Sepsis-induced SOCS-3 expression is immunologically restricted to phagocytes

Abstract

We have shown that immune cells from septic mice exhibit a suppressed response to exogenous stimuli in vitro. The suppressors of the cytokine signaling (SOCS) family are proteins that block intracellular signaling and can be induced by inflammatory mediators. Therefore, we hypothesized that SOCS-3 is up-regulated in immune cells in response to a septic challenge induced by cecal ligation and puncture (CLP). Mice were subjected to CLP or sham-CLP, and 2-48 h later, the blood, thymus, spleen, lung, and peritoneal leukocytes were harvested and examined. SOCS-3 was undetectable in thymocytes or blood leukocytes. In contrast, SOCS-3 was up-regulated in the spleen, lung, and peritoneal leukocytes in a time-dependent manner. Further examination revealed that only the macrophages and neutrophils expressed SOCS-3. These data suggest that cytokines and bacterial toxins present during sepsis have the ability to suppress the cytokine and/or lipopolysaccharide response and the function of immune cells by up-regulating SOCS-3.

Fig. 1

SOCS-3 expression is site-specific. Blood (Blood Leuk.), spleen, lung, liver (Thymus), and peritoneal leukocyte (P. Leuk.) samples were examined for SOCS-3 expression 24 h post sham-CLP (Sham) or CLP by Western blot analysis. RAW264.7 (Raw) stimulated with LPS for 4 h was used as a positive control. Each lane represents samples taken from individual animals. Blots shown are representative of three individual experiments (three different groups of mice, two to three sham or CLP mice analyzed per group).

Fig. 2

Time-dependent SOCS-3 expression in lung and spleen. SOCS-3 expression was examined over time in the lung and spleen by Western blot analysis (WB). Samples were collected 2, 4, 12, 24, and 48 h post-sham-CLP (Sham or S) or post-CLP (CLP or C). (A) Representative Western blots of SOCS-3. Protein bands were quantitated via densitometry and expressed as optical density (OD)/cm² for the spleen (B) and lung (C). Results are presented as mean ± sem, n = 6. *, Time points where SOCS-3 expression after CLP is significantly greater than that measured in sham controls (P<0.05).

Fig. 3

SOCS-3 expression is delayed in peritoneum. SOCS-3 expression was examined over time in the peritoneal leukocytes (P.Leuk.) by Western blot analysis. Samples were collected 4, 12, 24, and 48 h post-sham-CLP (Sham, S) or post-CLP (C). (A) Representive Western blot of SOCS-3. (B) Protein bands were quantitated via densitometry and expressed as OD/cm². Results are presented as mean ± sem, n = 6. *, Time points where SOCS-3 expression after CLP is significantly greater than that measured in sham controls (P<0.05).

Fig. 4

SOCS-3 expression is limited to phagocyte populations. Splenocytes and peritoneal leukocytes were separated by adherence to determine which cell types within each population expressed SOCS-3. (A) Splenocytes were separated into macrophage (MΦ)-, B cell (B)-, and T cell (T)-enriched populations. (B) Peritoneal leukocytes were separated into MΦ- and neutrophil (PMN)-enriched populations. Each set (underlined groups) represents samples taken from individual animals. Blots shown are representative of three individual experiments (three different groups of mice, two to three sham or CLP mice analyzed per group).

Fig. 5

SOCS-3 induction by endotoxin is limited to the peritoneum. SOCS-3 expression was examined in the lung, spleen, and peritoneal leukocytes (P. Leukocytes) 24 h post-CLP by Western blot analysis. Induction of SOCS-3 via endotoxin/Gram(-) bacteria was examined using HeJ mice that express a mutant TLR4 and are, thus, endotoxin-tolerant. Expression was compared with that observed in their background controls, HeN. (A) Representative Western blots for each cell population. (B) Protein bands were quantitated via densitometry and expressed as OD/cm². Results are presented as mean ± sem, n = 6. Gray bar/background denotes average level in sham controls. *, Expression levels that are significantly different than HeN controls (P<0.05).

Fig. 6

Gram(+) and Gram(-) bacteria induce SOCS-3. The ability of heat-killed E. coli and S. aureus to induce SOCS-3 expression was examined in vitro. TPMΦ, obtained from endotoxin-sensitive HeN and endotoxin-tolerant HeJ mice, were incubated with bacteria for 4 h, and SOCS-3 expression was detected by Western blot analysis. Control samples received no bacteria. (A) Representative Western blot of SOCS-3 expression. (B) Protein bands were quantitated via densitometry and expressed as OD/cm². Results are presented as mean ± sem, n = 5. *, Expression levels that are significantly different than HeN (P<0.05).

Fig. 7

Inhibition of IL-4 or IL-10 does not affect SOCS-3 expression, which was examined in the lung, spleen, and peritoneal leukocytes (P.Leukocytes) obtained from mice that were treated with neutralizing antibodies to IL-4 (αIL4) or IL-10 (αIL10) 12 h post-CLP. Control animals received rat IgG isotype-control antibody (rIgG). (A) Representative Western blots of SOCS-3 expression in each population. (B) Protein bands visualized by Western blot analysis were quantitated via densitometry and expressed as OD/cm². Results are presented as mean ± sem, n = 6.