Distinct role of p38 and c-Jun N-terminal kinases in IL-10-dependent and IL-10-independent regulation of the costimulatory molecule B7.2 in lipopolysaccharide-stimulated human monocytic cells

The costimulatory molecule B7.2 (CD86) plays a vital role in immune activation and development of Th responses. The molecular mechanisms by which B7.2 expression is regulated are not understood. We investigated the role of mitogen-activated protein kinases (MAPK) in the regulation of B7.2 expression in LPS-stimulated human monocytic cells. LPS stimulation of human monocytes resulted in the down-regulation of B7.2 expression that could be abrogated by anti-IL-10 Abs. Furthermore, SB202190, a specific inhibitor of p38 MAPK, inhibited LPS-induced IL-10 production and reversed B7.2 down-regulation, suggesting that LPS-induced B7.2 down-regulation may be mediated, at least in part, via regulation of IL-10 production by p38 MAPK. In contrast to human promonocytic THP-1 cells that are refractory to the inhibitory effects of IL-10, LPS stimulation enhanced B7.2 expression. This IL-10-independent B7.2 induction was not influenced by specific inhibitors of either p38 or p42/44 MAPK. To ascertain the role of the c-Jun N-terminal kinase (JNK) MAPK, dexamethasone, an inhibitor of JNK activation, was used, which inhibited LPS-induced B7.2 expression. Transfection of THP-1 cells with a plasmid expressing a dominant-negative stress-activated protein/extracellular signal-regulated kinase kinase 1 significantly reduced LPS-induced B7.2 expression, thus confirming the involvement of JNK. To study the signaling events downstream of JNK activation, we show that dexamethasone did not inhibit LPS-induced NF-kappaB activation in THP-1 cells, suggesting that JNK may not be involved in NF-kappaB activation leading to B7.2 expression. Taken together, our results reveal the distinct involvement of p38 in IL-10-dependent, and JNK in IL-10-independent regulation of B7.2 expression in LPS-stimulated monocytic cells.     

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.     

Lipopolysaccharide-induced c-Jun NH2-terminal kinase activation in human neutrophils: role of phosphatidylinositol 3-Kinase and Syk-mediated pathways

Polymorphonuclear leukocytes (neutrophils) respond to lipopolysaccharide (LPS) through the up-regulation of several pro-inflammatory mediators. We have recently shown that LPS-stimulated neutrophils express monocyte chemoattractant protein 1 (MCP-1), an AP-1-dependent gene, suggesting that LPS activates the c-Jun N-terminal kinase (JNK) pathway in neutrophils. Previously, we have shown the activation of p38 MAPK, but not JNK, in suspended neutrophils stimulated with LPS but have recently shown activation of JNK by TNF-alpha in an adherent neutrophil system. We show here that exposure to LPS activates JNK in non-suspended neutrophils and that LPS-induced MCP-1 expression, but not tumor necrosis factor-alpha (TNF-alpha) or interleukin-8 (IL-8), is dependent on JNK activation. In addition, LPS stimulation of non-suspended neutrophils activates Syk and phosphatidylinositol 3-kinase (PI3K). Inhibition of Syk with piceatannol or PI3K with wortmannin inhibited LPS-induced JNK activation and decreased MCP-1 expression after exposure to LPS, suggesting that both Syk and PI3K reside in a signaling pathway leading to LPS-induced JNK activation in neutrophils. This Syk- and PI3K-dependent pathway leading to JNK activation after LPS exposure in non-suspended neutrophils is specific for JNK, because inhibition of neither Syk nor PI3K decreased p38 activation after LPS stimulation. Furthermore we show that PI3K inhibition decreased LPS-induced Syk activation suggesting that PI3K resides upstream of Syk in this pathway. Finally, we show that Syk associates with Toll-like receptor 4 (TLR4) upon LPS stimulation further implicating Syk in the LPS-induced signaling pathway in neutrophils. Overall our data suggests that LPS induces JNK activation only in non-suspended neutrophils, which proceeds through Syk- and PI3K-dependent pathways, and that JNK activation is important for LPS-induced MCP-1 expression but not for TNF-alpha or IL-8 expression.     

Differential regulation of CD44 expression by lipopolysaccharide (LPS) and TNF-alpha in human monocytic cells: distinct involvement of c-Jun N-terminal kinase in LPS-induced CD44 expression

Alterations in the regulation of CD44 expression play a critical role in modulating cell adhesion, migration, and inflammation. LPS, a bacterial cell wall component, regulates CD44 expression and may modulate CD44-mediated biological effects in monocytic cells during inflammation and immune responses. In this study, we show that in normal human monocytes, LPS and LPS-induced cytokines IL-10 and TNF-alpha enhance CD44 expression. To delineate the mechanism underlying LPS-induced CD44 expression, we investigated the role of the mitogen-activated protein kinases (MAPKs), p38, p42/44 extracellular signal-regulated kinase, and c-Jun N-terminal kinase (JNK) by using their specific inhibitors. We demonstrate the involvement, at least in part, of p38 MAPK in TNF-alpha-induced CD44 expression in both monocytes and promonocytic THP-1 cells. However, neither p38 nor p42/44 MAPKs were involved in IL-10-induced CD44 expression in monocytes. To further dissect the TNF-alpha and LPS-induced signaling pathways regulating CD44 expression independent of IL-10-mediated effects, we used IL-10 refractory THP-1 cells as a model system. Herein, we show that CD44 expression induced by the LPS-mediated pathway predominantly involved JNK activation. This conclusion was based on results derived by transfection of THP-1 cells with a dominant-negative mutant of stress-activated protein/extracellular signal-regulated kinase kinase 1, and by exposure of cells to JNK inhibitors dexamethasone and SP600125. All these treatments prevented CD44 induction in LPS-stimulated, but not in TNF-alpha-stimulated, THP-1 cells. Furthermore, we show that CD44 induction may involve JNK-dependent early growth response gene activation in LPS-stimulated monocytic cells. Taken together, these results suggest a predominant role of JNK in LPS-induced CD44 expression in monocytic cells.     

Tumor necrosis factor-alpha induces functionally active hyaluronan-adhesive CD44 by activating sialidase through p38 mitogen-activated protein kinase in lipopolysaccharide-stimulated human monocytic cells

Interaction of CD44, an adhesion molecule, with its ligand, hyaluronan (HA), in monocytic cells plays a critical role in cell migration, inflammation, and immune responses. Most cell types express CD44 but do not bind HA. The biological functions of CD44 have been attributed to the generation of the functionally active, HA-adhesive form of this molecule. Although lipopolysaccharide (LPS) and cytokines induce HA-adhesive CD44, the molecular mechanism underlying this process remains unknown. In this study, we show that LPS-induced CD44-mediated HA (CD44-HA) binding in monocytes is regulated by endogenously produced tumor necrosis factor (TNF)-alpha and IL-10. Furthermore, p38 mitogen-activated protein kinase (MAPK) activation was required for LPS- and TNF-alpha-induced, but not IL-10-induced, CD44-HA-binding in normal monocytes. To dissect the signaling pathways regulating CD44-HA binding independently of cross-regulatory IL-10-mediated effects, IL-10-refractory promonocytic THP-1 cells were employed. LPS-induced CD44-HA binding in THP-1 cells was regulated by endogenously produced TNF-alpha. Our results also suggest that lysosomal sialidase activation may be required for the acquisition of the HA-binding form of CD44 in LPS- and TNF-alpha-stimulated monocytic cells. Studies conducted to understand the role of MAPKs in the induction of sialidase activity revealed that LPS-induced sialidase activity was dependent on p42/44 MAPK-mediated TNF-alpha production. Blocking TNF-alpha production by PD98059, a p42/44 inhibitor, significantly reduced the LPS-induced sialidase activity and CD44-HA binding. Subsequently, TNF-alpha-mediated p38 MAPK activation induced sialidase activity and CD44-HA binding. Taken together, our results suggest that TNF-alpha-induced p38 MAPK activation may regulate the induction of functionally active HA-binding form of CD44 by activating sialidase in LPS-stimulated human monocytic cells.     

Implications of proteasome inhibition: an enhanced macrophage phenotype

The objective of this study was to elucidate the role of the cellular proteasome on endotoxin-mediated activation of the macrophage. To study this role, THP-1 cells were stimulated with lipopolysaccharide (LPS) with selective cells being pretreated with the proteasome inhibitor, lactacystin or MG-132. LPS stimulation led to the phosphorylation and degradation of IRAK, followed by activation of JNK/SAPK, ERK 1/2, and p38. Subsequently, LPS induced the degradation of IkappaB, and the nuclear activation of NF-kappaB and AP-1. Activation of these pathways was associated with the production of IL-6, IL-8, IL-10, and TNF-alpha. Proteasome inhibition with either lactacystin or MG-132 attenuated LPS-induced IRAK degradation, and enhanced activation of JNK/SAPK, ERK 1/2, and p38. Proteasome inhibition, also, led to increased LPS-induced AP-1 activation, and attenuated LPS-induced IkappaB degradation resulting in abolished NF-kappaB activation. Proteasome inhibition led to significant modulation of LPS-induced cytokine production; increased IL-10, no change in IL-6, and decreased IL-8, and TNF-alpha. Thus, this study demonstrates that cellular proteasome is critical to regulation of LPS-induced signaling within the macrophage, and inhibition of the proteasome results in a conversion to an anti-inflammatory phenotype.     

Interleukin-10 inhibits tumor necrosis factor-alpha production in lipopolysaccharide-stimulated RAW 264.7 cells through reduced MyD88 expression

The mechanism of interleukin (IL)-10-mediated inhibition of tumor necrosis factor (TNF)-alpha production was studied by lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells. IL-10 inhibited TNF-alpha production transiently at an early stage after LPS stimulation. IL-10 inhibited the activation of nuclear factor (NF)-kappaB, p38 and stress-activated protein kinase (SAPK) in LPS-stimulated RAW 264.7 cells. Although the level of MyD88 protein increased in response to LPS, IL-10 prevented the LPS-induced MyD88 augmentation. There was no significant difference in the MyD88 mRNA expression between the cells pretreated with or without IL-10 in response to LPS. Therefore, IL-10 was suggested to inhibit LPS-induced TNF-alpha production via reduced MyD88 expression.     

Induction of heme oxygenase-1 expression in murine macrophages is essential for the anti-inflammatory effect of low dose 15-deoxy-Delta 12,14-prostaglandin J2

15-Deoxy-Delta 12,14-prostaglandin J2 (15d-PGJ2), a cyclopentenone prostaglandin, displays a potent anti-inflammatory effect at micromolar concentrations (>2 microM) through direct inhibition of nuclear factor (NF)-kappa B activation. Here we show that at submicromolar concentrations (0.1-0.5 microM) 15d-PGJ2 retains the ability to suppress the production of tumor necrosis factor-alpha (TNF-alpha) and nitric oxide (NO) in lipopolysaccharide (LPS)-activated murine J774 macrophages under the conditions of a prolonged incubation (>12 h). Western blot analysis revealed that the expression of the cytoprotective enzyme, heme oxygenase-1 (HO-1), was induced and coincident with the anti-inflammatory action of 15d-PGJ2. Inhibition of HO-1 activity or scavenging carbon monoxide (CO), a byproduct derived from heme degradation, significantly attenuated the suppressive activity of 15d-PGJ2. Furthermore, LPS-induced NF-kappa B activation assessed by the inhibitory protein of NF-kappa B(I kappa B) degradation and p50 nuclear translocation was diminished in cells subjected to prolonged treatment with the low concentration of 15d-PGJ2. Treatment of cells with the protein synthesis inhibitor, cycloheximide, or the specific p38 MAP kinase inhibitor, SB203580, blocked the induction of HO-1 and suppression of LPS-induced I kappa B degradation mediated by 15d-PGJ2. Likewise, HO inhibitor and CO scavenger were effective in abolishing the inhibitory effects of 15d-PGJ2 on NF-kappa B activation induced by LPS. The functional role of CO was further demonstrated by the use of a CO releasing molecule, tricarbonyldichlororuthenium(II) dimer, which significantly suppressed LPS-induced nuclear translocation of p50 as assessed by confocal immunofluorescence. Collectively, these data suggest that even at submicromolar concentrations 15d-PGJ2 can exert an anti-inflammatory effect in macrophages through a mechanism that involves the action of HO/CO.     

NF-kappa B regulates the LPS-induced expression of interleukin 12 p40 in murine peritoneal macrophages: roles of PKC, PKA, ERK, p38 MAPK, and proteasome

NF-kappa B plays a critical role in coordinating the control of gene expression during monocyte/macrophage activation. In this report we describe our investigation of the mechanisms of LPS-induced NF-kappa B activation and IL-12 expression in murine peritoneal suppressor macrophages. Treatment of these macrophages with LPS induced I kappa B alpha degradation and NF-kappa B activation. EMSAs demonstrated that NF-kappa B bound to a cis-acting element located in the murine IL-12 p40 promoter. LPS signal transduction has been shown to involve a variety of signal pathways. The results in this paper indicate that LPS-induced NF-kappa B binding activity was independent of PKC, PKA, ERK, and p38 MAPK, but was regulated by proteasome. Furthermore, Proteasome Inhibitor I abolished the LPS-induced mRNA expression of IL-12 p35 and p40, and SB203580 reduced these mRNA levels, whereas the blockade of PKC, PKA, and ERK had little effect. These data demonstrate that the LPS-induced activation of proteasome. I kappa B. NF-kappa B and p38 MAPK signal pathways regulate the IL-12 expression in murine peritoneal suppressor macrophages.     

Sphingosine kinase protects lipopolysaccharide-activated macrophages from apoptosis

Lipopolysaccharide (LPS) signaling is critical for the innate immune response to gram-negative bacteria. Here, evidence is presented for LPS stimulation of sphingosine kinase (SPK) in the RAW 264.7 murine macrophage cell line and rat primary hepatic macrophages (HMs). LPS treatment of RAW 264.7 cells resulted in a time- and dose-dependent activation of SPK and membrane translocation of SPK1. Further, LPS-induced SPK activation was blocked by SPK1-specific small interfering RNA (siRNA). Overexpression of Toll-like receptor 4 and MD2, the receptor and coreceptor of LPS, in HEK 293 cells activated SPK activity in the absence of LPS treatment. Inhibition of SPK by the pharmacological inhibitor N,N-dimethylsphingosine (DMS) or SPK1-specific siRNA blocked LPS stimulation of extracellular signal-regulated kinase 1/2 and p38 but enhanced LPS-induced c-Jun N-terminal kinase activation. The SPK inhibitor DMS and dominant-negative SPK1 also blocked LPS activation of Elk-1 and NF-kappaB reporters in RAW 264.7 cells. Inhibition of SPK sensitized RAW 264.7 cells and HMs to LPS-induced apoptosis. These data demonstrate the critical role of SPK1 in LPS signaling in macrophages and suggest that SPK1 is a potential therapeutic target to block hyperimmune responses induced by gram-negative bacteria.     

RelA/p65 is a molecular target for the immunosuppressive action of protein kinase A.

Stimulation of the protein kinase A (PKA) signalling pathway exerts an inhibitory effect on the proliferation of numerous cells, including T lymphocytes. In CD4+ T helper cells, stimulation of PKA leads to suppression of interleukin 2 (IL-2) induction, while induction of the genes coding for the lymphokines IL-4 and IL-5 is enhanced. We show that the differential effect of PKA activity on induction of the IL-2 and IL-4 genes is mediated through their promoters. One major target of the suppressive effect of PKA is the kappa B site in the IL-2 promoter. A kappa B site is missing in the IL-4 promoter. Mutations preventing factor binding to the IL-2 kappa B site result in a loss of PKA-mediated suppression of IL-2 promoter activity. Furthermore, activation of the PKA signalling pathway impairs the inducible activity of multiple kappa B sites of the IL-2 promoter, but not of other factor binding sites. The reduction in activity of kappa B sites in activated and PKA-stimulated T cells is accompanied by changes in the concentration and DNA binding of Rel/NF-kappa B factors. Stimulation of the PKA pathway in Jurkat T cells with the PKA activator forskolin leads to an increase in synthesis of c-Rel and p105/p50, while synthesis of p65/RelA remains unchanged. However, nuclear translocation and DNA binding of p65 is distinctly impaired, probably due to a retarded degradation of I kappa B-alpha. In a similar way, stimulation of the PKA signalling pathway inhibits nuclear translocation of p65 and generation of nuclear kappa B complexes in peripheral T lymphocytes from murine lymph nodes. These results indicate that PKA-mediated suppression of NF-kappa B activity plays an important role in the control of activation of peripheral T lymphocytes.     

Role of the cyclic AMP-protein kinase A pathway in lipopolysaccharide-induced nitric oxide synthase expression in RAW 264.7 macrophages. Involvement of cyclooxygenase-2.

The signaling pathway for lipopolysaccharide (LPS)-induced nitric oxide (NO) release in RAW 264.7 macrophages involves the protein kinase C and p38 activation pathways (Chen, C. C., Wang, J. K., and Lin, S. B. (1998) J. Immunol. 161, 6206-6214; Chen, C. C., and Wang, J. K. (1999) Mol. Pharmacol. 55, 481-488). In this study, the role of the cAMP-dependent protein kinase A (PKA) pathway was investigated. The PKA inhibitors, KT-5720 and H8, reduced LPS-induced NO release and inducible nitric oxide synthase (iNOS) expression. The direct PKA activator, Bt(2)cAMP, caused concentration-dependent NO release and iNOS expression, as confirmed by immunofluorescence studies. The intracellular cAMP concentration did not increase until after 6 h of LPS treatment. Two cAMP-elevating agents, forskolin and cholera toxin, potentiated the LPS-induced NO release and iNOS expression. Stimulation of cells with LPS or Bt(2)cAMP for periods of 10 min to 24 h caused nuclear factor-kappaB (NF-kappaB) activation in the nuclei, as shown by detection of NF-kappaB-specific DNA-protein binding. The PKA inhibitor, H8, inhibited the NF-kappaB activation induced by 6- or 12-h treatment with LPS but not that induced after 1, 3, or 24 h. The cyclooxygenase-2 (COX-2) inhibitors, NS-398 and indomethacin, attenuated LPS-induced NO release, iNOS expression, and NF-kappaB DNA-protein complex formation. LPS induced COX-2 expression in a time-dependent manner, and prostaglandin E(2) production was induced in parallel. These results suggest that 6 h of treatment with LPS increases intracellular cAMP levels via COX-2 induction and prostaglandin E(2) production, resulting in PKA activation, NF-kappaB activation, iNOS expression, and NO production.     

Selective suppression of neutrophil accumulation in ongoing pulmonary inflammation by systemic inhibition of p38 mitogen-activated protein kinase

The p38 mitogen-activated protein kinase (MAPK) signaling pathway regulates a wide range of inflammatory responses in many different cells. Inhibition of p38 MAPK before exposing a cell to stress stimuli has profound anti-inflammatory effects, but little is known about the effects of p38 MAPK inhibition on ongoing inflammatory responses. LPS-induced activation of p38 MAPK in human neutrophils was inhibited by poststimulation exposure to a p38 MAPK inhibitor (M39). Release of TNF-alpha, macrophage-inflammatory protein (MIP)-2 (MIP-1beta), and IL-8 by LPS-stimulated neutrophils was also reduced by poststimulation p38 MAPK inhibition. In contrast, release of monocyte chemoattractant protein-1 was found to be p38 MAPK independent. Ongoing chemotaxis toward IL-8 was eliminated by p38 MAPK inhibition, although the rate of nondirectional movement was not reduced. A murine model of acute LPS-induced lung inflammation was used to study the effect of p38 MAPK inhibition in ongoing pulmonary inflammation. Initial pulmonary cell responses occur within 4 h of stimulation in this model, so M39 was administered 4 h or 12 h after exposure of the animals to aerosolized LPS to avoid inhibition of cytokine release. Quantities of TNF-alpha, MIP-2, KC, or monocyte chemoattractant protein-1 recovered from bronchial alveolar lavage or serum were not changed. Recruitment of neutrophils, but not other leukocytes, to the airspaces was significantly reduced. Together, these data demonstrate the selective reduction of LPS-induced neutrophil recruitment to the airspaces, independent of suppression of other inflammatory responses. These findings support the feasibility of p38 MAPK inhibition as a selective intervention to reduce neutrophilic inflammation.