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Biomedical subjects

F Grinnell

Publications and source records attributed to F Grinnell.

At least 91 records · Page 5Linked to original sources
PubMed1984

Fibronectin and wound healing.

I have tried to briefly review the evidence (summarized in Table II) indicating that fibronectin is important in cutaneous wound healing. Fibronectin appears to be an important factor throughout this process. It promotes the spreading of platelets at the site of injury, the adhesion and migration of neutrophils, monocytes, fibroblasts, and endothelial cells into the wound region, and the migration of epidermal cells through the granulation tissue. At the level of matrix synthesis, fibronectin appears to be involved both in the organization of the granulation tissue and basement membrane. In terms of tissue remodeling, fibronectin functions as a nonimmune opsonin for phagocytosis of debris by fibroblasts, keratinocytes, and under some circumstances, macrophages. Fibronectin also enhances the phagocytosis of immune-opsonized particles by monocytes, but whether this includes phagocytosis of bacteria remains to be determined. In general, phagocytosis of bacteria has not appeared to involve fibronectin. On the contrary, the presence of fibronectin in the wound bed may promote bacterial attachment and infection. Because of the ease of experimental manipulations, wound healing experiments have been carried out on skin more frequently than other tissues. As a result, the possible role of fibronectin has not been investigated thoroughly in the repair of internal organs and tissues. Nevertheless, it seems reasonable to speculate that fibronectin plays a central role in all wound healing situations. Finally, the wound healing problems of patients with severe factor XIII deficiencies may occur because of their inability to incorporate fibronectin into blood clots.

Animals↗
PubMed1984

Fibroblast spreading and phagocytosis: similar cell responses to different-sized substrata.

Experiments were carried out to test the hypothesis that cell spreading and phagocytosis are similar cell responses to different-sized substrata. The following morphological and biochemical studies provided evidence for this supposition. Cells phagocytosed 1.09-micron and 5.7-micron latex beads, but were unable to completely ingest 15.8-micron or 25.7-micron beads. With the larger beads, the cells spread around the bead surfaces with an appearance typical of cells spread on culture dishes. Biochemical studies with cytochalasin D, azide, and iodoacetate, as well as temperature-dependence studies, demonstrated similar responses of cell spreading and phagocytosis to these treatments. Similar cell surface receptors were involved in cell spreading and phagocytosis based upon experiments using antibodies to baby hamster kidney (BHK) cell wheat germ agglutinin receptors. And finally, BHK cell variants with defective plasma fibronectin (pFN) receptors were unable to spread on pFN-coated dishes or ingest pFN-coated beads. Evidence also is presented concerning the "contact" process in cell adhesion. It was found that azide and low temperature inhibited cell attachment per se but did not block fibronectin-receptor interactions based upon cell binding of pFN-coated beads. A possible explanation for the contact process is presented based upon the resistance of cells and beads to shear forces.

Animals↗
PubMed1984

Calcium ions protect cell-substratum adhesion receptors against proteolysis. Evidence from immunoabsorption and electroblotting studies.

An antibody preparation against BHK cell wheat germ agglutinin receptors (anti-WGA-R), which inhibits fibronectin-mediated cell adhesion, was characterized by immunoabsorption and electroblotting experiments. The results indicated that a 48 kD cell surface WGA-R receptor is protected from proteolysis by Ca2+ ions and is important in fibronectin-mediated cell adhesion.

Animals↗
PubMed1984

BHK cell variant with defective fibronectin receptor function.

Baby hamster kidney cells were mutagenized with N-methyl-N'-nitro-N-nitrosoguanidine and selected to obtain a population of non-attaching cells. The cell variant FN-1 was cloned from the non-attaching cell population, recloned, and tested for cell adhesive interactions using four different assays of fibronectin (pFN) receptor function: cell attachment and spreading on culture dishes and cell binding and phagocytosis of latex beads. On pFN-coated culture dishes, FN-1 cells had decreased attachment compared to parental cells and were unable to spread. With pFN-coated beads, only one third as many pFN-bead binding sites could be detected on FN-1 cells as on the parental cells, and the FN-1 cells were unable to phagocytose the pFN-coated beads. In other studies, the variant cells were able to attach normally and spread partially on substrata coated with polycationic ferritin, concanavalin A, or anti-BHK cell surface antibody. The results suggest that the pFN-receptor function of FN-1 cells is defective.

Animals↗
PubMed1984

Human keratinocyte adhesion and phagocytosis promoted by fibronectin.

Epidermal cells from human foreskin or cadaver skin were found to bind and phagocytose fibronectin-coated latex beads but not serum albumin-coated beads. Three lines of evidence suggested that the beads actually were internalized by the cells and not bound only at the cell surfaces. First, the fibronectin coat on internalized beads could be detected with antifibronectin antibodies only if the cells were permeabilized prior to indirect immunofluorescence staining. Second, the internalized beads were not released from the cells by trypsin treatment. Finally, electron microscopic observations showed that the internalized beads were packaged individually and in clusters inside endocytic vesicles in the perinuclear region of the cells. These vesicles had the typical appearance of lysosomes, and, based on the release of trichloroacetic acid-soluble radioactive fragments of fibronectin into the incubation medium, it was concluded that degradation of the fibronectin on the bead surfaces occurred. The epidermal cells that phagocytosed the fibronectin-coated beads were confirmed as keratinocytes according to their electron microscopic appearance and prominent immunofluorescence staining with antikeratin but not anti-plasma fibronectin antibodies. Fibronectin also was found to promote the attachment and spreading of keratinocytes on culture dishes, although the concentration of fibronectin required for half-maximal activity (approximately 5 micrograms/ml) was severalfold higher than the concentration of fibronectin required for spreading of human fibroblasts (approximately 1 microgram/ml). The results suggest the possible importance of fibronectin in mediating adhesion and phagocytosis by keratinocytes, which may be important for the migration of these cells during wound repair.

Antibodies↗
PubMed1984

Manganese-dependent cell-substratum adhesion.

In the presence of manganese, baby hamster kidney cells attached and spread on substrata without added adhesion factors (e.g., fibronectin, lectins). This Mn-dependent adhesion occurred even when the substratum was coated with proteins, such as albumin, haemoglobin, immunoglobulin or ovalbumin, or a dried collagen film. Under similar conditions, cells without Mn in Mg/Ca-containing medium attached poorly and did not spread. Other divalent cations, including Mg, Ca, Fe, Co and Ni, could not replace Mn. Cell surface sites required for Mn-dependent adhesion were destroyed by brief proteolytic treatment of the cells with trypsin or Pronase under conditions where the fibronectin-receptor was unaffected. Also, addition to the incubations of antibodies that inhibited ligand-mediated cell adhesion (e.g., by fibronectin or lectins) inhibited adhesion of cells in Mn-containing medium and caused rounding of cells previously attached and spread in the presence of Mn. The continuous presence of Mn was required for adhesion. That is, cells that were attached and spread in Mn-containing medium and then switched to Mg/Ca-containing medium (which permitted cytoskeletal function) were found to round up and detach. In marked contrast, cells that were allowed to attach and spread on fibronectin-coated substrata in the presence of Mn did not round up when they were switched to Mg/Ca containing medium. Possible explanations for Mn-dependent cell adhesion are discussed.

Animals↗
PubMed1984

Reorganization of hydrated collagen lattices by human skin fibroblasts.

Fibroblasts were cultured on top of or at the bottom of hydrated collagen lattices. Shortly after initially interacting with the collagen lattices, fibroblasts appeared to attach to individual collagen fibrils and in many cases cell processes were found wrapped around clusters of collagen fibrils. Tension generated by cells during spreading resulted in proximal collagen fibrils becoming aligned distal in the plane of spreading and more densely packed. During subsequent culture, the collagen fibrils to the cells underwent a similar reorganization and the lattice thinned to one-tenth of its original thickness. The rate of thinning was similar regardless of whether the cells were originally above or at the bottom of the lattices. The presence of cells distributed throughout the lattice was unnecessary for lattice reorganization to occur. When the lattices were allowed to come off the underlying substratum, compaction of the collagen gels was observed, and the resulting matrix had the typical appearance of dermis as observed by both light and electron microscopy. Collagen fibrils associated with the cell surface often appeared to be under tension and, in regions of close fibril binding, there was a prominent reorganization of submembranous microfilaments. It is suggested that reorganization of the collagen lattice by fibroblasts may depend upon secreted cell factors as well as physical forces generated by the cells.

Cells, Cultured↗
PubMed1983

Deposition of fibronectin on material surfaces exposed to plasma: quantitative and biological studies.

Experiments were carried out to characterize plasma fibronectin deposition onto material surfaces exposed to plasma solutions. Under nonclotting conditions, the amount of fibronectin adsorption on the surfaces, determined by an indirect radioactive antibody assay, was maximal at low plasma concentrations (0.1%). At higher concentrations of plasma, other plasma proteins appeared to compete with and inhibit adsorption of fibronectin. Biological activity (fibronectin-promoted cell spreading) was also greatest at low plasma concentrations and decreased as the plasma concentration was raised. When surfaces were exposed to plasma under clotting conditions (i.e., addition of Ca2+ and thrombin), fibronectin deposition on the surfaces and biological activity remained constant or increased as the plasma concentration was raised. Based on indirect immunofluorescent antibody assays, the fibronectin deposited from clotting plasma appeared to be in a punctate distribution over the entire material surface and occasionally was associated with discrete fibrillar structures. The increased deposition of fibronectin from clotting plasma compared to nonclotting plasma (approximately a 10-fold difference with 10% plasma) was partially a result of covalent crosslinking of fibronectin to fibrin based upon studies with putrescine added to inhibit crosslinking during clotting. On the other hand, the increase in biological activity that occurred if the surfaces were exposed to clotting plasma was completely inhibited by putrescine, indicating that fibronectin had to be crosslinked to fibrin to have biological activity under these conditions. Finally, fibronectin deposition also occurred on surfaces exposed to whole blood and was markedly enhanced when clotting occurred.

Adsorption↗
PubMed1983

Fibronectin-mediated binding and phagocytosis of polystyrene latex beads by baby hamster kidney cells.

The binding and phagocytosis of fibronectin (pFN)-coated latex beads by baby hamster kidney (BHK) cells was studied as a function of fibronectin concentration and bead diameter. Cells were incubated with radioactive pFN-coated beads, and total bead binding (cell surface or ingested) was measured as total radioactivity associated with the cells. Of the bound beads, those that also were phagocytosed were distinguished by their insensitivity to release from the cells by trypsin treatment. In continuous incubations, binding of pFN-coated beads to cells occurred at 4 degrees C or 37 degrees C, but phagocytosis was observed only at 37 degrees C. In addition, degradation of 3H-pFN from ingested beads occurred at 37 degrees C, as shown by the release of trichloroacetic acid-soluble radioactivity into the incubation medium. When the fibronectin density on the beads was varied, binding at 4 degrees C and ingestion at 37 degrees C were found to have the same dose-response dependencies, which indicated that pFN densities that permitted bead binding were sufficient for phagocytosis to occur. The fibronectin density for maximal binding of ingestion was approximately 250 ng pFN/cm2. When various sized beads (0.085-1.091 micron), coated with similar densities of pFN, were incubated with cells at 4 degrees C, no variation in binding as a function of bead size was observed. Under these conditions, the absolute amount of pFN ranged from less than 100 molecules on the 0.085-micron beads to greater than 15,000 molecules on the 1.091-micron beads. Based upon these results it can be concluded that the critical parameter controlling fibronectin-mediated binding of latex beads by BHK cells is the spacing of the pFN molecules on the beads. Correspondingly, it can be suggested that the spacing between pFN receptors on the cell surface that is optimal for multivalent interactions to occur is approximately 18 nM. When phagocytosis of various sized beads was compared, it was found that the largest beads were phagocytosed slightly better (two fold) than the smallest beads. This occurred both in continuous incubations of cells with beads and when the beads were prebound to the cells. Finally, the kinetic constants for the binding of 0.085 microM pFN-coated beads to the cells were analyzed. There appeared to be approximately 62,000 binding sites and the KD was 4.03 X 10(-9) M. Assuming a bivalent interaction, it was calculated that BHK cells have approximately 120,000 pFN receptors/cell and the binding affinity between pFN and its receptor is approximately 6 X 10(-5) M.

Animals↗
PubMed1982

Thiol-sensitive sites in cell adhesion. Decreased entry of SH-binding reagents into attached BHK cells.

Studies were carried out to learn more about the critical SH groups involved in cell spreading. Pretreatment of suspended baby hamster kidney (BHK) cells with 3 mM-iodoacetate or iodoacetamide for 10 min at 4 degrees C completely inhibited the ability of the cells to spread on fibronectin-coated substrata. If, however, BHK cells were permitted to attach and spread before being treated with the SH-binding reagents, and then harvested by trypsinization and assayed for spreading on fibronectin-coated substrata, there was no inhibition of cell spreading. The extent of prior attachment required before the cells became insensitive to the SH-binding reagents was tested and was found to occur early during the cell adhesion process, before any cell spreading was observed. In analytical experiments, there did not appear to be any difference in the total number of SH groups between suspended or spread cells as determined with 5,5'-dithiobis-(2-nitrobenzoic acid). The uptake of radiolabelled iodoacetate into intact spread cells, however, was found to be 3.5 times less than that found with suspended cells. On the other hand, the distribution of incorporated radioactivity into suspended and spread cells was similar. Most of the radioactivity (approximately 70%) was incorporated into small molecules (e.g. glutathione and cysteine), less (approximately 20%) was incorporated into cytoplasmic proteins, and the least incorporation (approximately 10%) was into the cell cytoskeleton. The data are interpreted to indicate there is a decreased permeability of spread cells to the SH-binding reagents.

Animals↗
PubMed1982

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.

Animals↗
PubMed1982

Inhibition of fibronectin receptor function by antibodies against baby hamster kidney cell wheat germ agglutinin receptors.

Previous studies suggest that the baby hamster kidney (BHK) cell fibronectin receptor is also a wheat germ agglutinin receptor (WGA-R). To analyze this possibility further, IgG and Fab fragments of antibodies produced against a BHK cell WGA-R preparation were tested to determine their effects on cell adhesion mediated by fibronectin, wheat germ agglutinin, concanavalin A, and polycationic ferritin. The WGA-R preparation was isolated by octylglucoside extraction of BHK cells followed by chromatography of the extract on WGA-agarose. The antibodies against the WGA-R preparation reacted primarily with polypeptides of molecular weights 48, 61, 83, 105, 120, 165, 210, and 230 kilodaltons (kdaltons). It was concluded that the antibodies interfered with BHK cell fibronectin receptors on the basis of the ability of anti-WGA-R IgG or Fab fragments to (a) inhibit cell spreading on fibronectin-coated substrata; (b) cause rounding and detachment of cells previously spread on fibronectin-coated substrata; and (c) inhibit binding of fibronectin-coated latex beads to the cells. Antibody activity was blocked by treatment of anti-WGA-R with the WGA-R preparation or by absorption of anti-WGA-R with intact BHK cells. The antibodies also appeared to prevent coupling of ligand-receptor complexes (involving concanavalin A or polycationic ferritin) with the cytoskeleton. Finally, cell rounding and detachment caused by the antibodies were found to require metabolic energy since it did not occur in the presence of azide or at 4 degrees C.

Animals↗