Deletion of Tumor Necrosis Factor-alpha Receptor 1 (TNFR1) Protects against Diet-induced Obesity by Means of Increased Thermogenesis

In diet-induced obesity, hypothalamic and systemic inflammatory factors trigger intracellular mechanisms that lead to resistance to the main adipostatic hormones, leptin and insulin. Tumor necrosis factor-alpha (TNF-alpha) is one of the main inflammatory factors produced during this process and its mechanistic role as an inducer of leptin and insulin resistance has been widely investigated. Most of TNF-alpha inflammatory signals are delivered by TNF receptor 1 (R1); however, the role played by this receptor in the context of obesity-associated inflammation is not completely known. Here, we show that TNFR1 knock-out (TNFR1 KO) mice are protected from diet-induced obesity due to increased thermogenesis. Under standard rodent chow or a high-fat diet, TNFR1 KO gain significantly less body mass despite increased caloric intake. Visceral adiposity and mean adipocyte diameter are reduced and blood concentrations of insulin and leptin are lower. Protection from hypothalamic leptin resistance is evidenced by increased leptin-induced suppression of food intake and preserved activation of leptin signal transduction through JAK2, STAT3, and FOXO1. Under the high-fat diet, TNFR1 KO mice present a significantly increased expression of the thermogenesis-related neurotransmitter, TRH. Further evidence of increased thermogenesis includes increased O(2) consumption in respirometry measurements, increased expressions of UCP1 and UCP3 in brown adipose tissue and skeletal muscle, respectively, and increased O(2) consumption by isolated skeletal muscle fiber mitochondria. This demonstrates that TNF-alpha signaling through TNFR1 is an important mechanism involved in obesity-associated defective thermogenesis.

TUMOR NECROSIS FACTOR IS NOT ASSOCIATED WITH INTESTINAL ISCHEMIA/REPERFUSION-INDUCED LUNG INFLAMMATION

Intestinal ischemia-reperfusion (I/R) injury may cause acute systemic and lung inflammation. Here, we revisited the role of TNF-alpha in an intestinal I/R model in mice, showing that this cytokine is not required for the local and remote inflammatory response upon intestinal I/R injury using neutralizing TNF-alpha antibodies and TNF ligand-deficient mice. We demonstrate increased neutrophil recruitment in the lung as assessed by myeloperoxidase activity and augmented IL-6, granulocyte colony-stimulating factor, and KC levels, whereas TNF-alpha levels in serum were not increased and only minimally elevated in intestine and lung upon intestinal I/R injury. Importantly, TNF-alpha antibody neutralization neither diminished neutrophil recruitment nor any of the cytokines and chemokines evaluated. In addition, the inflammatory response was not abrogated in TNF and TNF receptors 1 and 2-deficient mice. However, in view of the damage on the intestinal barrier upon intestinal I/R with systemic bacterial translocation, we asked whether Toll-like receptor (TLR) activation is driving the inflammatory response. In fact, the inflammatory lung response is dramatically reduced in TLR2/4-deficient mice, confirming an important role of TLR receptor signaling causing the inflammatory lung response. In conclusion, endogenous TNF-alpha is not or minimally elevated and plays no role as a mediator for the inflammatory response upon ischemic tissue injury. By contrast, TLR2/4 signaling induces an orchestrated cytokine/chemokine response leading to local and remote pulmonary inflammation, and therefore disruption of TLR signaling may represent an alternative therapeutic target.

Hypothalamic Actions of Tumor Necrosis Factor alpha Provide the Thermogenic Core for the Wastage Syndrome in Cachexia

TNF alpha is an important mediator of catabolism in cachexia. Most of its effects have been characterized in peripheral tissues, such as skeletal muscle and fat. However, by acting directly in the hypothalamus, TNF alpha can activate thermogenesis and modulate food intake. Here we show that high concentration TNF alpha in the hypothalamus leads to increased O(2) consumption/CO(2) production, increased body temperature, and reduced caloric intake, resulting in loss of body mass. Most of the thermogenic response is produced by beta 3-adrenergic signaling to the brown adipose tissue (BAT), leading to increased BAT relative mass, reduction in BAT lipid quantity, and increased BAT mitochondria density. The expression of proteins involved in BAT thermogenesis, such as beta 3-adrenergic receptor, peroxisomal proliferator-activated receptor-gamma coactivator-1 alpha, and uncoupling protein-1, are increased. In the hypothalamus, TNF alpha produces reductions in neuropeptide Y, agouti gene-related peptide, proopiomelanocortin, and melanin-concentrating hormone, and increases CRH and TRH. The activity of the AMP-activated protein kinase signaling pathway is also decreased in the hypothalamus of TNF alpha-treated rats. Upon intracerebroventricular infliximab treatment, tumor-bearing and septic rats present a significantly increased survival. In addition, the systemic inhibition of beta 3-adrenergic signaling results in a reduced body mass loss and increased survival in septic rats. These data suggest hypothalamic TNF alpha action to be important mediator of the wastage syndrome in cachexia. (Endocrinology 151: 683-694, 2010)

Tumor necrosis factor-alpha-308G/A single nucleotide polymorphism and red-complex periodontopathogens are independently associated with increased levels of tumor necrosis factor-alpha in diseased periodontal tissues

Background and Objective: Inflammatory cytokines such as tumor necrosis factor-alpha are involved in the pathogenesis of periodontal diseases. A high between-subject variation in the level of tumor necrosis factor-alpha mRNA has been verified, which may be a result of genetic polymorphisms and/or the presence of periodontopathogens such as Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola (called the red complex) and Aggregatibacter actinomycetemcomitans. In this study, we investigated the effect of the tumor necrosis factor-alpha (TNFA) -308G/A gene polymorphism and of periodontopathogens on the tumor necrosis factor-alpha levels in the periodontal tissues of nonsmoking patients with chronic periodontitis (n = 127) and in control subjects (n = 177). Material and Methods: The TNFA-308G/A single nucleotide polymorphism was investigated using polymerase chain reaction-restriction fragment length polymorphism analysis, whereas the tumor necrosis factor-alpha levels and the periodontopathogen load were determined using real-time polymerase chain reaction. Results: No statistically significant differences were found in the frequency of the TNFA-308 single nucleotide polymorphism in control and chronic periodontitis groups, in spite of the higher frequency of the A allele in the chronic periodontitis group. The concomitant analyses of genotypes and periodontopathogens demonstrated that TNFA-308 GA/AA genotypes and the red-complex periodontopathogens were independently associated with increased levels of tumor necrosis factor-alpha in periodontal tissues, and no additive effect was seen when both factors were present. P. gingivalis, T. forsythia and T. denticola counts were positively correlated with the level of tumor necrosis factor-alpha. TNFA-308 genotypes were not associated with the periodontopathogen detection odds or with the bacterial load. Conclusion: Our results demonstrate that the TNFA-308 A allele and red-complex periodontopathogens are independently associated with increased levels of tumor necrosis factor-alpha in diseased tissues of nonsmoking chronic periodontitis patients and consequently are potentially involved in determining the disease outcome.

Expression of human papillomavirus type 16 E7 oncoprotein alters keratinocytes expression profile in response to tumor necrosis factor-alpha

Acute expression of E7 oncogene from human papillomavirus (HPV) 16 or HPV18 is sufficient to overcome tumor necrosis factor (TNF)-alpha cytostatic effect on primary human keratinocytes. In the present study, we investigated the molecular basis of E7-induced TNF resistance through a comparative analysis of the effect of this cytokine on the proliferation and global gene expression of normal and E7-expressing keratinocytes. Using E7 functional mutants, we show that E7-induced TNF resistance correlates with its ability to mediate pRb degradation and cell transformation. On the other hand, this effect does not depend on E7 sequences required to override DNA damage-induced cell cycle arrest or extend keratinocyte life span. Furthermore, we identified a group of 66 genes whose expression pattern differs between normal and E7-expressing cells upon cytokine treatment. These genes are mainly involved in cell cycle regulation suggesting that their altered expression may contribute to sustained cell proliferation even in the presence of a cytostatic stimulus. Differential expression of TCN1 (transcobalamin I), IFI44 (Interferon-induced protein 44), HMGB2 (high-mobility group box 2) and FUS [Fusion (involved in t(12; 16) in malignant liposarcoma)] among other genes were further confirmed by western-blot and/or real-time polymerase chain reaction. Moreover, FUS upregulation was detected in HPV-positive cervical high-grade squamous intraepithelial lesions when compared with normal cervical tissue. Further evaluation of the role of such genes in TNF resistance and HPVassociated disease development is warranted.

High frequency of immature dendritic cells and altered in situ production of interleukin-4 and tumor necrosis factor-alpha in lung cancer

Introduction Antigen-presenting cells, like dendritic cells (DCs) and macrophages, play a significant role in the induction of an immune response and an imbalance in the proportion of macrophages, immature and mature DCs within the tumor could affect significantly the immune response to cancer. DCs and macrophages can differentiate from monocytes, depending on the milieu, where cytokines, like interleukin (IL)-4 and granulocyte-macrophage colony-stimulating factor (GM-CSF) induce DC differentiation and tumor necrosis factor (TNF)-alpha induce DC maturation. Thus, the aim of this work was to analyze by immunohistochemistry the presence of DCs (S100+ or CD1a+), macrophages (CD68+), IL-4 and TNF-alpha within the microenvironment of primary lung carcinomas. Results Higher frequencies of both immature DCs and macrophages were detected in the tumor-affected lung, when compared to the non-affected lung. Also, TNF-alpha-positive cells were more frequent, while IL-4-positive cells were less frequent in neoplastic tissues. This decreased frequency of mature DCs within the tumor was further confirmed by the lower frequency of CD14-CD80+ cells in cell suspensions obtained from the same lung tissues analyzed by flow cytometry. Conclusion These data are discussed and interpreted as the result of an environment that does not oppose monocyte differentiation into DCs, but that could impair DC maturation, thus affecting the induction of effective immune responses against the tumor.

Fibroblast growth factor 2 restrains ras-driven proliferation of malignant cells by triggering RhoA-mediated senescence

Fibroblast growth factor 2 (FGF2) is considered to be a bona fide oncogenic factor, although results from our group and others call this into question. Here, we report that exogenous recombinant FGF2 irreversibly inhibits proliferation by inducing senescence in Ras-dependent malignant mouse cells, but not in immortalized nontumorigenic cell lines. We report the following findings in K-Ras-dependent malignant YI adrenocortical cells and H-Ras V12-transformed BALB-3T3 fibroblasts: (a) FGF2 inhibits clonal growth and tumor onset in nude and immunocompetent BALB/c mice, (b) FGF2 irreversibly blocks the cell cycle, and (c) FGF2 induces the senescence-associated -galactosidase with no accompanying signs of apoptosis or necrosis. The tyrosine kinase inhibitor PD173074 completely protected malignant cells from FGF2. In Yl adrenal cells, reducing the constitutively high levels of K-Ras-GTP using the dominant-negative RasN17 mutant made cells resistant to FGF2 cytotoxicity. In addition, transfection of the dominant-negative RhoA-N19 into either YI or 3T3-B61 malignant cell lines yielded stable clonal transfectants that were unable to activate RhoA and were resistant to the FGF2 stress response. We conclude that in Rasdependent malignant cells, FGF2 interacts with its cognate receptors to trigger a senescence-like process involving RboAGTP. Surprisingly, attempts to select FGF2-resistant cells from the Yl and 3T3-B61 cell lines yielded only rare clones that (a) had lost the overexpressed ras oncogene, (b) were dependent on FGF2 for proliferation, and (c) were poorly tumorigenic. Thus, FGF2 exerted a strong negative selection that Rasdependent malignant cells could rarely overcome.

Amyloid beta-peptide activates nuclear factor-kappa B through an N-methyl-D-aspartate signaling pathway in cultured cerebellar cells

Amyloid P-peptide (A beta) likely causes functional alterations in neurons well prior to their death. Nuclear factor-kappa B (NF-kappa B), a transcription factor that is known to play important roles in cell survival and apoptosis, has been shown to be modulated by A beta in neurons and glia, but the mechanism is unknown. Because A beta has also been shown to enhance activation of N-methyl-D-aspartate (NMDA) receptors, we investigated the role of NMDA receptor-mediated intracellular signaling pathways in A beta-induced NF-kappa B activation in primary cultured rat cerebellar cells. Cells were treated with different concentrations of A beta 1-40 (1 or 2 mu M) for different periods (6, 12, or 24 hr). MK-801 (NMDA antagonist), manumycin A and FTase inhibitor 1 (farnesyltransferase inhibitors), PP1 (Src-family tyrosine kinase inhibitor), PD98059 [mitogen-activated protein kinase (MAPK) inhibitor], and LY294002 [phosphatidylinositol 3-kinase (PI3-k) inhibitor] were added 20 min before A beta treatment of the cells. A beta induced a time- and concentration-dependent activation of NF-kappa B (1 mu M, 12 hr); both p50/p65 and p50/p50 NF-kappa B dimers were involved. This activation was abolished by MK-801 and attenuated by manumycin A, FTase inhibitor 1, PP1, PD98059, and LY294002. AP at 1 mu M increased the expression of inhibitory protein I kappa B, brain-derived neurotrophic factor, inducible nitric oxide synthase, tumor necrosis factor-alpha, and interleukin-1 beta as shown by RTPCR assays. Collectively, these findings suggest that AP activates NF-kappa B by an NMDA-Src-Ras-like protein through MAPK and PI3-k pathways in cultured cerebellar cells. This pathway may mediate an adaptive, neuroprotective response to A beta. (c) 2007 Wiley-Liss, Inc.

Toll-Like Receptor 4 Signaling Leads to Severe Fungal Infection Associated with Enhanced Proinflammatory Immunity and Impaired Expansion of Regulatory T Cells

Toll-like receptors (TLRs) present in innate immune cells recognize pathogen molecular patterns and influence immunity to control the host-parasite interaction. The objective of this study was to characterize the involvement of TLR4 in the innate and adaptive immunity to Paracoccidioides brasiliensis, the most important primary fungal pathogen of Latin America. We compared the responses of C3H/HeJ mice, which are naturally defective in TLR4 signaling, with those of C3H/HePas mice, which express functional receptors, after in vitro and in vivo infection with P. brasiliensis. Unexpectedly, we verified that TLR4-defective macrophages infected in vitro with P. brasiliensis presented decreased fungal loads associated with impaired synthesis of nitric oxide, interleukin-12 (IL-12), and macrophage chemotactic protein 1 (MCP-1). After intratracheal infection with 1 million yeasts, TLR4-defective mice developed reduced fungal burdens and decreased levels of pulmonary nitric oxide, proinflammatory cytokines, and antibodies. TLR4-competent mice produced elevated levels of IL-12 and tumor necrosis factor alpha (TNF-alpha), besides cytokines of the Th17 pattern, indicating a proinflammatory role for TLR4 signaling. The more severe infection of TLR4-normal mice resulted in increased influx of activated macrophages and T cells to the lungs and progressive control of fungal burdens but impaired expansion of regulatory T cells (Treg cells). In contrast, TLR4-defective mice were not able to clear their diminished fungal burdens totally, a defect associated with deficient activation of T-cell immunity and enhanced development of Treg cells. These divergent patterns of immunity, however, resulted in equivalent mortality rates, indicating that control of elevated fungal growth mediated by vigorous inflammatory reactions is as deleterious to the hosts as low fungal loads inefficiently controlled by limited inflammatory reactions.

Variants in the toll-like receptor signaling pathway and clinical outcomes of malaria

Background. Malaria is one of the most significant infectious diseases in the world and is responsible for a large proportion of infant deaths. Toll-like receptors (TLRs), key components of innate immunity, are central to countering infection. Variants in the TLR-signaling pathway are associated with susceptibility to infectious diseases. Methods. We genotyped single nucleotide polymorphisms ( SNPs) of the genes associated with the TLR-signaling pathway in patients with mild malaria and individuals with asymptomatic Plasmodium infections by means of polymerase chain reaction. Results. Genotype distributions for the TLR-1 I602S differed significantly between patients with mild malaria and persons with asymptomatic infection. The TLR-1 602S allele was associated with an odds ratio ( OR) of 2.2 ( P = .003; P(corrected) = .015) for malaria among patients with mild malaria due to any Plasmodium species and 2.1 ( P = .015; P(corrected) = .75) among patients with mild malaria due to Plasmodium falciparum only. The TLR-6 S249P SNP showed an excess of homozygotes for the TLR-6 249P allele in asymptomatic persons, compared with patients with mild malaria due to any Plasmodium species (OR 2.1; 95% confidence interval [CI], 1.1-4.2; P = .01; P(corrected) = .05), suggesting that the TLR-6 249S allele may be a risk factor for malaria ( OR, 2.0; 95% CI, 1.1-3.7; P = 0.01; P(corrected) = .05). The TLR-9-1486C allele showed a strong association with high parasitemia ( P < .001). Conclusions. Our findings indicate that the TLR-1 and TLR- 6 variants are significantly associated with mild malaria, whereas the TLR-9-1486C/T variants are associated with high parasitemia. These discoveries may bring additional understanding to the pathogenesis of malaria.

The effect of DMSA-functionalized magnetic nanoparticles on transendothelial migration of monocytes in the murine lung via a beta(2) integrin-dependent pathway

Magnetic nanoparticles surface-functionalized with meso-2,3-dimercaptosuccinic acid (MNPs-DMSA) constitute an innovative and promising approach for tissue- and cell-targeted delivery of therapeutic drugs in the lung. Transendothelial migration of leukocytes in the lung is a side effect of endovenous administration of MNPs-DMSA. Using cytologic and phenotypic analysis of murine bronchoalveolar lavage cells, we identified monocytes/macrophages as the main subpopulation of leukocytes involved in this process. Moreover, ultrastructural analysis revealed the presence of nanoparticles inside of numerous macrophages from bronchoalveolar lavage. MNPs-DMSA at concentrations as high as 1 X 10(15) nanoparticles/mL had no toxic effects on macrophages, as evidenced by 3-(4, 5-dimethylthiazolyi-2)-2,5-diphenyltetrazolium bromide (MTT) assay. Notably, MNPs-DMSA up-regulated the mRNA expression of E, L- and P-selectin and macrophage-1 antigen in the murine lung. Upregulation of these cell adhesion molecules was associated with an increased concentration of tumor necrosis factor-alpha in lung. Finally, the critical relevance of the beta(2) integrin-dependent pathway in leukocyte transmigration elicited by MNPs-DMSA was demonstrated by use of knockout mice. Our results characterize mechanisms of the pro-inflammatory effects of MNPs-DMSA in the lung, and identify beta(2) integrin-targeted interventions as promising strategies to reduce pulmonary side effects of MNPs-DMSA during biomedical applications. (C) 2009 Elsevier Ltd. All rights reserved.

Intermittent Therapy with 1,25 Vitamin D and Calcitonin Prevents Cyclosporin-Induced Alveolar Bone Loss in Rats

Bone loss associated with cyclosporin A (CsA) therapy can result in serious morbidity to patients. Intermittent administration of 1,25 Vitamin D and calcitonin reduces osteopenia in a murine model of postmenopausal osteoporosis. The purpose of this study was to evaluate the effects of this therapeutic approach on CsA-induced alveolar bone loss in rats. Forty male Wistar rats were allocated to four experimental groups according to the treatment received during 8 weeks: (1) CsA (10 mg/kg/day, s.c.); (2) 1,25 Vitamin D (2 mu g/kg, p.o.; in weeks 1, 3, 5, and 7) plus calcitonin (2 mu g/kg, i.p.; in weeks 2, 4, 6, and 8); (3) CsA concurrently with intermittent 1,25 Vitamin D and calcitonin administration; and (4) the control treatment group (vehicle). At the end of the 8-week treatment period, serum concentrations of bone-specific alkaline phosphatase, tartrate-resistant acid phosphatase (TRAP-5b), osteocalcin, interleukin (IL)-1 beta, IL-6, and tumor necrosis factor alpha (TNF-alpha) were measured and an analysis of bone volume, bone surface, number of osteoblasts, and osteoclasts was performed. CsA administration resulted in significant alveolar bone resorption, as assessed by a lower bone volume and an increased number of osteoclasts, and increased serum bone-specific alkaline phosphatase, TRAP-5b, IL-1 beta, IL-6, and TNF-alpha concentrations. The intermittent administration of calcitriol and calcitonin prevented the CsA-induced osteopenic changes and the increased serum concentrations of TRAP-5b and inflammatory cytokines. Intermittent calcitriol/calcitonin therapy prevents CsA-induced alveolar bone loss in rats and normalizes the production of associated inflammatory mediators.

BCR-ABL-mediated upregulation of PRAME is responsible for knocking down TRAIL in CML patients

Tumor necrosis factor-related apoptosis-inducing ligand-TNFSF10 (TRAIL), a member of the TNF-alpha family and a death receptor ligand, was shown to selectively kill tumor cells. Not surprisingly, TRAIL is downregulated in a variety of tumor cells, including BCR-ABL-positive leukemia. Although we know much about the molecular basis of TRAIL-mediated cell killing, the mechanism responsible for TRAIL inhibition in tumors remains elusive because (a) TRAIL can be regulated by retinoic acid (RA); (b) the tumor antigen preferentially expressed antigen of melanoma (PRAME) was shown to inhibit transcription of RA receptor target genes through the polycomb protein, enhancer of zeste homolog 2 (EZH2); and (c) we have found that TRAIL is inversely correlated with BCR-ABL in chronic myeloid leukemia (CML) patients. Thus, we decided to investigate the association of PRAME, EZH2 and TRAIL in BCR-ABL-positive leukemia. Here, we demonstrate that PRAME, but not EZH2, is upregulated in BCR-ABL cells and is associated with the progression of disease in CML patients. There is a positive correlation between PRAME and BCR-ABL and an inverse correlation between PRAME and TRAIL in these patients. Importantly, knocking down PRAME or EZH2 by RNA interference in a BCR-ABL-positive cell line restores TRAIL expression. Moreover, there is an enrichment of EZH2 binding on the promoter region of TRAIL in a CML cell line. This binding is lost after PRAME knockdown. Finally, knocking down PRAME or EZH2, and consequently induction of TRAIL expression, enhances Imatinib sensibility. Taken together, our data reveal a novel regulatory mechanism responsible for lowering TRAIL expression and provide the basis of alternative targets for combined therapeutic strategies for CML. Oncogene (2011) 30, 223-233; doi:10.1038/onc.2010.409; published online 13 September 2010

Modulation of inflammatory response by selective inhibition of cyclooxygenase-1 and cyclooxygenase-2 in acute kidney injury

This work explored the role of inhibition of cyclooxygenases (COXs) in modulating the inflammatory response triggered by acute kidney injury. C57Bl/6 mice were used. Animals were treated or not with indomethacin (IMT) prior to injury (days -1 and 0). Animals were subjected to 45 min of renal pedicle occlusion and sacrificed at 24 h after reperfusion. Serum creatinine and blood urea nitrogen, reactive oxygen species (ROS), kidney myeloperoxidase (MPO) activity, and prostaglandin E2 (PGE(2)) levels were analyzed. Tumor necrosis factor (TNF)-alpha, t-bet, interleukin (IL)-10, IL-1 beta, heme oxygenase (HO)-1, and prostaglandin E synthase (PGES) messenger RNA (mRNA) were studied. Cytokines were quantified in serum. IMT-treated animals presented better renal function with less acute tubular necrosis and reduced ROS and MPO production. Moreover, the treatment was associated with lower expression of TNF-alpha, PGE(2), PGES, and t-bet and upregulation of HO-1 and IL-10. This profile was mirrored in serum, where inhibition of COXs significantly decreased interferon (IFN)-gamma, TNF-alpha, and IL-12 p70 and upregulated IL-10. COXs seem to play an important role in renal ischemia and reperfusion injury, involving the secretion of pro-inflammatory cytokines, activation of neutrophils, and ROS production. Inhibition of COX pathway is intrinsically involved with cytoprotection.

Interleukin-10 attenuates vascular responses to endothelin-1 via effects on ERK1/2-dependent pathway

Giachini FR, Zemse SM, Carneiro FS, Lima VV, Carneiro ZN, Callera GE, Ergul A, Webb RC, Tostes RC. Interleukin-10 attenuates vascular responses to endothelin-1 via effects on ERK1/2-dependent pathway. Am J Physiol Heart Circ Physiol 296: H489-H496, 2009. First published December 12, 2008; doi:10.1152/ajpheart.00251.2008.-Interleukin-10 (IL-10) is an anti-inflammatory cytokine with protective actions on the vasculature. On the other hand, endothelin ( ET)-1 has potent vasoconstrictor, mitogenic, and proinflammatory activities, which have been implicated in the pathophysiology of a number of cardiovascular diseases. We hypothesized that, in a condition where ET-1 expression is upregulated, i.e., on infusion of TNF-alpha, IL-10 confers vascular protection from ET-1-induced injury. Aortic rings and first-order mesenteric arteries from male C57BL/6 (WT) and IL-10-knockout (IL-10(-/-)) mice were treated with human recombinant TNF-alpha (220 ng.kg(-1).day(-1)) or vehicle (saline) for 14 days. TNF-alpha infusion significantly increased blood pressure in IL-10(-/-), but not WT, mice. TNF-alpha augmented vascular ET-1 mRNA expression in arteries from WT and IL-10(-/-) mice. ET type A (ETA) receptor expression was increased in arteries from IL-10(-/-) mice, and TNF-alpha infusion did not change vascular ETA receptor expression in control or IL-10(-/-) mice. Aorta and mesenteric arteries from TNF-alpha-infused IL-10(-/-) mice displayed increased contractile responses to ET-1, but not the ET type B receptor agonist IRL-1620. The ETA receptor antagonist atrasentan completely abolished responses to ET-1 in aorta and mesenteric vessels, whereas the ERK1/2 inhibitor PD-98059 abrogated increased contractions to ET-1 in arteries from TNF-alpha-infused IL-10(-/-) mice. Infusion of TNF-alpha, as well as knockdown of IL-10 (IL-10(-/-)), induced an increase in total and phosphorylated ERK1/2. These data demonstrate that IL-10 counteracts ET(A)-mediated vascular responses to ET-1, as well as activation of the ERK1/2 pathway.