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PubMed2005

Autologous T-lymphocytes stimulate proliferation of orbital fibroblasts derived from patients with Graves' ophthalmopathy.

PURPOSE: Graves' ophthalmopathy (GO) affects 50% to 60% of patients with Graves' hyperthyroidism, resulting in exophthalmos, periorbital edema, pain, double vision, optic neuropathy, and loss of vision. Fibroblasts are a key autoimmune target in GO and have effector functions that contribute to GO-associated pathologic conditions, including proliferation, production of excess glycosaminoglycans, and fat deposition. GO is also characterized by autoimmune inflammation of orbital connective tissue with mononuclear cell infiltration, including T cells. METHODS: To determine whether autologous T cells can drive proliferation of orbital fibroblasts and thus contribute to GO, a novel reverse autologous mixed-cell reaction (rAMCR) was performed. Fibroblasts cultured from orbital tissue of patients with GO that was removed during orbital decompression surgery were mixed with autologous T cells, and fibroblast proliferation was determined. RESULTS: Autologous T cells stimulated proliferation of orbital fibroblasts. Fibroblasts derived from blepharoplasty fat of two different patients did not proliferate, demonstrating that the effect is specific to cells derived from deep orbital fat. Proliferation was dependent on cell contact and on major histocompatibility complex (MHC) class II and CD40-CD154 (CD40 ligand) signaling. CONCLUSIONS: The results suggest that T cells and orbital fibroblasts participate in an antigen-dependent positive feedback loop in which presentation of autoantigens by fibroblasts via MHC class II and CD40-CD40L signaling results in T-cell activation. These activated T cells stimulate fibroblast proliferation, leading to fibroblast-associated diseases in GO. Thus, therapies that interfere with CD40-CD40L signaling, antigen expression by fibroblasts, or T-cell function may be effective in preventing progression of GO symptoms.

Antigen Presentation↗

PubMed1982

Human alveolar macrophage growth factor for fibroblasts. Regulation and partial characterization.

The number of fibroblasts composing the alveolar structures in controlled within narrow limits by a strictly modulated rate of fibroblast replication. One possible source of growth-modulating signals for alveolar fibroblasts is the alveolar macrophage, a member of the mononuclear phagocyte family of cells, which collectively are known to be important sources of growth factors for a variety of target cells. To evaluate the role of alveolar macrophages in the control of alveolar fibroblast replication, macrophages from normal individuals obtained by bronchoalveolar lavage were maintained in suspension culture with and without added stimuli, and supernates were evaluated for fibroblast growth-promoting effect. Supernates from unstimulated macrophages contained no growth factor activity. In marked contrast, supernates from macrophages stimulated with particulates and immune complexes contained a growth factor that caused a significant increase in fibroblast replication rate. Maximum growth factor activity was observed 3-4 h after macrophage stimulation, at a concentration of 1-2 x 10(6) macrophages/ml. The alveolar macrophagederived growth factor eluted from DEAE-cellulose at 0.27 M NaCl at neutral pH had an apparent molecular weight of 18,000, and appeared to be distinct from other characterized growth factors. The alveolar macrophage-derived growth factor stimulated lung fibroblast DNA synthesis within 12 h, with cell division apparent within 48 h. In serum-free culture, the alveolar macrophage-derived growth factor by itself did not promote fibroblast replication, but rather acted as a progression factor causing a synergistic increase in fibroblast replication rate in the presence of competence factors such as fibroblast growth factor or platelet-derived growth factor. These studies suggest that when stimulated, human alveolar macrophages may modulate, in part, the replication rate of alveolar fibroblasts by releasing a growth factor within the alveolar microenvironment.

Cell Line↗

PubMed1996

Acute lung injury fibroblast migration and invasion of a fibrin matrix is mediated by CD44.

Fibrosis results when myofibroblasts invade the wound fibrin provisional matrix. Extracellular matrix receptors on the cell surface mediate cell adhesion, migration, and invasion. Recent work with transformed cells indicates that these cells use the cell surface matrix receptor CD44 for migration and invasion. In this study, we examine whether lung fibroblasts, isolated from patients dying with acute alveolar fibrosis, use CD44 to invade a fibrin matrix. Consistent with a role for CD44 in mediating fibroblast invasion and subsequent tissue fibrosis, immunohistochemical analysis of lung tissue from patients who died from acute alveolar fibrosis after lung injury reveals CD44-expressing mesenchymal cells throughout newly formed fibrotic tissue. PCR, Western, and immunoprecipitation analysis demonstrate that the 85-kD CD44 isoform is expressed by acute lung injury fibroblasts. Consistent with a role in mediating matrix adhesion and migration ultrastructurally, CD44 was found uniformly over the cell surface and was found densely labeling filopodia and lamellipodia, highly motile structures involved in cell migration. To determine if lung injury fibroblasts use CD44 to invade fibrin, a fibrin gel model of fibrosis was used. By blocking the function of CD44 with monoclonal antibodies, fibroblast invasion into a fibrin matrix was inhibited. To examine the mechanism by which CD44 mediates fibroblast invasion, the role of CD44 in fibroblast migration and adhesion was evaluated. Anti-CD44 antibody blocked fibroblast migration on the provisional matrix proteins fibronectin, fibrinogen, and hyaluronic acid. Additionally, fibroblast CD44 mediated adhesion to the provisional matrix proteins fibronectin, fibrin, and hyaluronic acid, but not to laminin, a component of the basement membrane. These findings support the hypothesis that fibroblast CD44 functions as an adhesion receptor for provisional matrix proteins and is capable of mediating fibroblast migration and invasion of the wound provisional matrix resulting in the formation of fibrotic tissue.

Cell Adhesion↗

PubMed1996

Osteopontin is produced by rat cardiac fibroblasts and mediates A(II)-induced DNA synthesis and collagen gel contraction.

Angiotensin II (AII) is a critical factor in cardiac remodeling which involves hypertrophy, fibroblast proliferation, and extracellular matrix production. However, little is known about the mechanism by which AII accelerates these responses. Osteopontin is an acidic phosphoprotein with RGD (arginine-glycine-aspartate) sequences that are involved in the vascular smooth muscle cell remodeling process. We identified the presence of osteopontin mRNA and protein in cultured rat cardiac fibroblasts and its prominent regulation by AII (10(-11) M). Osteopontin message levels were increased fourfold (P < 0.01) and protein fivefold (P < 0.05) at 24 h after addition of AII (10(-7) M). This response was inhibited by the AT1 receptor blocker, losartan. Osteopontin mRNA levels were increased in hypertrophied ventricles from animals with renovascular hypertension (1.6-fold, P < 0.05) and aortic banding (2.9-fold, P < 0.05). To examine the function of osteopontin, we determined its effects on (a) the ability of cardiac fibroblasts to contract three-dimensional collagen gels and (b) cardiac fibroblast growth. A monoclonal antibody against osteopontin partially blocked AII-induced three-dimensional collagen gel contraction by cardiac fibroblasts (64+/-4 vs. 86+/-5% in the presence of antibody, P < 0.05), while osteopontin itself promoted contraction of the gels by fibroblasts (71+/-5%, P < 0.05 compared with control). Either a monoclonal antibody against beta3 integrin which is a ligand for osteopontin or the RGD peptide blocked both AII and osteopontin-induced collagen gel contraction. Thus, the osteopontin RGD sequence binds to beta3 integrins on the fibroblast to promote fibroblast binding to collagen. All induced a threefold increase in DNA synthesis of cardiac fibroblasts, which was completely blocked by antibodies against osteopontin and beta3 integrin, or by RGD peptide, but not by controls. Thus, All-induced growth of cardiac fibroblasts also requires osteopontin engagement of the beta3 integrin. Taken together, these results provide the first evidence that osteopontin is a potentially important mediator of AII regulation of cardiac fibroblast behavior in the cardiac remodeling process.

Angiotensin II↗

PubMed1999

Endogenous IL-1alpha from systemic sclerosis fibroblasts induces IL-6 and PDGF-A.

It is reported that fibroblasts derived from clinically affected skin areas of patients with systemic sclerosis (SSc) have the ability to overproduce several cytokines and growth factors (i.e., IL-6, PDGF), an ability that might be involved in the pathogenesis of SSc. We have previously shown that the expression of IL-1alpha was constitutively observed in SSc fibroblasts, whereas this was not detected in normal fibroblasts. Although it was suggested that the aberrant IL-1alpha production could be associated with the fibrogenic phenotype of SSc fibroblasts, little is known about the roles of IL-1alpha in SSc fibroblasts. IL-1alpha induced IL-6 and PDGF-A, which are potent stimulators of collagen production and proliferation in normal fibroblasts. This article examines the proposal that IL-6 and PDGF-A are elevated through the action of endogenous IL-1alpha in SSc fibroblasts. An antisense oligodeoxynucleotide complementary to IL-1alpha mRNA was used to suppress endogenous IL-1alpha. Inhibition of endogenous IL-1alpha led to decreased levels of IL-6 and PDGF-A expression in SSc fibroblasts. Moreover, the blocking of the IL-6 response using anti-IL-6 antibody resulted in a significant reduction of procollagen type I in cultured SSc fibroblasts. These results suggest that endogenous IL-1alpha expressed by SSc fibroblasts may play a key role in the abnormal function of SSc fibroblasts through the expression of IL-6 and PDGF-A.

Base Sequence↗

PubMed2004

Gene expression profiling reveals novel TGFbeta targets in adult lung fibroblasts.

BACKGROUND: Transforming growth factor beta (TGFbeta), a multifunctional cytokine, plays a crucial role in the accumulation of extracellular matrix components in lung fibrosis, where lung fibroblasts are considered to play a major role. Even though the effects of TGFbeta on the gene expression of several proteins have been investigated in several lung fibroblast cell lines, the global pattern of response to this cytokine in adult lung fibroblasts is still unknown. METHODS: We used Affymetrix oligonucleotide microarrays U95v2, containing approximately 12,000 human genes, to study the transcriptional profile in response to a four hour treatment with TGFbeta in control lung fibroblasts and in fibroblasts from patients with idiopathic and scleroderma-associated pulmonary fibrosis. A combination of the Affymetrix change algorithm (Microarray Suite 5) and of analysis of variance models was used to identify TGFbeta-regulated genes. Additional criteria were an average up- or down- regulation of at least two fold. RESULTS: Exposure of fibroblasts to TGFbeta had a profound impact on gene expression, resulting in regulation of 129 transcripts. We focused on genes not previously found to be regulated by TGFbeta in lung fibroblasts or other cell types, including nuclear co-repressor 2, SMAD specific E3 ubiquitin protein ligase 2 (SMURF2), bone morphogenetic protein 4, and angiotensin II receptor type 1 (AGTR1), and confirmed the microarray results by real time-PCR. Western Blotting confirmed induction at the protein level of AGTR1, the most highly induced gene in both control and fibrotic lung fibroblasts among genes encoding for signal transduction molecules. Upregulation of AGTR1 occurred through the MKK1/MKK2 signalling pathway. Immunohistochemical staining showed AGTR1 expression by lung fibroblasts in fibroblastic foci within biopsies of idiopathic pulmonary fibrosis. CONCLUSIONS: This study identifies several novel TGFbeta targets in lung fibroblasts, and confirms with independent methods the induction of angiotensin II receptor type 1, underlining a potential role for angiotensin II receptor 1 antagonism in the treatment of lung fibrosis.

Cells, Cultured↗

PubMed2006

Lung fibroblasts from patients with emphysema show markers of senescence in vitro.

BACKGROUND: The loss of alveolar walls is a hallmark of emphysema. As fibroblasts play an important role in the maintenance of alveolar structure, a change in fibroblast phenotype could be involved in the pathogenesis of this disease. In a previous study we found a reduced in vitro proliferation rate and number of population doublings of parenchymal lung fibroblasts from patients with emphysema and we hypothesized that these findings could be related to a premature cellular aging of these cells. In this study, we therefore compared cellular senescence markers and expression of respective genes between lung fibroblasts from patients with emphysema and control patients without COPD. METHODS: Primary lung fibroblasts were obtained from 13 patients with moderate to severe lung emphysema (E) and 15 controls (C) undergoing surgery for lung tumor resection or volume reduction (n = 2). Fibroblasts (8E/9C) were stained for senescence-associated beta-galactosidase (SA-beta-Gal). In independent cultures, DNA from lung fibroblasts (7E/8C) was assessed for mean telomere length. Two exploratory 12 k cDNA microarrays were used to assess gene expression in pooled fibroblasts (3E/3C). Subsequently, expression of selected genes was evaluated by quantitative PCR (qPCR) in fibroblasts of individual patients (10E/9C) and protein concentration was analyzed in the cell culture supernatant. RESULTS: The median (quartiles) percentage of fibroblasts positive for SA-beta-Gal was 4.4 (3.2;4.7) % in controls and 16.0 (10.0;24.8) % in emphysema (p = 0.001), while telomere length was not different. Among the candidates for differentially expressed genes in the array (factor > or = 3), 15 were upregulated and 121 downregulated in emphysema. qPCR confirmed the upregulation of insulin-like growth factor-binding protein (IGFBP)-3 and IGFBP-rP1 (p = 0.029, p = 0.0002), while expression of IGFBP-5, -rP2 (CTGF), -rP4 (Cyr61), FOSL1, LOXL2, OAZ1 and CDK4 was not different between groups. In line with the gene expression we found increased cell culture supernatant concentrations of IGFBP-3 (p = 0.006) in emphysema. CONCLUSION: These data support the hypothesis that premature aging of lung fibroblasts occurs in emphysema, via a telomere-independent mechanism. The upregulation of the senescence-associated IGFBP-3 and -rP1 in emphysema suggests that inhibition of the action of insulin and insulin-like growth factors could be involved in the reduced in vitro-proliferation rate.

Biomarkers↗

PubMed2006

RhoA signaling modulates cyclin D1 expression in human lung fibroblasts; implications for idiopathic pulmonary fibrosis.

BACKGROUND: Idiopathic Pulmonary Fibrosis (IPF) is a debilitating disease characterized by exaggerated extracellular matrix deposition and aggressive lung structural remodeling. Disease pathogenesis is driven by fibroblastic foci formation, consequent on growth factor overexpression and myofibroblast proliferation. We have previously shown that both CTGF overexpression and myofibroblast formation in IPF cell lines are dependent on RhoA signaling. As RhoA-mediated regulation is also involved in cell cycle progression, we hypothesise that this pathway is key to lung fibroblast turnover through modulation of cyclin D1 kinetic expression. METHODS: Cyclin D1 expression was compared in primary IPF patient-derived fibroblasts and equivalent normal control cells. Quantitative real time PCR was employed to examine relative expression levels of cyclin D1 mRNA; protein expression was confirmed by western blotting. Effects of Rho signaling were investigated using transient transfection of constitutively active and dominant negative RhoA constructs as well as pharmacological inhibitors. Cellular proliferation of lung fibroblasts was determined by BrdU incorporation ELISA. To further explore RhoA regulation of cyclin D1 in lung fibroblasts and associated cell cycle progression, an established Rho inhibitor, Simvastatin, was incorporated in our studies. RESULTS: Cyclin D1 expression was upregulated in IPF compared to normal lung fibroblasts under exponential growth conditions (p < 0.05). Serum deprivation inhibited cyclin D1 expression, which was restored following treatment with fibrogenic growth factors (TGF-beta1 and CTGF). RhoA inhibition, using a dominant negative mutant and a pharmacological inhibitor (C3 exotoxin), suppressed levels of cyclin D1 mRNA and protein in IPF fibroblasts, with significant abrogation of cell turnover (p < 0.05). Furthermore, Simvastatin dose-dependently inhibited fibroblast cyclin D1 gene and protein expression, inducing G1 cell cycle arrest. Similar trends were observed in control experiments using normal lung fibroblasts, though exhibited responses were lower in magnitude. CONCLUSION: These findings report for the first time that cyclin D1 expression is deregulated in IPF through a RhoA dependent mechanism that influences lung fibroblast proliferation. This potentially unravels new molecular targets for future anti-IPF strategies; accordingly, Simvastatin inhibition of Rho-mediated cyclin D1 expression in IPF fibroblasts merits further exploitation.

ADP Ribose Transferases↗

PubMed1977

alpha-L-fucosidase in cultured skin fibroblasts from normal subjects and fucosidosis patients.

alpha-L-Fucosidase (EC 3.2.1.51) activity was studied in cultured skin fibroblasts obtained from 23 members of a family in which two cases of fucosidosis type 2 had occurred and in the fibroblasts of a patient with fucosidosis type 1. Both the 4-methylumbelliferyl glycoside and the p-nitrophenyl derivative were used as substrates. pH activity profiles showed two major peaks of activity. With the fluorogenic substrate pH optimum was at 4.5, whereas with the colorigenic substrate maximum activity was at pH 5.7. No activity was found in the fibroblasts of the three patients with the colorimetric assay. With the fluorometric assay the mean activity in the two patients with fucosidosis type 2 was 4.1 and 2.8 (range 1.3-9.9); activity in the patient with fucosidosis type 1 was 4.6 nmol 4-methylumbelliferone/mg protein/hr (range 1.0-8.6). The specific activity in the lysates of the patients' fibroblasts decreased as the amount of cellular protein used per assay increased. Fucosidosis fibroblasts cultured for 3 and 5 days in medium without fetal calf serum showed almost the same levels of apparent residual activity as fibroblasts cultured in medium containing fetal calf serum. Maximum activity in the deficient fibroblasts was at pH 4.5-4.75. Mixing experiments between lysates of both of fucosidosis and a normal fibroblast strain showed the expected enzymatic activities. The mean alpha-fucosidase activity in four heterozygotes for fucosidosis was 37.6 (range 24.1-48.7) and 30.3 (range 19.0-44.1) nmol final product split/mg protein/hr with the fluorogenic and the colorigenic substrate, respectively. In 12 normal fibroblast strains the mean activity +/- SD was 85.3 +/- 24.4 (range 50.8-129.3) and 67.6 +/- 21.1 (range 31.1-118.3). However, in four family members in which the alpha-L-fucosidase phenotype (by isoelectric focusing) was type 2-1, and who should therefore be carrying two normal alleles, the activity was within the heterozygote range. This indicates that occasional overlap between normal subjects and carriers may be present in cultured skin fibroblasts. Increased specific activity of alpha-L-fucosidase at pH 3.2-4.0 was observed after incubation of cell lysates with neuraminidase. The alpha-L-fucosidase activity did not show any increase after fusion between fucosidosis fibroblasts type 1 and 2.

Biopsy↗

PubMed1998

Activation of the signal transducers and activators of transcription signaling pathway by growth hormone (GH) in skin fibroblasts from normal and GH binding protein-positive Laron Syndrome children.

We have previously described two families (H and M) with GH binding protein-positive Laron Syndrome (LS), proposed to have one or more post GHR signaling defects. In the present study, we have examined whether the signal transducers and activators of transcription (STAT) pathway is activated by GH in skin fibroblast cultures established from these LS children, to determine the level(s) at which GH insensitivity has occurred. On immunoblots, both normal and LS fibroblasts express JAK2 and STATs 1, 3, and 5. GH induced rapid tyrosine phosphorylation of a protein at approximately 93 kDa in normal fibroblasts, and Western blotting with STAT-specific antibodies revealed STAT5 activation (phosphorylation) by GH. To determine further the identity and the DNA binding characteristics of the STAT proteins that were activated by GH, EMSAs were performed using three DNA elements known to bind STAT proteins; m67, the high affinity c-sis-inducible element (SIE), the interferon response element (IRE), and the lactogenic hormone-responsive region (LHRR). GH failed to induce protein binding to the SIE or IRE in normal skin fibroblasts but did induce the formation of a specific complex with the LHRR. Induction by GH of this LHRR/protein complex, which could be supershifted partially by anti-STAT1 antisera and completely by anti-STAT5 antisera, was transient, maximal between 10 and 30 min and reduced by 60 min. GH also induced distinct LHRR/protein complexes in mouse 3T3-F442A fibroblasts and in human IM-9 lymphocytes, but supershift analysis revealed that these complexes contained STAT5 but not STAT1. Whereas no binding to the LHRR was observed in GH-treated H fibroblasts, GH induced binding to this element in M fibroblasts. These results demonstrate that 1) the JAK-STAT pathway is activated by GH in normal fibroblasts and that STATs 1 and 5 have a role in GH-dependent signaling in these cells; 2) GH activation of DNA/STAT binding is cell type- and species-specific; and 3) GH failed to activate the STAT pathway in H fibroblasts but induced STAT signaling in M fibroblasts, indicating that the site of GH resistance in the latter is likely to be located within another GH signaling pathway. These fibroblast cultures therefore provide unique models with which to further our understanding of the mechanisms of human GH signaling.

Carrier Proteins↗

PubMed1987

Measurement of insulin-like growth factor I (IGF-I) responsiveness of fibroblasts of children with short stature: identification of a patient with IGF-I resistance.

Somatomedins are important mediators of GH action on skeletal tissues. A possible cause of growth failure, therefore, is impaired somatomedin (Sm) responsiveness of the target tissues. To investigate Sm responsiveness, we studied fibroblasts from 11 normal-statured individuals and 9 patients with short stature. Five of the short patients had normal serum GH and normal or increased Sm levels and, therefore, are considered possibly to be Sm resistant. The other short patients include 2 with Laron-type dwarfism and 2 with GH resistance of undefined type. As a measure of Sm responsiveness, we determined the ability of insulin-like growth factor I (IGF-I; Sm-C) to stimulate uptake of [3H]alpha-aminoisobutyric acid by fibroblasts. The mean ED50 of the 11 normal fibroblast cell lines was 3.2 +/- 0.9 (+/- SD) ng/ml. Fibroblasts from 8 of the 9 short-statured patients had ED50 values within the normal range. This included 2 fibroblast lines isolated from children with Laron-type dwarfism. Fibroblasts from 1 patient, however, were significantly less sensitive to IGF-I in 9 separate assays, with an ED50 of 10.7 +/- 1.8 ng/ml. Fibroblasts from the mother of the patient had an ED50 of 5.0 +/- 1.4 ng/ml, and fibroblasts from the father had an ED50 of 4.3 +/- 0.7 ng/ml, both above the normal mean. Measurements of [125I]IGF-I binding by suspended fibroblasts from this patient and her parents failed to demonstrate significant abnormalities in either the number of binding sites or the affinity of binding. We conclude that the ability of IGF-I to stimulate [3H]alpha-aminoisobutyric acid uptake by human fibroblasts provides a useful method of identifying short children with Sm resistance. Of five patients with clinical evidence of possible Sm resistance, fibroblasts from one consistently were hyporesponsive to IGF-I. Cells from two patients with Laron-type dwarfism were normally responsive to IGF-I.

Aminoisobutyric Acids↗

PubMed1991

Detection, cellular localization, and modulation of heat shock proteins in cultured fibroblasts from patients with extrathyroidal manifestations of Graves' disease.

We hypothesize that fibroblasts obtained from the retroocular space and the pretibial skin, sites affected by the peripheral manifestations of Graves' disease, share unique characteristics that may in part explain the site specificity of Graves' ophthalmopathy (GO) and pretibial myxedema (PTM). Heat shock proteins (HSPs), synthesized by cells undergoing stress, function to maintain cellular homeostasis and are probably involved in the intracellular processing and cell surface presentation of antigens. We investigated possible differences in the expression of 70-kDa HSPs between cultured fibroblasts obtained from patients with severe GO and normal individuals. In addition, we compared HSP expression in fibroblasts derived from tissues involved in the extrathyroidal manifestation of Graves' disease (GO and PTM) with that in fibroblasts from uninvolved tissues. HSPs were detected by both immunoblotting and indirect immunofluorescence, using monoclonal antibodies that are directed against HSP72, HSP72/73 (termed HSP70), and HSP90. HSP expression at baseline and after treatment with various cytokines and heat stress was examined. At baseline, HSP72 reactivity was exclusively detected in retroocular and pretibial fibroblasts from patients with severe GO and PTM, but was not observed in abdominal fibroblasts from these patients and was not detectable in fibroblasts from any anatomical site of normal individuals. The abundance of HSP70 expression at baseline and after treatment with certain cytokines was significantly greater in retroocular and pretibial fibroblasts from patients with GO than in normal individuals. In addition, characteristic changes in the cellular localization of HSPs before and after exposure to heat stress and cytokines were observed; cell surface expression of HSP70 was detected at baseline in fibroblasts from patients, but not in normal fibroblasts. These data provide the first evidence that HSPs are differentially expressed by fibroblasts derived from tissues affected by the extrathyroidal manifestations of GD. These proteins may have a role in localized immune processes, leading to the development of GO and PTM.

Biopsy↗

PubMed1995

Evidence for cellular heterogeneity in primary cultures of human orbital fibroblasts.

Orbital fibroblasts in culture display phenotypic attributes that distinguish them from fibroblasts derived from other anatomical regions. The current studies were conducted to define potential cellular heterogeneity among orbital fibroblasts with regard to 1) differential expression of Thy-1, a 25-kilodalton glycoprotein associated with cell signaling; 2) cells undergoing a change in shape in response to prostaglandin E2 (PGE2); and 3) differences in morphology and Thy-1 expression between single cell-derived clonal fibroblast strains. On the basis of flow cytometric analysis using an anti-Thy-1 monoclonal antibody, 65% of intact orbital fibroblasts expressed surface Thy-1 (n = 5; range, 54-71%). In contrast, greater than 95% of the fibroblasts present in the five dermal strains tested were Thy-1 positive. A total of six strains of orbital fibroblasts were assessed for their shape change response to a 4-h treatment with PGE2 (100 nmol/L). A mean of 37% of the fibroblasts present in each culture responded to PGE2 (range, 22-50%). In contrast, only 1% of dermal fibroblasts exhibited any change in morphology. Three separate clones were generated from a single parent strain of Graves' orbital fibroblasts. These clones consisted of homogeneous appearing cells; however, substantial clone to clone differences in morphology were stably expressed for several population doublings. Thy-1 was expressed uniformly in cells of two clones, whereas the third was Thy-1 negative. Factor VIII and smooth muscle-specific alpha-actin were undetectable in any of the orbital or dermal cultures examined. Thus, Thy-1 expression is uniform in fibroblasts from certain anatomical regions such as the skin and heterogeneous in cells derived from human lung and orbit. These findings suggest that human orbital connective tissue may have a complexity not previously appreciated.

Antigens↗

PubMed1994

Dermal fibroblasts activate keratinocyte outgrowth on collagen gels.

The effects of dermal fibroblasts on keratinocyte outgrowth on collagen substrata was studied using an in vitro keratinocyte-collagen gel composite model. Skin fibroblasts were seeded inside collagen gels, which remained attached to the cell culture plastic substratum. Fibroblasts incorporated in collagen gels were either kept viable throughout the study, or were lysed hypotonically with water at different time intervals (2 hours and 5 days). Results show that very little keratinocyte outgrowth occurred on either plain collagen gels or gels that had previously contained viable fibroblasts for 2 hours. A 3- to 4-fold increase in keratinocyte outgrowth occurred on collagen gels that had previously contained viable fibroblasts for 5 days. A striking increase (20-fold) in keratinocyte outgrowth was observed on collagen gels that contain viable fibroblasts. The effect of fibroblast diffusible factors on keratinocyte outgrowth was further studied with a co-culture system using Millicell inserts. It was found that the co-culture of fibroblasts with the composite enhanced keratinocyte outgrowth on collagen gels that had previously contained viable fibroblasts for 5 days. Among all, however, the keratinocyte outgrowth was far better on gels containing viable fibroblasts. Addition of keratinocyte growth factor or its neutralizing antibody did not affect keratinocyte outgrowth. These results suggest that dermal fibroblasts can activate keratinocyte outgrowth on collagen matrices through some diffusible factors other than keratinocyte growth factor, and epithelial-mesenchymal interactions exert some special effects on keratinocyte outgrowth on collagen gels.

Cell Communication↗

PubMed1997

Assembly of basement membrane in vitro by cooperation between alveolar epithelial cells and pulmonary fibroblasts.

To investigate basement membrane formation by cooperation between pneumocytes and pulmonary fibroblasts, we cultured type II alveolar epithelial cells obtained from rats transfected with SV40-large T antigen gene (SV40-T2 cells) on type I collagen matrices. On fibroblasts-embedded gel (T2-Fgel), SV40-T2 cells ultrastructurally formed a continuous and thin layer of lamina densa, while on collagen gel without fibroblasts (T2-gel) SV40-T2 cells produced only discontinuous and diffuse deposits. Stripping SV40-T2 cells off the tissues by H2O2 treatment revealed a continuous and plane surface of lamina densa assembled on the T2-Fgel tissue, whereas only amorphous deposits appeared on the T2-gel tissue. Immunolocalization of major basement membrane components showed that type IV collagen, laminin, perlecan and entactin (nidogen) were continuously integrated on the lamina densa in T2-Fgel. In T2-gel, all these components were discontinuously distributed beneath SV40-T2 cells. The contribution of pulmonary fibroblasts to the assembly of basement membrane through reorganization of collagen matrix and/or soluble factors was examined by the cultured of SV40-T2 cells on the freeze-thawed fibroblast-tissue and/or with the fibroblast-conditioned medium. Both SV40-T2 cells on the freeze-thawed fibroblast-tissue and SV40-T2 cells in T2-gel in the fibroblast-conditioned medium failed to produce a lamina densa. SV40-T2 cells could assemble a lamina densa only on the freeze-thawed fibroblast-tissue in the fibroblast-conditioned medium. These results show that the basement membrane components are assembled to a lamina densa by combination of the reorganization of collagen matrix and the supply of soluble factors by pulmonary fibroblasts.

Animals↗

PubMed1994

Effects of dibutyryl cyclic AMP on hyaluronan and proteoglycan synthesis by retroocular tissue fibroblasts in culture.

Deposition of glycosaminoglycan is one of the histological features of Graves' ophthalmopathy. Although retroocular tissue fibroblasts are considered to be responsible for glycosaminoglycan accumulation, it is not known what is stimulating the fibroblasts. There are studies which are in support of and against the role of anti-TSH receptor antibodies in the pathogenesis of Graves' ophthalmopathy. TSH-receptor antibodies increase cAMP as a second messenger in thyroid cells. We studied the effects of dibutyryl cyclic AMP (Bt2 cAMP) on glycosaminoglycan synthesis by retroocular tissue fibroblasts in order to known whether cAMP can modulate glycosaminoglycan synthesis. Retroocular tissue fibroblasts mainly synthesize hyaluronan, the large chondroitin sulfate proteoglycan and the small chondroitin sulfate proteoglycan as glycosaminoglycan in cell culture. The amount of hyaluronan synthesis was measured as [3H]glucosamine incorporation into macromolecule susceptible to hyaluronidase digestion (from Streptomyces hyaluronlyticus). The amount of proteoglycan synthesis was measured as [35S]sulfate incorporation into macromolecules in medium and cell layer fraction. Proteoglycans in medium were further separated into the large proteoglycan and the small proteoglycan on a Superose 6 column. Bt2 cAMP increased both hyaluronan and proteoglycan synthesis by retroocular tissue fibroblasts, especially stimulating the secretion of the large proteoglycan synthesis by retroocular tissue fibroblasts, especially stimulating the secretion of the large proteoglycan. Effects of Bt2 cAMP on glycosaminoglycan synthesis were then compared with those in adult skin fibroblasts. Although the magnitude of response between the two was indistinct, the stimulation of the large proteoglycan synthesis by Bt2 cAMP was more prominent in retroocular tissue fibroblasts. The results suggest that the regulation of glycosaminoglycan synthesis by retroocular tissue fibroblasts is different from that by adult skin fibroblasts. Although further studies are required to determine its actual role, cAMP stimulates glycosaminoglycan synthesis by retroocular tissue fibroblasts and underlies the mechanism in Graves' ophthalmopathy.

Adult↗

PubMed2005

Evidence for protein and mRNA TSHr expression in fibroblasts from patients with thyroid-associated ophthalmopathy (TAO) after adipocytic differentiation.

OBJECTIVE: Thyroid-associated ophthalmopathy (TAO) is a chronic autoimmune disorder characterized by an increased volume of adipose/connective tissue in the human orbit. DESIGN: The aim of this study was to investigate the thyrotropin receptor (TSHr) expression in orbital fibroblasts from TAO patients undergoing adipocytic differentiation. METHODS: Retro-ocular tissue and skin were obtained from five patients undergoing orbital decompression surgery for TAO and placed in culture. Proliferating fibroblasts were subjected to adipocytic differentiation for 10 days. Total RNA was isolated from fibroblasts and was reverse transcribed. TSHr mRNA levels were determined by real-time PCR. cAMP was determined by radioimmunoassay (RIA) after fibroblast incubation with the substances to test. RESULTS: Orbital differentiated fibroblasts became rounded and acquired lipid droplets. The amount of TSHr mRNA in these fibroblasts was higher than fibroblasts not subjected to adipocytic differentiation. Immunocytochemical analysis showed TSHr protein in differentiated orbital fibroblasts. Differentiated orbital fibroblasts stimulated with bovine (b) TSH showed a cAMP production greater than that in paired undifferentiated cultures. A specific thyroid-inhibiting antibody (TBAb) inhibited cAMP production after bTSH challenge, and a thyroid-stimulating antibody (TSAb) stimulated cAMP production in differentiated fibroblasts. CONCLUSIONS: We suggest that orbital fibroblasts subjected to adipocytic differentiation increase TSHr expression that responds specifically to bTSH and TSAb stimulation, and to TBAb inhibition.

Adipocytes↗

PubMed2000

Lack of correlation between DNA strand breakage and p53 protein levels in human fibroblast strains exposed to ultraviolet lights.

The contribution of DNA strand breaks accumulating in the course of nucleotide excision repair to upregulation of the p53 tumor suppressor protein was investigated in human dermal fibroblast strains after treatment with 254 nm ultraviolet (UV) light. For this purpose, fibroblast cultures were exposed to UV and incubated for 3 h in the presence or absence of l-beta-D-arabinofuranosylcytosine (araC) and/or hydroxyurea (HU), and then assayed for DNA strand breakage and p53 protein levels. As expected from previous studies, incubation of normal and ataxia telangiectasia (AT) fibroblasts with araC and HU after UV irradiation resulted in an accumulation of DNA strand breaks. Such araC/HU-accumulated strand breaks (reflecting nonligated repair-incision events) following UV irradiation were not detected in xeroderma pigmentosum (XP) fibroblast strains belonging to complementation groups A and G. Western blot analysis revealed that normal fibroblasts exhibited little upregulation of p53 (approximately 1.2-fold) when incubated without araC after 5 J/m2 irradiation, but showed significant (three-fold) upregulation of p53 when incubated with araC after irradiation. AraC is known to inhibit nucleotide excision repair at both the damage removal and repair resynthesis steps. Therefore, the potentiation of UV-induced upregulation of p53 evoked by araC in normal cells may be a consequence of either persistent bulky DNA lesions or persistent incision-associated DNA strand breaks. To distinguish between these two possibilities, we determined p53 induction in AT fibroblasts (which do not upregulate p53 in response to DNA strand breakage) and in XP fibroblasts (which do not exhibit incision-associated breaks after UV irradiation). The p53 response after treatment with 5 J/m2 UV and incubation with araC was similar in AT, XPA, XPG and normal fibroblasts. In addition, exposure of XPA and XPG fibroblasts to UV (5, 10 or 20 J/m2) followed by incubation without araC resulted in a strong upregulation of p53. We further demonstrated that HU, an inhibitor of replicative DNA synthesis (but not of nucleotide excision repair), had no significant impact on p53 protein levels in UV irradiated and unirradiated human fibroblasts. We conclude that upregulation of p53 at early times after exposure of diploid human fibroblasts to UV light is triggered by persistent bulky DNA lesions, and that incision-associated DNA strand breaks accumulating in the course of nucleotide excision repair and breaks arising as a result of inhibition of DNA replication contribute little (if anything) to upregulation of p53.

Ataxia Telangiectasia↗