Spermine protects mice against lethal sepsis partly by attenuating surrogate inflammatory markers

Abstract

The pathogenesis of sepsis is partly attributable to dysregulated inflammatory response mediated by pathogen-associated molecular patterns (PAMPs) (for example, endotoxin) and damage-associated molecular patterns (DAMPs) (for example, high-mobility group box 1 [HMGB1]). An endogenous ubiquitous polyamine, spermine, inhibits endotoxin-induced cytokine release in vitro, but its capacities to attenuate sepsis- and HMGB1-induced inflammatory responses was previously unknown. We thus tested the hypothesis that spermine protects mice against lethal sepsis by attenuating sepsis-induced local and systemic inflammatory responses. Intraperitoneal (i.p.) administration of spermine (10 mg/kg, twice daily, for 3 d) conferred a significant protection against lethal sepsis. The protective effects were associated with a significant reduction in peritoneal and serum levels of several surrogate markers of sepsis (for example, Interleukin-6 [IL-6], keratinocyte-derived chemokine [KC], monocytes chemoattractant protein-1 [MCP-1], macrophage inflammatory protein-2 [MIP-2], tissue inhibitor of metalloproteinase-1 [TIMP-1], soluble tumor necrosis factor-alpha receptor I [sTNFRI], and soluble tumor necrosis factor-alpha receptor II [sTNFRII]) during a late stage of sepsis. In vitro, spermine effectively inhibited HMGB1-induced release of the above surrogate markers in peritoneal macrophages. Thus, spermine confers protection against lethal sepsis partly by attenuating sepsis- and HMGB1-induced inflammatory responses.

Figure 1

Spermine protected mice against lethal sepsis. Balb/C mice were subjected to lethal sepsis (induced by CLP), and sper-mine was administered intraperitoneally (i.p., 10 mg/kg, twice a day, for 3 d) beginning at +24 h (A) or +0.5 h (B) post the onset of sepsis. The Kaplan–Meier method was used to compare mortality rates between groups of two independent experiments (with 13–14 mice per group) with similar results. Arrows indicate the time points of spermine administration. *P < 0.05 versus saline control group.

Figure 2

Spermine-attenuated, sepsis-induced local and systemic inflammatory responses. Balb/C mice were subjected to sepsis by CLP, and administered with control saline (0.2 mL/mouse) or spermine (10.0 mg/kg, twice a d) beginning at +0.5 h post CLP. At 52 h post CLP, levels of various cytokines in the peritoneal fluid (PF) or serum were determined by cytokine antibody array (A,B,D) and ELISA (C,E), respectively. (A) Representative cytokine antibody arrays. Peritoneal lavage fluid was pooled from three normal mice (− CLP), three septic mice receiving saline (+CLP), and three septic mice receiving spermine (+CLP +SPM), and assayed for cytokine levels by antibody arrays. (B) Relative peritoneal cytokine levels. The optical intensities (AU) for each pair of cytokine spots were determined using a Gel & Graph Digitizing Software, and expressed as mean ± SD of three independent experiments (n = 3). *P < 0.05 versus saline (+CLP). (C,E) ELISA analysis of IL-6 levels. (D) Relative serum cytokine levels. *P < 0.05 versus saline group (+CLP).

Figure 3

Spermine-attenuated peritoneal HMGB1 levels. Balb/C mice were subjected to sepsis by CLP, and administered with control saline (0.2 mL/mouse) or spermine (10 mg/kg, twice a d) beginning at + 0.5 h post CLP. At 52 h post CLP, levels of HMGB1 in the peritoneal fluid (PF) or serum were determined by Western blotting analysis. (A) Representative Western blots. − CLP, two normal mice; +CLP, two septic mice receiving saline; +CLP +SPM, two septic mice receiving spermine. (B) Relative HMGB1 levels. The relative band intensities were quantified using the NIH image 1.59 software to determine HMGB1 levels with reference to standard curves generated with purified HMGB1. *P< 0.05 versus saline group (+CLP).

Figure 4

HMGB1 and LPS induce distinct cytokine release profiles in macrophage cultures. Primary peritoneal macrophages (MuMAcs) were stimulated with HMGB1 (+HMGB1, 1.0 μg/mL) or LPS (+LPS, 100 ng/mL) for 16 h, and levels of various cytokines in the culture medium were determined by cytokine antibody arrays. (A) Representative cytokine antibody arrays. (B) Relative cytokine levels. The optical intensities (AU) for each pair of cytokine spots were determined using a Gel & Graph Digitizing Software, and expressed as mean ± SD of three independent experiments (n = 3). *P < 0.05 versus HMGB1 group (+HMGB1).

Figure 5

Spermine-attenuated, HMGB1-induced cytokine release. Primary peritoneal macrophages were stimulated with LPS (100 ng/mL), HMGB1 (1.0 μg/mL), either alone or in the presence of spermine at indicated doses for 16 h, and levels of various cytokines in the culture medium were assayed by ELISA (A,B) or cytokine antibody arrays (C,D). (A,B) ELISA assay of TNF and IL-6 levels. (C,D) Cytokine antibody arrays of various cytokines. The optical intensities (in arbitrary units, AU) for each pair of cytokine spots were expressed as mean ± SD of three independent experiments (n = 3). *P < 0.05 versus LPS or HMGB1 group, respectively.