Inhibition of apoptosis by Escherichia coli K1 is accompanied by increased expression of BclXL and blockade of mitochondrial cytochrome c release in macrophages

Escherichia coli K1 survival in the blood is a critical step for the onset of meningitis in neonates. Therefore, the circulating bacteria are impelled to avoid host defense mechanisms by finding a niche to survive and multiply. Our recent studies have shown that E. coli K1 enters and survives in both monocytes and macrophages in the newborn rat model of meningitis as well as in macrophage cell lines. Here we demonstrate that E. coli K1 not only extends the survival of human and murine infected macrophage cell lines but also renders them resistant to apoptosis induced by staurosporine. Macrophages infected with wild-type E. coli expressing outer membrane protein A (OmpA), but not with OmpA- E. coli, are resistant to DNA fragmentation and phosphatidylserine exposure induced by staurosporine. Infection with OmpA+ E. coli induces the expression of Bcl(XL), an antiapoptotic protein, both at the mRNA level as assessed by gene array analysis and at the protein level as evaluated by immunoblotting. OmpA- E. coli infection of macrophages induced the release of cytochrome c from mitochondria into the cytosol and the activation of caspases 3, 6, and 9, events that were significantly blocked in OmpA+ E. coli-infected macrophages. In addition, OmpA+ E. coli-infected cells were resistant to a decrease in the transmembrane potential of mitochondria induced by staurosporine as measured by the MitoCapture fluorescence technique. Complementation of OmpA- E. coli with a plasmid containing the ompA gene restored the ability of OmpA- E. coli to inhibit the apoptosis of infected macrophages, further demonstrating that E. coli OmpA expression is critical for inducing macrophage survival and thereby finding a safe haven for its growth.     

Outer membrane protein A of Escherichia coli contributes to invasion of brain microvascular endothelial cells.

Escherichia coli is the most common gram-negative bacteria causing meningitis during the neonatal period, but is unclear what microbial factors mediate traversal of E. coli across the blood-brain barrier. Outer membrane protein A (OmpA), a highly conserved 35-kDa protein, was examined for its role in E. coli K1 invasion of brain microvascular endothelial cells (BMEC). The invasive capability of the OmpA+ strains was 25- to 50-fold greater than that of OmpA- strains, and the invasive capability of OmpA- strains was restored to the level of the OmpA+ strain by complementation with the OmpA+ E. coli into BMEC. Two short synthetic peptides (a hexamer, Asn-27-Glu-32, and a pentamer, Gly-65-Asn-69) generated from the N-terminal amino acid sequence of OmpA exhibited significant inhibition of OmpA+ E. coli invasion, suggesting that these two sequences represent the OmpA domains involved in E. coli invasion of BMEC. These findings suggest that OmpA is the first microbial structure identified to enhance E. coli invasion of BMEC, an important event in the pathogenesis of E. coli meningitis.     

Increase of CCL20 expression by human gingival fibroblasts upon stimulation with cytokines and bacterial endotoxin

We have demonstrated recently that CCL20 was expressed in periodontal diseased tissues and abundant CCR6 positive T cells infiltrated in periodontally diseased tissue. However, it is uncertain which cells can elicit CCL20 production. In the present study, we examined the properties of CCL20 production by human gingival fibroblasts (HGF) culture. Here, we report that interleukin-1 beta (IL-1beta), tumour necrosis factor-alpha (TNF-alpha) and Escherichia coli lipopolysaccharide (LPS) can significantly induce the production of CCL20 by HGF. We found that TNF-alpha and E. coli LPS enhanced the production of CCL20 by HGF treated with IL-1beta. In contrast, interferon-gamma (IFN-gamma) dramatically diminished CCL20 production induced by IL-1beta. Moreover, we demonstrated that nuclear factor-kappaB (NF-kappaB), p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinases (ERK) play an important role in mediating the production of CCL20 induced by IL-1beta and TNF-alpha. On the other hand, we found that not only NF-kappaB, p38 MAPK and ERK but also c-Jun NH2-terminal kinase (JNK) are involved in CCL20 production induced by E. coli LPS. Finally, we found that HGF express CCR6, CCL20 receptor, and CCL20 induced vascular endothelial growth factor (VEGF) by HGF. Taken together, these findings that HGF will be a source of CCL20 in periodontal tissue, and the CCL20 production will be controlled by proinflammatory cytokine and bacterial LPS in periodontally diseased tissue. Thus, CCL20 by HGF might be involved in inflammatory cells infiltration, and promote the progression of periodontal disease.     

Curcumin attenuates the expression of IL-1beta, IL-6, and TNF-alpha as well as cyclin E in TNF-alpha-treated HaCaT cells; NF-kappaB and MAPKs as potential upstream targets

TNF-alpha induces some proinflammatory cytokines including IL-1beta, IL-6, IL-8, and itself by activation of NF-kappaB or MAPKs (p38, JNK, ERK). These cytokines play important roles in various inflammatory skin diseases, such as psoriasis. Recently it was also reported that expression of cyclin E is up-regulated by ERK pathway after TNF-alpha treatment. However, it was unknown whether curcumin, showing inhibitory effects on NF-kappaB and MAPKs, attenuates the expression of TNF-alpha-induced IL-1beta, IL-6, IL-8, and TNF-alpha as well as cyclin E expression in HaCaT cells. In this study, we investigated the inhibitory effect of curcumin on expression of proinflammatory cytokines and cyclin E in TNF-alpha-treated HaCaT cells. We found that curcumin inhibited the expression of TNF-alpha-induced IL-1beta, IL-6, and TNF-alpha, but not IL-8, in TNF-alpha-treated HaCaT cells as well as the TNF-alpha-induced cyclin E expression. In addition, curcumin inhibited the activation of MAPKs (JNK, p38 MAPK, and ERK) and NF-kappaB in TNF-alpha-treated HaCaT cells. Taken together, curcumin exerts anti-inflammatory and growth inhibitory effects in TNF-alpha-treated HaCaT cells through inhibition of NF-kappaB and MAPK pathways.     

Signaling events involved in cytokine and chemokine production induced by secretory phospholipase A2 in human lung macrophages

Secretory phospholipases A(2) (sPLA(2)) are enzymes released during inflammatory reactions. These molecules activate immune cells by mechanisms either related or unrelated to their enzymatic activity. We examined the signaling events activated by group IA (GIA) and group IB (GIB) sPLA(2) in human lung macrophages leading to cytokine/chemokine production. sPLA(2) induced the production of cytokines (TNF-alpha, IL-6 and IL-10) and chemokines (CCL2, CCL3, CCL4 and CXCL8), whereas no effect was observed on IL-12, CCL1, CCL5 and CCL22. sPLA(2) induced the phosphorylation of the MAPK p38 and ERK1/2, and inhibition of these kinases by SB203580 and PD98059, respectively, reduced TNF-alpha and CXCL8 release. Suppression of sPLA(2) enzymatic activity by a site-directed inhibitor influenced neither cytokine/chemokine production nor activation of MAPK, whereas alteration of sPLA(2) secondary structure suppressed both responses. GIA activated the phosphatidylinositol 3-kinase (PI3 K)/Akt system and a specific inhibitor of PI3 K (LY294002) reduced sPLA(2)-induced release of TNF-alpha and CXCL8. GIA promoted phosphorylation and degradation of IkappaB and inhibition of NF-kappaB by MG-132 and 6-amino-4-phenoxyphenylethylamino-quinazoline suppressed the production of TNF-alpha and CXCL8. These results indicate that sPLA(2) induce the production of cytokines and chemokines in human macrophages by a non-enzymatic mechanism involving the PI3 K/Akt system, the MAPK p38 and ERK1/2 and NF-kappaB.     

Roles of p38 mitogen-activated protein kinase,NF-kappaB,and protein kinase C in proinflammatory cytokine mRNA expression by human peripheral blood leukocytes,monocytes, and neutrophils in response to Anaplasma phagocytophila

Anaplasma phagocytophila, an obligately intracellular bacterium of granulocytes, causes human granulocytic ehrlichiosis. Within 2 h after addition of A. phagocytophila, interleukin-1beta (IL-1beta), tumor necrosis factor alpha (TNF-alpha), and IL-6 mRNAs are induced in human peripheral blood leukocytes (PBLs) or monocytes in vitro. However, neutrophils generate only IL-1beta mRNA. In the present study, signaling pathways for induction of these three cytokines were examined. TNF-alpha and IL-6 mRNA expression by PBLs was inhibited with SB 203580 (a p38 mitogen-activated protein kinase [MAPK] inhibitor), MG-132 (a proteasome inhibitor), and SN-50 (an NF-kappaB inhibitor). Activation of p38 MAPK and NF-kappaB mRNAs in monocytes was detectable within 15 to 30 min after addition of A. phagocytophila. Expression of these two cytokine mRNAs in PBLs and monocytes was also dependent on protein kinase C (PKC), protein kinase A (PKA), and protein tyrosine kinase (PTK). IL-1beta mRNA expression by neutrophils was not dependent on p38 MAPK, and p38 MAPK was not activated in neutrophils incubated with A. phagocytophila. IL-1beta mRNA induction by PBLs, monocytes, and neutrophils was dependent on PKC and PKA. Neutrophil expression of IL-1beta mRNA was dependent on transglutaminase, phospholipase C, and PTK, all of which are also required for internalization of A. phagocytophila. However, monocyte expression of IL-1beta mRNA was less dependent on these enzymes. These results suggest that A. phagocytophila transduces different signals between its host neutrophils and monocytes for proinflammatory cytokine generation.     

Aberrant activation profile of cytokines and mitogen-activated protein kinases in type 2 diabetic patients with nephropathy

Cytokine-induced inflammation is involved in the pathogenesis of type 2 diabetes mellitus (DM). We investigated plasma concentrations and ex vivo production of cytokines and chemokines, and intracellular signalling molecules, mitogen-activated protein kinases (MAPK) in T helper (Th) cells and monocytes in 94 type 2 diabetic patients with or without nephropathy and 20 healthy controls. Plasma concentrations of inflammatory cytokines tumour necrosis factor (TNF)-alpha, interleukin (IL)-6, IL-18 and chemokine CCL2 in patients with diabetic nephropathy (DN) were significantly higher than control subjects, while IL-10, CXCL8, CXCL9, CXCL10 and adiponectin concentrations of DN were significantly higher than patients without diabetic nephropathy (NDN) and control subjects (all P < 0.05). Plasma concentrations of TNF-alpha, IL-6, IL-10, IL-18, CCL2, CXCL8, CXCL9, CXCL10 and adiponectin exhibited significant positive correlation with urine albumin : creatinine ratio in DN patients. The percentage increases of ex vivo production of IL-6, CXCL8, CXCL10, CCL2 and CCL5 upon TNF-alpha activation were significantly higher in both NDN and DN patients than controls (all P < 0.05). The percentage increases in IL-18-induced phosphorylation of extracellular signal-regulated kinase (ERK) in Th cells of NDN and DN were significantly higher than controls (P < 0.05), while the percentage increase in TNF-alpha-induced phosphorylation of p38 MAPK in monocytes and IL-18-induced phosphorylation of p38 MAPK in Th cells and monocytes were significantly higher in NDN patients than controls. These results confirmed that the aberrant production of inflammatory cytokines and chemokines and differential activation of MAPK in different leucocytes are the underlying immunopathological mechanisms of type 2 DM patients with DN.     

Endothelial cell GlcNAc beta 1-4GlcNAc epitopes for outer membrane protein A enhance traversal of Escherichia coli across the blood-brain barrier.

Inadequate knowledge of pathogenesis and pathophysiology has contributed to the high mortality and morbidity associated with neonatal Escherichia coli meningitis. We have shown previously that outer membrane protein A (OmpA) contributes to E. coli K1 membrane invasion of brain microvascular endothelial cells. In this study we report that this OmpA+ K1 E. coli invasion of brain microvascular endothelial cells was inhibited by wheat germ agglutinin and chitooligomers prepared from the polymer of 1,4-linked GlcNAc, chitin. The specificity of the interaction between OmpA and GlcNAc beta 1-4GlcNAc epitopes was verified by the demonstration that chitotriose-bound OmpA and wheat germ agglutinin-bound brain microvascular endothelial cell membrane proteins inhibit E. coli K1 invasion. Of interest, OmpA+ E. coli invasion into systemic endothelial cells did not occur, but invasion similar to that of brain microvascular endothelial cells was observed when systemic cells were treated with alpha-fucosidase, suggesting that the GlcNAc beta 1-4GlcNAc moieties might be substituted with L-fucose on these cells. More importantly, the chitooligomers prevented entry of E. coli K1 into the cerebrospinal fluid of newborn rats with experimental hematogenous E. coli meningitis, suggesting that the GlcNAc beta 1-4GlcNAc epitope of brain microvascular endothelial cells indeed mediates the traversal of E. coli K1 across the blood-brain barrier. A novel strategy with the use of soluble receptor analog(s) may be feasible in the prevention of devastating neonatal E. coli meningitis.     

p38 MAPK is a critical regulator of the constitutive and the beta4 integrin-regulated expression of IL-6 in human normal thymic epithelial cells

Cytokines and adhesion receptors are key mediators in the dialog occurring between thymic epithelial cells (TEC) and thymocytes and regulating T cell maturation and epithelial embryonic differentiation. Among cytokines, IL-6 can be critical in the thymus, fostering proliferation, differentiation and/or survival of both TEC and thymocytes. We have previously reported in human normal TEC that clustering of the laminin receptor alpha6beta4 integrin induced by thymocyte contact or monoclonal antibody-mediated cross-linking regulates IL-6 gene expression via activation of NF-kappaB and NF-IL6 transactivators. Here we show that alpha6beta4 integrin activates p38 mitogen-activated protein kinase (MAPK) and that p38 is essential for IL-6 gene expression. In fact, beta4 cross-linking activated p38 and extracellular signal-regulated kinase (ERK) MAPK, Rac1, p21-activated protein kinase 1 (PAK1) and MAPK kinases (MKK) 3/MKK6. However, pharmacological blockade of p38 or ERK demonstrated that p38 inhibition abrogated both basal and beta4 integrin-induced production of IL-6 preventing NF-kappaB and NF-IL6 activation, whereas ERK inhibition reduced IL-6 production, hampering only NF-kappaB activation. Overall, our results indicate that p38 MAPK and alpha6beta4 integrin, expressed by TEC throughout their life, are critical regulators of the intrathymic availability of a cytokine controlling fate and functions of cells governing development and maintenance of thymic architecture and immune responses.     

IL-17 suppresses TNF-alpha-induced CCL27 production through induction of COX-2 in human keratinocytes

BACKGROUND: The chemokine CCL27 attracts skin-homing T cells. CCL27 production by keratinocytes is dependent on nuclear factor kappaB (NF-kappaB) activity and enhanced in lesions of patients with atopic dermatitis, psoriasis vulgaris, or allergic contact dermatitis. IL-17 is released from activated memory T cells and modulates skin inflammation. OBJECTIVE: We examined the in vitro effects of IL-17 on TNF-alpha-induced CCL27 production in human keratinocytes. METHODS: Keratinocytes were incubated with TNF-alpha, IL-17, or both. CCL27 secretion and mRNA levels were analyzed by means of ELISA and RT-PCR, respectively. COX-2 promoter and NF-kappaB activities were analyzed by using luciferase assays. COX-2 protein levels were analyzed by means of Western blotting. RESULTS: IL-17 suppressed TNF-alpha-induced CCL27 secretion and mRNA expression and NF-kappaB activity in keratinocytes. The COX-2 inhibitor NS398 counteracted the effects of IL-17, and prostaglandin E(2) prevented counteraction by NS398. IL-17 alone or synergistically with TNF-alpha increased prostaglandin E(2) release from keratinocytes, and the increase was suppressed by NS398. IL-17 alone or synergistically with TNF-alpha increased COX-2 mRNA and protein levels, promoter activity, and mRNA stability. The stimulatory effects of IL-17 on COX-2 expression were suppressed by inhibitors of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK) kinase. IL-17 alone or synergistically with TNF-alpha induced dual phosphorylation of p38 MAPK and ERK. CONCLUSION: IL-17 might suppress TNF-alpha-induced CCL27 production by inhibiting NF-kappaB through induction of COX-2. The induction of COX-2 might be mediated by activation of p38 MAPK and ERK. T cell-derived IL-17 might alleviate T-cell skin infiltration through inhibition of CCL27 production.     

Synergistic effect of SCF and TNF-alpha on the up-regulation of cell-surface expression of ICAM-1 on human leukemic mast cell line (HMC)-1 cells

Intercellular adhesion molecule-1 (ICAM-1) has been shown to play crucial roles in mast cell interaction with other inflammatory cells and recruitment into the inflamed tissue. In the present study, human mast cell line-1 (HMC-1) was stimulated with different cytokines including stem cell factor (SCF), tumor necrosis factor alpha (TNF-alpha), interleukin (IL)-13, IL-18, and IL-25. Cell-surface expression of ICAM-1 was assessed by flow cytometry. To elucidate the intracellular signal transduction regulating the ICAM-1 expression, phosphorylated extracellular signal-regulated kinase (ERK), phosphorylated p38 mitogen-activated protein kinase (MAPK), and nuclear factor (NF)-kappaB translocation were assessed by enzyme-linked immunosorbent assay. Results showed that SCF, TNF-alpha, and IL-13 but not IL-18 and IL-25 could up-regulate the surface expression of ICAM-1 on HMC-1 cells. A synergistic effect of SCF and TNF-alpha on ICAM-1 expression was demonstrated. This synergistic effect was shown to be dose-dependently enhanced by SCF but not TNF-alpha. Results indicated that SCF activated ERK, and TNF-alpha activated the p38 MAPK and NF-kappaB pathway. Selective inhibitor of ERK, PD098059, and c-kit inhibitors, STI571 and PP1, suppressed the combined SCF and TNF-alpha-induced ICAM-1 expression. BAY117082 but not SB203580, which are the inhibitors of NF-kappaB and p38 MAPK, respectively, suppressed the TNF-alpha-induced ICAM-1 expression. Therefore, SCF and TNF-alpha acted through ERK and the NF-kappaB pathway to regulate the ICAM-1 expression and elicited the synergistic effect. In conclusion, our results provide insight for cross-talk between different signaling pathways that can help in understanding the fine control of adhesion molecule expression under the concerted effects of cytokines.     

Lipopolysaccharide-Induced Tumor Necrosis Factor Alpha Production by Human Monocytes Involves the Raf-1/MEK1-MEK2/ERK1-ERK2 Pathway

During gram-negative sepsis, human monocytes are triggered to produce large quantities of proinflammatory cytokines such as tumor necrosis factor alpha (TNF-alpha) in response to endotoxin (lipopolysaccharide [LPS]). Several studies have identified signal transduction pathways that are activated by LPS, including activation of nuclear factor-kappaB (NF-kappaB) and activation of mitogen-activated protein kinases (MAPKs), including ERK1 and ERK2, c-Jun N-terminal kinase, and p38. In this study, the relevance of ERK1 and ERK2 activation for LPS-induced TNF-alpha production by primary human monocytes has been addressed with PD-098059, which specifically blocks activation of MAPK kinase (MEK) by Raf-1. TNF-alpha levels in the monocyte culture supernatant, induced by 10 ng of LPS/ml, were reduced by PD-098059 (50 microM). In addition, PD-098059 also reduced TNF-alpha mRNA expression when cells were stimulated for 1 h with LPS. On the other hand, LPS-induced interleukin-10 (IL-10) levels in the monocyte supernatant were only slightly inhibited by PD-098059. Ro 09-2210, a recently identified MEK inhibitor, completely abrogated TNF-alpha levels at nanomolar concentrations. IL-10 levels also were strongly reduced. To show the efficacy of PD-098059 and Ro 09-2210, ERK1 and -2 activation was monitored by Western blotting with an antiserum that recognizes the phosphorylated (i.e., activated) forms of ERK1 and ERK2. Addition of LPS to human monocytes resulted in activation of both ERK1 and ERK2 in a time- and concentration (50% effective concentration between 1 and 10 ng of LPS/ml)-dependent manner. Activation of ERK2 was blocked by PD-098059 (50 microM), whereas ERK1 seemed to be less affected. Ro 09-2210 completely prevented LPS-induced ERK1 and ERK2 activation. LPS-induced p38 activation also was prevented by Ro 09-2210. These data further support the view that the ERK signal transduction pathway is causally involved in the synthesis of TNF-alpha by human monocytes stimulated with LPS.     

Cytokine profiling using monocytes/macrophages cultured on common biomaterials with a range of surface chemistries

Cytokines, chemokines, and growth factors were assayed from the supernatants of monocytes and macrophages cultured on common biomaterials with a range of surface chemistries. TNF-alpha, MCP-1, MIP-1alpha, IL-8, IL-6, IL-1beta, VEGF, IL-1ra, and IL-10 were measured from monocyte/macrophage cultures at different stages of activation and differentiation seeded onto polyethylene, polyurethane, expanded polytetrafluoroethylene, polymethyl methacrylate, and a hydrogel copolymer of 2-hydroxyethyl methacrylate, 1-vinyl-2-pyrrolidinone, and polyethylene glycol acrylate in tissue culture polystyrene (TCPS) plates. Empty TCPS wells and organo-tin polyvinyl chloride served as "blanks" and positive controls, respectively. Results showed an overall increase in cytokine, chemokine, and growth factor production as monocytes are activated or differentiated into macrophages and that proinflammatory and anti-wound healing cytokines and chemokines dominate this profile. However, cytokine production was only modestly affected by the surface chemistry of these four stable and noncytotoxic biomaterials.     

Enhanced interleukin-1beta, interleukin-6 and tumor necrosis factor-alpha production by LPS stimulated human monocytes isolated from HIV+ patients

Periodontal disease and tooth loss is a common finding among advanced HIV+ patients. In addition to local oral lipopolysaccharide (LPS) stimulation, systemic up-regulation of monocyte pro-inflammatory cytokine secretion may also be involved in the pathogenesis of HIV disease. A study was undertaken to investigate IL-1beta, IL-6 and TNF-alpha production by resting and LPS stimulated monocytes isolated from HIV+ patients and also to investigate the relationship of the patient's HIV viral load status to the cytokine production. Whole blood samples in EDTA were collected from 39 HIV-1 infected patients and 20 age and sex matched uninfected controls. Plasma was separated by centrifugation. Viral load was determined using a quantitative RT-PCR. Monocytes were isolated by Ficoll-hypaque gradient separation followed by overnight plastic adherence. Cultured monocytes (1x10(6)/ml) were stimulated with LPS (1 microg/ml) of either P. gingivalis or F. nucleatum for 2, 8, 24 and 48 h and supernatant fluids were collected. IL-1beta, IL-6, and TNF-alpha levels in supernatant fluids were estimated by ELISA. Increased overall production of IL-1beta, IL-6 and TNF-alpha by LPS stimulated monocytes isolated from HIV-1 infected patients was observed when compared to HIV-1 uninfected controls. LPS stimulated monocytes from HIV-1 infected patients with high viral load (HVL) produced significant (p<0.05) elevations in these pro-inflammatory cytokines when compared to HIV-1 uninfected controls. Both LPS of P. gingivalis and F. nucleatum produced a comparable cytokine production by monocytes after 8 h of stimulation. These data suggest that enhanced IL-1beta, IL-6 and TNF-alpha is produced by monocytes/macrophages isolated from HVL HIV+ patients and may be involved in the overall pathogenesis of HIV-1 infection.     

Intracellular JNK, p38 MAPK and NF-kappaB regulate IL-25 induced release of cytokines and chemokines from costimulated T helper lymphocytes

Novel Th2 cytokine IL-25 has been shown to be elevated in allergic inflammation. We investigated the intracellular mechanisms regulating IL-25-induced Th2 cytokines and chemokines from human Th lymphocytes upon costimulation by anti-CD3 and anti-CD28 antibodies. Cytokines, chemokines, and phosphorylated p38 mitogen activated protein kinases (MAPK), c-Jun amino-terminal kinase (JNK) and extracellular signal-regulated protein kinase were analyzed by bead-based array using flow cytometry. Nuclear factor (NF)-kappaB and total MAPK were assessed by electrophoretic mobility shift assay and Western blot, respectively. IL-25 could synergistically induce the release of Th2 cytokines IL-4, IL-5 and IL-10, inflammatory cytokine IL-6, Th1 related chemokines CXCL9 and CXCL10, and chemokine CCL5 from anti-CD3 and anti-CD28 antibodies costimulated Th cells, especially memory Th cells. Costimulation could also upregulate the cell surface expression of IL-25 receptor on Th cells. Costimulation with or without IL-25 treatment could activate JNK, p38 MAPK and NF-kappaB. The upregulation of costimulation-induced IL-25 receptors and release of cytokines and chemokines from IL-25 treated costimulated Th cells were differentially regulated by intracellular JNK, p38 MAPK and NF-kappaB activity. Therefore, the optimal activation of Th cells by IL-25 for the release of Th2 cytokines and chemokines requires the CD3 and CD28 mediated costimulation of Th cells via the upregulation of IL-25 receptors and the activation of intracellular signaling pathways. This mechanistic study shows that IL-25 and CD28 costimulation can play pathophysiological roles by inducing inflammation and hyperresponsiveness through the production of both Th2 cytokines and chemokines from memory Th cells.