Antimicrobial actions of the NADPH phagocyte oxidase and inducible nitric oxide synthase in experimental salmonellosis. I. Effects on microbial killing by activated peritoneal macrophages in vitro

Abstract

The contribution of the NADPH phagocyte oxidase (phox) and inducible nitric oxide (NO) synthase (iNOS) to the antimicrobial activity of macrophages for Salmonella typhimurium was studied by using peritoneal phagocytes from C57BL/6, congenic gp91phox(-/)-, iNOS(-/)-, and doubly immunodeficient phox(-/)-iNOS(-/)- mice. The respiratory burst and NO radical (NO.) made distinct contributions to the anti-Salmonella activity of macrophages. NADPH oxidase-dependent killing is confined to the first few hours after phagocytosis, whereas iNOS contributes to both early and late phases of antibacterial activity. NO-derived species initially synergize with oxyradicals to kill S. typhimurium, and subsequently exert prolonged oxidase-independent bacteriostatic effects. Biochemical analyses show that early killing of Salmonella by macrophages coincides with an oxidative chemistry characterized by superoxide anion (O(2).(-)), hydrogen peroxide (H(2)O(2)), and peroxynitrite (ONOO(-)) production. However, immunofluorescence microscopy and killing assays using the scavenger uric acid suggest that peroxynitrite is not responsible for macrophage killing of wild-type S. typhimurium. Rapid oxidative bacterial killing is followed by a sustained period of nitrosative chemistry that limits bacterial growth. Interferon gamma appears to augment antibacterial activity predominantly by enhancing NO. production, although a small iNOS-independent effect was also observed. These findings demonstrate that macrophages kill Salmonella in a dynamic process that changes over time and requires the generation of both reactive oxidative and nitrosative species.

Figure 1

Both ROS and RNS contribute to the antimicrobial activity of macrophages for Salmonella. Intracellular bacterial counts of untreated and IFN-γ–activated periodate-elicited macrophages from C57BL/6, iNOS ^−/−, and gp91phox ^−/− mice were compared. Viable intracellular bacteria were quantified by plating 20 h after the macrophages were challenged with S. typhimurium 14028s. The data are the mean ± SEM of 12–18 independent observations obtained on at least four separate days.

Figure 2

Nitrite and superoxide production by untreated and IFN-γ–activated macrophages. NO₂ ⁻ (□) accumulated by macrophages (mφ) from C57BL/6 mice in response to S. typhimurium 14028s was determined by the Griess reaction at 20 h after challenge, and O₂·⁻ (○) production was measured by the reduction of cytochrome c over a 1-h interval 20 h after challenge. The data are the mean ± SEM of six independent observations obtained on at least two separate days.

Figure 4

The lucigenin- and luminol-dependent chemiluminescence and NO_X production of Salmonella-infected macrophages vary over time. The capacity of IFN-γ–activated macrophages (mφ) to produce ROS and RNS was measured at selected 1-h intervals over a 10-h period from independent wells. The respiratory burst and NO_X production were measured as (A) lucigenin- and (B) luminol-dependent chemiluminescence, and (C) by the Griess reaction, respectively. The data are the mean ± SEM of 3–11 independent observations obtained on at least four separate days.

Figure 3

The contribution of iNOS and the NADPH phagocyte oxidase to the antimicrobial activity of macrophages for S. typhimurium varies over time. The killing activity of IFN-γ–activated, periodate-elicited macrophages from (A–C) C57BL/6, (A) phox ^−/−, (B) iNOS ^−/−, and (C) iNOS ^−/−gp91phox ^−/− mice was recorded over a 1-h period after challenge with wild-type S. typhimurium strain 14028s. The data are the mean ± SEM of 3–11 independent observations obtained on at least two separate days.

Figure 5

Macrophage production of ROS and RNS in response to Salmonella challenge is largely dependent on the NADPH oxidase and iNOS. (A) O₂·⁻, (B) H₂O₂, and (C) NO_X production were measured by reduction of cytochrome c, horseradish peroxidase–dependent oxidation of phenol red, and the Griess reaction, respectively. A–C represent metabolite production during the first hour after challenge. D and E represent NO₂ ⁻ and NO₃ ⁻ accumulated over a 1-h interval at 1 and 10 h after challenge. The data are the mean ± SEM of 3–10 independent observations obtained on at least two separate days. mφ, macrophages.

Figure 6

Nitrotyrosine staining fails to colocalize with bacteria in Salmonella-containing macrophages. The presence of nitrotyrosine (red) was examined by immunofluorescence microscopy of IFN-γ–activated macrophages from (A) C57BL/6, (B) iNOS ^−/−, and (C) gp91phox ^−/− mice that were challenged in vitro with GFP-expressing S. typhimurium (green). The pictures are representative of data obtained on two separate days.

Figure 7

Uric acid improves killing of Salmonella by macrophages. 1 mmol uric acid enhances killing of Salmonella by (A) IFN-γ–activated macrophages, but this enhancement is iNOS dependent (B). The data are the mean ± SEM of six independent observations obtained on two separate days.