After 2 h at 37C, the J774.16 monolayers were extensively washed with PBS to remove ... compared after 2- and 24-h incubations for macrophage-yeast. 574.
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Vol. 63, No. 2
Antibodies to Cryptococcus neoformans Glucuronoxylomannan Enhance Antifungal Activity of Murine Macrophages SUSHOVITA MUKHERJEE,1 SUNHEE C. LEE,2
AND
ARTURO CASADEVALL1*
Departments of Medicine (Division of Infectious Diseases) and Microbiology and Immunology1 and Neuropathology,2 Albert Einstein College of Medicine, Bronx, New York 10461 Received 28 September 1994/Returned for modification 17 November 1994/Accepted 1 December 1994
Monoclonal antibodies (MAbs) to the capsular polysaccharide of the pathogenic fungus Cryptococcus neoformans can prolong survival and decrease organ fungal burden in experimental murine cryptococcosis. To investigate the mechanism of antibody-mediated protection, the interaction of C. neoformans and murine macrophage-like J774.16 cells was studied in the presence and absence of MAbs differing in isotype. Immunoglobulin G2a (IgG2a) and IgG2b isotype switch variants were isolated from an IgM hybridoma to complete the IgG subclass set. IgM, IgG1, IgG2a, IgG2b, IgG3, and IgA MAbs were studied for their ability to promote phagocytosis and reduce the number of CFU in C. neoformans and J774.16 cell cocultures. The MAbs in this set had similar if not identical fine specificities and were derived from a single B cell. All isotypes promoted phagocytosis; however, the IgG subclasses were more effective opsonins than IgM or IgA. All isotypes enhanced J774.16 anti-C. neoformans activity in vitro, as measured by a reduction in the number of CFU. The IgG1 MAbs were consistently more active in promoting opsonization and reducing the number of CFU. Addition of IgG1 MAb to C. neoformans and J774.16 cocultures resulted in rapid reduction in the number of CFU, which is consistent with fungal killing. Electron microscopy revealed that MAb-opsonized C. neoformans cells were internalized and appeared damaged. Administration of IgM, IgG1, IgG2a, and IgG2b isotype switch variant MAbs revealed that the IgG2a and IgG2b subclasses were the most and least effective isotypes, respectively, in prolonging survival in an intraperitoneal murine infection model. The results indicate that murine antibody subclasses differ in their ability to enhance macrophage anti-C. neoformans activity and suggest that antibody enhancement of macrophage function is a mechanism by which antibodies modify infection in vivo. 50, 51). Success of the antibody-based strategies may depend on providing the most effective isotype against C. neoformans. Here, we compare the efficacies of murine isotypes in enhancing murine macrophage function.
The fungus Cryptococcus neoformans is primarily a pathogen for persons with impaired cell-mediated immunity. Cryptococcal infections occur in 6 to 8% of patients with AIDS (13) and are accompanied by high mortality and frequent recurrences of treated infections (56). Genetic analysis of initial and relapse isolates from patients with recurrent infections suggested persistence of infection despite antifungal therapy (9, 53). The difficulty in eradicating C. neoformans infections in patients with AIDS underscores the importance of immunity in the success of antifungal therapy. Macrophages are important in protection against C. neoformans (1, 12, 16, 18, 22, 24, 28, 34, 35, 44, 45). Macrophages mediate anti-C. neoformans effects intracellularly and extracellularly (16, 44). C. neoformans, like the classical encapsulated bacteria Streptococcus pneumoniae and Haemophilus influenzae, has a polysaccharide capsule which is antiphagocytic (26). Complement- and capsule-specific antibodies provide important opsonins for phagocytosis (18, 26, 47). However, complement and antibody opsonins are probably scarce in patients with cryptococcosis, since capsular polysaccharide is poorly immunogenic (41), and complement is often depleted as a result of complement activation by the yeast capsule (31, 54). Recently, there has been renewed interest in antibody immunity for prevention (14) and treatment (55) of human infection. The rationale for antibody immunity is, in part, to provide opsonins which enhance cellular functions against C. neoformans. There is evidence that antibody isotypes differ in their ability to modify infection and promote phagocytosis (38,
MATERIALS AND METHODS C. neoformans. American Type Culture Collection (Rockville, Md.) C. neoformans 24067 was used for all experiments. Strain 24067 was used because prior studies of antibody efficacy in mice had used this strain (36–40, 55). Serotype D strains are pathogenic for humans, and D is the most prevalent serotype isolated in certain countries (27). Strain 24067 was maintained at 48C in Sabouraud dextrose agar (Difco Laboratories, Detroit, Mich.). For in vitro experiments, cultures were grown overnight at 308C in Sabouraud Dextrose broth (Difco) with moderate shaking (200 rpm). For animal experiments, the cultures were grown in Sabouraud dextrose broth (Difco) at 378C with moderate shaking for 4 to 5 days. Yeast cells were collected by centrifugation and washed three times with sterile 0.2 M phosphate-buffered saline (PBS). Inocula were determined by hemocytometer counting. Glucuronoxylomannan (GXM) from strain 24067 was isolated from culture supernatants by cetylmethylammonium bromide (Aldrich, Milwaukee, Wis.) complexation (11). Strain 24067 produces large capsules in vivo and small capsules when grown in Sabouraud dextrose broth. MAbs. Monoclonal antibodies (MAbs) 2H1 (immunoglobulin G1 [IgG1]), 2D10 (IgM), 3E5 (IgG3), 4H3 (IgG3), and 18G9 (IgA) have been described elsewhere (5, 7). MAbs 2H1, 2D10, 3E5, and 18G9 bind GXM from all serotypes and were generated in BALB/c mice immunized with a GXM-tetanus toxoid vaccine (5). The serological properties (5, 6), molecular analysis (36), and efficacy against C. neoformans (37, 38) of MAb 2H1 have been described elsewhere. MAbs 2H1, 2D10, 3E5, and 18G9 were derived from the same pre-B cell, use identical variable region genes, and have similar if not identical specificities (5, 36) and the same idiotype (4). Nevertheless, these four MAbs differ by several amino acid residues in the variable regions resulting from somatic mutations, and although no changes in fine specificity have been detected, subtle differences in binding cannot be ruled out (5, 36). MAb 36-65 is an IgG1 with specificity for p-azophenylarsonate (48) which does not bind to GXM or modify the course of C. neoformans infection in mice (37, 40). Generation of IgG2a and IgG2b antibodies. No IgG2a or IgG2b GXM-binding MAbs were recovered from mice infected with C. neoformans or immunized with the GXM-tetanus toxoid vaccine (5, 7). To complete the IgG subclass set, we isolated IgG2a and IgG2b isotypes in vitro from the IgM hybridoma 2D10 by sib
* Corresponding author. Arturo Casadevall, Department of Medicine, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461. Phone: (718) 430-4260. Fax: (718) 597-5814. 573
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selection with the enzyme-linked immunosorbent assay (ELISA) spot assay modification (52). An IgG1 switch variant from 2D10 has been described elsewhere (40). The 2D10 hybridoma IgG2a and IgG2b switch variants were cloned twice in soft agar and characterized by ELISA (5) and Southern blot analysis of heavy (VH) and light (VL) variable gene rearrangements by using the J11 and Jk1-5 DNA probes as described elsewhere (7). Antibody purification. MAb-containing ascites fluid was obtained by paracentesis after injection of hybridoma cells into the peritoneal cavity of pristaneprimed BALB/c mice. For unknown reasons, injection of 2D10 IgG2b hybridoma cells into BALB/c mice produced only low-MAb-concentration ascites during several attempts (i.e., ,200 mg/ml). Ascites fluid with a higher IgG2b content was obtained by injecting 2D10 IgG2b hybridoma cells into SCID mice. IgG1, IgG2a, IgG2b, and IgG3 MAbs were purified by protein G chromatography (Pierce, Rockford, Ill.). MAb 2D10 IgM was purified by mannose affinity chromatography (Immunopure IgM Purification kit; Pierce). MAb 18G9 IgA was purified from heat-inactivated ascites by size exclusion chromatography with a G-200 Sephadex column (Sigma). Purified MAbs were concentrated by centrifugation in Centriprep-100 columns (Amicon Inc., Beverly, Mass.) and sterilized by filtering through a 0.2-mm-pore-size membrane (Sigma). MAb concentrations were determined relative to isotype-matched standards with known concentrations by ELISA. After purification, MAbs were tested for GXM binding by ELISA as described elsewhere (6). Macrophage experiments. J774.16 is a murine macrophage-like cell line which has been extensively characterized (10). For phagocytosis assays, cells were grown in Dulbecco’s modified Eagle medium (Mediatech, Washington, D.C.) with 10% heat-inactivated fetal calf serum (Bioproducts for Science, Indianapolis, Ind.), 10% NCTC-109 medium (Gibco Laboratories, Life Technologies, Inc., Grand Island, N.Y.), and 1% nonessential amino acids (Cellgro; Mediatech, Washington, D.C.), plated at 105 cells per well in 96-well tissue culture plates (Falcon; Becton Dickinson, Mountain View, N.J.), and stimulated with recombinant murine gamma interferon (IFN-g; Genzyme, Cambridge, Mass.) and lipopolysaccharide (LPS; Sigma, St. Louis, Mo.) as described elsewhere (39, 55). Briefly, J774.16 cells were stimulated with 500 U of IFN-g per ml and incubated at 378C overnight. The medium in each well was then replaced with fresh medium containing 500 U of IFN-g per ml, 3 mg of LPS per ml, and C. neoformans cells. Phagocytosis was measured in the presence or absence of purified MAbs. Phagocytosis of C. neoformans by nonstimulated J774.16 cells was studied in the same manner, except that IFN-g and LPS were not added. After the addition of C. neoformans, the cells were incubated at 378C for 2 h, washed several times with sterile PBS to remove nonadherent yeast cells, fixed with cold absolute methanol, and stained with a 1:20 solution of Giemsa (Sigma). Phagocytic indices were determined with a microscope at a magnification of 3600 (Nikon Diaphot; Nikon Inc., Instrument Division, Garden City, N.J.). The phagocytic index is the number of macrophages attached and internalized yeast cells per number of macrophages per field. For each experiment, eight fields were counted. The antifungal efficacy of J774.16 cells was determined by counting the CFU (1 yeast colony 5 1 CFU) of C. neoformans after coculturing of yeast and macrophage cells in the presence and absence of MAb as described elsewhere (39). Briefly, J774.16 cells were mixed with C. neoformans as described for the phagocytosis assays (see above) in the presence and absence of 5 to 10 mg of MAb per ml, and the mixtures were incubated for either 2 or 24 h. The contents of each well were then removed, and the cells were lysed by the addition of 100 ml of sterile water, 30 min of incubation, and repeated vigorous aspiration and ejection of well contents with a pipette to complete disruption of cells while avoiding the generation of aerosols. Supernatants and the lysate were then pooled, diluted 1:10 in distilled water, and vortexed for 15 s. The lysate was then diluted again 1:100 and plated in Sabouraud dextrose agar. This procedure uses a combination of MAb dilution that is less than the agglutination threshold and mechanical dissociating to avoid the potential problem of antibody-mediated agglutination resulting in artifactual reduction of the number of CFU. Microscopic examination of the agar surface after plating of the vortexed suspension has shown that the overwhelming majority of yeasts are single cells without evidence of aggregation (39, 55). After 48 h of incubation, the colonies were homogeneous, which is consistent with origination from one cell. Control experiments using C. neoformans and MAbs in the absence of J774.16 cells revealed no decrease in the number of CFU, which is consistent with full disruption of clumped yeasts and no direct effect of MAbs on yeast growth. For example, values for mean CFU 6 standard deviations for C. neoformans incubated for 24 h in the presence and absence of MAb 2H1 were 141,000 6 25,239 and 131,000 6 25,580, respectively (P 5 0.706). Thus, CFU values reflect the number of viable cells deposited on the agar surface. EM. J774.16 monolayers in 4-well Lab-tek chamber slides containing ;50,000 cells per well were incubated with C. neoformans at an effector-to-target cell (E:T) ratio of 5:1 with and without 10 mg of either MAb 2H1 (IgG1), 3E5 (IgG3), or 18G9 (IgA) per ml. After 2 h at 378C, the J774.16 monolayers were extensively washed with PBS to remove extracellular unbound C. neoformans and were fixed with Trump’s solution (1% glutaraldehyde and 4% paraformaldehyde in PBS) for 24 h at 48C. The monolayers were then postfixed with osmium tetroxide, dehydrated by immersion in graded alcohol solutions, and embedded in Epon. One-micron Epon sections were stained with toluidine blue and examined by
INFECT. IMMUN. light microscopy. Ultrathin sections were stained with uranyl acetate and lead citrate and examined with a Siemens 102 electron microscope (EM). Mouse experiments. Female A/J mice aged 5 to 7 weeks from the National Cancer Institute (Bethesda, Md.) were given either 0.25 ml of sterile PBS (control) or 1 mg of 2D10 IgM, IgG1, IgG2a, or IgG2b MAb (ascites) by intraperitoneal (i.p.) injection and were infected i.p. about 30 min later with 5 3 107 C. neoformans cells. The i.p. model can distinguish protective from nonprotective MAbs (38) and has been used by others for the study of antibody efficacy against C. neoformans (15, 17). i.p. infection models have identified clinically useful sera against other pathogens (8). Statistical analysis. Pairwise comparisons between groups were done by the t test using Primer of Statistics-The Program (McGraw Hill Co., New York, N.Y.). Survival analysis was done by log rank analysis by C. J. Chang, a statistician at our institution.
RESULTS Ig2a and IgG2b isotype switch variants of IgM MAb 2D10. IgG2a and IgG2b isotype switch variants of MAb 2D10 were isolated by sib selection. The IgG2a and IgG2b hybridomas shared the same productive VH and VL gene rearrangements as the parent IgM hybridoma by Southern blot analysis (autoradiograms not shown). The 2D10 IgG2a and IgG2b MAbs bound C. neoformans GXM by ELISA and had binding characteristics similar to those of the IgG1 variant previously isolated (data not shown). The IgM, IgG1, IgG2a and IgG2b isotype switch variants of the 2D10 IgM line are referred to as 2D10m, 2D10g1, 2D10g2a, and 2D10g2b, respectively. Since 2D10m, 2H1 (IgG1), 3E5 (IgG3), and 18G9 (IgA) originated from one B cell (36) and since 2D10g1, 2D10g2a, and 2D10g2b were derived from 2D10m, all of the MAbs used here share the same B-cell ancestry. GXM-binding MAbs enhance C. neoformans phagocytosis. In the absence of capsule-specific MAbs, there was little or no phagocytosis or attachment of C. neoformans to J774.16 cells (Fig. 1). Experiments with MAb 2H1 (IgG1) revealed that the phagocytic index was dependent on the J774.16-to-C. neoformans ratio (E:T ratio) (Fig. 2). The maximal phagocytic index was measured at an E:T ratio of 1:1. The phagocytic index was dependent on the concentration for the two IgG1 MAbs 2H1 and 2D10g1 (Fig. 3). Since MAb 2H1 has greater apparent affinity for GXM than 2D10g1 (unpublished data), we attribute the differences in phagocytic indices to differences in affinity. In the absence of capsule-specific MAbs or in the presence of the irrelevant IgG1 MAb 36-65, C. neoformans phagocytic indices by J774.16 cells at E:T ratios of 1:1 were ,0.1. LPS and IFN-g stimulation of J774.16 cells resulted in higher phagocytic indices for MAbs 3E5 (IgG3), 2H1 (IgG1), and 18G9 (IgA) (Fig. 4). Among the 2D10 isotype variants, the relative opsonic efficacy was IgG1 . IgG2a . IgG2b and IgM (Fig. 5). Antifungal activity of J774.16 cells. The relative efficacies of MAbs in enhancing J774.16 antifungal activity were studied by measuring the number of CFU after 24 h of incubation of IFN-g- and LPS-activated J774.16 cells and C. neoformans. In the absence of GXM-binding MAbs or in the presence of the irrelevant MAb 36-65 (IgG1), there was little or no reduction in the number of CFU (Fig. 6). GXM-binding MAbs without J774.16 cells had no effect on C. neoformans CFU (see Materials and Methods). Addition of GXM-binding MAbs to cocultures of C. neoformans and J774.16 cells significantly reduced the number of CFU (Fig. 6). Although all isotypes reduced the number of CFU, the IgG1 MAbs 2H1 and 2D10g1 consistently produced the largest reductions in the number of CFU (three of three experiments). The 24-h incubation assays do not distinguish between fungicidal and fungistatic effects. To determine whether J774.16 cells were killing C. neoformans, the numbers of CFU were compared after 2- and 24-h incubations for macrophage-yeast
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FIG. 1. Giemsa-stained J774.16 cells after incubation with C. neoformans in the presence and absence of MAb 2H1 (5 mg/ml). The picture was taken after washing and staining, and hence the majority of extracellular yeast cells have been removed. Arrow points to yeast cells. In the absence of MAb, only one extracellular yeast cell is apparent in this field (magnification, 3600).
mixtures at an initial E:T ratio of 5:1 in the presence and absence of 10 mg of MAb 2H1 per ml (Fig. 7). After 2 h, there was a significant reduction in the number of CFU only in the presence of MAb 2H1 (Fig. 7). Since the incubation time was shorter than the replication time, the reduction in the number of CFU at 2 h suggested that approximately half of the yeast cells in the MAb-treated group were killed. However, the results were not conclusive, because some C. neoformans cells may have replicated within the 2-h incubation time, and we investigated the timing of CFU reduction in the first 2 h (Fig. 8). Surprisingly, a greater-than-twofold reduction occurred within the 20 min that it took to add the C. neoformans to the wells and then to remove the supernatant for CFU analysis (point 0 h in Fig. 8). At 1 and 2 h, there were increased numbers of CFU in wells containing C. neoformans alone and J774.16 cells with C. neoformans, but there was no increase in the numbers of CFU in wells containing C. neoformans and
FIG. 2. Phagocytic index as a function of E:T ratio for J774.16 cells in the presence of MAb 2H1 (5 mg/ml). Each bar represents the average of eight fields, and brackets denote standard deviations.
J774.16 cells with MAb 2H1. After 24 h, there were increased numbers of CFU in all three groups; however, the numbers of CFU in the presence of MAb 2H1 were significantly reduced (Fig. 8). This indicates that some yeast cells in the MAb 2H1treated group survive killing and continue to grow but reach numbers at 24 h lower than those found in the absence of MAb. Increasing the E:T ratio (by reducing the C. neoformans inoculum) resulted in larger reductions in the number of CFU at 24 h (Fig. 9). EM. Ultrastructural examination of C. neoformans opsonized by 2H1 (IgG1), 3E5 (IgG3), and 18G9 (IgA) revealed that the yeast cells were internalized in phagosomes by 2 h (Fig. 10). The appearances of C. neoformans opsonized by the IgG1, IgG3, and IgA MAbs were similar if not identical (not shown). Figure 10A shows a J774.16 cell with several internalized yeast cells in the process of phagocytosing additional yeast
FIG. 3. Phagocytic index as a function of MAb concentration for the IgG1 MAbs 2H1 and 2D10g1. Indices were determined at an E:T ratio of 1:1 after 2 h of incubation. Each point is the average of eight measurements, and brackets denote standard deviations. P values refer to the comparison between 2H1 and 2D10g1 at each concentration.
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FIG. 4. Phagocytic indices for MAbs 3E5 (IgG3), 2H1 (IgG1), and 18G9 (IgA) with IFN-g- and LPS-stimulated and nonstimulated J774.16 cells. Experimental conditions were as follows: 2 h of incubation, E:T ratio of 1:1, and MAb concentration of 5 mg/ml. Each point is the average of eight measurements, and brackets denote standard deviations. P values refer to comparison of phagocytic indices obtained with stimulated and nonstimulated J774.16 cells for each isotype.
cells. Intracellular C. neoformans showed evidence of cellular damage, including electron-dense cytoplasm and crenated cell walls. Figure 10B shows a J774.16 cell with six intracellular yeast cells in various stages of degeneration. Rare organisms with intact cellular morphology and buds were noted intracellularly (Fig. 10C). In the absence of MAb, no phagocytosis was noted by EM. The damaged and degenerating yeast forms noted by EM indicate killing of yeast cells by J774.16 and are consistent with the reduction in numbers of CFU at 2 h (Fig. 7 and 8). Antibody protection experiments. Administration of 2D10m, 2D10g1, 2D10g2a, and 2D10g2b shortly before i.p. inoculation of C. neoformans modified the course of infection by prolonging survival of lethally infected mice (Table 1). Two experiments were done with different challenge inocula. In each experiment, the most and least effective isotypes in prolonging survival were IgG2a and IgG2b, respectively. DISCUSSION J774.16 cells have many characteristics of murine macrophages, including capacity for phagocytosis, oxidative burst,
FIG. 5. Phagocytic indices for the MAb 2D10 isotype switch family variants. MAbs 2H1 and 36-65 are included as positive and negative controls, respectively. Experimental conditions were as follows: 2 h of incubation, E:T ratio of 1:1, and MAb concentration of 5 mg/ml. Each point is the average of eight fields, and brackets denote standard deviations. P values refer to comparison with J774.16 cells without MAbs.
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FIG. 6. C. neoformans (CN) CFU after incubation with J774.16 cells in the presence and absence of various MAbs. Experimental conditions were as follows: 24 h of incubation, E:T ratio of 5:1, and MAb concentration 10 mg/ml. Each bar is the average of eight measurements, and brackets denote standard deviations. P values are calculated relative to CFU obtained with J774.16 cells in the absence of MAbs.
nitric oxide synthase activity, and production of microbicidal proteins (10, 21). J774 cells have been used to study the interaction of murine macrophages with other pathogens, including Mycobacterium tuberculosis (10), Bacillus anthracis (20), and Brucella abortus (23). In the present study, J774.16 cells were used to compare the opsonic efficacies of murine isotypes for C. neoformans and to demonstrate the potential of MAbs in enhancing macrophage antifungal efficacy. Addition of IgM, IgG, and IgA isotypes to J774.16 cell-C. neoformans mixtures significantly enhanced phagocytosis. For the IgG subclasses, there are several Fc receptors which promote phagocytosis when cross-linked by antibody-antigen complexes (46). An IgA Fc receptor in rodent macrophages which provides an explanation for the opsonic efficacy of this isotype has been described elsewhere (43). However, the mechanism by which IgM promotes phagocytosis is unclear. IgM Fc receptors in various cells have been described and may exist in J774 cells (49). Alternatively, IgM binding may cause changes in the polysaccharide capsule which result in enhanced uptake through non-Fc receptor mechanisms.
FIG. 7. C. neoformans (CN) CFU in the presence and absence of J774.16 cells and MAb 2H1 after incubation for 2 and 24 h. Experimental conditions were as follows: E:T ratio of 5:1 and MAb concentration of 10 mg/ml. Each bar is the average of eight measurements, and brackets denote standard deviations. P1, comparison with CN alone; P2 comparison with CN plus J774.16 cells.
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FIG. 8. C. neoformans (CN) CFU in the presence and absence of J774.16 cells and MAb 2H1 after incubation for 0, 1, and 2 h. The 0-h time point in fact involved approximately a 20-min incubation, since this was the minimum time needed to add C. neoformans to all wells and then to remove the supernatants for CFU determination. Experimental conditions were as follows: E:T ratio of 10:1 and MAb concentration 10 mg/ml. Similar results were obtained in a repeat experiment (data not shown). Each bar is the average of eight measurements, and brackets denote standard deviations. P values above the hatched column refer to the comparison with the CN group. P values above the solid column refer to comparison with J774.16 cells plus the CN group.
IgG MAbs were consistently better opsonins than IgM or IgA. Among the four IgG subclasses, the relative opsonic efficacy was IgG1 and IgG3 . IgG2a . IgG2b. Our findings contrast with those of Schlagetter and Kozel (51), which placed the opsonic activity of the murine isotypes in the order IgG2a . IgG1 . IgG2b using a family of isotype switch variants of the MAb 471 with mouse peritoneal macrophages. The differences in relative opsonic activities between IgG1 and IgG2a are small, and the discrepancy between our findings and those of Schlagetter and Kozel (51) may have resulted from the use of different macrophage cell types, C. neoformans strains, and E:T ratios and/or the fine specificities of the MAbs used (4). IgG3 was a potent opsonin despite its relative ineffectiveness at prolonging survival of lethally infected mice (38). There was significant interexperimental variation in the magnitude of the phagocytic indices measured. For example, using a MAb 2H1 concentration of 5 mg/ml and an E:T ratio of 1:1, the phagocytic index in the experiments shown in Fig. 2, 3, 4, and 5 were 3.7, 2.1, 3.0, and 1.15, respectively. The cause of interexperimental variation is not understood but may reflect subtle changes in the state of J774.16 and/or yeast cells. Dif-
FIG. 9. C. neoformans CFU after incubation with J774.16 cells in the presence and absence of MAb 2H1 (10 mg/ml) for 24 h at various initial E:T ratios. Each bar is the average of eight measurements, and brackets denote standard deviations. P values refer to comparison of CFU in the presence and absence of MAb 2H1.
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ferences in C. neoformans culture age can affect adherence of yeast to rat cells (32). Phagocytic indexes were comparable to those reported by other investigators (51). The highest indices were measured at E:T ratios of ;1, and the index decreased at high or low E:T ratios. At high E:T ratios, the low phagocytic index may simply reflect a paucity of C. neoformans for internalization. At low E:T ratios, lower phagocytic indices may reflect capsule polysaccharide inhibition of phagocytosis (25) from the large number of yeast cells and/or MAb consumption. Phagocytic indices of .1.0 were measured despite initial E:T ratios of 1:1. Since the replication time of C. neoformans is 2.5 to 3 h, some increase in the number of yeast cells may have occurred after 2 h of incubation. Phagocytic indices of .1.0 may also reflect preferential phagocytosis by the subset of J774.16 which attached best to the plate. Phagocytic indices were measured after washing, fixing, and staining of cells, and it is likely that nonadherent or poorly adherent cells were removed. Surface attachment factors can affect C. neoformans phagocytosis by human macrophages (30), and the same may apply to murine macrophages. Phagocytic indices were higher in IFN-g- and LPS-stimulated macrophages, which is consistent with an increase in Fc receptors (19). Despite interexperimental variation, addition of MAbs resulted in increased phagocytosis in all experiments. In the absence of GXM-binding MAbs, there was little or no fungistatic and/or fungicidal activity by J774.16 cells. Addition of IgM, IgG, and IgA MAbs resulted in a significant reduction in the number of CFU after 24 h of incubation. For MAb 2H1, the effect of the antibody on the numbers of CFU at 0, 1, 2, and 24 h was studied. Significant reduction in the number of CFU occurred rapidly only in the presence of antibody. The rapidity of CFU reduction is striking and indicates that activated J774.16 cells produce antimicrobial products which are rapidly lethal for C. neoformans. J774.16 cells make nitric oxide, reactive oxygen intermediates, and fungicidal proteins (10, 21). EM revealed that the majority of internalized cells were damaged at 2 h, which is consistent with J774.16 fungicidal activity. Despite the MAb-mediated reduction in the number of CFU at 2 h, incubation of 24 h revealed increased numbers of CFU, indicating continued growth. Since the overwhelming majority of the internalized C. neoformans cells appeared damaged by EM, it is likely that continued replication reflects extracellular growth for yeast cells that escape phagocytosis. Our results are consistent with previous studies which showed that rodent macrophages can be fungicidal to C. neoformans (2). Internalization of C. neoformans has been shown to enhance macrophage antifungal activity (16). Thus, antibody may enhance killing by promoting internalization of C. neoformans cells. In this regard, the most effective isotypes in promoting phagocytosis (i.e., IgG1 and IgG2a) were also the most effective at reducing the number of CFU. IgM, IgG1, IgG2a, and IgG2b isotype variants of the IgM hybridoma 2D10 significantly prolonged survival (P , 0.05) in protection experiment I. In experiment II, the MAb-mediated prolongation in survival was significant at the 0.05 confidence level only for the IgM and IgG2a isotypes. Paradoxically, mice in experiment I survived longer than those in experiment II, despite infection with a higher inoculum. Interexperimental variation in mouse protection experiments is common and is not understood (see references 8, 39, and 40 for discussion of potential variables). In both experiments, IgG2a and IgG2b were the most and least effective IgG subclasses, respectively, in prolonging survival. The effectiveness of IgG2a is consistent with the observation that this IgG subclass was the most effective at reducing fungal burden in another mouse model (50). However, the finding that MAb 3E5 (IgG3) was an effective
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FIG. 10. Electron micrographs of J774.16 cells after the addition of C. neoformans in the presence of MAb 2H1 (5 mg/ml); several J774.16 cells with multiple internalized C. neoformans cells are shown (marked by asterisks). (A) A C. neoformans cell being phagocytosed (arrow). Note the cell pseudopod penetrating into the polysaccharide capsule. (B) A J774.16 cell with six C. neoformans cells in various stages of degeneration. Note the capsule and cell wall changes. (C) Two J774.16 cells with internalized C. neoformans cells. Arrow shows a budding organism. Magnifications were 37,500, 36,000, and 39,000 for panels A, B, and C, respectively.
opsonin which reduced the number of CFU in vitro is in contrast to the relative ineffectiveness of this MAb in vivo (38) and suggests caution in extrapolating in vitro results to in vivo effects. Differences in the Fc receptor types found in peritoneal macrophages and J774.16 cells could explain the fact that 3E5 (IgG3) is a potent opsonin in vitro but is relatively ineffective in prolonging survival in i.p. infection models (38). Alternatively, the relative ineffectiveness of 3E5 (IgG3) in vivo may result from failure of this MAb to enhance the antifungal activities of other effector cells. The mechanism by which antibodies modify C. neoformans infection in mice is not known. Antibodies can enhance the antifungal activity of NK cells (42), neutrophils (33), and monocytes (29), and it is likely that multiple cell types are
TABLE 1. Summary of survival data in passive antibody protection experiments Expt (inoculum) 7
I (10 )
II (108)
Groupa
nb
No. deadc
Survival (days) Median
Mean 6 SD
PBS 2D10m 2D10g1 2D10g2a 2D10g2b
9 10 9 9 10
9 10 8 5 10
7 13.5 10 8 8.5
6.0 6 2.6 13.1 6 2.3 21.2 6 34.1 54.2 6 54.8 9.3 6 3.1
PBS 2D10m 2D10g1 2D10g2a 2D10g2b
10 10 10 10 10
10 10 10 10 10
5.0 19.0 9.5 14.0 9.0
8.1 6 5.3 17.1 6 5.2 12.1 6 5.6 18.8 6 12.4 9.6 6 1.1
involved in antibody-mediated protection against C. neoformans in vivo. The finding that GXM-binding MAbs are potent opsonins which promote killing of C. neoformans by J774.16 macrophage-like cells suggests that enhancement of macrophage function is a potential mechanism for antibody protection. Opsonization may also be important for efficient antigen presentation by macrophages and development of effective T-cell immunity (12). Antibody-mediated reduction in tissue polysaccharide antigen may also improve host immune function and prolong survival (40). Differences in the phagocytic activities of the various murine isotypes suggest functional differences which could translate into differences in in vivo efficacy. Our results provide additional support for the usefulness of antibody immunity. Strategies to enhance macrophage function in immunocompromised patients by restoring antibody immunity through immunization (14) and/or passive antibody administration (3) may be useful in clinical practice. ACKNOWLEDGMENTS
Pd
0.0001 0.0020 0.0021 0.0269
A.C. is supported by a James S. McDonnell Scholar award and NIH grants RO1-AI33774 and RO1-AI13342. S.L. is supported by NIMH MH 47667. We are grateful to Barry Bloom and John Chan for advice on J774.16 assays and many helpful discussions and to Yvonne Kress for excellent performance of EM. REFERENCES
0.0006 0.0959 0.0217 0.7593
a Treatment groups. Control mice received PBS i.p. MAb groups received 1.0 mg of 2D10 switch variant MAb i.p. shortly before infection (15 to 30 min). b n, number of mice per group. c No. dead, the actual number of death events per group. In experiment 1, some mice (four in the 2D10g2a group and one in the 2D10g1 group) were still alive at the end of the observation period (155 days). For statistical analysis, these points were censored to decrease their contribution to the mean. d P values were calculated by log rank test. For experiment I, the overall comparison between groups (4 df) yielded a P of 0.0001. For experiment II, the overall comparison between groups yielded a P of 0.0004.
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