Biosynthesis of fibronectin by human embryonal fibroblasts transformed by SV-40 virus

Fibronectin biosynthesis by human embryonic fibroblasts transformed with virus SV-40 was studied in intact cells and in a cell-free protein synthesizing system on free and membrane-bound polyribosomes isolated from these cells. It was found that fibronectin release from transformed fibroblasts into the culturing medium was decreased 4.5-fold, while its per cent content--2-fold. The amount of fibronectin precipitated by antibodies in the course of an immunoprecipitation reaction in transformed cells appeared to be somewhat higher than in normal cells, although when expressed on a per cent basis this content was decreased only 1.5-fold. However, the content of fibronectin monomer with Mr = 220 kD exceeded that in normal fibroblast cell material 1.6 times. Study on fibronectin biosynthesis in a cell-free system revealed that in transformed cells 45% of fibronectin is synthesized on free polyribosomes as compared to 13% in normal fibroblasts. It is assumed that the decreased fibronectin biosynthesis in human fibroblasts transformed with virus SV-40 results in spatial uncoupling of polyribosomes and membrane structures responsible for protein transport from the cell, as a result of which a significant part of fibronectin synthesized by transformed fibroblasts undergoes intracellular degradation.     

Fibronectin expression is determined by the genotype of the transformed parental cells in heterokaryons between normal and transformed fibroblasts

The expression of fibronectin, a cell surface-associated transformation-sensitive glycoprotein, was studied in hetero- and homokaryons of normal and SV40-transformed human fibroblasts. In immunofluorescence, fibroblast homokaryons had an intense surface-associated and intracelluar fibronectin fluorescence similar to that of normal fibroblasts. Transformed cells and their homokaryons had a minimal surface-associated and a weak intracellular fibronectin fluorescence. In heterokaryons formed between transformed and normal fibroblasts, the expression of fibronectin fell within 24 h to the level of the transformed cell homokaryons. The change was detectable already at 3 h after fusion and was gene-dose dependent. These results show that the transformed genotype determines fibronectin expression in the heterokaryons.     

Fibronectin and cell shape in vivo: studies on the endometrium during pregnancy

The rat endometrium during pregnancy was used as a model system to study fibronectin in vivo. Fibronectin distribution on stromal fibroblasts, as determined by indirect immunofluorescence staining, was studied in relationship to cell shape during decidual transformation. Fibroblasts of the estrus endometrial stroma were elongated cells with a fibrillar pattern of fibronectin on their surfaces. During days 1-6 of pregnancy, as these elongated cells acquired a round morphology, fibronectin changed first to a patched distribution on the cells'a surfaces and then disappeared. The change in fibronectin was specific for the fibroblasts since over the same time period there was no decrease in fibronectin found associated with blood vessels or in the epithelial-stromal basement membrane. These results support the proposed relationship between cell surface fibronectin and cell shape that has been inferred from in vitro experiments. After implantation, fibronectin distribution was studied in relationship to the position of the conceptus. In the stroma proximal to the implanting conceptus, fibronectin was absent except around blood vessels, which may help explain how decidual tissue could act as a barrier to trophoblast invasion. Finally, fibronectin distribution was studied in the uterus after parturition. Debris in the uterine lumen was coated with fibronectin, which may be important in the rapid removal of this material by phagocytic cells. Also, fibronectin associated with the epithelial-stromal basement membrane was reorganized after reepithelialization had occurred.     

Loss of pericellular fibronectin matrix from heterokaryons of normal human fibroblasts and epithelial cells

The expression of fibronectin in heterokaryons of normal human fibroblasts and normal or malignant epithelial cells was studied by indirect immunofluorescence microscopy. Fibroblasts and their homokaryons showed a characteristic pericellular fibronectin matrix, whereas both normal (MDCK) and malignant (HeLa) epithelial cells, and their homokaryons, lacked such a matrix. The fibroblast homokaryons also showed a typical strong, perinuclear cytoplasmic, fibronectin-specific fluorescence. This was much weaker or absent in the MDCK and HeLa cells and their homokaryons. When human fibroblasts were fused with either normal or malignant epithelial cells, no pericellular matrix-like, fibronectin-specific fluorescence could be seen in the heterokaryons. Interestingly, however, a distinct intracellular fluorescence was seen in the heterokaryons, indicating continued production of fibronectin. The results of the present study indicate that both malignant and normal epithelial cells, which do not deposit fibronectin matrix, can cause its loss in heterokaryons with fibroblasts. Thus, discontinued fibronectin matrix formation does not point exclusively to malignancy, but may also reflect the state of differentiation of the parental cells.     

Initial adhesion of human fibroblasts in serum-free medium: possible role of secreted fibronectin.

Experiments were carried out to test the hypothesis that the initial attachment and spreading of human fibroblasts in serum-free medium occurs to cell fibronectin which has been secretd spread on tissue culture substrata in serum-free medium in 60 min. When potential protein adsorption sites on the substratum were covered with bovine serum albumin before initial human fibroblasts attachment, their subsequent attachment to the substratum was prevented. When substratum adsorption sites were covered immediately after initial attachment, subsequent cell spreading was prevented. The distribution of fibronectin on human fibroblast surfaces during initial attachment and spreading was studied by indirect immunofluorescence analysis using a monospecific anti-cold-insoluble globulin antiserum. The initial appearance (10 min) of fibronectin was in spots over the entire cell surface. Concomitant with human fibroblast spreading, the random distribution of sites disappeared, and most fibronectin was subsequently observed in spots at the cell substratum interface (60 min). A fibrillar pattern of fibronectin appeared later (2-8 hr). The sites beneath the cells could be visualized as footprints on the substratum following treatment of the attached human fibroblasts with 0.1 M NaOH. A second fluorescence pattern of fibronectin secreted on the substratum was characterized by a diffuse halo around the cells and a very faint, diffuse staining elsewhere on the substratum. Another cell type (baby hamster kideny cells) was used to assay biologically for the presence or absence of the factor secreted by human fibroblasts on the substratum. Human fibroblasts were found to secrete an adhesion factor for baby hamster kidney cells into the substratum in a time- and temperature-dependent fashion, and immunological studies indicated that the factor secreted by human fibroblasts was cross-reactive with cold-in-soluble globulin, the plasma form of fibronectin. The conditioning factor secreted by the human fibroblasts was also found to be an attachment and spreading factor for human fibroblasts in experiments measuring human fibroblast adhesion to fibronectin footprints of human fibroblasts. Substratum-adsorbed cold-insoluble globulin was also found to be an attachment and spreading factor for human fibroblasts. Based upon the timing of appearance of conditioning factors on the substratum and the immunofluorescence patterns, it seems that the diffusely organized fibronectin on the substratum constitutes the sites to which cell attachment occurs. The bright spots of fibronectin that appear beneath the cells may represent fibronectin reorganization during cell spreading.     

Membrane-type 1 matrix metalloproteinase regulates fibronectin assembly and N-cadherin adhesion

Fibronectin matrix formation requires the increased cytoskeletal tension generated by cadherin adhesions, and is suppressed by membrane-type 1 matrix metalloproteinase (MT1-MMP). In a co-culture of Rat1 fibroblasts and MT1-MMP-silenced HT1080 cells, fibronectin fibrils extended from Rat1 to cell–matrix adhesions in HT1080 cells, and N-cadherin adhesions were formed between Rat1 and HT1080 cells. In control HT1080 cells contacting with Rat1 fibroblasts, cell–matrix adhesions were formed in the side away from Rat1 fibroblasts, and fibronectin assembly and N-cadherin adhesions were not formed. The role of N-cadherin adhesions in fibronectin matrix formation was studied using MT1-MMP-silenced HT1080 cells. MT1-MMP knockdown promoted fibronectin matrix assembly and N-cadherin adhesions in HT1080 cells, which was abrogated by double knockdown with either integrin β₁ or fibronectin. Conversely, inhibition of N-cadherin adhesions by its knockdown or treatment with its neutralizing antibody suppressed fibronectin matrix formation in MT1-MMP-silenced cells. These results demonstrate that fibronectin assembly initiated by MT1-MMP knockdown results in increase of N-cadherin adhesions, which are prerequisite for further fibronectin matrix formation.     

The characteristics of actin cytoskeleton structure and its rearrangements by extracellular matrix proteins in normal, immortalized and transformed rat fibroblasts

Comparative analysis of actin cytoskeleton structure in rat embryonic fibroblasts, E1A-immortalized and E1A + cHa-ras-transformed cells has been carried out. A decrease in adhesiveness and the rate of changes in actin cytoskeleton structures was shown to correlate with the level of morphological transformation of cells. E1A + cHa-ras-transformants show the lowest adhesiveness and complete disorganization of actin structures. Cultivation on serum-free media promoted disassembling of actin cytoskeleton structures in a small part of normal fibroblast population, only in a few immortalized cells, but exerted no influence on transformed cells. The influence of immobilized extracellular matrix proteins fibronectin, laminin and collagens type I and III on actin cytoskeleton structure in normal, immortalized and transformed fibroblasts was studied. Transformed cells spread on fibronectin completely restored highly organized actin structures, displayed a lot of stress fibers and focal contacts. The use of laminin revealed differences in locomotion between normal and transformed cells. Normal, immortalized and transformed fibroblasts spread on fibronectin and laminin demonstrate some peculiarities in actin cytoskeleton structures as a result of specificity of ligand-receptor interaction. Cells spread on fibronectin have polygonal shapes, many stress fibers and focal contacts, whereas cells spread on laminin are highly polarized and develop broad lamellae filled with actin microfilament meshwork. Collagens type I and III can affect adhesive properties and actin cytoskeleton structure in all cell lines studied only slightly, in comparison with fibronectin and laminin.     

Fibronectin is overproduced by keloid fibroblasts during abnormal wound healing.

Wound healing in certain individuals leads to the development of keloid tumors which exhibit abnormal collagen metabolism and an increased abundance of extracellular matrix components. Comparison of fibronectin levels in fibroblasts derived from keloids and normal dermis revealed a relative increase in intracellular and extracellular fibronectin in the keloid-derived cells. While fibronectin was similarly processed, compartmentalized, and degraded by both cell types, fibronectin biosynthesis was found to be accelerated as much as fourfold in keloid fibroblasts due to a corresponding increase in the amount of accumulated fibronectin mRNA. These changes account for the elevated steady-state level of the molecule in keloid fibroblasts and suggest that increased fibronectin in keloid lesions is due to overproduction by the wound-healing fibroblasts. Glucocorticoid treatment stimulated fibronectin biosynthesis in both normal and keloid fibroblasts. However, the amount of stimulation was less for the keloid-derived cells, indicating a limitation on maximal rates of fibronectin biosynthesis. These observations suggest that separate mechanisms act to control basal and maximal rates of fibronectin production. Biosynthesis of the 140-kilodalton fibronectin receptor was also found to be increased in keloid fibroblasts, suggesting some level of coordinate regulation for fibronectin and fibronectin receptor expression.     

The adhesion modulating properties of tenascin-W

Tenascins are extracellular matrix glycoproteins associated with cell motility, proliferation and differentiation. Tenascin-C inhibits cell spreading by binding to fibronectin; tenascin-R and tenascin-X also have anti-adhesive properties in vitro. Here we have studied the adhesion modulating properties of the most recently characterized tenascin, tenascin-W. C2C12 cells, a murine myoblast cell line, will form broad lamellipodia with stress fibers and focal adhesion complexes after culture on fibronectin. In contrast, C2C12 cells cultured on tenascin-W fail to spread and form stress fibers or focal adhesion complexes, and instead acquire a multipolar shape with short, actin-tipped pseudopodia. The same stellate morphology is observed when C2C12 cells are cultured on a mixture of fibronectin and tenascin-W, or on fibronectin in the presence of soluble tenascin-W. Tenascin-W combined with fibronectin also inhibits the spreading of mouse embryo fibroblasts when compared with cells cultured on fibronectin alone. The similarity between the adhesion modulating effects of tenascin-W and tenascin-C in vitro led us to study the possibility of tenascin-W compensating for tenascin-C in tenascin-C knockout mice, especially during epidermal wound healing. Dermal fibroblasts harvested from a tenascin-C knockout mouse express tenascin-W, but dermal fibroblasts taken from a wild type mouse do not. However, there is no upregulation of tenascin-W in the dermis of tenascin-C knockout mice, or in the granulation tissue of skin wounds in tenascin-C knockout animals. Similarly, tenascin-X is not upregulated in early wound granulation tissue in the tenascin-C knockout mice. Thus, tenascin-W is able to inhibit cell spreading in vitro and it is upregulated in dermal fibroblasts taken from the tenascin-C knockout mouse, but neither it nor tenascin-X are likely to compensate for missing tenascin-C during wound healing.     

Fibronectin–collagen binding and requirement during cellular adhesion

Fibronectin isolated from human plasma and from the extracellular matrices of cell monolayers mediates the attachment in vitro and spreading of trypsin-treated cells on a collagen substratum. Fibronectin-dependent kinetics of cellular attachment to collagen were studied for several adherent cell types. It was shown that trypsin-treated human umbilical-cord cells, mouse sarcoma CMT81 cells, endothelial cells, and human fibroblasts from a patient with Glanzmann's disease were completely dependent on fibronectin for their attachment to collagen, whereas guinea-pig and monkey smooth-muscle cells and chick-embryo secondary fibroblasts displayed varying degrees of dependence on fibronectin for their attachment. Radiolabelled human plasma fibronectin possessed similar affinity for collagen types I, II and III from a variety of sources. The fibronectin bound equally well to the collagens with or without prior urea treatment. However, in the fibronectin-mediated adhesion assay using PyBHK fibroblasts, a greater number of cells adhered and more spreading was observed on urea-treated collagen. Fibronectin extracted from the extracellular matrix of chick-embryo fibroblasts and that purified from human plasma demonstrated very similar kinetics of complexing to collagencoated tissue-culture dishes. Fibronectin from both sources bound to collagen in the presence of 0.05–4.0m-NaCl and over the pH range 2.6–10.6. The binding was inhibited when fibronectin was incubated with 40–80% ethylene glycol, the ionic detergents sodium dodecyl sulphate and deoxycholate, and the non-ionic detergents Nonidet P-40, Tween 80 and Triton X-100, all at a concentration of 0.1%. From these results we proposed that fibronectin–collagen complexing is mainly attributable to hydrophobic interactions.     

Products of cultured neuroglial cells: II. The production of fibronectin by C6 glioma cells

The possibility of fibronectin production by C6 glioma cells was examined with assays which require protein synthesis. Proteins produced by C6 cells using radiolabeled amino acid precursors were tested for affinity to collagen by binding to immobilized gelatin. The predominant collagen binding protein made by C6 coelectrophoresed with fibronectin synthesized by control fibroblasts and with the larger of the two proteins in unlabeled fibronectin when applied to polyacrylamide gels with sodium dodecyl sulfate (SDS). In addition, C6 produced a larger collagen binding protein of approximately 270,000 molecular weight. Solubilities in urea solutions of the collagen-binding proteins made by C6 cells and fibroblasts were similar. Immunofluorescence showed fibronectin associated with the C6 cell monolayer, but less abundant than the fibronectin associated with fibroblasts. Results provide evidence for the production of fibronectin by the C6 glioma cell line.     

Adhesion, orientation, and movement of cells cultured on ultrathin fibronectin fibers

Summary This study examined the behavior of rat tendon fibroblasts, baby hamster kidney fibroblasts, macrophage-like P388D1 cells, and neurons from rat dorsal root ganglia, cultured on fibronectin strands 0.2–5 μm in diameter. We investigated cell spreading, orientation, formation of focal contacts, the speed of cell movement, and the speed of neurite outgrowth in cells cultured on fibronectin strands, glass covered with fibronectin, and plain, nontreated glass. Fibronectin strands significantly promoted cell spreading and caused a marked alignment of all kinds of cells to the direction of the fiber. The fibers caused the alignment of actin filaments in fibroblasts and focal contacts in fibroblasts and macrophages and increased polymerization of F-actin in cells. Fibronectin fibers also increased the speed and persistence of cell movement and the rate of neurite outgrowth. Macrophages grown on fibronectin fibers produced numerous actin-rich microspikes and adopted a polarized, migratory phenotype. These findings indicate that fibronectin strands, resembling natural components of the extracellular matrix, are more effective in activating various types of cells than two-dimensional, fibronectin-covered substrata. The results also confirm the suitability of the three-dimensionally oriented fibronectin form for use in clinical practice.     

Cellular and metabolic specificity in the interaction of adhesion proteins with collagen and with cells

Fibronectin mediates the adhesion of fibroblasts to collagen substrates, binding first to the collagen and then to the cells. We report here that the interaction of the cells with the fibronectin-collagen complex is blocked by specific gangliosides, GD_{1 a} and GT₁, and that the sugar moieties of these gangliosides contain the inhibitory activity. The gangliosides act by binding to fibronectin, suggesting that they may be the cell surface receptor for fibronectin. Evidence is presented that other adhesion proteins or mechanisms of attachment exist for chondrocytes, epidermal cells, and transformed tumorigenic cells, since adhesion of these cells is not stimulated by fibronectin. Chondrocytes adhere via a serum factor that is more temperature-sensitive and less basic than fibronectin. Unlike that of fibroblasts chondrocyte adhesion is stimulated by low levels of gangliosides. Epidermal cells adhere preferentially to type IV (basement membrane) collagen but at a much slower rate than fibroblasts or chondrocytes. This suggests that these epidermal cells synthesize their own specific adhesion factor. Metastatic cells cultured from the T241 fibrosarcoma adhere rapidly to type IV collagen in the absence of fibronectin and do not synthesize significant amounts of collagen or fibronectin. Their growth, in contrast to that of normal fibroblasts, is unaffected by a specific inhibitor of collagen synthesis. These data indicate the importance of specific collagens and adhesion proteins in the adhesion of certain cells and suggest that a reduction in the synthesis of collagen and of fibronectin is related to some of the abnormalities observed in transformed cells.     

HIV-infected lymphocytes regulate fibronectin synthesis by TGF beta 1 secretion

Alterations in lymph node architecture occur with HIV infection and contribute to immunological derangements. We previously showed that matrix fibronectin stabilized HIV and increased HIV infection of PBL. We showed increased fibronectin deposition in lymph nodes of HIV-infected patients. However, we did not detect a difference in fibronectin synthesis between uninfected and infected PBL. Therefore, we hypothesized that interactions of HIV-infected cells with fibroblasts resulted in increased fibronectin deposition. We detected increased fibronectin deposition by immunofluorescence on fibroblasts cocultured with HIV-infected PBL. We also found a 6-fold increase in fibronectin mRNA levels in fibroblasts cocultured with HIV-infected PBL by real-time PCR. Furthermore, when HIV-infected PBL were added to reporter fibroblasts stably transfected with a fibronectin promoter, we found a 1.5- to 2-fold increase in promoter activity. Since conditioned medium from HIV-infected PBL also increased fibronectin promoter activity, we hypothesized that a soluble factor such as TGFbeta was responsible for increased fibronectin secretion. Pretreatment of supernatant from HIV-infected PBL with a neutralizing Ab to TGFbeta1 abrogated the increased fibronectin promoter activity. We confirmed that HIV-infected PBL produced increased TGFbeta1 by ELISA. Using Mv1Lu reporter cells, we found a 2- to 3-fold increase in biologically active TGFbeta in supernatants of HIV-infected PBL. Finally, we determined that HIV infection did not change the percentage of active TGFbeta. Our data suggest that HIV-infected lymphocytes indirectly contribute to lymph node remodeling by secretion of TGFbeta1, which increases fibronectin synthesis by fibroblasts.     

Immunological characterization of a major transformation-sensitive fibroblast cell surface glycoprotein. Localization, redistribution, and role in cell shape

The major cell surface glycoprotein of chick embryo fibroblasts, cellular fibronectin (formerly known as CSP or LETS protein), was purified and used to produce monospecific antisera. After affinity purification, the anti-fibronectin was used to investigate fibronectin's localization, its transfer from intracellular to extracellular pools, its antibody-induced redistribution on the cell surface, and its role in cell shape. Anti-fibronectin localizes to extracellular fibrils located under and between sparse cells, and to a dense matrix that surrounds confluent cells. Cellular fibronectin is also present in granular intracytoplasmic structures containing newly synthesized fibronectin before secretion. This intracellular staining disappears 2 h after treatment with cycloheximide or puromycin, and returns after removal of these protein synthesis inhibitors. In pulse-chase experiments using cycloheximide, fibronectin was sequentially transferred from the intracellular to the fibrillar extracellular forms. Transformation of chick fibroblasts results in decreases in both extracellular and intracellular fibronectin, and in altered cell shape. Treatment of untransformed chick fibroblasts with anti-fibronectin results in rapid (30 min) alteration to a rounder cell shape resembling that of many transformed cells. These rapid shape changes are followed by a slow, antibody-induced redistribution of fibronectin to supranuclear caplike structures. This "capping" is inhibited by metabolic inhibitors. Reconstitution of cell surface fibronectin onto transformed cells restores a more normal fibroblastic phenotype. The reconstituted fibronectin on these cells organizes into fibrillar patterns similar to those of untransformed cells. As with untransformed cells, treatment of these reconstituted cells with anti-fibronectin also results in cell rounding and "capping" of fibronectin.     

Identification of fibronectin receptors on T lymphocytes

We report the identification of fibronectin receptors on thymocytes and T lymphoma cells. Affinity chromatography of extracts of the T cell lymphoma, WR16.1, on a fibronectin-Sepharose column combined with specific elution using a synthetic peptide containing the cell attachment-promoting sequence, arginine-glycine-aspartic acid, yielded two polypeptide components having apparent molecular masses of approximately 160 kD reduced and 175 and 150 kD nonreduced. Immunoprecipitations from surface-iodinated WR16.1 cells or fibronectin-adherent thymocytes using a rabbit antiserum raised against the fibronectin receptor that is present on human fibroblasts revealed, in each case, the same two radiolabeled components. In contrast, immunoprecipitation from fibronectin-nonadherent T lymphoma cells, designated WR2.3, revealed the presence of only the smaller subunit. Although the lymphocyte receptor and the fibronectin receptor identified on fibroblasts share immunologic determinants, they differ in that the molecular mass of the lymphocyte protein is larger. Moreover, trypsinization of either thymocytes or the WR16.1 T lymphoma cells resulted in a subsequent loss of their ability to adhere to fibronectin-coated substrates and a reduction in the electrophoretic mobility of each of the polypeptide chains of the fibronectin receptor present on their surfaces. These changes, however, were not observed with normal rat kidney fibroblasts or mouse 3T3 fibroblasts in response to trypsinization. The data establish the existence on normal lymphocytes of fibronectin receptors that are quite similar to those found on fibroblasts. The possible function of this molecule on thymocytes is discussed.     

Temporal relationships of F-actin bundle formation, collagen and fibronectin matrix assembly, and fibronectin receptor expression to wound contraction

Wound contraction can substantially reduce the amount of new tissue needed to reestablish organ integrity after tissue loss. Fibroblasts, rich in F-actin bundles, generate the force of wound contraction. Fibronectin-containing microfibrils link fibroblasts to each other and to collagen bundles and thereby provide transduction cables across the wound for contraction. The temporal relationships of F-actin bundle formation, collagen and fibronectin matrix assembly, and fibronectin receptor expression to wound contraction have not been determined. To establish these relationships, we used a cutaneous gaping wound model in outbred Yorkshire pigs. Granulation tissue filled approximately 80% of the wound space by day 5 after injury while wound contraction was first apparent at day 10. Neither actin bundles nor fibronectin receptors were observed in 5-d wound fibroblasts. Although fibronectin fibrils were assembled on the surfaces of 5-d fibroblasts, few fibrils coursed between cells. Day-7 fibroblasts stained strongly for nonmuscle-type F-actin bundles consistent with a contractile fibroblast phenotype. These cells expressed fibronectin receptors, were embedded in a fibronectin matrix that appeared to connect fibroblasts to the matrix and to each other, and were coaligned across the wound. Transmission EM confirmed the presence of microfilament bundles, cell-cell and cell-matrix linkages at day 7. Fibroblast coalignment, matrix interconnections, and actin bundles became more pronounced at days 10 and 14 coinciding with tissue contraction. These findings demonstrate that granulation tissue formation, F-actin bundle and fibronectin receptor expression in wound fibroblasts, and fibroblast-matrix linkage precede wound contraction.     

Connective tissue components in synovial fibroblast cultures exposed to interleukin 1 and prostaglandin E2

Long-term synovial fibroblast cultures were exposed to interleukin 1 (IL-1) or prostaglandin E2 (PGE2). The normally spindle-shaped fibroblasts changed to stellate-shaped cells, resembling the HLA-DR-positive, collagenase-producing cells which are normally seen only in primary cultures from enzyme-digested rheumatoid synovial tissue. However, the IL-1- or PGE2-induced fibroblasts were not HLA-DR-positive. This suggests that these cell populations represent originally different cell lines or that the expression of HLA-DR antigens is not induced by the agents used. For further characterization of these stellate cells, the location of fibronectin and type I collagen was studied by specific antibodies and the pericellular coat around fibroblasts was visualized by the erythrocyte exclusion method. Both IL-1 and PGE2 treatments destroyed the intercellular fibronectin network. Type I collagen was detected as intracellular granules. The stellate fibroblasts were usually full of these granules in contrast to intact fibroblasts in which the number of collagen fluorescence granules varied greatly. The pericellular coat known to be formed mainly by hyaluronic acid was similar around spindle and stellate-shaped fibroblasts. Rheumatoid arthritis-derived fibroblasts did not differ from their non-rheumatoid counterparts in any of the experiments. The effect of IL-1 and PGE2 on fibroblasts simulates the interaction between mononuclear cells and fibroblasts in synovial stroma and also potentially the interactions between different cell types in synovial lining.     

Spreading of human fibroblasts in serum-free medium: Inhibition by dithiothreitol and the effect of cold insoluble globulin (plasma fibronectin)

We have tested the effect of dithiothreitol (DTT) treatment on the initial spreading of human fibroblasts in serum-free medium in tissue culture dishes. Cell spreading was inhibited following treatment of these cells with 10 mM DTT. Inhibition occurred when the cells were treated at 37°C but not at 4° and was reversible metabolically but not by the addition of sulfhydryl oxidizing reagents. The inhibition was overcome when DTT-treated human fibroblasts were plated on cold insoluble globulin (plasma fibronectin)—coated dishes. Under these conditions spreading appeared to be completely normal, including the formation of focal adhesions. Analysis of the fibronectin concentrations in the human fibroblasts following DTT treatment indicated that there was little decrease in the absolute level of activity as determined in a biological assay for BHK cell spreading on culture dishes. Analysis of the fibronectin distribution on the DTT-treated human fibroblasts by indirect immunofluorescence using a specific anti-CIG antiserum revealed that fibronectin was no longer deposited onto the culture dish surfaces. Even when the DTT-treated human fibroblasts spread in the presence of fetal calf serum, the cell fibronectin remained for the most part in a perinuclear location. These results indicate that DTT treatment of human fibroblasts prevents the normal translocation of fibronectin from a perinuclear location to the surface of the culture dish. This study further supports our hypothesis that the initial spreading in serum-free medium of fibroblasts from cell strains depends upon secretion of fibronectin onto the culture dish surface.