Roles of Macrophage Fc and C3b Receptorsin Phagocytosis Of

Proc. NatL Acad. Sci. USA Vol. 78, No. 6, pp. 3853-3857, June 1981 Immunology

Roles of macrophage Fc and C3b receptors in phagocytosis of immunologically coated Cryptococcus neoformans (lympholdnes/lymphocyte-macrophage interactions) FRANK M. GRIFFIN, JR. Division ofInfectious Diseases, Department of Medicine, University ofAlabama in Birmingham, Birmingham, Alabama 35294 Communicated by Ray D. Owen, March 23, 1981

ABSTRACT I have studied the roles of macrophage Fc and tococci. Because interactions between the humoral and cellular C3b receptors in the cell's interaction with encapsulated Crjpto- immune systems are necessary for the generation of the lym- coccus neoformana and have defined the effects of a lymphokine phokine, these findings may account, at least in part, for the that enhances macrophage complement receptor function, the complexity of host defense against cryptococcosis. Moreover, effects of ingestion of soluble immune complexes, and the effects the mechanisms defined here for macrophages' interaction with of corticosteroid treatment upon the ability of macrophages to cryptococci may be relevant to host defense against various phagocytize cryptococci via these receptors. Neither uncoated nor other microbial pathogens as well. C3-coated cryptococci were phagocytized, whereas IgG-coated cryptococci were avidly phagocytized by mouse peritoneal macrophages. Treatment of macrophages with the lymphokine en- MATERIALS AND METHODS abled them to ingest C3-coated cryptococci. Prior ingestion ofsol- Cryptococci. The strain of C. neofomans used in all exper- uble immune complexes severely compromised macrophages' iments was originally isolated from the cerebrospinal fluid of a ability to phagocytize cryptococci via their Fc receptors but did patient. It was in mouse brains, which were removed not affect their ability to ingest cryptococci via their complement passaged receptors. Corticosteroid treatment severely impaired the ability 6 days after inoculation and frozen at -70°C until used. Cryp- of macrophages to respond to the lymphokine. Based upon these tococci from thawed brains were cultivated at 37°C for 4 days experimental observations, I have constructed a model for normal on slants ofcornmeal agar containing 1% Tween 80 and 1% pep- host defense mechanisms against disease due to C. neoformans. tone (12). The yeasts were harvested with a loop and suspended in medium at a concentration of 108 per ml. Only suspensions Cryptococcus neoformans is one of the most troublesome op- containing heavily encapsulated cryptococci, as determined by portunistic pathogens; it causes serious meningeal, pulmonary, examination by the wet-field India ink method, were used in and disseminated disease, primarily in immunocompromised experiments. patients (1). The host defense mechanisms required to prevent Preparation ofAnticryptococcal Antibodies. An ammonium disease caused by the yeast are not well defined. However, re- sulfate precipitate ofserum from a rabbit repeatedly immunized sults of previous studies suggest that components of both the with C. neoformans (strain B-551, originally obtained from the humoral and the cell-mediated immune systems are necessary. Center for Disease Control, Atlanta, GA) was kindly provided Cryptococci are heavily encapsulated yeasts. Their ingestion by by S. Polt (University ofAlabama, Birmingham, AL). The pre- phagocytic cells in vitro requires that they be coated with anti- cipitate was suspended in phosphate-buffered saline, dialyzed capsular IgG antibody (2, 3). Their clearance in vivo is enhanced against 5 mM phosphate buffer (pH 7.2), placed on a DEAE- by complement (4). These characteristics suggest that the hu- cellulose column, and eluted with the same buffer (13). The moral immune system is the prime defender against disease resulting IgG fraction, designated anticryptococcal IgG, was caused by the organism. However, from some in vitro studies dialyzed against phosphate-buffered saline and stored in small (5, 6) and especially from the clinical observation that patients portions at -20°C until used. It agglutinated encapsulated with disorders of the T lymphocyte-macrophage system are at cryptococci, suspended at a concentration of 106 per ml, at a greatest risk for cryptococcal disease (7, 8), it is apparent that dilution of 1:320. host defense against cryptococcosis is critically dependent upon An antiserum containing anticryptococcal IgM but depleted cell-mediated immunity. of IgG was prepared by immunizing a rabbit with 108 heavily Work from this laboratory has recently defined an in vitro encapsulated cryptococci intravenously daily for 3 days and har- system in which the humoral and cellular limbs ofimmunity are vesting serum on days 6 and 7 after the initial immunization. tightly interwoven in a cyclic amplification ofeach by the other The antiserum was absorbed with protein A-Sepharose CL-4B (9, 10). In that system, engagement ofmacrophage Fc receptors (Pharmacia) by incubating 1 ml of antiserum with a gel con- by immune complexes triggers the cells to signal T lympho- taining 500 mg ofdry protein A-Sepharose for 1 hr at 4°C (14). cytes, which in turn elaborate aunique lymphokine that imparts The absorption was repeated with fresh protein A-Sepharose to macrophages the ability to phagocytize via their complement until the serum contained no IgG detectable by Ouchterlony receptors, a function they are otherwise unable to perform (11). double immunodiffusion against goat anti-rabbit IgG. The ab- In experiments reported here, I found that, under conditions likely to prevail at sites of cryptococcal infection in vivo, acti- Abbreviations: CnAg-Ab, soluble immune complexes composed of C. vation ofmacrophage complement receptors by the lymphokine neoformans polysaccharide capsular antigen and anticryptococcal IgG; Cn-C, C. neoformans incubated with CS-deficient mouse serum; Cn- is required for phagocytosis of immunologically coated cryp- IgG, C. neoformans coated with anticryptococcal IgG; Cn-IgG-C, C. neoformans coated with both anticryptococcal IgG and C3b; Cn-IgM, The publication costs ofthis article were defrayed in part by page charge C. neoformans coated with anticryptococcal IgM; Cn-IgM-C, Cn-IgM payment. This article must therefore be hereby marked "advertise- incubated with C5-deficient mouse serum; oxE(IgG), ox erythrocytes ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. coated with anti-ox erythrocyte IgG; VBG, Veronal-buffered glucose. 3853 Downloaded by guest on September 23, 2021 3854 Immunology: Griffin Proc. Natl. Acad. Sci. USA 78 (1981) sorbed antiserum, designated anticryptococcal IgM, agglutin- in two ways. In the preincubation assay (9), freshly established ated encapsulated cryptococci, suspended at a concentration of macrophage monolayers were covered with experimental su- 106 per ml, at a dilution of 1:4. That agglutination was mediated pernates and incubated at 370C for 48 hr; supernates were re- by JgM was demonstrated by using goat anti-rabbit IgM (Iz- moved and monolayers were covered with fresh medium. The chain specific) and goat anti-rabbit IgG (y-chain specific). Anti- ability ofthese macrophages to ingest coated and uncoated cryp- IgM agglutinated C. neoformans that had been incubated with tococci was determined by adding 2 X 107 Cn, Cn-IgG, Cn-C, either a 1:8 or a 1:16 dilution of the absorbed antiserum, Cn-IgM, Cn-IgM-C, or Cn-IgG-C, incubating the cultures for whereas anti-IgG did not. 2 hr at 370C, and determining the fate of the cryptococci by Preparation of Immunologically Coated Cryptococci. En- phase-contrast microscopy. capsulated cryptococci (108) were incubated with 1 ml of either In the direct assay (10), 48-hr explanted mouse peritoneal a 1:2000 dilution ofanticryptococcal IgG or a 1:2 dilution ofanti- macrophages that had been maintained in medium were washed cryptococcal IgM in medium for 15 min at 370C to prepare IgG- and overlaid with supernates; 2 x 107 immunologically coated coated (Cn-IgG) and IgM-coated cryptococci (Cn-IgM), or uncoated cryptococci were added to cultures simultaneously respectively. with supernates. Incubation was continued for 2 hr at 37'C, and Cn-IgM (108) were incubated with 1 ml of a 1:2 dilution of the fate ofthe microorganisms was determined microscopically. fresh C5-deficient AKR mouse serum in Veronal-buffered glu- Previous studies have strongly suggested that both assays cose (VBG) containing Ca2e, Mg2', and gelatin (15) for 10 min measure the same effect of the same lymphokine (10). at 370C to prepare C3-coated Cn-IgM (Cn-IgM-C). The serum Presentation of Results. Each result given represents the had been previously absorbed at 40C with cryptococci to remove mean ofat least three separate determinations, each performed any trace amounts ofanticryptococcal antibodies that may have in duplicate. Phagocytic index is the number ofcryptococci in- been present. Complement-coated cryptococci (Cn-C) were gested by 100 macrophages and was obtained by multiplying also prepared by incubating 108 Cn with 1 ml of fresh C5-de- the percentage of macrophages that had ingested any crypto- ficient AKR mouse serum in VBG for 10 min at 370C. cocci by the mean number of cryptococci ingested per macro- Cryptococci coated with both C3 and IgG were prepared in phage. Each result is presented as mean ± SEM. two ways: (a) 108 Cn-IgM-C were incubated for 15 min at 370C in medium with 1 ml ofa 1:2000 dilution ofanticryptococcal IgG; RESULTS (b) 108 Cn-IgG were incubated with 1 ml ofa 1:2 dilution offresh Interaction of Untreated and Lymphokine-Treated Mac- C5-deficient AKR mouse serum in VBG for 10 min at 37°C. Both rophages with Immunologically Coated and with Uncoated preparations were handled identically by macrophages and Cryptococci. Macrophages were incubated for 2 hr at 370C with were therefore used in experiments interchangeably. They are Cn, Cn-IgG, Cn-C, Cn-IgM, or Cn-IgM-C. Uncoated organ- collectively designated Cn-IgG-C. isms were not ingested whereas organisms coated with IgG Other Antibodies and Immune Complexes. Rabbit anti-ox were efficiently phagocytized (Table 1). Cn-C were coated with erythrocyte IgG, IgG-coated ox erythrocytes [oxE(IgG)], ly- C3, as demonstrated by the ability of anti-C3 antiserum to op- ophilized human serum albumin, rabbit anti-human serum al- sonize them for ingestion by normal macrophages. However, bumin antiserum, rhodamine-tagged albumin, and soluble al- Cn-C were neither attached (data not shown) nor ingested by bumin-antialbumin complexes, some ofwhich were complement- any macrophage preparations, either because too little C3b was treated, were prepared as described (9, 10, 16). Goat anti-rabbit deposited or because C3b was deposited at a site (e.g., beneath IgM and goat anti-rabbit IgG were obtained from Cappel Lab- the capsule) inaccessible to the macrophages' complement oratories, Cochranville, PA. Rabbit anti-mouse C3 antiserum receptors. was a gift from K. Gollahon (University of Alabama, Birming- I therefore used anticryptococcal IgM as a means to deposit ham). It was raised by repeatedly injecting a rabbit with mouse C3b on the surfaces of cryptococci. Because mouse peritoneal serum-treated zymosan (17); it formed one major precipitin macrophages do not have a receptor for rabbit IgM (19), Cn-IgM band on double immunodiffusion and a single band in the region were neither bound (data not shown) nor ingested (Table 1) by of C3 on immunoelectrophoresis against mouse serum. macrophages. Nor were Cn-IgM-C ingested by normal mac- Cryptococcal capsular polysaccharide antigen, prepared as described (18), was a gift from J. Bennett (National Institutes of Health, Bethesda, MD). Soluble cryptococcal antigen-anti- Table 1. Interaction of normal and lymphokine-treated body complexes (CnAg-Ab) were prepared by incubating 0.33 macrophages with immunologically coated and mg of antigen with 1 ml of a 1:100 dilution of anticryptococcal uncoated cryptococci IgG for 1 hr at 370C. Phagocytic index of particle by macrophages Preparation ofSupernates that Augment Macrophage Com- plement Receptor Function and Assessment of Their Effects Treated, with lymphokine* upon Macrophages' Ability to Phagocytize Cryptococci. Ex- By perimental supernates were prepared as described (9). Briefly, Not treated with preincu- monolayers containing 106 mouse peritoneal macrophages were Particle presented lymphokine bation Directly fed immune complexes in the form of either oxE(IgG), CnAg- Cryptococci 4 1 9 3 13 ± 2 Ab, or albumin-antialbumin (there was no difference in potency Cn-IgG 347 ± 55 337 ± 26 370 ± 45 among supernates prepared by using the different immune Cn-C 4 2 12 6 3 ± 1 complexes; therefore, they were used in experiments inter- Cn-C + anti-C3t 149 ± 44 ND ND changeably). Cultures were washed, 5 x 106 thymocytes were Cn-IgM 6±2 3±1 1±1 added to each dish, and incubation was continued for 24 hr at Cn-IgM-C 13 ± 6 254 ± 19 310 ± 75 37°C. Supernatant medium from each culture was harvested Cn-IgM-C + anti-C3t 145 + 10 ND ND and centrifuged at 750 x g for 10 min to remove cells and cel- * ND, not done. lular debris. t Cn-C and Cn-IgM-C were incubated with rabbit anti-mouse C3 anti- The effect of supernates upon macrophages' ability to ingest serum (25 gl/ml) for 15 min at37C. Similartreatment didnot render immunologically coated and uncoated cryptococci was assessed uncoated organisms ingestible. Downloaded by guest on September 23, 2021 Immunology: Griffin Proc. Natl. Acad. Sci. USA 78 (1981) 3855

rophages. However, IgM served efficiently to deposit C3b on munologically Coated Cryptococci. Macrophages were fed sol- the surfaces of cryptococci, as evidenced by enhanced attach- uble immune complexes; they were also treated with lympho- ment ofCn-IgM-C to the macrophage surface (data not shown), kine-containing supernates, either by preincubation or by by the ability ofanti-C3 antiserum to promote ingestion of Cn- inclusion in the subsequent assay, and their ability to ingest IgM-C by normal macrophages, and by ingestion of Cn-IgM-C immunologically coated cryptococci was determined. The strik- by lymphokine-treated macrophages (Table 1). ing impairment by immune complexes of macrophages' ability Effect of Ingestion of Soluble Immune Complexes upon to ingest Cn-IgG (Fig. 1) was not overcome by lymphokine Macrophages' Ability to Ingest IgG-Coated Cryptococci. Mac- treatment (Fig. 2). However, ingestion of immune complexes rophages were incubated for 1 hr at 370C with soluble immune had no effect upon the ability ofmacrophages to respond to the complexes composed of either albumin-antialbumin or CnAg- lymphokine and ingest complement-coated cryptococci, Cn- Ab. Examination, by fluorescence microscopy, of parallel cul- IgM-C, or Cn-IgG-C. Identical results were obtained when tures of macrophages incubated with rhodamine-tagged com- macrophages were preincubated with immune complexes that plexes revealed that more than 90% of macrophages ingested had been incubated with 50% mouse complement (data not large quantities of the material. Cultures were washed and in- shown). These results indicate that the ability of macrophage cubated for 2 hr at 370C with Cn-IgG. Prior uptake ofimmune complement receptors to promote phagocytosis of opsonized complexes severely impaired macrophages' ability to ingest cryptococci is critically important to macrophages that have in- these IgG-coated cryptococci (Fig. 1). gested immune complexes, even when the cryptococci are Because the interaction of macrophage Fc receptors with coated with both IgG and C3b. particle-bound IgG is greatly strengthened when the particle Effect of Hydrocortisone upon the Ability of Macrophages is coated with both IgG and C3b and bound by both Fc and C3b to Ingest Immunologically Coated Cryptococci. Macrophages receptors (20) and because cryptococci are likely to be coated were incubated for 48 hr in the presence of 10 nM hydrocor- with both ligands in vivo, it seemed important to determine the tisone. Some cultures were treated with lymphokine-containing effect of ingestion of immune complexes upon macrophages' supernates, either by preincubation or by inclusion in the sub- ability to ingest cryptococci coated with both ligands. Macro- sequent assay. Macrophages not treated with lymphokine were phages that had ingested soluble immune complexes were in- then incubated with Cn-IgG, and lymphokine-treated macro- cubated for 2 hr at 370C with Cn-IgG-C. These macrophages phages were incubated with Cn-IgM-C, both for 2 hr at 37°C. were not able to ingest cryptococci coated with both ligands any Particle uptake was assessed microscopically. Hydrocortisone better than cryptococci coated with only IgG (Fig. 1). Thus, pretreatment markedly impaired the ability of lymphokine- immune complexes efficiently block the interaction of macro- treated macrophages to ingest Cn-IgM-C but had no effect upon phages' Fc receptors with cryptococcal-bound IgG, even when macrophages' ability to ingest Cn-IgG (Fig. 3). the yeast is coated with C3b as well. The selective inhibition, by hydrocortisone, of the ability of Effect ofIngestion of Soluble Immune Complexes upon the lymphokine-treated macrophages to ingest Cn-IgM-C could Ability of Lymphokine-Treated Macrophages to Ingest Im- have been due either to the drug's directly blocking some aspect of complement receptor-mediated phagocytosis itself or to its preventing macrophages from responding to the lymphokine. 40C r T The complement receptor function of thioglycollate-elicited 400

x la ._ w x ._- 200 '0 0 cc c0 'a) >, 200-

tobe cu

o0 A B C 0 FIG. 1. Effect ofingestion ofsoluble immune complexes upon mac- A B C rophages' ability to ingest IgG-coated C. neoformans. Some macrophages were fed immune complexes (0.5 ml perdish) composed ofeither FIG. 2. Effect of ingestion of soluble immune complexes upon the albumin-antialbumin (B) or CnAg-Ab (C); others were not fed com- ability oflymphokine-treated macrophages to ingest immunologically plexes (A). The cells were then incubated for 2 hr at 3700 with either coated cryptococci. Macrophages were fed CnAg-Ab complexes either Cn-IgG (solid bars) or Cn-IgG-C (hatched bars), and the phagocytic in- after preincubation in supernates (open bars) or before exposure to su- dex was determined. Preincubation of macrophages with albumin pernates (hatched bars). The cells' ability to ingest immunologically alone, antialbumin antiserum alone, cryptococcal antigen alone, or coated cryptococci was then assessed. Virtually identical results were anticryptococcal IgG alone had no effect upon the cells' ability to ingest obtained when albumin-antialbumin complexes were used in place of either Cn-IgG or Cn-IgG-C. CnAg-Ab. A, Cn-IgG; B, Cn-IgM-C; C, Cn-IgG-C. Downloaded by guest on September 23, 2021 3856 Immunology: Griffin Proc. Nati. Acad. Sci. USA 78 (1981) to contamination of the antiserum with anticryptococcal IgG. First, the absorbed antiserum failed to react with anti-rabbit IgG by double immunodiffusion. Second, anti-rabbit IgM, but not anti-rabbit IgG, agglutinated cryptococci that had been treated with the absorbed antiserum. Third, cryptococci coated with the absorbed antiserum were not ingested by normal macrophages, even when coated with C3b as well (Table 1). The results presented indicate that macrophage Fc and C3b receptors have different, but cooperative, functions. Fc receptors are capable ofpromoting phagocytosis quite efficiently but are susceptible to blockade by soluble immune complexes. Al- 200 - though innately incapable of promoting phagocytosis, C3b re- .... ceptors can be activated to do so by a unique lymphokine, the first step in the generation of which is engagement of macrophage Fc receptors by immune complexes (9). In contrast to Fc receptor-mediated phagocytosis, C3b receptor-mediated phagocytosis of cryptococci is not impaired by the ingestion of immune complexes. The mechanisms defined here in vitro maybe relevant to host defense against cryptococcosis in vivo. At the time ofinitial infection with C. neoformans, the yeasts cannot be ingested because their capsules do not bear recognition sites to which macrophages can bind. The microorganisms therefore replicate in - + - + - + tissues and shed large quantities of their capsular antigen (26). Within several days, anticapsular IgG is produced, and the A B yeasts then become coated with both IgG and C3b. However, anticryptococcal antibody also combines with shed cryptococcal FIG. 3. Effect of hydrocortisone upon the ability of macrophages to ingest immunologically coated cryptococci. Macrophages were antigen, forming soluble immune complexes, ingestion ofwhich preincubated in either medium alone (-) or medium containing 10 nM would block macrophage Fc receptors. Phagocytosis of cryp- hydrocortisone (+) for 48 hr at 37TC. Some macrophages were treated tococci would therefore depend upon the macrophage's com- with supernates either by preincubation (open bars) or by inclusion in plement receptors, which can mediate ingestion only when ac- the subsequent assay (hatched bars); others were not treated with su- tivated by the lymphokine described (9, 10). Generation ofthe pernates (stippled bars). The ability of normal macrophages (stippled mac- bars) to ingest Cn-IgG (A) and of supernate-treated macrophages to lymphokine requires the uptake ofimmune complexes by ingest Cn-IgM-C (B) was determined. rophages (9). Thus, cryptococcal antigen-antibody complexes may exert both the detrimental effect of preventing macrophages from ingesting immunologically coated cryptococci via macrophages, cells that phagocytize via their complement re- their Fc receptors and the beneficial effect ofinitiating cellular ceptors (21), was not affected by treatment with hydrocortisone events that result in elaboration ofthe lymphokine that enables at concentrations as high as 0.1 mM (data not shown), indicating macrophages to ingest cryptococci via their complement that hydrocortisone does not block complement receptor-me- receptors. diated phagocytosis per se but that it does impair the ability of Most microorganisms that cause disease in patients with dis- macrophages to respond to the lymphokine that augments their orders ofcell-mediated immunity are intracellular parasites (1). complement receptor function. They are easily ingested by macrophages but can be killed only by macrophages whose intracellular microbicidal machinery has DISCUSSION been activated. Patients who are immunosuppressed are unu- The present experiments were designed to define the roles of sually susceptible to disease caused by these organisms because macrophage Fc and C3b receptors in phagocytosis of immu- the intracellular killing capabilities oftheir macrophages cannot .nologically coated cryptococci in vitro. Essential to discerning be activated. C. neoformans is an extracellular parasite that is separate roles for the receptors was the development ofa means difficult for macrophages to ingest and whose ability to survive by which C3b could be deposited, in the absence ofIgG, on the within phagocytic cells is suspect (2). Therefore, a defect in in- surfaces of cryptococci. Attempts to opsonize cryptococci di- tracellular killing would not explain the increased frequency of rectly with C3b via the alternative complement pathway, by cryptococcosis in immunocompromised patients. My results incubating the microbes with C5-deficient mouse serum, failed. suggest that the major factor predisposing corticosteroid-treated This was not surprising in light of recent work showing that C3b patients (and perhaps patients with other disorders of cell-me- is generated less efficiently by the alternative than by the clas- diated immunity as well) to cryptococcosis may be the inability sical complement pathway (22) and that C3b generated directly of their macrophages to respond to the lymphokine that aug- by the surface structures of encapsulated microorganisms is ments their complement receptor function. deposited beneath their capsules (23-25). I was able to deposit I thank Ms. Peggy M. Rains for excellenttechnical assistance and Ms. sufficient C3b at a site on the surface of cryptococci that was Susan J. Lamb for typing the manuscript. This work was supported by accessible to macrophage complement receptors by first coating Grant IM-173 from the American Cancer Society, Inc., and by Research the yeasts with anticryptococcal IgM and then incubating them Career Development Award AI-00135 from the National Institutes of with C5-deficient mouse serum, in a manner analogous to that Health. used to prepare C3b-coated sheep erythrocytes (9, 10, 16, 19, 1. Williams, D. M., Krick, J. A. & Remington, J. S. (1976) Am. Rev. 20). Several lines of evidence suggest that the protein A-ab- Resp. Dis. 114, 359-394, 593-627. sorbed anticryptococcal antiserum used deposited IgM on the 2. Diamond, R. D., Root, R. K. & Bennett, J. E. (1972) J. Infect. organism's surface and that the results obtained were not due Dis. 125, 367-376. Downloaded by guest on September 23, 2021 Immunology: Griffin Proc. Nati. Acad. Sci. USA 78 (1981) 3857

3. Kozel, T. R. & McGaw, T. G. (1979) Infect. Immun. 25, 255-261. 15. Rapp, H. J. & Borsos, T. (1970) Molecular Basis ofComplement 4. Diamond, R. D., May, J. E., Kane, M. A., Frank, M. M. & Ben- Action (Appleton-Century-Crofts, New York), pp. 75-78. nett, J. E. (1973) Proc. Soc. Exp. Biol. Med. 144, 312-315. 16. Griffin, F. M., Jr. (1980) J. Exp. Med. 152, 905-919. 5. Graybill, J. R. & Drutz, D. J. (1978) Cell. Immunol. 40, 263-274. 17. Mardiney, M. R., Jr. & Muller-Eberhard, H. J. (1965)J. Immu- 6. Gentry, L. 0. & Remington, J. S. (1971)J. Infect. Dis. 123, 22- nol 94, 877-882. 31. 18. Bhattacharjee, A. K., Kwon-Chung, K. J. & Glaudemans, C. P. 7. Diamond, R. D. & Bennett, J. E. (1973)J. Infect. Dis. 127, 694- J. (1978) Immunochemistry 15, 673-679. 697. 19. Griffin, F. M., Jr., Bianco, C. & Silverstein, S. C. (1975)J. Exp. 8. Schimpff, S. C. & Bennett, J. E. (1975)J. Allergy Clin. Immunol. Med. 141, 1269-1277. 55, 430-441. 20. Ehlenberger, A. G. & Nussenzweig, V. (1977)J. Exp. Med. 145, 9. Griffin, J. A. & Griffin, F. M., Jr. (1979)J. Exp. Med. 150, 653- 357-371. 675. 21. Bianco, C., Griffin, F. M., Jr. & Silverstein, S. C. (1975)J. Exp. 10. Griffin, F. M., Jr. & Griffin, J. A. (1980)J. Immunol. 125, 844- Med. 141, 1278-1290. 849. 22. Verbrugh, H. A., van Dijk, W. C., van Erne, M. E., Peters, R., 11. Mantovani, B., Rabinovitch, M. & Nussenzweig, V. (1972) J. Peterson, P. K. & Verhoef, J. (1979) Infect. Immun. 26, 808-812. Exp. Med. 135, 780-792. 23. Wilkinson, B. J., Sisson, S. P., Kim, Y. & Peterson, P. K. (1979) 12. Finegold, S. M., Martin, W. J. & Scott, E. G. (1978) Bailey and Infect. Immun. 26, 1159-1163. Scott's Diagnostic Microbiology (Mosby, St. Louis, MO), p. 450. 24. Winklestein, J. A., Abramovitz, A. S. & Tomasz, A. (1980)J. Im- 13. Williams, C. A. & Chase, M. W. (1967) Methods in Immunology munol. 124, 2502-2506. and Immunochemistry (Academic, New York), Vol. 1, pp. 322- 25. Laxalt, K. A. & Kozel, T. R. (1979) Infect. Immun. 26, 435-440. 326. 26. Diamond, R. D. & Bennett, J. E. (1974) Ann. Intern. Med. 80, 14. Horwitz, M. A. & Silverstein, S. C. (1980)J. Clin. Invest. 65, 82- 176-181. 94. Downloaded by guest on September 23, 2021