Macrophage-fibroblast interactions in collagenase production and cartilage degradation.

Rabbit bone-marrow macrophages and fibroblasts were cultured, independently or together, with pieces of 35S-labelled cartilage or at the surface of dried [14C]collagen gels. Each type of cell, cultivated alone, rapidly degraded the proteoglycan of cartilage, but only the fibroblasts degraded collagen. The co-culture of both types of cell had no consistent effect on the rate of proteoglycan degradation, but it stimulated the rate of collagen degradation. In parallel, the accumulation of collagenase in the culture fluid was enhanced but not that of neutral proteinase. Coinditioned media from macrophage cultures added to cultures of fibroblasts had the same effect as the living macrophages in stimulating the production of collagenase. Their action was itself enhanced when the macrophages had been activated by concanavalin A-stimulated spleen-cell factors. These data suggest that fibroblasts may act as effector cells in producing collagenase and degrading collagen in response to soluble factors released by macrophages under the control of lymphocyte factors.     

Anti-inflammatory effects of Stephania tetrandra S. Moore on interleukin-6 production and experimental inflammatory disease models

Deregulation of interleukin-6 (IL-6) expression caused the synthesis and release of many inflammatory mediators. It is involved in chronic inflammation, autoimmune diseases, and malignancy. Stephania tetrandra S. Moore is a Chinese medicinal herb which has been used traditionary as a remedy for neuralgia and arthritis in China. To investigate the anti-inflammatory effects of S. tetrandra S. Moore in vitro and in vivo, its effects on the production of IL-6 and inflammatory mediators were analysed. When human monocytes/macrophages stimulated with silica were treated with 0.1-10 mug/ml S. tetranda S. Moore, the production of IL-6 was inhibited up to 50%. At these concentrations, it had no cytotoxicity effect on these cells. It also suppressed the production of IL-6 by alveolar macrophages stimulated with silica. In addition, it inhibited the release of superoxide anion and hydrogen peroxide from human monocytes/macrophages. To assess the anti-fibrosis effects of S. tetrandra S. Moore, its effects on in vivo experimental inflammatory models were evaluated. In the experimental silicosis model, IL-6 activities in the sera and in the culture supernatants of pulmonary fibroblasts were also inhibited by it. In vitro and in vivo treatment of S. tetrandra S. Moore reduced collagen production by rat lung fibroblasts and lung tissue. Also, S. tetrandra S. Moore reduced the levels of serum GOT and GPT in the rat cirrhosis model induced by CCL(4), and it was effective in reducing hepatic fibrosis and nodular formation. Taken together, these data indicate that it has a potent anti-inflammatory and antifibrosis effect by reducing IL-6 production.     

Pulmonary macrophages alter the collagen phenotype of lung fibroblasts

Cultured lung fibroblasts produced and secreted interstitial collagen types I and III. The relative proportion of type III collagen increased as a linear function of cell density, with confluent cultures producing 8.6% type III collagen. When human lung fibroblasts were cultured in the presence of newly harvested lung macrophages, the proportion of type III collagen secreted rose to 15.5%. This high level of type III collagen synthesis was greater than could be induced by withdrawal of serum, a perturbation known to alter the proportion of types I and III collagen synthesized by fibroblasts. This effect on fibroblast phenotype was independent of cell density, as both low and high density cultures of fibroblasts responded similarly when cultured with macrophages. There was no evidence that fibroblasts synthesize new or different collagen types (such as type I trimer) in response to macrophages. Optimal conditions for eliciting an effect on fibroblast connective tissue metabolism required interaction of the two cell types for 5–8 days. These in vitro changes are analogous to the sequence of interactions and changes in connective tissue metabolism seen during recovery from tissue injury.     

Collagen resorption by macrophages and fibroblasts in liver cirrhosis

Intracellular lysis of collagen by macrophages and fibroblasts in liver cirrhosis and during its reverse development has been shown in experiments on mice by electron microscopy and electron histochemistry. Based on the data obtained it is concluded that in the process under consideration, there occur phagocytosis and intracellular resorption of collagen by macrophages and fibroblasts and that intracellular collagen lysis in the cells in question occurs in the presence of active involvement in the process of lysosomal enzymes.     

Macrophages produce TGF-beta-induced (beta-ig-h3) following ingestion of apoptotic cells and regulate MMP14 levels and collagen turnover in fibroblasts

Phagocytic clearance of apoptotic cells by macrophages is an essential part in the resolution of inflammation. It coincides with activation of repair mechanisms, including accumulation of extracellular matrix. A possible link between clearance of apoptotic debris and accumulation of extracellular matrix has not been investigated. Production of collagen was measured in primary fibroblasts cocultured with macrophages. Ingestion of apoptotic cells by monocyte-derived macrophages led to up-regulation of collagen. Direct contact between macrophages and fibroblasts was not required for collagen up-regulation. Macrophages produced TGF-beta following ingestion of apoptotic cells, but the levels of this cytokine were lower than those required for a significant up-regulation of collagen. Simultaneously, the levels of TGF-beta-induced (TGFBI), or keratoepithelin/BIGH3, mRNA and protein were increased. In contrast, primary alveolar macrophages stimulated collagen production without exposure to apoptotic cells; there was no further increase in the levels of TGFBI, mRNA or protein, or collagen after ingestion of apoptotic cells. Stimulation of fibroblasts with TGFBI down-regulated MMP14 levels, decreased DNA binding by p53, increased DNA binding by PU.1, and up-regulated collagen protein but not mRNA levels. Overexpression of MMP14 or p53, or small interfering RNA-mediated inhibition of PU.1 led to an increase in MMP14 and a decline in collagen levels, whereas small interfering RNA-mediated inhibition of MMP14 led to elevation of collagen levels. In conclusion, monocyte-derived but not alveolar macrophages produce TGFBI following ingestion of apoptotic cells, leading to the down-regulation of MMP14 levels in fibroblasts through a mechanism involving p53 and PU.1, and to subsequent accumulation of collagen.     

Effects of intravenous silica on immune and non-immune functions of the murine host.

Silica, an agent toxic for macrophages, administered i.v. to DBA/2 mice rapidly depresses the clearance of colloidal carbon by the reticuloendothelial system and reduces the in vitro phagocytic activity of peritoneal macrophages harvested 3 days after silica injection. Silica blocks the humoral immune response to sheep erythrocytes and the cell-mediated immune response to allogeneic fibroblasts when given before antigen. Silica also induces complex alterations in spleen cell responsiveness to concanavalin A involving both local and serum factors. Silica had no significant effect on the induction of interferon by statolon or Newcastle disease virus. No unequivocal evidence was obtained that silica has a direct depressive effect on cells other that macrophages, but indirect effects on lymphocytes were produced most likely by factors released from silica-lysed macrophages. Intravenous silica may prove useful for the separation of interferon induction and immune response stimulation in studies of host resistance to infection and oncogenesis. Considerable variation exists in the immunodepressive effects of different preparations of silica.     

Cysteine cathepsins and caspases in silicosis

Silicosis is an occupational pneumoconiosis caused by inhalation of crystalline silica. It leads to the formation of fibrohyalin nodes that result in progressive fibrosis. Alternatively, emphysema may occur, with abnormal destruction of collagen fibres in the advanced stages. Although the pathophysiological mechanisms remain unclear, it has been established that the lung responds to silica by massive enrollment of alveolar macrophages, triggering an inflammatory cascade of reactions. An imbalance in the expression of lung proteases and their inhibitors is implicated in extracellular matrix remodelling and basement membrane disruption. Moreover, exposure to silica can initiate apoptotic cell death of macrophages. This review summarises the current knowledge on cysteine cathepsins that have been ignored so far during silicosis and outlines the recent progress on cellular pathways leading to silica-induced caspase activation, which have been partly delineated.     

Macrophage-stimulated cardiac fibroblast production of IL-6 is essential for TGF β/Smad activation and cardiac fibrosis induced by angiotensin II

Interleukin-6 (IL-6) is an important cytokine participating in multiple biologic activities in immune regulation and inflammation. IL-6 has been associated with cardiovascular remodeling. However, the mechanism of IL-6 in hypertensive cardiac fibrosis is still unclear. Angiotensin II (Ang II) infusion in mice increased IL-6 expression in the heart. IL-6 knockout (IL-6-/-) reduced Ang II-induced cardiac fibrosis: 1) Masson trichrome staining showed that Ang II infusion significantly increased fibrotic areas of the wild-type mouse heart, which was greatly suppressed in IL-6-/- mice and 2) immunohistochemistry staining showed decreased expression of α-smooth muscle actin (α-SMA), transforming growth factor β1 (TGF-β1) and collagen I in IL-6-/- mouse heart. The baseline mRNA expression of IL-6 in cardiac fibroblasts was low and was absent in cardiomyocytes or macrophages; however, co-culture of cardiac fibroblasts with macrophages significantly increased IL-6 production and expression of α-SMA and collagen I in fibroblasts. Moreover, TGF-β1 expression and phosphorylation of TGF-β downstream signal Smad3 was stimulated by co-culture of macrophages with cardiac fibroblasts, while IL-6 neutralizing antibody decreased TGF-β1 expression and Smad3 phosphorylation in co-culture of macrophage and fibroblast. Taken together, our results indicate that macrophages stimulate cardiac fibroblasts to produce IL-6, which leads to TGF-β1 production and Smad3 phosphorylation in cardiac fibroblasts and thus stimulates cardiac fibrosis.     

Cleavage of Type I Collagen by Fibroblast Activation Protein-α Enhances Class A Scavenger Receptor Mediated Macrophage Adhesion

Pathophysiological conditions such as fibrosis, inflammation, and tumor progression are associated with modification of the extracellular matrix (ECM). These modifications create ligands that differentially interact with cells to promote responses that drive pathological processes. Within the tumor stroma, fibroblasts are activated and increase the expression of type I collagen. In addition, activated fibroblasts specifically express fibroblast activation protein-α (FAP), a post-prolyl peptidase. Although FAP reportedly cleaves type I collagen and contributes to tumor progression, the specific pathophysiologic role of FAP is not clear. In this study, the possibility that FAP-mediated cleavage of type I collagen modulates macrophage interaction with collagen was examined using macrophage adhesion assays. Our results demonstrate that FAP selectively cleaves type I collagen resulting in increased macrophage adhesion. Increased macrophage adhesion to FAP-cleaved collagen was not affected by inhibiting integrin-mediated interactions, but was abolished in macrophages lacking the class A scavenger receptor (SR-A/CD204). Further, SR-A expressing macrophages localize with activated fibroblasts in breast tumors of MMTV-PyMT mice. Together, these results demonstrate that FAP-cleaved collagen is a substrate for SR-A-dependent macrophage adhesion, and suggest that by modifying the ECM, FAP plays a novel role in mediating communication between activated fibroblasts and macrophages.     

Localization of endothelin receptors in bleomycin-induced pulmonary fibrosis in the rat

Pulmonary fibrosis is characterized by excessive extracellular matrix deposition with concomitant loss of gas exchange units, and endothelin-1 (ET-1) has been implicated in its pathogenesis. Increased levels of ET-1 from tissues and bronchoalveolar lavage have been reported in patients with pulmonary fibrosis and in animal models after intratracheal bleomycin. We characterized the cellular distribution of alveolar ET receptors by immunohistochemistry in bleomycin-induced pulmonary fibrosis in the rat and determined the regulation by bleomycin of ET receptor mRNA expression in isolated alveolar macrophages and rat lung fibroblasts. We found significant increases in the numbers of fibroblasts and macrophages at day 7 compared to day 28 and control animals. ET(B) receptor immunoreactivity was observed on fibroblasts and invading monocytes. Isolated fibroblasts expressed both ET(A) and ET(B) receptor mRNA, and ET(A) receptor mRNA was upregulated by bleomycin. Isolated resident alveolar macrophages expressed neither ET(A) nor ET(B) receptor mRNA which were also not induced by bleomycin. We conclude that, while ET(B) receptor stimulation of fibroblasts and monocytes recruited during bleomycin-induced lung injury exerts antagonistic effects on fibroblast collagen synthesis, the observed increase in the number of fibroblasts in vivo and upregulation of fibroblast ET(A) receptor mRNA by bleomycin in vitro point to a predominance of the profibrotic effects of ET receptor engagement.     

Bacillus Calmette Guerin induces fibroblast activation both directly and through macrophages in a mouse bladder cancer model

Background

Bacillus Calmette-Guerin (BCG) is the most effective treatment for non-muscle invasive bladder cancer. However, a failure in the initial response or relapse within the first five years of treatment has been observed in 20% of patients. We have previously observed that in vivo administration of an inhibitor of nitric oxide improved the response to BCG of bladder tumor bearing mice. It was described that this effect was due to a replacement of tumor tissue by collagen depots. The aim of the present work was to clarify the mechanism involved in this process.

Methodology/Principal Findings

We demonstrated that BCG induces NIH-3T3 fibroblast proliferation by activating the MAPK and PI3K signaling pathways and also differentiation determined by alpha-smooth muscle actin (alpha-SMA) expression. In vivo, intratumoral inoculation of BCG also increased alpha-SMA and collagen expression. Oral administration of L-NAME enhanced the pro-fibrotic effect of BCG. Peritoneal macrophages obtained from MB49 tumor-bearing mice treated in vivo with combined treatment of BCG with L-NAME also enhanced fibroblast proliferation. We observed that FGF-2 is one of the factors released by BCG-activated macrophages that is able to induce fibroblast proliferation. The involvement of FGF-2 was evidenced using an anti-FGF2 antibody. At the same time, this macrophage population improved wound healing rate in normal mice and FGF-2 expression was also increased in these wounds.

Conclusions/Significance

Our findings suggest that fibroblasts are targeted by BCG both directly and through activated macrophages in an immunotherapy context of a bladder murine model. We also described, for the first time, that FGF-2 is involved in a dialog between fibroblasts and macrophages induced after BCG treatment. The fact that L-NAME administration improves the BCG effect on fibroblasts, NO inhibition, might represent a new approach to add to the conventional BCG therapy.     

Receptor-mediated endocytosis of fibroblast beta-glucuronidase by peritoneal macrophages

beta-Glucuronidase secreted by mouse 3T3 fibroblasts in vitro was taken up into mouse peritoneal macrophages and into human fibroblasts by a process which was rapid and saturable. High concentrations of mannose-containing compounds inhibited uptake into macrophages but had no effect on uptake into fibroblasts. Mannose-6-phosphate inhibited uptake into both types of cell, reducing uptake into macrophages by 34% and abolishing uptake into fibroblasts completely at a concentration of 5 mM. Fructose-1-phosphate was almost equally as effective at inhibiting uptake into fibroblasts but had no effect on macrophages. Pre-treatment of beta-glucuronidase with alkaline phosphatase totally prevented its uptake into fibroblasts but had no effect on its uptake into macrophages. These results indicate that fibroblasts can secrete a lysosomal enzyme in a form recognised as a high uptake ligand not only by other fibroblasts but also by peritoneal macrophages and that endocytosis appears to be mediated by different receptors present on each type of cell. This has important implications for the potential treatment of mucopolysaccharidoses by fibroblast transplants.     

Induction of early alveolar injury by inhaled asbestos and silica

Inhaled asbestos fibers and silica crystals are known to cause interstitial fibrotic lung disease in animals and humans. The initial cellular events and biochemical mechanisms that lead to development of disease are poorly understood. In ongoing studies reviewed here it has been shown that inhaled particulates small enough to pass through the conducting airways are deposited initially at the bifurcations of alveolar ducts. Within hours after brief exposure, alveolar epithelial cells phagocytose inhaled particles that subsequently are translocated to interstitial matrix and fibroblasts. Within 48 h after exposure, inhaled asbestos on alveolar surfaces activates a complement-dependent chemotactic factor for macrophages that accumulate at duct bifurcations. Epithelial cells, macrophages, fibroblasts, and the interstitial matrix are significantly altered by brief (1- 5-h) exposure to chrysotile asbestos. The basic mechanisms that mediate these initial events remain to be defined.     

Receptor-mediated endocytosis of fibroblast β-glucuronidase by peritoneal macrophages

β-Glucuronidase secreted by mouse 3T3 fibroblasts in vitro was taken up into mouse peritoneal macrophages and into human fibroblasts by a process which was rapid and saturable. High concentrations of mannose-containing compounds inhibited uptake into macrophages but had no effect on uptake into fibroblasts. Mannose-6-phosphate inhibited uptake into both types of cell, reducing uptake into macrophages by 34% and abolishing uptake into fibroblasts completely at a concentration of 5 mM. Fructose-1-phosphate was almost equally as effective at inhibiting uptake into fibroblasts but had no effect on macrophages. Pre-treatment of β-glucuronidase with alkaline phosphatase totally prevented its uptake into fibroblasts but had no effect on its uptake into macrophages. These results indicate that fibroblasts can secrete a lysosomal enzyme in a form recognised as a high uptake ligand not only by other fibroblasts but also by peritoneal macrophages and that endocytosis appears to be mediated by different receptors present on each type of cell. This has important implications for the potential treatment of mucopolysaccharidoses by fibroblast transplants.     

Bronchial epithelial cell matrix production in response to silica and basic fibroblast growth factor

BACKGROUND: Previous studies show that macrophages, lung fibroblasts, and their soluble mediators are responsible for the onset and development of pulmonary fibrosis. This study was conducted to determine whether airway epithelial cells are also directly involved in response to fibrogenic agents and consequently in the pathogenesis of lung fibrosis. To verify the hypothesis, we determined whether silica acts directly on human bronchial epithelial cells by stimulating cytokine and growth factor release and by modifying matrix production. MATERIALS AND METHODS: An SV40 large T antigen-transformed human airway epithelial cell line, 16HBE14o (16HBE), was used. The expression profile of some proinflammatory interleukins (ILs), such as IL-1alpha, IL-1beta and IL-6 and their modulation by silica, were evaluated by polymerase chain reaction (PCR) analysis. Transforming growth factor beta (TGFbeta) and basic fibroblast growth factor (bFGF) mRNA levels were tested by Northern blotting in the presence and in the absence of silica. The silica- and/or bFGF-induced effects on matrix components (total proteins, collagen, and fibronectin) were also evaluated using radio-labeled precursors. RESULTS: The results demonstrated 16HBE internalized silica particles. Silica induced a little IL-6 secretion, without affecting IL-1 and TGFbeta isoform production and strongly stimulated bFGF mRNA level and bFGF protein secretion. Silica also induced changes in 16HBE production of total proteins, collagen, and fibronectin production. When added in combination with the growth factor, it strengthened bFGF stimulation of matrix component secretion. CONCLUSIONS: These results support the hypothesis that the changes in matrix components are due to a direct effect of silica on bronchial epithelial cells. Silica-induced over-secretion of bFGF suggests that autocrine and paracrine differentiation loops for bFGF may also be operative and that these mechanisms may be involved in the pathogenesis of pulmonary fibrosis. In the future, cytokine-directed therapeutic strategies might find a place in clinical practice.     

Localization of endothelin receptors in bleomycin-induced pulmonary fibrosis in the rat

Pulmonary fibrosis is characterized by excessive extracellular matrix deposition with concomitant loss of gas exchange units, and endothelin-1 (ET-1) has been implicated in its pathogenesis. Increased levels of ET-1 from tissues and bronchoalveolar lavage have been reported in patients with pulmonary fibrosis and in animal models after intratracheal bleomycin. We characterized the cellular distribution of alveolar ET receptors by immunohistochemistry in bleomycin-induced pulmonary fibrosis in the rat and determined the regulation by bleomycin of ET receptor mRNA expression in isolated alveolar macrophages and rat lung fibroblasts. We found significant increases in the numbers of fibroblasts and macrophages at day 7 compared to day 28 and control animals. ET_B receptor immunoreactivity was observed on fibroblasts and invading monocytes. Isolated fibroblasts expressed both ET_A and ET_B receptor mRNA, and ET_A receptor mRNA was upregulated by bleomycin. Isolated resident alveolar macrophages expressed neither ET_A nor ET_B receptor mRNA which were also not induced by bleomycin. We conclude that, while ET_B receptor stimulation of fibroblasts and monocytes recruited during bleomycin-induced lung injury exerts antagonistic effects on fibroblast collagen synthesis, the observed increase in the number of fibroblasts in vivo and upregulation of fibroblast ET_A receptor mRNA by bleomycin in vitro point to a predominance of the profibrotic effects of ET receptor engagement.     

Degradation of collagen and proteoglycan by macrophages and fibroblasts Individual potentialities of each cell type and cooperative effects through the activation of fibroblasts by macrophages

Fibroblasts and macrophages of various sources (peritoneal, alveolar or bone marrow-derived), from either rabbit or mouse, were cultured, independently or together, at the surface of [³H]proteoglycan/[¹⁴C]collagen-coated plates to evaluate their capacities for proteoglycan and collagen degradation. The various macrophage populations differed widely in their potentialities for proteoglycan and particularly, for collagen degradation, native collagen being significantly degraded, in this model only by rabbit alveolar macrophages. Fibroblasts were as active in proteoglycan degradation as the most active macrophage preparations, but their potential for collagen degradation appeared much higher than that of macrophages. Moreover, all types of macrophages secreted a factor, a monokine, that activated collagen and proteoglycan degradation by fibroblasts. Thus, fibroblasts might well be a major effector cell, active in connective tissue degradations occurring under chronic inflammatory situations.     

Acute downregulation of miR-155 leads to a reduced collagen synthesis through attenuating macrophages inflammatory factor secretion by targeting SHIP1

Fibrosis, tightly associated with fibroblasts collagen synthesis, is related closely with inflammatory response. Our previously study found that acute downregulation of miR-155 at wound sites leads to a reduced fibrosis, however its particular mechanism is unclear. Herein, we aimed to explore the mechanism of miR-155 in reducing fibrosis. We first found that down-regulation of miR-155 inhibited macrophages transforming growth factor-β1 (TGF-β1) and IL-1β secretion. Next, we found that co-cultured with macrophages increased the proliferation and collagen synthesis of fibroblasts, and downregulation of miR-155 in macrophages could effectively attenuate the accelerative effects. We further identified SH2 domain containing inositol-5-phosphatase 1 (SHIP1) as a direct target of miR-155 in macrophages, and the expression of SHIP1 was negatively correlated with the level of miR-155. We further confirmed that PI3K/Akt pathway was involved in this process. Last, we found that downregulation of miR-155 leads to a reduced fibrosis in sever burn rat. Taken together, these results indicate that down-regulation of miR-155 leads to a reduced fibroblasts proliferation and collagen synthesis through attenuating macrophages TGF-β1 and IL-1β secretion by targeting SHIP1 via PI3K/Akt pathway, suggesting its potential therapeutic effects on the treatment of skin fibrosis.     

WOUND HEALING AND COLLAGEN FORMATION : I. Sequential Changes in Components of Guinea Pig Skin Wounds Observed in the Electron Microscope

The regular sequence encountered in healing guinea pig skin wounds has been examined by methods of light and electron microscopy. Observations on cell populations, their fine structure, and fibril formation in the connective tissue have been made. Linear incisions in the skin of normal female guinea pigs weighing 300 to 350 grams were allowed to heal. The wounds were then excised, fixed with buffered 2 per cent osmium tetroxide, and postfixed in neutral buffered formalin, at 16 and 24 hours and at 3, 5, 9, and 14 days after wounding. They were then embedded in epoxy resin. In the inflammatory phase the exudate observed in the early wounds consists largely of polymorphonuclear neutrophilic leukocytes, macrophages, fibrin, and free extracellular organelles from the disrupted inflammatory cells. These organelles later appear in vacuoles in the cytoplasm of the macrophages. Fibroblasts first appear at 24 hours, and show extensive development and dilatation of the endoplasmic reticulum, which sometimes contains moderately dense flocculent material. In addition, these fibroblasts have enlarged mitochondria and condensations of filamentous material within the cytoplasm near the cell surface. Occasional myelin figures and moderately dense, 0.5 to 1.0 micron bodies are found within the cytoplasm of the early fibroblasts. Collagen fibrils are first seen at 3 days extracellularly near the cell surfaces. They appear at the later times in two populations of sizes. With increasing wound age the fibroblasts retain their morphology and the wounds decrease in cellularity concomitantly with the formation of increasing amounts of collagen. Several proposed mechanisms of collagen fibril formation are discussed in relation to the observed phenomena. The problem of correlating fibril diameter with the appearance of the periodic structure of collagen in relation to the minimal size fibril which would be anticipated to display this appearance is discussed.