Roles of Macrophage Fc and C3b Receptorsin Phagocytosis Of
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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 mac- rophages. 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