Fn Production Research Articles

Targeting Alternative Splicing of Fibronectin (Fn) to Reduce Extra Domain A (EDA)+ Fn Production and Inhibit Kidney Fibrosis

Targeting Alternative Splicing of Fibronectin (Fn) to Reduce Extra Domain A (EDA)+ Fn Production and Inhibit Kidney Fibrosis

TLR4 signaling modulates extracellular matrix production in the lamina cribrosa.

The optic nerve head (ONH) is a place of vulnerability during glaucoma progression due to increased intraocular pressure damaging the retinal ganglion cell axons. The molecular signaling pathways involved in generating glaucomatous ONH damage has not been fully elucidated. There is a great deal of evidence that pro-fibrotic TGFβ2 signaling is involved in modulating the ECM environment within the lamina cribrosa (LC) region of the ONH. Here we investigated the role of signaling crosstalk between the TGFβ2 pathway and the toll-like receptor 4 (TLR4) pathway within the LC. ECM deposition was examined between healthy and glaucomatous human ONH sections, finding increases in fibronectin and fibronectin extra domain A (FN-EDA) an isoform of fibronectin known to be a damage associated molecular pattern (DAMP) that can activate TLR4 signaling. In human LC cell cultures derived from healthy donor eyes, inhibition of TLR4 signaling blocked TGFβ2 induced FN and FN-EDA expression. Activation of TLR4 by cellular FN (cFN) containing the EDA isoform increased both total FN production and Collagen-1 production and this effect was dependent on TLR4 signaling. These studies identify TGFβ2-TLR4 signaling crosstalk in LC cells of the ONH as a novel pathway regulating ECM and DAMP production.

ED-B-Containing Isoform of Fibronectin in Tumor Microenvironment of Thymomas: A Target for a Theragnostic Approach.

Simple SummaryThe extra-domain B fibronectin (ED-B FN) is highly expressed in thymic epithelial tumors (TETs), as demonstrated by in vivo targeting using 131I-labeled L19 small immunoprotein (131I-L19-SIP) and immunohistochemistry with a predominant expression by stromal cells of a thymoma microenvironment rather than epithelial cells. Such high expression derived from the induction of stromal cells shifts FN production to the ED-B subtype. Our results suggest that Radretumab radioimmunotherapy (R-RIT) inefficacy is not related to low TET ED-B expression but to multifactorial aspects including patients’ inherent characteristics, the pattern expression of the target, the biological characteristics of the tumor, and the format of the target agent, which contribute to the resistance of tumor cells to treatment.Aim: to exploit tissue-specific interactions among thymic epithelial tumor (TETs) cells and extra-domain B fibronectin (ED-B FN). Material and methods: The stromal pattern of ED-B FN expression was investigated through tumor specimen collection and molecular profiling in 11 patients with recurrent TETs enrolled in prospective theragnostic phase I/II trials with Radretumab, an ED-B FN specific recombinant human antibody. Radretumab radioimmunotherapy (R-RIT) was offered to patients who exhibited the target expression. Experiments included immunochemical analysis (ICH), cell cultures, immunophenotypic analysis, Western blot, slot-blot assay, and quantitative RT-PCR of two primary thymoma cultures we obtained from patients’ samples and in the Ty82 cell line. Results: The in vivo scintigraphic demonstration of ED-B FN expression resulted in R-RIT eligibility in 8/11 patients, of which seven were treated. The best observed response was disease stabilization (n = 5/7) with a duration of 4.3 months (range 3–5 months). IHC data confirmed high ED-B FN expression in the peripherical microenvironment rather than in the center of the tumor, which was more abundant in B3 thymomas. Further, there was a predominant expression of ED-B FN by the stromal cells of the thymoma microenvironment rather than the epithelial cells. Conclusions: Our data support the hypothesis that thymomas induce stromal cells to shift FN production to the ED-B subtype, likely representing a favorable hallmark for tumor progression and metastasis. Collectively, results derived from clinical experience and molecular insights of the in vitro experiments suggested that R-RIT inefficacy is unlikely related to low target expression in TET, being the mechanism of R-RIT resistance eventually related to patients’ susceptibility (i.e., inherent characteristics), the pattern expression of the target (i.e., at periphery), the biological characteristics of the tumor (i.e., aggressive and resistant phenotypes), and/or to format of the target agent (i.e., 131I-L19-SIP).

Proteasomal Degradation of Enhancer of Zeste Homologue 2 in Cholangiocytes Promotes Biliary Fibrosis.

During biliary disease, cholangiocytes become activated by various pathological stimuli, including transforming growth factor β (TGF-β). The result is an epigenetically regulated transcriptional program leading to a pro-fibrogenic microenvironment, activation of hepatic stellate cells (HSCs), and progression of biliary fibrosis. This study evaluated how TGF-β signaling intersects with epigenetic machinery in cholangiocytes to support fibrogenic gene transcription. We performed RNA sequencing in cholangiocytes with or without TGF-β. Ingenuity pathway analysis identified "HSC Activation" as the highly up-regulated pathway, including overexpression of fibronectin 1 (FN), connective tissue growth factor, and other genes. Bioinformatics identified enhancer of zeste homologue 2 (EZH2) as an epigenetic regulator of the cholangiocyte TGF-β response. EZH2 overexpression suppressed TGF-β-induced FN protein in vitro, suggesting FN as a direct target of EZH2-based repression. Chromatin immunoprecipitation assays identified an FN promoter element in which EZH2-mediated tri-methylation of lysine 27 on histone 3 is diminished by TGF-β. TGF-β also caused a 50% reduction in EZH2 protein levels. Proteasome inhibition rescued EZH2 protein and led to reduced FN production. Immunoprecipitation followed by mass spectrometry identified ubiquitin protein ligase E3 component N-recognin 4 in complex with EZH2, which was validated by western blotting in vitro. Ubiquitin mutation studies suggested K63-based ubiquitin linkage and chain elongation on EZH2 in response to TGF-β. A deletion mutant of EZH2, lacking its N-terminal domain, abrogates both TGF-β-stimulated EZH2 degradation and FN release. In vivo, cholangiocyte-selective knockout of EZH2 exacerbates bile duct ligation-induced fibrosis whereas MDR2-/- mice are protected from fibrosis by the proteasome inhibitor bortezomib. Conclusion: TGF-β regulates proteasomal degradation of EZH2 through N-terminal, K63-linked ubiquitination in cholangiocytes and activates transcription of a fibrogenic gene program that supports biliary fibrosis.

Effect of transforming growth factor -β1 on α-smooth muscle actin and collagen expression in equine endometrial fibroblasts

Transforming growth factor (TGF)-β1 not only regulates cell growth, development, and tissue remodeling, but it also participates in the pathogenesis of tissue fibrosis. In the equine endometrium, the concentration of TGF-β1 is correlated with endometrosis (equine endometrial fibrosis). In other tissues, TGF-β1 induces differentiation of many cell types into myofibroblasts. These cells are characterized by α-smooth muscle actin (α-SMA) expression and an ability to deposit excessive amounts of extracellular matrix (ECM) components. The aim of the study was to determine whether TGF-β1 plays a role in the development of equine endometrosis. In Exp. 1, endometrial expression of α-SMA in different stages of endometrosis was determined. In endometrial tissues from the mid luteal phase (n = 6 for each stages of endometrosis) and the follicular phase of the estrous cycle (n = 5 for each stages of endometrosis), mRNA transcription and protein expression of α-Sma were evaluated by Real-time PCR and Western-blot, respectively. The α-Sma mRNA transcription and protein expression levels were correlated with the severity of endometrosis (P < 0.05). In both phases of the estrous cycle, α-SMA protein expression was up-regulated in final stage of endometrosis compared to initial stage (P < 0.05). In Exp. 2, the dose- and time-dependent effects of TGF-β1 on expression of α-SMA and ECM components were determined, as well as cell proliferation of equine fibroblasts. Equine endometrial fibroblasts (n = 6, Kenney and Doig category I) were stimulated with vehicle or TGF-β1 (1, 5, 10 ng/ml) for 24, 48 or 72 h. Then, mRNA transcription of α-Sma, collagen type I (Col1a1), collagen type III (Col3a1) and fibronectin 1 (Fn1) were determined by Real-time PCR. The production of ECM components was determined by ELISA. Transforming growth factor-β1 increased the mRNA transcription of α-Sma and ECM components in a dose- and time-dependent manner in cultured endometrial fibroblasts (P < 0.05). Additionally, TGF-β1 at a dose of 10 ng/ml increased α-SMA protein expression and COL1, COL3, FN production after 72 h of stimulation (P < 0.05). The data showed a positive linkage between the presence of myofibroblasts and severity of endometrosis. We conclude that TGF-β1 may participate in pathological fibrotic changes in equine endometrial tissue by induction of myofibroblast differentiation, increased production of ECM components and fibroblast proliferation.

The effects of orally administered Bacillus coagulans and inulin on prevention and progression of rheumatoid arthritis in rats

BackgroundProbiotics have been considered as an approach to addressing the consequences of different inflammatory disorders. The spore-forming probiotic strain Bacillus coagulans has demonstrated anti-inflammatory and immune-modulating effects in both animals and humans. The prebiotic inulin also potentially affects the immune system as a result of the change in the composition or fermentation profile of the gastrointestinal microbiota.ObjectiveIn the present study, an in vivo model was conducted to investigate the possible influences of probiotic B. coagulans and prebiotic inulin, both in combination and/or separately, on the downregulation of immune responses and the progression of rheumatoid arthritis (RA), using arthritis-induced rat model.DesignForty-eight healthy male Wistar rats were randomly categorized into six experimental groups as follows: 1) control: normal healthy rats fed with standard diet, 2) disease control (RA): arthritis-induced rats fed with standard diet, 3) prebiotic (PRE): RA+ 5% w/w long-chain inulin, 4) probiotic (PRO): RA+ 109 spores/day B. coagulans by orogastric gavage, 5) synbiotic (SYN): RA+ 5% w/w long-chain inulin and 109 spores/day B. coagulans, and 6) treatment control: (INDO): RA+ 3 mg/kg/day indomethacin by orogastric gavage. Feeding with the listed diets started on day 0 and continued to the end of study. On day 14, rats were injected with complete Freund's adjuvant (CFA) to induce arthritis. Arthritis activity was evaluated by the biochemical parameters and paw thickness. Biochemical assay for fibrinogen (Fn), serum amyloid A (SAA), and TNF-α and alpha-1-acid glycoprotein (α1AGp) was performed on day 21, 28, and 35 (7, 14 and 21 days post RA induction), respectively.ResultsPretreatment with PRE, PRO, and SYN diets significantly inhibits SAA and Fn production in arthritic rats (P < 0.001). A significant decrease in the production of pro-inflammatory cytokines, such as TNF-α, was seen in the PRE, PRO, and SYN groups (P < 0.001), which was similar to the anti-inflammatory effect of indomethacin. Furthermore, no significant anti-inflammatory effects were observed following different treatments using α1AGp as an RA indicator. Pretreatment with all supplied diets significantly inhibited the development of paw swelling induced by CFA (P < 0.001).ConclusionThe results of this study indicate that the oral intake of probiotic B. coagulans and prebiotic inulin can improve the biochemical and clinical parameters of induced RA in rat.

Incorporation of Mg particles into PDLLA regulates mesenchymal stem cell and macrophage responses.

In this work, we investigated a new approach to incorporate Mg particles within a PDLLA matrix using a solvent-free commercially available process. PDLLA/Mg composites were manufactured by injection moulding and the effects of Mg incorporated into PDLLA on MSC and macrophage responses were evaluated. Small amounts of Mg particles (≤ 1 wt %) do not cause thermal degradation of PDLLA, which retains its mechanical properties. PDLLA/Mg composites release hydrogen, alkaline products and Mg(2+) ions without changing pH of culture media. Mg-containing materials provide a noncytotoxic environment that enhances MSC viability. Concentration of Mg(2+) ions in extracts of MSCs increases with the increment of Mg content in the composites. Incorporation of Mg particles into PDLLA stimulates FN production, ALP activity, and VEGF secretion in MSCs, an effect mediated by degradation products dissolved from the composites. Degradation products of PDLLA induce an increase in MCP-1, RANTES, and MIP-1α secretion in macrophages while products of composites have minimal effect on these chemokines. Regulation of MSC behavior at the biomaterial's interface and macrophage-mediated inflammatory response to the degradation products is related to the incorporation of Mg in the composites. These findings suggest that including small amounts of Mg particles into polymeric devices can be a valuable strategy to promote osseointegration and reduce host inflammatory response.

Differential deposition of fibronectin by asthmatic bronchial epithelial cells

Altered ECM protein deposition is a feature in asthmatic airways. Fibronectin (Fn), an ECM protein produced by human bronchial epithelial cells (HBECs), is increased in asthmatic airways. This study investigated the regulation of Fn production in asthmatic or nonasthmatic HBECs and whether Fn modulated HBEC proliferation and inflammatory mediator secretion. The signaling pathways underlying transforming growth factor (TGF)-β1-regulated Fn production were examined using specific inhibitors for ERK, JNK, p38 MAPK, phosphatidylinositol 3 kinase, and activin-like kinase 5 (ALK5). Asthmatic HBECs deposited higher levels of Fn in the ECM than nonasthmatic cells under basal conditions, whereas cells from the two groups had similar levels of Fn mRNA and soluble Fn. TGF-β1 increased mRNA levels and ECM and soluble forms of Fn but decreased cell proliferation in both cells. The rate of increase in Fn mRNA was higher in nonasthmatic cells. However, the excessive amounts of ECM Fn deposited by asthmatic cells after TGF-β1 stimulation persisted compared with nonasthmatic cells. Inhibition of ALK5 completely prevented TGF-β1-induced Fn deposition. Importantly, ECM Fn increased HBEC proliferation and IL-6 release, decreased PGE2 secretion, but had no effect on VEGF release. Soluble Fn had no effect on cell proliferation and inflammatory mediator release. Asthmatic HBECs are intrinsically primed to produce more ECM Fn, which when deposited into the ECM, is capable of driving remodeling and inflammation. The increased airway Fn may be one of the key driving factors in the persistence of asthma and represents a novel, therapeutic target.

Fibronectin (FN) decreases glomerular lesions and synthesis of tumour necrosis factor-alpha (TNF-alpha), platelet-activating factor (PAF) and FN in proliferative glomerulonephritis.

We have studied the effect of therapy with plasma FN on glomerular synthesis of PAF, TNF-alpha and FN, in experimental proliferative glomerulonephritis. Glomerular PAF, TNF-alpha and FN production were increased in rats with nephritis. Peak glomerular PAF production preceded, while peak glomerular TNF-alpha bioactivity coincided with maximal proteinuria. Rats treated with FN (5 mg/kg per 48 h) for 15 days had less proteinuria, glomerular and interstitial cell infiltration and glomerular PAF, TNF-alpha and FN synthesis than non-treated rats. In order to characterize further the mechanisms of action of FN, healthy rats were injected with either FN or saline. Peripheral blood mononuclear cells and neutrophils from healthy rats injected with FN secreted less TNF-alpha and PAF, respectively, than those obtained from saline-treated rats. Our data suggest that the beneficial effect of FN may be related to decreased number of glomerular leucocytes and decreased synthesis of inflammatory mediators and extracellular matrix.

Psoriasis and extra domain A fibronectin loops

We have previously postulated that as well as T-helper (Th) 1 and Th17 cells, the transforming growth factor (TGF)-beta/fibronectin (FN)/alpha5beta1 pathway is central to psoriasis pathogenesis. EDA+ FN refers to an alternatively spliced isoform of FN with an additional domain known as extra domain A. EDA+ FN has two important properties pertinent to psoriasis lesions: it stimulates keratinocyte hyperproliferation, and, through stimulation of Toll-like receptor (TLR) 4, stimulates production of proinflammatory cytokines. EDA+ FN production induced by TGF-beta stimulation can be maintained in psoriasis lesions via two main feedback loops. Firstly, EDA+ FN stimulates proliferation of keratinocytes, which, in an autocrine fashion, will release more EDA+ FN. Secondly, EDA+ FN stimulates TLR4 expressed by antigen-presenting cells resulting in the production of proinflammatory cytokines such as tumour necrosis factor-alpha, interleukin (IL)-1, IL-6 and IL-12. The resultant promotion of cutaneous inflammation results in the recruitment of Th1 cells, which also produce EDA+ FN. We propose that these 'FN loops' contribute to the maintenance and progression of psoriatic lesions. Finally, although the association between psoriasis and heart/thrombotic disease remains unclear one plausible link may be the promotion of atherosclerosis and thrombotic heart disease by EDA+ FN.

Ascorbic acid and uric acid suppress glucose-induced fibronectin and vascular endothelial growth factor production in human endothelial cells

Human umbilical vein endothelial cells (HUVECs) were used to investigate the individual effect of ascorbic (0.01 and 0.05 mM) and uric acid (3, 5, and 7 mg/dl), as well as the combined effect of these two agents on glucose-induced overexpression of fibronectin (Fn), glutathione peroxidase (GPx) activity, H 2O 2 level and vascular endothelial growth factor (VEGF) protein levels. Under 30 mM glucose stress, GPx activity significantly decreased, and lactate dehydrogenase (LDH) release, H 2O 2 level, and Fn and VEGF production significantly increased in HUVECs ( P<.05). The addition of ascorbic acid at 0.05 mM or uric acid at 5 and 7 mg/dl significantly reduced LDH release, recovered GPx activity, suppressed H 2O 2 production, and decreased Fn and VEGF production ( P<.05). The combination of 0.05 mM ascorbic acid and 3, 5, or 7 mg/dl provided significantly greater effect in enhancing GPx activity and lowering H 2O 2 level than ascorbic acid or uric acid treatment alone did ( P<.05). These data suggest that ascorbic acid alone or combined with uric acid in these concentrations may be beneficial to ameliorate glucose-induced diabetic deterioration.

Fibronectin in malignancy: Effect of aging

Fibronectin (Fn) was the first ‘structural’ glycoprotein intensively studied as an ubiquitous matrix component of early phylogenetic appearance. Its age-dependent increase in plasma and tissues may be accompanied in pathological states, especially in tumor growth, by its proteolytic breakdown by a number of neutral proteases. It was also shown that several of its proteolytic breakdown products exhibit unexpected and mostly harmful biological activities. The first of these effects was a potentiation of malignant transformation. Some fragments had proteolytic activity, others behaved as proinflammatory agents stimulating IL-1 and collagenolytic MMP up-regulation. This matricryptic potential of Fn was followed by several other examples of proteolytic production of biologically active peptides. The study of solid human tumors showed among the early signs of malignant transformation the fragmentation of pericellular Fn, concommitent with the increase of its production by the peritumoral stroma. These results should encourage further investigations concerning the potential importance of Fn production and breakdown during cancer progression.

Activation of integrin receptors is required for growth factor-induced smooth muscle cell dysfunction

Purpose: Growth factors and cytokines such as platelet-derived growth factor (PDGF), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and transforming growth factor beta (TGF) stimulate smooth muscle cell (SMC) proliferation and extracellular matrix (ECM) protein production by binding and activating their respective receptors. Recent investigations suggest that simultaneous activation of integrins, which are heterodimeric receptors for ECM, may also be required for growth factor and cytokine function. In this study, we tested the hypothesis that activation of two integrins, ggavβ3 and α2β1, both previously identified in vascular SMCs, is necessary for growth factor- and cytokine-induced vascular SMC dysfunction.Methods. DNA synthesis was measured after stimulation of SMCs derived from human saphenous vein with the growth factors PDGF-BB, EGF, and bFGF. SMC fibronectin (Fn) production was measured (by means of Western blotting) in SMCs stimulated for 72 hours with TGF-β1 or EGF. Both endpoints were measured in the presence and absence of antibodies that block the function of the αvβ3 and α2β1 integrins as well as the α2 and βl subunits.Results: The αvβ3 integrin blocking antibody significantly inhibited PDGF-BB-, EGF-, and bFGF-induced SMC proliferation. The αvβ3 integrin antibody also markedly inhibited TGF-1- and EGF-induced SMC Fn production. Neither the a2 1 integrin nor the α2 or the β1 subunits inhibited either proliferation or matrix protein production in response to any of these agonists.Conclusion: The αvβ3 integrin is required for growth factor- and cytokine-induced SMC proliferation and FN production, whereas α2β1 is not. Since activation of αvβ3 is required for the activity of at least four distinct growth factors and cytokines, inhibition of this integrin might be used as a therapeutic tool for the prevention of intimal hyperplasia.

The Role of Mitogen-Activated Protein Kinase and Protein Kinase C in Fibronectin Production in Human Vascular Smooth Muscle Cells

Background. After evaluating various growth factors, cytokines, and extracellular matrix (ECM) proteins, we found that the most potent agonists of smooth muscle cell (SMC) fibronectin (Fn) production were transforming growth factor-beta (TGF-β) and epidermal growth factor (EGF). To determine the possible signaling pathways involved in the production of this matrix protein, we investigated the role of the intracellular proteins, protein kinase C (PKC) and mitogen-activated protein kinase (MAP-K), in TGF-β- and EGF-induced human vascular SMC Fn production.Materials and Methods. After stimulation of human SMCs with TGF-β (10 ng/ml) and EGF (100 ng/ml), Fn in the cell medium was assayed by immunoblotting using a specific antibody. PKC was activated by brief stimulation of SMC with phorbol 12,13-dibutyrate (PDBu) and inhibited by downregulation with PDBu or the inhibitor, GF109203X. MAP-K was inhibited with PD098059.Results. PKC activation increased basal and synergistically enhanced TGF-β- and EGF-induced Fn production. However, inhibition of PKC by downregulation and GF109203X did not diminish Fn production by TGF-β and EGF. Surprisingly, these two methods of inhibition slightly increased basal and agonist-induced Fn production. The MAP-K kinase inhibitor, PD098059, produced an almost complete inhibition of EGF and a partial inhibition of TGF-β-induced Fn production.Conclusions. Activation of PKC stimulates Fn production; however, neither TGF-β nor EGF produce Fn through a PKC-dependent pathway. EGF and TGF-β both stimulate Fn production at least in part through the intracellular signaling protein MAP-K. Understanding the signaling pathways involved in extracellular matrix protein production will allow the design of specific inhibitors of intimal hyperplasia.

Effects of Staphylococcus aureus protein A on the production of primary host defense factors against S. aureus infection

In vitro examination was carried out to investigate the effects of protein A of Staphylococcus aureus (S. aureus) on the production of fibronectin (Fn) and the third component of complement (C3) by macrophages and that of CD11b, CD49e and H2O2 by granulocytes. Fn production of cultured murine peritoneal macrophages (M phi ) increased significantly (P < 0.01) by treatment for 4 hr at 37 degrees C with recombinant PA (rPA) and the supernatant of overnight culture of S. aureus Cowan I strain (CoCS) (p < 0.01), not that of Wood 46 strain (WoCS), in comparison with that of control. The activity of rPA was inhibited strongly in the presence of CYH (1.0 microgram/ml). The production of C3 by cultured M phi did not increased by treatment for 4 hr at 37 degrees C with rPA, CoCS and WoCS. In these cells treated with CoCS and WoCS, however, the production increased by cultivation in serum free medium for a further 20 hr at 37 degrees C after the treatment. But increase was not found in rPA treated cells. On the other hand, the production of Mac-1, VLA-5 and H2O2 by granulocytes did not increase by treatment with rPA and CoCS. These results show that rPA and PA in CoCS are major components which stimulates Fn synthesis and secretion by cultured M phi some component(s) contained in the supernatant of overnight culture of S. aureus increased the production C3 by cultured M phi , and the supernatant may not contain stimulators to induce production of CD49e, CD11b and H2O2. Increase of Fn production of M phi by PA stimulation may play an important role in the primary host defense against S. aureus infection.

Modulation of fibronectin and tenascin production in human bronchial epithelial cells by inflammatory cytokines in vitro.

Fibronectin (Fn) and tenascin (Tn) are two major extracellular matrix glycoproteins participating in tissue morphogenesis and repair. The regulation of their synthesis and deposition during airway inflammation and their possible contribution in asthma are poorly understood. In this study, modulation of Fn and Tn production was investigated in transformed human bronchial epithelial cells in culture. The cells were treated with interferon gamma (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), a combination of these cytokines, interleukins 3 and 6 (IL-3 and IL-6), granulocyte macrophage-colony stimulating factor (GM-CSF), and a combination of IL-3 and IL-6 for 48 h. Immunofluorescence and immunoblotting methods with monoclonal antibodies to Fn and Tn antibodies suggested the production of some Fn and Tn in the untreated cells. Fn was minimally induced in response to IFN-gamma and TNF-alpha, when compared with the untreated cells, whereas TNF-alpha and especially the IFN-gamma plus TNF-alpha combination resulted in a prominent Tn induction. Interleukins and GM-CSF did not induce Fn or Tn in any case. These results show that human bronchial epithelial cells are capable of producing Fn and Tn. The modulation of Fn and Tn may have an important impact on the pathology of epithelial cells during airway inflammation in vivo.

Modulation of Fibronectin Production of Bovine Bronchial Epithelial Cells by Transforming Growth Factor-β

Regulation of airway repair after injury is poorly understood but is thought to be important in the development of airway diseases such as chronic bronchitis and asthma. There is evidence that fibronectin (Fn), an extracellular matrix glycoprotein, has a role in repair processes. In addition, transforming growth factor-beta (TGF-beta) is also likely involved in would healing and is known to influence extracellular matrix constituents in other cell systems. We postulated that TGF-beta may effect airway repair by modulating Fn production from airway epithelial cells. To examine this hypothesis, we studied the effect of TGF-beta 1 on Fn production by bovine bronchial epithelial cells in culture. Fn, released into the media of cultures exposed to TGF-beta 1, increased in a dose- and time-responsive fashion. Fn in the cell layer also increased in response to TGF-beta 1. De novo protein synthesis was demonstrated by an increase in [35S]methionine incorporation into Fn immunoprecipitated from media of TGF-beta-treated cultures. TGF-beta 1 also induced an increase in expression of Fn mRNA from cultured bronchial epithelial cells, suggesting that TGF-beta modulates Fn production of these cells, at least in part, through modulation of Fn gene expression. These data support a role for TGF-beta in airway repair through modulation of Fn production by airway epithelium.

Chrysotile asbestos exposures can produce an alveolitis with limited fibrosing activity in a subset of high fibre retainer sheep

Inhalation of fibrogenic mineral dust may enhance fibronectin (Fn) production by alveolar macrophages and increase fibroblast growth activity (FGA) in lung lavage fluid. To investigate the relationship of these changes to fibre retention and the development of asbestosis, we exposed 15 sheep to 100 mg Canadian chrysotile fibres in 100 ml saline, and ten sheep to 100 ml saline only, at ten day intervals. The animals were studied at 3 month intervals. At month 18, ten sheep had abnormal chest radiograph (category greater than or equal to 1) (group B) and five had normal radiograph (category 0) (group A). Pulmonary function data indicated restrictive patterns of abnormalities in both groups, more severe in group B. Sheep in group A had bronchoalveolar lavage (BAL) cellularity and biochemistry comparable to controls; sheep in group B had significant increases in total BAL cells (x2), macrophages (x2), neutrophils (x4) and eosinophils (x3), increased BAL lactate dehydrogenase and Fn, but FGA and procollagen 3 comparable to controls. Fibre retention was significantly increased in all exposed sheep and 2.5 x higher in group B vs group A despite similar exposures (70 intratracheal, 100 mg chrysotile infusions). Enhanced fibre retention in group B preceded the appearance of disease. This study confirms our earlier observation linking individual susceptibility to development of alveolitis to individual dust burden and provides evidence that the excess of fibre retention can be observed before detectable disease. In addition, we report a chrysotile-induced early alveolitis with incompletely expressed fibrosing activity at the time of initial radiographic detection. The intensity of the alveolitis is related to the degree of fibre retention.

Stimulation of rat hepatocyte fibronectin production by monocyte-conditioned medium is due to interleukin 6.

In this report, conditioned media from LPS-stimulated monocytes increased rat hepatocyte production of fibronectin (Fn) in a dose-dependent manner. Preincubation of the conditioned media with anti-IL-6, but not with anti-IL-1 alpha, anti-IL-1 beta, or anti-TNF, completely neutralized the Fn-stimulating activity. 10-100 pg/ml of rIL-6 was sufficient to increase Fn production. Neither IL-1 nor TNF had an effect on Fn production. The Fn-stimulating activity of IL-6 could be specifically neutralized only with anti-IL-6, but not with anti-IL-1 or anti-TNF. The increased Fn produced was shown to be of the plasma rather than the cellular form. These results demonstrate that IL-6 is the factor in monocyte-conditioned media that stimulates Fn production, and that IL-6 is the monokine tested with such activity.

Modulation of macrophage fibronectin secretion by LPS.

The secretion of fibronectin (Fn) by rat peritoneal macrophages was found to be down-regulated by LPS. A sensitive ELISA was used to quantitate both substrate-attached and supernatant Fn. Thioglycollate-elicited peritoneal exudate cells were observed to release a considerable amount of Fn during the adherence procedure for macrophage purification. After this procedure, macrophage Fn levels peaked within 2 hr and then declined steadily to baseline levels by 96 hr. Fn release by exudate cells during adherence purification was less affected by cycloheximide treatment than was subsequent Fn secretion by purified macrophages. Macrophages elicited with thioglycollate and P. acnes displayed enhanced Fn secretory activity when compared with resident unstimulated cells. Exposure to lipopolysaccharide (LPS) suppressed the levels of immunoreactive Fn in supernatants of elicited cells. This inhibition was shown to be dose-dependent and most significant after 24 hr of incubation. The inclusion of polymyxin B in the culture medium did not reverse the LPS-induced inhibition of Fn production but did prevent LPS stimulation of interleukin-1 secretion in the macrophage cultures. These observations demonstrate that Fn secretion by macrophages is regulated according to the functional state of the cell.