Morphology and microfilament organization in human blood lymphocytes : Effects of substratum and mitogen exposure

During culture in serum-containing medium normal human blood lymphocytes, depleted of phagocytic and adherent cells, do not attach to adhesive surfaces. Concanavalin A (ConA) or phytohemagglutinin (PHA) in appropriate concentrations mediate adhesion of these lymphocytes to tissue culture plastic or glass. This process consists of two phases.

1. 1. The mitogen-mediated contact with a surface induces an almost instantaneous alteration of cell shape and a simultaneous redistribution of actin in the majority of the cells.

2. 2. The initial morphological changes are accompanied by an accumulation of actin-containing material in prominent peripheral cytoplasmic outgrowths formed by the spread cells. The contact-induced spreading and rearrangement of actin are inhibited by cytochalasin B (CB) but not by colchicine or vinblastine. The distribution of detectable actin in spread lymphocytes is similar to the distribution of footprints of actin after detachment of spread cells suggesting that actin is involved in the attachment of lymphocytes to substratum. In contrast to lymphocytes on glass or tissue culture plastic which show morphological changes and redistribution of actin cells cultured with ConA on non-adhesive surfaces of bacterial plastic or poly-2-hydroxy-methacrylate do not exhibit any morphological alterations and no rearrangement of actin.

The present approach enables visualization of cytoskeletal structures in lymphocytes to an extent which is not possible using conventional methods with the cells in suspension. The results indicate that contact is a regulator of lymphocyte shape and that actin-containing structures mediate and maintain contact-induced changes of lymphocyte morphology.     

Degree of cell spreading on a substrate as the factor determining the nature of cell microrelief in suspension

Quantitative ratio of various types of cell surface microrelief was determined in suspensions prepared from mouse monolayer cultures of embryo fibroblasts grown on different solid substrates: with high (Falcon) or low poly(2-hydroxyethylmethacrylate) adhesiveness; with flat or cylindrical (53-mu curvature radius) surfaces (polyvinylchloride). The electron microscopy revealed that poorly spread cells (on low adhesive or cylindrical substrata) in suspensions had the microvillous surface relief much more often than the cells brought to suspension from highly adhesive or flat substrata. Thus, the lower the degree of cell spreading on the substratum, the higher the probability for the cell to acquire the microvillous relief in suspended state. The microvillous relief of transformed cells in suspensions is, probably, due to their poor spreading on substrata in the monolayer cultures.     

Adhesive responses of fibroblast and neuroblastoma cells to substrata coated with polyvalent or monoclonal antibody to fibronectin

Both polyvalent and hybridoma-produced antibodies to fibronectin (Fn) were used to ‘map’ the immunoaccessible subsets of cell surface fibronectin on virus-transformed murine fibroblast SVT2 and rat neuroblastoma B104 cells. As one approach to this end, attachment and spreading responses of cells were measured on tissue culture substrata coated with antibody or with plasma fibronectin to compare their adhesive responses. Both SVT2 and B104 cells adhere poorly to polyvalent anti-Fn-coated substrata over short time intervals, but within several hours changes occur which permit cells to attach and spread as well on anti-Fn as on Fn (post-adsorption of the anti-Fn with Fn also generates a maximal response). This adhesive response could be completely prevented by predigesting the cells with Flavobacterium heparanase, but not with chondroitinase ABC, indicating that the cell surface Fn responsible for antibody-mediated adhesion is associated with heparan sulfate proteoglycans on the cell surface. The compositions of the substratum-attached material (left bound after EGTA-mediated detachment of cells) from cells attaching to anti-Fn or Fn were analysed by SDS-PAGE and found to be identical within the same cell type for the two different substrata. Three hybridoma-produced antibodies, which recognize different determinants on Fn, generated different adhesive responses for SVT2 or B104 cells when adsorbed to the substratum. SVT2 cells adhered well to antibody no. 32-coated substrata but poorly to antibodies 92 or 136; on the other hand, B104 cells responded similarly to all three antibodies over short times of attachment but much better to no. 32 after a several hour incubation. These experiments indicate that (1) much of the cell surface fibronectin is complexed with heparan sulfate proteoglycan and is initially inaccessible to bind to polyvalent antibody on the substratum to promote adhesion; (2) the surface of neuroblastoma cells contains a fibronectin-like molecule which is important in their substratum adhesion; and (3) monoclonal antibodies are valuable tools in ‘mapping’ the orientation of cell surface molecules like fibronectin by measuring adhesive responses to antibody-coated substrata.     

Inhibition of morphogenetic cell interactions by 6-diazo-5-oxo-norleucine (DON).

Inasmuch as cell adhesion and spreading are fundamental properties involved in a variety of vital processes, the dependence of the expression of spontaneous macrophage-mediated cytotoxicity on these attributes was investigated using unique means by which substratum adhesiveness of effector and target cells could be either markedly diminished or varied within a large range. Spontaneous cytotoxicity by activated macrophages against various tumor targets growing in adherent or in suspension culture was unaltered, irrespective of whether interaction took place on plastic, on Teflon, or on plastic coated with varying concentrations of the polymer, poly(HEMA). The findings indicate that whatever the nature is of spontaneous macrophage-mediated target cell killing, it is quite independent of cell adhesion and cell shape considerations.     

Fibrinogen induces adhesion, spreading, and microfilament organization of human endothelial cells in vitro

Human umbilical vein endothelial cells (ECs) have been shown to attach to a substratum of fibrinogen (fg). Later, ECs undergo spreading, organization of thick microfilament bundles of the stress fiber type, and formation of focal contacts (adhesion plaques) that correspond to accumulation of vinculin at the cytoplasmic aspect of the ventral membrane. The rate of attachment to fg and the type of spreading is virtually identical to that obtained on substrata coated with fibronectin (FN). Antibodies to fg, but not to FN, prevent EC adhesion to fg; conversely, antibodies to FN, but not to fg, prevent adhesion of ECs to a FN-coated substratum. The removal of residual FN contamination from fg preparations by means of DEAE-cellulose chromatography does not result in any difference in EC adhesion on fg. Moreover, pretreatment of cells with inhibitors of synthesis and release of proteins does not impair their adhesion capacity on an fg-coated substratum. In contrast, human arterial smooth muscle cells do not adhere and spread on fg substrata but do so on FN. The synthetic peptides (Gly-Arg-Gly-Asp[GRGD] and Gly-Arg-Gly-Asp-Ser-Pro[GRGDSP]) containing the tripeptide Arg-Gly-Asp (RGD), originally found to be responsible for the cell binding activity of FN, have been found to inhibit EC spreading and the redistribution of their cytoskeleton, including the formation of stress fibers and the localization of vinculin either on fg or on FN. Conversely, the synthetic peptide Arg-Gly-Gly (RGG) was completely uneffective in inhibiting the adhesion and the sequence of events leading to spreading and cytoskeletal organization. These results indicate that ECs, but not smooth muscle cells, specifically adhere and spread on an fg substratum and this occurs by recognition mechanisms similar to those reported for FN.     

Mechano-transduction mediated secretion and uptake of galectin-3 in breast carcinoma cells: implications in the extracellular functions of the lectin

In the following experiments, we sought to understand the triggering mechanism which propels galectin-3 to be secreted into the extracellular compartment from its intracellular stores in breast carcinoma cells. We also wanted to analyze in greater details the role of galectin-3 in cellular adhesion and spreading. To do this, we made use of two pairs of breast carcinoma cell lines where one of the pair has high expression of galectin-3 and the other low expression of the lectin. We determined that galectin-3 secreted into the conditioned medium of sub-confluent and spread cells in culture was quite low, almost negligible. However, once the cells were detached and rounded up, a mechano-sensing mechanism triggered the rapid secretion of galectin-3 into the conditioned medium. The secretion was constitutive as long as the cells remained detached. Galectin-3 was shown to be actively taken up from the conditioned medium by spreading cells. The cells which express and secrete high levels of galectin-3 adhered and spread much faster on plastic than those with reduced expression. The uptake of galectin-3 according to our data was important in cell spreading because if this process was compromised significantly, cells failed to spread. The data suggested that galectin-3 uptake modulates the adhesion plaques in that cells which express high levels of galectin-3 have thin-dot like plaques that may be suited for rapid adhesion and spreading while cells in which galectin-3 expression is reduced or knocked-down, have thick and elongated plaques which may be suited for a firmer adhesion to the substratum. Recombinant galectin-3 added exogenously reduced the thickness of the adhesion plaques of tumor cells with reduced galectin-3 expression. Taken together, the present data suggest that galectin-3 once externalized, is a powerful modulator of cellular adhesion and spreading in breast carcinoma cells.     

Cell to substratum adhesion-promoting activity released by normal and virus-transformed cells in culture

It is demonstrated here that cultured fibroblasts release into their medium a nondialyzable, protease-sensitive factor(s) capable of promoting the adhesion and spreading of virus-transformed rat fibroblasts on a plastic substratum. A relatively sensitive biological assay is described for quantitation of the adhesion-promoting factor (APF) activity in serum-free, conditioned medium harvested from the cultures. Evidence is presented which indicates that the primary mode of action of the APF is by binding to and modifying the properties of the substratum. Conditioned media harvested after 24 h of incubation in similarly populated cultures of normal fibroblasts of diverse animal species exhibited similar levels of APF activity. However, conditioned media obtained from Rous sarcoma virus (Prague strain)-transformed and avian sarcoma virus B77-transformed rat fibroblasts exhibited three- to sixfold lower levels of APF activity than media conditioned in parallel cultures of heterologous or homologous normal fibroblasts. Cultivation of B77 virus-transformed rat cells in the presence of dibutyryl cyclic AMP and theophylline led to as much as a sevenfold increase in the level of APF activity appearing in the culture medium, with a concomitant increase in the adhesiveness of the cells to the culture substratum. The results support the role of extracellular macromolecules in cell to substratum adhesion. It is postulated that the reduced adhesiveness of transformed cells to a substratum may be at least partially owing to a deficiency in the production and/or release of APF-like macromolecules.     

Plasminogen activator is an apparent lymphocyte mitogen

Culture fluids of avian sarcoma virus (ASV)-transformed but not normal chicken embryo cells frequently elicited a mitogenic response in normal avian and murine lymphocytes. We examined the possibility that plasminogen activator (PA) might be responsible for the observed mitogenic effect. PA activity, present in culture medium, was correlated positively with lymphocyte mitogenic capacity. Treatment of cells with phorbol myristate acetate, which elevates PA levels, increased mitogenesis. Similar treatment with dexamethasone, which inhibits PA biosynthesis and/or secretion, reduced lymphocyte mitogenic activity. Addition to culture fluids of either benzamidine or diisopropylfluorophosphate, both specific PA inhibitors, blocked lymphocyte proliferative responsiveness to culture fluids. In contrast, neither epsilon-amino-caproic acid nor trasylol, which inhibits plasmin esterase activity but not PA, abrogated lymphocyte responsiveness. Furthermore, purified urokinase, an enzyme of similar substrate specificity to PA, had lymphocyte stimulatory activity. These results strongly suggest that PA can function as a lymphocyte mitogen.     

A Yolk-granule Component Acts as an Adhesive Material for Dissociated Gastrula Cells of the Newt, Cynops pyrrhogaster

Yolk granules were collected from full-grown ovarian oocytes of the newt, Cynops pyrrhogaster , and dissolved in 3% acetic acid or 8 M urea solution. Culture dishes were then coated with either of the yolk-granule solutions. The yolk-coated surfaces acted as adhesive substrata for dissociated gastrula cells, which showed active locomotion when spread on the surfaces. Divalent cation was required for cell adhesion and spreading on the yolk-coated surface. Trypsin and glycosidase digestions of dissociated cells or the yolk-coated surfaces inhibited cell adhesion and spreading. Addition of concanavalin A to the culture medium inhibited cell spreading on the yolk-coated surfaces, while high concentration (50 mM) of the saccharides, D-galactose, D-lactose and D-mannose, had a slightly inhibitory effect on cell spreading.
Two yolk components (30-kD and 108-kD proteins) were isolated from yolk granules and applied to the culture dish surfaces. Surfaces coated with the 30-kD protein showed strong adhesiveness for dissociated gastrula cells.     

A labile antigen complex at the lymphocyte surface: the effect of the substratum on its expression

Human T lymphocytes carry a surface antigen, detectable by a monoclonal antifibronectin antibody, which appears to consist of 150 and 55 kd components as revealed by SDS-PAGE. After in vitro culture of the lymphocytes on a plastic substratum for 48 hr comparatively few cells (40 +/- 18% in separate individuals) express the antigen. In contrast, the vast majority of lymphocytes cultured on a collagen matrix for the same time period maintains surface expression of the antigen (76 +/- 14% in separate individuals). Conditioned media from lymphocytes on plastic contain substantial amounts of antigenicity detectable by the same antibody, whereas conditioned media from lymphocytes on collagen are devoid of such antigenicity. The expression at the cell surface of other T lymphocyte antigens (Leu-4, Leu-3, and OKT8) is identical during culture on plastic and collagen for 48 hr. Collagen does not activate the cells to DNA synthesis or expression of IL 2 receptors, and consequently the potentiation of antigen expression by this substratum cannot be attributed to a mitogenic effect. The composition of subsets of T lymphocytes and the viability of the cells are the same on plastic and collagen, which excludes that the substratum-dependent variations in antigenicity reflect selection or loss of antigen-bearing cells. Thus, substratum-dependent regulation of the expression at the cell surface appears to be a unique property of the 150/55 kd T cell surface antigen. Culture on collagen substrata augments the number of lymphocytes showing motile behavior two to four times compared with culture on plastic.     

Contact formation by fibroblasts adhering to heparan sulfate-binding substrata (fibronectin or platelet factor 4)

The process of cell-substratum adhesion of BALB/c 3T3 fibroblasts on fibronectin (FN)-coated substrata was compared with that of cells adhering to substrata coated with the heparan sulfate (HS)-binding protein, platelet factor four (PF4). FN has binding domains for HS and an unidentified cell surface receptor, whereas PF4 binds to only HS on the surface of the cell. The attachment and early spreading sequences of cells on either substratum were similar as shown by scanning electron microscopy (SEM). Within 2 h of spreading, cells on FN developed typical fibroblastic morphologies, whereas those on PF4 lacked polygonal orientations and formed numerous broadly spread lamellae. Interference reflection microscopic analysis indicated that PF4-adherent cells formed only close adhesive contacts, whereas FN-adherent cells formed both close contacts and tight focal contacts. Cells on either substratum responded to Ca2+ chelation with EGTA by rounding up, but remained adherent to the substratum by relatively EGTA-resistant regions of the cell's undersurface, demonstrating that cell surface HS by binding to an appropriate substratum is capable of initiating a Ca2+-dependent spreading response. The EGTA-resistant substratum-attached material on PF4 was morphologically similar to that on FN, the latter of which was derived from both tight focal contacts and discrete specializations within certain close contacts. These studies show that heparan sulfate proteoglycans on the surface of these cells can participate in the formation of close contact adhesions by binding to an appropriate substratum and suggest that sub-specializations within close contact adhesions may evolve into tight focal contacts by the participation of an unidentified cell surface receptor which binds specifically to fibronectin but not to PF4. In addition, the functional role of FN in tight focal contact formation is demonstrated.     

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.     

Lymphocyte adhesion to cultured Peyer's patch high endothelial venule cells is mediated by organ-specific homing receptors and can be regulated by cytokines

Adhesion of lymphocytes to high endothelial venule (HEV) cells is the first step in the migration of these cells from blood into lymph nodes and Peyer's patches (PP). In the present study, we isolated and cultured HEV cells from PP of the rat and assessed their capacity to interact with lymphocytes. Flow cytometric analysis with a rat HEV-specific mAb KJ-4 revealed that greater than 90% of the cultured cells were stained by the antibody. Furthermore, confluent monolayers of PP HEV cells retained the capacity to support the adhesion of lymphocytes from spleen, thoracic duct, and lymph nodes but not binding of immature cells from thymus and bone marrow, which are deficient in cells capable of binding to HEV in vivo. In addition, intraepithelial lymphocytes that preferentially migrated into mucosal lymphoid tissues were also enriched in cells that adhered to the endothelial monolayers. The binding process required energy, was calcium-dependent, and could be inhibited by cytochalasin D, trypsin, and mixed glycosidase. Interestingly, pretreatment of PP HEV cells with rTNF, IFN-gamma, or granulocyte-macrophage CSF significantly increased the endothelial adhesiveness for thoracic duct lymphocytes in a time- and dose-dependent manner. In contrast, stimulation of lymphocytes with phorbol ester or TNF resulted in the rapid modulation of the surface expression of the PP homing receptor and decrease in lymphocyte binding to normal or TNF-stimulated HEV cells. The adhesion of lymphocytes to normal or cytokine-stimulated HEV cells can be blocked by pretreatment of lymphocytes, but not HEV cells, with the PP homing receptor-specific 1B.2.6 antibody. Taken together, these experiments provide strong evidence that the interaction between lymphocytes and cultured HEV cells are mediated by adhesive mechanisms that regulate lymphocyte entry into PP in vivo and that cytokines can promote HEV adhesiveness for lymphocytes through increased expression of organ-specific ligands on HEV cells.     

Regulation of integrin affinity on cell surfaces

Lymphocyte activation triggers adhesiveness of lymphocyte function-associated antigen-1 (LFA-1; integrin α(L)β(2)) for intercellular adhesion molecules (ICAMs) on endothelia or antigen-presenting cells. Whether the activation signal, after transmission through multiple domains to the ligand-binding αI domain, results in affinity changes for ligand has been hotly debated. Here, we present the first comprehensive measurements of LFA-1 affinities on T lymphocytes for ICAM-1 under a broad array of activating conditions. Only a modest increase in affinity for soluble ligand was detected after activation by chemokine or T-cell receptor ligation, conditions that primed LFA-1 and robustly induced lymphocyte adhesion to ICAM-1 substrates. By stabilizing well-defined LFA-1 conformations by Fab, we demonstrate the absolute requirement of the open LFA-1 headpiece for adhesiveness and high affinity. Interaction of primed LFA-1 with immobilized but not soluble ICAM-1 triggers energy-dependent affinity maturation of LFA-1 to an adhesive, high affinity state. Our results lend support to the traction or translational motion dependence of integrin activation.     

Behavior of cultured cells on substrata of variable adhesiveness

The locomotory behavior of tissue cells cultured on various artificial substrata was studied by time-lapse cinemicrography. Cells were able to spread more completely on certain more wettable substrata and to accumulate preferentially on these substrata according to a consistent hierarchy of cell-substratum affinity, which was the same for all cell types. Cell responses to variations in substrata suggest that substratum adhesiveness is the determining factor, but that cells accumulate on more adhesive substrata as the result of unequal competition between several actively locomotory ruffled lamellae around their margin. The increased overlapping between cells cultured on less adhesive substrata was found to be attributable to factors other than a decrease of contact inhibition of locomotion.     

Basement membrane and its components on lymphocyte adhesion, migration, and proliferation.

During inflammation and recirculation, lymphocytes migrate into tissues by traversing the capillary endothelium, a process known as extravasation. After crossing the endothelial cells, lymphocytes come into contact with the basement membrane, which is a specialized layer of extracellular matrix containing predominantly laminin, collagen type IV, entactin, and heparan sulfate proteoglycans. In tissue invasion by inflammatory cells and metastatic tumor cells, the basement membrane serves as a substratum for cell adhesion and migration. However, the role of basement membrane in lymphocyte extravasation remains unclear. In this study, we investigated the effect of basement membrane on lymphocyte adhesion, migration, and proliferation, using matrigel as a model for basement membrane. We observed that matrigel promotes both lymphocyte adhesion and migration, with entactin primarily responsible for promoting adhesion and laminin for promoting migration. In addition, activation of lymphocytes by anti-CD3 enhances their adhesion and migration on matrigel-coated substratum. We also observed that matrigel inhibits the proliferation of lymphocytes stimulated by Con A. Furthermore, we demonstrated that laminin is the matrigel component responsible for inhibiting lymphocyte proliferation. However, matrigel has no effect on the proliferation of lymphocytes stimulated by LPS. These results suggest that matrigel has different effects on lymphocyte subpopulations. In agreement with the results on proliferation, matrigel also inhibits the production of IL-2 by Con A-stimulated lymphocytes.     

Rodent myoblast interactions with laminin require cell surface glycoconjugates but not laminin glycosyl groups

Laminin glycosyl groups are necessary for the spreading of murine melanoma cells which become attached to this glycoprotein. Laminin has been implicated in myogenesis but the potential role of its glycosyl groups in this process has not been examined. In this study we report the effects of the carbohydrate moieties of laminin on myoblast adhesion, spreading, and differentiation. Unglycosylated laminin from tunicamycin-treated cultures of a mouse cell line, M1536 B3, was used in the experiments. Glycosylated laminin from a murine tumor and from cultures of M1563 B3 cells served as controls. Cell binding experiments with C2C12 mouse myoblasts showed that the cells preferred a laminin-coated surface, compared to the uncoated plastic surface (nontissue culture wells). Myoblasts did not distinguish between glycosylated and unglycosylated laminin substrates. Both glycosylated and unglycosylated forms of laminin promoted myoblast growth and differentiation. In contrast, cells on uncoated plastic surfaces grew very slowly and did not further differentiate. The L6 rat myoblast response to glycosylated and unglycosylated laminin was the same. These results indicate that although rodent myoblasts in culture require a laminin substratum for spreading, growth, and differentiation on a proprietary plastic surface, laminin carbohydrates are not implicated in those cellular responses. In contrast, parallel studies using the lectin, Con A, indicate that cell surface glycoconjugates of myoblasts are implicated in the response of these cells to a laminin substratum.     

Effect of retinoic acid on cell-cell adhesiveness in cloned BHK21/C13 cells which form piling-up colonies

The effect was studied of retinoic acid (RA) on cell-cell adhesiveness in Ag8-1 cells, which are piling-up colony-forming cells cloned from a Syrian hamster kidney fibroblastic cell line BHK21/C13. From the piled-up part of the colonies grown with RA (10 microM), many cells were dissociated by mere shaking or pipetting. The dissociated cells soon adhered to and spread on plastic surfaces in the presence of RA. The number of cells per colony increased almost at the same rate in the presence or absence of RA. The effect of RA on the appearance of cells dissociable from colonies was noticeable above 0.1 microM, prominent from 1 to 10 microM, greater when added in the earlier stages of colony formation and negligible when added just before the dissociation assay. Single cells from the monolayer culture grown with RA (10 microM) had less tendency to aggregate than did those from the control culture. Cells from the colonies grown with RA adhered to and spread on a plastic dish for bacterial use, but control cells seldom adhered. These results indicate that RA decreases the cell-cell adhesiveness or suppresses the development of it but increases cell-substratum adhesiveness.     

Tumor promoter and fibronectin induce actin stress fibers and focal adhesion sites in spreading human erythroleukemia (HEL) cells

The effects of plasma fibronectin (pFn) and the tumor promoter 12-0-tetradecanoyl-phorbol-13-acetate (TPA) on adhesion and cytoskeletal organization of human erythroleukemia (HEL) cells were studied. HEL cells, that normally grow in suspension, attached rapidly on pFn-coated growth substratum and some cells showed spreading. Upon exposure to TPA most of the cells adhered and showed some degree of spreading also when plated on plastic. The spread cells showed mostly peripheral accumulations of F-actin in addition to actin fibers seen in some of the cells. When the cells were plated in the presence of TPA on pFn or on pFn-fragments, containing the cell binding site, all the cells adhered rapidly, spread extensively, organized prominent F-actin stress fibers and typical ventral plaques of vinculin and alpha-actinin. Both proteins were revealed also in the suspended cells by Western blot analysis. When plated on substratum coated with other pFn-fragments or laminin, the HEL cells did not adhere or spread. Both adhesion on pFn as well as formation of stress fibers in the presence of TPA could be prevented by the synthetic peptide Arg-Gly-Asp-Ser (RGDS). HEL cells were also able to organize typical ventral fibrillar arrays of Fn. Immunostaining and metabolic labeling experiments showed that the cells did not contain or synthesize Fn, indicating that the plaques were formed from exogenous pFn by the cells. The results suggest that Fn and TPA synergistically induce the organization of the actomyosin system in HEL cells by promoting the formation of prominent actin stress fibers and focal adhesion sites.     

Effects of C-reactive protein on the lymphoid system. II. Inhibition of mixed lymphocyte reactivity and generation of cytotoxic lymphocytes.

C-reactive protein (CRP), an acute phase reactant which increases in concentration during inflammation, has been found to bind to human T cells and to inhibit certain of their functions. In the present study CRP was found to display a binding specificity for theta-bearing cells from mouse peripheral lymphoid tissue but not for thymus cells. CRP inhibited the proliferative response in a similar manner in both murine and human mixed lymphocyte reactions. This inhibition was prevented by the addition of the CRP substrate, pneumococcal C-polysaccharide (CPS), and was not a result of toxicity of CRP for lymphocytes. By contrast the response of spleen lymphocytes to mitogenic Con A concentrations was not altered by CRP. CRP also exerted an inhibitory effect on the in vitro generation of cytolytic T lymphocytes (CL) in mixed lymphocyte reactions of mouse spleen cells. The expression of the cytolytic process by T cells sensitized either in vivo or in mixed lymphocyte cultures was not altered in the presence of CRP. Therefore, CRP appears to influence the inductive phase of the allograft response and perhaps exerts a regulatory effect on cellular immune responsiveness during inflammatory reactions.