Injury-induced GR-1+ macrophage expansion and activation occurs independently of CD4 T-cell influence

Abstract

Burn injury initiates an enhanced inflammatory condition referred to as the systemic inflammatory response syndrome or the two-hit response phenotype. Prior reports indicated that macrophages respond to injury and demonstrate a heightened reactivity to Toll-like receptor stimulation. Since we and others observed a significant increase in splenic GR-1 F4/80 CD11b macrophages in burn-injured mice, we wished to test if these macrophages might be the primary macrophage subset that shows heightened LPS reactivity. We report here that burn injury promoted higher level TNF-α expression in GR-1, but not GR-1 macrophages, after LPS activation both in vivo and ex vivo. We next tested whether CD4 T cells, which are known to suppress injury-induced inflammatory responses, might control the activation and expansion of GR-1 macrophages. Interestingly, we found that GR-1 macrophage expansion and LPS-induced TNF-α expression were not significantly different between wild-type and CD4 T cell-deficient CD4(-/-) mice. However, further investigations showed that LPS-induced TNF-α production was significantly influenced by CD4 T cells. Taken together, these data indicate that GR-1 F4/80 CD11b macrophages represent the primary macrophage subset that expands in response to burn injury and that CD4 T cells do not influence the GR-1 macrophage expansion process, but do suppress LPS-induced TNF-α production. These data suggest that modulating GR-1 macrophage activation as well as CD4 T cell responses after severe injury may help control the development of systemic inflammatory response syndrome and the two-hit response phenotype.

Figure 1. Burn injury induces an increase in the percentage of F4/80+ CD11b+ GR-1+ splenic macrophages

Spleens from WT sham or burn-injured mice were harvested at day 7 and splenocyte suspensions were prepared. Cells were stained with fluorescently-labeled anti-GR-1, -F4/80, and -CD11b specific antibodies. The dot plots shown in (A) represent the GR-1 and CD11b staining profiles of FACS-gated live and F4/80+ spleen cells. Cells were prepared from the blood, bone marrow, and spleens of WT sham or burn mice at day 7 post-injury and stained for cell-surface GR-1+/F4/80+/CD11b+. The combined FACS data from all mice is plotted in Figure 1B. There was a significant increase in GR-1+/F4/80+/CD11b+ blood and spleen macrophages that were harvested from burn as compared to sham mice (* p<0.05, Student’s t test). Data expressed as mean ± SEM of n=4 mice per group and are representative of 3 independent experiments.

Figure 2. GR-1+ F4/80+ macrophages are principal producers of TNFα following burn injury with LPS stimulation at day 7 following injury

Spleens from WT sham or burn injured mice were harvested at day 7 and splenocyte suspensions prepared. LPS, followed by Brefeldin A, was added to cultures to induce an in vitro two-hit effect. Cells were stained with fluorescently-labeled anti-GR-1, -F4/80, and intracellular -TNFα specific antibodies. The data plotted in (A) shows the percentage TNFα-expressing cells in GR-1+ versus GR-1− macrophages. The data plotted in (B) shows the MFI for TNFα staining in GR-1+ and GR-1− macrophages. There is a significantly higher percentage TNFα expression and expression levels (MFI) in GR-1+ F4/80+ macrophages from burn-injured mice as compared to GR-1+ F4/80+ sham (*p<0.01). There was no significant difference observed between sham or burn-injured mice in GR-1 F4/80+ macrophages (# p>0.05 ns). Overall, GR-1+ F4/80+ macrophages expressed higher levels of TNFα as compared withGR-1− F4/80+ macrophages (** p<0.001, One-Way ANOVA with Tukey’s multiple comparison). Data expressed as mean ± SEM of n=4 mice per group and are representative of 3 independent experiments.

Figure 3. GR-1+ macrophages from burn-injured mice express significantly higher TNFα following in vivo challenge with LPS

Groups of mice underwent sham or burn injury. Seven days later, mice were challenged with LPS (10 mg/kg) by i.p. injection. After 30 minutes, spleens were harvested into medium containing Brefeldin A (10 μg/ml) to prevent cytokine release. Cells were plated in C-5 medium and cultured at 37° C with Brefeldin A (10 μg/ml) for an additional 3 hours. Cells were stained with fluorescently-labeled anti-GR-1, -F4/80, and intracellular -TNFα specific antibodies. The FACS plots in (A) illustrate the intracellular TNFα staining profile in GR-1+ and GR-1− macrophages for day 7 sham versus burn mice. The data plotted in (B) represents combined TNFα expression staining in GR-1+ versus GR-1− macrophages from the spleens of sham versus burn mice at day 7 after injuries. Data are expressed as mean ± SEM of n=8 mice per group. GR-1+ macrophages from burn mice showed a significant increase (*, p<0.05) in intracellular TNFα expression as compared to sham mice.

Figure 4. CD4+ T cells do not influence the burn-induced increase in GR-1+ macrophages

Spleen cells were prepared from sham- or burn-injured WT or CD4 −/− mice at 7 days after injury. Cells were stained with fluorescently-labeled anti-GR-1, anti-F4/80, and anti-CD11b antibodies. After staining, cells were fixed and stained for FACS analysis. Burn injury induced a significant increased in GR-1+F4/80+CD11b+ macrophages in WT and CD4−/− (*p<0.05 by One-Way ANOVA). The percentage of GR-1+F4/80+CD11b+ macrophages was significantly lower in burn-injured CD4−/− mice than burn WT mice (#, p<0.05 by One-Way ANOVA). Data are expressed as mean ± SEM of n=3 mice per group from 2 independent experiments.

Figure 5. CD4+ T cells do not influence the injury-induced increase in TNF α expression by LPS-stimulated GR-1+ macrophages

Spleen cells were prepared from WT or CD4 −/− sham or burn-injured mice at days 1 and 7. As a control, splenocytes from sham or burn-injured WT mice were depleted of CD4 T cells. Following in vitro LPS stimulation, we measured TNFα expression in FACS-gated F4/80+ GR-1+ or GR-1− macrophages. Panel (A) shows representative FACS plots of gated GR-1+F4/80+ spleen cells stimulated with LPS from sham or burn injured WT, CD4 depleted WT spleen cells or CD4−/− mice at day 1 and day 7 after injury. Panel (B) shows TNFα expression in live, F4/80+ GR-1+ macrophages, while (C) shows TNFα expression in F4/80+ GR-1− macrophages. Our results demonstrate that LPS-stimulated GR-1+ macrophages from day 7 burn mice expressed significantly higher levels of TNFα as compared with GR-1+ macrophages from day 7 sham or day 1 burn mice. The in vivo or in vitro absence of CD4 T cells did not influence GR-1+ macrophage TNFα expression levels, as confirmed by results in CD4−/− and CD4 T cell depleted splenocytes (#p>0.05 ns, one way ANOVA with Tukey’s multiple comparison). In contrast, GR-1− macrophages were more inflammatory at day 1 as compared to day 7 after burn injury (**p<0.001). Data are expressed as mean ± SEM of n=3 mice per group and are representative of 3 independent experiments.

Figure 6. Mice lacking CD4 T cells produce higher levels of TNFα following injury

WT or CD4−/− mice were randomized to sham or burn injury. At day 7 after injury, mice were killed and splenocyte suspensions were prepared. Spleen cells were cultured with increasing doses of LPS (0–1μg/ml, five concentrations) for 48 hours and supernatants were tested for TNFα levels using a Luminex assay. The plots show LPS-induced TNFα levels in supernatants from (A) WT, (B) WT/CD4 T cell depleted, and (C) CD4−/− spleen cells. Spleen cells from all groups of burn mice produced significantly higher TNFα at 7 days as compared to spleen cells from sham mice; *p<0.05 by One-Way ANOVA. In addition, splenocytes from CD4−/− mice and CD4 T cell depleted spleen cells exhibited higher TNFα production at day 7 after burn injury as compared to spleen cells from burn WT mice; #p<0.001 by One-Way ANOVA. Data are expressed as mean ± SEM of n=4 mice per group and are representative of 2 independent experiments.