DAP12 (KARAP) amplifies inflammation and increases mortality from endotoxemia and septic peritonitis

Abstract

DAP12 (KARAP) is a transmembrane signaling adaptor for a family of innate immunoreceptors that have been shown to activate granulocytes and monocytes/macrophages, amplifying production of inflammatory cytokines. Contrasting with these data, recent studies suggest that DAP12 signaling has an inhibitory role in the macrophage response to microbial products (Hamerman, J.A., N.K. Tchao, C.A. Lowell, and L.L. Lanier. 2005. Nat. Immunol. 6:579-586). To determine the in vivo role for DAP12 signaling in inflammation, we measured the response of wild-type (WT) and DAP12-/- mice to septic shock. We show that DAP12-/- mice have improved survival from both endotoxemia and cecal ligation and puncture-induced septic shock. As compared with WT mice, DAP12-/- mice have decreased plasma cytokine levels and a decreased acute phase response during sepsis, but no defect in the recruitment of cells or bacterial control. In cells isolated after sepsis and stimulated ex vivo, DAP12 signaling augments lipopolysaccharide-mediated cytokine production. These data demonstrate that, during sepsis, DAP12 signaling augments the response to microbial products, amplifying inflammation and contributing to mortality.

Figure 1.

DAP12 signaling augments mortality and inflammatory cytokine levels during endotoxemia. (A) Survival of WT and DAP12^−/− mice after endotoxemia was measured at three different doses: 5, 6.25, and 10 mg/kg (n = 5 for all doses). At both 5 and 6.25 mg/kg, DAP12^−/− mice had improved survival as compared with WT mice (P ≤ 0.05 by the log-rank test). At 10 mg/kg, both strains died. (B) Plasma was harvested from WT and DAP12^−/− mice 2 (n = 5–6), 4 (n = 3), or 24 (n = 3) h after injection of 5 mg/kg LPS. At 2 h, WT mice had increased levels of TNF-α and IL-10 (*, P < 0.05 vs. WT by the Mann-Whitney test).

Figure 2.

DAP12 signaling augments mortality and inflammatory cytokine levels during bacterial sepsis. WT and DAP12^−/− mice were subjected to CLP, and survival (A) and cytokine production (B) were assessed. DAP12^−/− mice (n = 19) are resistant to CLP as compared with WT mice (n = 20; P < 0.001 by the log-rank test). Plasma was harvested from WT or DAP12^−/− mice before CLP (n = 5) and either 6 (n = 6) or 24 (n = 12–15) h after CLP, and cytokine levels were measured. At 6 h, we found elevated but equal levels of MCP-1, IL-6, and TNF-α in WT and DAP12^−/− mice. By 24 h, WT mice had significantly higher levels of MCP-1, IL-6, TNF-α, and IL-10 (P < 0.05 by the Mann-Whitney test). Values shown are SEM.

Figure 3.

DAP12 signaling does not contribute to cellular recruitment or bactericidal activity. 24 h after CLP, peritoneal exudate was harvested by peritoneal lavage. Total cell numbers (A; n = 16–17), bacterial load (B; n = 11–12), and distribution of cell types (C; n = 4–5) were measured. We found no difference between WT and DAP12^−/− mice. Each asterisk represents a measurement for a single mouse.

Figure 4.

DAP12 signaling augments ex vivo cytokine production by macrophages after sepsis but not sterile peritonitis. WT and DAP12^−/− mice were subjected to CLP, and peritoneal cells were harvested after 24 h (n = 8–12). (A) Cells were cultured ex vivo with or without stimulation with 1 μg/ml LPS, and levels of cytokine in the supernatant were measured. With no stimulation, WT cells (black bars) produced more IL-6, MCP-1, TNF-α, and IL-10 as compared with DAP12^−/− cells (hatched bars; *, P < 0.05; **, P < 0.01 by the Mann-Whitney test). After LPS stimulation, WT cells produced increased amounts of TNF-α, MCP-1, and IL-10. (B) Cells were also harvested 72 h after i.p. injection of thioglycollate broth and cultured ex vivo with (10, 100, or 1,000 ng/ml) or without LPS (n = 3 for all cultures). There was no statistically significant difference between WT and DAP12^−/− cells, although there was a trend toward increased IL-10 by the DAP12^−/− cells with maximal stimulation. Values shown are SEM.