Modulation of interleukin signalling and gene expression in cardiac myocytes by endothelin-1

The related inflammatory cytokines, interleukin- (IL-) 1β and IL-33, are both implicated in the response of the heart to injury. They also activate mitogen-activated protein kinases (MAPKs) in cardiac myocytes. The hypertrophic Gq protein-coupled receptor agonist endothelin-1 is a potentially cardioprotective peptide and may modulate the inflammatory response. Endothelin-1 also stimulates (MAPKs) in cardiac myocytes and promotes rapid changes in expression of mRNAs encoding intercellular and intracellular signalling components including receptors for IL-33 (ST2) and phosphoprotein phosphatases. Prior exposure to endothelin-1 may specifically modulate the response to IL-33 and, more globally, influence MAPK activation by different stimuli. Neonatal rat ventricular myocytes were exposed to IL-1β or IL-33 with or without pre-exposure to endothelin-1 (5 h) and MAPK activation assessed. IL-33 activated ERK1/2, JNKs and p38-MAPK, but to a lesser degree than IL-1β. Endothelin-1 increased expression of soluble IL-33 receptors (sST2 receptors) which may prevent binding of IL-33 to the cell-surface receptors. However, pretreatment with endothelin-1 only inhibited activation of p38-MAPK by IL-33 with no significant influence on ERK1/2 and a small increase in activation of JNKs. Inhibition of p38-MAPK signalling following pretreatment with endothelin-1 was also detected with IL-1β, H2O2 or tumour necrosis factor α (TNFα) indicating an effect intrinsic to the signalling pathway. Endothelin-1 pretreatment suppressed the increase in expression of IL-6 mRNA induced by IL-1β and decreased the duration of expression of TNFα mRNA. Coupled with the general decrease in p38-MAPK signalling, we conclude that endothelin-1 attenuates the cardiac myocyte inflammatory response, potentially to confer cardioprotection.

The role of proliferator-activated receptor gamma coactivator-1 alpha in the fatty-acid -dependent transcriptional control of interleukin-10 in hepatic cells of rodents

Interleukin-10 (IL-10) is an endogenous factor that restrains hepatic insulin resistance in diet-induced steatosis Reducing IL-10 expression increases proinflammatory activity in the steatotic liver and worsens insulin resistance As the transcriptional coactivator proliferator-activated receptor gamma coactivator-1 alpha (PGC-1 alpha) plays a central role in dysfunctional hepatocytic activity in diet-induced steatosis, we hypothesized that at least part of the action of PGC-1 alpha could be mediated by reducing the transcription of the IL-10 gene Here, we used immunoblotting, real-time polymerase chain reaction, immunocytochemistry, and chromatin immunoprecipitation assay to investigate the role of PGC-1 alpha in the control of IL-10 expression in hepatic cells First, we show that, in the intact steatotic liver, the expressions of IL-10 and PGC-1 alpha are increased Inhibiting PGC-1 alpha expression by antisense oligonucleotide increases IL-10 expression and reduces the steatotic phenotype. In cultured hepatocytes, the treatment with saturated and unsaturated fatty acids increased IL-10 expression. This was accompanied by increased association of PGC-1 alpha with c-Maf and p50-nuclear factor (NF) kappa B, 2 transcription factors known to modulate IL-10 expression In addition, after fatty acid treatment. PGC-1 alpha, c-Maf, and p50-NF kappa B migrate from the cytosol to the nuclei of hepatocytes and bind to the IL-10 promoter region Inhibiting NF kappa B activation with salicylate reduces IL-10 expression and the association of PGC-1 alpha with p50-NF kappa B Thus, PGC-1 alpha emerges as a potential transcriptional regulator of the inflammatory phenomenon taking place in the steatotic liver (C) 2010 Elsevier Inc All rights reserved

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.

Interleukin-10 secreted by B-1 cells modulates the phagocytic activity of murine macrophages in vitro

As demonstrated previously in our laboratory, B-1 cells migrate from the peritoneal cavity of mice and home to a distant site of inflammation to become macrophage-like cells. However, the influence that these cells might have on the kinetics and fate of the inflammatory process is not known. Considering that macrophages are pivotal in the inflammatory reaction, we decided to investigate the possible influence B-1 cells could have on macrophage activities in vitro. Our results show that peritoneal macrophages from Xid mice, a mouse strain deprived of B-1 cells, have higher phagocytic indexes for zymozan particles when compared with macrophages from wild-type mice. Moreover, macrophages from wild-type mice have a lower ability to release nitric oxide and hydrogen peroxide when compared with macrophages from Xid mice. Experiments using cocultures of B-1 cells and macrophages from Xid mice in transwell plates demonstrated that B-1 cells down-regulate macrophage activities. These observations also indicate that this phenomenon is not due to a physical interaction between these two cell populations. As B-1 cells are one of the main sources of interleukin (IL)-10, we demonstrate in this study that adherent peritoneal cells from Xid mice produce significantly less amounts of this cytokine in culture when compared with IL-10 production by cells from wild-type mice. When B-1 cells from IL-10 knock-out mice and macrophages from wild-type mice were cocultured in transwell plates, the phagocytic index of macrophages was not altered demonstrating that B-1 cells can influence the effector functions of macrophages in vitro via IL-10 secretion.

Evaluation of transformation growth factor beta(1), interleukin-10, and interferon-gamma in male symptomatic and asymptomatic dogs naturally infected by Leishmania (Leishmania) chagasi

The aims of this study were to evaluate the immunomodulatory role of TGF-beta(1), 1L-10, and INF-gamma in spleen and liver extracts and supernatant cultures of white spleen cells from male symptomatic and asymptomatic dogs, naturally infected by Leishmania (Leishmania) chagasi. Thirty dogs from Aracatuba, São Paulo, Brazil, an endemic leishmaniosis area, were selected by positive ELISA serological reaction for Leishmania sp. and divided into two groups: asymptomatic (n=15) and symptomatic (n=15) consisting of animals with at least three characteristic signs (fever, dermatitis, lymphoadenopathy, onychogryphosis, weight loss, cachex a, locomotion problems, conjunctivitis, epistaxis, hepatosplenomegaly, edema, and apathy). After euthanasia, spleen and liver fragments were collected for ex vivo quantification of TGF-beta(1), IL-10, and INF-gamma. Naturally active in vitro produced TGF-beta(1) was also evaluated in spleen cell culture supernatant. Spleen and liver extract of asymptomatic dogs had higher mean TGF-beta(1) levels than symptomatic dogs. High concentrations of IL-10 were found in spleen, and mainly in liver extract of both groups. Higher INF-gamma concentrations were found in spleen extracts of symptomatic dogs, and in liver extracts of asymptomatic dogs. Extract of this cytokire was lower in spleen extract. Although INF-gamma is being produced in canine infection, mean levels of TGF-beta(1) and IL-10 from spleen and liver extracts were quantitatively much higher; suggesting that immune response in both asymptomatic and symptomatic dogs A as predominantly type Th2. (c) 2006 Elsevier B.V. All rights reserved.

Toll-like receptor 2 knockdown modulates interleukin (IL)-6 and IL-8 but not stromal derived factor-1 (SDF-1/CXCL12) in human periodontal ligament and gingival fibroblasts

Background: Fibroblasts are now seen as active components of the immune response because these cells express Toll-like receptors (TLRs), recognize pathogen-associated molecular patterns, and mediate the production of cytokines and chemokines during inflammation. The innate host response to lipopolysaccharide (LPS) from Porphyromonas gingivalis is unusual inasmuch as different studies have reported that it can be an agonist for Toll-like receptor 2 (TLR2) and an antagonist or agonist for Toll-like receptor 4 (TLR4). This study investigates and compares whether signaling through TLR2 or TLR4 could affect the secretion of interleukin (IL)-6, IL-8, and stromal derived factor-1 (SDF-1/CXCL12) in both human gingival fibroblasts (HGF) and human periodontal ligament fibroblasts (HPDLF). Methods: After small interfering RNA-mediated silencing of TLR2 and TLR4, HGF and HPDLF from the same donors were stimulated with P. gingivalis LPS or with two synthetic ligands of TLR2, Pam2CSK4 and Pam3CSK4, for 6 hours. IL-6, IL-8, and CXCL12mRNA expression and protein secretion were evaluated by quantitative polymerase chain reaction and enzymelinked immunosorbent assay, respectively. Results: TLR2 mRNA expression was upregulated in HGF but not in HPDLF by all the stimuli applied. Knockdown of TLR2 decreased IL-6 and IL-8 in response to P. gingivalis LPS, or Pam2CSK4 and Pam3CSK4, in a similar manner in both fibroblasts subpopulations. Conversely, CXCL12 remained unchanged by TLR2 or TLR4 silencing. Conclusion: These results suggest that signaling through TLR2 by gingival and periodontal ligament fibroblasts can control the secretion of IL-6 and IL-8, which contribute to periodontal pathogenesis, but do not interfere with CXCL12 levels, an important chemokine in the repair process.

Interleukin-18 and interferon-gamma polymorphisms are implicated on proviral load and susceptibility to human T-lymphotropic virus type 1 infection

Interleukin-18 (IL-18) and interferon-gamma (IFN-?) exert important functions in both innate and adaptive immune responses against intracellular pathogens and viruses. Previous studies suggested that host genetic factors, including cytokines gene polymorphisms, could be involved in the pathogenesis of human T-cell leukemia virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Thus, we analyzed -137C/G and -607A/C of the IL-18 promoter and +874T/A of the IFN-? in DNA samples from 98 HTLV-1-infected individuals exhibiting or not clinical symptoms and 150 healthy control individuals. The IL-18 promoter -607CC genotype was significantly lower in HTLV-1 asymptomatic carriers (HAC) and HTLV-1-infected individuals (HAC + HAM/TSP) than healthy control group. In contrast, the -607AC genotype was significantly higher in HAC and HTLV-1-infected individuals group compared to the healthy control group. The -137G/-607A IL-18 haplotype was higher in infected group than healthy control group, and the -137C/-607C IL-18 haplotype was increased in the healthy control group compared to the others. Finally, the IFN-? polymorphism analysis showed that the HTLV-1-infected individuals with +874AT genotype presented higher proviral load than +874AA genotype. These data indicate that the IL-18-607AC genotype and -137G/-607A haplotype could be a risk factor for HTLV-1 infection, whereas the protective effect could be conferred by -607CC genotype and -137C/-607C haplotype. Also, the IFN-? could be implicated on the proviral load levels.

Toll-like receptor 2 knockdown modulates Interleukin (IL)-6 and IL-8 but not Stromal Derived Factor-1 (SDF-1/CXCL12) in human periodontal ligament and gingival fibroblasts

Background: Fibroblasts are now seen as active components of the immune response because these cells express Toll-like receptors (TLRs), recognize pathogen-associated molecular patterns, and mediate the production of cytokines and chemokines during inflammation. The innate host response to lipopolysaccharide (LPS) from Porphyromonas gingivalis is unusual inasmuch as different studies have reported that it can be an agonist for Toll-like receptor 2 (TLR2) and an antagonist or agonist for Toll-like receptor 4 (TLR4). This study investigates and compares whether signaling through TLR2 or TLR4 could affect the secretion of interleukin (IL)-6, IL-8, and stromal derived factor-1 (SDF-1/CXCL12) in both human gingival fibroblasts (HGF) and human periodontal ligament fibroblasts (HPDLF). Methods: After small interfering RNA-mediated silencing of TLR2 and TLR4, HGF and HPDLF from the same donors were stimulated with P. gingivalis LPS or with two synthetic ligands of TLR2, Pam2CSK4 and Pam3CSK4, for 6 hours. IL-6, IL-8, and CXCL12 mRNA expression and protein secretion were evaluated by quantitative polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. Results: TLR2 mRNA expression was upregulated in HGF but not in HPDLF by all the stimuli applied. Knockdown of TLR2 decreased IL-6 and IL-8 in response to P. gingivalis LPS, or Pam2CSK4 and Pam3CSK4, in a similar manner in both fibroblasts subpopulations. Conversely, CXCL12 remained unchanged by TLR2 or TLR4 silencing. Conclusion: These results suggest that signaling through TLR2 by gingival and periodontal ligament fibroblasts can control the secretion of IL-6 and IL-8, which contribute to periodontal pathogenesis, but do not interfere with CXCL12 levels, an important chemokine in the repair process.

Response of interleukin-6 during euglycaemic and hyperglycaemic exercise in patients with type 1 diabetes mellitus

The aim of this randomized, single-blinded cross-over study was to investigate the response of interleukin-6 (IL-6) during moderate aerobic exercise in stable euglycaemia and hyperglycaemia in seven male patients with type 1 diabetes mellitus. IL-6 increased significantly over the entire study period in euglycaemia, but not in hyperglycaemia.

Interleukin-17A stimulates granulocyte-macrophage colony-stimulating factor release by murine osteoblasts in the presence of 1,25-dihydroxyvitamin D(3) and inhibits murine osteoclast development in vitro

OBJECTIVE To investigate the effects of interleukin-17A (IL-17A) on osteoclastogenesis in vitro. METHODS Bone marrow cells (BMCs) were isolated from the excised tibia and femora of wild-type C57BL/6J mice, and osteoblasts were obtained by sequential digestion of the calvariae of ddY, C57BL/6J, and granulocyte-macrophage colony-stimulating factor-knockout (GM-CSF(-/-)) mice. Monocultures of BMCs or cocultures of BMCs and osteoblasts were supplemented with or without 1,25-dihydroxyvitamin D(3)(1,25[OH](2)D(3)), recombinant human macrophage colony-stimulating factor (M-CSF), RANKL, and IL-17A. After 5-6 days, the cultures were fixed with 4% paraformaldehyde and subsequently stained for the osteoclast marker enzyme tartrate-resistant acid phosphatase (TRAP). Osteoprotegerin (OPG) and GM-CSF expression were measured by enzyme-linked immunosorbent assay, and transcripts for RANK and RANKL were detected by real-time polymerase chain reaction. RESULTS In both culture systems, IL-17A alone did not affect the development of osteoclasts. However, the addition of IL-17A plus 1,25(OH)(2)D(3) to cocultures inhibited early osteoclast development within the first 3 days of culture and induced release of GM-CSF into the culture supernatants. Furthermore, in cocultures of GM-CSF(-/-) mouse osteoblasts and wild-type mouse BMCs, IL-17A did not affect osteoclast development, corroborating the role of GM-CSF as the mediator of the observed inhibition of osteoclastogenesis by IL-17A. CONCLUSION These findings suggest that IL-17A interferes with the differentiation of osteoclast precursors by inducing the release of GM-CSF from osteoblasts.

Interleukin 18 stimulates HIV type 1 in monocytic cells

The cytokine interleukin (IL) 18 (formerly interferon γ-inducing factor) induces the T helper type 1 response. In the present studies, IL-18 increased HIV type 1 (HIV-1) production from 5- to 30-fold in the chronically infected U1 monocytic cell line. Inhibition of tumor necrosis factor (TNF) activity by the addition of TNF-binding protein reduced IL-18-stimulated HIV-1 production by 48%. In the same cultures, IL-18-induced IL-8 was inhibited by 96%. Also, a neutralizing anti-IL-6 mAb reduced IL-18-induced HIV-1 by 63%. Stimulation of U1 cells with IL-18 resulted in increased production of IL-6, and exogenous IL-6 added to U1 cells increased HIV-1 production 4-fold over control. A specific inhibitor of the p38 mitogen-activated protein kinase reduced IL-18-induced HIV-1 by 73%, and a 50% inhibition was observed at 0.05 μM. In the same cultures, IL-8 was inhibited by 87%. By gel-shift and supershift analyses, increased binding activity of the transcription factor NF-κB was measured in nuclear extracts from U1 cells 1 h after exposure to IL-18. These results demonstrate induction of HIV-1 by IL-18 in a monocyte target associated with an intermediate role for TNF and IL-6, activation of p38 mitogen-activated protein kinase, and nuclear translocation of NF-κB.

Interleukin 12 and B7-1 costimulatory molecule expressed by an adenovirus vector act synergistically to facilitate tumor regression

Stimulation of antitumor immune mechanisms is the primary goal of cancer immunotherapy, and accumulating evidence suggests that effective alteration of the host–tumor relationship involves immunomodulating cytokines and also the presence of costimulatory molecules. To examine the antitumor effect of direct in vivo gene transfer of murine interleukin 12 (IL-12) and B7-1 into tumors, we developed an adenovirus (Ad) vector, AdIL12–B7-1, that encodes the two IL-12 subunits in early region 1 (E1) and the B7-1 gene in E3 under control of the murine cytomegalovirus promoter. This vector expressed high levels of IL-12 and B7-1 in infected murine and human cell lines and in primary murine tumor cells. In mice bearing tumors derived from a transgenic mouse mammary adenocarcinoma, a single intratumoral injection with a low dose (2.5 × 107 pfu/mouse) of AdIL12–B7-1 mediated complete regression in 70% of treated animals. By contrast, administration of a similar dose of recombinant virus encoding IL-12 or B7-1 alone resulted in only a delay in tumor growth. Interestingly, coinjection of two different viruses expressing either IL-12 or B7-1 induced complete tumor regression in only 30% of animals treated at this dose. Significantly, cured animals remained tumor free after rechallenge with fresh tumor cells, suggesting that protective immunity had been induced by treatment with AdIL12–B7-1. These results support the use of Ad vectors as a highly efficient delivery system for synergistically acting molecules and show that the combination of IL-12 and B7-1 within a single Ad vector might be a promising approach for in vivo cancer therapy.

Three distinct domains of SSI-1/SOCS-1/JAB protein are required for its suppression of interleukin 6 signaling

Cytokine-inducible protein SSI-1 [signal transducers and activators of transcription (STAT)-induced STAT inhibitor 1, also referred to as SOCS-1 (suppressor of cytokine signaling 1) or JAB (Janus kinase-binding protein)] negatively regulates cytokine receptor signaling by inhibition of JAK kinases. The SSI family of proteins includes eight members that are structurally characterized by an SH2 domain and a C-terminal conserved region that we have called the SC-motif. In this study, we investigated the roles of these domains in the function of SSI-1. Results of reporter assays demonstrated that the pre-SH2 domain (24 aa in front of the SH2 domain) and the SH2 domain of SSI-1 were required for the suppression by SSI-1 of interleukin 6 signaling. Coexpression studies of COS7 cells revealed that these domains also were required for inhibition of three JAKs (JAK1, JAK2, and TYK2). Furthermore, deletion of the SH2 domain, but not the pre-SH2 domain, resulted in loss of association of SSI-1 with TYK2. Thus, SSI-1 associates with JAK family kinase via its SH2 domain, and the pre-SH2 domain is required for the function of SSI-1. Deletion of the SC-motif markedly reduced expression of SSI-1 protein in M1 cells, and this reduction was reversed by treatment with proteasome inhibitors, suggesting that this motif is required to protect the SSI-1 molecule from proteolytic degradation. Based on these findings, we concluded that three distinct domains of SSI-1 (the pre-SH2 domain, the SH2 domain, and the SC-motif) cooperate in the suppression of interleukin 6 signaling.

Local production of the p40 subunit of interleukin 12 suppresses T-helper 1-mediated immune responses and prevents allogeneic myoblast rejection.

The p40 subunit of interleukin 12 (IL-12p40) has been known to act as an IL-12 antagonist in vitro. We here describe the immunosuppressive effect of IL-12p40 in vivo. A murine myoblast cell line, C2C12, was transduced with retro-virus vectors carrying the lacZ gene as a marker and the IL-12p40 gene. IL-12p40 secreted from the transfectant inhibited the IL-12-induced interferon gamma (IFN-gamma) production by splenocytes in vitro. Survival of C2C12 transplanted into allogeneic recipients was substantially prolonged when transduced with IL-12p40. Cytokine (IL-2 and IFN-gamma) production and cytotoxic T lymphocyte induction against allogeneic C2C12 were impaired in the recipients transplanted with the IL-12p40 transfectant. Delayed-type hypersensitivity response against C2C12 was also diminished in the IL-12p40 recipients. Furthermore, serum antibodies against beta-galactosidase of the T-helper 1-dependent isotypes (IgG2 and IgG3) were decreased in the IL-12p40 recipients. These results indicate that locally produced IL-12p40 exerts a potent immunosuppressive effect on T-helper 1-mediated immune responses that lead to allograft rejection. Therefore, IL-12p40 gene transduction would be useful for preventing the rejection of allografts and genetically modified own cells that are transduced with potentially antigenic molecules in gene therapy.