Polymicrobial sepsis enhances clearance of apoptotic immune cells by splenic macrophages

Abstract

Background: Macrophage phagocytosis of apoptotic cells induces an anti-inflammatory macrophage phenotype. Immune cell apoptosis is widespread in sepsis; however, it is unknown whether sepsis alters the capacity of macrophages to clear this expanded population. We hypothesize that sepsis will enhance splenic macrophage phagocytosis of apoptotic immune cells, potentially contributing to immunosuppression.

Methods: Sepsis was induced in C57BL/6J mice by cecal ligation and puncture (CLP). Apoptosis was induced in mouse thymocytes by dexamethasone incubation. At multiple time points after CLP/sham, splenic and peritoneal macrophages were isolated, plated on glass coverslips, co-incubated with apoptotic thymocytes, and fixed and the coverslips were then Giemsa stained. Splenic macrophages were also isolated 48 hours after CLP/sham, stained with the red fluorescent dye PKH26, and co-incubated with green fluorescent dye CFSE-stained apoptotic thymocytes and then coverslips were fixed and counterstained with DAPI. The macrophage phagocytic index (PI) was calculated for both staining methods.

Results: The PI of CLP splenic macrophages was significantly higher than sham by 24 hours, and this difference was sustained through 48 hours.

Conclusions: Studies suggest that apoptotic cell clearance leads to an anti-inflammatory macrophage condition, which together with our findings in septic macrophages, may point at a process that contributes to septic immune suppression.

Figure 1

Typical flow cytograms are given for thymocytes that were stained with Annexin V and 7-amino-actinomycin D (7-AAD) following dexamethasone incubation (a) or freshly isolated without dexamethasone incubation (c). The cumulative results of 3 separate experiments are presented as mean +/- SEM (b) (d). Total [Annexin V +], early [Annexin V+/ 7-AAD-], and late [Annexin V+/ 7-AAD+] apoptotic cell populations are presented.

Figure 2

Splenic macrophages (arrow) that have phagocytized apoptotic thymocytes (arrowheads) as typically seen following May-Grunwald staining (a). Phagocytic index of splenic macrophages harvested at given time points after CLP/sham and co-incubated with apoptotic thymocytes (b). N = 4-8 mice/ group/ time point. * P<0.05, CLP vs. Sham, One-way ANOVA. # SEM=0.53. Phagocytic index of splenic macrophages harvested 48 hours after CLP/sham and co-incubated with either apoptotic thymocytes, non-apoptotic thymocytes, or no thymocytes (c). N = 4 mice per group.

Figure 3

Peritoneal macrophages (arrow) that have phagocytized apoptotic thymocytes (arrowheads) as typically seen following May-Grunwald staining (a). Phagocytic index of peritoneal macrophages harvested at given time points after CLP/sham and co-incubated with apoptotic thymocytes (b). N = 4-8 mice/ group/ time point. * P<0.05, CLP vs. Sham, One-way ANOVA.

Figure 4

Typical morphology of PKH26-stained splenic macrophages isolated 48 hours after CLP/sham that have phagocytized CFSE-stained apoptotic thymocytes (a). Cumulative phagocytic index of splenic macrophages isolated 48 hours after CLP, stained with the red fluorescent viable cell dye PKH26, and co-incubated with CFSE-labeled apoptotic thymocytes (b). N = 4 mice per group. * P<0.05, CLP vs. Sham, Mann-Whitney Rank Sum Test. A typical PKH26-stained splenic macrophage that has phagocytized CFSE-stained apoptotic thymocytes (c). 5um consecutive horizontal sections starting at the slide surface (i) and moving through the macrophage cytoplasm (ii-viii). Images are from one representative of multiple examined macrophages.

Figure 5

Typical flow cytograms of splenic macrophages isolated from mice 48 hours after CLP (a) or sham (b) and stained with CD11b and Gr-1. Percentages of all splenocytes stained with either CD11b or Gr-1 alone, and for both CD11b as well as Gr-1 as delineated by flow cytometry are presented (c). *P<0.05, CLP vs. Sham, Student’s t-test. The percentage of CD11b positive splenocytes that are Gr-1 positive is presented (d). Cumulative results of 5 separate experiments are presented as mean +/- SEM.