Modulation of Immune Responses in Murine Pulmonary Histoplasmosis

Ruth Allendoerfer 0 1 2 Gregory P. Boivin 0 1 2 George S. Deepe 0 1 2 Jr. 0 1 2 0 The Journal of Infectious Diseases 1997;175:905-14 q 1997 by The University of Chicago. All rights reserved. 0022-1899/97/7504-0024$01.00 1 Received 16 September 1996; revised 31 October 1996. Presented in part: 96th general meeting, American Society for Microbiology , New Orleans, May 1996 (abstract F 106). All animal experiments were done in accordance with NIH Animal Welfare Act guidelines. Grant support: NIH (AI-23017). Diseases, Dept. of Medicine, University of Cincinnati College of Medicine , P.O. Box 670560, Cincinnati, OH 45267-0560 2 Division of Infectious Diseases, University of Cincinnati College of Medicine, and Research Service, VA Medical Center , Cincinnati, Ohio The influence of endogenous interleukin (IL)-12 on the course of pulmonary histoplasmosis was examined in naive and immune mice. All naive animals pretreated with anti - IL-12 monoclonal antibody (MAb) died by day 14. All mice died when anti - IL-12 MAb was initiated as late as postinfection day 3. Unlike those of controls, lungs of naive mice given anti - IL-12 MAb had depressed levels of interferon (IFN)-g and increased tumor necrosis factor (TNF)-a. The 2 groups had similar IL-4 levels. Administration of anti - IL-4 MAb rescued mice from the inimical effects of anti - IL-12 MAb. Survival of mice given both anti - IL-12 and anti - IL-4 MAb was associated with a blunted TNF-a response. In reinfection histoplasmosis, treatment with anti - IL-12 MAb did not alter survival. Fungus burden in lungs, livers, and spleens differed at week 2, but not at week 1, of infection. Thus, endogenous IL-12 is critical for optimal generation of a protective immune response in pulmonary histoplasmosis. - Histoplasma capsulatum is a dimorphic fungus that causes a self-limited infection in immunocompetent hosts [1]. In contrast, in individuals with an impaired immune system, infection with the fungus is often progressive [2]. Although macrophages are the principal effector cells in infection with H. capsulatum [3], there is ample evidence that both CD4 and CD8 T cells play important roles in host resistance to this fungus. Depletion of CD4 cells increases the severity of infection in a murine model and results in unrestricted growth of the fungus, leading to death of animals. b2-microglobulin knockout mice that lack CD8 cells and class I major histocompatibility complex molecules have impaired clearance of the organism [4, 5]. Thus, resolution of infection is dependent upon the coordinated responses between both T cell subsets and macrophages [6, 7]. One principal mechanism by which immunocompetent cells contribute to clearance of H. capsulatum is by release of cytokines that arm macrophages to inhibit intracellular replication. Three cytokines have been implicated in the protective immune response to H. capsulatum: interferon (IFN)-g, tumor necrosis factor (TNF)-a, and interleukin (IL)-12. IFN-g production is one signal for murine macrophage activation and has been implicated in the growth restriction of the fungus [7, 8]. Several studies have indicated that treatment with either polyclonal or monoclonal anti TNF-a antibodies alters host susceptibility to infection with H. capsulatum [9, 10] (Allendoerfer R, unpublished data). A recent study found that recombinant IL-12 promoted the generation of a protective immune response and that the effect of this cytokine is mediated through the induction of IFN-g [10]. However, most studies have used intravenous models of disseminated histoplasmosis, and the influence of these cytokines in pulmonary infection has not been established. IL-12 plays an important role in the immune response. It is produced primarily by macrophages and is thought to be a principal regulator of T cell differentiation and function in vivo [11]. It induces the production of IFN-g from T cells and NK cells and enhances their proliferation [11, 12]. Moreover, IL12 increases the cytolytic activity of NK and CD8 T cells [13, 14]. The outcome of infection with intracellular pathogens, such as Leishmania major and Toxoplasma gondii, which depends on a relative Th1 response, can be altered by treatment with IL-12 or neutralizing antibodies to IL-12 [15 18]. We describe a series of studies that examined the influence of endogenous IL-12 on the protective immune response in murine pulmonary histoplasmosis in naive and immune animals. Materials and Methods Animals. Male C57BL/6 mice were purchased from Jackson Laboratories (Bar Harbor, ME). Preparation of H. capsulatum and infection of mice. H. capsulatum cells were grown at 377C in Hams F12 medium supplemented with glucose (18.2 g/L), glutamic acid (1 g/L), HEPES (6 g/L), and cysteine (8.4 mg/L) for 36 h. Yeast cells were washed three times, suspended in balanced salt solution, and counted. To produce a sublethal infection in naive mice, mice were infected intranasally with 2.5 1 106 H. capsulatum cells in a 50-mL volume. Mice were immunized by intranasal inoculation of 104 yeast cells. Immune and age-matched controls were challenged with 2.5 1 106 yeast cells 6 8 weeks later. Antibodies. The C15.1 and C15.6 cell lines that produce anti IL-12 monoclonal antibody (MAb; rat IgG1) were provided by G. Trinchieri (Wistar Institute, Philadelphia). Cells were inoculated into serum-free Opti-MEM (GIBCO BRL, Gaithersburg, MD) containing 10 mg/mL gentamicin, 5 1 1005 M 2-mercaptoethanol, and 1% Nutridoma NS (Boehringer Mannheim Biochemicals, Indianapolis). Cells were grown for 6 days in 5% CO2 . Supernatants were harvested by centrifugation, concentrated by ultrafiltration through a YM 10 membrane (Amicon, Beverly, MA) to 0.01 of the original volume, filter-sterilized, and stored at 0207C until used. The concentration of rat IgG in supernatants was assessed by ELISA and calculated by linear regression from a rat IgG (Organon Teknika, Durham, NC) standard curve. All antibodies were assayed for possible endotoxin contamination by limulus amebocyte lysate test (BioWhittaker, Walkersville, MD); all antibody preparations contained 5 ng/mL endotoxin. Rat anti-mouse IL-4 (11B11) MAb was provided by the Biological Response Modifiers Program (National Cancer Institute, Frederick, MD). Treatment of mice with anticytokine MAb. Mice were injected intraperitoneally with 500 mg of anti IL-12 MAb (C15.1 and C15.6; 250 mg each) on days 02, 1, 3, and 5 of infection and every week thereafter. In studies with anti IL-4 MAb, mice received one of the following regimens. One group received 500 mg of anti IL-12 MAb intraperitoneally on days 02, 1, 3, 5, and 7 plus 1 mg of rat IgG on days 02 and 7. The second group received 1 mg of anti IL-4 MAb and 500 mg of anti IL-12 MAb intraperitoneally on day 02 of infection followed by anti IL-12 MAb on days 1, 3, 5, and 7 of infection. On day 7, mice were given 1 mg of anti IL-4 MAb. Each week thereafter, mice received 1 mg of anti IL-4 MAb and 500 (...truncated)