Induction of IL-13 triggers TGF-beta1-dependent tissue fibrosis in chronic 2,4,6-trinitrobenzene sulfonic acid colitis

To investigate the immunopathogenesis of inflammation-associated fibrosis, we analyzed the chronic colitis and late-developing fibrosis occurring in BALB/c mice administered weekly doses of intrarectal 2,4,6-trinitrobenzene sulfonic acid. We showed first in this model that an initial Th1 response involving IL-12p70 and IFN-gamma subsides after 3 wk to be supplanted by an IL-23/IL-25 response beginning after 4-5 wk. This evolution is followed by gradually increasing production of IL-17 and cytokines ordinarily seen in a Th2 response, particularly IL-13, which reaches a plateau at 8-9 wk. In vitro stimulation studies suggest that this IL-13 production is dependent on IL-23 and IL-25, but not on IL-12p70. We then show that IL-13 production results in the induction of an IL-13R formerly thought to function only as a decoy receptor, IL-13Ralpha(2), and this receptor is critical to the production of TGF-beta(1) and the onset of fibrosis. Thus, if IL-13 signaling through this receptor is blocked by administration of soluble IL-13Ralpha(2)-Fc, or by administration of IL-13Ralpha(2)-specific small interfering RNA, TGF-beta(1) is not produced and fibrosis does not occur. These studies show that in chronic 2,4,6-trinitrobenzene sulfonic acid colitis, fibrosis is dependent on the development of an IL-13 response that acts through a novel cell surface-expressed IL-13R to induce TGF-beta(1). A similar mechanism may obtain in certain forms of human inflammatory bowel disease.     

Interleukin-13 (IL-13)/IL-13 receptor alpha1 (IL-13Ralpha1) signaling regulates intestinal epithelial cystic fibrosis transmembrane conductance regulator channel-dependent Cl- secretion

Interleukin-13 (IL-13) has been linked to the pathogenesis of inflammatory diseases of the gastrointestinal tract. It is postulated that IL-13 drives inflammatory lesions through the modulation of both hematopoietic and nonhematopoietic cell function in the intestine. To delineate the relevant contribution of elevated levels of intestinal IL-13 to intestinal structure and function, we generated an intestinal IL-13 transgenic mouse (iIL-13Tg). We show that constitutive overexpression of IL-13 in the small bowel induces modification of intestinal epithelial architecture (villus blunting, goblet cell hyperplasia, and increased epithelial proliferation) and epithelial function (altered basolateral → apical Cl(-) ion conductance). Pharmacological analyses in vitro and in vivo determined that elevated Cl(-) conductance is mediated by altered cystic fibrosis transmembrane conductance regulator expression and activity. Generation of iIL-13Tg/Il13rα1(-/-), iIL-13Tg/Il13rα2(-/-), and iIL-13Tg/Stat6(-/-) mice revealed that IL-13-mediated dysregulation of epithelial architecture and Cl(-) conductance is dependent on IL-13Rα1 and STAT-6. These observations demonstrate a central role for the IL-13/IL-13Rα1 pathway in the regulation of intestinal epithelial cell Cl(-) secretion via up-regulation of cystic fibrosis transmembrane conductance regulator, suggesting an important role for this pathway in secretory diarrhea.     

Hypoxia modulates the effects of transforming growth factor-beta isoforms on matrix-formation by primary human lung fibroblasts

Chronic hypoxia is implicated in lung fibrosis, which is characterized by enhanced deposition of extracellular matrix (ECM) molecules. Transforming growth factor-beta (TGF-beta) plays a key role in fibroblast homeostasis and is involved in disease states characterized by excessive fibrosis, such as pulmonary fibrosis. In this study, we investigated if hypoxia modulates the effects of TGF-beta on the expression of gelatinases: matrix metalloproteinase (MMP)-2 and MMP-9, interstitial collagenases: MMP-1 and MMP-13, tissue inhibitors of MMP (TIMP), collagen type I and interleukin-6 (IL-6). Primary human lung fibroblasts, established from tissue biopsies, were cultivated under normoxia or hypoxia in the presence of TGF-beta1, TGF-beta2 or TGF-beta3. Gelatinases were assessed by gelatin zymography and collagenases, TIMP, collagen type I and IL-6 by ELISA. Under normoxia fibroblasts secreted MMP-2, collagenases, TIMP, collagen type I and IL-6. TGF-betas significantly decreased MMP-1 and increased TIMP-1, IL-6 and collagen type I. Hypoxia significantly enhanced MMP-2, and collagenases. Compared to normoxia, the combination of TGF-beta and hypoxia reduced MMP-1, and further amplified the level of TIMP, IL-6, and collagen type I. Thus, in human lung fibroblasts hypoxia significantly increases the TGF-betas-induced secretion of collagen type I and may be associated to the accumulation of ECM observed in lung fibrosis.     

IL-13 induces disease-promoting type 2 cytokines, alternatively activated macrophages and allergic inflammation during pulmonary infection of mice with Cryptococcus neoformans

In the murine model of Cryptococcus neoformans infection Th1 (IL-12/IFN-gamma) and Th17 (IL-23/IL-17) responses are associated with protection, whereas an IL-4-dependent Th2 response exacerbates disease. To investigate the role of the Th2 cytokine IL-13 during pulmonary infection with C. neoformans, IL-13-overexpressing transgenic (IL-13Tg(+)), IL-13-deficient (IL-13(-/-)), and wild-type (WT) mice were infected intranasally. Susceptibility to C. neoformans infection was found when IL-13 was induced in WT mice or overproduced in IL-13Tg(+) mice. Infected IL-13Tg(+) mice had a reduced survival time and higher pulmonary fungal load as compared with WT mice. In contrast, infected IL-13(-/-) mice were resistant and 89% of these mice survived the entire period of the experiment. Ag-specific production of IL-13 by susceptible WT and IL-13Tg(+) mice was associated with a significant type 2 cytokine shift but only minor changes in IFN-gamma production. Consistent with enhanced type 2 cytokine production, high levels of serum IgE and low ratios of serum IgG2a/IgG1 were detected in susceptible WT and IL-13Tg(+) mice. Interestingly, expression of IL-13 by susceptible WT and IL-13Tg(+) mice was associated with reduced IL-17 production. IL-13 was found to induce formation of alternatively activated macrophages expressing arginase-1, macrophage mannose receptor (CD206), and YM1. In addition, IL-13 production led to lung eosinophilia, goblet cell metaplasia and elevated mucus production, and enhanced airway hyperreactivity. This indicates that IL-13 contributes to fatal allergic inflammation during C. neoformans infection.     

Transforming growth factor (TGF)-beta1 stimulates pulmonary fibrosis and inflammation via a Bax-dependent, bid-activated pathway that involves matrix metalloproteinase-12

Fibrosis, apoptosis, and the exaggerated production of transforming growth factor (TGF)-beta(1) are juxtaposed in a variety of pulmonary diseases including the interstitial lung diseases and asthma. In these disorders, the relationships between these responses are not well defined. In addition, the apoptosis pathways that contribute to these responses and the mechanism(s) of their contribution have not been described. We hypothesized that BH3 domain-only protein-induced apoptosis plays an important role in the pathogenesis of TGF-beta(1)-induced pulmonary responses. To test this hypothesis, we characterized the effects of transgenic TGF-beta(1) in mice with wild type (WT) and null Bax loci. To investigate the mechanisms of Bax activation and its effector functions, we also compared the effects of TGF-beta(1) in mice with WT and null Bid and matrix metalloproteinase (MMP)-12 loci, respectively. These studies demonstrate that TGF-beta(1) is a potent stimulator of Bax, Bid, and MMP-12. The studies also demonstrate that Bax and Bid play key roles in the pathogenesis of TGF-beta(1)-induced inflammation, fibrosis, and apoptosis; that TGF-beta(1) stimulates MMP-12, TIMP-1, and cathepsins and inhibits MMP-9 and p21 via Bax- and Bid-dependent mechanisms; and that TGF-beta(1)-stimulated pulmonary fibrosis is ameliorated in MMP-12-deficient animals. Finally, they demonstrate that Bax, Bid, and MMP-12 play similar roles in bleomycin-induced fibrosis, thereby highlighting the importance of this Bid-activated, Bax-mediated pathway and downstream MMP-12 in a variety of fibrogenic settings.     

PAI-1 deficiency reduces liver fibrosis after bile duct ligation in mice through activation of tPA

Plasminogen activator inhibitor-1 (PAI-1) increases injury in several liver, lung and kidney disease models. The objective of this investigation was to assess the effect of PAI-1 deficiency on cholestatic liver fibrosis and determine PAI-1 influenced fibrogenic mechanisms. We found that PAI-1(-/-) mice had less fibrosis than wild type (WT) mice after bile duct ligation. This change correlated with increased tissue-type plasminogen activator (tPA) activity, and increased matrix metalloproteinase-9 (MMP-9), but not MMP-2 activity. Furthermore, there was increased activation of the tPA substrate hepatocyte growth factor (HGF), a known anti-fibrogenic protein. In contrast, there was no difference in hepatic urokinase plasminogen activator (uPA) or plasmin activities between PAI-1(-/-) and WT mice. There was also no difference in the level of transforming growth factor beta 1 (TGF-beta1), stellate cell activation or collagen production between WT and PAI-1(-/-) animals. In conclusion, PAI-1 deficiency reduces hepatic fibrosis after bile duct obstruction mainly through the activation of tPA and HGF.     

Effect of adenovirus-mediated overexpression of decorin on metalloproteinases, tissue inhibitors of metalloproteinases and cytokines secretion by human gingival fibroblasts.

Decorin is a small leucine-rich proteoglycan that plays a role in control of cell proliferation, cell migration, collagen fibrillogenesis and modulation of the activity of TGF-beta. In the present study, we investigated the effects of decorin on the production of metalloproteinases (MMP-1, -2, -3, -9 and -13), tissue inhibitors of metalloproteinases (TIMP-1, -2) and cytokines (TGF-beta, IL-1beta, IL-4 and TNF-alpha). Decorin was overexpressed in cultured human gingival fibroblasts using adenovirus-mediated gene transfer. Decorin infection resulted in decreased protein levels of MMP-1 and MMP-3 whereas MMP-2 and TIMP-2 secretion was increased. MMP-9, MMP-13 and TIMP-1 were not affected by decorin infection. Cytokine measurements by ELISA showed that decorin overexpression reduced TGF-beta and IL-1beta. In contrast, IL-4 and TNF-alpha levels were markedly increased in decorin-infected cells. These results suggest that decorin could modulate the expression of certain metalloproteinases and their inhibitors, as well as the production of cytokines. Altogether, our data suggest that decorin might play a pivotal role in tissue remodeling by acting on the balance between extracellular matrix synthesis and degradation.     

Existence of autocrine loop between interleukin-6 and transforming growth factor-beta1 in activated rat pancreatic stellate cells

Interleukin (IL)-6 is a proinflammatory cytokine assumed to participate in pancreatic fibrosis by activating pancreatic stellate cells (PSCs). Autocrine TGF-beta1 is to central in PSC functional regulation. In this study, we examined IL-6 secretion from culture-activated rat PSCs and its regulatory mechanism. Activated PSCs express and secrete IL-6. When anti-TGF-beta1 neutralizing antibody was added in the culture medium, IL-6 secretion from activated PSCs was inhibited, whereas exogenous TGF-beta1 added in the culture medium enhanced IL-6 expression and secretion by PSCs in a dose dependent manner. Infection of PSCs with an adenovirus expressing dominant-negative Smad2/3 attenuated basal and TGF-beta1-stimulated IL-6 expression and secretion of PSCs. We also demonstrated the reciprocal effect of PSCs-secreted IL-6 on autocrine TGF-beta1. Anti-IL-6 neutralizing antibody inhibited TGF-beta1 secretion from PSCs. Preincubation of cells with 10 nM PD98059, an extracellular signal-regulated kinase (ERK)-dependent pathway inhibitor, attenuated IL-6-enhanced TGF-beta1 expression and secretion of PSCs. In addition, IL-6 activated ERK in PSCs. These data indicate the existence of autocrine loop between IL-6 and TGF-beta1 through ERK- and Smad2/3-dependent pathways in activated PSCs.     

Inhibition of matrix metalloproteinase-3 and -13 synthesis induced by IL-1beta in chondrocytes from mice lacking microsomal prostaglandin E synthase-1

Joint destruction in arthritis is in part due to the induction of matrix metalloproteinase (MMP) expression and their inhibitors, especially MMP-13 and -3, which directly degrade the cartilage matrix. Although IL-1β is considered as the main catabolic factor involved in MMP-13 and -3 expression, the role of PGE(2) remains controversial. The goal of this study was to determine the role of PGE(2) on MMP synthesis in articular chondrocytes using mice lacking microsomal PGE synthase-1 (mPGES-1), which catalyses the rate-limiting step of PGE(2) synthesis. MMP-3 and MMP-13 mRNA and protein expressions were assessed by real-time RT-PCR, immunoblotting, and ELISA in primary cultures of articular chondrocytes from mice with genetic deletion of mPGES-1. IL-1β-induced PGE(2) synthesis was dramatically reduced in mPGES-1(-/-) and mPGES-1(+/-) compared with mPGES-1(+/+) chondrocytes. A total of 10 ng/ml IL-1β increased MMP-3 and MMP-13 mRNA, protein expression, and release in mPGES-1(+/+) chondrocytes in a time-dependent manner. IL-1β-induced MMP-3 and MMP-13 mRNA expression, protein expression, and release decreased in mPGES-1(-/-) and mPGES-1(+/-) chondrocytes compared with mPGES-1(+/+) chondrocytes from 8 up to 24 h. Otherwise, MMP inhibition was partially reversed by addition of 10 ng/ml PGE(2) in mPGES-1(-/-) chondrocytes. Finally, in mPGES-1(-/-) chondrocytes treated by forskolin, MMP-3 protein expression was significantly decreased compared with wild-type, suggesting that PGE(2) regulates MMP-3 expression via a signaling pathway dependent on cAMP. These results demonstrate that PGE(2) plays a key role in the induction of MMP-3 and MMP-13 in an inflammatory context. Therefore, mPGES-1 could be considered as a critical target to counteract cartilage degradation in arthritis.     

Eosinophils and T lymphocytes possess distinct roles in bleomycin-induced lung injury and fibrosis

Leukocyte infiltration is characteristic of lung injury and fibrosis, and its role during tissue repair and fibrosis is incompletely understood. We found that overexpression of IL-5 in transgenic mice (IL-5(TG)) or by adenoviral gene transfer increased bleomycin (blm)-induced lung injury, fibrosis, and eosinophilia. Surprisingly, blm-treated IL-5-deficient (IL-5(-/-)) mice also developed pronounced pulmonary fibrosis but characterized by marked T lymphocyte infiltration and absence of eosinophilia. In both murine strains however, induction of lung TGF-beta expression was evident. Purified lung eosinophils from blm-treated IL-5(TG) mice stimulated alpha-smooth muscle actin and collagen expression in mouse lung fibroblasts, without affecting proliferation. Furthermore instillation of purified eosinophils into murine lungs resulted in extension of blm-induced lung fibrosis, thus confirming a role for eosinophils. However, lung T lymphocytes from blm-treated IL-5(-/-) mice were able to stimulate fibroblast proliferation but not alpha-smooth muscle actin or collagen expression. Blocking T cell influx by anti-CD3 Abs abrogated lung fibrosis, thus also implicating T lymphocytes as a key participant in fibrosis. Pulmonary fibrosis in IL-5(TG) mice was preferentially associated with type 2 cytokines (IL-4 and IL-13), whereas fibrotic lesions in IL-5(-/-) animals were accompanied by proinflammatory cytokine (TNF-alpha, IL-1beta, and IFN-gamma) expression. We suggest that eosinophils and T cells contribute distinctly to the development of blm-induced lung fibrosis potentially via their production of different cytokine components, which ultimately induce TGF-beta expression that is intimately involved with the fibrosis.     

Interleukin-6 plays a critical role in aldosterone-induced macrophage recruitment and infiltration in the myocardium

Macrophages play an important role in aldosterone-induced myocardial fibrosis, in which the first key steps are macrophage recruitment and infiltration. We hypothesized that IL-6 may be a key mediator of aldosterone-induced macrophage recruitment and infiltration. To test this hypothesis, we designed cell studies with a human monocytic cell line THP-1 that with monocyte/macrophage functions to explore the signaling pathway of aldosterone-induced macrophage infiltration, and further investigated the phenomenon and consequent pathway in aldosterone-infused mice studies. The results showed that aldosterone induced the expression of IL-6 via mineralocorticoid receptors, and enhanced THP-1 cell migration and infiltration. Further experiments using a protease array and siRNA revealed that expressions of MMP-1 and MMP-9 were associated with aldosterone-induced macrophage infiltration. In addition, aldosterone-induced MMP-1 and MMP-9 expressions were mediated via cyclooxygenase-II and prostaglandin E2/EP-2 and EP-4 receptors. In aldosterone-infused mice, mRNA expressions of MMP-1, MMP-9 and COX-2 in peripheral blood monocytic cells were significantly increased. Moreover, the number of mouse macrophage-restricted F4/80 protein-positive cells in the myocardium was significantly higher in the aldosterone-infused mice compared with control mice. The increase in F4/80-positive cells in the myocardium was suppressed in the aldosterone-infused mice with the aldosterone antagonist eplerenone or anti-IL-6 antibody treatment. In conclusion, interleukin-6 played an important role in aldosterone-induced macrophage recruitment and infiltration in the myocardium.     

Smad3 null mice develop airspace enlargement and are resistant to TGF-beta-mediated pulmonary fibrosis

Transforming growth factor-beta 1 plays a key role in the pathogenesis of pulmonary fibrosis, mediating extracellular matrix (ECM) gene expression through a series of intracellular signaling molecules, including Smad2 and Smad3. We show that Smad3 null mice (knockout (KO)) develop progressive age-related increases in the size of alveolar spaces, associated with high spontaneous presence of matrix metalloproteinases (MMP-9 and MMP-12) in the lung. Moreover, transient overexpression of active TGF-beta 1 in lungs, using adenoviral vector-mediated gene transfer, resulted in progressive pulmonary fibrosis in wild-type mice, whereas no fibrosis was seen in the lungs of Smad3 KO mice up to 28 days. Significantly higher levels of matrix components (procollagen 3A1, connective tissue growth factor) and antiproteinases (plasminogen activator inhibitor-1, tissue inhibitor of metalloproteinase-1) were detected in wild-type lungs 4 days after TGF-beta 1 administration, while no such changes were seen in KO lungs. These data suggest a pivotal role of the Smad3 pathway in ECM metabolism. Basal activity of the pathway is required to maintain alveolar integrity and ECM homeostasis, but excessive signaling through the pathway results in fibrosis characterized by inhibited degradation and enhanced ECM deposition. The Smad3 pathway is involved in pathogenic mechanisms mediating tissue destruction (lack of repair) and fibrogenesis (excessive repair).     

Mechanisms of cardiac fibrosis induced by urokinase plasminogen activator

Human hearts with end-stage failure and fibrosis have macrophage accumulation and elevated plasminogen activator activity. However, the mechanisms that link macrophage accumulation and plasminogen activator activity with cardiac fibrosis are unclear. We previously reported that mice with macrophage-targeted overexpression of urokinase plasminogen activator (SR-uPA+/o mice) develop cardiac macrophage accumulation by 5 weeks of age and cardiac fibrosis by 15 weeks. We used SR-uPA+/o mice to investigate mechanisms through which macrophage-expressed uPA causes cardiac macrophage accumulation and fibrosis. We hypothesized that: 1) macrophage accumulation and cardiac fibrosis in SR-uPA+/o mice are dependent on localization of uPA by the uPA receptor (uPAR); 2) activation of plasminogen by uPA and subsequent activation of transforming growth factor-beta1 (TGF-beta1) and matrix metalloproteinase (MMP)-2 and -9 by plasmin are critical pathways through which uPA-expressing macrophages accumulate in the heart and cause fibrosis; and 3) uPA-induced cardiac fibrosis can be attenuated by treatment with verapamil. To test these hypotheses, we bred the SR-uPA+/o transgene into mice deficient in either uPAR or plasminogen and measured cardiac macrophage accumulation and fibrosis. We also measured cardiac TGF-beta1 protein (total and active), Smad2 phosphorylation, and MMP activity after the onset of macrophage accumulation but before the onset of cardiac fibrosis. Finally, we treated mice with verapamil. Our studies revealed that plasminogen is necessary for uPA-induced cardiac fibrosis and macrophage accumulation but uPAR is not. We did not detect plasmin-mediated activation of TGF-beta1, MMP-2, or MMP-9 in hearts of SR-uPA+/o mice. However, verapamil treatment significantly attenuated both cardiac fibrosis and macrophage accumulation.     

Regulation of hepatic fibrosis and extracellular matrix genes by the th response: new insight into the role of tissue inhibitors of matrix metalloproteinases.

Hepatic fibrosis is the hallmark of Schistosoma mansoni infection and often results in portal hypertension and bleeding from esophageal varices. The fibrotic process is highly dependent on type 2 cytokines, yet their role in the regulation of extracellular matrix remodeling genes remains largely unknown. Here, we examined the expression of matrix metalloproteases (MMP) -2, -3, -9, -12, and -13 and their inhibitors, tissue inhibitor of metalloproteases (TIMP) -1, -2, and -3, in the livers of infected mice and correlated their expression profiles with fibrosis and type 2 cytokine production. Expression of MMP-2, -3, -9, -12, and -13 and of TIMP-1 and -2 mRNA rapidly increased at the onset of egg laying in infected mice, while TIMP-3 was unchanged. Because TIMP are presumed to be important regulators of the extracellular matrix, and their expression correlated with the development of fibrosis, we studied their role in fibrogenesis by infecting TIMP-1- and TIMP-2-deficient mice. Strikingly, our data revealed no role for TIMP-1 or -2 in the fibrotic pathology induced by S. mansoni eggs. Because of these findings, we infected IL-10/IFN-gamma-deficient mice that develop an exaggerated fibrotic response to determine whether changes in type 2 cytokine dominance influence the pattern of MMP and TIMP expression. Fibrosis and type 2 cytokine production correlated with increased MMP-2/MMP-9 vs TIMP-1/TIMP-2 expression. These data, in addition to our knockout studies, demonstrate that TIMP-1/TIMP-2 play no essential role in fibrogenesis in schistosomiasis. Indeed, our findings suggest that inhibiting profibrotic cytokines or specific MMP may be a more effective strategy to ameliorate fibrotic pathology.     

Protection from fluorescein isothiocyanate-induced fibrosis in IL-13-deficient, but not IL-4-deficient, mice results from impaired collagen synthesis by fibroblasts

Intratracheal injection of FITC results in acute lung injury and progresses to fibrosis by day 21 postchallenge. In response to FITC, BALB/c mice produce IL-4 and IL-13 in the lung. To investigate whether IL-4 and/or IL-13 were important profibrotic mediators in this model, we examined the fibrotic response to FITC in mice that were genetically deficient in IL-4 (IL-4(-/-)), IL-13 (IL-13(-/-)), or IL-4 and IL-13 combined (IL-4/13(-/-)). Baseline levels of collagen were similar in all mice. In response to FITC, both BALB/c and IL-4(-/-) mice developed fibrosis, whereas the IL-13(-/-) and IL-4/13(-/-) mice were significantly protected, as measured by total lung collagen levels and histology. Total leukocyte recruitment to the lung was similar in all four strains of mice when measured on days 7, 14, and 21 post-FITC. BALB/c mice showed prominent eosinophilia on day 7 that was absent in IL-4(-/-), IL-13(-/-), and IL-4/13(-/-) mice, suggesting that eosinophilia is not necessary for development of a fibrotic response. There were no significant differences in the percentages of any other leukocytes analyzed between the genotypes. Similarly, protection in IL-13(-/-) mice was not associated with alterations in cytokine or eicosanoid profiles. Interestingly, TGF-beta1 production was not reduced in IL-13(-/-) mice. Analyses of fibroblasts isolated from the four genotypes demonstrated that although there were similar numbers of fibroblasts present in cultures of lung minces, fibroblasts from IL-13-deficient strains have reduced basal and stimulated levels of collagen production. IL-13Ralpha1 expression increases on fibroblasts during fibrotic responses in vivo, and IL-13 increases collagen synthesis in fibroblasts. Thus, IL-13 mediates its profibrotic actions through direct effects on fibroblast production of extracellular matrix.     

Interleukin-1 Receptor Antagonist Has a Novel Function in the Regulation of Matrix Metalloproteinase-13 Expression

Interleukin-1 receptor antagonist (IL-1Ra) is an IL-1 family member, which binds to IL-1 receptors but does not induce any intracellular signaling. We addressed whether IL-1Ra has a novel function in regulation of the extracellular matrix or adhesion molecules. Polymerase chain reaction array analysis demonstrated a ~5-fold increase in matrix metalloproteinase 13 (MMP-13) mRNA expression of IL-1Ra siRNA-transfected Ca9-22 human oral squamous epithelial carcinoma cells compared with the control. In fact, MMP-13 mRNA and protein expression as well as its activity in IL-1Ra siRNA-transfected Ca9-22 cell lines were significantly higher than those in the control. IL-1Ra siRNA treatment resulted in strong elevation of MMP-13 expression, whereas addition of rhIL-1Ra (40 ng/ml) suppressed MMP-13 expression, suggesting that IL-1Ra had a specific effect on MMP-13 induction. IL-1Ra siRNA could potently suppress IL-1α. No significant difference was found between the MMP-13 mRNA expression of IL-1Ra siRNA-transfected cells and those treated with anti-IL-1α or anti-IL-1β antibodies. These results suggested that continuous supply of IL-1 had no effect on the induction of MMP-13 by IL-1Ra siRNA. Histopathological investigation of MMP-13 in periodontal tissue showed specific localization in the junctional epithelial cells of IL-1Ra knockout (KO) mice. Furthermore, infection with Aggregatibacter actinomycetemcomitans to establish an experimental periodontitis model resulted in predominant localization of MMP-13 along apical junctional epithelial cells. Laminin-5, which is degraded by MMP-13, was found in the internal basal lamina of wild-type mice, whereas the internal basal lamina of IL-1Ra KO mice did not show obvious laminin-5 localization. In particular, laminin-5 localization almost disappeared in the internal basal lamina of IL-1Ra KO mice infected with A. actinomycetemcomitans, suggesting that the suppression of IL-1Ra resulted in strong induction of MMP-13 that degraded laminin-5. In conclusion, IL-1Ra is associated with MMP-13 expression and has a novel function in such regulation without interference of the IL-1 signaling cascade.     

TGF-beta and Smad3 signaling link inflammation to chronic fibrogenesis

Transient adenovirus-mediated gene transfer of IL-1beta (AdIL-1beta), a proinflammatory cytokine, induces marked inflammation and severe and progressive fibrosis in rat lungs. This is associated with an increase in TGF-beta1 concentration in bronchoalveolar lavage (BAL) fluid. TGF-beta1 is a key cytokine in the process of fibrogenesis, using intracellular signaling pathways involving Smad2 and Smad3. In this study we investigate whether inflammation induced by IL-1beta is able to independently induce lung fibrosis in mice deficient in the Smad3 gene. Seven days after AdIL-1beta administration, similar levels of IL-1beta transgene are seen in BAL in both wild-type (WT) and knockout (KO) mice, and BAL cell profiles demonstrated a similar marked neutrophilic inflammation. Phospho-Smad2 staining was positive in areas of inflammation in both WT and KO mice at day 7. By day 35 after transient IL-1beta expression, WT mice showed marked fibrosis in peribronchial areas, quantified by picrosirius red staining and morphometry. However, there was no evidence of fibrosis or collagen accumulation in IL-1beta-treated KO mice, and peribronchial areas were not different from KO mice treated with the control adenovector. TGF-beta1 and phospho-Smad2 were strongly positive at day 35 in fibrotic areas observed in WT mice, but no such staining was detectable in KO mice. The IL-1beta-induced chronic fibrotic response in mouse lungs is dependent on Smad3. KO and WT animals demonstrated a similar inflammatory response to overexpression of IL-1beta indicating that inflammation must link to the Smad3 pathway, likely through TGF-beta, to induce progressive fibrosis.     

Th3 cells in peripheral tolerance. I. Induction of Foxp3-positive regulatory T cells by Th3 cells derived from TGF-beta T cell-transgenic mice

TGF-beta has been shown to be critical in the generation of CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs). Because Th3 cells produce large amounts of TGF-beta, we asked whether induction of Th3 cells in the periphery was a mechanism by which CD4(+)CD25(+) Tregs were induced in the peripheral immune compartment. To address this issue, we generated a TGF-beta1-transgenic (Tg) mouse in which TGF-beta is linked to the IL-2 promoter and T cells transiently overexpress TGF-beta upon TCR stimulation but produce little or no IL-2, IL-4, IL-10, IL-13, or IFN-gamma. Naive TGF-beta-Tg mice are phenotypically normal with comparable numbers of lymphocytes and thymic-derived Tregs. We found that repeated antigenic stimulation of pathogenic myelin oligodendrocyte glycoprotein (MOG)-specific CD4(+)CD25(-) T cells from TGF-beta Tg mice crossed to MOG TCR-Tg mice induced Foxp3 expression in both CD25(+) and CD25(-) populations. Both CD25 subsets were anergic and had potent suppressive properties in vitro and in vivo. Furthermore, adoptive transfer of these induced regulatory CD25(+/-) T cells suppressed experimental autoimmune encephalomyelitis when administrated before disease induction or during ongoing experimental autoimmune encephalomyelitis. The suppressive effect of TGF-beta on T cell responses was due to the induction of Tregs and not to the direct inhibition of cell proliferation. The differentiation of Th3 cells in vitro was TGF-beta dependent as anti-TGF-beta abrogated their development. Thus, Ag-specific TGF-beta-producing Th3 cells play a crucial role in inducing and maintaining peripheral tolerance by driving the differentiation of Ag-specific Foxp3(+) regulatory cells in the periphery.