Eosinophil-Cryptococcus neoformans Interactions In Vivo and In Vitro

INFECTION AND IMMUNITY,0019-9567/97/$04.0010

May 1997, p. 1899–1907 Vol. 65, No. 5

Copyright q 1997, American Society for Microbiology

Eosinophil-Cryptococcus neoformans Interactions In Vivoand In Vitro

MARTA FELDMESSER,1* ARTURO CASADEVALL,1,2 YVONNE KRESS,3

GADI SPIRA,4 AND AMOS ORLOFSKY3

Departments of Medicine (Division of Infectious Diseases),1 Microbiology and Immunology,2 and Pathology,3 AlbertEinstein College of Medicine, Bronx, New York 10461-1602, and Laboratory of Cell Biology, The Rappaport FamilyInstitute for Research in the Medical Sciences, The Bruce Rappaport Faculty of Medicine, Technion, Haifa, Israel4

Received 6 November 1996/Returned for modification 18 December 1996/Accepted 10 February 1997

Eosinophils are components of inflammatory responses to a variety of pathogens. Although a variety ofbeneficial and harmful functions have been ascribed to these cells, their role in protection against infectiousagents remains uncertain. Previous studies have reported eosinophilic pneumonia in mice infected intratra-cheally with Cryptococcus neoformans. We confirmed this observation and studied the inflammatory response inthe lung at day 14 by light and electron microscopy. Immunostaining for glucuronoxylomannan showedisolated cryptococci inside the eosinophilic cuffs. Eosinophils were found to be in close association with C.neoformans in vivo. Cryptococci were associated with eosinophils within eosinophilic perivascular cuffs, withingranulomas, and lining the alveolar space. To further investigate this phenomenon in vitro, we isolated ratperitoneal eosinophils and studied cryptococcus-eosinophil interactions in the presence and absence of anti-capsular immunoglobulin G1 (IgG1) and IgE monoclonal antibody (MAb). Eosinophils phagocytosed C.neoformans only in the presence of specific antibody. Phagocytosis was rapid, and dense rings that appeared toconsist of granule contents were formed around the organisms. Mast cells were observed to occasionallyphagocytose C. neoformans in vitro in the presence of IgE MAb. Our observations suggest that eosinophils maybe effector cells against C. neoformans.

Eosinophils are encountered in a broad range of patholog-ical processes, but their physiologic function is largely un-known. Among the functions proposed for eosinophils areenhanced expulsion and killing of extracellular parasites (2,64), tumoricidal effects (65), antigen (Ag) presentation (23, 51,73), and assistance in wound healing (68, 74). Eosinophils caningest small particles, including Ag-antibody (Ab) complexes(3, 46, 61), and phagocytose a variety of organisms, includingcomplement-coated Candida albicans (16, 26, 38), bacteria(16), and mycobacteria (15). Eosinophils also produce a widevariety of cytokines, some of which upregulate their functionand prolong their own survival (reviewed in reference 53).Eosinophils are prominent components of the inflammatoryresponse to allergens and are involved in the pathogenesis ofasthma (29, 69). Because of their toxic granule contents andtheir ability to generate superoxide radicals and leukotrienes(especially LTC4), eosinophils are potentially toxic to host tis-sues. Eosinophil products have been shown to be particularlytoxic to respiratory epithelial cells (20, 31). Thus, it is difficultto distinguish between eosinophils as antimicrobial effectorcells and eosinophils as participants in tissue damage afterrecruitment by inflammatory signals.Eosinophils are recruited to inflammatory sites in a variety

of infections. Although particularly prominent in helminthicinfections, in which they are believed to contribute to theimmune response by Ab-mediated extracellular killing (10),they are also present in inflammatory responses to fungi andmycobacteria (15, 37, 45). Little is known about the role ofeosinophils in fungal infections. The most-studied fungal in-fection is bronchopulmonary aspergillosis, in which eosinophils

are recruited as part of a hypersensitivity reaction to AspergillusAgs (reviewed in reference 71).Cryptococcus neoformans is an encapsulated yeast that

causes meningitis in 6 to 8% of patients with AIDS (19).Cryptococcal infection is presumed to be acquired by inhala-tion, with the lung being the primary site of infection (44). Theprimary host immune response to cryptococcus is believed tobe nonspecific, involving macrophages and NK cells, and thedevelopment of cell-mediated immunity is essential for hostprotection (57). Eosinophils are also observed during the in-flammatory response to C. neoformans infection. Eosinophilsare rarely found in the cerebrospinal fluid of patients withcryptococcal meningitis (1, 56), but patients with peripheralblood eosinophilia and C. neoformans pulmonary infectionshave been described (25, 39). Eosinophils were reported in apleural effusion due to C. neoformans infection (25) and in theinflammatory response of an epidural abscess (9). In experi-mental models, eosinophils are components of the inflamma-tory response to pulmonary cryptococcal infection in rats (32)and in several mouse strains (36, 37, 45). Eosinophils are re-cruited following infection with both serotype D and serotypeA cryptococcal strains (36). Eosinophil recruitment in crypto-coccal infection in BALB/c mice is dependent on the presenceof CD41 cells and is associated with interleukin-5 (IL-5) pro-duction by cultured murine lung cells (37). The discrepancybetween the frequencies with which eosinophils are observedin human and rodent tissues may reflect the following: (i) whileT-cell immunity is intact in rodent models, humans with cryp-tococcal disease most commonly have T-cell deficiencies; (ii)while many cases of human disease are chronic infections ormay reflect reactivation of latent infection, experimental infec-tion in rodents is usually a primary and acute infection; and(iii) human tissue is frequently obtained from patients whohave received antifungal therapy, which could alter the im-mune response, or from autopsy specimens, which represent

* Corresponding author. Mailing address: Albert Einstein Collegeof Medicine, Ullmann Bldg. Room 1219, 1300 Morris Park Ave.,Bronx, NY 10461. Phone: (718) 430-4259. Fax: (718) 430-8968. E-mail:[email protected].

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end stage disease. Hence, the paucity of reports implicatingeosinophils in human cryptococcosis does not necessarily ne-gate an important role for these cells in host defense.The present study was undertaken to explore the role of

eosinophils in murine infection and the potential for eosino-phils to function as effector cells against C. neoformans. Weexamined the interaction between eosinophils and C. neofor-mans in vivo by light microscopy and electron microscopy(EM) of intratracheally infected mice and in vitro by using ratperitoneal eosinophils. Additionally, we studied the ability ofmonoclonal antibody (MAb) to capsular polysaccharide topromote eosinophil phagocytosis of C. neoformans.

MATERIALS AND METHODS

C. neoformans. Strain 24067 (serotype D) was obtained from the AmericanType Culture Collection (Rockville, Md.). This strain was used because it hasbeen extensively studied by several groups (37, 55, 63) and elicits an eosinophilicpneumonia in mice (37). Cultures were grown in Sabouraud dextrose broth(Difco Laboratories, Detroit, Mich.) at 308C overnight with moderate shaking.Yeast cells were washed three times and suspended in sterile 0.02 M phosphate-buffered saline (PBS; pH 7.2). Yeast cells were counted in a hemacytometer.MAbs. Two MAbs which bind to the C. neoformans glucuronoxylomannan

(GXM) were used in this study. The immunoglobulin G1 (IgG1) MAb 2H1 hasbeen described elsewhere (13). The IgE MAb is an isotype switch of the IgG3MAb 3E5 (66). MAbs 2H1 and 3E5 use the same variable-region genes, werederived from the same B cell in vivo, and are believed to have similar if notidentical epitope specificities (54). MAb-containing ascites was obtained by para-centesis of pristane-primed BALB/c mice following intraperitoneal injection ofhybridoma cells. The IgG1 MAb was purified by protein G affinity chromatog-raphy (Pierce, Rockford, Ill.). The IgE MAb was purified by affinity chromatog-raphy on Sepharose 4B coupled to rat anti-mouse kappa serum. MAb concen-trations relative to those of isotype-matched standards were determined byenzyme-linked immunosorbent assay (14, 66).In vivo experiments. Eight female C57BL/6 mice were obtained from Jackson

Laboratories (Bar Harbor, Maine) and from Charles River Laboratories(Charles River, Mass.). Mice were 6 to 14 weeks old at the time of infection. Twomice received 100 mg of IgG1 MAb intraperitoneally on the day prior to infec-tion. Mice were anesthetized with 65 mg of sodium pentobarbital (Anpro Phar-maceutical, Arcadia, Calif.) per kg of body weight and infected by inoculation of104 or 106 organisms in 0.05 ml of PBS into the trachea, as described elsewhere(28). On day 14, mice were killed by cervical dislocation. The lungs were removedand fixed in 10% neutral buffered formalin (Sigma) (for light microscopy) orovernight in Trump’s fixative (4% paraformaldehyde and 1% glutaraldehyde in0.1 M phosphate buffer). In a separate experiment, the lungs of two mice werehomogenized in 10 ml of PBS for determination of cryptococcal capsule size.Light microscopy and immunohistochemistry. Paraffin-embedded tissue sec-

tions 4 mm thick were stained with hematoxylin and eosin. Tissue GXM immu-nohistochemistry was done as described elsewhere (55). Deparaffinized tissuesamples were treated with 3% H2O2 for 30 min and incubated with 5% goatserum in PBS for 1 h. Primary GXM-binding MAb 2H1 was applied at 2.6 mg/ml,and the tissue was incubated overnight. Alkaline phosphatase-conjugated goatanti-mouse IgG (Southern Biotechnology Associates, Inc., Birmingham, Ala.)was diluted 1:200 and applied. The mixture was incubated for 2 h at roomtemperature. Color was developed with 5-bromo-4-chloro-3-indolyl phosphateand nitroblue tetrazolium (BCIP/NBT) (Sigma). Tissue was counterstained by a2-s incubation in eosin Y with 1% phloxin, ethanol, and acetic acid.Cryptococcal capsule size in vivo and in vitro. India ink preparations of lung

homogenates were examined under oil immersion (magnification,31,000), usinga grid with resolution to 0.001 mm. Measurements of the distance from thecapsule edge to the cell wall were averaged (n 5 20 for each of two mice). Thecapsule sizes of organisms grown overnight in Sabouraud dextrose broth werealso measured.Rat peritoneal eosinophils. Male Sprague-Dawley rats (Charles River Labo-

ratories) were killed in a CO2 chamber. Eosinophils were isolated by using amodification of a previously described method (18, 48). Briefly, 25 ml of coldHanks balanced salt solution without Ca21, Mg21, HCO32, or phenol red(Gibco Life Technologies, Grand Island, N.Y.) but with 10 U of heparin (Sigma)per ml and 0.2% bovine serum albumin (Sigma) was injected into the perito-neum. The abdomen was massaged for 10 to 20 s, and the wash was recoveredafter the peritoneum was opened by ventral incision. Lavage fluids were pooled,and cells were collected by centrifugation at 3303 g for 15 min at 48C. Cells weresuspended in warm RPMI (Gibco) with 10% heat-inactivated fetal calf serum(HyClone, Logan, Utah) containing penicillin and streptomycin (Gibco) andincubated in tissue culture dishes (Falcon, Lincoln Park, N.J.) for 1.5 h at 378Cwith 5% CO2. The same medium was used for subsequent procedures. Nonad-herent cells were pooled and spun at 330 3 g for 15 min. Vital dye staining withtrypan blue showed .99% viability. Cytospin preparations were spun at 553 gfor 5 min in a Cytospin 3 cytocentrifuge (Shandon, Inc., Pittsburgh, Pa.), dried in

a laminar-flow hood for 15 min, fixed with methanol for 1 min, and stained withLeukostat (Sigma). Approximately 50% of the cells were eosinophils (range, 42to 51%). Contaminating cells consisted of mast cells (8 to 18%), macrophages(21 to 38%), lymphocytes (5 to 10%), and neutrophils (0 to 1%).Eosinophil stimulation. In one experiment, cells from rat eosinophil prepara-

tions of some groups were incubated in 1 nM recombinant human IL-5 (rhIL-5)(R & D Systems, Minneapolis, Minn.) (27, 47), 15 ng of bacterially expressedrecombinant mouse granulocyte-macrophage colony-stimulating factor rmGM-CSF (27, 52), and 0.1 mg of lipopolysaccharide (LPS) from Escherichia coliO1127:B8 (Sigma) (67) for 30 min prior to their use in a phagocytosis assay (seebelow) to promote activation of cells. In a second experiment, 200 U of rmIL-5per ml in COS-conditioned medium (Genzyme, Cambridge, Mass.) (42) was alsoadded. In all other experiments, cells were used without addition of cytokines orLPS on the day they were obtained.Eosinophil phagocytosis assays. Cells were plated at a density of 106/ml in

media on 24- or 96-well tissue culture plates (Costar Corp., Cambridge, Mass.)with 106 yeast cells/ml (effector/target cell ratio of 1:1) and 0 or 5 mg of IgG1 orIgE MAb per ml. Plates were incubated at 378C for 2 h in all experiments exceptone, in which cells were also incubated for 0, 15, 30, and 60 min. The cells werethen collected from the wells, suspended in Trump’s fixative for 30 min, centri-fuged, resuspended in fixative, and stored at 48C overnight.EM. Tissue blocks or cells were postfixed with 1% osmium for 1 h, dehydrated

in ascending ethanol (50 to 100%), cleared in two changes of acetonitrile, andinfiltrated with and embedded in Araldite-Epon. Ultrathin sections were stainedwith uranyl acetate followed by lead citrate. After light microscopic review of1-mm-thick toluidine blue-stained sections, selected regions were examined witha JEOL (Tokyo, Japan) 100S electron microscope. Eosinophils were identified bythe presence of specific granules with crystalline cores in the cytoplasms of intactcells. For some of the in vitro phagocytosis experiments, the number of crypto-cocci ingested by eosinophils per 100 yeast cells was determined by EM. Twofields on each of five grids were counted for each measurement. For two exper-iments, the number of eosinophils with ingested cryptococci per 100 eosinophilswas determined. One measurement was made for each of five grids. Mast cellswere identified by their relative sizes, single nuclei, narrow surface folds, andlarge numbers of electron-dense secretory granules (24). Eosinophil and cryp-tococcal sizes were determined in vivo and in vitro by measurement of thediameter of the long axes of individual cells or organisms (including the capsule).Ten determinations were made for each measurement. Eosinophil size in vitrowas determined with unstimulated cells.Statistical analysis. Statistical analysis was performed using Primer of Biosta-

tistics: The Program, Version 3.01 (McGraw-Hill Inc., New York, N.Y.). Multi-variate analysis was performed by analysis of variation, with pairwise compari-sons made by an unpaired two-tailed Student t test. The a level in each case wasadjusted by the Bonferroni method for the number of comparisons made.

RESULTS

In vivo observations. Light microscopic review of paraffin-embedded lung sections showed the presence of numerouseosinophils by 14 days after infection. Eosinophils were com-ponents of mixed inflammatory infiltrates. In some mice, largeperivascular cuffs of eosinophils in which occasional yeast cellswere present on hematoxylin-and-eosin-stained tissue (Fig. 1).Immunostaining for GXM more clearly demonstrated isolatedcryptococci inside the eosinophilic cuffs. Cryptococci were alsoseen in close association with eosinophils that were insidegranulomas and that lined the alveolar space. The cytoplasmsof some eosinophils stained for GXM (Fig. 2). EM showedoccasional eosinophils which appeared to be directly interact-ing with C. neoformans, and yeast cells were present in aggre-gates of eosinophils (Fig. 3). Yeast cells that were in directcontact with eosinophils had on their surfaces electron-densematerial that was more highly concentrated along the border ofcontact and that may represent discharged granule contents.Eosinophils in the tissue of one of the IgG1 MAb-treated micevery rarely contained phagocytosed yeast cells. No phagocyto-sis of yeast cells by eosinophils was observed in the tissues ofcontrol mice. Each phagocytosed yeast cell was surrounded bya thin membrane, consistent with being in a phagosome. Themean capsule sizes of cryptococci (6 standard deviations) ob-tained by light microscopy of lung homogenates from each oftwo mice were 8.4 6 1.8 and 8.3 6 1.6 mm. The capsule sizeof this strain in vitro after overnight growth in Sabourauddextrose broth at 308C is 1.6 6 0.9 mm (28). Thus, thecapsules of cryptococci were significantly larger in vivo in

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the lung than in vitro (P , 0.0001). The mean diameter ofcryptococci in vivo (including the capsules) as measured by EMwas 11.5 6 4.2 mm, and that of murine lung eosinophils was9.56 1.9 mm (P5 0.13; Student’s t test). The measurements byEM and light microscopy are not directly comparable, becauselight microscopy was done in fluid phase, while tissue for EMwas fixed and dehydrated.In vitro phagocytosis assays. Rat peritoneal eosinophils

were used for in vitro studies because they are a convenientsource of eosinophils, and eosinophils are components of theinflammatory response to C. neoformans infection in the ratlung (32). There was no difference in the electron microscopicappearances of yeast cells coincubated with unstimulated eo-sinophils or with eosinophils that were incubated in IL-5, GM-CSF, and LPS without MAb. EM of heat-killed cryptococcishowed dead organisms to be indistinguishable from those inthe experimental assays. Thus, we could not distinguish livefrom dead C. neoformans by EM.EM showed minimal phagocytosis of cryptococci when eo-

sinophils and yeast cells were coincubated without MAb for 2 h(Table 1). The percentage of eosinophils with ingested yeastwas slightly increased when rmIL-5 was used for stimulation.Cells incubated with either IgG1 or IgE MAb showed signifi-cant phagocytosis of yeast cells. Both the number of crypto-cocci ingested and the percentage of eosinophils with ingestedorganisms significantly increased for both MAbs, but theamount of phagocytosis appeared to be greater in the presenceof IgG1 MAb than in the presence of IgE MAb in unstimu-lated cells or when rhIL-5 was used for stimulation. When

rmIL-5 was added, both MAbs promoted phagocytosis equally.There was no significant difference in phagocytosis when cellswere incubated with IL-5, GM-CSF, and LPS versus mediumalone in the presence of IgG1 or IgE MAb. In the presence ofIgE MAb, mast cells (present as contaminants) occasionallyphagocytosed cryptococci (Fig. 4). This phenomenon was notseen with IgG1 MAb or when no MAb was present.When yeast cells and eosinophils were coincubated in the

presence of IgG1 MAb, eosinophils ingested C. neoformanswithin 15 min. Ingested cryptococci were most often sur-rounded by a rim of electron-dense material lining the inside ofthe phagosome. This rim may begin to form before the organ-ism is internalized (Fig. 5). The exact nature of this material isunknown. However, specific granules appeared to empty intophagosomes, and the density of the granule contents is similarto the density of the rim. The appearances of ingested yeastcells and of eosinophils with ingested cryptococci were thesame at 15 min as at 1 h. However, by 2 h, phagocytic eosin-ophils were largely degranulated, and eosinophils with dis-rupted cell membranes were seen. Ingestion in the presence ofIgG1 MAb increased with time (Fig. 6). The mean diameter ofcryptococci in vitro was 3.96 1.0 mm and that of rat peritonealeosinophils was 7.9 6 1.0 mm (P , 0.0001; Student’s t test).

DISCUSSION

The present study demonstrates for the first time a closeinteraction between C. neoformans and eosinophils in murinepulmonary infection. Eosinophil recruitment in murine pulmo-

FIG. 1. (A) Light microscopy of lungs of C57BL/6 mice stained with hematoxylin and eosin, showing perivascular cuffs of eosinophils 14 days after infection. Arrowspoint to scattered cryptococci seen within these cuffs (magnification,3400). (B) Eosinophils (arrows) were also present as components of mixed inflammatory infiltratesin granulomas containing C. neoformans (magnification, 3400).

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nary cryptococcal infection had previously been reported andshown to be dependent on the presence of CD41 cells (37).EM examination of lung tissue revealed that eosinophils caninteract directly with C. neoformans and may release granulecontents extracellularly in response to contact with yeast cells.Immunohistochemistry revealed the presence of GXM insideeosinophils, strongly suggesting that eosinophils ingestedGXM. We could not determine whether GXM was taken up asfree polysaccharide or as components of Ag-Ab complexes.The use of EM was crucial to our study because it allowedspecific distinction of eosinophils from other phagocytic cells,even when the eosinophils were extensively degranulated.The main function of eosinophils has been proposed to be

defense against helminthic infection. Indeed, granule productsare cytotoxic to Trichinella spiralis larvae and to Schistosomamansonii schistosomula (72) and can mediate enhanced intes-tinal motility and mucous secretion, which may contribute toparasite expulsion and gut protection (64). However, theirfunction in helminthic infection has been questioned, sinceprevention of accumulation of eosinophils by anti-IL-5 MAbsdoes not alter worm burden or immune resistance (35). Incontrast, the function of eosinophils in antibacterial and anti-fungal responses has received comparatively less attention.The ultrastructural and immunohistochemical demonstrationof eosinophils in intimate contact with C. neoformans in theinflammatory response of C57BL/6 mice suggests that eosino-phils may be able to damage the organism by extracellularmechanisms. Polycationic proteins in eosinophil granules arehighly toxic to protozoa and bacteria (43, 70) as well as tomammalian cells such as pneumocytes, tracheal epithelial cells,

and endothelial cells (31). Thus, the interaction between eo-sinophils and C. neoformans may result in toxicity to the yeastcell or to the lung. Eosinophils may function to clear Ag-Abcomplexes. Alternatively, they may function as Ag presentersor cytokine producers in this infection. Though eosinophilsperform these functions in other contexts, determination ofwhether they do so in cryptococcal infection requires furtherstudy.In vitro studies were done with rat peritoneal eosinophils.

These cells were unable to significantly phagocytose C. neofor-mans in the absence of MAb. When stimulated briefly withIL-5, GM-CSF, and LPS, factors that prime or activate a va-riety of eosinophil functions (27, 47, 67), a slightly higherpercentage of eosinophils phagocytosed cryptococci in one ex-periment. We initially explored the interaction of eosinophilsand C. neoformans in the presence of specific Ab to determinewhether interaction between the organisms and the cells wasenhanced. Eosinophils constitutively express a variety of Fcreceptors, including FcgRII, FcεRII, and FcRa (11, 21, 34, 41,69), and the expression of these can vary with cytokine stimu-lation. Eosinophils can be induced to express FcgRIII and thehigher-affinity FcgRI and FcεRI (21, 34). Adherence to micro-organisms, such as Schistosoma mansonii schistosomules, isFcR mediated (12), and cross-linking of these receptors resultsin a variety of effects, including induction of cytotoxicity (33)and selective discharge of granule contents (11, 30). Otherconsequences of receptor cross-linking include phagocytosis(38, 62), immune complex binding, and induction of the respi-ratory burst (22, 34). In the presence of specific MAb, initialphagocytosis was rapidly completed, and there was a slower

FIG. 2. Light microscopy of lungs of C57BL/6 mice immunostained for GXM (blue) and counterstained with eosin (red) 14 days after infection. (A) Perivascularcuffs of eosinophils (E) were seen to contain isolated cryptococci (arrowhead) (magnification, 3250). (B) Control section (the secondary Ab was eliminated) showedno blue staining inside eosinophils (arrows) or around cryptococci (arrowheads) (magnification, 31,000). (C and D) GXM was present around eosinophils (arrows)in contact with collections of cryptococci (arrowheads), and eosinophil cytoplasm stained purple (combination of eosin with BCIP/NBT), which is consistent witheosinophil uptake of GXM (arrowheads) (magnification, 31,000).



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continued increase in phagocytic indices. Both IgG1 and IgEstimulated eosinophils to phagocytose cryptococci. With bothMAbs, granule contents appeared to empty into phagosomes.Eosinophils can ingest a variety of organisms, including se-

rum-opsonized Candida albicans and baker’s yeast (16, 26, 47),Mycobacterium avium (15), Staphylococcus aureus, and E. coli(16). In contrast to phagocytosis of C. neoformans, efficientphagocytosis of C. albicans requires both Ab and complement(38). When Trypanosoma cruzi amastigotes are phagocytosed,the phagosomes contain an electron-dense core which stains

for a-major basic protein (MBP) Ab, and amastigotes degen-erate (40, 62). Eosinophils also effectively ingest Ag-Ab com-plexes (4, 61). Because Ag-Ab complexes are chemotactic foreosinophils, one function proposed for the eosinophil is re-moval of these complexes from tissue (5, 61). CR3-dependentphagocytosis of serum-treated zymosan is increased by stimu-lation with platelet-activating factor, GM-CSF, IL-5, and IL-3and results in triggering of the respiratory burst (7, 8, 75). Inthe present study, Ab was sufficient to opsonize C. neoformanswithout complement, and the contribution of complement to

FIG. 3. (A) EM of mouse lung 14 days after infection showed an eosinophil directly abutting a yeast cell (C). The electron-dense material (arrow) may representeosinophil granule contents discharged in response to contact with the polysaccharide capsule (magnification, 310,000). (B) Eosinophils (white crosses) recruited tothe site of infection surround C. neoformans (magnification, 33,000).

TABLE 1. Eosinophil phagocytosis assays

Stimulation Opsonin

Phagocytic index (mean6 SD)a

Expt 1 Expt 2

Method 1 Method 2 Method 1 Method 2

None None ND ND 0.5 6 0.7 0.6 6 0.9IgG1 7.3 6 3.6b 46.3 6 4.0b 4.2 6 2.0b 11.8 6 1.1b

IgE ND ND 4.5 6 1.5b 7.4 6 0.02b,d

IL-5,c GM-CSF, LPS None 0.3 6 0.7 0.6 6 0.9 1.9 6 1.6 3.6 6 1.5e

IgG1 13.0 6 3.6b 47.0 6 4.7b 4.6 6 1.4b 12.8 6 2.5b

IgE 5.6 6 1.6b,d 22.9 6 7.3b,d 5.6 6 1.8b 12.1 6 2.5b

a Eosinophils and yeast were coincubated for 2 h at 378C. Means are of five measurements. At least 100 cells were counted for each measurement. Method 1 measuredthe number of C. neoformans cells ingested per 100 C. neoformans cells. Two measurements were made on each of five grids for each group. Method 2 measured thenumber of eosinophils with ingested C. neoformans per 100 eosinophils. One measurement was made for each of five grids for each group. ND, not done.b Statistically significant difference (P , 0.013 in experiment 1; P , 0.008 in experiment 2) in phagocytosis from group without MAb.c In experiment 1, rhIL-5 was used; in experiment 2, rhIL-5 and rmIL-5 were added.d Statistically significant difference from groups incubated with IgG1 MAb.e Statistically significant difference from unstimulated group with corresponding opsonin by two-tailed Student’s t test.



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eosinophil phagocytosis of C. neoformans was not studied.Short-term incubation with IL-5, GM-CSF, and LPS did notappear to enhance phagocytosis over that seen with untreatedcells.In vivo phagocytosis has rarely been reported, and it has

been commented that eosinophil phagocytosis is largely an invitro phenomenon (6). However, in the air pouch model, eo-sinophils have been shown to ingest M. avium and Mycobacte-rium smegmatis (15). Guinea pig eosinophils phagocytose S.aureus, E. coli, and Ag-Ab complexes in vivo in animals stim-ulated with diphtheria toxoid (59), and eosinophils from thesputa of patients with asthma have been reported to containintracellular bacteria within phagosomes (17). In the presentstudy, close association between C. neoformans and eosinophilswas observed in the lung, but phagocytosis was very rare.Phagocytosis of cryptococci by eosinophils was seen in only onemouse that received systemic IgG1 MAb prior to infection,though only two IgG1 MAb-treated mice were studied. Nophagocytosis was seen in the six mice that did not receive MAb.When phagocytosis was observed, the electron-dense materialseen surrounding phagocytosed yeast in vitro was not present.Thus, the in vivo significance of the finding that specific Ab caninduce eosinophil phagocytosis of C. neoformans in vitro isunclear. In vivo strain 24067 has a significantly larger capsule,and the absence of in vivo phagocytosis may reflect the inabilityof eosinophils to ingest well-encapsulated yeast cells. In vivo,strain 24067 cells and eosinophils were approximately the samesize, and this may preclude phagocytosis. In contrast, C. neo-formans cells were significantly smaller than eosinophils in

vitro, where more frequent phagocytosis was seen. Alterna-tively, the presence of multiple eosinophils surrounding a sin-gle organism could alter the eosinophil-cryptococcus interac-tion and prevent phagocytosis. Partial engulfment may besufficient for eosinophil degranulation.A higher percentage of eosinophils appeared to phagocytose

C. neoformans in the presence of IgG than of IgE when theeosinophils were unstimulated or when rhIL-5, GM-CSF, andLPS were used for stimulation. However, this remains a pre-liminary observation because the presence of mast cells (whichexpress high-affinity IgE receptors) in the eosinophil prepara-tion may have resulted in mast cell-mediated effects specific toone of the two MAbs (60). Since our eosinophil preparationcontained macrophages, which are known to kill C. neoformansin the presence of MAb, we did not attempt to study eosino-phil-mediated killing in vitro. Furthermore, the finding that theeosinophil-C. neoformans interaction was qualitatively differ-ent in vivo and in vitro diminished our enthusiasm for pursuingkilling studies in vitro. Hence, the question of whether eosin-ophils can kill or inhibit C. neoformans in vivo or in vitroremains unanswered.We observed rare phagocytosis of cryptococci by mast cells

in the presence of IgE. As for eosinophils, many functions havebeen proposed for mast cells, but the relative importance invivo of the potential functions is unknown. Mast cells are alsobelieved to be involved in the regulation of the immune re-sponse through mediator release. Mast cells produce cytokinesinvolved in B- and T-cell responses and can present Ag bymajor histocompatibility complex class I and II pathways (50).

FIG. 4. Mast cell phagocytosis of C. neoformans (magnification, 38,000).



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FIG. 5. In vitro phagocytosis of C. neoformans (C) in the presence of IgG1 MAb. (A) Eosinophil beginning to encircle yeast (t 5 1 h; magnification, 35,000). (B)Eosinophil almost engulfing C. neoformans (t 5 30 min; magnification, 36,000). (C and D) Eosinophil with intracellular yeast cell discharging electron-dense granules(arrow) into phagosome (C, t 5 2 h and magnification 5 38,000; D, t 5 1 h and magnification 5 36,000). (E and F) Intracellular organisms with electron-dense rings(arrowheads) surrounding capsule. Many eosinophils phagocytosed multiple organisms (E, t 5 2 h and magnification 5 35,000; F, t 5 30 min and magnification 536,000).



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Mast cell phagocytosis has generally been studied in in vitrosystems and can be mediated by either FcgRII or CR3 (58).Mast cells phagocytose FimH-expressing members of the fam-ily Enterobacteriaceae through a mannose-binding protein.Bacterial phagocytosis results in killing of the organisms by aprocess involving phagosomal acidification and superoxide re-lease (49). Bacterial Ags can be processed through a phago-cytic route for class I major histocompatibility complex pre-sentation (50). As with eosinophils, little is known about mastcell antimicrobial function in vivo. Because of the rarity withwhich we observed cryptococcal phagocytosis by mast cells invitro, we conclude only that it can occur.In summary, we show that direct interaction between eosin-

ophils and C. neoformans occurs in vivo in experimental murineintratracheal infection. Specific Ab is sufficient for phagocyto-sis by rat peritoneal eosinophils and results in degranulationwith granular proteins placed in contact with the organism.Although eosinophils are unlikely to be the predominant ef-fector cells in the immune response to this organism, the find-ing of eosinophils in intimate association with C. neoformanssuggests a potential function for eosinophils as effector cells.As in many other infectious processes, elucidation of the “pur-pose” of the presence of eosinophils in pulmonary cryptococcalinfection requires further study.

ACKNOWLEDGMENTS

M.F. is supported by NIH grant K08AI01341; A.C. is supported byNIH grants AI22774 and AI13342 and a Burroughs Wellcome Devel-opmental Therapeutics Award; G.S. and A.C. are supported by NIH-Fogarty grant 1 R03 TW00498-01; A.O. is supported by YU grant4-526-0522. This support is gratefully acknowledged.We thank Jorge Bermudez for outstanding histopathology. We are

grateful to Sunhee Lee and Jacques Padower for many valuable dis-cussions.

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Eosinophil-Cryptococcus neoformans Interactions In Vivo and In Vitro

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