Lipopolysaccharide and monophosphoryl lipid A differentially regulate interleukin-12, gamma interferon, and interleukin-10 mRNA production in murine macrophages.

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-gamma), 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-gamma 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-gamma 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-gamma. 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.

CD14-dependent and CD14-independent signaling pathways in murine macrophages from normal and CD14 knockout mice stimulated with lipopolysaccharide or taxol.

The antitumor agent, Taxol, shares with bacterial LPS the ability to activate murine macrophages, and its LPS-mimetic effects are blocked by LPS analogue antagonists. Since CD14 is central to the recognition of LPS by macrophages, we sought to examine a role for CD14 in the response to Taxol vs LPS. A comparison of responses of macrophages from wild-type mice with those from mice lacking CD14 due to a targeted disruption of the CD14 gene (CD14-deficient knockout (CD14KO)) revealed that like LPS, Taxol induces both CD14-dependent and -independent pathways of gene activation, although the CD14 dependency of Taxol stimulation is much less striking than that observed with LPS. The macrophage interaction with low concentrations of LPS (< or = 10 ng/ml) is largely CD14 dependent, as evidenced by the lack of induction of TNF-alpha, IL-1beta, and interferon-inducible protein-10 (IP-10) genes by CD14KO macrophages cultured in the absence of soluble CD14 (i.e., in autologous CD14KO -/- mouse serum). However, at high concentrations of LPS or Taxol, a CD14-independent pathway of activation is observed: this pathway leads to minimal IP-10 gene induction, even though induction of TNF-alpha and IL-1beta occurs. Measurements of TNF secretion followed a similar pattern to that observed at the level of steady state mRNA. These data suggest the existence of two pathways of activation by both LPS and Taxol: one that is CD14 dependent and leads to induction of TNF-alpha, IL-1beta, and IP-10 gene induction, and a CD14-independent pathway that results in the induction of TNF-alpha and IL-1beta, with minimal induction of IP-10.

Differential dysregulation of nitric oxide production in macrophages with targeted disruptions in IFN regulatory factor-1 and -2 genes.

Recent studies have reported that IFN regulatory factor-1 (IRF-1) regulates nitric oxide (NO.) production in murine macrophages (M phi). Since IRF-2 recognizes the same consensus sequence as IRF-1, we postulated that IRF-2 may also regulate NO.. Therefore, the ability of M phi from INF-2 homozygous (IRF-2-/-) and heterozygous (IRF-2+/-) knockout mice to produce NO. following LPS and/or IFN-gamma stimulation was compared with the responses of IRF-1-/-, IRF-1+/-, and C57BL/6+/+ M phi. IRF-2-/- M phi produced less LPS-induced NO2- than IRF-2+/- or C57BL/6 M phi. LPS and IFN-gamma synergized to increase NO2- production from IRF-2-/- M phi to approximately 50% of IRF-2+/- and C57BL/6 levels. Unexpectedly, IRF-2-/-, IRF-2+/- and C57BL/6 M phi produced comparable levels of inducible NO synthase mRNA in response to treatment with LPS and IFN-gamma. IRF-1-/- M phi produced barely detectable NO2- and low, but detectable, inducible NO. synthase mRNA in response to IFN-gamma and LPS. These results indicate that IRF-1 and IRF-2 differ in their mechanism of NO. regulation and that IRF-2 regulates inducible NO. synthase post-transcriptionally.