of Leukocyte



Inflammatory Responses to Cryptococcosis Congenitally Athymic Mice Cindy University of Wisconsin

A. Salkowski






Medical School, Departments of Surgery (E.B.) and Medical Microbiology and Immunology (C.A.S., E.B.), Madison

Histopathology revealed that nu/nu mice developed both acute and chronic inflammatory responses following infection with Cr,pfococcus neoformans. In comparison to inflammatory responses In nu/+ mice, the responses in nu/nu mice were delayed, less intense, contained predominantly more polymorphonuclear Ieukocytes (PMNs) than macrophages (M4s), and did not develop Into granulomas. In addition, nu/nu mice developed cryptococcal skin lesions demonstrating that C. neoformans is dermatotropic in a T-cell deficient host. Quantitative culturing of infected organs confirmed that delayed and Incomplete inflammatory responses observed in nu/nu mice correlated with their enhanced susceptibility to C. neoformans. Key words:

histopathology, Crypfococcus cosis, gnotoblotic

INTRODUCTION Cryptococcus neoformans is an encapsulated yeast that causes pulmonary, central nervous system, or systemic disease in man. A composite of both innate and acquired immune responses is required to control this fungus in vivo. The high frequency of man’s exposure to C. neoformans in conjunction with the low incidence of cryptococcosis in the general population indicates that innate host defense mechanisms, such as the complement cascade, phagocytic cells, and natural killer (NK) cells are relatively efficient in protecting against cryptococcal infection [10,11,13,15,21,27]. Despite the importance of innate immunity, the capacity of the host to mount an adequate cell-mediated immune (CMI) response also plays a pivotal role in resistance to cryptococcosis [6,17,22]. The importance of CMI in resistance to cryptococcosis has been particularly evident in patients with acquired immune deficiency syndrome (AIDS), a disease which affects T-helper (TH) lymphocytes. In AIDS patients, cryptococcosis is the fourth most common cause of life-threatening infection [7]. Because cryptococcosis is frequently observed in immunodeficient or immunocompromised individuals, animal models that mimic the immunologic defects in man provide a mechanism to study pathogenesis and susceptibility to C. neoformans in the immunocompromised host. Nude mice have congenital dysplasia of the thymus, lack thymus-matured T cells, and mimic the immune deficiencies observed in individuals with defects in T-cell mediated immunity. Previous studies have associated the nude gene with increased susceptibility to cryptococcosis and have provided strong experimental evidence that T cells play an important role in host defense against this © 1991 Wiley-Liss,







disease [6,16,24,28]. Histopathology of C. neoformansinfected tissues from nu/nu mice in the latter studies indicated that neither an acute nor chronic inflammatory response was elicited following infection [24,28]. The lack of an inflammatory response in nu/nu mice was noted at early and late times after cryptococcal infection [24,28]. The lack of inflammatory responses in nu/nu mice is perplexing in light of work by Cauley and Murphy [6] which showed that nu/nu mice had lower numbers of C. neoformans in their tissues during the first 14 days of infection than their immunocompetent nu/+ littermates. Early resistance of nulnu mice to cryptococcosis was attributed to macrophages (M4s), NK cells, or both [6,18] since the activity of these cells is heightened in nude mice [3,8]. To date, the cells responsible for this early resistance to C. neoformans in nu/nu mice have not been identified. In this study, histopathology was used to determine whether nu/nu mice were capable of eliciting an inflammatory response to C. neoformans and to assess whether differences in inflammatory responses correlated with differences in the capacity of C. neoformans to grow in tissues of infected nu/nu and nu/+ mice.









weeks 2, 1990:

nu/nu of age







in this



mice study.


Reprint requests: Edward Balish. University of Wisconsin Medical School. Departments of Surgery and Medical Microbiology and Immunology. 4638 Medical Sciences Center. 1300 University Avenue. Madison, WI 53706.



and Balish

Animals were obtained from the University of Wisconsin Gnotobiotic Research Laboratory (Madison, WI) and were maintained in accordance with NIH guidelines. On the day each experiment was started, mice were removed from the OF isolator and were maintained in sterile cages with filter bonnets in a laminar flow hood.


of Immunodeficiencies

To confirm T-cell defects in athymic mice, spleen cells from uninfected OF-nu/nu and nu/+ mice were assayed for their ability to respond to the T- and B-cell mitogens, concanavalin A (ConA), and lipopolysaccharide (LPS), as previously described [2]. In contrast to spleen cells from flu! + mice, spleen cells from nulnu BALB/c mice did not respond to ConA in vitro. Spleen cells from both nu/nu and nu!+ mice had strong proliferative responses to LPS in vitro. A standard 4-h Cr-release assay, previously described [3], was used to confirm that nu/nu mice had elevated NK-cell activity. Athymic mice had significantly greater (P < 0.05) splenic NK-cell activity than nu!+ mice (16.2% and 9.1%, respectively at effector: target ratio of 100:1).

Yeast Cultures

and Animal


An encapsulated strain of C. neoformans serotype A (strain SLHA) was maintained on Sabouraud’s dextrose agar (SDA). Yeast cells were transferred to Sabouraud’s dextrose broth and incubated at 37#{176}C for 48 h. Cryptococci were harvested, washed 3 times by centnfugation (1 ,000g, 15 mm), resuspended in non-pyrogenic saline, counted on a hemacytometer, and adjusted to either 1 X io cells/mi or 1 X 106 cells/mI. Mice were infected by injecting 0. 1 ml of the yeast cell suspension into the tail vein. To verify the number of viable cells, the inoculum was serially diluted in phosphate-buffered saline (PBS), plated on SDA, incubated at 37#{176}C for 48 h, and colony forming units (CFU) were determined. In mortality studies, 8-10 mice were used per genotype and deaths were recorded on a daily basis. Mortality studies were terminated at 60 days after intravenous (i.v.) challenge and the number of C. neoformans CFU in the internal organs was assessed as described below. To calculate mean survival time (MST) when mice survived to day 60, each surviving mouse was arbitrarily assigned day 61. Statistical differences in MST were determined by analysis of variance (ANOVA).



Mice were killed at 1, 3, 7, 14, and 21 days or at 1, 3, 7, 10, and 14 days after i.v. challenge with 1 X io or 1 x l0 C. neoformans, respectively. The kidneys, liver, lungs, spleen, and brain were removed and homogenized in 5 ml PBS. Homogenates were serially diluted in PBS, plated in duplicate on SDA, and colonies were counted

after incubation for 48 h at 37#{176}C. Data are expressed as the mean log o number of C. neoformans per gram (dry weight) of each tissue homogenate from 3 mice per group for each culture interval. Dry weight was assessed by placing 1 ml of tissue homogenate in an aluminum pan and drying it at 60#{176}C for 24 h. Statistical differences in C. neoformans CFU between nulnu mice and their nu/+ littermates were determined using the Student’s t-test and ANOVA.

Histopathology Tissue biopsies were collected from 3 mice per group on days 14 and 21 after i.v. challenge with 1 x l0 C. neoformans. After fixation for 48 h in HollandeBouin’s fixative, specimens were dehydrated through increasing concentrations of ethanol (50%, 70%, 80%, 95%) and embedded in glycol methyl-acrylate (Biorad, Richmond, CA). Sections (2-2.5 j.t) were cut on a JB-4 microtome (Sorvall, Newton, CT), and stained with periodic acid-Schiff followed by either azure A-eosin B or hematoxylin and eosin. At least 3 sets of 3-4 consecutive sections per sample were examined by light microscopy. In addition, histopathology was confirmed by a pathologist in a masked fashion.

RESULTS Histopathology and nu/+ Mice

of C. neoformans



To assess whether nu/nu mice were capable of mounting in vivo inflammatory responses to C. neoformans, histopathology of various tissues from flu/flu and nu! + mice was assessed after i.v. challenge with 1 x i04 cryptococci. In these experiments, tissue samples were collected 14 and 21 days after challenge since preliminary studies indicated these time points were optimal for detecting differences in populations of C. neoformans in infected nu/nu and nu/+ mice.

Lung On day 14 after i. v. challenge, interstitial pneumonia, distributed throughout the lungs, was the major histopathologic feature observed in nu/nu mice (Fig. 1A). Morphologically, alveolar septa of nu/nu mice were congested with polymorphonuclear leukocytes (PMNs), M4s, lymphocytes, encapsulated yeasts, and some fibrin (Fig. 1A); inflammatory cells were also observed ringing yeast cells (Fig. 1A1). By day 21, focal areas of bronchopneumonia (Fig. 1B) with patchy interstitial inflammation predominated in nulnu mice and inflammatory cells were primarily PMNs and a few Ms. In contrast to the lung pathology in nulnu mice, nul+ mice (day 14) had several discrete inflammatory foci that contained encapsulated and/or nonencapsulated yeasts

Cryptococcosis surrounded by PMNs, Ms, lymphocytes, fibrin, and collagen (Fig. 1C). By day 21, lesions in nu!+ mice were primarily chronic inflammatory foci with fewer yeasts and increased mononuclear infiltrate and fibroblastic proliferation than in nu/nu mice (Fig. 1D). Alveolar M4s in these chronic foci contained ingested nonencapsulated yeast cells (Fig. LD). In general, inflammatory foci in nu/nu mice on day 21 contained more PMNs than Ms while primarily Ms were observed in nu/+ mice.

Brain Both nu/nu and nu/+ mice developed cryptococcal meningitis. Meningeal lesions in nu/nu mice contained numerous encapsulated organisms and little to no cellular infiltrate (days 14, 21; Fig. 1E). In contrast to nu/nu mice, meningeal lesions in nul+ mice contained encapsulated yeasts interspersed with PMNs and Ms (days 14, 21; Fig. lF). Both nulnu and nul+ mice also had large foci of encapsulated yeasts scattered throughout the cerebrum and cerebellum. No cellular infiltrate was observed in or around cryptococcal foci in nu/nu mice (days 14, 21; Fig. 10). Minimal cellular infiltrate consisting of PMNs, M4s, and occasional lymphocytes was observed surrounding cryptococcal foci in nu!+ mice (days 14, 21; Fig. lH).

Kidney The predominant histopathologic feature in the kidneys of nu/nu mice on day 14 was foci of encapsulated yeasts with no host inflammatory response (Fig. 2A). In contrast, lesions in nul+ mice on day 14 were primarily chronic inflammatory foci that contained encapsulated, nonencapsulated, and degraded yeasts interspersed with numerous Ms, proliferating fibroblasts, and early collagen deposition (Fig. 2B). By day 21, these chronic inflammatory foci in nu/+ mice had enlarged and collagen deposition was increased.

Liver Large foci of encapsulated yeasts were the predominant histopathologic feature in the liver of nu/nu mice on day 14. While some foci evoked no host inflammatory response (Fig. 2C), other foci were surrounded by a low grade chronic inflammatory response consisting of M4s and some fibroblasts (Fig. 2D). Abscesses that contained encapsulated and/or nonencapsulated yeast cells interspersed with numerous PMNs and cellular debris (Fig. 2E) were also observed, but less frequently than foci. While the types of lesions in the liver of nu/nu mice on day 21 were similar to that observed on day 14 (foci, abscesses, chronic inflammation), the number of yeast cells in all lesion types had increased and some cryptococcal foci still evoked no inflammatory response. Although increased inflammatory responses were also observed in

in Athymic



both abscesses and chronic inflammatory foci (Fig. 2F) of nu/nu mice on day 21, granuloma formation was not observed in the liver. In contrast to nu/nu mice, the lesions in the liver of nu/+ mice on day 14 had already progressed to either chronic inflammation or chronic granulomatous inflammation. When compared to nu/nu mice, chronic inflammatory foci in nu/+ were smaller, contained fewer yeast cells (both nonencapsulated and encapsulated), numerous Ms and lymphocytes, and extensive fibroblast proliferation and collagen deposition (Fig. 2G). Granulomas containing giant cells with ingested yeast cells were also observed in the liver of nu/+ mice on day 21 (Fig. 2H).

Spleen Microscopically, the spleen of nu/nu mice on days 14 and 21 contained either large foci of encapsulated yeasts with no cellular response (Fig. 21) or small foci of encapsulated and nonencapsulated yeasts surrounded by acute (PMN5) and chronic (Ms, fibroblasts, and collagen) inflammation (Fig. 2J). No lesions were observed in the spleen of nu/+ mice on either days 14 or 21.

Skin Macroscopically, nu/nu mice began developing skin nodules by day 14. Histopathology of skin nodules revealed large collections of encapsulated yeasts in the dermis (Fig. 2K) with a minimal inflammatory response consisting of M4s. Yeast cells were also observed in the stratum corneum of the skin. Conversely, skin lesions were not observed in nu/+ mice.

Pathogenesis nu/+ BALB/c

of C. neoformans Mice

for flu/flu


To determine whether differences in histopathology observed between nu/nu and nu/+ mice correlated with enhanced susceptibility, the number of yeasts in the internal organs of infected mice was quantitated. The numbers of C. neoformans in the internal organs of nu/nu mice i.v. challenged with 1 x l0 yeasts increased throughout the 21 day study (Fig. 3). Nude mice died 17-21 days after i.v. challenge. Similar increasing numbers of C. neoformans were cultured from the livers of both nu/nu and nu/+ mice on days 1 and 3 and from the kidneys and lungs of nu/nu and nu/+ mice on days 1, 3, and 7 after i.v. challenge. By day 7 in the liver or by day 14 in the kidneys and lungs, the numbers of C. neoformans in nu!+ mice had stabilized and were significantly lower (P C,)











Fig. 5. Mortality in flu/flu and nu/+ BALB/c mice i.v. challenged with C. neoformans.

mice developed cutaneous cryptococcosis coincident with progressive disseminated disease suggesting that C. neoformans may be dermatotropic in a T-cell deficient host. This association between cutaneous cryptococcosis and the absence of thymus-matured T cells has also been observed in bg/bg-nu/nu mice [32]. Investigators have demonstrated, however, that cutaneous cryptococcosis is not always linked to suppressed host immunity. Cutaneous cryptococcal lesions have been established in immunocompetent mice [12, 14] suggesting that some C. neoformans strains may possess a characteristic which is required for the expression of the dermatotropic phenotype. Results from our study confirm the association of the athymic state with increased susceptibility to cryptococcosis reported by other investigators [16,24,28]. The absence of thy mus-matured I cells shortened the lifespan of C. neoformans challenged (1 04) nu/nu mice by 34%. In general, differences in number of C. neoformans in the internal organs of i.v. challenged (10) nu/nu and nu/+ mice were not evident until day 7 after infection. Interestingly, nu/nu mice had higher numbers of C. neoformans in the spleen than nu/+ mice as early as day 3. These results were unexpected since T-cell responses to C. neoformans antigens are typically not detectable until 14 days after challenge with viable organisms [23]. The spleen is an organ rich in lymphoid cells, and it is likely that 1 cells can impact on the course of cryptococcal infection in the microenvironment of infected tissues before T-cell populations have expanded enough to be detected by either in vitro or in vivo measures of CMI responsiveness. Our study also yielded data that conflicted with other studies. Cauley and Murphy [6] reported that nu/nu mice were more resistant to C. neoformans on days 7 and 14 after intraperitoneal challenge than nu/+ mice. This early enhanced resistance to ciyptococcosis was attributed to either Ms [6], NK cells [18], or both since these

activities are enhanced in nu/nu mice [3,8]. While the lack of early resistance in nulnu mice in our study could be due to variations in experimental design, it could also be due to differences in the levels of M4 activation and/or NK-cell activity present in the nu/nu mice used in this study since they were originally GF and were maintained under “clean” conditions after challenge. Several investigators have shown that Ms from conventionallyreared nu/nu mice have increased bactericidal [9], tumoiicidal [25], or antiviral [29] activity compared to Ms from GF nu/nu mice. Additionally, in previous studies, we have shown that GF nu/nu mice have lower NK-cell activity than conventionally-reared nu/nu mice [3]. Gnotobiotic immunodeficient mice are excellent models in which to study pathogenesis and immunity to cryptococcosis. The lifespan of conventionally-reared immunodeficient animals is often shortened [1,20] and they are highly susceptible to chronic wasting disease, infection with viruses, and Pneumocystis carinii pneumonia [1,20,34]. This is important when one considers that infection with C. neoformans is immunosuppressive, both specifically for cryptococcal antigens [26] and nonspecifically for unrelated antigens [4]. The nonspecific immunosuppression associated with cryptococcal infection may increase the susceptibility of conventionally-reared immunodeficient mice to secondary infections which could alter both mortality and disease expression. Based on culture data and histopathology, the mice used in this study remained free of secondary bacterial infections. Thus, our data represent the host’s response to infection with a pure culture of C. neoformans.

ACKNOWLEDGMENTS We thank Donna Brackett for typing this manuscript. We are also grateful to JoAnne Croft and her staff at the University of Wisconsin Gnotobiotic Laboratory for supplying the animals used in this study. We also thank Terry Oberley of the Department of Pathology for reviewing histopathology slides and results. These studies were supported by the National Institutes of Health Grant CA-36695.

REFERENCES 1. Azar, HA., Hansen, CT., and Costa, J. N:NIH(S)LI-nu/nu mice with combined immunodeficiency: A new model for human tumor transplantation. J. Nail. Cancer Inst. 65,421, 1980. 2. Balish, E., Filutowicz, H., and Oberley, TO. Correlates of cell-mediated immunity in Candida albicans-colonized gnotobiotic mice. Infect. Immun. 58,107, 1990. 3. Bartizal, K.F., Salkowski, C.A., Pleasants, J.R., and Balish, E. The effect of microbial flora, diet and age on the tumoricidal activity of natural killer cells. J. Leukoc. Biol. 36,739, 1984. 4. Blackstock, R, and Hall, N.K. Non-specific immunosuppression by Cryptococcus neoformans infection. Mycopathologia 86,35, 1984.

Cryptococcosis 5. Borten, L.K., and Wintroub, B.U. Disseminated cryptococcosis presenting as herpetiform lesions in a homosexual man with acquired immunodeficiency syndrome. J. Am. Acad. Dermatol. 10,387, 1984. 6. Cauley, L.K., and Murphy, J.W. Response of congenitally athymic (nude) and phenotypically normal mice to Cryptococcus neoformans infection. Infect. Immun. 23,644, 1979. 7. Chandler, F.W. Pathology of the mycoses in patients with the acquired immunodeficiency syndrome (AIDS). In Current Topics in Medical Mycology, (McGinnis, MR., Ed.). New York: Springer-Verlag, Vol. 1, p 1, 1985. 8. Cheers, C., and Wailer, R. Activated macrophages in congenitally athymic “nude” mice and in lethally irradiated mice. J. Immunol. 115,884, 1975. 9. Czuprynski, C.J., and Brown, J.F. Phagocytes from flora-defined and germfree athymic nude mice do not demonstrate enhanced antibacterial activity. Infect. Immun. 50,425, 1985. 10. Diamond, R.O., May, i.E., Kane, M.A., Frank, M.M., and Bennett, J.E. The role of the classical and alternate complement pathway in host defenses against Cryptococcus neoformans infection. J. Immunol. 112,2260, 1974. 11. Diamond, R.O., Root, R.K., and Bennett, J.E. Factors influencing killing of Cryptococcus neoformans by human leukocytes in vitro. I. Infect. Dis. 123,367, 1972. 12.

Dixon, D.M., and Polak, A. In vivo and in vitro studies atypical, rhinotropic isolate of Cryplococcus neoformans. pathologia 96,33, 1986.

with an Myco-

13. Flesch, I.E., Schwamberger, G., and Kaufmann, S.H.E. Fungicidal activity of IFN-y-activated macrophages. Extracellular killing of Cryptococcus neoformans. J. Immunol. 142,3219, 1989. 14. Fromtling, R.A., Abruzzo, G.K., and Ruiz, A. Cryptococcus neoformans: A central nervous system isolate from an AIDS patient that is rhinotropic in a normal mouse model. Mycopathologia 102,79, 1988. 15. Granger, DL., Perfect, J.R., and Durack, D.T. Macrophagemediated fungistasis in vitro: Requirements for intracellular and extracellular cytotoxicity. J. Immunol. 136,672, 1986. 16. Graybill, J.R., and Drutz, D.J. Host defenses in cryptococcosis. II. Cryptococcosis in the nude mouse. Cell. Immuno!. 40,263, 1978. 17. Graybill, J.R., and Mitchell, L. Host defense in cryptococcosis. 111./n vivo alteration in immunity. Mycopathologia 69,171, 1979. 18. Hidore, M.R., and Murphy, J.W. Correlation of natural killer cell activity and clearance of Cryptococcus neofor,nans from mice after adoptive transfer of splenic nylon wool-nonadherent cells. Infect. Immun. 51,547, 1986. 19. lacobellis, F.W. Primary cutaneous cryptococcosis. Arch. Dermatol. 115,984, 1979. 20.

Karre, K., Klein, G.O., Kiessling, R., Klein, G., and Roder, J.C. In vitro NK-activity and in vivo resistance to leukemia: Studies of
















in Athymic



beige, beige/nude and wild-type hosts of C57BL background. Int. J. Cancer 26,789, 1980. Levitz, S.M., and DiBenedetto, D.J. Differential stimulation of murine resident peritoneal cells by selectively opsonized encapsulated and acapsular Cryptococcus neoformans. Infect. Immun. 56,2544, 1988. Lim, T.S., and Murphy, J.W. Transfer of immunity to cryptococcosis by 1-enriched splenic lymphocytes from Cryptococcus neoformans-sensitized mice. Infect. Immun. 30,5, 1980. Lim, T.S., Murphy, J.W., and Cauley, L.K. Host-etiological agent interactions in intranasally and intraperitoneally-induced cryptococcosis in mice. Infect. Immun. 29,633, 1980. Marquis, G., Montplaisir, S., Pelletier, M., Mousseau, S., and Auger, P. Genetic resistance to murine cryptococcosis: Increased susceptibility in CBA/N xid mutant strain of mice. Infect. Immun. 47,282, 1985. Meltzer, M.S. Tumoricidal responses in vitro of peritoneal macrophages from conventionally housed and germ-free nude mice. Cell. Immunol. 22,176, 1976. Murphy, J.W., and Cox, R.A. Induction of antigen-specific suppression by circulating Cryptococcus neoformans antigen. Clin. Exp. Immunol. 73,174, 1988. Murphy, J.W., and McDaniel, DO. In vitro reactivity of natural killer (NK) cells against Crvprococcus neoformans. J. Immunol. 28,1577, 1982. Nishimura, K., and Miyaji, M. Histopathological studies on experimental cryptococcosis in nude mice. Mycopathologia 68, 145, 1979. Rama Rao, G., Rawls, WE., Perey, DYE., and Tompkins, W.A.F. Macrophage activation in congenitally athymic mice raised under conventional or germ-free conditions. J. Reticuloendothel. Soc. 21,13, 1977. Ray, T.L. Fungal infections in the immunocompromised host: Symposium on Cutaneous Signs of Systemic Disease. Med. Clin. North Am. 64,955, 1980. Rico, M.J., and Pennys, N.S. Cutaneous cryptococcosis resembling molluscum contagiosum in a patient with AIDS. Arch. Dermatol. 121,901, 1985. Salkowski, C.A., and Balish, E. Pathogenesis of Cryptococcus neoformans in congenitally immunodeficient beige athymic mice. Infect. Immun. 58, 3300, l990. Schupbach, C.W., Wheeler, CE., Briggaman, R.A., Warner, N.A., Kanof, E.P. Cutaneous manifestations of disseminated cryptococcosis. Arch. Dermatol. 112,1734, 1976. Walzer, P.D., Kim, C.K., Linke, Mi., Pogue, C.L., Huerkamp, Mi., Chrisp, CE., Lerro, A.V., Wixson, S.K., Hall, E., and Shultz, L.D. Outbreaks of Pneumocvstis carinii pneumonia in colonies of immunodeficient mice. Infect. Immun. 57,62, 1989. West, M.A. Role of cytokines in leukocyte activation: Phagocytic cells. In Current Topics in Membrane Transport (Kleinzeller, A., Ed.). San Diego: Academic Press. Vol. 35. p. 537, 1990.

Inflammatory responses to cryptococcosis in congenitally athymic mice.

Histopathology revealed that nu/nu mice developed both acute and chronic inflammatory responses following infection with Cryptococcus neoformans. In c...
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