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Biomedical subjects

F Grinnell

Publications and source records attributed to F Grinnell.

At least 109 records · Page 6Linked to original sources

Migration of human neutrophils in hydrated collagen lattices.

The migration of human neutrophilic granulocytes in hydrated collagen lattices was studied by a combination of cinemicroscopy, and scanning and transmission electron microscopy. The basic pattern of cell migration in collagen was similar to that observed previously for these cells on inert material surfaces; i.e., a cycle of cell extension and cytoplasmic flow into the leading extension. In general, however, neutrophils in collagen were less spread than on glass or plastic surfaces. Thin lamellipodia were absent and the leading extension of the cells was often an elaborately folded pseudopodium. In addition, neutrophils migrating in collagen were never observed to have retraction fibres at the tail end of the cells, although a uropod was usually seen. In the region of the uropod, extensive blebbing of the cells often occurred, and when this happened, forward movement of the cells ceased. At the ultrastructural level, both the leading pseudopodium and the blebs at the tail of the cell were found to contain a dense cytoskeletal network from which cell organelles were excluded. Finally, the cells were found to be coated with an extensive glycocalyx, and individual collagen fibres were sometimes observed within the glycocalyx.

Cell Movement↗

Adsorption characteristics of plasma fibronectin in relationship to biological activity.

We studied the adsorption properties of plasma fibronectin (pFN) on wettable tissue culture (TC) dishes and nonwettable bacteriological (BAC-T) dishes in relationship to its biological activity of promoting fibroblast spreading. The binding of pFN to the dish surfaces was found to be very tight and partially resistant to treatments with 1M NaOH, 2% SDS, 8M urea, or 6M guanidine HCl. Only the combination of trypsin (1 mg/ml) followed by 1M NaOH resulted in complete recovery of surface bound material. The amount of surface bound pFN did not directly correlate with its activity when comparing TC and BAC-T dishes. At low concentrations, more pFN adsorbed onto the surfaces of BAC-T dishes than TC dishes, but the adsorbed material was biologically less active on BAC-T dishes. At high concentrations, pFN adsorbed similarly on both dish surfaces reaching a maximum level of 320 ng/cm2 or 4.4 x 10(11) molecules/cm2. The possibility was considered that pFN unfolded into an inactive conformation when adsorbed on BAC-T dishes at a low concentration but that at high concentrations, molecular packing requirements prevented unfolding. Evidence in favor of this hypothesis was the observation that addition of 50 micrograms/ml of serum albumin simultaneously with low pFN concentrations resulted in adsorption of pFN on BAC-T dishes in an active conformation even though the amount of adsorbed pFN decreased. Competition experiments between pFN and other proteins were carried out, and it was found that pFN has a much higher affinity for BAC-T or TC dish surfaces than albumin or fibrinogen. It was also found that mild heat denaturation of albumin increased its affinity for the surface by an order of magnitude.

Adsorption↗

Distribution of fibronectin during wound healing in vivo.

The distribution of fibronectin during wound healing has been studied. Full thickness wounds were made in the skin on the sides of guinea pigs' trunks. Biopsy specimens were taken from the normal skin, from the 5-hr-old wound, and on days 1-18 after wounding. Unfixed frozen sections were analyzed for fibronectin distribution by indirect immunofluorescence analysis with a specific antiserum prepared in rabbits against guinea pig plasma fibronectin. Tissue samples were also embedded in methacrylate and sections cut and stained with hematoxylin and eosin for general histology or with a silver stain for reticulin. Fibronectin was prominent in the basement membranes of normal skin epidermis. It was also present in the papillary dermis and to a lesser extent in the reticular dermis. After wounding, fibronectin was part of the fibrin clot and distributed along fibrin strands. Fibronectin was also deposited along newly synthesized collagen in the granulation tissue, which was at least in part collagen type III based upon staining for reticulin. Eventually, the entire granulation tissue was transformed into aligned collagen fibrils coated with fibronectin. Throughout the period of wound healing, the level of fibronectin associated with what appeared to be type I collagen in the reticular dermis adjacent to the wound area stayed about the same. When fibrils with the histological characteristics of type I collagen were within the granulation tissue, however, they were coated with fibronectin. The results indicate that fibronectin is a major component present during wound healing.

Animals↗

Fibroblast adhesion on collagen substrata in the presence and absence of plasma fibronectin.

We have used scanning electron microscopy and transmission electron microscopy to compare the initial adhesion of baby hamster kidney (BHK) cells and early passage human skin fibroblasts on glass, dried collagen, and hydrated collagen substrata. On glass or dried collagen substrata, BHK cell spreading required fibronectin and the cells were extensively flattened, with broad lamellipodia. In marked contrast, BHK cell spreading on hydrated collagen substrata did not require fibronectin and the cell bodies tended to be rounded, with one or several large filipodia. These filipodia generally penetrated into collagen lattice where they interwove with the collagen fibrils. Proteolysis of the collagen was not evident. In the presence of fibronectin, the morphology of BHK cell spreading on hydrated collagen substrata was similar, except that there was an increase in the number of cells that spread with lamellipodia that did not penetrate the collagen lattice. Human fibroblasts also demonstrated marked differences in cell spreading on glass or dried collagen compared to hydrated collagen substrata. Spreading was characterized by lamellipodia on glass or dried collagen but by multiple filipodia on hydrated collagen. In the latter case, the filipodia penetrated just beneath the surface of the collagen lattice. Added fibronectin was not required for spreading on human fibroblasts on any of the substrata and the presence of added fibronectin had no effect on cell morphology. Finally, the human fibroblasts were observed to form adhesion plaques in some cases where the fibroblasts made close contacts with individual collagen fibrils. The results are discussed in terms of connective tissue organization.

Animals↗

Effects of plant lectins on the adhesive properties of baby hamster kidney cells.

We studied the effects of different lectins on the adhesive properties of baby hamster kidney (BHK) cells. The purpose of these studies was to learn more about the cell surface receptors involved in cell adhesion. Three adhesive phenomena were analyzed: 1) the adhesion of BHK cells to lectin-coated substrata; 2) the effects of lectins on the adhesion of cells to substrata coated by plasma fibronectin (pFN); and 3) the effects of lectins on the binding of pFN-coated beads to cells. Initial experiments with fluorescein-conjugated lectins indicated that concanavalin A (Con A), ricinus communis agglutinin I (RCA I), and wheat germ agglutinin (WGA) bound to BHK cells but peanut agglutinin (PNA), soybean agglutinin (SBA), and ulex europaeus agglutinin I (UEA I) dod not bind. All three of the lectins which bound to the cells promoted cell spreading on lectin substrata, and the morphology of the spread cells was similar to that observed with cells spread on pFN substrata. Protease treatment of the cells, however, was found to inhibit cell spreading on pFN substrata or WGA substrata more than on Con A substrata or RCA I substrata. In the experiment of cells with Con A or WGA inhibited cell spreading on pFN substrata, but RCA I treatment had no effect. Finally, treatment of cells with WGA inhibited binding to cells of pFN beads, but neither Con A nor RCA I affected this interaction. These results indicate that the lectins modify cellular adhesion in different ways, probably by interacting with different surface receptors. The possibility that the pFN receptor is a WGA receptor is discussed.

Animals↗

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 degrees C but not at 4 degrees 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 cells 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 perinulear 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.

Animals↗

Fibroblast adhesion to fibrinogen and fibrin substrata: requirement for cold-insoluble globulin (plasma fibronectin).

We carried out experiments to determine conditions for fibroblast adhesion to fibrinogen and fibrin substrata. Baby hamster kidney (BHK) cells did not attach to substrata composed of purified fibrinogen or fibrin. When cold-insoluble globulin (CIG) (plasma fibronectin) was bound to fibrinogen or fibrin substrata, adhesion of BHK sells was observed and the extent of adhesion was dependent upon the CIG conecntration. Binding of CIG to fibrinogen or fibrin substrata in the presence of Factor XIII (factor) under covalent crosslinking conditions resulted in a marked increased in the ability of the substrata to support cell adhesion. Control experiments indicated that CIG formed the sites on the fibrinogen and fibrin substrata to which the cells were attaching. In addition, the effect of factor XIII was shown to require covalent crossliking of CIG to the fibrinogen or fibrin, which involved a glutamine residue on the CIG molecule and could be prevented by prior crosslinking of CIG with putrescine or with itself. The enhanced ability of Factor XIII-crosslinked CIG substrata to support cell adhesion could not be accounted for by the absolute amount of CIG bound to the substrata. We present in this paper the possibility that the orientation of CIG on the substrata is the critical factor.

Animals↗

Visualization of cell-substratum adhesion plaques by antibody exclusion.

We report on an antibody exclusion method for detecting cell adhesion plaques. Substrata coated by plasma fibronectin )pFN) appeared uniformly fluorescent when analyzed by indirect immunofluorescence using a specific anti-pFN antibody. When cells that had spread on the substrata were permeabilized and analyzed by indirect immunofluorescence, areas of non-fluorescence were apparent beneath the cells and in focal streaks along the cell margins. The nonfluorescent focal streaks were coincident with adhesion plaques visualized by interference reflection microscopy.

Cell Adhesion↗

Fibroblast receptor for cell-substratum adhesion: studies on the interaction of baby hamster kidney cells with latex beads coated by cold insoluble globulin (plasma fibronectin).

Studies were carried out on the interactions of uncharged latex beads (0.76 micrometer) with baby hamster kidney cells. Binding of beads to the cells occurred if the beads were coated by cold insoluble globulin (CIG) (plasma fibronectin) but not if the beads were coated by bovine albumin. Bovine albumin-coated beads did not bind to the cells even in the presence of excess CIG in the incubation medium. Binding of beads occurred randomly over the entire surfaces of cells in suspension. However, cell receptors for CIG beads were no longer detectable on the upper surface of cells spread onCIG-coated tissue culture dishes. Binding of CIG beads to cells occurred at all temperatures tested from 4 degrees to 37 degrees C but the rate was lowest at 4 degrees C. At 37 degrees C, binding was accompanied by endocytosis and the beads were found inside vesicles which appeared to be lysosomes. There was also release of radioactivity from radiolabeled CIG beads during incubation with the cells at 37 degrees C. Binding of CIG beads to cells did not require divalent cations. Finally, the cell receptor for CIG beads was lost after cell trypsinization. The data are discussed in terms of current ideas about the basis for cell adhesion.

Animals↗

Cell adhesion and spreading factor: chemical modification studies.

The purified fetal calf serum factor that promotes cell adhesion and spreading of baby hamster kidney cells on tissue culture substrata has been subjected to a variety of chemical modifications and then tested for activity. These studies have shown that modification of the carbohydrate portions of the factor by glycosidic enzymes or by periodate oxidation did not alter its ability to promote cell spreading. On the other hand, modification of some protein portions of the factor by proteolytic enzymes or by specific modification of -COOH groups, tyrosine residues, or tryptophan residues resulted in a marked inhibition of factor activity. Modification of protein -SH groups, -NH2 groups, or methionine residues did not affect factor activity. Control experiments indicate that the various modifications were directed at the activity of the factor and not its adsorption onto the substrata.

Adsorption↗

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.

Animals↗

The influence of cold insoluble globulin on platelet morphological response to substrata.

Light microscopic studies have been carried out on the attachment and morphological responses of washed human platelets in serum-free medium to fibrinogen-coated, collagen-coated and uncoated tissue culture plastic substrata. Platelets were observed to attach to the substratum, extend filipodia and undergo spreading. Subsequently, lysis of platelets occurred. On uncoated tissue culture plastic substrata, the addition of cold insoluble globulin to the incubations had no effect on the above morphological changes. On the other hand, on the protein coated substrata, there was very little platelet spreading or lysis without the addition of cold insoluble globulin.

Blood Platelets↗