Inhibitor of apoptosis protein cIAP2 is essential for lipopolysaccharide-induced macrophage survival

Abstract

The cellular inhibitor of apoptosis 2 (cIAP2/HIAP1) is a potent inhibitor of apoptotic death. In contrast to the other members of the IAP family, cIAP2 is transcriptionally inducible by nuclear factor-kappaB in response to multiple triggers. We demonstrate here that cIAP2-/- mice exhibit profound resistance to lipopolysaccharide (LPS)-induced sepsis, specifically because of an attenuated inflammatory response. We show that LPS potently upregulates cIAP2 in macrophages and that cIAP2-/- macrophages are highly susceptible to apoptosis in a LPS-induced proinflammatory environment. Hence, cIAP2 is critical in the maintenance of a normal innate immune inflammatory response.

FIG. 1.

Disruption of the ciap2 gene by homologous recombination. (A) Structure of the 5′ end of the mouse ciap2 locus, targeting vector, and the targeted ciap2 allele. The open black box shows the position of the probe used for the genomic Southern blot analysis. (B) A Southern blot analysis of EcoRV-digested genomic DNA from embryonic stem cells shows the presence of the targeted (15 kb) and the wild-type (27 kb) alleles. (C) A Western blot analysis of lung protein extracts indicates the absence of the 66-kDa cIAP2 full-length polypeptide and the presence of cIAP1 and Survivin protein. (D) Relative quantitative levels of cIAP2, cIAP1, and xiap mRNA, which was derived from mouse embryonic fibroblasts of cIAP2^−/− and wild-type littermate mice. The results are means ± the standard deviations. n = 6, average of triplicate wells per mouse, P < 0.05.

FIG. 2.

cIAP2 is highly upregulated in response to LPS. The iap mRNA levels of LPS-treated peritoneal macrophages derived from wild-type C57BL/6 mice relative to untreated controls were assayed. (A) xiap, ciap1, and ciap2 message levels of macrophages exposed to a range of LPS doses relative to untreated controls. (B) The ciap2 mRNA message of macrophages exposed to LPS was assayed over 24 h. The results are means ±thestandard deviation. n = 5, average of triplicate wells per mouse.

FIG. 3.

cIAP2^−/− mice are resistant to LPS-induced endotoxic shock. Mice were injected i.p. with a range of indicated LPS doses (n = 15). (A) cIAP2^−/− mice (solid shapes; all cIAP2^−/− mice survived for up to 7 days at an LPS dose of 40 mg/kg and lower) and littermate controls (open shapes) were treated with a range of LPS doses. (B) cIAP2^−/− mice (solid shapes) and littermate controls (open shapes; all littermate control mice did not survive past the first day at an LPS dose of 60 mg/kg and above) were given a higher range of LPS doses (P < 0.001).

FIG. 4.

Macrophage cell counts and function is not impaired in cIAP2^−/− mice. (A) Spleen- and peritoneum-derived macrophage cell count numbers for cIAP2^−/−, cIAP2^+/−, and cIAP2^+/+ mice (n = 10). (B) Both cIAP2^−/− mouse- and littermate control-derived macrophages stained for the LPS-binding surface receptor CD14. (C) Proliferation of B cells for cIAP2^−/− mice and littermate controls cIAP2^+/+ after culture with the indicated range of LPS doses (n = 6). (D and E) Cultured macrophages from cIAP2^−/− or littermate control cIAP2^+/+ mice were exposed to LPS for 10 h, and the IL-1β (D) or TNF-α (E) levels were measured by enzyme-linked immunosorbent assay (n = 6). (F and G) In addition, macrophages were exposed to the indicated range of LPS doses for 24 h, and the IL-1β (F) or TNF-α (G) levels were determined (n = 6). The results are means ± the standard deviations in triplicate per mouse (P < 0.01).

FIG. 5.

cIAP2^−/−-mouse-derived macrophages display an increased sensitivity to apoptosis. (A) Peritoneum-derived macrophages from cIAP2^−/− mice (lower panels) and cIAP2^+/+ mice (upper panels) were either pretreated with LPS for 4 h or not pretreated prior to exposure to α-Fas antibody and then TUNEL stained to assess cell viability. The percentages of viable cells (n = 5; average of triplicate wells per mouse, P < 0.01) are shown within each TUNEL-stained panel along with the standard deviation. (B) cIAP2^−/−-derived (open shapes) and cIAP2^+/+-derived (solid shapes) T cells were preincubated with a range of IL-7 concentrations and then exposed to dexamethasone, and T-cell survival was monitored over a 12-h period (n = 3, average of triplicate wells per mouse; results are means ± the standard deviation, P_12 h < 0.05). (C) Peritoneum-derived cell numbers are shown for macrophages of cIAP2^−/− mice and littermate control cIAP2^+/+ mice injected i.p. with LPS. (D) Combined number of T and B cells of cIAP2^−/− mice and littermate control mice that had been i.p. injected with LPS.

FIG. 6.

Reduced liver-derived macrophage cell counts in cIAP2^−/− mice treated with LPS. (A and B) Liver tissue derived from cIAP2^−/− (upper panels) and cIAP2^+/+ mice (lower panels) pretreated with LPS (35 mg/kg) for 2 h (A) or 6 h (B). Cell counts (n = 5; average of three sections [average of five fields per section] per mouse, P < 0.01]) are shown within each anti-F4/80 FITC-conjugated antibody-stained panel, along with the standard deviation.