IL-13 signaling through the IL-13alpha2 receptor is involved in induction of TGF-beta1 production and fibrosis

Interleukin (IL)-13 is a major inducer of fibrosis in many chronic infectious and autoimmune diseases. In studies of the mechanisms underlying such induction, we found that IL-13 induces transforming growth factor (TGF)-beta(1) in macrophages through a two-stage process involving, first, the induction of a receptor formerly considered to function only as a decoy receptor, IL-13Ralpha(2). Such induction requires IL-13 (or IL-4) and tumor necrosis factor (TNF)-alpha. Second, it involves IL-13 signaling through IL-13Ralpha(2) to activate an AP-1 variant containing c-jun and Fra-2, which then activates the TGFB1 promoter. In vivo, we found that prevention of IL-13Ralpha(2) expression reduced production of TGF-beta(1) in oxazolone-induced colitis and that prevention of IL-13Ralpha(2) expression, Il13ra2 gene silencing or blockade of IL-13Ralpha(2) signaling led to marked downregulation of TGF-beta(1) production and collagen deposition in bleomycin-induced lung fibrosis. These data suggest that IL-13Ralpha(2) signaling during prolonged inflammation is an important therapeutic target for the prevention of TGF-beta(1)-mediated fibrosis.     

Sphingosine potentiates IL-1-mediated prostaglandin E2 production in human fibroblasts

IL-1 stimulates PGE2 production in human fibroblasts by stimulating arachidonic acid (AA) mobilization and cyclooxygenase synthesis. Cyclooxygenase is the first enzyme in the pathway that converts AA to PGE2. To examine the role of protein kinase C (PKC) in IL-1-mediated PGE2 production, we treated cells with PMA, which stimulated PGE2 production suggesting a positive role for PKC activation in the regulation of PGE2 synthesis. Therefore, we tested the effect of sphingosine, a PKC inhibitor, on IL-1-induced PGE2 production. Alone, sphingosine had little effect on PGE2 production. However, when sphingosine was added with IL-1, or IL-1 was added to sphingosine-pretreated cells, PGE2 production increased severalfold, suggesting that the inhibition of PKC results in enhanced IL-1-mediated PGE2 production; structural analogs of sphingosine did not potentiate the IL-1 effect. In cells made deficient in PKC by prolonged exposure to PMA, IL-1-mediated PGE2 production was enhanced compared with normal cells, further suggesting that functional PKC is not required for, and may down-modulate, IL-1-mediated PGE2 production. These findings also suggest that PMA and IL-1 stimulate PGE2 synthesis via fundamentally different pathways. In separate studies on the effect of IL-1 on AA mobilization, we found that IL-1 induced an increase in phospholipase A2 (PLA2) activity and that cycloheximide blocked the increase, suggesting the requirement for new protein synthesis. We also found that the PLA2 activity increased as a result of IL-1 exposure was further stimulated by sphingosine. Thus, in addition to its primary effects on the cell, which are likely mediated via PKC, we present evidence suggesting that sphingosine may also play a role in potentiating an IL-1-induced PLA2 activity, resulting in increased availability of AA for conversion to PGE2.     

Leukotriene d(4) and interleukin-13 cooperate to increase the release of eotaxin-3 by airway epithelial cells

Introduction

Airway epithelial cells play a central role in the physiopathology of asthma. They release eotaxins when treated with T_H2 cytokines such as interleukin (IL)-4 or IL-13, and these chemokines attract eosinophils and potentiate the biosynthesis of cysteinyl leukotrienes (cysLTs), which in turn induce bronchoconstriction and mucus secretion. These effects of cysLTs mainly mediated by CysLT₁ and CysLT₂ receptors on epithelial cell functions remain largely undefined. Because the release of inflammatory cytokines, eotaxins, and cysLTs occur relatively at the same time and location in the lung tissue, we hypothesized that they regulate inflammation cooperatively rather than redundantly. We therefore investigated whether cysLTs and the T_H2 cytokines would act in concert to augment the release of eotaxins by airway epithelial cells.

Methods

A549 cells or human primary bronchial epithelial cells were incubated with or without IL-4, IL-13, and/or LTD₄. The release of eotaxin-3 and the expression of cysLT receptors were assessed by ELISA, RT-PCR, and flow cytometry, respectively.

Results

IL-4 and IL-13 induced the release of eotaxin-3 by airway epithelial cells. LTD₄ weakly induced the release of eotaxin-3 but clearly potentiated the IL-13-induced eotaxin-3 release. LTD₄ had no effect on IL-4-stimulated cells. Epithelial cells expressed CysLT₁ but not CysLT₂. CysLT₁ expression was increased by IL-13 but not by IL-4 and/or LTD₄. Importantly, the upregulation of CysLT₁ by IL-13 preceded eotaxin-3 release.

Conclusions

These results demonstrate a stepwise cooperation between IL-13 and LTD₄. IL-13 upregulates CysLT₁ expression and consequently the response to cysLTs This results in an increased release of eotaxin-3 by epithelial cells which at its turn increases the recruitment of leukocytes and their biosynthesis of cysLTs. This positive amplification loop involving epithelial cells and leukocytes could be implicated in the recruitment of eosinophils observed in asthmatics.     

Concerted expression of eotaxin-1, eotaxin-2, and eotaxin-3 in human bronchial epithelial cells

Eotaxin-1/CCL11, eotaxin-2/CCL24, and eotaxin-3/CCL26 bind specifically and exclusively to CC chemokine receptor (CCR) 3, which is a potential therapeutic target in treating the peribronchial eosinophilia associated with allergic airway diseases. Bronchial epithelial cells represent an important source of chemokines, and thus we investigated in vitro and in vivo expression of eotaxin-2 and eotaxin-3 in bronchial epithelial cells in comparison with that of eotaxin-1. Immunohistochemistry showed increased expression of both eotaxin-2 and eotaxin-3 in addition to eotaxin-1 in asthmatics. Considerable amounts of eotaxins were secreted by bronchial epithelial lineage. As with eotaxin-1 production, generation of eotaxin-2 and eotaxin-3 by bronchial epithelial cells was up-regulated by IL-4 and IL-13, and attenuated by IFN-gamma and glucocorticoids. In addition to eotaxin-1 expression, but also eotaxin-2 and eotaxin-3 expression in the bronchial epithelium should be taken into consideration when developing the therapeutic strategies to treat eosinophilic airway diseases.     

The stimulatory effects of alpha1-adrenergic receptors on TGF-beta1, IGF-1 and hyaluronan production in human skin fibroblasts

Skin fibroblasts modulate tissue repair, wound healing and immunological responses. Adrenergic receptors (ARs) mediate important physiological functions, such as endocrine, metabolic and neuronal activity. In this study, the expression α1A-ARs in human skin fibroblasts is examined and verified. Regulatory effects of α1-agonist cirazoline on cell migration and the production of transforming growth factor β1 (TGF-β1), insulin-like growth factor 1 (IGF-1), hyaluronan (HA), fibronectin and procollagen type I carboxy-terminal peptide (PIP) by human skin fibroblasts are assessed and validated. α1A-AR mRNA and protein were found in human skin fibroblasts WS1. Exposure of cirazoline doubled skin fibroblast migration and the increase in cell migration was attenuated by α1-antagonist prazosin. TGF-β1 mRNA and production were enhanced after exposure to cirazoline and IGF-1 production was also increased after treatment with cirazoline. Exposure to cirazoline also enhanced HA and PIP production. The increases in TGF-β1, IGF-1, HA and PIP production were partially abolished in fibroblasts transfected with α1A-AR short interfering RNAs, indicating that α1A-ARs are involved in the cirazoline-induced increases in TGF-β1, IGF-1, HA and PIP production. Thus, α1A-ARs are stably expressed and stimulate cell migration and TGF-β1, IGF-1, HA and PIP production in human skin fibroblasts. Moreover, TGF-β1, IGF-1, HA and PIP production and the cell migration of human skin fibroblasts are possibly modulated by natural catecholamines produced by the endocrine system or sympathetic innervation, which could directly or indirectly participate in cytokine secretion, fibroblast migration and matrix production of wound healing in the skin.     

Smad3 mediates TGF-beta1 induction of VEGF production in lung fibroblasts

Transforming growth factor-beta1 (TGF-beta1) is a key factor in a variety of physiological and pathological processes. Vascular endothelial growth factor (VEGF) is a key angiogenic factor, and vascular change is one of the features of airway remodeling. We examined the effect of TGF-beta1 on VEGF production by fibroblasts from mice lacking expression of Smad2 or Smad3 as well as human lung fibroblasts treated with or without Smad2 or Smad3 siRNA. TGF-beta1 stimulated VEGF production by fibroblasts from Smad2 deficient animals and wildtype animals. In contrast, TGF-beta1 did not affect VEGF production by fibroblasts from Samd3 deficient mice. Similarly, TGF-beta1 failed to stimulate VEGF production by HFL-1 cells treated with Samd3 siRNA but significantly increased VEGF production by the cells treated with Smad2 siRNA. These result suggest that TGF-beta1 stimulation of VEGF production by fibroblasts is regulated by Smad3 but not by Smad2 signaling.     

TNF-alpha and IL-4 regulate expression of IL-13 receptor alpha2 on human fibroblasts

Two interleukin 13 receptors (IL-13Rs) have been identified as IL-13Ralpha1 and IL-13Ralpha2. IL-13Ralpha1 is composed of a heterodimer consisting of IL-13Ralpha1 and IL-4 receptor alpha (IL-4Ralpha) as a signaling subunit. In contrast, IL-13Ralpha2 is known as a decoy receptor for IL-13. In this study, we investigated the expression of IL-13Rs on human fibroblasts. IL-13Ralpha2 was significantly up-regulated after stimulation with tumor necrosis factor-alpha (TNF-alpha) and/or IL-4. In contrast, IL-13Ralpha1 was constitutively detectable and was not up-regulated. After the induction of IL-13alpha2 by IL-4, STAT6 phosphorylation through IL-13Ralpha1 by IL-13 was inhibited. We also detected large intracellular pools of IL-13Ralpha2 in fibroblasts quantitatively. Furthermore, mobilization of the IL-13Ralpha2 protein stores from the cytoplasm to the cell surface was prevented by an inhibitor of protein transport, brefeldin-A. These results indicate that TNF-alpha and IL-4 synergistically up-regulate the expression of IL-13Ralpha2 decoy receptor on human fibroblasts by inducing gene expression and mobilizing intracellular receptors, and thus may down-regulate the IL-13 signaling.     

Differential regulation of CCL-11/eotaxin-1 and CXCL-8/IL-8 by Gram-positive and Gram-negative bacteria in human airway smooth muscle cells

Background

Bacterial infections are a cause of exacerbation of airway disease. Airway smooth muscle cells (ASMC) are a source of inflammatory cytokines/chemokines that may propagate local airway inflammatory responses. We hypothesize that bacteria and bacterial products could induce cytokine/chemokine release from ASMC.

Methods

Human ASMC were grown in culture and treated with whole bacteria or pathogen associated molecular patterns (PAMPs) for 24 or 48 h. The release of eotaxin-1, CXCL-8 or GMCSF was measured by ELISA.

Results

Gram-negative E. coli or Gram-positive S. aureus increased the release of CXCL-8, as did IL-1β, LPS, FSL-1 and Pam₃CSK4, whereas FK565, MODLys18 or Poly I:C did not. E. coli inhibited eotaxin-1 release under control conditions and after stimulation with IL-1β. S. aureus tended to inhibit eotaxin-1 release stimulated with IL-1β. E. coli or LPS, but not S. aureus, induced the release of GMCSF.

Conclusion

Gram-positive or Gram-negative bacteria activate human ASMC to release CXCL-8. By contrast Gram-negative bacteria inhibited the release of eotaxin-1 from human ASMCs. E. coli, but not S. aureus induced GMCSF release from cells.Our findings that ASMC can respond directly to Gram-negative and Gram-positive bacteria by releasing the neutrophil selective chemokine, CXCL-8, is consistent with what we know about the role of neutrophil recruitment in bacterial infections in the lung. Our findings that bacteria inhibit the release of the eosinophil selective chemokine, eotaxin-1 may help to explain the mechanisms by which bacterial immunotherapy reduces allergic inflammation in the lung.     

TGF-beta enhances the production of hyaluronan in human lung but not in skin fibroblasts

Transforming growth factor-beta (TGF-beta) enhances the production of extracellular matrix components, such as type I and type III collagen, fibronectin, proteoglycans, in various cell types. The effect on hyaluronan synthesis in relation to proteoglycan synthesis has not been investigated. Human lung or skin fibroblast cultures were treated with TGF-beta in serum-free medium for various periods of time. 35SO4 or [3H]glucosamine was then added to the cultures in the absence of TGF-beta for up to 48 h. Hyaluronan and proteoglycans were isolated by ion-exchange chromatography and quantitated. TGF-beta induced a three- to fourfold increase in hyaluronan production by lung cells but had no effect on skin fibroblasts. In contrast, proteoglycan synthesis was enhanced in both cell types, although skin fibroblasts responded at lower concentrations of TGF-beta. Increased accumulation of hyaluronan was noted only in the cell medium, whereas proteoglycan accumulation was observed both in the medium and in the cell layer. The ED50 for TGF-beta on hyaluronan accumulation in lung cells was the same as that for proteoglycan accumulation, i.e., 40 pM. In skin fibroblasts the ED50 was considerably lower (4 pM). The induction time needed to attain full effect of TGF-beta was 6 h for both hyaluronan and proteoglycan synthesis. These results indicate that TGF-beta has tissue-specific effects on matrix production which may be of importance for control of cell proliferation in various disease states.     

6-Mer hyaluronan oligosaccharides increase IL-18 and IL-33 production in mouse synovial fibroblasts subjected to collagen-induced arthritis

Hyaluronan (HA) oligosaccharides stimulate pro-inflammatory responses in different cell types by modulating both cluster determinant 44 (CD44) and TLR4. The activation of these receptors is also mediated by collagen-induced arthritis (CIA) that, via two different pathways, culminates in the liberation of NF-κB. This then stimulates the production of pro-inflammatory cytokines, including IL-18 and IL-33, that are greatly involved in rheumatoid arthritis. The aim of this study was to investigate the effects of 6-mer HA oligosaccharides on mouse synovial fibroblasts obtained from normal DBA/J1 mice or mice subjected to CIA. Compared with normal synovial fibroblasts (NSF), rheumatoid arthritis synovial fibroblasts (RASF) showed no up-regulation of CD44 and TLR4 mRNA expression and the related proteins, as well as no activation of NF-κB. Very low levels of both mRNA and related proteins were also detected for IL-18 and IL-33. Treatment of NSF and RASF with 6-mer HA oligosaccharides significantly increased all the parameters in both fibroblast groups, although to a greater extent in RASF. The addition of hyaluronan binding protein to both NSF and RASF inhibited HA activity and was able to reduce the effects of 6-mer HA oligosaccharides and the consequent inflammatory response.     

Depressed IL-1 production by chronic GVHD dermal fibroblasts

Chronic Graft-versus-Host disease (GVHD) is characterized by overt immunosuppression. In addition, the skin is a major anatomical site affected in chronic GVHD for reasons not yet known. Increased collagen deposition, a mononuclear cell infiltrate in the dermis as well as loss of fat and appendages, are observed in the skin. The inflammatory cytokine IL-1 was shown to affect fibroblast proliferation and secretory activities. In the present study, IL-1 generation by dermal fibroblasts, of chronic GVHD or control mice, was assessed. It was shown that two sequential signals are needed for IL-1 generation by dermal fibroblasts; priming by lymphokines/cytokines followed by a challenge with LPS. A variety of recombinant lymphokines and cytokines (G/M-CSF, IL-2, TNF, IL-1 beta and IFNs alpha, beta and gamma) were shown to be efficient in priming dermal fibroblasts for IL-1 generation. IL-1 activity in dermal fibroblasts, most probably of the IL-1 alpha species, was located in frozen-thawed cell lysates or associated to the cell membrane, though not secreted into the culture fluids. Dermal fibroblasts from chronic GVHD mice manifested a pronounced depression in IL-1 generation upon stimulation with exogenous lymphokines/cytokines and LPS. This was observed over a wide range of concentrations of lymphokines/cytokines and LPS. The depressed ability of chronic GVHD fibroblasts to generate IL-1 was pronounced even after few passages of the cells in vitro, and upon stimulation in culture outside the suppressive milieu of the animal.(ABSTRACT TRUNCATED AT 250 WORDS)     

IL-21 effects on human IgE production in response to IL-4 or IL-13

In human atopic disease, IgE sensitizes the allergic response, while IgG4 is protective. Because IL-4 and IL-13 trigger switch recombination to both IgE and IgG4, additional agents must regulate the balance between these isotypes to influence susceptibility or tolerance to atopy. In this report, we define in vitro conditions leading to activation or inhibition of human IgE and IgG4 production by IL-21. IL-21 reduced IL-4-driven IgE synthesis by mitogen-stimulated human PBMC. IL-21 inhibition of human IgE production was not a direct effect on B cells, was not seen following B cell activation with IL-13, and was overcome by CD40 ligation. Neither IFN-gamma, IL-10, IL-12, CD40L expression, nor apoptosis was responsible for the inhibitory effect. In contrast, IL-21-stimulated secretion of IgG4 from PBMC. Our findings indicate that IL-21 may influence the production of both human IgE and IgG4, and thus contribute to the regulation of atopic reactions.     

Platelet-derived growth factor mediates interleukin-13-induced collagen I production in mouse airway fibroblasts

Interleukin-13 (IL-13) is associated with the production of collagen in airway remodelling of asthma. Yet, the molecular mechanisms underlying IL-13 induction of collagen remain unclear; the aim of this study is to address this issue. IL-13 dose- and time-dependently-induced collagen I production in primary cultured airway fibroblasts; this was accompanied with the STAT6 phosphorylation, and pre-treatment of cells with JAK inhibitor suppressed IL-13-induced collagen I production. Further study indicated that IL-13 stimulated JAK/STAT6-dependent PDGF production and subsequent ERK1/2 MAPK activation in airway fibroblasts, and the presence of either PDGF receptor blocker or MEK inhibitor partially suppressed IL-13-induced collagen I production. Taken together, our study suggests that activation of JAK/STAT6 signal pathway and subsequent PDGF generation and resultant ERK1/2 MAPK activation mediated IL-13-induced collagen I production in airway fibroblasts.