American Jou-nal of Pathology, Vol. 141, No. 6, December 1992 Copyright X) American Association of Patbologist

Animal Model of Human Disease Pulmonary Cryptococcosis

Gary B. Huffnagle and Mary F. Lipscomb From the Department ofPathology, University of Texas Southwestern Medical Center, Dallas, Texas

Infection by the encapsulated yeast Cryptococcus neoformans is one of the most frequent fungal infections occurring in acquired immune deficiency syndrome (AIDS) patients.12 In addition to AIDS, cryptococcal infections frequently are seen in a number of other conditions that impair immunity such as lymphoreticular malignancies, sarcoidosis, chronic corticosteroid therapy, Cushing's syndrome, and alchoholism.3 The incidence of cryptococcal infections among apparently immunocompetent patients is extremely low, thereby illustrating the opportunistic nature of this organism. T-cell.-mediated immunity is the most important component of host defenses against C. neoformans infections, most likely through T-cell-mediated fungicidal activation of macrophages.4 6The significant incidence of cryptococcosis in AIDS patients demonstrates the protective role of CD4 T cells in human anti-cryptococcal immunity.1 2 7-9 Results from animal studies also suggest that CD8 T cells may have a protective role against the disease in humans.8,9 Cryptococcosis generally manifests as meningitis, but the respiratory tract is the portal of entry for the organism.3 The incidence of pulmonary cryptococcosis is most likely underestimated in the literature because the meningitis can be fatal if not treated, whereas the pneumonia is rarely debilitating and is usually self-limiting.1011 Studies have shown, however, that C. neoformans readily disseminates from the lungs to the central nervous system in immunodeficient patients, whereas the infection often remains localized in the lungs of immunocompetent patients and resolves.12'13 The localization of the infection in the lungs of immunocompetent individuals raises a question as to whether an initial pulmonary cryptococcal infection that resolves but in which a small number of residual organisms are present could be reactivated by an immunodeficiency.

Animal Model Mice provide an excellent model in which to study the pathogenesis of C. neoformans because of the availability of numerous inbred strains and the large number of immunologic reagents. Many of the murine cryptococcal infection models reported in the literature, however, are descriptions of the pathogenesis of a disease that is ultimately fatal even in immunocompetent mice. Mice were usually infected by a nonrespiratory route (intraperitoneal, intravenous, or subcutaneous) with extremely large inoculating doses (usually >105 organisms). Our laboratory has described a murine cryptococcal infection model that more closely mimics the human disease: intratracheal infection of BALB/c or C.B-1 7 mice with C. neoformans strain 52D.

Biologic Features Intratracheal inoculation of BALB/c or C.B-17 mice with C. neoformans 52D results in a pulmonary infection that initially grows progressively but then resolves after the development of T-cel-mediated immunity.14 If the infected mice are rendered immunodeficient by depletion of their T cells using monoclonal antibody treatments, the progressive pulmonary infection disseminates systemically rather than resolving.89 By manipulating the immune response of the infected mice through T cell depletion at different points during the infection, the pathogenesis of the pulmonary disease in immunocompetent mice can be tentatively divided into four phases (Figure 1): Publication sponsored by the Registry of Comparative Pathology, Armed Forces Institute of Pathology and supported by Grant RR 00301 from the National Center for Research Resources, NIH, under the auspices of Universities Associated for Research and Education in Pathology, Inc., and by Hazelton Laboratories America, Inc. Address reprint requests to Dr. Mary F. Lipscomb, Department of Pathology, University of Texas Southwestern Medical Center, Dallas, TX 75235-9072.

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4) CD4 T-cel[-dependent final clearance (after week

10) 1-6

Macroscopically, nodules can be observed in the lungs of the immunocompetent mice from about 2 to 4 weeks (Figure 2A). Microscopic examination shows that

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42 M 1 54 55 Days After Infection Figure 1. Pulmonary clearance in normal and T-cell depleted Balbic and CB-17 mice after intratracheal inoculation with C. neoformans 52D. The dark line (--) represents the time course of pulmonary clearance in normal mice. The regions are referred to in the text. The T cell depletions shown were initiated before the infection and the pulmonary clearance followedfor 35 days after the start of T-cell depletion. Regions three and four have been arbitranly demarcated based on preliminary results from T-cell depletions beginning 28 or 66 days post-infection (data not *

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shown) Groups -0- nonal, -0- CD4 depleted, -A- CD8 depleted, -C- CD4 and CD8 double depleted. *P < .01 compared with the "normal" infected group.

1) T-cellindependent initial, rapid increase in yeast (week 1) 2) T-cel-dependent progressive pulmonary clearance (weeks 2 through 4) 3) T-cell-independent stasis of infection (weeks 5 through 10)

these nodules consist of granulomatous inflammation (Figure 2B). The nodules are histologically identical to cryptococcomas seen in humans and contain large foamy macrophages, multinucleated giant cells, lymphocytes, and some neutrophils.3 Initially, the lesions also contain numerous cryptococci, but as the immune response continues, the cryptococci are eliminated from the granulomatous lesions. At the peak of the cellular influx (2 weeks after infection), approximately half to three quarters of alveoli are filled with inflammatory cells. As a result of the infection, perhaps from signals generated by resident pulmonary cells (alveolar macrophages, endothelial cells, or epithelial cells), both CD4 and CD8 T cells are recruited into the lungs.8'9 These recruited T cells then mediate the influx of inflammatory cells from the circulation into the airways while at the same time activating the infiltrating phagocytes.9 These activated phagocytes are most likely the cells responsible for eliminating the cryptococci from the airways.4 6 After 4 weeks, a chronic low-level infection remains in the lungs. Preliminary results indicate that induction of T cell deficiency during this phase of the infection does not

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Figure 2. Left: Left upper lobe from a 21-daly C. tneofotr-mnis 52D inqfected Bal3alh/c luIZg. The light circular areas are nodular-lesionJsl

fbrmed in re.sponse to the crmptococcal infrction (two noduzles ar-e indicated by the an-'ous). Right: Photomicrograph of the infammatot-v influx in the lungs (fC C. ineoformaiis 52D infected Balbc/ mice. Note the cgl2 8oli ar-e filled with lairge Jbanq inacropbages, nuIlt iucleated g ian t cells and cnrptoc cci and the alveolear septae are intact (the lesions gene(rally heal without residual scaniing). Tbe edge of a perivescular large/lv l/wnphocvtic infiltracte can-i be seen in the uippetr right cor-ner qf the micn1rograph. (IC-I&E x300)

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reactivate the infection (data not shown). Additional preliminary studies suggest that after 10 weeks, the low-level infection begins to be cleared to below detectable levels through a CD4 T-cell-dependent process (data not shown).

Comparison with Human Disease The pulmonary cryptococcosis that occurs in BALB/c and C.B-17 mice after an intratracheal inoculation of C. neoformans 52D appears very similar to human pulmonary cryptococcosis. The organism is opportunistic, with host defenses dependent on T-cell-mediated immunity. Control of dissemination from the lungs to the CNS appears to be CD4 T cell mediated,79 consistent with the high incidence of cryptococcal meningitis seen in AIDS patients.1' 2 Macroscopically and microscopically, the lesions appearing in the lungs of infected mice are identical to human cryptococcomas.3

Usefulness of This Model Although a considerable amount is known about the immune response to C. neoformans, the exact mechanism of how the immune system orchestrates the protective response is still unknown. One intriguing issue has always been that although the organism enters the body by inhalation, why does the infection usually manifest as meningitis rather than pneumonia? The recent findings that CD8 T cells in addition to CD4 T cells may be involved in the protective response in the lungs whereas CD4 T cells seem predominantly responsible for extrapulmonary defenses may shed light on this issue. The model can be used to address the question of whether cryptococcal meningitis can result from reactivation of an infection held in check at a low level by the immune system (similar to many other infections seen in AIDS patients) or is always a newly acquired infection. More experiments at later stages of the disease will be required to answer this question. The model also promises to provide important information on how T cells are recruited and what cytokines they produce to initiate phagocytic influx and activation in any pulmonary opportunistic infection. In terms of exploring therapy in human disease, the model offers opportunities to study not only new fungal antibiotics but the use of systemic versus intrapulmonary cytokine administration to facilitate clearance of an

opportunistic infectious agent in the lungs of T-cellsuppressed hosts.

Availability BALB/c mice are available from a number of vendors. The C.B-17 mice (BALB/c congenic for C57BL/6 IgH) used in our studies were from the breeding facilities of the animal resource center, University of Texas Southwestern Medical Center at Dallas, but C.B-1 7 mice are now available through a vendor (Taconic Labs, Philadelphia, PA). The 52D strain of C. neoformans was purchased from the American Type Culture Collection (24067; Rockville, MD). Other investigators have reported results similar to ours after intratracheal or intranasal inoculation of BALB/c mice with C. neoformans strain 1 84A.815

References 1. Kovacs JA, Kovacs AA, Polis M, Wright WC, Gill VJ, Tuazon CU, Gelman EP, Lane HC, Longfield R, Overturf G, Macher AM, Fauci AS, Parrillo J, Bennett JE, Masur H: Cryptococcosis in the acquired immunodeficiency syndrome. Ann Intern Med 1985, 103:533-538 2. Eng RHK, Bishburg E, Smith SM, Kapila R: Cryptococcal infections in patients with the acquired immune deficiency syndrome. Am J Med 1986, 81:19-23 3. Diamond RD: Cryptococcus neoformans, Principles and Practice of Infectious Diseases, 3rd edition. Edited by GL Mandell, RG Douglas, Jr, JE Bennett. New York, Churchill Livingstone, 1990, pp 1980-1989 4. Murphy JW: Cryptococcosis, Immunology of the Fungal Diseases. Edited by RA Cox. Boca Raton, FL, CRC Press, 1989, pp 93-138 5. Granger DL, Perfect JR, Durack DT: Macrophage-mediated fungistasis in vitro: Requirements for intracellular and extracellular cytoxicity. J Immunol 1986,136:672-680 6. Mody CH, Tyler CL, Sitrin RG, Jackson C, Toews GB: Interferon-gamma activates rat alveolar macrophages for anticryptococcal activity. Am J Respir Cell Mol Biol 1991, 5:19-

26 7. Mody CH, Lipscomb MF, Street NE, Toews GB: Depletion of CD4 + (L3T4 +) lymphocytes in vivo impairs murine host defense to Cryptococcus neoformans. J Immunol 1990, 144: 1472-1 477 8. Hill JO, Harmsen AG: Intrapulmonary growth and dissemination of an avirulent strain of Cryptococcus neoformans in mice depleted of CD4+ and CD8+ T cells. J Exp Med

1991, 173:755-758 9. Huffnagle GB, Yates JL, Lipscomb MF: Immunity to a pulmonary Cryptococcus neoformans infection requires both CD4 + and CD8 + T cells. J Exp Med 1991, 173:793-800

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10. Balmes JR, Hawkins JG: Pulmonary cryptococcosis. Semin Respir Med 1987, 9:180-186 11. Cameron ML, Bartlett JA, Gallis HA, Waskin HA: Manifestations of pulmonary cryptococcosis in patients with acquired immunodeficiency syndrome. Rev Infect Dis 1991, 13:6467 12. Kerkering TM, Duma RJ, Shadomy S: The evolution of pulmonary cryptococcosis: Clinical implications from a study of 41 patients with and without compromising host factors. Ann Intern Med 1981, 94:611-616

13. Wasser L, Talavera W: Pulmonary cryptococcosis in AIDS. Chest 1987, 92:692-695 14. Huffnagle GB, Yates JL, Lipscomb MF: T cell-mediated immunity in the lung: A Cryptococcus neoformans pulmonary infection model using SCID and athymic nude mice. Infect Immun 1991, 59:1423-1433 15. Lim TS, Murphy JW, Cauley LK: Host-etiological agent interactions in intranasally and intraperitoneally induced cryptococcosis in mice. Infect Immun 1980, 29:633-641

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Pulmonary cryptococcosis.

American Jou-nal of Pathology, Vol. 141, No. 6, December 1992 Copyright X) American Association of Patbologist Animal Model of Human Disease Pulmonar...
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