Polymicrobial sepsis induced by cecal ligation and puncture (CLP) reproduces many of the pathophysiologic features of septic shock. In this study, we demonstrate that mRNA for a broad range of pro- and anti-inflammatory cytokine and chemokine genes are temporally regulated after CLP in the lung and liver. We also assessed whether prophylactic administration of monophosphoryl lipid A (MPL), a nontoxic derivative of lipopolysaccharide (LPS) that induces endotoxin tolerance and attenuates the sepsis syndrome in mice after CLP, would alter tissue-specific gene expression post-CLP. Levels of pulmonary interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α), granulocyte colony-stimulating factor (G-CSF), IL-1 receptor antagonist (IL-1ra), and IL-10 mRNA, as well as hepatic IL-1β, IL-6, gamma interferon (IFN-γ), G-CSF, inducible nitric oxide synthase, and IL-10 mRNA, were reduced in MPL-pretreated mice after CLP compared to control mice. Chemokine mRNA expression was also profoundly mitigated in MPL-pretreated mice after CLP. Specifically, levels of pulmonary and hepatic macrophage inflammatory protein 1α (MIP-1α), MIP-1β, MIP-2, and monocyte chemoattractant protein-1 (MCP-1) mRNA, as well as hepatic IFN-γ-inducible protein 10 and KC mRNA, were attenuated in MPL-pretreated mice after CLP. Attenuated levels of IL-6, TNF-α, MCP-1, MIP-1α, and MIP-2 in serum also were observed in MPL-pretreated mice after CLP. Diminished pulmonary chemokine mRNA production was associated with reduced neutrophil margination and pulmonary myeloperoxidase activity. These data suggest that prophylactic administration of MPL mitigates the sepsis syndrome by reducing chemokine production and the recruitment of inflammatory cells into tissues, thereby attenuating the production of proinflammatory cytokines.

Monophosphoryl lipid A (MPL) is a nontoxic derivative of the lipid A region of lipopolysaccharide (LPS) that is being developed as both an adjuvant and prophylactic drug for septic shock. We compared the ability of LPS and MPL to induce interleukin-10 (IL-10), IL-12 p35, IL-12 p40, gamma interferon (IFN-␥), glucocorticoid receptor (GR), IL-1 receptor antagonist (IL-1ra), and inducible nitric oxide synthase mRNA expression in murine peritoneal macrophages. These genes were chosen for their ability to positively or negatively regulate the host immune response and thus for their potential involvement in MPL-induced adjuvanticity or in its ability to protect against sepsis. LPS was a more potent inducer of IL-12 p35, IL-12 p40, and IFN-␥ mRNA, as well as of IL-12 protein, than MPL. In contrast, MPL induced higher levels of IL-10 mRNA than did LPS from 1 to 1,000 ng/ml. In general, MPL was not a more potent inducer of negative regulatory genes, since MPL and LPS induced similar levels of GR and IL-1ra mRNA. Addition of anti-IL-10 antibody to cultures increased the induction of MPL-induced IL-12 p35, IL-12 p40, and IFN-␥ mRNA, suggesting that the enhanced production of IL-10 by MPL-stimulated macrophages contributes to decreased production of mRNA for IL-12 (p35 and p40) and IFN-␥. Conversely, the addition of exogenous IL-10 to LPS-treated macrophages reduced the mRNA expression of these cytokine genes. These studies suggest that enhanced production of IL-10 by MPL-stimulated macrophages may contribute to the reduced toxicity of MPL through its negative action on induction of cytokines shown to enhance endotoxicity.

Acyloxyacyl hydrolase (AOAH) is an enzyme found in macrophages and neutrophils that specifically cleaves the acyloxyacyl moieties of lipopolysaccharide (LPS), thus rendering it non-toxic for human cells. In the present study, we demonstrate that LPS augments AOAH mRNA expression (10-20-fold) in murine macrophages. Following LPS treatment (100 ng/m]), AOAH mRNA was induced by 2 h, peaked at 6 h, and was sustained over 72 h. Optimal induction of AOAH mRNA was observed with as little as 0.1 ng/ml LPS. LPS also induced a concomitant increase in AOAH enzymatic activity in cytosolic extracts from murine macrophages and the ability of macrophages to deacylate LPS was not diminished in endotoxin-tolerized macrophages. LPS-stimulated AOAH mRNA expression was cycloheximide sensitive, indicating that de novo protein synthesis is required for AOAH mRNA production. Moreover, AOAH mRNA expression was also induced by IFN-γ. LPS-stimulated mRNA expression was not suppressed by either dexamethasone or IL-10. Finally, intraperitoneal challenge of mice with 25 μg of LPS resulted in increases in AOAH mRNA in both the lung (∼3-fold) and in the liver (∼6-fold). A possible role for LPS-inducible AOAH in the elimination of LPS is discussed.

Recent studies have suggested that lipopolysaccharide (LPS) stimulates cells by mimicking the secondmessenger function of ceramide, a lipid generated in the cell by the action of sphingomyelinase (SMase). To examine this possibility further, we compared the abilities of LPS, SMase, and/or ceramide analogs to induce cytokine secretion, modulate gene expression, and induce endotoxin tolerance in macrophages. SMase and LPS induced secretion of tumor necrosis factor alpha (TNF-␣) to comparable degrees; however, unlike LPS, SMase failed to stimulate detectable interferon activity. Cell-permeable analogs of ceramide induced the expression of many LPS-inducible genes; however, the expression of interferon-inducible protein 10 (IP-10) and interferon consensus sequence-binding protein (ICSBP) mRNAs was significantly lower than that induced by LPS. Both SMase-induced TNF-␣ secretion and LPS-induced TNF-␣ secretion were inhibited by pretreatment with a serine/threonine phosphatase inhibitor, calyculin A. Macrophages preexposed in vitro to LPS to induce a well-characterized state of endotoxin tolerance secreted little or no TNF-␣ upon secondary challenge with either LPS or SMase, whereas macrophages preexposed to SMase secreted high levels of TNF-␣ upon secondary stimulation with LPS or SMase. Collectively, these results suggest that ceramide activates a subset of LPS-induced signaling pathways in murine peritoneal exudate macrophages.

To evaluate potential roles for macrophages, IFN-gamma, and TNF receptor 1 (TNFR1) in the regulation of LPS-induced inducible nitric oxide synthase (iNOS) mRNA expression, we used a model of macrophage depletion as well as IFN-gamma (GKO) and TNFR1 (TNFR1 -/-) knockout mice. LPS-induced iNOS mRNA in spleen was ablated in both macrophage-depleted and GKO mice. In livers of macrophage-depleted mice, LPS-induced iNOS mRNA was reduced by 55 to 85%, with the most profound reductions detected 6 and 8 h after LPS injection. In GKO mice, peak iNOS mRNA expression in liver (3 h) was unaffected by the loss of endogenous IFN-gamma. By 6 to 12 h after LPS challenge, however, hepatic LPS-induced iNOS mRNA and serum nitrate/nitrite levels were reduced substantially in GKO mice. Residual LPS-induced iNOS mRNA in livers of GKO mice was nearly ablated by macrophage depletion, indicating that induction of iNOS mRNA in liver requires both endogenous IFN-gamma and either macrophages and/or macrophage-derived factors. TNFR1-mediated signaling was involved in the induction of LPS-induced iNOS mRNA in liver at 3 and 6 h, but not in its maintenance at 8 h. Conversely, induction of iNOS mRNA in spleen by LPS was independent of TNFR1-mediated signaling. Our results indicate that macrophages and/or their secreted products, endogenous IFN-gamma production, and TNFR1-mediated signaling play key roles in the in vivo regulation of iNOS mRNA expression and that the extent of their involvement is both time and organ specific.