Mitogenic activity for fibroblasts induced by silica and titanium dioxide particles in vitro and in vivo.

Experimental studies on particle-induced pulmonary fibrosis have not provided consistent evidence for the specific induction of fibroblast-regulating cytokines by pulmonary macrophages in response to fibrogenic as compared to non-fibrogenic particles. Using an optimized, wholly serum-free bioassay, we assessed mitogenic activity for pulmonary fibroblasts in supernatants of short-term cultures of alveolar macrophages exposed to either fibrogenic silica or non-fibrogenic titanium dioxide ducts. The responses to these supernatants were influenced by the replicative status of the target cells, in that samples which stimulated non-cycling fibroblasts caused inhibition of DNA synthesis by cycling cells when tested at the same concentration. However, both silica and titanium dioxide elicited comparable secretion of growth factor activity by macrophages, following either in-vitro or in-vivo administration of particles. In contrast, bronchoalveolar lavage fluids from animals that received intratracheal injections of silica, but not from those that received titanium dioxide, exhibited a sustained reduction in fibroblast-stimulating activity. We conclude that secretion of growth factor activity by alveolar macrophages in culture is induced by particles in a non-specific manner. However, alterations in mitogenic activity in bronchoalveolar lavage fluid may constitute a biological marker of the pattern of pulmonary injury which progresses to fibrosis.     

Fibroblast-stimulating factor 1, a novel lymphokine produced in schistosomal egg granulomas, stimulates liver fat-storing cells in vitro.

We report that the novel lymphokine fibroblast-stimulating factor 1, produced within hepatic schistosomal egg granulomas, stimulates liver fat-storing cells (FSC) in vitro to proliferate and express fibronectin genes. Because FSC are critical in hepatic fibrogenesis generally, we propose that in vivo stimulation of FSC by fibroblast-stimulating factor 1 may be an important step in schistosomal liver fibrosis.     

Schistosomal egg granuloma-derived fibroblast-stimulating factor is apparently distinct from interleukin-1.

We have previously reported that egg granulomas isolated from livers of Schistosoma mansoni-infected euthymic mice in vitro elaborate a factor(s) that stimulates a variety of fibroblast responses including fibroblast proliferation and enhanced synthesis of extracellular matrix proteins. We have postulated that these factors play a role in the pathogenesis of hepatic fibrosis in schistosomiasis mansoni. Serum-free supernatants from egg granuloma cultures also stimulate thymocyte proliferation in an assay that defects interleukin-1 (IL-1). Thymocytes and fibroblasts are stimulated to proliferate by the same fractions of egg granuloma culture supernatant separated by gel filtration, isoelectric focusing, and ion-exchange chromatography. This suggested that granuloma-derived IL-1 is responsible for the observed fibroblast stimulation. Here we report that the ability of granuloma culture supernatants to stimulate the IL-1-sensitive D10.G4.1 cells but not fibroblasts is removed by treatment with immobilized anti-IL-1 antibody. We also observed that dialyzed culture supernatants from egg granulomas obtained from infected congenitally athymic (nude) mice also stimulate fibroblast proliferation. Treatment with anti-IL-1 antibody did not abrogate this response. In contrast to our experience with egg granulomas isolated from euthymic mice, IL-1- and fibroblast-stimulating activity could be separated by gel filtration and isoelectric focusing. We conclude that the fibroblast growth-stimulating activities elaborated by egg granulomas from S. mansoni-infected euthymic and athymic mice may be different but both appear to be distinct from IL-1.     

Direct and indirect effects of soluble extracts of Schistosoma mansoni eggs on fibroblast proliferation in vitro.

The possibility that soluble products of Schistosoma mansoni eggs might participate in the pathogenesis of hepatic fibrosis in schistosomiasis was investigated. Both crude saline extracts of eggs (soluble egg antigen [SEA]) and a partially purified SEA fraction contained activity which stimulated guinea pig and human dermal fibroblasts to proliferate in vitro, as measured by uptake of [3H]thymidine. Maximum activity was present in fractions which eluted from Sephacryl S-200 with an apparent molecular weight of less than or equal to 12,500 and in fractions which had an estimated pI 8, as determined by preparative isoelectric focusing of partially purified SEA. Activity in crude SEA was not removed by chromatography on concanavalin A-Sepharose 4B. When concanavalin A-binding glycoproteins lacking intrinsic fibroblast-stimulating activity were incubated with spleen cells from infected or uninfected mice, fibroblasts-stimulating activity was detected in the culture supernatants. Thus, SEA contains two functionally distinct molecular species. One of these directly stimulates fibroblasts, whereas the other induces the release of a fibroblast-stimulating activity from lymphocytes or macrophages or both. Since these fibroblast-stimulating factors might be elaborated in the livers of infected individuals, these observations suggest a potential role of soluble schistome products in the pathogenesis of hepatic fibrosis in schistosomiasis.     

Expression and regulation of tissue inhibitor of metalloproteinase-1 and matrix metalloproteinases by intestinal myofibroblasts in inflammatory bowel disease

Intestinal fibrosis and strictures frequently occur in Crohn's disease but not ulcerative colitis. We have recently shown that, compared to myofibroblasts obtained from normal and ulcerative colitis tissue, myofibroblasts isolated from fibrotic Crohn's disease mucosal samples express significantly lower amounts of transforming growth factor (TGF)-beta 3, but the expression of TGF-beta 2 was significantly greater. We now report that in myofibroblast cultures established from fibrotic Crohn's disease mucosal samples there is significantly higher constitutive expression of tissue inhibitor of metalloproteinase (TIMP)-1 compared to similar cells isolated from normal or ulcerative colitis tissue. Myofibroblasts derived from normal mucosa and from mucosa affected by ulcerative colitis or Crohn's disease also expressed matrix metalloproteinase (MMP)-1, MMP-2, and MMP-3 but did not express MMP-9. Recombinant (r) TGF-beta 1 and rTGF-beta 2, but not rTGF-beta 3, induced expression of TIMP-1 in normal intestinal myofibroblasts. These studies illustrate a potential mechanism by which differential expression of isoforms of TGF-beta may lead to excessive deposition of extracellular matrix and stricture formation via TIMP-1-mediated inhibition of MMP activity.     

Production of human T cell growth factor

We provide here a protocol for production of T cell growth factor (TCGF) using cells isolated from defibrinated blood. Whether combined in allogeneic pools or tested as single donors, these cells consistently yield high activity TCGF, following PHA stimulation. Protocols using cells isolated from heparinized blood have often included addition of indomethacin or removal of adherent cells, or both, to overcome the problem of frequent nonproducing cultures. Our failure to find nonproducing cultures using this source of cells suggests that inhibitory cells or factors are removed by the blood clot formed during defibrination.

A distincteconomic advantage is gained by using these cells, not only because of the reliability of obtaining active supernatants, but also because the same blood can be used for preparation of a serum pool for cell cultures and the erythrocytes are useful for absorption of contaminating PHA present in the TCGF-containing media. Not only can defibrinated blood leukocytes be stimulated by PHA to release TCGF, but when cultured in allogeneic mixtures containing no PHA, they also release active TCGF.     

Partial characterization of a fibroblast-stimulating factor produced by cloned murine T lymphocytes.

T cells may regulate tissue fibrosis through the elaboration of soluble factors that stimulate fibroblast growth. The authors previously identified a factor produced by cloned Schistosoma mansoni antigen-specific T cells which served as a competence factor for murine fibroblasts. In the present report, they further characterize this fibroblast-stimulating factor (FsF) and differentiate it from a number of other T-cell-derived lymphokine activities. Crude supernatants from concanavalin-activated cloned T cells were fractionated by gel filtration, ion exchange, or reversed-phase high-pressure liquid chromatography. FsF has an apparent molecular weight of 17,000 and could be differentiated from colony-stimulating factor (CSF), interleukin-3 (IL-3), and interferon (IFN) on the basis of chromatographic characteristics. Highly purified or recombinant IL-2, IL-3, CSF, and IFN had no significant effect on fibroblast proliferation. Furthermore, a monoclonal anti-B-cell-stimulating factor-1 antibody only partially blocked the fibroblast proliferation induced by T-cell supernatants.     

Exaggerated spontaneous release of platelet-derived growth factor by alveolar macrophages from patients with idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis is a fibrotic lung disease characterized by an increased number of mesenchymal cells in the alveolar walls. Alveolar macrophages constitutively express low levels of c-sis, the protooncogene coding for the B chain of platelet-derived growth factor, a protein with chemotactic and mitogenic activity toward mesenchymal cells. We therefore hypothesized that alveolar macrophages in patients with idiopathic pulmonary fibrosis may release increased amounts of platelet-derived growth factor, which might help to explain the accumulation of mesenchymal cells and the fibrosis of the lower respiratory tract in the disease. Evaluation of alveolar macrophages recovered from the lungs of patients with idiopathic pulmonary fibrosis demonstrated that these cells spontaneously released four times more platelet-derived growth factor than did alveolar macrophages recovered from normal persons (P less than 0.01). That the platelet-derived growth factor molecules were potentially active was shown by their chemotactic activity for smooth-muscle cells and their ability to act as a "competence" factor for fibroblast growth. These observations suggest the possibility that the accumulation of mesenchymal cells within the alveolar walls in patients with idiopathic pulmonary fibrosis may result partly from the exaggerated release of the potent mitogen platelet-derived growth factor by mononuclear phagocytes in the lower respiratory tract.     

Interleukin-1 stimulation of human B-lymphoblast differentiation

Transformed B-cell lines have provided useful tools for analyzing B-cell responses to growth and differentiation factors. SKW 6.4 is a human B-lymphoblastoid line that differentiates in response to B-cell differentiation factor (BCDF) but not to other T-cell-derived lymphokines. The present studies were undertaken to determine whether monocyte-derived factors such as interleukin-1 (IL-1) could also promote the maturation of SKW 6.4 cells. Cells were cultured with and without supernatants derived from adherent monocytes of normal individuals. The number of Ig-producing plaque-forming cells in control media cultures gradually increased over a period of 4 days. Monocyte supernatants caused a 3- to 10-fold further increase in the number of IgM-secreting cells over that found in control media cultures. This stimulation by monocytes supernatants was maximal on the third or fourth day of culture and was not accompanied by an increase in proliferation. Recombinant IL-1 added to cultures of SKW 6.4 also produced a dose-related increase in the number of IgM-secreting cells without stimulating proliferation. A polyclonal antiserum against IL-1 prevented the increase in IgM-secreting cells which occurred with the addition of monocyte supernatants. Finally, IL-1 activity was detected in unstimulated SKW 6.4 supernatants. We conclude from these studies that SKW 6.4 cells both secrete IL-1 and differentiate in response to exogenous IL-1. SKW 6.4 may therefore provide a useful tool for future studies on mechanisms of IL-1-induced B cell maturation. It is possible that the basal numbers of IgM-secreting cells seen in unstimulated cultures results from an autocrine mechanism of IL-1-induced differentiation. Finally, care should be taken to exclude IL-1 from supernatants that are being tested for BCDF activity using SKW 6.4 cells.     

Dog mastocytoma cells secrete a growth factor for fibroblasts.

It is suspected that mast cells play a part in the pathogenesis of fibrotic diseases, but the mediators that might be involved in induction of fibrosis have not been identified. We asked whether cultured dog mast cell lines produced growth factor(s) for fibroblasts. Three mastocytoma cell lines were found to secrete proliferative activity for human, hamster, and rabbit fibroblasts. Both mastocytoma cell-conditioned medium and cell extract served as competence factors for induction of DNA synthesis in confluent mouse Swiss 3T3 fibroblasts. The mitogenic activity in the conditioned medium was stable to heat, acid, and high concentrations of chaotropic agents or organic solvents but was decreased by treatment with proteases or reducing agents. The activity had an apparent molecular mass of 10 kD and did not bind to heparin. Activity eluted in a single peak from reverse-phase HPLC, and retention time differed from that of typical mesenchymal mitogens. We offer the hypothesis that mast cells produce growth factors for fibroblasts, possibly including a novel growth factor, and that this may contribute to pathologic fibrosis.     

Hepatocyte growth factor/scatter factor and MET are involved in arterial repair and atherogenesis

Several studies have shown that in the arterial wall hepatocyte growth factor/scatter factor (HGF/SF) is expressed by smooth muscle cells (SMCs) but acts on endothelial cells, not SMCs. Other studies, however, have indicated that SMCs can respond to HGF/SF. We have reinvestigated expression and activity of HGF/SF and its receptor MET in arterial SMC and endothelial cell cultures and in whole arteries after superficial or deep injury or atherogenesis. High-density cultures of SMCs produced HGF/SF but did not express MET, whereas SMCs, at the leading edge of injured cultures, expressed both ligand and receptor and showed a dramatic motility and growth response to HGF/SF. In line with these results, HGF/SF and MET expression was undetectable in the media of uninjured carotid arteries but was induced after deep arterial injury in areas of SMC migration in the neointima. Strong MET expression was also observed in the SMCs of the atherosclerotic lesions of homozygous apoE^−/− mice, whereas HGF/SF was expressed by macrophage-derived foam cells. These results demonstrate that MET is induced in migrating and proliferating SMCs and that HGF/SF and MET are key mediators of the SMC response in atherogenesis.     

Cystic fibrosis: Synthesis of ciliary inhibitor by amniotic cells

The presence of a ciliary inhibitor in media of cultured amniotic cells obtained from a fetus heterozygous for cystic fibrosis has been observed by the oyster gill cilia assay. The chromatographic fraction containing the inhibitor corresponded to eluted fractions chromatographed from cystic fibrosis fibroblast media and serum. An analogous chromatographic fraction from media of cultured amniotic cells from two proportedly normal fetuses did not inhibit cilia. The chromatographic fraction from media of cultured amniotic cells of a fetus at high risk for cystic fibrosis did not inhibit ciliary activity. Serum was collected from this baby seven weeks after birth and also did not inhibit ciliary action, indicating a homozygous normal genotype. These observations may lead to the development of an antenatal test for cystic fibrosis.     

A macrophage-dependent factor that stimulates the proliferation of fibroblasts in vitro.

Whole blood serum (HBS) stimulates the proliferation of fibroblasts in vitro, while platelet-poor plasma serum (PPPS) does not. Fibroblasts grown in the presence of PPPS are truly quiescent in that they are not deprived of nutrients in the culture medium and less than 3% of the cells synthesize DNA and divide. In vivo experiments have suggested that macrophages are necessary for stimulation of fibroplasia during wound repair. We have utilized the difference in growth-promoting activity between HBS and PPPS to study the ability of macrophages to produce growth-promoting activity in cell culture. Guinea pig peritoneal macrophages cultured in vitro in medium containing PPPS release into the medium, either directly or indirectly, a factor (or factors) that stimulates the proliferation of guinea pig wound fibroblasts. This macrophage-dependent, fibroblast-stimulating activity (MFSA) is nondialyzable, heat stable (56 C for 30 minutes), and requires culture in vitro for demonstration of activity. The relationship between MFSA and other growth factor(s) has not yet been determined. In contrast to the macrophage, lymphocytes prepared from mesenteric lymph nodes produced no figroblast-stimulating activity.     

Small molecular weight secretory factors from Pseudomonas aeruginosa have opposite effects on IL-8 and RANTES expression by human airway epithelial cells

Pseudomonas aeruginosa is an opportunistic human pathogen that causes both an acute lung disease in patients with hospital-acquired pneumonia and a chronic lung disease in individuals with cystic fibrosis. Many of the pathophysiologic effects of P. aeruginosa infection are due to factors secreted by the bacterium. Conditioned media from cultures of P. aeruginosa increased interleukin-8 expression and decreased regulated on activation, normal T cells expressed and secreted (RANTES) expression by human airway epithelial cells. Both of these activities were present in heat-treated, protease-treated, small molecular weight fractions. The activities were not inhibited by polymyxin B and were not extracted into ethyl acetate, suggesting that they were not due to endotoxin or autoinducer. Conversely, results from chloroform extractions and studies with a phenazine-minus mutant suggested that the blue pigment pyocyanin contributes to these activities when present. In addition to the effects of small molecular weight factors on cytokine expression, proteases in bacterial-conditioned media further decreased levels of RANTES. By altering expression, release, and/or activity of inflammatory cytokines, secretory factors from P. aeruginosa could disrupt the delicate balance that constitutes the immune response to bacterial infection and thus could contribute to the lung damage that occurs in P. aeruginosa-infected airways.     

G蛋白偶联受体30的特点及其在纤维化疾病中的作用机制

纤维化是一种组织受到破坏后的修复反应,主要由于炎症因子过度刺激引发实质细胞坏死、细胞外基质(extracellular matrix,ECM)分泌过剩,大量沉积于细胞间质,形成胶原蛋白排列紊乱的病理表现,严重者导致器官结构紊乱和功能障碍,甚至发生器官衰竭[1].     

Purification of a novel B cell growth and differentiation factor associated with lupus syndrome.

We have previously reported that KML1-7 cells cloned from a lupus-prone MRL/l mice produced a soluble factor that preferentially expanded anti-DNA antibody production across the H-2 barrier. We purified this factor, a 55-kDa protein that we termed nucleobindin (Nuc). Nuc showed not only induction of anti-ssDNA IgG antibody in cultures of B cells from MRL/l mice (greater than 16 weeks old), but also growth activity. Furthermore, antibodies against existing cytokines have so far not been shown to block Nuc activity on these B cells. In view of the fact that Nuc did not boost anti-ssDNA IgG antibody production in cultures of spleen cells of comparable age from MRL/n mice, which develop a mild form of lupus after the age of one year, Nuc may act on pre-activated B cells to help IgG anti-DNA antibody production. Taken together, Nuc is a new kind of growth and differentiation factor associated with lupus syndrome.     

An immunohistochemical study of feline myocardial fibrosis

The aim of the present study was to investigate the pathology of feline myocardial fibrosis. The hearts from 40 cats with myocardial fibrosis were compared with the hearts from 25 normal cats. Clinical data were available in 11 cases. Hearts with myocardial fibrosis were hypomotile and there were hyperechoic areas in the ventricular wall on echocardiography. The presence of myocardial fibrosis was correlated significantly with hypertrophy of the ventricles, atrial dilation and angiosclerosis. Immunohistochemical studies demonstrated that normal feline cardiomyocytes expressed matrix metalloproteinase (MMP)-2, MMP-9, MMP-14, tissue inhibitor of matrix metalloproteinase (TIMP)-2 and transforming growth factor (TGF)-β2. Fibroblasts in normal hearts expressed only TIMP-2. In the hearts with myocardial fibrosis, expression of MMP-2, TIMP-3 and TGF-β2 by cardiomyocytes was significantly increased, but TIMP-2 expression was diminished. Fibroblasts in the affected hearts showed expression of MMP-14 in several cases. These findings suggest that a complex fibrotic remodelling of the feline myocardium occurs in this disease and that cardiomyocytes are involved in this process.     

Senescent mesenchymal cells accumulate in human fibrosis by a telomere-independent mechanism and ameliorate fibrosis through matrix metalloproteinases

Fibrosis can occur in many organs, where it is a debilitating and preneoplastic condition. The senescence of activated fibroblasts has been proposed to ameliorate fibrosis via the innate immune system but its role in humans has not been investigated. The availability of oral submucous fibrosis (OSMF) biopsies at different stages of disease progression allowed us to test the hypothesis that senescent fibroblasts accumulate with the progression of human fibrosis in vivo, and also to examine the mechanism of senescence. We tested the hypothesis that senescent cells may ameliorate fibrosis by increasing the secretion of matrix metalloproteinases (MMPs). We have used a combination of in situ immunodetection techniques, drug treatments, fluorescence-activated cell sorting and enzyme-linked absorbance assays on tissue samples and fibroblast cultures. We report a novel panning technique, based on fibronectin adhesion rates, to enrich and deplete senescent cells from fibroblast populations. Senescent fibroblasts, as determined by the presence of senescence-associated heterochromatic foci, accumulated with OSMF progression (R(2) = 0.98) and possessed a reduced replicative lifespan in vitro. Unlike wounds, however, OSMF fibroblasts were quiescent in vivo and consistent with this observation, possessed functional telomeres of normal length. Senescence was associated in vivo and in vitro with oxidative damage, DNA damage foci and p16(INK4A) accumulation and required the production of reactive oxygen species (ROS), perhaps from damaged mitochondria, but not the continuous presence of the disease stimulus (areca nut and tobacco), the tissue environment or other cell types. Depletion of OSMF fibroblasts of senescent cells showed that these cells accounted for 25-83 times more MMP-1 and -2 than their pre-senescent counterparts. The results show that the accumulation of senescent fibroblasts in human fibrosis occurs by a telomere-independent mechanism involving ROS and may locally ameliorate the condition by the increased expression of MMPs prior to clearance by the immune system.     

Stimulation of the differentiation of osteogenic rat bone marrow stromal cells by osteoblast cultures

The effect of conditioned medium (CM) from rat calvaria (RC) cel cultures on the growth and differentiation of osteogenic cells in rat bone marrow stromal cell (BMSC) cultures was investigated. Control cultures received either CM from periodontal ligament fibroblast cultures or fresh medium. RCCM stimulated the formation of nodules of bonelike tissue in bone marrow stromal cell cultures in a dose-dependent manner,and the maximal stimulation was associated with the osteoblast-enriched cell populations of the RC cultures. Ultrafiltration demonstrated that activity was confined to a CM fraction of 10- to 30-kilodalton molecular size. The activity was sensitive to boiling and trypsin treatments, but was not affected by neutralizing antibodies to transforming growth factor beta or insulin-like growth factor I or II. RCCM was found to initially increase the number and proportion of cells that expressed alkaline phosphatase activity, although the proportion of alkaline phosphatase-positive cells subsequently declined. These data were consistent with an initial stimulation of proliferation of a subpopulation of osteoprogenitor cells within the cultures, followed by their differentiation. The results suggest that mature osteoblasts may produce a paracrine growth factor that can stimulate the differentiation of osteoblasts from precursor cells.     

Steroid Sensitivity of the Response of Human Lymphocytes to Phytohemagglutinin and Pokeweed Mitogen: Role of Phagocytic Cells

Populations of human peripheral blood lymphocytes were stimulated in vitro with phytohemagglutinin (PHA) or pokeweed mitogen (PWM) The presence of various concentrations of water-soluble prednisolone inhibited this response in a dose-dependent fashion Removal of phagocytic cells from the cell preparations increased steroid sensitivity of both the PHA and PWM responses a 100-fold or more Medium obtained from cultures of unpurified populations of lymphoid cells, but not from purified populations, rendered purified lymphocytes more resistant to steroids both when PHA and PWM were used as stimulants A similar protective activity against steroids was observed in media conditioned by adherent cells obtained from peripheral blood. The results indicate that the monocyte-macrophage cell line produces a factor that protects lymphocytes from the effects of corticotds.