capping of complement receptors on human ... - John D. Lambris

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CR,, the C3b and iC3b receptors, respectively, on human neutrophils. Incubation of monolayers or cell suspensions of neutrophils with PG-PS opsonized with ...
0022-1767/83/1304-1674$02.00/0 JOURNALOF IMMUNOLOGY Copyrlght 0 1983 by The Amerlcan Assoclatlon of lmmunologlsts

Vol. 130, No. 4, Aprd 1983 Pmted In U. S.A.

THE

CAPPING OF COMPLEMENT RECEPTORS ON HUMAN NEUTROPHILS INDUCED BY GROUP A STREPTOCOCCAL CELL WALLS‘ KATHERINE B. PRYZWANSKY,2 EDITH K. MAcRAE, AND JOHN D. LAMBRIS3 From the Departmentof Bacteriology and Immunology, the Department of Anatomy, and the Department of Medicine, University of North Carolina, Chapel Hil,NC 27574

Peptidoglycan-polysaccharidepolymersderivedfrom 4-6). group A streptococcal cell walls (PG-PS) were opsonized Activation of the classical or alternative C pathways by target with either purified C3 or normal human serum and were particles or soluble complexes leadsto covalent binding of C3b used as a probe to investigate the mobility ofCR,and to these components. The bound C3bitsor degradation product, CR,, the C3b and iC3b receptors, respectively, on human iC3b, can mediate several important biologic functions, including neutrophils. Incubation of monolayers orcell suspensions enhancement of immune adherence, phagocytosis, and activaof neutrophils with PG-PS opsonized with C3b or serum tion of B cells (6-14). It is suggested that a primary role of the resulted in cappingof PG-PS, as detected by fluorescein- C3b receptor is to mediate efficient binding of particles to neulabeled antibody to PS.No binding of PG-PS to neutrophils trophils during phagocytosis (7, 10). Both C3b and iC3b recepwas observed with heat-inactivatedserum. By 30 min the tors can efficiently promote phagocytosis in monocytes (1 5), and cell walls were internalized and observed in one to three the mobility of these receptors appears to correlate with macvacuoles. Capping was totally inhibited when PG-PS oprophage activation (15, 16). Fearon et a/. (1 7), using immunoflusonized with C3b or serum was preincubated with Fab’orescence, have demonstrated clusters of C3b receptors on the anti-C3b. Similar inhibition was observed when C3b-opsonized PG-PS was incubated with neutrophils that were neutrophilmembrane.InternalizationofCR,canresultfrom cross-linking of the respective receptor by F(ab’)* anti-CR1 with preincubated with anti-CR, or fluid-phase C3b; only partial subsequent pinocytosis. Similar evidence was obtained in studinhibitionofneutrophilcappingwasobservedbyusing ies with C3b-fixed sheep erythrocyte membrane proteins (C3bserum-opsonizedPG-PS.Becauseanti-CR,blocksonly OR) and F(ab’), anti-C3. Capping of receptors was not reported the C3b receptor, the cap formation observed with serumopsonized PG-PS is probably due to CR,. These results (17, 18). Thepeptidoglycanfrom group A streptococcus hasbeen suggest that both CR, and CR, on neutrophils cap after stimulation by groupA streptococcal cell wall fragments. reported both in vitro (19) and in vivo (20) as the most active cell wall component of this species for activation of the alternative C pathway. Furthermore, these wall fragments are postulated to Neutrophilic polymorphonuclear leukocytes (neutrophils) and play a role in rheumatic-like heart lesions in mice (21, 22) and monocytes have been shown to express two different types of chronic polyarthritis in rats (23, 24). The opsonic recognition of membrane complement (C) receptors, which have been desigstreptococcal cell wall by neutrophil C receptors offers another nated as C receptor type one (CR1)4 and C receptor type three approach to study the distribution and endocytosis of CR1 and (CR,). These receptors have specificities for different sites on CR, receptorsand the mechanism of clearance of bacterial debris the C3 molecule (1-3). The CRl binds to both C4b and C3b, by neutrophils. In the present study, peptidoglycan-polysacchawhereas CR3 binds to iC3b. In addition to these receptors, B ride sonicates of polymers derived from group A streptococcal lymphocytes express another C receptor, C receptor type two cellwalls(PG-PS)wereopsonizedwitheitherC3ornormal (CR,), which bindsto the C3d or d region of iC3b (3,4). Recently human serum and were used to investigate the mobility of CRl the CR1and CR, receptors were isolated andshown to be and CR3on human neutrophil membranes. The data demonstrate glycoproteins with m.w. of 205,000 and 72,000, respectively (2, that C3b and iC3b, fixed to PG-PS, induce capping of CR, and CR, on the neutrophil membrane. Received for publication October 25, 1982. Accepted for publicatlon December 8, 1982. MATERIALSANDMETHODS The costs of publication of this article were defrayed In part by the payment of of serum components to streptococcal cell walls.The physical, Fixation pagecharges.Thisarticlemusttherefore be herebymarkedadvertisementin chemcal, and arthropathic properties of a lOOP fraction of purified cell walls accordance wlth 18 U.S.C. Section 1734 solely to indicate this fact. ‘This workwassupportedbyResearchGrantAM25733fromtheNational derived from group A streptococci have been described (24). This preparation to Dr. Institute of Arthritis, Diabetes, and Digestive and Kidney Diseases Awarded has a m.w. average of 5.0 x lo7 and is composed of peptidcglycanJohn H. Schwab. and by the Unlverslty Research Council. Porttons of this report polysaccharide polymers. This fraction can be sedimented at 100,000 X G will be presented in preliminary form at the Cell Biology meetings in Baltimore, MD,for 1 h In a T-865 rotor in a Sorvall OTD-2 ultracentrifuge. For these studies December 2,1982. the pellet was resuspended in phosphate-buffered saline (PBS) to a concenCorrespondence to Dr. Katherlne 6. Pryzwansky, Department of Pathology, tration of 2 mg rhamnose/ml. Rhamnose content of cell wall fragments was Unlversity of North Carolina, Chapel Hill, NC 27514. determined by the method of Dische and Shettles (25). These cell wall Presentaddress:DepartmentofMolecularImmunology,ScrippsClinicand fragments will be referred to as PG-PS. Research Foundation, LaJolla,CA 92037 PG-PS was incubated with normal human serum or C3 as follows: Four Abbreviations used In this paper: PG-PS, peptidoglycan-polysaccharlde polytotal volume of 2 ml of PBS were incubated for 15 mers derived from group A streptococcal cell walls; PS. polysaccharide polymers milligrams of PG-PS in a min at 37OC with whole human type AB serumto achieve afinal concentration derived from group A streptococcal cell walls; serum-(PG-PS), serum-opsonized of 10% serum (serum-(PG-PS)). The pellet was washed three times with cold peptidoglycan-polysaccharide polymers of group A streptococcal cell walls; C3bPBS at 4OC by centrifugation at 100,000 x G and resuspended in PBS to a (PG-PS). C3b-opsonlzed peptidoglycan-polysaccharide polymers of group A strepfinal concentratlon of 2 mg rhamnose/ml. A control fraction was prepared as tococcalcellwalls; CR,, C-receptortypeone,theC4b-C3breceptor;CR3,Cnot C3d; above, but incubated in PBS. For C3b fixation to PG-PS (C3b-(PG-PS)). 4mg receptor type three, C-receptor specific for iC3b (inactivated C3b) but ofPG-PS in a total volumeof2ml were incubated with 5 mg C3 in the FITC, fluorescein isothiocyanate; EDTA, ethylene diamlne tetraacetic acid. 1674

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CAPPING OF COMPLEMENT NEUTROPHILS RECEPTORS ON presence of 100 p g trypsln for 2 mln at 37OC. The reaction was stopped by the addition of a threefold molar excess of soybean trypsin inhibitor (300 p g ) and put on ice. The C3b-(PG-PS) was washed three times by centrlfugation at 100,000 x G with cold PBS at 4°C. Preparation of C3b fragments. Purification of C3 has been described (26). C3 was cleaved to C3b with trypsin in the presence of a 50% suspension of activated thlol-Sepharose (ATS; Pharmacia Flne Chemicals), generating disulfide-linked C3b-Sepharose. Briefly, a ratio of 8 mg of C3 to 1 ml of 50% ATS in 1 ml of 10 mM EDTA4/PBS pH 7.5 was treated with a 0.24% weight ratio of trypsin to C3 for 15 mln at 37OC wtth stirrmg. Trypsin was then inhibited by addition of a threefold molar excess of soybean trypsin inhibitor, and the C3b-ATS was washed by centrifugation three tunes with PBS. C3b was eluted with 10 mM lcysteine(raised to pH 7.0 with NaOH) and dialyzed extensively against PBS at 4°C. Preparation of antibodies specific for CR,. C3b. and group A streptococci. Antisera to CR, and C3b were raised in rabbits Immunized with purified CR, or C3b and were used as Fab fragments (2). Antiserum to group A streptococcus was raised in rabbits immunlzed with group A streptococcal PS. The antibody was characterized for specificity and conjugated wlth fluorescein isothiccyanate (FITC) as previously descrlbed (27. 28). Preparation of neutrophrl monolayers and suspensions. Human peripheral venous blood was allowed to clot onto 8-mm glass coverslips as described (29).The cells were layered withGey's balanced salts plus 10% human serum and were reincubated for 10 mm at 37OC. Monolayers consisted of 90% neutrophils, 8 to 10% monocytes, and 1 to 2% eosinophils, as determined by Wrlght's stain. Neutrophils for suspension studles were purified by Ficoll-Hypaque sedimentation of heparmuzedblood as described (30). Erythrocytes were removed by hypotonic lys~sin distilled water. lsotonlcity was restored with 3.6% sodium chloride. Cells were resuspended in Gey's plus gelatin at a concentratlon of 2 x 1O6 cells/ml. Cell viablllty was routinely assessed by trypan blue excluslon; in all cases, this was at least 95%. lncubation of neutrophils with PG-PS fragments. A 1-ml suspenslon of 2 x lo6 neutrophils in Gey's plus 1% gelatin was Incubated with shaking for 5 to 30 min at 37°C with 100 p g of C3b-(PG-PS),serum-(PG-PS), or PG-PScontrol. The cells were immediately fixed with 9 ml of 1% paraformaldehyde, placed on Ice for 30 min. washed two times in PBS by centrifugation (125 x G). and stained with FITC-labeledantibody to group A PS. Monolayers of cells were washed carefully wlth Gey's containing 10% bovine serum albumln (BSA) to remove all traces of serum. The cells were incubated as above with 100pg/ml of the various PG-PS preparations. The reaction was stopped by removing the medlum and simultaneously flxlng and washlng the cells wlth 1% paraformaldehyde. Cells were elther fixed for 30 mln at 4'C in paraformaldehyde to detect surface bindmg. or further fixed wlth buffered formalm acetone for 1 mln to detect Intracellular bmding (29). In some Instances. monolayers containing 1 ml Gey's plus 10% human serum or 10% heat-inactivated serum (serum heated for 30 min at 56OC) were Incubated for 30 min at 37OC wlth 100 p g ofserum-(PG-PS) or heatmactwated serum-(PG-PS). In this Instance. the PG-PS components were incubated for 10 min with 10% serum or 10% heat-inactivated serum before incubation with the cells. Cells were foxed and processed for fluorescence microscopy as above. The cells were then stained for 30 mln at 4OC. washed in PBS. and mounted. The cells were viewed wlth a Leltz Orthoplan fluorescent microscope wlth the use of incident light excitation with filter pack H and edge filter K480 to minimize quenching. In some instances, cells were counterstained with 0.01% methyl green to visualize the nucleus (31). RESULTS

Capping of PG-PS on neutrophils was observed in both monolayers(Fig. 1) andcellsuspensions(Fig.2)incubated with serum-opsonized PG-PS. The cap was distributed at the posterior end (uropod) of polarized cells. No surface binding of PG-PS to neutrophils was detected in the absence of fresh serum or whenheat-inactivatedserumwasused.Thekineticsofcap formation is shown in Table I. The PG-PS was observed within 5 min as clusters randomly distributed around the cell periphery A plateau of about and as capsdistributed at one pole of the cell. 80% capped cells was reached by 10 min. The distribution of PG-PS during cap formation is shown in Figure 2. When monolayers were fixed with formalin-acetone and subsequently stained for PG-PS, internalized PG-PS was observed (Fig. 3). Within20min,approximately22%ofthecellshad endocytic vacuoles; the number of cellswith vacuoles increased to 88% by 30 min. Cells were observed that had both caps and endocytic vacuoles. Because serum-opsonized PG-PS probably has both C3b and iC3b on its surface, binding of PG-PS opson-

Flgure 1 Capplng of neutrophll monolayers Induced byserum-(PG-PS)Neutrophlls were Incubated wlth serum-(PG-PS) for30 mln. flxed. and stalned for surface blndlng of PG-PS. The cells were counlerstalned wlth methyl green lo reveal the nucleus (n).Arrows lndlcale dlrectlon of locomollon of polanzed cells. The caps are dlslrlbuted at the posterlor endof the cell The lnlensltyof fluorescence vanes wlth the focal plane.

ized with purified C3b was investigated.As indicated in Table11, similar observations were noted when neutrophil suspensions were incubated for 30 min at 37°C with CSb-(PG-PS) or serum(PG-PS).No binding was observed on neutrophils incubated with heat-inactivatedserum-(PG-PS) or non-opsonized PG-PS. Inhibition studies were subsequently performed to further identify theneutrophilreceptorsinvolvedincapping.Theresultsare presented in Table111. Capping was totally abolished when C3b. opsonized or serum-opsonized PG-PS was incubated with Fab anti-C3bbeforeexposure to thecells.Similarinhibitionwas observedwhenCSb-(PG-PS)wasincubated withneutrophils that were preincubated with anti-CR1 or fluid-phase C3b. These observations suggest that CSb-(PG-PS) and serum-(PG-PS) induce capping of CR,. To determine whether CR3 also caps when cells are incubated with serum-(PG-PS), neutrophils were preincubated with antiserum to CR, or fluid-phase C3b and subsequently exposed to serum-(PG-PS). As shown in Table 111, only partial inhibition of PG-PSbindingwasobserved with serum(PG-PS). Approximately85% of the neutrophils showed caps or clusters ofPG-PS on the cell surface after prior treatment of cells with anti-CR,. In addition, 27% of the neutrophils displayed clusters of PG-PS after preincubation of neutrophils with C3b. Because anti-CR1 blocks only the C3b receptor, the cap formation observedwith serum-(PG-PS) must be dueto CR3. DISCUSSION

Group A streptococcal cell wall fragments consisting of p e p tidoglycan-polysaccharidepolymers with m.w. of 5.0 x 10' (24) have been used as a probe to study the mobility of C receptors on human neutrophils. Peptidoglycan activates the alternativeC pathway both in vitro (19)and invivo (20),with subsequent fixation of C3b molecules on its surface. The fixation of C by PG-PS is probably favorable because SIH, a cofactor required for cleavage of C3b, is minimally active with molecules deficient in sialic acid residues (32). Theinvolvementof C receptors in neutrophil recognition of PG-PS is supported by the fact that heat-inactivated serum-(PG-PS) is not bound by neutrophils. By comparingC3b-(PG-PS) with serum-(PG-PS),themobilityof receptors to C3b (CR,) and iC3b (CR3) was investigated. Both receptors have the ability to cap. C3b-(PG-PS) did not bind to

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CAPPING OF COMPLEMENTRECEPTORSONNEUTROPHILS

Figure 2. Cap formation of neutrophil suspensions induced byserum-(PG-PS).Neutrophils were incubated with serum-(PG-PS) for 10 mln.fixedwlth 1% paraformaldehyde.and stainedforsurfacebindingofPG-PS.PG-PS was observed as clusters( A ) and caps (B).

TABLE I Time course of cap formationon neutrophils induced by serum-(PG-PS) ~~

"

Dlstrlbutlon of

Fluorescence(percent of neutrophils)

TABLE Ill lnhibirion of capping of PG-PS by Fab' anti-C3b. Fab' anti-CR,. and fluid-phase C3b

Tlme (rnln)

Clusters 1 5 10 20 30

Negative 98

0

8 80

5 2 0

Capped

2 6 27 12 27 21

0 0 65 71 79

PBS Anti-C3b Anti-CR, C3b

3 100 100 100

3 100 1' 5 73O

Caps and clusters Clusters.

to permit cross-linking of receptors and redistributionof recepFlgure 3. Internallzed PG-PS. Endocytlc vacuoles contalnlng PG-PS were o b served wlthln20 rnm after lncubatlon of neutrophlls wtth serum-(PG-PS). Both capstors to a cap. The PG-PS is first distributed on the neutrophil as (arrows) and vacuoles were observedIn lndlvldual cells. patches or as clusters and is finally distributed to the uropod as a cap, where it is probably internalized. Internalizationof the PGPS appears to be different than that induced by cross-linkage of ~. " . C receptors by antibody. The antibody C receptor complexes Percent of C a w l w are pinocytosed(17), whereas the PG-PS is concentrated at the Serum-(PG-PS) uropod. It seems that C3b-(PG-PS) a3 the distance between receptor clusters serum-(PG-PS) Heat-Inactivated cannot be bridged by IgG, but can be connected with PG-PS. 4 Control-(PG-PS) This bridging of receptor clusters, which is probably similar to a Neutrophil suspenslons were incubated with PG-PS for 30 min at 37°C and cross-linking and aggregation of IgG on lymphocytes, results in stained with FITC-labeled antibody to Group A streptococcal PS. capping and endocytosis of PG-PS by neutrophils. Hafemanet a/. (33) have foundthat the distribution and motion neutrophils after cells were preincubated with excess C3b or anti-CR1 or after preincubation of C3qPG-PS) with anti-C3b. of the C3b receptoris affected by the natureof the substratum to whichthecellsareattached.Ourdatasuggestthatthe These inhibition studies support the involvement of CR1 in the recognition of this bacterial component by neutrophils. Complete distribution and motion of the C3b receptor does not appearto be affected once the receptors are bound by the antigen. Capinhibition of capping was also noted when serum-(PG-PS) was TABLE II Serum factor mediating PG-PS binding to neutrophils'

~~

2

" "

~~

CAPPING OF COMPLEMENT NEUTROPHILS RECEPTORS ON

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14. Newman, S. L., N. J. Dobson, J. D. Lambris, G. D. Ross, and P. M. Henson. ping wasobserved in both suspensionsandglass-attached 1981. Specificity and function of human macrophage complement receptors neutrophils. Thus, both polarized and nonpolarized cells cap. for different fragments of C3. Fed. Proc. 40:1017. Differences in the mechanism of capping on neutrophils be- 15. Wright, S. D.. and S. C. Silverstein. 1981. Complement receptor modulation and function in cultured monocytes. J. Cell Biol. 91:255a. tween C and Con A should be noted. ConA capping is observed 16.Shaw. D. R., andF.M.Griffin,Jr.1981.Phagocytosisrequiresrepeated only on neutrophils that are polarized (34, 35) or preincubated triggering of macrophage phagocytic receptors during particle ingestlon. Nature 289:409. with colchicine or cold to (36) disassemble microtubules. Capping 17. Fearon. 0. T., I. Kaneko. and G. G. Thompson. 1981. Membrane distrlbution of C receptors occurs in cells with intact microtubule networks and adsorptive endocytosis by C3b receptors on human polymorphonuclear leukoyctes. J. Exp. Med. 153:1615. and on both polarized and nonpolarized neutrophils. Similarly, H. R., L. M. Smith, D. T. Fearon, and H. M. McConnell. 1980. Lateral capping of IgG on lymphocytes occurs incells with intact micro- 18. Petty, distribution and diffusion of the C3b receptor of complement, HLA antigens, A onlymphocytesis tubules(37),whereascappingofCon and lipid probes in peripheral blood leukocytes. Proc. Natl. Acad. Sci. USA 77:6587. achieved after disassembly of microtubules (38). ConA binds to R. J.Boackle,andJ.H.Schwab.1978.Activationofthe J.J., all glycoproteins with a-D-glycosyl and sterically related residues19.Greenblatt. alternate complement pathway by peptidoglycan from streptococcal cell wall. (39). The significance of the different mechanisms involved in theInfect. Immun. 19:296. 6. Allen,and J. H.Schwab.1982. In vivochangesin 20.Lambris,J.H.,J. capping of specific receptors by their specific ligands and that complement induced with peptidoglycan-polysaccharide polymers from strepA remains to be induced by nonspecific ligands such as Con toCoCCal cell walls. Infect. Immun. 35377. 21. Cromartie, W. J., and J. G. Craddock. 1966. Rheumatic-like cardiac lesions in understood. mice. Science 154:285. PG-PShasbeen reported to produce chronic inflammatory 22. Ohanian. S. H., J. H. Schwab,andW. J. Cromartie. 1969. Relatlon of rheudiseases such as rheumatoidarthritis and rheumatic carditis (21, matic-like cardiac lesions of the mouse to the localization of group A streptccoccal cell wall. J. Exp. Med. 129:37. 23). In addition, C appears to have a role in early joint inflamJ. H. 1979. Acute and chronic inflammation induced by bacterial cell mation in rats injected i.p. with aqueous suspensions ofPG-PS 23. Schwab. wall structures. In 1979 Microbiology. American Society of Microbiologists, (40). Streptococcal cell wall fragments are apparently not readily Washington, DC. P. 209. degraded by macrophages and persist within cultured macro- 24. Fox. A.. R. R. Brown, S. K. Anderle. et a/. 1982. Arthropathic properties related to the molecular weight of peptidoglycan-polysaccharide polymers of streptoA streptococciand their phages for 40 days (28). Similarly, group coccal cel walls. Infect. Immun. 351003. isolated cell walls are minimally degraded by neutrophils (4125. Dische, F.. and L. 8. Shettles. 1948. A specific color reaction of methylpentoses and a spectrophotometric micromethod for their determination. J. Biol. Chem. 43).Wehaveshown that C plays a keyroleinclearance of 175:595. peptidogycan-polysaccharidecomplexes by neutrophils. Recent 26. Lambris, J. D., N. J. Dobson, and G. D. Ross. 1980. Release of endogenous C3b inactivator from lymphocytes In response to triggering membrane recepobservations suggest that thesecell wall fragments elicit aggretors for plH globulin. J. Exp. Med. 152:1625. gation of leukocytes and erythrocytes and that this aggregation 27. Ohanian, S. H., and J. H. Schwab. 1967. Persistence of groupA streptococcal is C3b dependent (Pryzwansky, manuscript submitted for publicell wall relatedto chronic inflammation of rabbit dermal connective tissue. J. Exp. Med. 1251137. cation). The implications for the clearance or dissemination of 28. Smialowicz. R. J.. and J. H. Schwab. 1977. Processing of streptococcal cell C3b-bound cell wall debris are being pursued. wallsbyratmacrophagesandhumanmonocytes in vitro.Infect.Immun.

Acknowledgments. We thank Drs. John Schwab and W. Cromartie for many helpful suggestions, review of the manuscript, and support for pursuit of this study. REFERENCES 1. Fearon, D. T. 1980. ldentificatlon of the membrane glycopcftem that is the C3breceptor of thehumanerythrocyte,polymorphonuclearleukocyte,B lymphocyte and monocyte. J. Exp. Med. 152:20. 2. Dobson, N. J., J. 0. Lambris. and G. D.Ross. 1981. Characteristics of isolated erythrocyte complement receptor type one (CR,. C4bC3b receptor) and CR,specific antibodies. J. Immunol. 126:693. 3. Ross, G. D., and J. D. Lambris. 1982. Identification of a C3bl-specific membrane complement receptor (CR,) that is expressed on lymphocytes, monocytes, neutrophils, and erythrocytes. J Exp. Med.15596. 4. Lambris. J. D., N. J. Dobson, and G. D. Ross. 1981. Isolation of lymphocyte membrane complement receptor typetwo (the C3d receptor) and preparation of receptor-specificantibody. Proc. Natl. Acad. Sci.USA 78:1828. 5. Fearon, D. T. 1979. Regulation of the amplification C3 convertase of human complementbyaninhibitoryproteinisolatedfromthehumanerythrocyte membrane. Proc. Natl. Acad. Sci.USA 765867. is the 6.Fearon, D. T. 1980. Identification of the membrane glycoprotein that C3breceptorofthehumanerythrocyte,polymorphonuclearleukocyte. Blymphocyte and monocyte. J. Exp. Med. 152:20. 7. Roos. D., A. A. M. Bot, M. L. J. van Schaik, M. deBoer, and M.R. Daha. 1981, Interactionbetweenhumanneutrophilsandzymosanparticles:theroleof opsonins and divalent cations. J. Immunol. 126:433. 8. Mantovani, 8. 1975.DifferentrolesofIgGandcomplementreceptorsin phagocytosis by polymorphonuclear leukocytes. J. Irnmunol. 115:l 5. 9. Ehlenberger, A. G., and V. Nussenzweig. 1977. The role of membranerecep tors for C3b andC3d in phagocytosis. J. Exp. Med.145357. 10. Newman. S. L.,and R. 8. Johnston, Jr. 1979. Role of binding through C3b and IgG in polymorphonuclear neutrophil function: studies with trypsin-generated C3b. J. Immunol. 123:1839. 11. Gigli, I.,and R. A. Nelson, Jr. 1968. Complementdependent immune phage cytosis. I. Requirements forC1, C'4, and C'3. Exp. Cell Res. 51 :45. 12. Eden, A.. G. W. Miller, and V. Nussenzweig. 1973. Human lymphocytes bear receptors for C3b and C3d. J. Clin. Invest. 52:3239. 13. Ross, G. D.. and M. J. Polley. 1975. Speclficity of human lymphocyte complement receptors. J. Exp. Med. 141:1163.

17591. 29. Pryzwansky, K. B., E. K. MacRae, J. K. Spltznagel, and M. H. Cooney. 1979. Earlydegranulationofhumanneutrophils:immunocytochemicalstudiesof surface and intracellular phagocytic events. Cell 18:1025. 30. Pryzwansky, K. B.. L. E. Martin, andJ. K. Spltznagel. 1978. Immunocytochemicallocalizationofmyeloperoxidase,lactoferrin.lysozymeandneutralproteases in human monocytes and neutrophilic granulocytes. J. Reticuloendothel. Soc. 24:295. 31. Pryzwansky, K. B. 1982. Applications of double-label immunofluorescence. In Techniques in Immunocytochemistry, Vol. 1. Edited by G. R. Bullock and P. Petrusz. Academic Press, London P. 77. 32. Kazatchkine, M. D.. D. T. Fearon, and K. F. Austen. 1979. Human alternative complement pathway: membrane-associated sialic acid regulates the competition between B andPIH for cell-bound C3b. J. Immunol. 122:75. 33. Hafeman, D. G., L. M. Smith, D. T. Fearon. and H. M. McConnell. 1982. Lipid monolayercoated solid surfaces do not perturb the lateral motion and distnbution of C3b receptors on neutrophils. J. Cell Biol. 94:224. 34.Ryan,G. 6..J. 2. Borysenko, and M. J. Karnovsky. 1974. Factors affecting the redistribution of surface-bound concanavalin A on human polymorphonuclear leukocyctes. J. Cell Biol. 62:351. 35.Weinbaum.D. L., J. A. Sullivan,andG.L.Mandell.1980.Receptorsfor concanavalin A cluster at the front of polartzed neutrophils. Nature 286:725. of 36.Albertini, D. F.. R. D.Berlin,andJ.M.Oliver.1977.Themechanism Concanavalin A cap formation in leukocytes.J. Cell Sci. 2657. 37.Rogers,K.A..M.A.Khoshbaf,andD.L.Brown.1981.Relatlonshipof microtubule organizatlon in lymphocytes to the capping of immunoglobulin. Eur. J. Cell Biol.24:l. R. Pfeiffer, and H. M. Dosch. 38. Oliver, J. M., E. W. Welfand. C. B. Pearson, J. 1980.MicrotubuleassemblyandconcanavalinAcappinginlymphocytes: reappraisal using normal and abnormal human peripheral blood cells. Proc. Nat. Acad. Sa. USA 77:3499. 39. Goldstein, I. J.. C. E. Hollerman, and E. E. Smith. 1965. Proteincarbohydrate A with interactions. 2. Inhibition studies on the interaction of concanavalin polysaccharides. Biochemistry 4:876. J. 6. Allen, S. K. Anderle, D. R . Eisenberg. and W. J. Cromartie. 40. Schwab. J. H.. 1982. RelationshipOf complement to experimental arthritis induced in rats with streptococcal cell walls. Immunology 46:83. 41. Ayoub. E. M., and M. McCarty. 1968. lntraphagocytic p-N-acetylglucosamlnidase. J. Exp. Med. 127:833. streptococci 42. Ayoub. E. M., and L. W. Wannamaker. 1967. The fate of group A following phagocytosis. J. Immunol. 99:1099. 43. Glick, A. D., J. M. Ranhand. and R. M. Cole. 1972. Degradation of group A streptococcal cell walls by egg-white lysozyme and human lysosomal enzymes. Infect. Immun. 6:403.