Cryptococcus neoformans fails to induce nitric oxide synthase in primed murine macrophage-like cells

Nitric oxide (NO) is a microbiostatic gas generated by activated murine macrophages. Cytokine signals, gamma interferon (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha) act synergistically to induce production of a macrophage nitric oxide synthase (NOS). A variety of intracellular pathogens, when recognized by macrophages primed with IFN-gamma, induce NOS by eliciting TNF-alpha secretion, which then functions as a positive autocrine signal. In cell culture assays, a murine macrophage cell line (J774), primed with IFN-gamma, was tested for NOS induction upon challenge with virulent Cryptococcus neoformans. C. neoformans failed to induce macrophage NOS as measured by nitrite production. This was true irrespective of the C. neoformans-to-J774 ratio. Other nonpathogenic Cryptococcus species likewise failed to induce NOS, yet Saccharomyces cerevisiae, Histoplasma capsulatum, and Candida albicans were efficient inducers of NOS. Conditions which promoted attachment and/or phagocytosis of C. neoformans did not lead to NOS induction (including opsonization with specific antibodies against C. neoformans). Assays for transcriptional repressors of NOS were negative. Tests for consumption of nitrite by measurement of additional products of NOS induction were negative. No TNF-alpha was detected by enzyme-linked immunosorbent assay in supernatants from C. neoformans-J774 cocultures. A mutant C. neoformans strain with a minimal, but visible, polysaccharide capsule also failed to induce NOS; however, several nonencapsulated mutants of C. neoformans did induce NOS. Failure of C. neoformans to act as an inducer of NOS may be related to the virulence of this pathogen in mice; C. neoformans is a unique example of a facultative intracellular pathogen which fails to induce NOS in primed macrophages. The mechanism appears to involve the failure of TNF-alpha secretion once the macrophage comes in contact with the fungus. The presence of the polysaccharide capsule appears to mask the signal necessary for TNF-alpha secretion and, ultimately, NOS induction.