In vivo blockage of nitric oxide with aminoguanidine inhibits immunosuppression induced by an attenuated strain of Salmonella typhimurium, potentiates Salmonella infection, and inhibits macrophage and polymorphonuclear leukocyte influx into the spleen

Abstract

Our laboratory has previously shown that after immunization with a strain of Salmonella typhimurium, SL3235, made avirulent by a blockage in the pathway of aromatic synthesis, murine splenocytes were profoundly suppressed in their capacity to mount an in vitro antibody plaque-forming cell (PFC) response to sheep erythrocytes. Evidence indicated that suppression was mediated by nitric oxide (NO), since the in vitro addition of NG-monomethyl-L-arginine blocked suppression. The present studies examined the effect of blocking NO production on Salmonella-induced immunosuppression by in vivo administration of aminoguanidine hemisulfate (AG). AG was administered to C3HeB/FeJ mice in their drinking water (2.5% solution) for 7 days prior to intraperitoneal inoculation with SL3235. AG treatment inhibited the increase in nitrate and nitrite levels in plasma and nitrite levels in the spleen seen in immunized mice. Importantly, AG treatment completely blocked suppression of the splenic PFC response and markedly attenuated the suppression of the response to concanavalin A in immunized mice, providing further evidence that Salmonella-induced immunosuppression is mediated by NO. AG treatment also alleviated the majority of the splenomegaly associated with SL3235 inoculation, which correlated with a blockage of influx of neutrophils and macrophages into spleens, as assessed by flow cytometry. AG treatment unexpectedly resulted in 90% mortality in mice injected with the highly attenuated vaccine strain of Salmonella, SL3235. Increased mortality in AG-treated mice correlated with inability to clear organisms from the spleen by day 15 postinoculation and with persistent bacteremia, compared with control mice. Collectively, these in vivo results underscore the dual biological consequences of NO production following Salmonella infection, with NO being necessary for host defense, but also having the potentially adverse effect of immunosuppression. A unifying hypothesis to explain how these seemingly paradoxical effects could both result from NO production is presented.

FIG. 1

Effect of AG treatment on nitrate and nitrite levels in plasma. Mice were treated for 7 days with AG in their drinking water prior to i.p. inoculation with saline (AG-saline) or SL3235 (AG-SL3235). AG-treated animals continued to receive AG in their drinking water until the time of sacrifice. Control groups received water without AG and were given SL3235 (H₂O-SL3235) or saline (H₂O-saline) i.p. At 5 days postinfection, plasma was obtained by cardiac puncture and nitrate and nitrite levels were assessed. Data are pooled from four experiments, except for the AG-saline group, where data are from two experiments. The total number of animals included in each group was 23 for H₂O-saline, 22 for H₂O-SL3235, 21 for AG-SL3235, and 11 for AG-saline. Horizontal lines indicate mean values. P ≤ 0.0001, AG-SL3235 versus H₂O-SL3235; not significant, AG-SL3235 versus H₂O-saline or AG-saline.

FIG. 2

Effect of in vivo AG treatment on nitrite production by spleen cells in vitro. The nitrite level in spleen cells was determined in mice treated for 7 days with AG in their drinking water prior to i.p. inoculation with SL3235 (AG-SL3235). AG-treated animals continued to receive AG in their drinking water until the time of sacrifice. Controls were given water and inoculated with SL3235 (H₂O-SL3235) or saline (H₂O-saline). Splenocytes were isolated 5 days after i.p. injection and cultured for 2 days at 2 × 10⁶ cells/well. The nitrite concentrations in cell-free supernatants were assessed. Data are expressed as the mean ± standard error of the mean for a minimum of triplicate wells from four experiments. P ≤ 0.0001, AG-SL3235 versus H₂O-SL3235 or H₂O-saline.

FIG. 3

Effect of in vivo AG treatment on SL3235-induced suppression of the response to heterologous antigens. Mice were treated for 7 days with AG in their drinking water prior to i.p. inoculation with SL3235 (AG-SL3235). AG-treated animals continued to receive AG in their drinking water until the time of sacrifice. Controls were given water and inoculated with SL3235 (H₂O-SL3235) or saline (H₂O-saline). Splenocytes were isolated 5 days postinjection. (A) Suppression of the in vitro PFC response. Splenocytes were cultured with SRBC. Five days later, the number of PFC/10⁷ cells was determined. Data are expressed as a ratio of H₂O-SL3235 or AG-SL3235 compared with H₂O-saline. Data are the mean and standard error of the mean for a minimum of triplicate wells from four experiments. P ≤ 0.0001, AG-SL3235 versus H₂O-SL3235; not significant, AG-SL3235 versus H₂O-saline (the H₂O-saline group has a suppression index of 1.0). (B) Suppression of responses to ConA. SL3235-infected splenocytes (2 × 10⁵/well) were cultured with splenocytes from H₂O-saline mice (6 × 10⁵/well) for 48 h in the presence of ConA. Splenocytes were pulsed with [³H]thymidine to assess proliferative responses. Data are expressed as a ratio of H₂O-SL3235 or AG-SL3235 compared with H₂O-saline and are the mean and standard error of the mean for a minimum of triplicate wells from four experiments. P ≤ 0.05, AG-SL3235 versus H₂O-SL3235 or H₂O-saline (the H₂O-saline group has a suppression index of 1.0).

FIG. 4

Effect of AG treatment on spleen weight on day 5 after i.p. immunization. Mice were treated for 7 days with AG in their drinking water prior to i.p. inoculation with saline (AG-saline) or SL3235 (AG-SL3235). AG-treated animals continued to receive AG in their drinking water until the time of sacrifice. Control groups received water without AG and were given SL3235 (H₂O-SL3235) or saline (H₂O-saline) i.p. The mice were sacrificed 5 days postinjection, and their spleens were weighed. Data were collected from individual mice, at least five per experiment, and are the pool of seven experiments, with the exception of the data for the AG-saline group, which is the pool of two experiments. The horizontal lines indicate mean values. P ≤ 0.05, AG-SL3235 versus H₂O-SL3235, AG-saline, and H₂O-saline.

FIG. 5

Effect of AG treatment on mouse survival. Mice were treated for 7 days with AG in their drinking water prior to i.p. inoculation with saline (AG-saline) or 7 × 10⁵ SL3235 organisms (AG-SL3235). AG treatment was continued through the course of the experiment. Control groups received water without AG and were given 7 × 10⁵ SL3235 organisms (H₂O-SL3235) or saline (H₂O-saline) i.p. The mice were observed daily, and deaths were recorded. Results are scored as percent survival. The data represent combined results from two experiments.

FIG. 6

Effect of AG treatment on bacterial burden over time after i.p. immunization. Mice were treated for 7 days with AG in their drinking water prior to i.p. inoculation with SL3235 (AG-SL3235). AG-treated animals continued to receive AG in their drinking water until the time of sacrifice. The control group received water without AG and was given SL3235 (H₂O-SL3235) i.p. At various days postinoculation, at least five mice were anesthetized and blood was obtained by cardiac puncture. The mice were then sacrificed, and the spleens and livers were aseptically removed, weighed, and homogenized. Appropriate dilutions in sterile saline were plated on EMB agar plates and grown overnight, and the number of Salmonella colonies was counted. Data represent combined results from two experiments. In one of the experiments, no mice were sacrificed on day 5. Lines connect the median values for each group. (A) Splenic bacterial burden per organ. P ≤ 0.016, AG-SL3235 versus H₂O-SL3235 on day 10; P ≤ 0.0001, AG-SL3235 versus H₂O-SL3235 on day 15; not significant, AG-SL3235 versus H₂O-SL3235 on day 5. (B) Liver bacterial burden per organ. P ≤ 0.0001, AG-SL3235 versus H₂O-SL3235 on days 10 and 15; not significant, AG-SL3235 versus H₂O-SL3235 on day 5. (C) Blood bacterial burden/0.1 ml of blood. P ≤ 0.0001, AG-SL3235 versus H₂O-SL3235 on days 10 and 15; not significant, AG-SL3235 versus H₂O-SL3235 on day 5.