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)-gamma and increased tumor necrosis factor (TNF)-alpha. 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-alpha 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.
HIS-62 is a glycoprotein that has been isolated from the cell wall and cell membrane fraction of the pathogenic fungus Histoplasma capsulatum. It is a target of the cellular immune response to this fungus, and it protects mice against a lethal intravenous inoculum of H. capsulatum yeast cells. In this study, we cloned the gene encoding this antigen to reveal its biological nature and studied the immunological activity of recombinant antigen. The amino acid sequences of the NH 2 terminus and internal peptides were obtained by Edman degradation. Degenerate oligonucleotides were used to isolate a gene fragment of HIS-62 by PCR. One 680-bp segment that corresponded to the known peptide sequence was amplified from H. capsulatum DNA. This DNA was used to screen a genomic library, and the full-length gene was isolated and sequenced. The deduced amino acid sequence of the gene demonstrated ϳ70 and ϳ50% identity to heat shock protein 60 (hsp 60) from Saccharomyces cerevisiae and hsp 60 from Escherichia coli, respectively. A cDNA was synthesized by reverse transcription PCR and was expressed in E. coli. Recombinant protein reacted with a monospecific polyclonal rabbit antiserum raised against native HIS-62, with monoclonal HIS-62-reactive T cells, and with splenocytes from mice immunized with viable yeast cells. Moreover, vaccination with the recombinant protein conferred protection in mice against a lethal intranasal inoculation with yeast cells. Thus, HIS-62 is a member of the hsp 60 family, and the recombinant hsp 60 is protective against pulmonary histoplasmosis in mice. MATERIALS AND METHODS Animals. BALB/c (H-2 d) mice were purchased from Jackson Laboratory, Bar Harbor, Maine. Female New Zealand White rabbits were purchased from Myrtle Rabbitry, Thompson's Station, Tenn. Antigen. HIS-62 was isolated from CW/M as described previously (10). Antibodies. To prepare anti-HIS-62 antibody, a rabbit was initially phlebotomized to obtain preimmune control serum. The rabbit was then given an intradermal injection of 50 g of HIS-62 emulsified in Titermax (Cyt-Rx Corp., Norcross, Ga.). Four weeks later, the immunization was repeated in the absence of adjuvant. Serum was collected and tested for reactivity against HIS-62. Monospecific antibody was prepared as described by Smith and Fisher (25); 50 g of HIS-62 was electrophoresed in a sodium dodecyl sulfate-10% polyacrylamide gel electrophoresis (SDS-PAGE) gel and electroblotted onto a nitrocellulose membrane. The membrane was stained with Ponceau S, and the band corresponding to HIS-62 was excised. Subsequently, the nitrocellulose strip was blocked with 5% dried milk in Tris-buffered saline (pH 7.4), air dried, and then incubated with the rabbit polyclonal serum for 24 h at 4ЊC. The strip was washed three times with 0.1% Tween 20 in Tris-buffered saline, and the bound antibody was eluted with 2 ml of a 200 mM glycine buffer, pH 3.0. Eluted antibody was neutralized with 0.1 volume of 1 M Tris, pH 7.4. Anti-GroEL antibody was a kind gift of Roger Hendrix, University of Pittsburgh. ...
The concerted action of several cytokines is necessary for resolution of both primary and secondary infection with Histoplasma capsulatum. Among the soluble factors that contribute to tissue sterilization, TNF-α stands as a central mediator of protective immunity to this fungus. In this study, we explored the regulation of protective immunity by TNFR1 and -2. In primary pulmonary infection, both TNFR1−/− and -2−/− mice manifested a high mortality after infection with H. capsulatum, although TNFR1−/− mice were more susceptible than TNFR2 −/− mice. Overwhelming infection in the former was associated with a pronounced decrement in the number of inflammatory cells in the lungs and elevated IFN-γ and TNF-α levels in the lungs. In contrast, IFN-γ levels were markedly decreased in TNFR2−/− mice, and treatment with this cytokine restored protective immunity. Lung macrophages from both groups of knockout mice released substantial amounts of NO. Upon secondary infection, TNFR2−/− mice survived rechallenge and cleared infection as efficiently as C57BL/6 animals. In contrast, mice given mAb to TNFR1 succumbed to reexposure, and the high mortality was accompanied by a significant increase in fungal burden in the lungs. Both IL-4 and IL-10 were elevated in the lungs of these mice. The results demonstrate the pivotal influence of TNFR1 and -2 in controlling primary infection and highlight the differences between these receptors for regulation reexposure histoplasmosis.
T cells are essential for controlling infection with
We examined the immunobiological responses to Histoplasma capsulatum in lungs of gamma interferon (IFN-␥) knockout mice (GKO mice). Naive GKO mice succumbed by day 9 to intranasal challenge with 2.5 ؋ 10 6 yeasts, whereas all wild-type (WT) mice survived for 45 days. Compared to lungs of WT mice, the lungs of acutely infected GKO mice exhibited dramatically elevated numbers of CFU in lungs and significantly higher levels of tumor necrosis factor alpha (TNF-␣) and granulocyte-macrophage colony-stimulating factor (GM-CSF) but not interleukin-12 (IL-12) or IL-4. To determine if IFN-␥ is necessary in reexposure histoplasmosis, GKO and WT mice were inoculated with 10 4 yeasts intranasally and given amphotericin B for 3 weeks. Six weeks later, mice were rechallenged with 2.5 ؋ 10 6 yeasts. All GKO mice died by day 6, whereas all WT mice survived for 45 days. Lungs of GKO mice contained substantially elevated numbers of CFU and higher TNF-␣ and GM-CSF levels but not IL-12 or IL-4. Thus, IFN-␥ is requisite for control of pulmonary histoplasmosis in naive and reexposed mice.
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