Treatment of Paracoccidioides brasiliensis-Infected Mice with a Nitric Oxide Inhibitor Prevents the Failure of Cell-Mediated Immune Response Aname ´lia L. Bocca,* ‡ Emı ´lia E. Hayashi,* Artur G. Pinheiro,* Alessandra B. Furlanetto,* Ana P. Campanelli,* Fernando Q. Cunha, ² and Flore ˆncio Figueiredo* § The activation of the nitric oxide (NO) production system and its involvement in the control of the lung fungal burden and in immunosuppression mechanisms were studied during the course of Paracoccidioides brasiliensis-infected mice. Mice that had been infected with the fungus were treated daily with a specific inhibitor of NO synthesis, Nv-nitro-L-arginine, or with buffered saline (control); NO production was assessed on the basis of spontaneous NO 2 2 production by bronchoalveolar and peritoneal macro- phages (Mf) and of serum NO 3 2 levels. The infection coursed with an elevation of NO 3 2 levels. The Mf produced NO 2 2 and released TNF-a only after stimulation with LPS. In addition, the immunoproliferative responses of spleen cells that had been stimulated with the fungus Ag or with Con A were depressed. An examination of the lungs of infected animals showed a pro- gressive increase in the size of the lesions. Treatment of the animals, which resulted in an inhibition of NO 2 2 production by Mf and a reduction of serum NO 3 2 levels, caused the spontaneous release of TNF-a from infected animals and prevented the failure of the lymphoproliferative capacity of spleen cells. Furthermore, the treatment resulted in less pulmonary damage despite the fact that the lung fungal burden increased. It was also demonstrated that the NO donors S-nitroso-acetyl penicillamine and 3-mor- pholino-sydnonimine-hydrochloride were able to inhibit the growth of P. brasiliensis in vitro. These results suggest that although NO is important for the killing of the fungi, the activation of NO production in P. brasiliensis infection contributes to the occurrence of the immunosuppression observed during the course of the infection. The Journal of Immunology, 1998, 161: 3056 –3063. P aracoccidioidomycosis, which is caused by Paracoccid- ioides brasiliensis, is a deep mycosis of chronic evolution and of granulomatous pattern. The disease manifests as multiple forms, ranging from benign and localized to severe and disseminated forms depending upon the extent of the depression of cell immunity (1, 2). Among the control mechanisms that have been reported for this infection, macrophages (Mf) 3 appear to play a fundamental role, acting as the first line of defense for the organism. This function depends upon their state of activation, which permits them to per- form microbicidal activity (3). As has been extensively demonstrated for different infectious agents such as Leishmania major, Mycobacterium bovis, Toxo- plasma gondii, Schistosoma mansoni, and Trypanosoma cruzi, the microbicidal activity of Mf correlates with the production of nitric oxide (NO) or related nitrogen oxides by inducible NO synthase (iNOS); iNOS depends upon nicotinamide adenine dinucleotide phosphate and tetrahydrobiopterin as cofactors (4 –7). The induc- tion of this enzyme depends upon the synergism of Th1 cytokines, especially IFN-g and TNF-a, with the products released by the microorganisms, such as LPS (5– 8). In contrast, TGF-b and Th2- type cytokines, especially IL-4 and IL-10, inhibit nitric oxide syn- thase (NOS) induction (9 –11). The microbicidal activity of NO was also demonstrated in vivo in murine models whose resistance to certain microorganisms such as T. cruzi and L. major is corre- lated with the level of NO production (12–13). Furthermore, mouse strains that are resistant to L. major infection (14) or to Listeria monocytogenes infection (15) become susceptible to in- fection by these parasites when they are genetically deprived of iNOS (iNOS 2/2 ). Besides having host-protecting effects, NO is involved in the immunosuppression that is mediated by Mf. Recently, NO pro- duction was shown to be critical for murine Mf to suppress mi- togen- or Ag-stimulated T cell proliferation (16, 17) and also to mediate immune suppression in some strains of mice infected with T. brucei (18, 19), Plasmodium vinckei (20), and Salmonella ty- phimurium (21). Moreover, there is evidence that NO reduces the Ag-presenting ability of pulmonary dendritic cells (22) and inhib- its Ia expression in IFN-g-stimulated murine Mf (23). Recently, it was demonstrated that the in vivo inhibition of NO synthesis by treatment with L-arginine methyl ester leads to a reduction of T. brucei parasitemia in C3H/He mice, (19) despite the fact that NO inhibits trypanosome proliferation in vitro. The authors concluded that the decreased parasitemia in L-arginine methyl ester-treated mice may be a consequence of an inhibition of the immunosup- pressive effects of NO. Departments of *Pathology and ² Pharmacology, Faculty of Medicine of Ribeira ˜o Preto, Sa ˜o Paulo, Brazil; and Departments of ‡ Cell Biology, Instituto de Biologia, and § Pathology, Faculdade de Cie ˆncias da Sau ´de, Universidade de Brasilia, Brazil Received for publication January 30, 1998. Accepted for publication May 22, 1998. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This research was supported by Fundac ¸a ˜o de Amparo a ` Pesquisa do Estado de Sa ˜o Paulo (FAPESP); Conselho Nacional de Pesquisa (CNPq); and Fundac ¸a ˜o Apoio ao Ensino, Pesquisa e Assiste ˆncia do Hospital das Clı ´nicas da Faculdade de Medicina de Ribeira ˜o Preto da Universidade da Sa ˜o Paulo (FAEPA). 2 Address correspondence and reprint requests to Dr. Flore ˆncio Figueiredo, Depart- ment of Pathology, Faculdade de Cie ˆncias da Sau ´de, University of Brasilia, Brasilia, DF, Brazil, 70900-910. 3 Abbreviations used in this paper: Mf, macrophage(s); NO, nitric oxide; NOS, nitric oxide synthase; iNOS, inducible nitric oxide synthase; H&E, hematoxylin and eosin; SNAP, S-nitroso-acetyl penicillamine; SIN-1, 3-morpholino-sydnonimine-hydrochlo- ride, Nitro-Arg, Nv-nitro-L-arginine. Copyright © 1998 by The American Association of Immunologists 0022-1767/98/$02.00