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Plasma fibronectin (pFN) adhesion mechanisms on inert substrata were evaluated for murine fibroblasts (3T3) and human neuroblastoma (Platt) cells using glass coverslips chemically derivatized with a self-assembled monolayer of aliphatic chains terminated with a specific endgroup to interact with adsorbed pFN: [CH3], [SH], [SCOCH3], [NH2], [SO3H], or underivatized glass [SiOH]. All surfaces bound similar amounts of pFN and facilitated attachment of both cell types within narrow ranges. However, spreading/differentiation responses of cells differed considerably among the surfaces. While 3T3 cells spread and developed microfilament stress fibers comparably on all surfaces, inclusion of an RGDS-containing synthetic peptide in the medium revealed variation in resistance to stress fiber formation mediated by an RGDS-recognizing integrin: [NH2] greater than [CH3] much greater than [SiOH],[SH],[SCOCH3]. Different patterns of neurite formation were observed for neuroblastoma cells: [SiOH], [SO3H] greater than [SCOCH3],[SH] much greater than [CH3] greater than [NH2]. Similarity in cell responses to both [CH3] and [NH2] surfaces argues against a pattern dependent upon the hydrophobicity of substrata. When pFN was diluted to a subsaturable concentration with albumin for adsorption, neuroblastoma responses changed significantly from those observed on pFN-saturated surfaces, for both spreading and neurite generation: [NH2],[SO3H] much greater than [SH], [SCOCH3] greater than [SiOH],[CH3]. Responses to the pFN: albumin mixture were markedly improved from responses after sequential adsorptions, demonstrating "optimization" of pFN conformation (not merely binding) by coadsorption of albumin molecules. In most cases, the [NH2] surface yielded responses distinctively different from the other surfaces. Overall, these data suggest many variations in the conformation of pFN molecules adsorbed to specific inert surfaces, as well as variations in the responses of cell surface receptors to conformationally specific pFNs. They also reveal cell-type-specific changes in differentiated cell responses on derivatized substrata, mediated by different classes of cell surface receptors for the two cell types, and provide optimism for regulating FN-dependent adhesion mechanisms in either positive or negative contexts on biomaterial surfaces derivatized with one or more of these chemical end-groups.

Abstract. Dermatan sulfate proteoglycans (DS-PGs) isolated from bovine articular cartilage have been examined for their effects on the adhesive responses of BALB/c 3T3 cells and bovine dermal fibroblasts on plasma fibronectin (pFN) and/or type I collagen matrices, and compared to the effects of the chondroitin sulfate/keratan sulfate proteoglycan monomers (CS/KSPGs) from cartilage. DS-PGs inhibited the attachment and spreading of 3T3 cells on pFN-coated tissue culture substrata much more effectively than the cartilage CS/KS-PGs reported previously; in contrast, dermal fibroblasts were much less sensitive to either proteoglycan class unless they were pretreated with cycloheximide. Both cell types failed to adhere to substrata coated only with the proteoglycans; binding of the proteoglycans to various substrata has also been quantitated. While a strong inhibitory effect was obtained with the native intact DS-PGs, little inhibitory effect was obtained with isolated DS chains (liberated by alkaline-borohydride cleavage) or with core protein preparations (liberated by chondroitinase ABC digestion). In marked contrast, DS-PGs did not inhibit attachment or spreading responses of either 3T3 or dermal fibroblasts on type I collagen-coated substrata when the collagen was adsorbed with pFN alone, DS-PGs alone, or the two in combination. These results support evidence for (a) collagen-dependent, fibronectin-independent mechanisms of adhesion of fibroblasts, and (b) different sites on the collagen fibrils where DS-PGs bind and where cell surface "receptors" for collagen bind.Experiments were developed to determine the mechanism(s) of inhibition. All evidence indicated that the mechanism using the intact pFN molecule involved the binding of the DS-PGs to the glycosaminoglycan (GAG)-binding sites of substratum-bound pFN, thereby inhibiting the interaction of the fibronectin with receptors on the cell surface. This was supported by affinity chromatography studies demonstrating that DS-PGs bind completely and effectively to pFN-Sepharose columns whereas only a subset of the cartilage CS/KS-PG binds weakly to these columns. In contrast, when a 120-kD chymotrypsin-generated cell-binding fragment of pFN (CBF which has no detectable GAG-binding activity as a soluble ligand) was tested in adhesion assays, DS-PGs inhibited 313 adherence on CBF more effectively than on intact pFN. A variety of experiments indicated that the mechanism of this inhibition also involved the binding of DS-PGs to only substratum-bound CBF due to the presence of a cryptic GAG-binding domain not observed in the soluble CBF. When a series of complementary cell-binding fragments generated from pFN by thermolysin digestion and subsequent affinity chromatography (Castellani, P., A. Siri, C. Rosellini, E. Infusini, L. Borsi, and L. , J. Cell Biol., 103:1671-1677 were tested, a graded response to inhibition by DS-PGs was observed revealing the proximity of the cryptic GAGbinding domain to the cell-binding domain of the fibronectin molecule.All of these results taken...

Adhesion responses of fibroblasts (Balb/c 3T3 cells) and human neuron-derived (Platt neuroblastoma) cells have been examined with plasma fibronectin (pFN) adsorbed to glass surfaces derivatized with an alkyl chain and six chemical end groups interfacing with the bound pFN to test regulation of pFN function. Using new derivatization protocols, the following surfaces have been tested in order of increasing polarity: [CH3], [C = C], [Br], [CN], [Diol], [COOH], and underivatized glass [( SiOH]). For all substrata, pFN bound equivalently using either a supersaturating amount of pFN or a subsaturating amount in competition with bovine albumin. Attachment of both cell types was also equivalent on all substrata. However, spreading/differentiation responses varied considerably. F-actin reorganization was tested in 3T3 cells with rhodamine-phalloidin staining. While stress fibers formed effectively on pFN-coated [SiOH] and [Br] substrata, only small linear bundles of F-actin and a few thin stress fibers were observed on the [COOH], [Diol], and [CN] substrata; the hydrophobic substrata [( CH3] and [C = C]) gave an intermediate response. When a synthetic peptide containing the Arg-Gly-Asp-Ser sequence required for integrin binding to FNs was included in the medium as an inhibitor, additional differences were noted: Stress fiber formation was completely inhibited on [SiOH] but not on [Br] and stress fiber formation was very sensitive to inhibition on the hydrophobic substrata while the F-actin patterns on the [CN] and [COOH] substrata were unaffected. Evaluation of neurite outgrowth by neuroblastoma cells on these substrata revealed both qualitative and quantitative differences as follows: [Diol] = [COOH] greater than [SiOH] much greater than [CN] = [Br] greater than [CH3] = [C = C]. While there was poor cytoplasmic spreading and virtually no neurites formed on the hydrophobic surfaces when pFN alone was adsorbed, neurite formation could be "rescued" if a mixture of pFN with an excess of bovine albumin was adsorbed, demonstrating complex conformational interactions between substratum-bound pFN and adhesion-inert neighboring molecules. In summary, these studies demonstrate that different chemical end groups on the substratum modulate pFN functions for cell adhesion, principally by affecting the conformation of these molecules rather than the amounts bound. Furthermore, these studies confirm multiple-receptor interactions with the FN molecules in cell type-specific adhesion patterns.(ABSTRACT TRUNCATED AT 400 WORDS)

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The surface of titanium has been modified by covalent attachment of an organic monolayer anchored by a siloxane network. This coating completely covers the metal and allows controlled modification of surface properties by the exposed chemical endgroups of the monolayer forming surfactant. The attachment of such a film allows different bulk materials (e.g., glass and titanium) to have identical surface properties and this can be used in regulating cell adhesion responses. This control over surface functionality can modulate the functions of fibronectin in regulating attachment and neurite formation by neuronal cells. The effect on bacterial adherence that is achieved by using such monolayers to vary surface hydrophilicity is also assessed.

Dim expression of CD5 on human B lymphocytes has been used to delineate B1 and B2 subsets. Nevertheless, others have suggested that the molecule is an activation marker and does not predicate a subset distinction. We have used enzymatic amplification staining, a technology that enhances the resolution of flow cytometric analysis of cell surface molecules by as much as 100-fold, to determine that essentially all human B cells express CD5. Furthermore, we show that this expression is regulated during EpsteinBarr virus transformation. C D5, a 67-kDa surface glycoprotein of the scavenger receptor cysteine-rich family, appears on T lymphocytes early in their development and is abundantly expressed on all mature T cells. The expression of the molecule on B lymphocytes and its role in defining separate lineages of these cells is controversial (1, 2). It has been postulated that the low-level expression of CD5 by a subset of B lymphocytes defines a separate lineage that is associated with autoreactive antigenic specificity (2, 3). Alternatively, others have shown that CD5 expression on B cells can be enhanced by various activating agents, prompting the suggestion that CD5 is a B cell activation antigen and that B cells comprise a single lineage (4, 5).We have developed a powerful technology, enzymatic amplification staining (EAS), that significantly enhances the resolution of flow cytometric analysis of cell surface molecules (6). By using EAS, we have achieved a 100-fold enhancement in the fluorescent signal. The enhanced signal allows for the detection of molecules that could not be observed previously. Thus, we have been able to define a subpopulation of peripheral blood cells stimulated in vitro that express Fas ligand (CD178), and we have demonstrated the validity of EAS in this case by correlating the staining with functional activity (6). EAS has also been used to provide a high-resolution immunophenotype of leukemic cells from patient samples. It was shown that enzymatic amplification gives a more powerful assessment of the clonality of the leukemic cells (7).Some investigators have proposed that human B lymphocytes can be divided into B1 and B2 subsets based on the expression of CD5 (2, 3); however, others have shown that CD5 expression on B cells can be up-regulated by various activating agents, which suggests that CD5 is a B cell activation antigen (4, 5). To address these alternative models, we used EAS to assess the expression of CD5 on human B lymphocytes. We found that essentially all B cells express CD5 constitutively at low levels, and we also found that CD5 expression is regulated by Epstien-Barr virus (EBV) transformation. Flow Cytometric Analysis. For standard amplification staining (indirect staining), we incubated cells with biotinylated primary antibodies followed by an incubation with streptavidin conjugated to fluorescein isothiocyanate. For EAS, we obtained kits from Flow-Amp Systems (Cleveland), and followed the manufacturer's instructions. The incubations were all performed for 10 min at room...