Interleukin-10 protects cultured fetal rat type II epithelial cells from injury induced by mechanical stretch

Mechanical ventilation plays a central role in the pathogenesis of bronchopulmonary dysplasia. However, the mechanisms by which excessive stretch of fetal or neonatal type II epithelial cells contributes to lung injury are not well defined. In these investigations, isolated embryonic day 19 fetal rat type II epithelial cells were cultured on substrates coated with fibronectin and exposed to 5% or 20% cyclic stretch to simulate mechanical forces during lung development or lung injury, respectively. Twenty percent stretch of fetal type II epithelial cells increased necrosis, apoptosis, and proliferation compared with control, unstretched samples. By ELISA and real-time PCR (qRT-PCR), 20% stretch increased secretion of IL-8 into the media and IL-8 gene expression and inhibited IL-10 release. Interestingly, administration of recombinant IL-10 before 20% stretch did not affect cell lysis but significantly reduced apoptosis and IL-8 release compared with stretched samples without IL-10. Collectively, our studies suggest that IL-10 may play an important role in protection of fetal type II epithelial cells from injury secondary to stretch.     

Suppressor of cytokine signaling 1 inhibits IL-10-mediated immune responses

IL-10 has proved to be a key cytokine in regulating inflammatory responses by controlling the production and function of various other cytokines. The suppressor of cytokine signaling (SOCS) gene products are a family of cytoplasmic molecules that are essential mediators for negatively regulating cytokine signaling. It has been previously shown that IL-10 induced SOCS3 expression and that forced constitutive expression of SOCS3 inhibits IL-10/STAT3 activation and LPS-induced macrophage activation. In this report, we show that, in addition to SOCS3 expression, IL-10 induces SOCS1 up-regulation in all cell lines tested, including Ba/F3 pro-B cells, MC/9 mast cells, M1 leukemia cells, U3A human fibroblasts, and primary mouse CD4(+) T cells. Induction of SOCS molecules is dependent on STAT3 activation by IL-10R1. Cell lines constitutively overexpressing SOCS proteins demonstrated that SOCS1 and SOCS3, but not SOCS2, are able to partially inhibit IL-10-mediated STAT3 activation and proliferative responses. Pretreatment of M1 cells with IFN-gamma resulted in SOCS1 induction and a reduction of IL-10-mediated STAT3 activation and cell growth inhibition. IL-10-induced SOCS is associated with the inhibition of IFN-gamma signaling in various cell types, and this inhibition is independent of C-terminal serine residues of the IL-10R, previously shown to be required for other anti-inflammatory responses. Thus, the present results show that both SOCS1 and SOCS3 are induced by IL-10 and may be important inhibitors of both IL-10 and IFN-gamma signaling. IL-10-induced SOCS1 may directly inhibit IL-10 IFN-gamma signaling, while inhibition of other proinflammatory cytokine responses may use additional IL-10R1-mediated mechanisms.     

IL-22-mediated liver cell regeneration is abrogated by SOCS-1/3 overexpression in vitro

The IL-10-like cytokine IL-22 is produced by activated T cells. In this study, we analyzed the role of this cytokine system in hepatic cells. Expression studies were performed by RT-PCR and quantitative PCR. Signal transduction was analyzed by Western blot experiments and ELISA. Cell proliferation was measured by MTS and [(3)H]thymidine incorporation assays. Hepatocyte regeneration was studied in in vitro restitution assays. Binding of IL-22 to its receptor complex expressed on human hepatic cells and primary human hepatocytes resulted in the activation of MAPKs, Akt, and STAT proteins. IL-22 stimulated cell proliferation and migration, which were both significantly inhibited by the phosphatidylinositol 3-kinase inhibitor wortmannin. IL-22 increased the mRNA expression of suppressor of cytokine signaling (SOCS)-3 and the proinflammatory cytokines IL-6, IL-8, and TNF-alpha. SOCS-1/3 overexpression abrogated IL-22-induced STAT activation and decreased IL-22-mediated liver cell regeneration. Hepatic IL-22 mRNA expression was detectable in different forms of human hepatitis, and hepatic IL-22 mRNA levels were increased in murine T cell-mediated hepatitis in vivo following cytomegalovirus infection, whereas no significant differences were seen in an in vivo model of ischemia-reperfusion injury. In conclusion, IL-22 promotes liver cell regeneration by increasing hepatic cell proliferation and hepatocyte migration through the activation of Akt and STAT signaling, which is abrogated by SOCS-1/3 overexpression.     

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.     

Effect of recombinant IL-10 on cultured fetal rat alveolar type II cells exposed to 65%-hyperoxia

Background

Hyperoxia plays an important role in the genesis of lung injury in preterm infants. Although alveolar type II cells are the main target of hyperoxic lung injury, the exact mechanisms whereby hyperoxia on fetal alveolar type II cells contributes to the genesis of lung injury are not fully defined, and there have been no specific measures for protection of fetal alveolar type II cells.

Objective

The aim of this study was to investigate (a) cell death response and inflammatory response in fetal alveolar type II cells in the transitional period from canalicular to saccular stages during 65%-hyperoxia and (b) whether the injurious stimulus is promoted by creating an imbalance between pro- and anti-inflammatory cytokines and (c) whether treatment with an anti-inflammatory cytokine may be effective for protection of fetal alveolar type II cells from injury secondary to 65%-hyperoxia.

Methods

Fetal alveolar type II cells were isolated on embryonic day 19 and exposed to 65%-oxygen for 24 h and 36 h. Cells in room air were used as controls. Cellular necrosis was assessed by lactate dehydrogenase-release and flow cytometry, and apoptosis was analyzed by TUNEL assay and flow cytometry, and cell proliferation was studied by BrdU incorporation. Release of cytokines including VEGF was analyzed by ELISA, and their gene expressions were investigated by qRT-PCR.

Results

65%-hyperoxia increased cellular necrosis, whereas it decreased cell proliferation in a time-dependent manner compared to controls. 65%-hyperoxia stimulated IL-8-release in a time-dependent fashion, whereas the anti-inflammatory cytokine, IL-10, showed an opposite response. 65%-hyperoxia induced a significant decrease of VEGF-release compared to controls, and similar findings were observed on IL-8/IL-10/VEGF genes expression. Preincubation of recombinant IL-10 prior to 65%-hyperoxia decreased cellular necrosis and IL-8-release, and increased VEGF-release and cell proliferation significantly compared to hyperoxic cells without IL-10.

Conclusions

The present study provides an experimental evidence that IL-10 may play a potential role in protection of fetal alveolar type II cells from injury induced by 65%-hyperoxia.     

In vitro inhibitory effects of cirsiliol on IL-6-induced STAT3 activation through anti-inflammatory activity

Many studies have identified and described various medicinal effects of cirsiliol. Here, we investigated the signaling pathway involved in the anti-inflammatory effects of cirsiliol on IL-6-induced activity. Cirsiliol showed no cytotoxicity and inhibited pSTAT3-induced luciferase activity. At the molecular level, cirsiliol suppressed the expression of IL-6-induced inflammatory marker genes such as CRP, IL-1β, ICAM-1 and SOCS3, IL-6-induced activation of Jak2, gp130, STAT3 and ERK and nuclear translocation of STAT3, as measured by PCR, immunofluorescence staining and western blot analysis. However, the interaction between IL-6 and its receptor was not affected by cirsiliol treatment. These results indicate that cirsiliol attenuates IL-6-induced cellular signaling by regulating Jak2 phosphorylation. Therefore, cirsiliol could be a therapeutic agent for IL-6-related inflammatory diseases.     

Human Mesenchymal Stem Cells Suppress the Stretch–Induced Inflammatory miR-155 and Cytokines in Bronchial Epithelial Cells

Current research in pulmonary pathology has focused on inflammatory reactions initiated by immunological responses to allergens and irritants. In addition to these biochemical stimuli, physical forces also play an important role in regulating the structure, function, and metabolism of the lung. Hyperstretch of lung tissues can contribute to the inflammatory responses in asthma, but the mechanisms of mechanically induced inflammation in the lung remain unclear. Our results demonstrate that excessive stretch increased the secretion of inflammatory cytokines by human bronchial epithelial cells (hBECs), including IL-8. This increase of IL-8 secretion was due to an elevated microRNA-155 (miR-155) expression, which caused the suppression of Src homology 2 domain–containing inositol 5-phosphatase 1 (SHIP1) production and the subsequent activation of JNK signaling. In vivo studies in our asthmatic mouse model also showed such changes in miR-155, IL-8, and SHIP1 expressions that reflect inflammatory responses. Co-culture with human mesenchymal stem cells (hMSCs) reversed the stretch-induced hBEC inflammatory responses as a result of IL-10 secretion by hMSCs to down-regulate miR-155 expression in hBECs. In summary, we have demonstrated that mechanical stretch modulates the homeostasis of the hBEC secretome involving miR-155 and that hMSCs can be used as a potential therapeutic approach to reverse bronchial epithelial inflammation in asthma.     

Dual function of interleukin-1beta for the regulation of interleukin-6-induced suppressor of cytokine signaling 3 expression

Interleukin-6 (IL-6) exerts pro- as well as anti-inflammatory activities in response to infection, injury, or other stimuli that affect the homeostasis of the organism. IL-6-induced expression of acute-phase protein genes in the liver is tightly regulated through both IL-6-induced feedback inhibitors and the activity of pro-inflammatory cytokines such as tumor necrosis factor alpha and interleukin-1beta. In previous studies mechanisms for how IL-1beta counteracts IL-6-dependent acute-phase protein gene induction have been proposed. Herein we analyzed IL-1beta-mediated regulation of IL-6-induced expression of the feedback inhibitor SOCS3. In hepatocytes IL-1beta alone does not induce SOCS3 expression, but it counteracts SOCS3-promoter activation in long term studies. Surprisingly, short term stimulation revealed IL-1beta to be a potent enhancer of SOCS3 expression in concert with IL-6. This activity of IL-1beta does not depend on IL-1beta-dependent STAT1-serine phosphorylation but on NF-kappaB-dependent gene induction. Such a regulatory network allows IL-1beta to counteract IL-6-dependent expression of acute-phase protein genes without inhibiting IL-6-induced SOCS3 expression and provides a reasonable mechanism for the IL-1beta-dependent inhibition of acute-phase gene induction, because reduced SOCS3 expression would lead to enhanced IL-6 activity.     

Unique expression of suppressor of cytokine signaling 3 is essential for classical macrophage activation in rodents in vitro and in vivo

On infiltrating inflamed tissue, macrophages respond to the local microenvironment and develop one of two broad phenotypes: classically activated (M1) macrophages that cause tissue injury and alternatively activated macrophages that promote repair. Understanding how this polarization occurs in vivo is far from complete, and in this study, using a Th1-mediated macrophage-dependent model of acute glomerulonephritis, nephrotoxic nephritis, we examine the role of suppressor of cytokine signaling (SOCS)1 and SOCS3. Macrophages in normal kidneys did not express detectable SOCS proteins but those infiltrating inflamed glomeruli were rapidly polarized to express either SOCS1 (27 +/- 6%) or SOCS3 (54 +/- 12%) but rarely both (10 +/- 3%). Rat bone marrow-derived macrophages incubated with IFN-gamma or LPS expressed SOCS1 and SOCS3, whereas IL-4 stimulated macrophages expressed SOCS1 exclusively. By contrast, incubation with IFN-gamma and LPS together suppressed SOCS1 while uniquely polarizing macrophages to SOCS3 expressing cells. Macrophages in which SOCS3 was knocked down by short interfering RNA responded to IFN-gamma and LPS very differently: they had enhanced STAT3 activity; induction of macrophage mannose receptor, arginase and SOCS1; restoration of IL-4 responsiveness that is inhibited in M1 macrophages; and decreased synthesis of inflammatory mediators (NO and IL-6) and costimulatory molecule CD86, demonstrating that SOCS3 is essential for M1 activation. Without it, macrophages develop characteristic alternatively activated markers when exposed to classical activating stimuli. Lastly, increased glomerular IL-4 in nephrotoxic nephritis inhibits infiltrating macrophages from expressing SOCS3 and was associated with attenuated glomerular injury. Consequently, we propose that SOCS3 is essential for development of M1 macrophages in vitro and in vivo.     

Prostaglandin E2 augments IL-10 signaling and function

In inflamed joints of rheumatoid arthritis, PGE(2) is highly expressed, and IL-10 and IL-6 are also abundant. PGE(2) is a well-known activator of the cAMP signaling pathway, and there is functional cross-talk between cAMP signaling and the Jak-STAT signaling pathway. In this study, we evaluated the modulating effect of PGE(2) on STAT signaling and its biological function induced by IL-10 and IL-6, and elucidated its mechanism in THP-1 cells. STAT phosphorylation was determined by Western blot, and gene expression was analyzed using real-time PCR. Pretreatment with PGE(2) significantly augmented IL-10-induced STAT3 and STAT1 phosphorylation, as well as suppressors of cytokine signaling 3 (SOCS3) and IL-1R antagonist gene expression. In contrast, PGE(2) suppressed IL-6-induced phosphorylation of STAT3 and STAT1. These PGE(2)-induced modulating effects were largely reversed by actinomycin D. Pretreatment with dibutyryl cAMP augmented IL-10-induced, but did not change IL-6-induced STAT3 phosphorylation. Misoprostol, an EP2/3/4 agonist, and butaprost, an EP2 agonist, augmented IL-10-induced STAT3 phosphorylation and SOCS3 gene expression, but sulprostone, an EP1/3 agonist, had no effect. H89, a protein kinase A inhibitor, and LY294002, a PI3K inhibitor, diminished PGE(2)-mediated augmentation of IL-10-induced STAT3 phosphorylation. In this study, we found that PGE(2) selectively regulates cytokine signaling via increased intracellular cAMP levels and de novo gene expression, and these modulating effects may be mediated through EP2 or EP4 receptors. PGE(2) may modulate immune responses by alteration of cytokine signaling in THP-1 cells.