Cell adhesion and phagocytosis promoted by monoclonal antibodies not directed against fibronectin receptors

1988 ◽  
Vol 90 (2) ◽  
pp. 201-214 ◽  
Author(s):  
F. Grinnell ◽  
C.H. Ho ◽  
T.L. Tuan
Keyword(s):  
Monoclonal Antibodies ◽  
Cell Adhesion ◽  
Binding Sites ◽  
Cell Spreading ◽  
The Other ◽  
Immunofluorescence Staining ◽  
Baby Hamster Kidney ◽  
Bhk Cells ◽  
Reducing Conditions ◽  
Latex Beads

In this report we describe cell adhesion and phagocytosis promoted by two monoclonal antibodies that were selected for immunofluorescence staining of non-permeabilized baby hamster kidney (BHK) cells. Anti-BHK1 staining was heaviest along cell margins, whereas anti-BHK2 staining was continuous along cell margins. Neither antibody stained elongated plaque structures such as were observed when cells were reacted with antibodies to fibronectin (FN) receptors. The monoclonal antibodies functioned as adhesion ligands in four different assays: attachment to culture dishes, spreading, binding of latex beads and phagocytosis. Anti-BHK1 and anti-BHK2 promoted attachment to culture dishes similarly, but anti-BHK2 was more effective at promoting cell spreading. Antibody-promoted cell spreading was inhibited by the peptides Ser-Asp-Gly-Arg and Gly-Arg-Gly-Asp-Ser-Pro but not by other, related, peptides tested. The monoclonal antibodies also promoted binding of latex beads, and the bead binding sites were motile, on the basis of their ‘capping’ response. Nevertheless, anti-BHK2 beads were phagocytosed by cells 5- to 20-fold more efficiently than anti-BHK1 beads. The binding sites for anti-BHK1 and anti-BHK2 were characterized by immunoprecipitation experiments. Anti-BHK1 binding sites contained 50K (K = 10(3) Mr) and 88K components under non-reducing conditions that migrated as a 51/53K doublet and a 93K component under reducing conditions. On the other hand, anti-BHK2 binding sites contained 88K and 110K components under non-reducing conditions that shifted to apparent 107K and 128K values when measured under reducing conditions.

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

1982 ◽  
Vol 208 (2) ◽  
pp. 473-478 ◽  
Author(s):  
D D McAbee ◽  
F Grinnell
Keyword(s):  
Cell Adhesion ◽  
Small Molecules ◽  
Cell Spreading ◽  
The Other ◽  
Bhk Cells ◽  
Sh Groups ◽  
Nitrobenzoic Acid ◽  
Cytoplasmic Proteins ◽  
Adhesion Process ◽  
Suspended 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.

scholarly journals Collagen can modulate cell interactions with fibronectin.

1985 ◽  
Vol 101 (2) ◽  
pp. 386-394 ◽  
Author(s):  
K Nagata ◽  
M J Humphries ◽  
K Olden ◽  
K M Yamada
Keyword(s):  
Cell Spreading ◽  
Inhibitory Effect ◽  
The Other ◽  
Baby Hamster Kidney ◽  
Cell Surfaces ◽  
Cell Binding ◽  
Bhk Cells ◽  
Soluble Collagen ◽  
D Cell ◽  
Direct Attachment

We have examined the effects of soluble collagen on the function of fibronectin in baby hamster kidney (BHK) cells. Collagen and its purified alpha1(l) chain noncompetitively inhibited cell spreading on substrates precoated with fibronectin or a 75,000-D cell-binding fragment of fibronectin. Neither preincubation of cells with collagen followed by washing nor the addition of collagen to previously spread cells had any inhibitory effect on cell spreading, which indicates a requirement for the concurrent presence of collagen during the process of spreading. Treatment of collagen or alpha1(l) chain with collagenase abolished the inhibitory effect on fibronectin-mediated cell spreading. However, direct attachment of BHK cells to fibronectin-coated or 75,000-D fragment-coated substrates was not inhibited by collagen or by the alpha1(l) chain. Moreover, the binding of [3H]fibronectin or the 3'-75,000-D fragment to cell surfaces was not inhibited by the presence of soluble collagen, whereas soluble fibronectin inhibited binding. Although the binding of [3H]fibronectin-coated beads to BHK cell surfaces was also not inhibited by collagen, the phagocytosis of such beads was inhibited by the presence of collagen. On the other hand, soluble fibronectin partially inhibited the binding of fibronectin-coated beads but did not inhibit phagocytosis of the beads that did bind. The mechanism of the inhibition of fibronectin function by collagen and the possible interactions of two different kinds of receptors on the cell surface are discussed.

THE MOLECULAR ORGANIZATION OF HUMAN ALPHA 2-MACROGLOBULIN. AN IMMUNO ELECTRON MICROSCOPIC STUDY WITH MONOCLONAL ANTIBODIES

1987 ◽  
Author(s):  
E Delain ◽  
M Barrav ◽  
J Tapon-Bretaudière ◽  
F Pochon ◽  
F Van Leuven
Keyword(s):  
Monoclonal Antibodies ◽  
Binding Sites ◽  
Divalent Cations ◽  
The Other ◽  
Molecular Organization ◽  
Cellular Receptor ◽  
Electron Microscopic ◽  
Microscopic Method ◽  
Bait Region ◽  
Electron Microscopic Method

Electron microscopy is a very convenient method to localize the epitopes of monoclonal antibodies (mAbs) at the surface of macromolecules for studying their tree-dimensional organization.We applied this immuno-electron microscopic method to human ct2-macroglobulin (ct2M). 29 anti-α2M mAbs have been tested with the four different forms of a2M : native and chymotrypsin-transformed tetramers, and the corresponding dimers, obtained by dissociation with divalent cations. These mAbs can be classified in three types : those which are specific for 1) the H-like transformed molecules, 2) the native molecules, and 3) those which can react with both forms of α2M.1) Among the H-like α2M specific mAbs, several react with the 20 kD-domain which is recognized by the cellular receptor of transformed a2M. This domain is located at the carboxyterminal end of each monomer. One IgG binds to the end of two adjacent tips of the H-like form.The other mAbs of this type bind to the α2M tips at non-terminal positions. Intermolecular connections built polymers of alternating α2M and IgG molecules.2) Among the native a2M-specific mAbs some are able to inhibit the protease-induced transformation of the native α2M. The binding sites of these mAbs are demonstrated on the native half-molecules. One of these mAbs was also able to react with transformed dimers, in a region corresponding very likely to an inaccessible epitope in the tetrameric transformed α2M molecule.3) Among the mAbs of this type, only two were able to inhibit the protease-induced transformation of α2M. Obviously, their epitopes should be close to the bait region of α2M. The other mAbs reacting with both α2M forms did not inhibit the α2M transformation.All these mAbs can be distinguished by the structure of the immune complexes formed with all forms of α2M. The epitopes are more easily located on the dimers and on the H-like transformed α2M than on the native molecules.From these observations, we propose a new model of the tree-dimensional organization of the human α2M in its native and transformed configurations, and of its protease-induced transformation.

Effects of polystyrene surface chemistry on the biological activity of solid phase fibronectin and vitronectin, analysed with monoclonal antibodies

1993 ◽  
Vol 104 (3) ◽  
pp. 793-803 ◽  
Author(s):  
P.A. Underwood ◽  
J.G. Steele ◽  
B.A. Dalton
Keyword(s):  
Monoclonal Antibodies ◽  
Cell Adhesion ◽  
Solid Phase ◽  
Divalent Cations ◽  
Biological Activities ◽  
Optimal Growth ◽  
Cell Types ◽  
Cell Binding ◽  
Urea Treatment ◽  
Bhk Cells

The conformation and biological activities of fibronectin (FN) and vitronectin (VN) coated on different plastic surfaces were investigated using cell adhesion and a panel of domain-specific monoclonal antibodies (mAbs). The adhesion of BHK fibroblasts was markedly better on FN coated on hydrophilic tissue culture polystyrene (TCPS) than on hydrophobic, untreated polystyrene (PS). mAbs A17 and 3E3, which inhibit the binding of BHK cells to the RGD-containing sequence within the cell binding region of FN, also bound preferentially to FN on TCPS. In contrast, two anti-FN mAbs, which have no effect on cell adhesion (A35 and A3), bound preferentially to the conformation of FN on the more hydrophobic PS. Mouse melanoma cells utilise an additional cell-binding site in the Hep II domain of FN and their preference for FN coated on TCPS was less marked than that of BHK cells. This reduced preference was again mimicked by the binding of a mAb, A32, which inhibits the binding of B16 cells to the Hep II domain of FN. In contrast, BHK cell adhesion to VN did not display a preference for TCPS over PS. The cell-binding activity of adsorbed VN was matched by the binding of a cell adhesion-inhibitory mAb, A18, which, unlike mAbs A17 and A32, displayed slightly increased binding to VN coated on PS, rather than TCPS. When the denaturating effect of coating FN and VN to PS in the presence of urea was investigated, similar correlations between BHK cell adhesion and the binding of inhibitory mAbs were observed. Urea treatment of FN significantly reduced both BHK cell adhesion and the binding of both cell adhesion-inhibitory mAbs, whereas the binding of A35 and A3 was unaffected. There was no significant effect of urea treatment of VN upon either BHK cell adhesion or mAb binding. A larger panel of anti-FN mAbs was used, together with the anti-VN mAbs, to determine whether there were differences in mAb recognition of FN and VN adsorbed on three different brands of TCPS. The mAbs segregated into four reactivity patterns, of which A17, A32, A35 and A18 respectively were representative. Significant differences were observed in mAb recognition of FN and VN adsorbed to different brands of TCPS. These may reflect differences in the ability of these surfaces to support optimal growth of different cell types. The effect of divalent cations upon adsorbed FN and VN was also investigated.(ABSTRACT TRUNCATED AT 400 WORDS)

MONOCLONAL ANTIBODIES THAT RECOGNIZE Ca2+-INDUCED CONFORMER OF PROTEIN C, INDEPENDENT OF GLA RESIDUES

1987 ◽  
Author(s):  
T Sugo ◽  
S Tanabe ◽  
K Shinoda ◽  
M Matsuda
Keyword(s):  
Monoclonal Antibodies ◽  
Light Chain ◽  
Metal Binding ◽  
Binding Sites ◽  
Plasma Proteins ◽  
Protein C ◽  
The Other ◽  
Metal Binding Sites ◽  
Direct Elisa ◽  
Gla Domain

Monoclonal antibodies (MCA’s) were prepared against human protein C (PC) according to Köhler & Milstein, and those that recognize the Ca2+-dependent PC conformers were screened by direct ELISA in the presence of 2 mM either CaCl2 or EDTA. Out of nine MCAߣs thus screened, five MCA's designated as HPC-1˜5, respectively, were found to react with PC in the presence of Ca2+ but not EDTA. By SDS-PAGE coupled with Western Blotting performed in the presence of 2 mM CaCl2, we found that two MCA’s HPC-1 and 2, recognized the light chain, and two others, HPC-3 and 4, recognized the heavy chain of PC. But another MCA, HPC-5 was found to react with only non-reduced antigens. Further study showed that HPC-1 and 5 failed to react with the Gla-domainless PC, i.e. PC from which the N-terminal Gla-domain of the light chain had been cleaved off by α-chymotrypsin. However, all the other three MCA's retained the reactivity with the antigen in the presence of Ca2+ even after the Gla-domain had been removed. The binding of these MCA’s to PC in the presence of Ca2+ was found to be saturable with respect to the Ca2+ concentration and the half maximal binding for each MCA was calculated to be about 0.5+mM. Moreover, many other divalent cations such as Mg2+, Mn2+ , Ba2+, Zn2+, Co2+, Sr2+, were found to substitute for Ca2+ in inducing the metal ion-dependent but Gla-domain-independent conformer of PC.Cross-reactivity to other vitamin K-aependent plasma proteins was examined by direct ELISA; HPC-2 and 3 reacted solely to PC, but HPC-1 and 4 also reacted with prothrombin and HPC-5 with both prothrombin and factor X.These findings indicated that there are two or more metal binding sites besides the Gla-domain, possibly one in the light chain and the other(s) in the heavy chain. The presence of these metal binding sites may contribute to the unique conformer of vitamin K-dependent plasma proteins including protein C.

THE PHOSPHOLIPID-BINDING SITE OF FACTOR VIII IS LOCATED ON THE 80 kD LIGHT CHAIN

1987 ◽  
Author(s):  
G Kemball-Cook ◽  
S J A Edwards ◽  
K Sewerin ◽  
L-O Andersson ◽  
T W Barrowcliffe
Keyword(s):  
Binding Site ◽  
Light Chain ◽  
Binding Sites ◽  
Immunoaffinity Chromatography ◽  
The Other ◽  
Immunological Methods ◽  
Electrophoretic Separation ◽  
Antigenic Sites ◽  
Reducing Conditions ◽  
Sds Page

The binding of Factoi. VIII (F.VIII) peptides to phospholipid (PL) vesicles has been studied by two different methods involving the use of fractionated anti-F.VIII:C I-Fab123’pre viously reported, i-Fab123’ was fractionated by immunoadsorptionwith F.VIII-PL complexes into two pools:one binding only to PL-binding sites on F.VIIIsAg (PL-site antibody), the other directed against other antigenic sites (non-PL-site antibody).The first technique used was a modification of the method of Weinstein et al. (Proc.Natl.Acad.Sci.USA, 78, 5137-5141, 1981), and involved incubation of the two anti-F.VIII pool swith F.VIII-containing samples, followed by electrophoretic separation of the complexes on the basis of size in non-denaturing SDS gels: this technique allows qualitative analysis of antibody reactive peptides in highly impure samples. Non-PL-site pool reacted with a range of peptides with MrMapparent Mr 90 kD up to 280 kD, a similar pattern to that of ’heavy chain’(HC) peptides of F.VIII seen on SDS-PAGE under reducing conditions; the PL-site antibody, however, reacted only with peptides at apparent Mrs of 80 kD and sometimes150 kD, but not with bands of higher Mr a pattern more consistent with binding to light chain (LC) peptides. Thesame patterns with the two labels were seen in both plasma and F.VIII concentrateThe second approach employed the two labels described above in direct immunoradiometric assays (IFMA’s) on purified human F.VIII peptides prepared by immunoaffinity chromatography and ion exchange on Mono Q gel. Both PL-site and non-PL-site labels measured similar amounts of F.VIII m a sample containing both HC and LC peptides; however, on assaying a sample containing purified HC peptides alone, PL-site antibody measured only 2% of F.VIII:Ag found by non-PL-site label, indicating that PL-binding sites present in samples containing both HC and LC are absent in HC alone.Results from both these immunological methods indicate that the 80 kD LC peptide of F.VIII carries the PL-binding site.

scholarly journals Two Drosophila melanogaster proteins related to intermediate filament proteins of vertebrate cells.

1981 ◽  
Vol 91 (1) ◽  
pp. 175-183 ◽  
Author(s):  
F G Falkner ◽  
H Saumweber ◽  
H Biessmann
Keyword(s):  
Drosophila Melanogaster ◽  
Monoclonal Antibodies ◽  
Heat Shock ◽  
Indirect Immunofluorescence ◽  
Intermediate Filament ◽  
Cross Reaction ◽  
Cytoplasmic Protein ◽  
Baby Hamster Kidney ◽  
Intermediate Filament Proteins ◽  
Bhk Cells

Monoclonal antibodies were prepared against a 46,000 mol wt major cytoplasmic protein from Drosophila melanogaster Kc cells. These antibodies reacted with the 46,000 and a 40,000 mol wt protein from Kc cells. Some antibodies showed cross-reaction with 55,000 (vimentin) and 52,000 mol wt (desmin) proteins from baby hamster kidney (BHK) cells that form intermediate sized filaments in vertebrate cells. In indirect immunofluorescence, the group of cross reacting antibodies stained a filamentous meshwork in the cytoplasm of vertebrate cells. In Kc cells the fluorescence seemed to be localized in a filamentous meshwork that became more obvious after the cells had flattened out on a surface. These cytoskeletal structures are heat-labile; the proteins in Kc or BHK cells rearrange after a brief heat shock, forming juxtanuclear cap structures.

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

1983 ◽  
Vol 97 (5) ◽  
pp. 1515-1523 ◽  
Author(s):  
D D McAbee ◽  
F Grinnell
Keyword(s):  
Cell Surface ◽  
Total Radioactivity ◽  
Polystyrene Latex ◽  
Absolute Amount ◽  
Baby Hamster Kidney ◽  
Bhk Cells ◽  
Latex Beads ◽  
Bead Diameter ◽  
Bivalent Interaction ◽  
Binding Cell

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.

scholarly journals STUDIES ON CELL ADHESION

1973 ◽  
Vol 56 (3) ◽  
pp. 659-665 ◽  
Author(s):  
Frederick Grinnell ◽  
Mary Milam ◽  
Paul A. Srere
Keyword(s):  
Cell Adhesion ◽  
Rate Constant ◽  
Adhesive Strength ◽  
Order Rate Constant ◽  
Baby Hamster Kidney ◽  
Bhk Cells ◽  
First Order ◽  
High Concentrations ◽  
Reversible Loss ◽  
Suspended Cells

Normal and transformed baby hamster kidney (BHK) cells attach to Falcon polystyrene with the same first order rate constant. The longer the cells are attached to the bottles, the more difficult they are to remove. Sulfhydryl (—SH) binding reagents inhibit both the attachment of BHK cells and the increase in adhesive strength of attached cells. Attached BHK cells bind fewer molecules of [1-14C]N-ethylamleimide (an —SH binding reagent) than do suspended cells. Incubation of cells with high concentrations of trypsin results in a reversible loss of cell adhesiveness. The recovery of adhesiveness of trypsin-treated cells is inhibited by cycloheximide.

The apparent infection of NA-C1300 cell cultures by nucleocapsid material of the Canadian Arctic strain of rabies virus

1989 ◽  
Vol 35 (8) ◽  
pp. 811-813
Author(s):  
W. A. Webster ◽  
K. M. Charlton
Keyword(s):  
Rabies Virus ◽  
Cell Cultures ◽  
Virus Strain ◽  
Canadian Arctic ◽  
The Other ◽  
Baby Hamster Kidney ◽  
Bhk Cells ◽  
Murine Neuroblastoma ◽  
Other Hand

Murine neuroblastoma (NA-C1300) and baby hamster kidney (BHK-21/C13) cell cultures were infected with the Canadian Arctic strain of rabies virus. Subcultures were passed following incubation for 3 to 4 days at 35 °C. The supernatant fluids from the BHK cultures demonstrated increasing infectivity in both NA and BHK cells concomitantly with an increase in the number of parent cells staining with an anti-glycoprotein stain. On the other hand, the supernatant fluids from the NA cultures initially showed higher infectivity in NA cells than in BHK cells. This feature was related to a low production of glycoprotein-staining cells in the parent NA cultures. The reduction of infectivity in NA cells of some NA supernatant fluids (and brain suspensions) by anti-nucleoprotein antibodies suggests that nucleocapsid material is, in some manner, capable of infecting NA cells. Infectivity of this virus strain in experimental mice is also related to the production of glycoprotein and may not be correlated with the degree of infection in NA cell cultures.Key words: rabies, nucleocapsid, infection, cells.

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