement activation productsC5a/C5a(desArg), C3a and the terminal SC5b-9 complementcomplex (TCC) were determined by specific ELISA procedureswhich have been developed in our laboratory. Briefly, mAbwith specificity for the respective complement activationproduct were adsorbed (16 hr/4°) into wells of microtitre plates(Immunoplate IIF; Nunc, Wiesbaden, Germany) in 50mMcarbonate buffer, pH 10-6. After blocking with phosphate-buffered saline (PBS)-1% gelatine, the wells were incubated(2 hr/room temperature) with plasma samples or purifiedstandard proteins which were diluted in PBS-0-05%Tween-20mm EDTA. Complement activation products were detectedby adding, in sequence, biotinylated antibodies and a 1000-fold dilution of peroxidase-labelled streptavidin (Amersham,Braunschweig, Germany) in PBS-Tween for 1 hr. Wells ofthe microtitre plate were washed three times with PBS-Tween between all incubations. Finally, 2,2-azino-di-(3-ethyl-benzthiazoline sulphonate) (Boehringer Mannheim, Mannheim,Germany) in 0-1 M sodium acetate, 50mm sodium phosphate,pH 4-2, 2 5mm H202 was added and the absorbances at 410 nm(reference wavelength 490 nm) were recorded using a micro-plate photometer (Dynatech MR 600; Dynatech, Denkendorf,Germany).

The assay for the quantification of CSa/CSa(desArg) is basedon the mAb C17/5.9 This antibody recognizes a neoantigenicdeterminant which is expressed on C5a and CSa(desArg), butwhich is absent from its precursor protein C5. The C5/CSa-specific mAb G25/2 in its biotinylated form was applied as thedetecting second antibody. Both C5-derived anaphylatoxinsare detected by this assay with identical sensitivities.

The mAb WU 7-2 also reacts with an activation-dependentneoepitope on C9 which is not expressed on the nativeprecursor protein. Solid-phase bound TCC was detected bybiotinylated polyclonal goat anti-C6 IgG fractions. This assay

allows direct quantification of plasma TCC concentrations.10The standard curve was constructed using a zymosan-activatedhuman serum pool where the standard had been defined tocontain 1000 AU (arbitrary units)/ml.

Quantification ofplasma C3a/C3a(desArg) was achieved byan ELISA which is based on two mAb (K13/16 and D17/1),which recognize two different C3a epitopes that are alsoexpressed on the C3 precursor protein.11 Native C3 wasremoved from the plasma samples prior to the analysis by aprecipitation step using 15% (w/v) PEG 4000 at pH 4-0.

Isolation ofgranulocytes and mononuclear cellfractions fromhuman blood and cell cultureThe polymorphonuclear leucocyte (PMN) and peripheralblood mononuclear cell (PBMC) fractions were freshlyisolated from EDTA blood by density gradient centrifugationon Lymphoprep (density = 1-077 g/cm3) (Nycomed Pharma,Oslo, Norway). The mononuclear cell fractions were isolatedfrom the interphases and consisted of 21 ± 8% C5aR-positivecells, as determined by flow cytometry using the fluorescein-labelled C5aR-specific mAb S5/1.8 It has been shownpreviously that within the mononuclear cell fraction onlyCD14+ monocytes express C5aR.8 Granulocytes were isolatedfrom the cell pellets after density gradient centrifugation andremoval of erythrocytes by incubation with Gey's lysis buffer. 12As determined by flow cytometry, 92 ± 5% of the PMNfraction expressed the C5aR, 91 i 10% were CD15+.

The promyelocytic leukaemia cell line U937 was cultured inRPMI-1640 medium (Seromed, Muinchen, Germany) sup-plemented with 20% 199 Earle's medium (Seromed) and 10%fetal bovine serum (FBS; Biochrom, Berlin, Germany). TheU937 cells were induced to express C5aR by incubation for3 days in the presence of 2mm dibutyryl cyclic (db)-AMP. Theexpression of C5aR by db-cAMP-induced U937 cells was

demonstrated by their binding of either human recombinantCSa or the C5aR-specific mAb S5/1, which were applied as

FITC conjugates. Analyses were done by fluorescence-activated flow cytometry.

In vitro complement activation ofplasmaHeparin plasma was obtained by centrifugation from freshhuman blood. The sample contained 3-9ng C5a(desArg)/ml,less than 0 1 AU TCC/ml and 358 ng C3a(desArg)/ml. Com-plement activation was initiated by the addition of 2 Mg CVF/ml plasma in the presence or absence of 1 mm MERGETPA.The carboxypeptidase inhibitor MERGETPA prevents thetransformation by serum carboxypeptidase N of the C5a or

C3a anaphylatoxins into their desArg forms.'3 After incuba-tion at 370 for 15 min, the reaction was stopped by the additionof 100 HM p-APMSF. Aliquots were kept frozen at -80° untilthey were used in the clearance assays. The sample which hadbeen activated by CVF in the presence of the carboxypeptidaseinhibitor MERGETPA contained 69 ng C5a/ml, 57 AU TCC/ml and 3450 ng C3a/ml. Complement activation of plasma inthe absence of MERGETPA resulted in C0a(desArg) concen-

trations of 83 ng/ml.

Clearance assay

The clearance of complement activation products from plasmaby their binding to leucocytes was determined by the incubationof plasma samples in the presence of purified leucocytepreparations. Plasma which contained elevated concentrationsof complement activation products was added to leucocytes,which were suspended in Hank's balanced salt solution. Afterthe incubation at room temperature for the indicated time

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intervals and end-over-end rotation, the leucocytes were pel-letted by centrifugation (30 seconds/3000 g). Plasma concen-trations of C5a/C5a(desArg), TCC and C3a were determined inthe supernatants.

Statistical analysisAll experiments were done in duplicate. Values are given asmean + SD. Results were analysed for statistical significanceby an unpaired Student's t-test.

RESULTS

Clearance of plasma C5a by co-incubation with leucocytesuspensions

The addition of freshly purified leucocytes to plasma that hadbeen activated by CVF resulted in the partial depletion of C5afrom these samples. Figure 1 shows that the co-incubation ofplasma with 2 5 x 107 granulocytes/ml resulted in the removalof 50% of the C5a content in the sample. Plasma C5a concen-trations were reduced to 20% of the original values by incu-bation with 108 granulocytes/ml. No effect of the co-incubationwith granulocytes on the TCC plasma levels was observed inthese experiments.

Both the granulocyte and the mononuclear cell fractionswere capable of depleting C5a from plasma samples (Fig. 2).However, mononuclear cell preparations which contained 21%C5aR-positive cells removed C5a less efficiently from plasmacompared to granulocytes.

Preincubation of leucocyte suspensions with the C5aR-specific mAb S5/1, which at this concentration (3 3 uM) hasbeen shown to inhibit C5a binding to its receptor,8 almostcompletely abolished the depletion of the CSa anaphylatoxinby leucocytes (Fig. 2). In contrast, the incubation of granu-locytes or of mononuclear cells with identical concentrations ofthe isotypic control mAb P21/15 (anti-human complement

100-

80-

0a 60-

.' 40-

20 -

0

I

I

0 2o 2 4 6 8Granulocytes (x 107/ml)

10

Figure 1. Clearance of C5a (e) and of the terminal C5b-9 complementcomplex (A) from plasma by granulocytes. Complement-activatedplasma was co-incubated with granulocytes for 5 min at room

temperature and complement activation products in the supernatantwere determined by ELISA. Clearance is expressed as (c1 -c2)/c1] x

100 (%), where cI is the plasma concentration ofcomplement fragmentsbefore the addition of granulocytes and c2 the concentration afterincubation with the cells. * Values significantly (P < 0005) differentfrom the C5a plasma clearance in the absence of leucocytes.

100

80

2 60

at 40

20'

I I

12 3I

1 2 3

Figure 2. Effects of anti-C5aR antibodies and of monensin on the C5aclearance by granulocytes or monocytes. Purified granulocytes (openbars) or mononuclear cell fractions (hatched bars) were preincubated(10 min/20°) with (1) buffer, (2) 10M monensin or with (3) 3-3 gM anti-C5aR mAb S5/1. The CSa clearance from plasma by incubation (5 min/200) with these cells (108/ml) was determined as described in the legendto Fig. 1.

fragment Ba) had no effect on the C5a uptake by these cells.This finding demonstrates that peripheral blood leucocytesavidly bind this complement-split product via specific receptorsand thereby remove CSa from plasma. The treatment of leuco-cytes with the carboxylic ionophore monensin, which preventsC5aR internalization and recycling in granulocytes, did notaffect the ability of granulocytes or of mononuclear leucocytesto remove the C5-derived anaphylatoxin from plasma. Thus,binding of fluid-phase CSa to the CSaR on the leucocyte mem-brane, rather than the internalization of the ligand-receptorcomplex, accounts for the clearance of CSa by leucocytes.

Clearance of C5a by co-incubation with U937 cells

The co-incubation with U937 cells that had been induced toexpress C5aR by db-cAMP, also resulted in the depletion ofC5a from plasma. In an experiment designed to study thekinetics of this reaction, it was found that CSa rapidly bound todb-cAMP-differentiated U937. Even after the incubation ofcells with plasma for only 5 seconds, the CSa clearance wasvirtually complete (Fig. 3). The same result was obtained whenthe kinetics of C5a binding and depletion by granulocytes wasexamined (data not shown).The finding that only differentiated,but not untreated, U937 cells were capable of depleting CSafrom plasma lends further support to the notion that these cellsbind CSa via specific receptors. Undifferentiated U937 cells didnot express the C5aR.

Clearance of complement activation products C5a(desArg), C3aand TCC from plasmaThe effect of the co-incubation of leucocytes with plasma on theclearance of other complement activation products besides C5awas studied by applying ELISA procedures for the quantifi-cation of C3a/C3a(desArg) and SC5b-9 (TCG) in plasmasupernatants. Plasma which had been activated by CVF in theabsence of the carboxypeptidase inhibitor MERGETPA was usedto study the clearance of CSa(desArg). Serum carboxypeptidasesconvert C3a and CSa anaphylatoxins into their desArg-forms.

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Clearance of C5afrom plasma

S 40-

20 -

0

0 60 120

Time (seconds)

Figure 3. Kinetics of the C5a clearance from plasma by incubation withU937 cells. Untreated U937 cells (o) or differentiated U937 cells whichhad been treated with 2 mm db-cAMP for 3 days (s) were co-incubatedat 108 cells/ml with complement-activated plasma for the indicated timeperiods. C5a levels in the supernatants were determined by ELISA.

As shown in Table 1 the co-incubation with granulocytesmediated the clearance from plasma only of the C5-derivedanaphylatoxins C5a and CSa(desArg), whereas the plasma con-centrations of other complement activation products remainedunaffected. Uptake and removal of C5a(desArg) from plasmaby granulocytes was less effective compared to the eliminationof CSa by these cells. Mononuclear leucocytes did not have astatistically significant effect on the clearance of plasmaCSa(desArg) at the cellular concentrations employed in theseexperiments.

DISCUSSION

Several mechanisms contribute to the inactivation of the CSaanaphylatoxin and thereby protect the organism from injuryinduced by this phlogistic mediator. The concept that CSa, once

it is generated under in vivo conditions, rapidly binds to CSaR

Table 1. Plasma clearance of complement activation products byco-incubation of plasma with granulocytes or with mononuclear

cell fractions*

Complement % clearance by % clearance byfragment granulocytes mononuclear cells

C5a, 78t 56tC5a(desArg) 32t 16C3a 4 (-8)SC5b-9 (TCC) 5 (-2)

* Plasma which had been activated by CVF in the presenceor absence of the carboxypeptidase inhibitor MERGETPA wasco-incubated for 5min with purified granulocytes or mono-nuclear cells (108 cells/ml). Complement activation products inthe supernatants were determined by ELISA and the clearancewas expressed as described in the legend to Fig. 1.

tConcentration of complement fragments in plasma super-natants after incubation with leucocyte fractions were signifi-cantly (P < 0-005) reduced compared to pre-clearance values.

on leucocytes and is thereby removed from the circulation ismainly based on experiments which were performed todetermine the catabolism of C5a in rabbits.5'6 In these studiesautologous complement-activated serum or 125I-labeled C5awas injected into rabbits and the half-life of C5a, as measuredby a bioassay, was determined to be 2-3 min. The finding of asignificantly prolonged half-life of C5a in neutropenic animalsimplied an important role of the high-affinity binding to C5aRon granulocytes in the clearance of C5a from plasma.

Studies which have directly addressed the catabolism ofC5ain humans have not been performed yet. However, the analysisof complement activation which is induced by the contact ofhuman blood with artificial membranes such as cuprophandialyzer membranes, has provided insights into the metabolismof complement activation products under in vivo conditionsand, more specifically, the interactions between C5a and neu-trophilic granulocytes.14,15 It was noted that highly elevatedC5a/C5a(desArg) concentrations were present in plasmasamples taken 15min after starting the treatment.16 Theseelevated C5a plasma concentrations rapidly declined to almostnormal values at the end of the 3-5 hr treatment period, andwere detectable only in the venous but not the arterial bloodline. Complement activation in these patients was accompaniedby pulmonary sequestration of granulocytes and monocytesand resulted in transient leukopenia.17 C5a-induced alterationsin the membrane expression of cell surface molecules whichmediate leucocyte adherence to the vascular endothelium havebeen implicated in these findings.18 In summary, these clinicalfindings are thought to support the concept of a rapidelimination of C5a from the circulation by the binding toleucocyte C5aR.

It was the aim of this study to directly address the possiblerole of leucocyte C5a receptors for the elimination of C5a fromhuman blood plasma. To this end the in vitro clearance of C5ain plasma by purified leucocyte preparations was examined.Plasma concentrations of C5a were determined by a specificand sensitive ELISA procedure.9 A mAb (S5/1) with specificityfor the C5aR, that reacts with a heptameric peptide sequencewithin the amino terminal domain of this receptor,8 wasanother prerequisite for these studies. This mAb recognizes apotential ligand-binding site on the C5aR and thereby preventsC5a and C5a(desArg) binding to myeloid blood cells.8 Theconcentration of C5a in CVF-activated plasma, which was usedin this study, amounted to 69 ng C5a/ml. This value correspondsto a fraction of approximately 1% (mol/mol) C5 activation andequals maximal C5a values which have been determined inpatients undergoing haemodialysis with cuprophan dialysers. 16

The results presented herein clearly demonstrate the rapidclearance ofCSa from plasma by leucocyte C5aR. Granulocytepreparations which consist of highly enriched C5aR-expressingcells were more effective in this respect than mononuclear cells,which contain only a minor fraction of C5aR+ monocytes.Furthermore, monocytes express fewer C5aR copies thanneutrophilic granulocytes.8 Binding of plasma C5a to thecellular receptor was very rapid and virtually complete after anincubation period of 5 seconds. The compound monensin,which acts on the Golgi apparatus to interfere with the pro-

cessing of proteins in this cellular compartment,'9 was used toinhibit the internalization of ligand-receptor complexes. Leu-cocytes which had been treated with monensin did not differ intheir capacity to eliminate C5a from plasma compared to

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520 M. Oppermann & 0. Gatze

untreated cells. This result implies that the endocytosis ofreceptor-bound C5a is not critical for C5a clearance from thefluid-phase. This assumption is also supported by the finding ofdifferent kinetics of C5a binding to the receptor, which iscomplete within a few seconds, and internalization of theligand-receptor complex, which has a half-life of several

20,21minutes.Preincubation of leucocytes with the C5aR-specific mAb

S5/1, which inhibits C5a binding to its receptor, completelyabrogated the elimination of CSa from plasma. It was furtherdemonstrated in this study that promyelocytic U937 cellsacquire the ability to remove C5a from plasma after treatmentwith db-cAMP. Differentiated, but not untreated, U937 cellsexpress C5aR on their cell surface.22 These results show thatspecific binding to the C5aR, rather than unspecific binding ofthe cationic ligand to the leucocyte membrane accounts for theclearance of C5a by these cells. The C5a uptake via specific cell-surface receptors therefore differs from the binding of theclosely related C3a anaphylatoxin to rat mast cells by unspecificmechanisms. 23,24

In dose-effect experiments we noticed that removal of asignificant proportion of plasma CSa was achieved only atleucocyte concentrations which were higher than the physio-logical leucocyte count in peripheral blood. Even the additionof 108 granulocytes/ml plasma, which equals 20 times thephysiological cellular concentration, resulted in the removal ofonly 80% of plasma C5a. Assuming a molecular weight of C5aof 11,000 and 6 x 04 GSa binding sites on granulocytes,8 thiscorresponds to 50% occupancy of granulocyte C5aR by CSafrom plasma. Although the experimental design which waschosen in this study does not fully resemble physiologicalconditions where leucocytes are dynamically supplied to aninflammatory site, this result may indicate that other cellsbesides blood leucocytes may also participate in the clearanceof CSa. Endothelial cells and tissue macrophages also expressCSaR and are therefore candidate cell types that may functionin the binding and elimination ofCSa. In support of this notion,we observed that plasma C5a/C5a(desArg) concentrations inpatients which undergo chemotherapy were not significantlyelevated during neutropenic episodes ( < 100 PMN/ M1 plasma),nor were CSa plasma levels during cuprophan haemodialysisinversely correlated with granulocyte counts in these patients(M. Oppermann, unpublished observations). We interpret thefinding of incomplete CSa removal from plasma even at highleucocyte concentrations as evidence for the partial structuralmodification of CSa, which may then be less avidly taken up byleucocyte CSaR. Background levels of C5a(desArg) which arepresent in plasma prior to complement activation by CVF andtraces of carboxypeptidase which escape inactivation byMERGETPA, may contribute to the generation of significantconcentrations of C5a(desArg) in the sample. It was shown inthis study that C5a(desArg) which binds to the C5aR withlower affinity is cleared less efficiently from plasma than C5a.

The kinetics of C3a elimination both in haemodialysispatients and in experimental animal models closely resemblesthe clearance of plasma C5a.15 As functional and bindingstudies have implied the expression of C3a receptors by humangranulocytes,25'26 we speculated that these cells may alsofunction in the plasma clearance of the C3a anaphylatoxin.However, by applying the clearance assay described in thispaper we could not demonstrate C3a uptake from plasma by

granulocytes. This negative result may be due to the largeexcess of C3a in activated plasma compared to the number ofC3a binding sites on granulocytes, which may have obscuredremoval of a minor fraction of plasma C3a. Furthermore, theexistence of specific C3a-binding sites on human granulocytes isstill controversially discussed and divergent values for dis-sociation constants and C3a receptor copies on these cells havebeen reported in the literature.25'27

In summary, this study provides direct evidence for a role ofthe leucocyte C5aR in the clearance of C5a from plasma. Thehigh-affinity receptor for the C5a anaphylatoxin is essentiallyinvolved not only in the signal transduction cascade whicheventually leads to C5a-induced cellular activation, but alsofunctions as a scavenger that helps to protect the organismfrom excessive inflammatory reactions.

ACKNOWLEDGMENTS

This work was supported by grants Op 42/5-1 and SFB 236, B6 fromthe Deutsche Forschungsgemeinschaft. The authors thank Ms G.Sonntag for excellent technical assistance.

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