Attenuated Salmonella vaccine-induced suppression of murine spleen cell responses to mitogen is mediated by macrophage nitric oxide: quantitative aspects

Abstract

Previous reports from our laboratory have shown that 7 days after infection of C3HeB/FeJ mice with an attenuated strain of Salmonella typhimurium, there is profound suppression of responses to B- and T-cell mitogens and suppression of the capacity of spleen cells to mount a primary, in vitro plaque-forming-cell (PFC) response to sheep erythrocytes. Inhibition of the PFC response was shown to be mediated by nitric oxide (NO), as NG-monomethyl-L-arginine (NMMA) gave complete reversal of suppression. The experiments reported here examined the role of NO in suppression of the response to the mitogen concanavalin A (ConA). In contrast to the PFC system, it was found that addition of NMMA to ConA-stimulated immune spleen cells resulted in less than 20% reversal of suppression. However, addition to NMMA resulted in a 50% reversal of suppression in cocultures of immune and normal spleen cells at a ratio of 1:4. A complete restoration of ConA-induced responses was achieved in cocultures incubated in medium containing a reduced concentration of L-arginine plus 1.25 mM NMMA. Investigation of why NMMA alone was not 100% effective in reversing suppression showed that addition of ConA significantly augmented production of nitrite and gamma interferon (IFN-gamma) in cocultures containing immune cells. Addition of anti-IFN-gamma reduced nitrite levels in the cultures, although results with the combination of anti-IFN-gamma and NMMA were not significantly better than results with NMMA alone. These findings suggest that suppression in cultures stimulated with ConA is difficult to reverse completely with NMMA alone because of an overproduction of NO, which can be offset by either reducing the L-arginine concentration or blocking IFN-gamma. The quantitative relationship between nitrite levels and suppression in cocultures was examined. It was found that suppression did not correlate directly with the nitrite concentration but rather with the log10 of the nitrite concentration. Nitrite levels above 15 microM gave almost complete suppression, and levels between 1 and 10 microM gave a wide range of suppression. These results strongly support NO as the suppressor factor in Salmonella-induced immunosuppression of responses to ConA and, by inference, suppression of responses to mitogens induced by other microbes. The results show that involvement of NO cannot always be demonstrated by simple addition of NMMA to suppressed mitogen-stimulated spleen cell cultures.