Helicobacter pylori-secreted factors inhibit dendritic cell IL-12 secretion: a mechanism of ineffective host defense

Helicobacter pylori evades host immune defenses and causes chronic gastritis. Immunity against intestinal pathogens is largely mediated by dendritic cells, yet the role of dendritic cells in acute H. pylori infection is largely unknown. We observed the recruitment of dendritic cells to the gastric mucosa of H. pylori-infected mice. Bone marrow-derived dendritic cells from mice responded to live H. pylori by upregulating the expression of proinflammatory cytokine mRNA (i.e., IL-1alpha, IL-1beta, and IL-6). The supernatant from dendritic cells stimulated with H. pylori for 18 h contained twofold higher levels of IL-12p70 than IL-10 and induced the proliferation of syngeneic splenocytes and type 1 T helper cell cytokine release (IFN-gamma and TNF-alpha). These responses were significantly lower compared with those induced by Acinetobacter lwoffi, another gastritis-causing pathogen more susceptible to host defenses. Analysis of whole H. pylori sonicate revealed the presence of a heat-stable factor secreted from H. pylori that specifically inhibited IL-12 but not IL-10 release from dendritic cells activated by A. lwoffi. Our findings suggest that dendritic cells participate in the host immune response against H. pylori and that their suppression by H. pylori may explain why infected hosts fail to prevent bacterial colonization.     

Somatostatin inhibits dendritic cell responsiveness to Helicobacter pylori

Somatostatin is a regulatory peptide found in abundance in the stomach. We have previously shown that somatostatin is required for IL-4-mediated resolution of Helicobacter pylori gastritis. In the current study, we hypothesize that somatostatin acts directly on antigen-presenting cells in the stomach to lessen the severity of gastritis. To test this hypothesis, we first show that CD11c+ dendritic cells are present in the infected tissue of mice with H. pylori-induced gastritis. Pretreatment of bone marrow-derived dendritic cells with somatostatin results in decreased IL-12 production, and lower splenocyte proliferation induced by H. pylori-stimulated dendritic cells. Furthermore, octreotide, a somatostatin analogue, is more potent than somatostatin in suppressing IL-12 release by H. pylori-stimulated dendritic cells through an NF-kappaB-independent pathway. In addition, IL-4 stimulates somatostatin secretion from dendritic cells. In conclusion, somatostatin inhibits dendritic cell activation by H. pylori; a possible mechanism by which IL-4 mediates resolution of gastritis. We suggest that octreotide may be effective in treating immune-mediated diseases of the stomach.     

Modulatory effect of HHV-6 on MCP-1 production by human monocytes

Chemokines represent a large family of proinflammatory proteins that orchestrate leukocyte trafficking to sites of viral infection. Human Herpes virus type 6 (HHV-6) is a typical immunosuppressive agent, as suggested by its tropism. In this study the production of monocyte chemoattractant protein-1 (MCP-1) and interleukin-10 (IL-10) by human peripheral blood monocytes was evaluated during HHV-6 infection. Our results demonstrate that HHV-6 infection triggers monocytes to release MCP-1 and IL-10. The addition of exogenous recombinant MCP-1 upregulates the release of extracellular virus, whereas does not influence the percentage of viral-antigen positive cells. Furthermore, the addition of monoclonal antibodies anti-IL-10 down-regulates MCP-1 release induced by HHV-6. These findings indicate that IL-10 and MCP-1 production was closely related and that the marked amounts of MCP-1 were supported not only by virus but also by virus-induced IL-10.     

Interferon-gamma Stimulates Monocyte Chemotactic Protein-1 Expression by Monocytes

Monocyte chemotactic protein (MCP-1) is a specific monocyte chemoattractant and activating factor produced by both immune cells (mononuclear phagocytes and lymphocytes) and non-immune cells (parenchymal and stromal cells). In order to define the conditions under which human monocytes express MCP-1, monocytes were exposed to IFN-gamma, IL- lbeta, TNF-alpha, IL-4 or PHA under serum free conditions. There was no significant MCP-1 production by monocytes following exposure to IL-lbeta, TNF-alpha or IL-4. In contrast, stimulation with IFN-gamma resulted in a dose dependent increase in MCP-1 protein and mRNA expression. Simultaneous stimulation with IFN-gamma and IL-1beta or TNF-alpha resulted in no further increase in MCP-1 production. It is concluded that IFN-gamma, primarily a product of T(H)1 T lymphocytes, stimulates the expression of MCP-1 by monocytes.     

Evidence for interleukin-1-independent stimulation of interleukin-12 and down-regulation by interleukin-10 in Helicobacter pylori-infected murine dendritic cells deficient in the interleukin-1 receptor

Helicobacter pylori infection is characterized by infiltration of cells of the immune system, including dendritic cells, into the gastric mucosa. During chronic inflammation with Helicobacter pylori infection, a variety of cytokines are secreted into the mucosa, including interleukin-1beta (IL-1beta). The role of IL-1 in H. pylori infection was investigated using bone-marrow-derived dendritic cells from wild-type and IL-1 receptor-deficient (IL-1R-/-) mice. Dendritic cells were incubated with H. pylori at a multiplicity of infection of 10 and 100, and cytokine production evaluated. Helicobacter pylori SS1, H. pylori SD4, and an isogenic cagE mutant of SD4 stimulated IL-12, IL-6, IL-1beta, IL-10, and tumor necrosis factor-alpha at comparable levels in dendritic cells from both wild-type and IL-1R-/- mice. IL-10 production required the higher inoculum, while IL-12 was decreased at this bacterial load. Pretreatment of dendritic cells with an antibody to IL-10 resulted in an increased production of IL-12, confirming the down-regulation of IL-12 by IL-10. cagE was required for maximum stimulation of IL-12 by H. pylori. We speculate that the down-regulation of IL-12 by IL-10 at the higher multiplicity of infection represents the modulation of the host inflammatory response in vivo by H. pylori when the bacterial load is high, allowing for persistent colonization of the gastric mucosa.     

The Helicobacter pylori plasticity region locus jhp0947-jhp0949 is associated with duodenal ulcer disease and interleukin-12 production in monocyte cells

Colonization with Helicobacter pylori always results in chronic gastritis, which is controlled by infiltration of mononuclear cells and the subsequent release of cytokines like interleukin (IL)-12. To identify H. pylori factors involved in inducing cytokine production in mononuclear cells, a random H. pylori mutant library was screened for the inability to induce IL-12 production in monocyte THP-1 cells. Of the 231 random mutants screened, one mutant (M1) showed a consistent twofold decrease in the amount of IL-12 induction compared to the parental strain 1061 (P <0.01). Further characterization of mutant M1 revealed that the kanamycin resistance cassette had integrated in the jhp0945 gene, which is situated in an H. pylori strain-specific plasticity region. Three reference strains possessing this plasticity region induced significantly higher amounts of IL-12 when compared to the H. pylori 26695 reference strain, which does not possess this plasticity region. The role in disease outcome of jhp0945 as well as the neighbouring plasticity region genes jhp0947 and jhp049 was assessed in a Dutch population cohort. Firstly, the presence of jhp0947 was completely linked with that of jhp0949 and was roughly associated with jhp0945 (P=0.072), but not with the cag pathogenicity island (PAI) (P=0.464). The presence of the jhp0947 and jhp0949 genes, but not of jhp0945, was significantly associated with duodenal ulcer disease when compared to gastritis (P=0.027). Therefore, the jhp0947-jhp0949 locus may be a novel putative H. pylori marker for disease outcome independent of the cag PAI.     

Immunological activity of lipopolysaccharide of Helicobacter pylori on human peripheral mononuclear blood cells in comparison to lipopolysaccharides of other intestinal bacteria

Abstract Lipopolysaccharide of Helicobacter pylori was tested for its mitogenicity and for its ability to stimulate cytokine release in human peripheral blood mononuclear cells (PBMC) of healthy and H. pylori -infected blood donors. Mitogenicity in PBMC induced by H. pylori LPS was similar to that induced by Campylobacter jejuni lipopolysaccharide, but lower than that induced by Escherichia coli lipopolysaccharide in the H. pylori negative blood donor group. Furthermore, H. pylori LPS was able to induce tumour necrosis factor (TNF) interleukin 1 (IL-1) and interleukin 6 (IL-6) secretion of PBMC. Compared with the ability of C. jejuni and E. coli lipopolysaccharides to stimulate cytokine release, H. pylori lipopolysaccharide induced a significantly lower TNF and IL-1 secretion of PBMC than the other tested bacterial lipopolysaccharides. Similar amounts of IL-6 release were obtained by stimulation of PBMC with H. pylori and C. jejuni lipopolysaccharides, whereas a higher IL-6 release was measured by stimulation with E. coli lipopolysaccharide. The results of this study suggest that H. pylori lipopolysaccharide has a lower immunological activity than lipopolysaccharides of other intestinal bacteria. This is probably due to its unusual acylation and phosphorylation pattern of lipid A.     

Proinflammatory response of alveolar epithelial cells is enhanced by alveolar macrophage-produced TNF-alpha during pulmonary ischemia-reperfusion injury

Pulmonary ischemia-reperfusion (IR) injury entails acute activation of alveolar macrophages followed by neutrophil sequestration. Although proinflammatory cytokines and chemokines such as TNF-alpha and monocyte chemoattractant protein-1 (MCP-1) from macrophages are known to modulate acute IR injury, the contribution of alveolar epithelial cells to IR injury and their intercellular interactions with other cell types such as alveolar macrophages and neutrophils remain unclear. In this study, we tested the hypothesis that following IR, alveolar macrophage-produced TNF-alpha further induces alveolar epithelial cells to produce key chemokines that could then contribute to subsequent lung injury through the recruitment of neutrophils. Cultured RAW264.7 macrophages and MLE-12 alveolar epithelial cells were subjected to acute hypoxia-reoxygenation (H/R) as an in vitro model of pulmonary IR. H/R (3 h/1 h) significantly induced KC, MCP-1, macrophage inflammatory protein-2 (MIP-2), RANTES, and IL-6 (but not TNF-alpha) by MLE-12 cells, whereas H/R induced TNF-alpha, MCP-1, RANTES, MIP-1alpha, and MIP-2 (but not KC) by RAW264.7 cells. These results were confirmed using primary murine alveolar macrophages and primary alveolar type II cells. Importantly, using macrophage and epithelial coculture methods, the specific production of TNF-alpha by H/R-exposed RAW264.7 cells significantly induced proinflammatory cytokine/chemokine expression (KC, MCP-1, MIP-2, RANTES, and IL-6) by MLE-12 cells. Collectively, these results demonstrate that alveolar type II cells, in conjunction with alveolar macrophage-produced TNF-alpha, contribute to the initiation of acute pulmonary IR injury via a proinflammatory cascade. The release of key chemokines, such as KC and MIP-2, by activated type II cells may thus significantly contribute to neutrophil sequestration during IR injury.     

Macrolide-affected Toll-like receptor 4 expression from Helicobacter pylori-infected monocytes does not modify interleukin-8 production

Macrolide antibiotics have an anti-inflammatory effect by suppressing lipopolysaccharide-induced IL-8 production. IL-8 secretion from monocytes is observed in Helicobacter pylori infection. Although cag gene products are known to induce IL-8 secretion, whether other bacterial substances can initiate the reaction is not determined. In this study, we show that clarithromycin induced down-regulation of Toll-like receptor 4 expression and did not lead to a decrease in IL-8 production and H. pylori lipopolysaccharide. However, Toll-like receptor 4 activation was possibly not the main cause in the induction of inflammation during H. pylori infection.     

Suppression of IL-8 production in gastric epithelial cells by MUC1 mucin and peroxisome proliferator-associated receptor-γ

MUC1 is a membrane-tethered mucin expressed on the apical surface of epithelial cells. Our previous report (Guang W, Ding H, Czinn SJ, Kim KC, Blanchard TG, Lillehoj EP. J Biol Chem 285: 20547-20557, 2010) demonstrated that expression of MUC1 in AGS gastric epithelial cells limits Helicobacter pylori infection and reduces bacterial-driven IL-8 production. In this study, we identified the peroxisome proliferator-associated receptor-γ (PPARγ) upstream of MUC1 in the anti-inflammatory pathway suppressing H. pylori- and phorbol 12-myristate 13-acetate (PMA)-stimulated IL-8 production. Treatment of AGS cells with H. pylori or PMA increased IL-8 levels in cell culture supernatants compared with cells treated with the respective vehicle controls. Prior small interfering (si)RNA-induced MUC1 silencing further increased H. pylori- and PMA-stimulated IL-8 levels compared with a negative control siRNA. MUC1-expressing AGS cells pretreated with the PPARγ agonist troglitazone (TGN) had reduced H. pylori- and PMA-stimulated IL-8 levels compared with cells treated with H. pylori or PMA alone. However, following MUC1 siRNA knockdown, no differences in IL-8 levels were seen between TGN/H. pylori and H. pylori-only cells or between TGN/PMA and PMA-only cells. Finally, TGN-treated AGS cells had increased Muc1 promoter activity, as measured using a Muc1-luciferase reporter gene, and greater MUC1 protein levels by Western blot analysis, compared with vehicle controls. These results support the hypothesis that PPARγ stimulates MUC1 expression by AGS cells, thereby attenuating H. pylori- and PMA-induced IL-8 production.     

Human dendritic cells discriminate between viable and killed Toxoplasma gondii tachyzoites: dendritic cell activation after infection with viable parasites results in CD28 and CD40 ligand signaling that controls IL-12-dependent and -independent T cell production of IFN-gamma

We studied how the interaction between human dendritic cells (DC) and Toxoplasma gondii influences the generation of cell-mediated immunity against the parasite. We demonstrate that viable, but not killed, tachyzoites of T. gondii altered the phenotype of immature DC. DC infected with viable parasites up-regulated the expression of CD40, CD80, CD86, and HLA-DR and down-regulated expression of CD115. These changes are indicative of DC activation induced by T. gondii. Viable and killed tachyzoites had contrasting effects on cytokine production. DC infected with viable T. gondii rather than DC that phagocytosed killed parasites induced secretion of high amounts of IFN-gamma by T cells from T. gondii-seronegative donors. IFN-gamma production in response to DC infected with viable parasites required CD28 and CD40 ligand (CD40L) signaling. In addition, this IFN-gamma response was dependent in part on IL-12 secretion. Production of IL-12 p70 occurred after interaction between T cells and DC infected with viable T. gondii, but not after incubation of T cells with DC plus killed tachyzoites. IL-12 synthesis was inhibited by blockade of CD40L signaling. IL-12-independent IFN-gamma production required CD80/CD86-CD28 interaction and, to a lesser extent, CD40-CD40L signaling. Taken together, T. gondii-induced activation of human DC is associated with T cell production of IFN-gamma through CD40-CD40L-dependent release of IL-12 and through CD80/CD86-CD28 and CD40-CD40L signaling that mediate IFN-gamma secretion even in the absence of bioactive IL-12.     

Selective suppression of IL-12 production by chemoattractants

We investigated the ability of chemoattractants to affect IL-12 production by human monocytes and dendritic cells. We found that pretreatment of monocytes with macrophage chemoattractant proteins (MCP-1 to -4), or C5a, but not stromal-derived factor-1, macrophage inflammatory protein-1alpha, RANTES, or eotaxin, inhibited IL-12 p70 production in response to stimulation with Staphylococcus aureus, Cowan strain 1 (SAC), and IFN-gamma. The production of TNF-alpha and IL-10, however, was minimally affected by any of the chemoattractants. The degree of inhibition of IL-12 p70 production by MCP-1 to -4 was donor dependent and was affected by the autocrine inhibitory effects of IL-10. In contrast, C5a profoundly suppressed IL-12 production in an IL-10-independent fashion. Neither TGF-beta1 nor PGE2 was important for the suppression of IL-12 by any of the chemoattractants tested. The accumulation of mRNA for both IL-12 p35 and p40 genes was inhibited by chemokine pretreatment. Interestingly, MCP-1 to -4 and C5a did not suppress IL-12 production by monocyte-derived dendritic cells (DC) stimulated with CD40 ligand and IFN-gamma or by SAC and IFN-gamma, suggesting that these factors may act at the site of inflammation to suppress IL-12 and IFN-gamma production rather than in the lymph node to affect T cell priming. Despite the inability of C5a to inhibit IL-12 production by DCs, the receptor for C5a (CD88) was expressed by these cells, and recombinant C5a induced a Ca2+ flux. Taken together, these results define a range of chemoattractant molecules with the ability to suppress IL-12 production by human monocytes and have broad implications for the regulation of immune responses in vivo.     

Different modulation by live or killed Helicobacter pylori on cytokine production from peripheral blood mononuclear cells

The mechanisms by which H. pylori colonizes and persists within the gastric mucosa are poorly understood. The induction and maintenance of gastric inflammation appear to depend on the complex interaction between a number of cytokines and chemokines. The gastric immune response observed "in vivo", during H. pylori infection, is characterized by a polarization of Th1 cell type that seems to be responsible for gastric pathology. The purpose of this study was to test the direct effect of H. pylori (live or gentamicin-killed) on human PBMC in order to evaluate the "in vitro" Th1-Th2 balance by monitoring IL-18, IFNgamma and IL-10 production. This study demonstrates for the first time that "in vitro" pretreatment with gentamicin-killed H. pylori of PBMC, followed by infection with live bacteria, downregulates the production of inflammatory cytokines such as IL-18 and IFNgamma Our results provide a possible strategy to restore the immunological disorders determined by H. pylori infection.     

Dendritic cells express CCR7 and migrate in response to CCL19 (MIP-3beta) after exposure to Helicobacter pylori

Helicobacter pylori infection induces chronic inflammation in the gastric mucosa with a marked increase in the number of lymphoid follicles consisting of infiltrating B and T cells, neutrophils, dendritic cells (DC) and macrophages. It has been suggested that an accumulation of mature DC in the tissue, resulting from a failure of DC to migrate to lymph nodes, may contribute to this chronic inflammation. Migration of DC to lymph nodes is regulated by chemokine receptor CCR7, expressed on mature DC, and the CCR7 ligands CCL19 and CCL21. In this study we analysed the maturation, in vitro migration and cytokine production of human DC after stimulation with live H. pylori. For comparison, DC responses to non-pathogenic Escherichia coli bacteria were also evaluated. Stimulation with H. pylori induced maturation of DC, i.e. up-regulation of the chemokine receptors CCR7 and CXCR4 and the maturation markers HLA-DR, CD80 and CD86. The H. pylori-stimulated DC also induced CD4(+) T-cell proliferation. DC stimulated with H. pylori secreted significantly more interleukin (IL)-12 compared to DC stimulated with E. coli, while E. coli-stimulated DC secreted more IL-10. Despite low surface expression of CCR7 protein following stimulation with H. pylori compared to E. coli, the DC migrated equally well towards CCL19 after stimulation with both bacteria. Thus, we could not detect any failure in the migration of H. pylori stimulated DC in vitro that may contribute to chronic gastritis in vivo, and our results suggest that H. pylori induces maturation and migration of DC to lymph nodes where they promote T cell responses.     

Intraperitoneal immunization led to T cell hyporesponsiveness to Helicobacter pylori infection in mice

During Helicobacter pylori infection, T cell response is critical in the development of active gastritis and in protective immunity against infection. We studied gastric inflammation and T cell response in H. pylori-challenged mice following an intraperitoneal immunization, using whole H. pylori lysate (HpAg) in the absence of adjuvants. H. pylori-challenged mice without immunization developed moderate to severe gastric inflammation, and splenocytes from these mice produced Th1 polarizing cytokines in response to HpAg and Con A during the acute infection. On the other hand, immunized-challenged mice (those inoculated with H. pylori following immunization) had little or no gastric inflammation despite persistent H. pylori colonization. Our immunization primed splenocytes to produce IL-2, IFN-gamma, and IL-4 in response to HpAg and Con A before infection. However, these cells became hyporesponsive to both stimulants immediately after live bacterial challenge in terms of the production of these cytokines, especially IL-2 and IFN-gamma. CTLA-4 has been documented to be a negative regulator of IL-2 production and lymphoproliferation that induces peripheral tolerance and functions 24-72 hr after the initiation of T cell activation. Compared with challenged mice, T cells from immunized-challenged mice showed higher levels of CTLA-4 expression at 72 hr after oral challenge. These data suggested that our immunization inhibited the development of H. pylori-associated gastritis and induced T cell hyporesponsiveness to H. pylori infection, which might be mediated by the early induction of CTLA-4 following challenge.     

A change of IL-2 and IL-4 production in patients with Helicobactor pylori infection

Hellcobacter pylori is the most common cause of gastroduodenal inflammation. However, the exact immune pathogenesis is not fully understood. To look for evidence of the immunological mechanism in H. pylori associated disease, we measured cytokine interleukin-2 (IL-2) and IL-4 levels produced by peripheral blood lymphocytes (PBL) and gastric biopsies in 20 subjects with or without H. pylori infection. H. pylori can stimulate IL-2 and IL-4 production from PBL in H. pylori negative as well as H. pylori positive individuals. The spontaneous IL-2 production by PBL and gastric biopsies was greater (p < 0.0025, <0.001)in H. pylori negative individuals than that in H. pylori infected patients. Increased IL-4 levels from PBL in H. pylori infected patients were found in the presence of H. pylori (p < 0.0025). An increased spontaneous production of IL-4 from gastric biopsies was also observed in H. pylori infected patients (p < 0.025). In conclusion, an enhanced type 2 cytokine production was observed in H. pylori infected patients, which may be responsible for H. pylori chronic infection.     

Activation of human neutrophils with Helicobacter pylori and the role of Toll-like receptors 2 and 4 in the response

The innate immune response to Helicobacter pylori infection is beginning to be understood and recent works support a role for Toll-like receptors (TLRs). Our aim was to study the response of human neutrophils to H. pylori and to elucidate the role of TLR2 and TLR4. Neutrophils from healthy H. pylori-negative volunteers were cocultured with H. pylori 26695 strain. The release of IL-8, IL-1beta, tumor necrosis factor (TNF)-alpha and IL-10 was measured. The role of TLR2 and TLR4 was investigated with blocking assays using monoclonal antibodies against TLRs. Neutrophils produced a significant increase of IL-8, IL-1beta and TNF-alpha after 3, 6 and 24 h of H. pylori challenge, respectively, whereas IL-10 increased after 24 h. Helicobacter pylori challenge increased TLR2 and TLR4 expression; and antibodies against TLR2 and TLR4 diminished significantly the H. pylori-induced production of IL-8 and IL-10. In human neutrophils, H. pylori induces an early inflammatory response, partially mediated via TLR2 and TLR4 activation.     

Key role of regulated upon activation normal T-cell expressed and secreted, nonstructural protein1 and myeloperoxidase in cytokine storm induced by influenza virus PR-8 (A/H1N1) infection in A549 bronchial epithelial cells

Influenza virus infection causes severe respiratory disease such as that due to avian influenza (H5N1). Influenza A viruses proliferate in human epithelial cells, which produce inflammatory cytokines/chemokines as a "cytokine storm" attenuated with the viral nonstructural protein 1 (NS1). Cytokine/chemokine production in A549 epithelial cells infected with influenza A/H1N1 virus (PR-8) or nonstructural protein 1 (NS1) plasmid was examined in vitro. Because tumor necrosis factor-α (TNF-α) and regulated upon activation normal T-cell expressed and secreted (RANTES) are predominantly produced from cells infected with PR-8 virus, the effects of mRNA knockdown of these cytokines were investigated. Small interfering (si)TNF-α down-regulated RANTES expression and secretion of RANTES, interleukin (IL)-8, and monocyte chemotactic protein-1 (MCP-1). In addition, siRANTES suppressed interferon (IFN)-γ expression and secretion of RANTES, IL-8, and MCP-1, suggesting that TNF-α stimulates production of RANTES, IL-8, MCP-1, and IFN-γ, and RANTES also increased IL-8, MCP-1, and IFN-γ. Furthermore, administration of TNF-α promoted increased secretion of RANTES, IL-8, and MCP-1. Administration of RANTES enhanced IL-6, IL-8, and MCP-1 production without PR-8 infection. These results strongly suggest that, as an initial step, TNF-α regulates RANTES production, followed by increase of IL-6, IL-8, and MCP-1 and IFNs concentrations. At a later stage, cells transfected with viral NS1 plasmid showed production of a large amount of IL-8 and MCP-1 in the presence of the H(2)O(2)-myeloperoxidse (MPO) system, suggesting that NS1 of PR-8 may induce a "cytokine storm" from epithelial cells in the presence of an H(2)O(2)-MPO system.     

Src family kinases regulate p38 MAPK-mediated IL-6 production in Kupffer cells following hypoxia

Tissue hypoxia is a common sequel of trauma-hemorrhage but can occur even without blood loss under hypoxic conditions. Although hypoxia is known to upregulate Kupffer cells (KC) to release cytokines, the precise mechanism of release remains unknown. We hypothesized that Src family kinases play a role in mediating KC mitogen-activated protein kinase (MAPK) signaling and their cytokine production after hypoxia. Male C3H/HeN mice received either Src inhibitor PP1 (1.5 mg/kg body wt) or vehicle 1 h before hypoxia. KCs were isolated 1 h after hypoxia, lysed, and immunoblotted with antibodies to Src, p38, ERK1/2, or JNK proteins. In addition, KCs were cultured to measure interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1) production. Hypoxia produced a significant increase in KC Src and MAPK (p38, ERK, JNK) activity compared with normoxic controls. This was associated with an increase in IL-6 and MCP-1 production. Treatment with PP1 abolished the increase in KC Src activation as well as p38 activity. However, PP1 did not prevent the increase in KC ERK1/2 or JNK phosphorylation. Furthermore, administration of PP1 prevented the hypoxia-induced increase in IL-6 but not MCP-1 release by KC. Additional in vitro results suggest that p38 but not ERK1/2 or JNK are critical for KC IL-6 production. In contrast, the production of MCP-1 by KC was found to be independent of MAPK. Thus hypoxia increases KC IL-6 production by p38 MAPK activation via Src-dependent pathway. Src kinases may therefore be a novel therapeutic target for preventing immune dysfunction following low-flow conditions in trauma patients. innate immunity; macrophages; cell signaling