Induction of IL-4 release and upregulated expression of protease activated receptors by GM-CSF in P815 cells

GM-CSF has been showed to be able to induce up-regulated receptor and cytokine expression in mast cells in inflammatory conditions. However, little is known of its effects on protease activated receptor (PAR) expression and Th2 cytokine secretion from mast cells. In the present study, we examined potential influence of GM-CSF on mast cell PAR expression and IL-4 and IL-10 release by using flow cytometry analysis, quantitative real time PCR, ELISA and cellular activation of signaling ELISA (CASE) techniques. The results showed that GM-CSF induced up to 3.0-fold increase in IL-4 release from P815 cells, and FSLLRY-NH₂ and trans-cinnamoyl (tc)-YPGKF-NH₂ did not affect GM-CSF induced IL-4 release. GM-CSF reduced tryptase and trypsin induced IL-4 release by up to approximately 55.8% and 70.3%, respectively. GM-CSF elicited the upregulated expression of PAR-1, PAR-2, PAR-3 and PAR-4 mRNAs, but enhanced only PAR-4 protein expression in P815 cells. U0126, PD98059 and LY204002 almost completely abolished GM-CSF induced IL-4 release when they were preincubated with P815 cells for 30 min, indicating ERK and Akt cell signaling pathways may be involved in the event. In conclusion, GM-CSF can stimulate IL-4 release from mast cells through an ERK and Akt cell signaling pathway dependent, but PAR independent mechanism. GM-CSF may serve as a regulator for IL-4 production in mast cells and through which participates in the mast cell related inflammation.     

PAR-1-dependent and PAR-independent pro-inflammatory signaling in human lung fibroblasts exposed to thrombin

Proteinase-activated receptors (PARs) are crucial in orchestrating cellular responses to coagulation proteinases, such as thrombin and FXa. Four PARs have been characterized and have been shown to be differentially expressed in mice and humans and between tissues. We have previously shown that in murine lung fibroblasts, PAR-1 is solely responsible for all cellular responses to thrombin and FXa. In contrast, we report here that in primary human lung fibroblasts (pHLFs), known PARs fail to account for all of the cellular responses to thrombin, in particular in the presence of high, but physiologically achievable concentrations of thrombin. We report that pHLFs secrete CCL2 in a PAR-1-dependent manner at low thrombin concentration (～0.3 nM). At or above 10 nM thrombin, pharmacological antagonism (RWJ-58259) fails to block thrombin-induced CCL2 release; whereas PAR-1 cleavage-blocking monoclonal antibodies (ATAP2 and WEDE15) only partially inhibit thrombin-induced CCL2 secretion. In addition, activation of PAR-3, PAR-4, and transactivation of either PAR-2 or EGFR were ruled out as being responsible for thrombin-mediated CCL2 secretion at high yet standard concentrations of the proteinase. We further provide evidence that PAR-1-dependent and PAR-independent signaling involves the rapid phosphorylation of ERK, which in turn is absolutely required for thrombin-induced CCL2 secretion at both low and standard concentration of the proteinase. Our findings suggest the existence of a PAR-independent signaling mechanism in human lung fibroblasts and have important implications for the design of therapeutic strategies aimed at blocking pro-inflammatory signaling responses associated with excessive thrombin generation.     

Mast cell tryptase stimulates human lung fibroblast proliferation via protease-activated receptor-2

Mast cells play a potentially important role in fibroproliferative diseases, releasing mediators including tryptase that are capable of stimulating fibroblast proliferation and procollagen synthesis. The mechanism by which tryptase stimulates fibroblast proliferation is unclear, although recent studies suggest it can activate protease-activated receptor (PAR)-2. We therefore investigated the role of PAR-2 in tryptase-induced proliferation of human fetal lung and adult lung parenchymal and airway fibroblasts and, for comparative purposes, adult dermal fibroblasts. Tryptase (0.7-70 mU/ml) induced concentration-dependent increases in proliferation of all fibroblasts studied. Antipain, bis(5-amidino-2-benzimidazolyl)methane, and benzamidine inhibited tryptase-induced fibroblast proliferation, demonstrating that proteolytic activity is required for the proliferative effects of tryptase. RT-PCR demonstrated the presence of PAR-2 mRNA, and immunohistochemical staining localized PAR-2 to the cell surface of lung fibroblasts. In addition, specific PAR-2 activating peptides, SLIGKV and SLIGRL, mimicked the proliferative effects of tryptase. In contrast, human dermal fibroblasts only weakly stained with the PAR-2 antibody, PAR-2 mRNA was almost undetectable, and fibroblasts did not respond to PAR-2 activating peptides. These results suggest that tryptase induces lung, but not dermal, fibroblast proliferation via activation of PAR-2 and are consistent with the hypothesis that the release of tryptase from activated mast cells may play an important role in the fibroproliferative response observed in asthma, chronic obstructive pulmonary disease, and patients with pulmonary fibrosis.     

Modulation of mast cell proteinase-activated receptor expression and IL-4 release by IL-12

It has been recognized that protease-activated receptors (PARs), interleukin (IL)-4 and IL-6 are involved in the pathogenesis of allergic diseases, and that IL-12 plays a role in adaptive immune response. However, little is known of the effect of IL-12 on protease-induced cytokine release from mast cells. In the present study, we examined potential influence of IL-12 on mast cell PAR expression and IL-4 and IL-6 release. The results showed that IL-12 downregulated the expression of PAR-2 and upregulated expression of PAR-4 on P815 cells. It also downregulated expression of PAR-2 mRNA, and upregulated expression of PAR-1, PAR-3 and PAR-4 mRNAs. However, IL-12 enhanced trypsin- and tryptase-induced PAR-2 and PAR-2 mRNA expression. It was observed that IL-12 induced release of IL-4, but reduced trypsin- and tryptase-stimulated IL-4 secretion from P815 cells. PD98059, U0126 and LY294002 not only abolished IL-12-induced IL-4 release but also inhibited IL-12-induced phosphorylation of extracellular signal-regulated kinase and Akt. In conclusion, IL-12 may serve as a regulator in keeping the balance of Th1 and Th2 cytokine production in allergic inflammation.     

Oncostatin M regulates eotaxin expression in fibroblasts and eosinophilic inflammation in C57BL/6 mice

Oncostatin M (OSM) is a member of the IL-6/LIF (or gp130) cytokine family, and its potential role in inflammation is supported by a number of activities identified in vitro. In this study, we investigate the action of murine OSM on expression of the CC chemokine eotaxin by fibroblasts in vitro and on mouse lung tissue in vivo. Recombinant murine OSM stimulated eotaxin protein production and mRNA levels in the NIH 3T3 fibroblast cell line. IL-6 could regulate a small induction of eotaxin in NIH 3T3 cells, but other IL-6/LIF cytokines (LIF, cardiotrophin-1 (CT-1)) had no effect. Cell signaling studies showed that murine OSM, LIF, IL-6, and CT-1 stimulated the tyrosine phosphorylation of STAT-3, suggesting STAT-3 activation is not sufficient for eotaxin induction in NIH 3T3 cells. OSM induced ERK-1,2 and p38 mitogen-activated protein kinase phosphorylation in NIH 3T3 cells, and inhibitors of ERK (PD98059) or p38 (SB203580) could partially reduce OSM-induced eotaxin production, suggesting partial dependence on mitogen-activated protein kinase signaling. OSM (but not LIF, IL-6, or CT-1) also induced eotaxin release by mouse lung fibroblast cultures derived from C57BL/6 mice. Overexpression of murine OSM in lungs of C57BL/6 mice using an adenovirus vector encoding murine OSM resulted in a vigorous inflammatory response by day 7 after intranasal administration, including marked extracellular matrix accumulation and eosinophil infiltration. Elevated levels of eotaxin mRNA in whole lung were detected at days 4 and 5. These data strongly support a role of OSM in lung inflammatory responses that involve eosinophil infiltration.     

Human airway trypsin-like protease induces amphiregulin release through a mechanism involving protease-activated receptor-2-mediated ERK activation and TNF alpha-converting enzyme activity in airway epithelial cells

Human airway trypsin-like protease (HAT), a serine protease found in the sputum of patients with chronic airway diseases, is an agonist of protease-activated receptor-2 (PAR-2). Previous results have shown that HAT enhances the release of amphiregulin (AR); further, it causes MUC5AC gene expression through the AR-epidermal growth factor receptor pathway in the airway epithelial cell line NCI-H292. In this study, the mechanisms by which HAT-induced AR release can occur were investigated. HAT-induced AR gene expression was mediated by extracellular signal-regulated kinase (ERK) pathway, as pretreatment of cells with ERK pathway inhibitor eliminated the effect of HAT on AR mRNA. Both HAT and PAR-2 agonist peptide (PAR-2 AP) induced ERK phosphorylation; further, desensitization of PAR-2 with a brief exposure of cells to PAR-2 AP resulted in inhibition of HAT-induced ERK phosphorylation, suggesting that HAT activates ERK through PAR-2. Moreover, PAR-2 AP induced AR gene expression subsequent to protein production in the cellular fraction through the ERK pathway indicating that PAR-2-mediated activation of ERK is essential for HAT-induced AR production. However, in contrast to HAT, PAR-2 AP could not cause AR release into extracellular space; it appears that activation of PAR-2 is not sufficient for HAT-induced AR release. Finally, HAT-induced AR release was eliminated by blockade of tumour necrosis factor alpha-converting enzyme (TACE) by the TAPI-1 and RNA interference, suggesting that TACE activity is necessary for HAT-induced AR release. These observations show that HAT induces AR production through the PAR-2 mediated ERK pathway, and then causes AR release by a TACE-dependent mechanism.     

Mold allergen, pen C 13, induces IL-8 expression in human airway epithelial cells by activating protease-activated receptor 1 and 2

Allergenic serine proteases are important in the pathogenesis of asthma. One of these, Pen c 13, is the immunodominant allergen produced by Penicillium citrinum. Many serine proteases induce cytokine expression, but whether Pen c 13 does so in human respiratory epithelial cells is not known. In this study, we investigated whether Pen c 13 caused IL-8 release and activated protease-activated receptors (PARs) in airway epithelial cells. In airway-derived A549 cells and normal human airway epithelial cells, Pen c 13 induced IL-8 release in a dose-dependent manner. Pen c 13 also increased IL-8 release in a time-dependent manner in A549 cells. Pen c 13 cleaved PAR-1 and PAR-2 at their activation sites. Treatment with Pen c 13 induced intracellular Ca(2+) mobilization and desensitized the cells to the action of other proteases and PAR-1 and PAR-2 agonists. Moreover, Pen c 13-mediated IL-8 release was significantly decreased in Ca(2+)-free medium and was abolished by the protease inhibitors, PMSF and 4-(2-aminoethyl) benzenesulfonyl fluoride. Blocking Abs against the cleavage sites of PAR-1 and PAR-2, but not of PAR-4, inhibited Pen c 13-induced IL-8 production, as did inhibition of phospholipase C. Pen c 13 induced IL-8 expression via activation of ERK 1/2, and not of p38 and JNK. In addition, treatment of A549 cells or normal human airway epithelial cells with Pen c 13 increased phosphorylation of ERK 1/2 by a Ca(2+)-dependent pathway. These finding show that Pen c 13 induces IL-8 release in airway epithelial cells and that this is dependent on PAR-1 and PAR-2 activation and intracellular calcium.     

Differential regulation of cytokine release and leukocyte migration by lipopolysaccharide-stimulated primary human lung alveolar type II epithelial cells and macrophages

Bacterial colonization is a secondary feature of many lung disorders associated with elevated cytokine levels and increased leukocyte recruitment. We hypothesized that, alongside macrophages, the epithelium would be an important source of these mediators. We investigated the effect of LPS (0, 10, 100, and 1000 ng/ml LPS, up to 24 h) on primary human lung macrophages and alveolar type II epithelial cells (ATII; isolated from resected lung tissue). Although macrophages produced higher levels of the cytokines TNF-alpha and IL-1beta (p < 0.0001), ATII cells produced higher levels of chemokines MCP-1, IL-8, and growth-related oncogene alpha (p < 0.001), in a time- and concentration-dependent manner. Macrophage (but not ATII cell) responses to LPS required activation of ERK1/2 and p38 MAPK signaling cascades; phosphorylated ERK1/2 was constitutively up-regulated in ATII cells. Blocking Abs to TNF-alpha and IL-1beta during LPS exposure showed that ATII cell (not macrophage) MCP-1 release depended on the autocrine effects of IL-1beta and TNF-alpha (p < 0.003, 24 h). ATII cell release of IL-6 depended on autocrine effects of TNF-alpha (p < 0.006, 24 h). Macrophage IL-6 release was most effectively inhibited when both TNF-alpha and IL-1beta were blocked (p < 0.03, 24 h). Conditioned media from ATII cells stimulated more leukocyte migration in vitro than conditioned media from macrophages (p < 0.0002). These results show differential activation of cytokine and chemokine release by ATII cells and macrophages following LPS exposure. Activated alveolar epithelium is an important source of chemokines that orchestrate leukocyte migration to the peripheral lung; early release of TNF-alpha and IL-1beta by stimulated macrophages may contribute to alveolar epithelial cell activation and chemokine production.     

Modulation of matrix metalloproteinase production from human lung fibroblasts by type 4 phosphodiesterase inhibitors

Over-expression of matrix metalloproteinases by lung fibroblasts has been blamed for much of the tissue destruction associated with airway inflammation. Because cyclic AMP is known to regulate fibroblast proliferation, as well as cytokine and extracellular matrix protein production, the current study was designed to evaluate the ability of three selective phosphodiesterase (PDE) type 4 inhibitors, rolipram, cilomilast and CI-1044, to inhibit extracellular matrix degradation. Using zymography and ELISA, we found that pro-MMP-2 release was enhanced following 24 h treatment of human lung fibroblast (MRC-5) with TGF-beta1 (10 ng/ml) or TNF-alpha (10 ng/ml), whereas PMA (0.02 microM) had no effect. One hour of pre-incubation with PDE4 inhibitors (10 microM) induced an inhibition of TNF-alpha-stimulated pro-MMP-2 release. Zymography and immunoblotting revealed that fibroblasts cultured with PMA or TNF-alpha released increased amounts of pro-MMP-1, whereas TGF-beta1 had no effect. Incubation with CI-1044 or cilomilast significantly prevented the TNF-alpha increase in pro-MMP-1. These results suggest that PDE4 inhibitors are effective in inhibiting the pro-MMP-2 and pro-MMP-1 secretion induced by TNF-alpha and might underline a potential therapeutic benefit of selective PDE4 inhibitors in lung diseases associated with abnormal tissue remodelling.     

Sphingosine kinase 1 is essential for proteinase-activated receptor-1 signalling in epithelial and endothelial cells

There is accumulating evidence that activation of sphingosine kinase 1 (SPHK1) is an important element in intracellular signalling cascades initiated by stimulation of multiple receptors, including certain growth factor, cytokine, and also G-protein coupled receptors. We here report that stimulation of the lung epithelial cell line A549 by thrombin leads to transient increase of SPHK1 activity and elevation of intracellular sphingosine-1-phosphate (S1P); abrogation of this stimulation by SPHK1-specific siRNA, pharmacological inhibition, or expression of a dominant-negative SPHK1 mutant blocks the response to thrombin, as measured by secretion of MCP-1, IL-6, IL-8, and PGE₂. Using selective stimulation of proteinase-activated receptors (PARs) a specific involvement of SPHK1 in the PAR-1 induced responses in A549 cell, including activation of NFκB, was evident, while PAR-2 and PAR-4 responses were independent of SPHK1. Moreover, PAR-1 or thrombin-induced cytokine production and adhesion factor expression of human umbilical vein endothelial cells was also seen to depend on SPHK1. Using dermal microvascular endothelial cells from SPHK1-deficient mice, we showed that absence of the enzyme abrogates MCP-1 production induced in these cells upon treatment with thrombin or PAR-1 activating peptide. We propose SPHK1 inhibition as a novel way to block PAR-1 mediated signalling, which could be useful in treatment of a number of diseases, in particular in atherosclerosis.     

白细胞介素-6在急性胰腺炎中的作用及其机制研究

目的:明确白细胞介素-6(IL-6)在小鼠急性胰腺炎中的作用及其机制研究。方法:通过胰胆管结扎的方法诱导小鼠急性胰腺炎;分离小鼠胰腺腺泡细胞。采用ELISA方法检测胰腺组织或腺泡细胞裂解物中的细胞因子;通过western blot分析检测组织或细胞中IL-6或ERK表达。结果:IL-6浓度在胰腺组织和腺泡细胞中显著增加(P0.05)。在离体原代小鼠腺泡细胞,TNF-α刺激增加IL-6释放(P0.05);与此同时,IL-6刺激可增加其它促炎性细胞因子的释放,两者都涉及ERK MAP激酶通路。黄酮类化合物木犀草素抑制IL-6刺激引起白细胞介素-6(IL-6)和人巨嗜细胞激活蛋白-1(CCL2/MCP-1)释放。最后进一步证实,IL-6激活人胰腺组织中的ERK。结论:IL-6在急性胰腺炎中增加,激活炎症通路并加重急性胰腺炎。     

The Novel Cytokine Interleukin-33 Activates Acinar Cell Proinflammatory Pathways and Induces Acute Pancreatic Inflammation in Mice

Background

Acute pancreatitis is potentially fatal but treatment options are limited as disease pathogenesis is poorly understood. IL-33, a novel IL-1 cytokine family member, plays a role in various inflammatory conditions but its role in acute pancreatitis is not well understood. Specifically, whether pancreatic acinar cells produce IL-33 when stressed or respond to IL-33 stimulation, and whether IL-33 exacerbates acute pancreatic inflammation is unknown.

Methods/Results

In duct ligation-induced acute pancreatitis in mice and rats, we found that (a) IL-33 concentration was increased in the pancreas; (b) mast cells, which secrete and also respond to IL-33, showed degranulation in the pancreas and lung; (c) plasma histamine and pancreatic substance P concentrations were increased; and (d) pancreatic and pulmonary proinflammatory cytokine concentrations were increased. In isolated mouse pancreatic acinar cells, TNF-α stimulation increased IL-33 release while IL-33 stimulation increased proinflammatory cytokine release, both involving the ERK MAP kinase pathway; the flavonoid luteolin inhibited IL-33-stimulated IL-6 and CCL2/MCP-1 release. In mice without duct ligation, exogenous IL-33 administration induced pancreatic inflammation without mast cell degranulation or jejunal inflammation; pancreatic changes included multifocal edema and perivascular infiltration by neutrophils and some macrophages. ERK MAP kinase (but not p38 or JNK) and NF-kB subunit p65 were activated in the pancreas of mice receiving exogenous IL-33, and acinar cells isolated from the pancreas of these mice showed increased spontaneous cytokine release (IL-6, CXCL2/MIP-2α). Also, IL-33 activated ERK in human pancreatic tissue.

Significance

As exogenous IL-33 does not induce jejunal inflammation in the same mice in which it induces pancreatic inflammation, we have discovered a potential role for an IL-33/acinar cell axis in the recruitment of neutrophils and macrophages and the exacerbation of acute pancreatic inflammation.

Conclusion

IL-33 is induced in acute pancreatitis, activates acinar cell proinflammatory pathways and exacerbates acute pancreatic inflammation.     

Modulation of PAR expression and tryptic enzyme induced IL-4 production in mast cells by IL-29

Interleukin (IL)-29 is a relatively newly discovered cytokine, which has been shown to be actively involved in the pathogenesis of allergic inflammation. However, little is known of the effects of IL-29 on protease activated receptor (PAR) expression and potential mechanisms of cytokine production in mast cells. In the present study, we examined potential influence of IL-29 on PAR expression and cytokine production in P815 and bone marrow derived mast cells (BMMCs) by using flow cytometry analysis, quantitative real time PCR, and ELISA techniques. The results showed that IL-29 downregulated the expression of PAR-1 by up to 56.2%, but had little influence on the expression of PAR-2, PAR-3 and PAR-4. IL-29 also induced downregulation of expression of PAR-1 mRNA. However, when mast cells were pre-incubated with IL-29, thrombin-, trypsin- and tryptase-induced expression of PAR-2, PAR-3 and PAR-4 was upregulated, respectively. IL-29 provoked approximately up to 1.9-fold increase in IL-4 release when mast cells was challenged with IL-29. Administration of IL-29 blocking antibody, AG490 or LY294002 abolished IL-29-induced IL-4 release from P815 cells. It was found that IL-29 diminished trypsin- and tryptase-induced IL-4 release from P815 cells following 16 h incubation. In conclusion, IL-29 can regulate expression of PARs and tryptase- and trypsin-induced IL-4 production in mast cells, through which participates in the mast cell related inflammation.     

Paracoccidioides brasiliensis induces secretion of IL-6 and IL-8 by lung epithelial cells. Modulation of host cytokine levels by fungal proteases

Paracoccidioides brasiliensis is a pathogenic, dimorphic fungus that causes paracoccidioidomycosis, a systemic human mycosis that is highly prevalent in Latin America. In this study, we demonstrated that P. brasiliensis yeasts induced interleukin (IL)-8 and IL-6 secretion by human lung epithelial A549 cells. However, tumor necrosis factor-α and interferon-γ were undetectable in these cultures. Moreover, P. brasiliensis yeasts induced activation of p38 mitogen-activated protein kinase (MAPK), c-Jun NH(2)-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) 1/2 in A549 cells, and IL-8 and IL-6 secretion promoted by this fungus was dependent on activation of p38 MAPK and ERK 1/2. In addition, IL-8 and IL-6 levels were significantly higher in culture supernatants of A549 cells that were incubated with formaldehyde-fixed P. brasiliensis compared to cultures of cells that were infected with live yeasts. Our results indicate that the observed cytokine level differences were due to protease expression, in live yeasts, that degraded these cytokines. Degradation of human recombinant IL-8 and IL-6 by live P. brasiliensis was inhibited by AEBSF and aprotinin, suggesting that these proteases belong to a family of serine proteases. This is the first report showing that P. brasiliensis may modulate host inflammation by expressing proteases that degrade proinflammatory cytokines.     

Serine protease inhibitors modulate chemotactic cytokine production by human lung fibroblasts in vitro

Chemotactic chemokines can be released from lung fibroblasts in response to interleukin (IL)-1beta and tumor necrosis factor (TNF)-alpha. An imbalance between proteases and antiproteases has been observed at inflammatory sites, and, therefore, protease inhibitors might modulate fibroblast release of chemotactic cytokines. To test this hypothesis, serine protease inhibitors (FK-706, alpha(1)-antitrypsin, or N(alpha)-p-tosyl-L-lysine chloromethyl ketone) were evaluated for their capacity to attenuate the release of neutrophil chemotactic activity (NCA) or monocyte chemotactic activity (MCA) from human fetal lung fibroblasts (HFL-1). Similarly, the release of the chemoattractants IL-8, granulocyte colony-stimulating factor, monocyte chemoattractant protein-1, macrophage colony-stimulating factor, and granulocyte/macrophage colony-stimulating factor, from HFL-1, were evaluated in response to IL-1beta and TNF-alpha. NCA, MCA, and chemotactic cytokines were attenuated by FK-706. However, matrix metalloproteinase inhibitors were without effect, and cysteine protease inhibitors only slightly attenuated chemotactic or cytokine release. These data suggest that IL-1beta and TNF-alpha may stimulate lung fibroblasts to release NCA and MCA by a protease-dependent mechanism and that serine protease inhibitors may attenuate the release.