Suppressor of cytokine signaling-2 modulates the fibrogenic actions of GH and IGF-I in intestinal mesenchymal cells

Growth hormone (GH) and IGF-I play important roles in wound healing during intestinal injury and inflammation, but there is also indirect evidence that locally expressed IGF-I may act to induce excessive collagen deposition, which can lead to intestinal fibrosis. Factors that dictate the balance between normal wound healing and excessive healing responses are unknown. Using RNase protection assay and in situ hybridization, we determined whether GH and/or IGF-I increase type I collagen deposition in the intestine of rats fed by total parenteral nutrition (TPN), a feeding modality used for many patients following intestinal surgery and resection. We also used an in vitro model system to confirm our in vivo effects and to directly evaluate the relative potency of GH and IGF-I on DNA synthesis and collagen deposition in intestinal myofibroblasts. Both GH and IGF-I stimulated collagen production in vivo and in vitro, and IGF-I, but not GH, stimulated DNA synthesis in vitro. In collagen production, GH was less potent than IGF-I. Suppressors of cytokine signaling (SOC) are cytokine-inducible proteins that negatively feedback to inhibit the actions of cytokines and we recently found that GH selectively upregulates SOC-2 in the intestine of TPN-fed rats. We examined whether SOC-2 may be responsible for the difference in magnitude of action of GH and IGF-I on collagen accumulation. GH, but not IGF-I, induced SOC-2 in isolated myofibroblasts, and overexpression of SOC-2 led to a suppression of GH- and IGF-I-induced collagen accumulation. SOC-2 null mice infused with IGF-I showed greater collagen gene expression compared with wild-type (WT) mice. Myofibroblasts isolated from SOC-2 null mice showed increased IGF-I-stimulated DNA synthesis compared with WT cells. Taken together, these findings suggest that SOC-2 induced by GH may play an important role in suppressing collagen accumulation and mesenchymal cell proliferation induced by GH or GH-induced IGF-I, providing a mechanism for the differing potencies of GH and IGF-I on intestinal mesenchyme and collagen synthesis.     

Characterisation of receptor-specific TNFalpha functions in adipocyte cell lines lacking type 1 and 2 TNF receptors

Tumour necrosis factor-alpha (TNFalpha) is a multifunctional cytokine that exerts a myriad of biological actions in numerous different tissues including adipocytes through its two distinct cell surface receptors. To address the role of each TNF receptor in the biological actions of TNFalpha in adipocytes, we have developed four new preadipocyte cell lines. These were established from wild type controls (TNFR1(+/+)R2(+/+)) and from mice lacking TNFR1 (TNFR1(-/-)), TNFR2 (TNFR2(-/-)) or both (TNFR1(-/-)R2(-/-)). All four new cell lines can fully differentiate to form mature adipocytes, under appropriate culture conditions, as judged by cell morphology, expression of multiple adipogenic markers and the ability to mediate agonist-stimulated lipolysis and insulin-stimulated glucose transport. In wild type (TNFR1(+/+)R2(+/+)) and TNFR2(-/-) adipocytes, TNFalpha stimulated lipolysis and inhibited insulin-stimulated glucose transport as well as insulin receptor autophosphorylation. In contrast, these activities were completely lost in the TNFR1(-/-)R2(-/-) and TNFR1(-/-) cells. Taken together, these studies demonstrate that TNFalpha-induced lipolysis, as well as inhibition of insulin-stimulated glucose transport are predominantly mediated by TNFR1 and that the presence of TNFR2 is not necessary for these functions. This new experimental system promises to be useful in dissecting the molecular pathways activated by each TNF receptor in mediating the biological functions of TNFalpha in differentiated adipocytes.     

Genetic disregulation of TNF alpha and TNF alpha type II receptors in colon cancer at the II and III stage of disease

The expression of the genes coding TNFalpha and TNF RII receptors (TNF RII: TNFR2 membrane and soluble domain, TNFR2/R7 soluble domain) was analysed in colon cancer at the II and III stage of disease, by estimation of mRNA expression. The study included 80 patients with histopathologically confirmed adenocarcinoma. The number of TNFalpha mRNA, TNFR2 mRNA and TNFR2/R7 mRNA copies were estimated in tumour and healthy tissue. The highest number of mRNA TNF-alpha copies were investigated in all samples of tissue and independently of the stage of disease. Simultaneously, we noticed the largest number of mRNA copies for TNFalpha and TNF R2/R7 in healthy cells at stage III of the disease. It is possible to draw a hypothetical line separating the anti-cancer activity of TNFalpha and its influence on cancer progression.     

Lack of tumor necrosis factor receptor type 1 inhibits liver fibrosis induced by carbon tetrachloride in mice

Chronic liver injury causes liver regeneration, resulting in fibrosis. The proinflammatory cytokine tumor necrosis factor (TNF) is involved in the pathogenesis of many acute and chronic liver diseases. TNF has pleiotropic functions, but its role in liver fibrosis has not been clarified. Chronic repeated injection of CCl4 induces liver fibrosis in mice. We examined whether signaling through TNF receptors was critical for this process, using mice lacking either TNF receptor (TNFR) type 1 or TNFR type 2 to define the pathophysiologic role of TNFR signals in liver fibrosis. Liver fibrosis caused by chronic CCl4 exposure was TNF-dependent; histological fibrosis was seen in wild-type (WT) and TNFR-2 knockout (KO) mice, but not in TNFR-1 KO mice. Furthermore, a marked reduction in procollagen and TGF-beta synthesis was observed in TNFR-1 KO mice, which also had little detectable NF-kappa B, STAT3, and AP1 binding, and reduced levels of liver interleukin-6 (IL-6) mRNA compared to WT and TNFR-2 KO mice. In conclusion, our results indicate the possibility that NF-kappa B, STAT3, and AP1 binding by signals transduced through TNFR-1 plays an important role in liver fibrosis formation.     

IGF-I and TGF-beta1 have distinct effects on phenotype and proliferation of intestinal fibroblasts

Insulin-like growth factor I (IGF-I) and transforming growth factor-beta1 (TGF-beta1) are upregulated in myofibroblasts at sites of fibrosis in experimental enterocolitis and in Crohn's disease (CD). We compared the sites of expression of IGF-I and TGF-beta1 in a rat peptidoglycan-polysaccharide (PG-PS) model of chronic granulomatous enterocolitis and fibrosis. We used the human colonic CCD-18Co fibroblast/myofibroblast cell line to test the hypothesis that TGF-beta1 and IGF-I interact to regulate proliferation, collagen synthesis, and activated phenotype typified by expression of alpha-smooth muscle actin and organization into stress fibers. IGF-I potently stimulated while TGF-beta1 inhibited basal DNA synthesis. TGF-beta1 and IGF-I each had similar but not additive effects to induce type I collagen. TGF-beta1 but not IGF-I potently stimulated expression of alpha-smooth muscle actin and stress fiber formation. IGF-I in combination with TGF-beta1 attenuated stress fiber formation without reducing alpha-smooth muscle actin expression. Stress fibers were not a prerequisite for increased collagen synthesis. TGF-beta1 upregulated IGF-I mRNA, which led us to examine the effects of IGF-I in cells previously activated by TGF-beta1 pretreatment. IGF-I potently stimulated proliferation of TGF-beta1-activated myofibroblasts without reversing activated fibrogenic phenotype. We conclude that TGF-beta1 and IGF-I both stimulate type I collagen synthesis but have differential effects on activated phenotype and proliferation. We propose that during intestinal inflammation, regulation of activated phenotype and proliferation may require sequential actions of TGF-beta1 and IGF-I, but they may act in concert to increase collagen deposition.     

Deficiency of TNFR1 protects myocardium through SOCS3 and IL-6 but not p38 MAPK or IL-1beta

Tumor necrosis factor-alpha (TNF-alpha) plays an important role in the development of heart failure. There is a direct correlation between myocardial function and myocardial TNF levels in humans. TNF may induce local inflammation to exert tissue injury. On the other hand, suppressors of cytokine signaling (SOCS) proteins have been shown to inhibit proinflammatory signaling. However, it is unknown whether TNF mediates myocardial inflammation via STAT3/SOCS3 signaling in the heart and, if so, whether this effect is through the type 1 55-kDa TNF receptor (TNFR1). We hypothesized that TNFR1 deficiency protects myocardial function and decreases myocardial IL-6 production via the STAT3/SOCS3 pathway in response to TNF. Isolated male mouse hearts (n = 4/group) from wild-type (WT) and TNFR1 knockout (TNFR1KO) were subjected to direct TNF infusion (500 pg.ml(-1).min(-1) x 30 min) while left ventricular developed pressure and maximal positive and negative values of the first derivative of pressure were continuously recorded. Heart tissue was analyzed for active forms of STAT3, p38, SOCS3 and SOCS1 (Western blot analysis), as well as IL-1beta and IL-6 (ELISA). Coronary effluent was analyzed for lactate dehydrogenase (LDH) activity. As a result, TNFR1KO had significantly better myocardial function, less myocardial LDH release, and greater expression of SOCS3 (percentage of SOCS3/GAPDH: 45 +/- 4.5% vs. WT 22 +/- 6.5%) after TNF infusion. TNFR1 deficiency decreased STAT3 activation (percentage of phospho-STAT3/STAT3: 29 +/- 6.4% vs. WT 45 +/- 8.8%). IL-6 was decreased in TNFR1KO (150.2 +/- 3.65 pg/mg protein) versus WT (211.4 +/- 26.08) mice. TNFR1 deficiency did not change expression of p38 and IL-1beta following TNF infusion. These results suggest that deficiency of TNFR1 protects myocardium through SOCS3 and IL-6 but not p38 MAPK or IL-1beta.     

Tumor necrosis factor inhibits ligand-stimulated EGF receptor activation through a TNF receptor 1-dependent mechanism

Tumor necrosis factor (TNF) and epidermal growth factor (EGF) are key regulators in the intricate balance maintaining intestinal homeostasis. Previous work from our laboratory shows that TNF attenuates ligand-driven EGF receptor (EGFR) phosphorylation in intestinal epithelial cells. To identify the mechanisms underlying this effect, we examined EGFR phosphorylation in cells lacking individual TNF receptors. TNF attenuated EGF-stimulated EGFR phosphorylation in wild-type and TNFR2(-/-), but not TNFR1(-/-), mouse colon epithelial (MCE) cells. Reexpression of wild-type TNFR1 in TNFR1(-/-) MCE cells rescued TNF-induced EGFR inhibition, but expression of TNFR1 deletion mutant constructs lacking the death domain (DD) of TNFR1 did not, implicating this domain in EGFR downregulation. Blockade of p38 MAPK, but not MEK, activation of ERK rescued EGF-stimulated phosphorylation in the presence of TNF, consistent with the ability of TNFR1 to stimulate p38 phosphorylation. TNF promoted p38-dependent EGFR internalization in MCE cells, suggesting that desensitization is achieved by reducing receptor accessible to ligand. Taken together, these data indicate that TNF activates TNFR1 by DD- and p38-dependent mechanisms to promote EGFR internalization, with potential impact on EGF-induced proliferation and migration key processes that promote healing in inflammatory intestinal diseases.     

TNF transactivation of EGFR stimulates cytoprotective COX-2 expression in gastrointestinal epithelial cells

TNF and epidermal growth factor (EGF) are well-known stimuli of cyclooxygenase (COX)-2 expression, and TNF stimulates transactivation of EGF receptor (EGFR) signaling to promote survival in colon epithelial cells. We hypothesized that COX-2 induction and cell survival signaling downstream of TNF are mediated by EGFR transactivation. TNF treatment was more cytotoxic to COX-2(-/-) mouse colon epithelial (MCE) cells than wild-type (WT) young adult mouse colon (YAMC) epithelial cells or COX-1(-/-) cells. TNF also induced COX-2 protein and mRNA expression in YAMC cells, but blockade of EGFR kinase activity or expression inhibited COX-2 upregulation. TNF-induced COX-2 expression was reduced and absent in EGFR(-/-) and TNF receptor-1 (TNFR1) knockout MCE cells, respectively, but was restored upon expression of the WT receptors. Inhibition of mediators of EGFR transactivation, Src family kinases and p38 MAPK, blocked TNF-induced COX-2 protein and mRNA expression. Finally, TNF injection increased COX-2 expression in colon epithelium of WT, but not kinase-defective EGFR(wa2) and EGFR(wa5), mice. These data indicate that TNFR1-dependent transactivation of EGFR through a p38- and/or an Src-dependent mechanism stimulates COX-2 expression to promote cell survival. This highlights an EGFR-dependent cell signaling pathway and response that may be significant in colitis-associated carcinoma.     

Cytokine induction of tumor necrosis factor receptor 2 is mediated by STAT3 in colon cancer cells

The IL-6/STAT3 and TNFα/NFκB pathways are emerging as critical mediators of inflammation-associated colon cancer. TNF receptor (TNFR) 2 expression is increased in inflammatory bowel diseases, the azoxymethane/dextran sodium sulfate (AOM/DSS) model of colitis-associated cancer, and by combined interleukin (IL) 6 and TNFα. The molecular mechanisms that regulate TNFR2 remain undefined. This study used colon cancer cell lines to test the hypothesis that IL-6 and TNFα induce TNFR2 via STAT3 and/or NFκB. Basal and IL-6 + TNFα-induced TNFR2 were decreased by pharmacologic STAT3 inhibition. NFκB inhibition had little effect on IL-6 + TNFα-induced TNFR2, but did inhibit induction of endogenous IL-6 and TNFR2 in cells treated with TNFα alone. Chromatin immunoprecipitation (ChIP) revealed cooperative effects of IL-6 + TNFα to induce STAT3 binding to a -1,578 STAT response element in the TNFR2 promoter but no effect on NFκB binding to consensus sites. Constitutively active STAT3 was sufficient to induce TNFR2 expression. Overexpression of SOCS3, a cytokine-inducible STAT3 inhibitor, which reduces tumorigenesis in preclinical models of colitis-associated cancer, decreased cytokine-induced TNFR2 expression and STAT3 binding to the -1,578 STAT response element. SOCS3 overexpression also decreased proliferation of colon cancer cells and dramatically decreased anchorage-independent growth of colon cancer cells, even cells overexpressing TNFR2. Collectively, these studies show that IL-6- and TNFα-induced TNFR2 expression in colon cancer cells is mediated primarily by STAT3 and provide evidence that TNFR2 may contribute to the tumor-promoting roles of STAT3.     

Changes in the expression of tumor necrosis factor (TNF) alpha,TNFalpha receptor (TNFR) 2, and TNFR-associated factor 2 in granulosa cells during atresia in pig ovaries

Tumor necrosis factor (TNF) alpha can induce both cell death and cell proliferation and exerts its effects by binding to either TNF receptor (TNFR) 1 or 2. When TNFalpha-bound TNFR2 interacts with TNFR-associated factor 2 (TRAF2), expression of survival/antiapoptotic genes is up-regulated. In the present study we determined the changes in localization of TNFalpha and TRAF2 and their mRNAs and the expression of TNFR2 in granulosa cells during follicular atresia in pig ovaries. In healthy follicles, intense signals for TNFalpha and TRAF2 and their mRNAs were demonstrated in the outer zone of the granulosa layer, where many proliferating cells and no apoptotic cells were observed. In atretic follicles, decreased or trace staining for TRAF2 and its mRNA and decreased expression of TNFR2 were observed in the granulosa layer, where many apoptotic cells were seen. These findings suggested that TNFalpha acts as a survival factor in granulosa cells during follicular atresia in pig ovaries.     

A mouse model of airway disease: oncostatin M-induced pulmonary eosinophilia, goblet cell hyperplasia, and airway hyperresponsiveness are STAT6 dependent, and interstitial pulmonary fibrosis is STAT6 independent

Oncostatin M (OSM), a pleiotropic cytokine of the gp130 cytokine family, has been implicated in chronic allergic inflammatory and fibrotic disease states associated with tissue eosinophilia. Mouse (m)OSM induces airway eosinophilic inflammation and interstitial pulmonary fibrosis in vivo and regulates STAT6 activation in vitro. To determine the requirement of STAT6 in OSM-induced effects in vivo, we examined wild-type (WT) and STAT6-knockout (STAT6(-/-)) C57BL/6 mouse lung responses to transient ectopic overexpression of mOSM using an adenoviral vector (AdmOSM). Intratracheal AdmOSM elicited persistent eosinophilic lung inflammation that was abolished in STAT6(-/-) mice. AdmOSM also induced pronounced pulmonary remodeling characterized by goblet cell hyperplasia and parenchymal interstitial fibrosis. Goblet cell hyperplasia was STAT6 dependent; however, parenchymal interstitial fibrosis was not. OSM also induced airway hyperresponsiveness in WT mice that was abolished in STAT6(-/-) mice. OSM stimulated an inflammatory signature in the lungs of WT mice that demonstrated STAT6-dependent regulation of Th2 cytokines (IL-4, IL-13), chemokines (eotaxin-1/2, MCP-1, keratinocyte chemoattractant), and extracellular matrix modulators (tissue inhibitor of matrix metalloproteinase-1, matrix metalloproteinase-13), but STAT6-independent regulation of IL-4Rα, total lung collagen, collagen-1A1, -1A2 mRNA, and parenchymal collagen and α smooth muscle actin accumulation. Thus, overexpression of mOSM induces STAT6-dependent pulmonary eosinophilia, mucous/goblet cell hyperplasia, and airway hyperresponsiveness but STAT6-independent mechanisms of lung tissue extracellular matrix accumulation. These results also suggest that eosinophil or neutrophil accumulation in mouse lungs is not required for OSM-induced lung parenchymal collagen deposition and that OSM may have unique roles in the pathogenesis of allergic and fibrotic lung disease.     

Both lymphotoxin-alpha and TNF are crucial for control of Toxoplasma gondii in the central nervous system

Immunity to Toxoplasma gondii critically depends on TNFR type I-mediated immune reactions, but the precise role of the individual ligands of TNFR1, TNF and lymphotoxin-alpha (LTalpha), is still unknown. Upon oral infection with T. gondii, TNF(-/-), LTalpha(-/-), and TNF/LTalpha(-/-) mice failed to control intracerebral T. gondii and succumbed to an acute necrotizing Toxoplasma encephalitis, whereas wild-type (WT) mice survived. Intracerebral inducible NO synthase expression and-early after infection-splenic NO levels were reduced. Additionally, peritoneal macrophages produced reduced levels of NO upon infection with T. gondii and had significantly reduced toxoplasmastatic activity in TNF(-/-), LTalpha(-/-), and TNF/LTalpha(-/-) mice as compared with WT animals. Frequencies of parasite-specific IFN-gamma-producing T cells, intracerebral and splenic IFN-gamma production, and T. gondii-specific IgM and IgG titers in LTalpha(-/-) and TNF/LTalpha(-/-) mice were reduced only early after infection. In contrast, intracerebral IL-10 and IL-12p40 mRNA expression and splenic IL-2, IL-4, and IL-12 production were identical in all genotypes. In addition, TNF(-/-), LTalpha(-/-), and TNF/LTalpha(-/-), but not WT, mice succumbed to infection with the highly attenuated ts-4 strain of T. gondii or to a subsequent challenge infection with virulent RH toxoplasms, although they had identical frequencies of IFN-gamma-producing T cells as compared with WT mice. Generation and infection of bone marrow reconstitution chimeras demonstrated an exclusive role of hematogeneously produced TNF and LTalpha for survival of toxoplasmosis. These findings demonstrate the crucial role of both LTalpha and TNF for control of intracerebral toxoplasms.     

TIMP-1, -2, -3, and -4 in idiopathic pulmonary fibrosis. A prevailing nondegradative lung microenvironment?

Fibroblast proliferation and extracellular matrix accumulation characterize idiopathic pulmonary fibrosis (IPF). We evaluated the presence of tissue inhibitor of metalloproteinase (TIMP)-1, -2, -3, and -4; collagenase-1, -2, and -3; gelatinases A and B; and membrane type 1 matrix metalloproteinase (MMP) in 12 IPF and 6 control lungs. TIMP-1 was found in interstitial macrophages and TIMP-2 in fibroblast foci. TIMP-3 revealed an intense staining mainly decorating the elastic lamina in vessels. TIMP-4 was expressed in IPF lungs by epithelial and plasma cells. TIMP-2 colocalized with Ki67 in fibroblasts, whereas TIMP-3 colocalized with p27 in inflammatory and epithelial cells. Collagenase-1 was localized in macrophages and alveolar epithelial cells, collagenase-2 was localized in a few neutrophils, and collagenase-3 was not detected. MMP-9 was found in neutrophils and subepithelial myofibroblasts. Myofibroblast expression of MMP-9 was corroborated in vitro by RT-PCR. MMP-2 was noticed in myofibroblasts, some of them close to areas of basement membrane disruption, and membrane type 1 MMP was noticed in interstitial macrophages. These findings suggest that in IPF there is higher expression of TIMPs compared with collagenases, supporting the hypothesis that a nondegrading fibrillar collagen microenvironment is prevailing.     

c-Jun N-terminal kinase attenuates TNFα signaling by reducing Nox1-dependent endosomal ROS production in vascular smooth muscle cells

Tumor necrosis factor-α (TNFα), a proinflammatory cytokine, causes vascular smooth muscle cell (VSMC) proliferation and migration and promotes inflammatory vascular lesions. Nuclear factor-kappa B (NF-κB) activation by TNFα requires endosomal superoxide production by Nox1. In endothelial cells, TNFα stimulates c-Jun N-terminal kinase (JNK), which inhibits NF-κB signaling. The mechanism by which JNK negatively regulates TNFα-induced NF-κB activation has not been defined. We hypothesized that JNK modulates NF-κB activation in VSMC, and does so via a Nox1-dependent mechanism. TNFα-induced NF-κB activation was TNFR1- and endocytosis-dependent. Inhibition of endocytosis with dominant-negative dynamin (DynK44A) potentiated TNFα-induced JNK activation, but decreased ERK activation, while p38 kinase phosphorylation was not altered. DynK44A attenuated intracellular, endosomal superoxide production in wild-type (WT) VSMC, but not in NADPH oxidase 1 (Nox1) knockout (KO) cells. siRNA targeting JNK1 or JNK2 potentiated, while a JNK activator (anisomycin) inhibited, TNFα-induced NF-κB activation in WT, but not in Nox1 KO cells. TNFα-stimulated superoxide generation was enhanced by JNK1 inhibition in WT, but not in Nox1 KO VSMC. These data suggest that JNK suppresses the inflammatory response to TNFα by reducing Nox1-dependent endosomal ROS production. JNK and endosomal superoxide may represent novel targets for pharmacologic modulation of TNFα signaling and vascular inflammation.     

Interaction of TNF with TNF receptor type 2 promotes expansion and function of mouse CD4+CD25+ T regulatory cells

Although TNF is a major proinflammatory cytokine, increasing evidence indicates that TNF also has immunosuppressive feedback effects. We have demonstrated in this study that, in both resting and activated states, mouse peripheral CD4(+)CD25(+) T regulatory cells (Tregs) expressed remarkably higher surface levels of TNFR2 than CD4(+)CD25(-) T effector cells (Teffs). In cocultures of Tregs and Teffs, inhibition of proliferation of Teffs by Tregs was initially transiently abrogated by exposure to TNF, but longer exposure to TNF restored suppressive effects. Cytokine production by Teffs remained continually suppressed by Tregs. The profound anergy of Tregs in response to TCR stimulation was overcome by TNF, which expanded the Treg population. Furthermore, in synergy with IL-2, TNF expanded Tregs even more markedly up-regulated expression of CD25 and FoxP3 and phosphorylation of STAT5, and enhanced the suppressive activity of Tregs. Unlike TNF, IL-1beta and IL-6 did not up-regulate FoxP3-expressing Tregs. Furthermore, the number of Tregs increased in wild-type mice, but not in TNFR2(-/-) mice following sublethal cecal ligation and puncture. Depletion of Tregs significantly decreased mortality following cecal ligation and puncture. Thus, the stimulatory effect of TNF on Tregs resembles the reported costimulatory effects of TNF on Teffs, but is even more pronounced because of the higher expression of TNFR2 by Tregs. Moreover, our study suggests that the slower response of Tregs than Teffs to TNF results in delayed immunosuppressive feedback effects.     

Leptin stimulates tissue inhibitor of metalloproteinase-1 in human hepatic stellate cells: respective roles of the JAK/STAT and JAK-mediated H2O2-dependant MAPK pathways

Leptin is recognized as a profibrogenic hormone in the liver, but the mechanisms involved have not been clarified. The tissue inhibitor of metalloproteinase (TIMP)-1, which acts through inhibition of collagen degradation, is synthesized by activated hepatic stellate cells (HSC) in response to fibrogenic substances. The capacity of leptin to induce TIMP-1 and its signaling molecules were investigated in a human HSC cell line, LX-2. Leptin stimulated TIMP-1 protein, mRNA, and promoter activity. JAK1 and -2, as well as STAT3 and -5, were activated. After leptin, there was increased expression of tyrosine 1141-phosphorylated leptin receptor, which may contribute to STAT3 activation. AG 490, a JAK inhibitor, blocked JAK phosphorylation with concomitant inhibition of STAT activation, TIMP-1 mRNA expression, and promoter activity. Leptin also induced an oxidative stress, which was inhibited by AG 490, indicating a JAK mediation process. ERK1/2 MAPK and p38 were activated, which was prevented by catalase, indicating an H2O2-dependent mechanism. Catalase treatment resulted in total suppression of TIMP-1 mRNA expression and promoter activity. SB203580, a p38 inhibitor, prevented p38 activation and reduced TIMP-1 message half-life with down-regulation of TIMP-1 mRNA. These changes were reproduced by overexpression of the dominant negative p38alpha and p38beta mutants. PD098059, an ERK1/2 inhibitor, opposed ERK1/2 activation and TIMP-1 promoter activity, leading to TIMP-1 mRNA down-regulation. Thus, leptin has a direct action on liver fibrogenesis by stimulating TIMP-1 production in activated HSC. This process appears to be mediated by the JAK/STAT pathway via the leptin receptor long form and the H2O2-dependent p38 and ERK1/2 pathways via activated JAK.     

Differential role of TNF receptors in cellular trafficking of intact TNF.

BACKGROUND/AIMS: Although ligand signaling and degradation within the cell have received much attention, few studies have quantified the role of receptors on the transcytosis of ligand into and out of the cell in intact form. Accordingly, we determined the differential role of the two receptors for tumor necrosis factor alpha (TNFR1, TNFR2) on cellular transcytosis. METHODS: TNFR1 and TNFR2 were overexpressed in HEK293 cells by transient transfection. Cell surface binding, endocytosis, and exocytosis of (125)I-TNF were quantified. Degradation was determined by acid precipitation and size-exclusion chromatography. RESULTS: TNFR1- mediated uptake of TNF was faster than TNFR2-mediated uptake of TNF. TNFR2, however, exhibited greater capacity, leading to a higher percentage release of TNF into the exocytosis medium. Rather than being degraded, most of the TNF inside the cell remained intact for 1 h. Both receptors exerted protective roles against degradation, but there was no cooperativity between them. CONCLUSION: The effects of TNFR1 and TNFR2 in shepherding TNF across the cell illustrate the differential roles of receptors on the cellular trafficking of the ligand in intact form so as to facilitate its biological effects.     

Early response cytokines and innate immunity: essential roles for TNF receptor 1 and type I IL-1 receptor during Escherichia coli pneumonia in mice

The early response cytokines, TNF and IL-1, have overlapping biologic effects that may function to propagate, amplify, and coordinate host responses to microbial challenges. To determine whether signaling from these early response cytokines is essential to orchestrating innate immune responses to intrapulmonary bacteria, the early inflammatory events induced by instillation of Escherichia coli into the lungs were compared in wild-type (WT) mice and mice deficient in both TNF receptor 1 (TNFR1) and the type I IL-1 receptor (IL1R1). Neutrophil emigration and edema accumulation induced by E. coli were significantly compromised by TNFR1/IL1R1 deficiency. Neutrophil numbers in the circulation and within alveolar septae did not differ between WT and TNFR1/IL1R1 mice, suggesting that decreased neutrophil emigration did not result from decreased sequestration or delivery of intravascular neutrophils. The nuclear translocation of NF-kappa B and the expression of the chemokine macrophage inflammatory protein-2 did not differ between WT and TNFR1/IL1R1 lungs. However, the concentration of the chemokine KC was significantly decreased in the bronchoalveolar lavage fluids of TNFR1/IL1R1 mice compared with that in WT mice. Thus, while many of the molecular and cellular responses to E. coli in the lungs did not require signaling by either TNFR1 or IL1R1, early response cytokine signaling was critical to KC expression in the pulmonary air spaces and neutrophil emigration from the alveolar septae.