c-Jun N-terminal kinase negatively regulates lipopolysaccharide-induced IL-12 production in human macrophages: role of mitogen-activated protein kinase in glutathione redox regulation of IL-12 production

Although c-Jun N-terminal kinase (JNK) plays an important role in cytokine expression, its function in IL-12 production is obscure. The present study uses human macrophages to examine whether the JNK pathway is required for LPS-induced IL-12 production and defines how JNK is involved in the regulation of IL-12 production by glutathione redox, which is the balance between intracellular reduced (GSH) and oxidized glutathione (GSSG). We found that LPS induced IL-12 p40 protein and mRNA in a time- and concentration-dependent manner in PMA-treated THP-1 macrophages, and that LPS activated JNK and p38 mitogen-activated protein (MAP) kinase, but not extracellular signal-regulated kinase, in PMA-treated THP-1 cells. Inhibition of p38 MAP kinase activation using SB203580 dose dependently repressed LPS-induced IL-12 p40 production, as described. Conversely, inhibition of JNK activation using SP600125 dose dependently enhanced both LPS-induced IL-12 p40 production from THP-1 cells and p70 production from human monocytes. Furthermore, JNK antisense oligonucleotides attenuated cellular levels of JNK protein and LPS-induced JNK activation, but augmented IL-12 p40 protein production and mRNA expression. Finally, the increase in the ratio of GSH/GSSG induced by glutathione reduced form ethyl ester (GSH-OEt) dose dependently enhanced LPS-induced IL-12 p40 production in PMA-treated THP-1 cells. GSH-OEt augmented p38 MAP kinase activation, but suppressed the JNK activation induced by LPS. Our findings indicate that JNK negatively affects LPS-induced IL-12 production from human macrophages, and that glutathione redox regulates LPS-induced IL-12 production through the opposite control of JNK and p38 MAP kinase activation.     

The IL-12p70/IL-10 interplay is differentially regulated by free heme and hemozoin in murine bone-marrow-derived macrophages

The outcome of malarial anemia is determined by a complex interplay between pro-inflammatory and anti-inflammatory cytokines, its severity associated with accumulation of hemozoin (Hz) in macrophages, elevated IL-10 responses and impaired IL-12 production. Although free heme contributes to malarial anemia by inducing oxidative damage of red blood cells (RBCs) and enhancing their clearance by phagocytes, its impact on IL-12/IL-10 interactions has not been fully characterized. Herein, the effect of hemin (HE) on IL-12 and IL-10 responses was studied in murine bone marrow-derived macrophages (BMDM) and compared with synthetic Hz. Our data reveal that HE induces modest inhibition of IL-12p70 responses to lipopolysaccharide (LPS) whereas Hz significantly impairs IL-12p70 responses to IFNγ/LPS through down-regulation of IL-12p35 and p40 gene expression. Although reactive oxygen species (ROS) are generated after short-term exposure to HE and Hz, prolonged exposure to these iron protoporphyrins has opposite effects on the cellular redox status, HE being the only compound able to promote persistent ROS production. Accordingly, the inhibitory effect of HE on IL-12p70 seems sustained by redox-dependent induction of IL-10 and is partially controlled by the p38 mitogen-activated protein kinase (MAPK) signalling pathway. Indeed, treatment with n-acetylcysteine (NAC) or with the p38 MAPK inhibitor SB203580 inhibits IL-10 responses and significantly restores IL-12p70 responses to IFNγ/LPS in HE-conditioned BMDM. Our results suggest that oxidant stress induced by free heme may potentially contribute to sustained production of IL-10 and down-regulation of IL-12 responses in malaria.     

Rac1 negatively regulates lipopolysaccharide-induced IL-23 p19 expression in human macrophages and dendritic cells and NF-kappaB p65 trans activation plays a novel role

IL-23 is a heterodimeric cytokine composed of a unique p19 subunit and of a p40 subunit that is also common to IL-12. We defined the distinct signaling mechanisms that regulate the LPS-mediated induction of IL-23 p19 and p40 in human macrophages and dendritic cells. We found that the overexpression of dominant-negative Rac1 (N17Rac1) enhanced LPS-induced IL-23 p19 expression but did not alter p40 expression or IL-12 p70 production in PMA-treated THP-1 macrophages and in human monocyte-derived dendritic cells. Although the inhibition of either p38 MAPK or JNK enhanced LPS-induced p19 expression, N17Rac1 did not influence either p38 MAPK or JNK activation. By contrast, N17Rac1 augmented both NF-kappaB gene expression and p65 trans activation stimulated by LPS without affecting the degradation of IkappaB-alpha or DNA binding to NF-kappaB. Furthermore, small interference RNA of NF-kappaB p65 attenuated cellular amounts of p65 and suppressed LPS-induced p19 expression but did not affect p40 expression. Our findings indicate that Rac1 negatively controls LPS-induced IL-23 p19 expression through an NF-kappaB p65 trans activation-dependent, IkappaB-independent pathway and that NF-kappaB p65 regulates LPS-induced IL-23 p19, but not p40, expression, which causes differences in the control of IL-23 p19 and p40 expression by Rac1.     

IL-12 p40 homodimer-dependent macrophage chemotaxis and respiratory viral inflammation are mediated through IL-12 receptor beta 1

Leukocyte recruitment to the airway lumen is a central feature of inflammatory conditions such as asthma and respiratory viral infection. Characterization of mediators that regulate leukocyte recruitment in these conditions revealed increased IL-12 p40 homodimer (p80) levels were associated with enhanced airway macrophage accumulation. To examine this association, we used in vivo and in vitro assays to demonstrate p80, but not IL-12 or p40, provided a macrophage chemoattractant signal. Macrophages from genetically deficient mice indicated p80-dependent chemotaxis was independent of IL-12 and required IL-12Rbeta1 (Rbeta1) expression. Furthermore, analysis of murine cell lines and primary culture macrophages revealed Rbeta1 expression, with an intact cytoplasmic tail, was necessary and sufficient to mediate p80-dependent chemotaxis. To examine the role for Rbeta1 in mediating macrophage accumulation in vivo, we contrasted Sendai virus-driven airway inflammation in wild-type and Rbeta1-deficient mice. Despite similar viral burden and production of the macrophage chemoattractant p80, the Rbeta1-deficient mice displayed a selective decrease in airway macrophage accumulation and resistance to viral-dependent mortality. Thus, Rbeta1 mediates p80-dependent macrophage chemotaxis and inhibition of the p80-Rbeta1 interaction may provide a novel anti-inflammatory strategy to manipulate the inflammation associated with asthma and respiratory viral infection.     

Paclitaxel enhances macrophage IL-12 production in tumor-bearing hosts through nitric oxide.

Tumor-induced macrophages (Mphis) mediate immunosuppression, in part, through increased production of factors that suppress T cell responsiveness and underproduction of positive regulatory cytokines. Pretreatment of tumor-bearing host (TBH) Mphis with the anticancer agent paclitaxel (Taxol) partially reverses tumor-induced Mphi suppressor activity, suggesting that paclitaxel may restore TBH Mphi production of proimmune factors. Because paclitaxel demonstrates LPS-mimetic capabilities and increased production of the LPS-induced immunostimulatory cytokine IL-12 could account for enhanced T cell responsiveness, we investigated whether paclitaxel induces Mphi IL-12 production. Tumor growth significantly down-regulated Mphi IL-12 p70 production through selective dysregulation of IL-12 p40 expression. LPS stimulation failed to overcome tumor-induced dysregulation of p40 expression. In contrast, paclitaxel significantly enhanced both normal host and TBH Mphi IL-12 p70 production in vitro, although TBH Mphi IL-12 production was lower than that of similarly treated normal host Mphis. Paclitaxel enhanced p40 expression in a dose-dependent manner. Through reconstituted Mphi IL-12 expression, paclitaxel pretreatment relieved tumor-induced Mphi suppression of T cell alloreactivity. Blocking Mphi NO suppressed paclitaxel's ability to induce IL-12 production. This suggests that paclitaxel-induced activities may involve a NO-mediated autocrine induction pathway. Collectively, these data demonstrate that paclitaxel restores IL-12 production in the TBH and ascribe a novel immunotherapeutic component to the pleiotropic activities of NO. Through its capacity to induce IL-12 production, paclitaxel may contribute to the correction of tumor-induced immune dysfunction.