Repair phenotype in corneal fibroblasts is controlled by an interleukin-1 alpha autocrine feedback loop

PURPOSE: To explore the role of autocrine interleukin-1 alpha (IL-1 alpha) as a central regulator of the repair phenotype in corneal fibroblasts. METHODS: Disruption of the actin cytoskeleton with cytochalasin B (CB), which mimics changes in shape that occur in repair tissues, was used to stimulate repair gene expression in early-passage fibroblasts. Changes in expression of IL-1 alpha, IL-8, collagenase, and ENA-78 were determined by Northern blot analysis, radioimmunoassay, and an enzyme-amplified sensitivity immunoassay (EASIA). Expression of repair genes was also examined in repair fibroblasts, isolated from healing, penetrating keratectomy wounds in rabbits. RESULTS: Blocking IL-1 alpha activity prevented both constitutive and stimulated increases in synthesis of IL-8 and collagenase in early-passage cultures of corneal fibroblasts, demonstrating the role of IL-1 alpha as a necessary intermediate for expression of these genes. Evidence is also presented that the IL-1 alpha autocrine controls expression of an IL-8 related factor, ENA-78. Unlike early-passage fibroblasts, fibroblasts freshly isolated from the uninjured cornea did not express IL-1 alpha. However, fibroblasts freshly isolated from remodeling corneal repair tissue 3 weeks after injury were found to express substantial levels of IL-1 alpha, regulated through an autocrine feedback loop. Neutralization experiments demonstrated that the IL-1 alpha autocrine is largely responsible for controlling both collagenase and IL-8 synthesis in repair fibroblasts, as it is in early-passage fibroblasts. CONCLUSIONS: These findings provide evidence that activation of an autocrine IL-1 alpha feedback loop is an important mechanism by which fibroblasts adopt a repair phenotype during remodeling of the cornea.     

Role of serum amyloid A as an intermediate in the IL-1 and PMA-stimulated signaling pathways regulating expression of rabbit fibroblast collagenase

The matrix metalloproteinase collagenase is expressed by resident tissue cells only when needed for biological remodeling. Exogenous addition of inflammatory and growth-promoting cytokines stimulates collagenase expression in early passage fibroblast cultures. In addition, the signal for collagenase expression in response to phorbol-12 myristate-13 acetate (PMA) or to agents which alter cell shape in early passage fibroblast cultures is routed extracellularly to an autocrine cytokine intermediate, IL-1 alpha. Importantly, fibroblasts, when freshly isolated from the tissue, are not competent for IL-1 alpha gene expression and, therefore, cannot produce collagenase in response to shape change agents. However, they do make a small amount of collagenase in response to PMA via an IL-1-independent pathway that has not been further characterized. In this paper, we investigate the role of a second autocrine, serum amyloid A3 (SAA3), in IL-1-dependent and -independent collagenase gene expression. We demonstrate that SAA3 is required for effective stimulation of collagenase expression by either exogenous or endogenous IL-1. Furthermore, while freshly isolated fibroblasts cannot express IL-1 alpha they can express SAA3, and this autocrine mediator acts independently of IL-1 alpha to control the low level of collagenase expression that can be stimulated by PMA. These results provide further evidence for a newly emerging paradigm of collagenase regulation which emphasizes the requirement for extracellular routing of signals. They also suggest that SAA3 might be utilized independently of IL-1 alpha to control tissue remodeling in vivo.     

Interferon gamma decreases hepatic stellate cell activation and extracellular matrix deposition in rat liver fibrosis

Interferon gamma (IFN-gamma) inhibits in vitro the activation of hepatic stellate cells (HSC), the primary extracellular matrix-producing cells in liver fibrosis. This study was undertaken to determine in vivo the effect of IFN-gamma in the rat model of liver fibrosis induced by dimethylnitrosamine (DMN), where HSC activation represents an early response to cell injury. Rats were killed after 1 or 3 weeks of treatment with DMN, IFN-gamma, DMN + IFN-gamma, or saline. Immunohistochemistry was used to identify proliferating (desmin-positive/bromodeoxyuridine (BrdU)-positive cells) and activated (alpha-smooth-muscle actin [alpha-SMA]-positive cells) HSCs. Collagen deposition was determined colorimetrically and by morphometry. The parenchymal extension of desmin- and actin-positive cells and of fibrotic tissue was measured by point-counting technique and expressed as a percentage of area. Western blot was used to determine laminin and fibronectin accumulation. The levels of messenger RNA (mRNA) for procollagen type I, fibronectin, and laminin were evaluated by Northern blot. No differences were observed in rats treated with either saline or IFN-gamma alone. IFN-gamma reduced HSC activation induced by liver injury, as shown by the decreased number of proliferating HSC and the reduction of parenchymal area occupied by alpha-SMA-positive cells observed in DMN + IFN-gamma-treated animals compared with the DMN group. This was associated with reduced collagen, laminin, and fibronectin accumulation and lower levels of mRNA for procollagen type I, fibronectin, and laminin in the DMN + IFN-gamma group. Thus, this study indicates that IFN-gamma reduces extracellular matrix deposition in vivo by inhibition of HSC activation.     

Radiology of skeletal tuberculosis

The process of hepatic fibrosis, and the changes in contents of hepatic hyproxyproline (HYP) and serum procollagen type III peptide (PIIINP) were examined in two rat models for hepatic fibrosis, i.e. bile duct ligation/scission (BDL/s)- and dimethylnitrosamine (DMN)-induced models. In addition, an expression of type III collagen mRNA in the liver of BDL/s model was also examined. In BDL/s model, hepatic fibrosis started at 2 weeks after operation (WAO) and cirrhosis with prominent bile duct hyperplasia was detected at and after 5 WAO. Serum PIIINP content measured using a modified double armed inhibition enzyme-linked immunosorbent assay (ELISA) method proposed by us started to increase at 1 WAO and continued to increase thereafter. Hepatic HYP content measured colorimetrically started to increase at 3 WAO and it continued to increase until 7 WAO. An expression of type III collagen mRNA in the liver was enhanced at and after 2 WAO, especially at 4 and 5 WAO. In DMN model, marked hepatic fibrosis was detected at 1 week after the last DMN administration (WAA), and the degree of fibrosis was apparently reduced at 4 WAA. Serum PIIINP content prominently increased at 1 WAA and decreased at and after 3 WAA. Hepatic HYP content showed a marked increase at 1 WAA and decreased thereafter. The present results indicated that the sequences of hepatic fibrosis, hepatic HYP content and serum PIIINP content were well correlated with each other in both BDL/s and DMN models. In conclusion, ELISA system for the detection of serum PIINP content is considered to be reliable method for assessment of cirrhotic liver, and the present two rat models for liver fibrosis/cirrhosis seems to be a good tool for researching antifibrotic agents.     

Enhanced IL-1 beta and tumor necrosis factor-alpha release and messenger RNA expression in macrophages from idiopathic pulmonary fibrosis or after asbestos exposure

Idiopathic pulmonary fibrosis (IPF) and asbestosis are fibrotic interstitial lung diseases characterized by alveolar wall fibrosis with accumulation of extracellular matrix, interstitial remodeling, and increased numbers of activated alveolar macrophages. Animal models and in vitro studies have shown that macrophage cytokines, namely IL-1 beta and TNF-alpha, play significant roles in the development of fibrosis. We found significant increases for TNF-alpha release in both diseases (p < 0.01) and a significant increase for IL-1 beta release in asbestosis compared to normal controls (p < 0.01). Also, the mRNA expression of these cytokines was increased in alveolar macrophages from patients with IPF or asbestosis compared with normals. The level of TNF-alpha release in macrophage supernatants correlated with the number of neutrophils per milliliter bronchoalveolar lavage fluid returned. Chrysotile, crocidolite, amosite asbestos, and silica stimulated IL-1 beta and TNF-alpha release and up-regulated their respective mRNA in macrophages or monocytes. To evaluate the role of IL-1 beta and TNF-alpha in the accumulation of extracellular matrix, we studied collagen types I and III and fibronectin gene expression in human diploid lung fibroblasts after short term (2 h) serum-free exposure to recombinant cytokines. Both cytokines up-regulated these genes 1.5- to 3.6-fold. These cytokines have the potential to influence the remodeling and fibrosis observed in the lower respiratory tract in IPF and asbestosis.     

Regulated production of type I collagen and inflammatory cytokines by peripheral blood fibrocytes

We recently described a novel population of blood-borne cells, termed fibrocytes, that display a distinct cell surface phenotype (collagen+/CD13+/CD34+/CD45+), rapidly enter sites of tissue injury, and contribute to scar formation. To further characterize the role of these cells in vivo, we examined the expression of type I collagen and cytokine mRNAs by cells isolated from wound chambers implanted into mice. Five days after chamber implantation, CD34+ fibrocytes but not CD14+ monocytes or CD90+ T cells expressed mRNA for type I collagen. Fibrocytes purified from wound chambers also were found to express mRNA for IL-1beta, IL-10, TNF-alpha, JE/MCP, MIP-1alpha, MIP-1beta, MIP-2, PDGF-A, TGF-beta1, and M-CSF. The addition of IL-1beta (1-100 ng/ml), a critical mediator in wound healing, to fibrocytes isolated from human peripheral blood induced the secretion of chemokines (MIP-1alpha, MIP-1beta, MCP-1, IL-8, and GRO alpha), hemopoietic growth factors (IL-6, IL-10, and macrophage-CSF), and the fibrogenic cytokine TNF-alpha. By contrast, IL-1beta decreased the constitutive secretion of type I collagen as measured by ELISA. Additional evidence for a role for fibrocytes in collagen production in vivo was obtained in studies of livers obtained from Schistosoma japonicum-infected mice. Mouse fibrocytes localized to areas of granuloma formation and connective matrix deposition. We conclude that fibrocytes are an important source of cytokines and type I collagen during both the inflammatory and the repair phase of the wound healing response. Furthermore, IL-1beta may act on fibrocytes to effect a phenotypic transition between a repair/remodeling and a proinflammatory mode.     

Retinoic acid-induced type II collagen degradation does not correlate with matrix metalloproteinase activity in cartilage explant cultures

OBJECTIVE: To determine the role of matrix metalloproteinases (MMPs) in retinoic acid (RetA)-induced degradation of type II collagen in cartilage. METHODS: Bovine nasal cartilage explants were cultured with 1 microM RetA or in 3 nM interleukin-1alpha (IL-1alpha). Release of proteoglycan and type II collagen into the medium was measured by colorimetric assay and immunoassay, respectively. MMP activity in the medium was determined using a quenched fluorescent substrate assay, while specific collagenases were identified by Western immunoblotting. In some cases the effects of low molecular mass synthetic MMP inhibitors and serum on collagen degradation were studied. RESULTS: RetA promoted maximal breakdown of type II collagen after 4 or 5 weeks in culture, compared with 3 weeks in culture with IL-1alpha. In IL-1alpha-stimulated cultures, collagen degradation was coincident with a large increase in MMP activity in the culture medium, whereas in RetA-stimulated cultures, there was only a small increase. In Western immunoblots of culture media containing RetA, prointerstitial collagenase and active collagenase 3 were sometimes detected, but not in all experiments. In IL-1alpha cultures, active interstitial collagenase was always detected, and active collagenase 3 was detectable in some experiments. Neutrophil collagenase was not detected in any cultures. IL-1alpha-stimulated collagen degradation was effectively inhibited by a potent, broad-spectrum inhibitor of MMPs, whereas it was poorly inhibited by a weak MMP inhibitor. The same 2 compounds were both only weak inhibitors of RetA-induced collagen degradation. When fetal calf serum was included in cartilage cultures, MMP activity in the culture medium was reduced to low levels. This resulted in a marked inhibition of IL-1alpha-induced type II collagen degradation, whereas there was no inhibition of RetA-induced collagen degradation. CONCLUSION: Unlike IL-1alpha, RetA induces degradation of type II collagen in cartilage explants by a mechanism that is mainly independent of those MMPs that can be detected in the culture medium.     

Integrin alpha 2 beta 1 is a positive regulator of collagenase (MMP-1) and collagen alpha 1(I) gene expression

A classical model for studying the effects of extracellular matrix is to culture cells inside a three-dimensional collagen gel. When surrounded by fibrillar collagen, many cell types decrease the production of type I collagen, and the expression of interstitial collagenase (matrix metalloproteinase-1; MMP-1) is simultaneously induced. To study the role of the collagen-binding integrins alpha 1 beta 1 and alpha 2 beta 1 in this process, we used three different osteogenic cell lines with distinct patterns of putative collagen receptors: HOS cells, which express only alpha 1 beta 1 integrin, MG-63 cells, which express only alpha 2 beta 1 integrin, and KHOS-240 cells, which express both. Inside collagen gels, alpha 1 (I) collagen mRNA levels were decreased in HOS and KHOS-240 cells but not in MG-63 cells. In contrast, MMP-1 expression was induced in KHOS-240 and MG-63 cells but not in HOS cells. Transfection of MG-63 cells with alpha 2 integrin cDNA in an antisense orientation reduced the expression level of alpha 2 integrin. These cell clones showed induction and reduction of mRNA levels for MMP-1, respectively. HOS cells normally lacking alpha 2 beta 1 integrin were forced to express it, and this prevented the down-regulation in the levels of alpha 1 (I) collagen mRNA when cells were grown inside collagen gels. The data indicate that the level of MMP-1 expression is regulated by the collagen receptor alpha 2 beta 1 integrin. The down-regulation of collagen alpha 1 (I) is mediated by another receptor. Integrin alpha 2 beta 1 may compete with it and thus be a positive regulator of collagen synthesis.     

Hepatic stellate cells are not subjected to oxidant stress during iron-induced fibrogenesis in rodents

Oxidant stress plays a key role in hepatic fibrogenesis. This study was undertaken to assess whether, during iron overload-associated liver fibrosis in vivo, oxidant stress occurs in hepatic stellate cells (HSC) during active fibrogenesis. Gerbils were treated with iron-dextran, and, after hepatic fibrosis developed, livers were subjected to various combination of in situ hybridization and immunocytochemistry analyses. In iron-treated animals, no specific accumulation of ferritin protein was found in collagen mRNA-expressing cells. Moreover, the activity of the iron regulatory protein, the main sensor of cellular iron status, was unchanged in HSC from iron-treated animals. Although a significant amount of malondialdehyde-protein adducts was detected in gerbil liver during fibrogenesis, accumulation of these lipid peroxidation by-products was restricted to iron-laden cells adjacent to activated HSC. In cultured gerbil HSC, iron, aldehydes, and other pro-oxidants were able to enhance the expression of an oxidant stress-responsive gene, heme oxygenase (HO), with no change in collagen mRNA accumulation. In keeping with these findings, we found that, in vivo, activation of HO gene was present in iron-filled nonparenchymal cell aggregates, but absent in HSC. In conclusion, the data indicate that during iron overload-associated fibrogenesis, HSC are not directly subjected to oxidant stress, but are likely to be activated by paracrine signals arising in neighboring cells.     

Remodeling of alveolar walls after elastase treatment of hamsters. Results of elastin and collagen mRNA in situ hybridization

Treatment of hamster lungs with porcine pancreatic elastase (PPE) causes emphysema and a decrease in lung elastin content, which returns to control level by Day 30. To explore the mechanism of alveolar wall remodeling after elastolytic injury, we examined the expression of elastin and alpha1(I) collagen mRNAs by in situ hybridization at 1, 2, 3, 5, 7, and 30 d after intratracheal PPE. The lungs of control animals displayed weak signals for elastin and alpha1(I) collagen mRNA in pleura, large arteries, veins, and airways. There was little or no signal in respiratory air space walls. Increased expression of elastin and alpha1(I) collagen mRNA began by Day 1 after PPE and reached an asymptote by Day 3 that was maintained by elastin until Day 7; expression of alpha1(I) collagen mRNA waned earlier. Elastin and, to a lesser extent, alpha1(I) collagen mRNA were heavily expressed in pleura, blood vessels, and airways. Analysis of serial sections showed elastin message was minimal in the walls of respiratory air spaces and when present, at 3, 5, and 7 d, was primarily found at the free margins of alveolar septa. Collagen message was very sparse in respiratory air space walls. By 30 d, elastin mRNA expression was reduced but still above control levels and emphysema was widespread and severe. Rank score of elastin mRNA expression in individual subpleural air spaces showed a positive correlation with air space size. In conclusion, most expression of elastin and alpha1(I) collagen mRNA occurs in the pleura, airway, and vascular walls. In respiratory air space walls, expression of elastin mRNAs occurs in damaged tissue at free septal margins.     

Ornithine decarboxylase and polyamines in familial adenomatous polyposis

The effects of concanavalin A (Con A) on liver cytokine gene expression was studied in mice. The CD4 mRNA expression in normal liver suggests the presence of CD4+ T cells. The administration of Con A induced interleukin (IL)-1beta, IL-2 and IL-2 receptor mRNAs, which implies lymphocyte activation in the liver. Interferon-gamma and tumor necrosis factor-alpha mRNA expressions were increased gradually. The present results showed that Con A induced liver cytokine genes. This cytokine gene induction might have been the result of lymphocyte activation in the liver.     

Targeted expression of IL-11 in the murine airway causes lymphocytic inflammation, bronchial remodeling, and airways obstruction

Interleukin-11 is a pleotropic cytokine produced by lung stromal cells in response to respiratory viruses, cytokines, and histamine. To further define its potential effector functions, the Clara cell 10-kD protein promoter was used to express IL-11 and the airways of the resulting transgene mice were characterized. In contrast to transgene (-) littermates, the airways of IL-11 transgene (+) animals manifest nodular peribronchiolar mononuclear cell infiltrates and impressive airways remodeling with subepithelial fibrosis. The inflammatory foci contained large numbers of B220(+) and MHC Class II(+) cells and lesser numbers of CD3(+), CD4(+), and CD8(+) cells. The fibrotic response contained increased amounts of types III and I collagen, increased numbers of alpha smooth muscle actin and desmin-containing cells and a spectrum of stromal elements including fibroblasts, myofibroblasts, and smooth muscle cells. Physiologic evaluation also demonstrated that 2-mo-old transgene (+) mice had increased airways resistance and non-specific airways hyperresponsiveness to methacholine when compared with their transgene (-) littermates. These studies demonstrate that the targeted expression of IL-11 in the mouse airway causes a B and T cell-predominant inflammatory response, airway remodeling with increased types III and I collagen, the local accumulation of fibroblasts, myofibroblasts, and myocytes, and obstructive physiologic dysregulation. IL-11 may play an important role in the inflammatory and fibrotic responses in viral and/or nonviral human airway disorders.     

Isolation of antigens recognized by coeliac disease autoantibodies and their use in enzyme immunoassay of endomysium and reticulin antibody-positive human sera

BACKGROUND & AIMS: Smooth muscle cells resident in the intestinal wall play a significant role in the healing of the injured intestine and in the fibrosis that complicates Crohn's disease. The cytokine interleukin 1 beta (IL-1 beta) is involved in inflammatory bowel disease. The aim of this study was to determine the action of IL-1 beta on proliferation and collagen metabolism in human intestinal smooth muscle cells. RESULTS: IL-beta caused a three-fold increase in [3H]thymidine uptake at 100 pmol/L. This mitogenic effect was equipotent with that of platelet-derived growth factor when cells were exposed to IL-beta for 48 vs. 24 hours. IL-beta inhibited the secretion of procollagen into culture medium by 70% and the accumulation of newly synthesized procollagen in cells by 55%. In addition, IL-beta caused a concentration-dependent inhibition of steady-state levels of procollagen I and III messenger RNA (85% inhibition at 100 pmol/L) and a 3-5-fold augmentation of collagenase messenger RNA levels. CONCLUSIONS: IL-beta is mitogenic for human intestinal smooth muscle cells, but this action is associated with a concomitant down-regulation of collagen synthesis and secretion and an augmention of collagenase expression.     

Interleukin-6, hepatocyte growth factor, and their receptors in biliary epithelial cells during a type I ductular reaction in mice: interactions between the periductal inflammatory and stromal cells and the biliary epithelium

The interleukin-6 (IL-6)/gp-80 and hepatocyte growth factor (HGF)/met ligand/receptor systems have been shown to stimulate biliary epithelial cell (BEC) DNA synthesis in vitro. The mRNA and protein production of these two in vitro mitogens were mapped in vivo during the first week after bile duct ligation (BDL) when peak BEC DNA synthesis is seen. Changes around the biliary tree were compared with those seen in the peripheral liver using a combination of Northern blotting and a unique biliary tree isolation technique, in which the bile ducts and the surrounding portal stroma and inflammatory cells are separated from the hepatocytes by perfusion digestion. Further localization was performed with in situ hybridization and immunohistochemistry. In the normal liver, there is low-level expression of HGF mRNA by periportal stellate cells, and HGF protein localizes to these cells and to neutrophils; extracellular HGF protein is present in the bile. There is no detectable IL-6 mRNA by Northern analysis or IL-6 protein expression in the normal liver, but both met and IL-6 receptor (IL-6R) mRNA are detectable; met mRNA is expressed strongly in the biliary tree, and met protein is expressed weakly on hepatocytes and strongly on BEC. IL-6R mRNA is weakly expressed in the biliary tree, and IL-6R protein is detectable on hepatocytes, with a periportal-to-perivenular gradient, but not on BEC. During the first 3 days after BDL, HGF mRNA expression is increased in both the biliary tree and in the peripheral liver, and production is localized to stellate cells, periductal neutrophils, and stromal cells, which typically accompany the proliferating ductules. IL-6 mRNA and protein were detected only near the biliary tree after BDL, and not in the peripheral liver, and the production was localized to periductal hematolymphoid cells, which had the morphological appearance of macrophages and/or dendritic cells. There is also a distinct up-regulation of met and gp-80 mRNA and protein in the biliary tree, which is stronger than that seen in the peripheral liver. Met protein expression is increased, and IL-6R(gp-80) protein is induced on the proliferating BEC, consistent with the participation of both the HGF/met and IL-6/gp-80 systems in the early phases of type I ductular reactions. These observations show that periductal hematolymphoid and stromal cells are the source of BEC growth factors, and receptors for these factors are up-regulated on BEC during active ductular proliferation. Complex interactions between the inflammatory, stromal, and BEC results in a dysmorphogenic repair response that eventually leads to cirrhosis.