Lymphocyte alpha-actinin. Relationship to cell membrane and co-capping with surface receptors

Mouse spleen lymphocytes synthesize a protein which comigrates with skeletal muscle alpha-actinin on two-dimensional gel electrophoresis and is immunoprecipitated by an antibody directed against skeletal muscle alpha-actinin. Mouse lymphocyte alpha-actinin is present in membrane fractions, and is immunoprecipitated from lymphocyte detergent lysates by an antiserum made against these purified membranes. The anti- alpha-actinin activity of this antiserum is not adsorbed after incubation with fixed intact lymphocytes. Lymphocyte alpha-actinin does not bind concanavalin A and it is inaccessible to lactoperoxidase- catalyzed surface iodination. Double immunofluorescence shows that alpha-actinin moves concurrently along the cell membrane with redistributed surface immunoglobulins and Thy-1 antigen, and remains associated up to 30 min with surface aggregates of these receptors. Our results suggest that lymphocyte alpha-actinin, as defined by molecular weight and cross reactivity with the antibody against the muscle protein, (a) is associated with the cell membrane, (b) is not expressed at the cell surface, and (c) participates in the movement of surface receptors.     

Modulation of cell surface iron transferrin receptors by cellular density and state of activation

This report describes investigations of plasma membrane transferrin receptors on a variety of lymphoid cell lines and normal peripheral blood lymphocytes during activation and cell growth cycles. Transformed lymphoid cell lines have as many as 1,000 times the number of receptors found on normal resting lymphocytes. The number of iron transferrin receptors on continuous cell lines as well as normal human fibroblasts is down-regulated during the transition from log-phase growth to stationary plateau growth. When normal lymphocytes are transformed by mixed lymphocyte culture or mitogens, they rapidly express a 50-fold increase in the number of transferrin binding sites. This appearance of iron transferrin receptors anticipates nuclear changes during cell activation and subsequent mitosis of normal cells.     

Sulfated glycolipids and cell adhesion

The adhesive glycoproteins laminin, thrombospondin, and von Willebrand factor bind specifically and with high affinity to sulfatides, and it is this binding that probably accounts for their ability to agglutinate glutaraldehyde-fixed erythrocytes. The three proteins differ, however, in the inhibition of their binding to sulfatides by sulfated polysaccharides. Fucoidan strongly inhibits binding of both laminin and thrombospondin, but not of von Willebrand factor, suggesting the involvement of laminin or thrombospondin, or other unknown sulfatide-binding proteins in specific cell interactions that are also inhibited by fucoidan. Thrombospondin adsorbed on plastic promotes the attachment and spreading of some melanoma cells. Interestingly, fucoidan and an antibody against the sulfatide-binding domain of thrombospondin selectively inhibit spreading but not attachment to thrombospondin-coated surfaces. Sulfatides, but not neutral glycolipids or gangliosides, when adsorbed on plastic also promote attachment and spreading of some cultured cell lines. Direct adhesion of melanoma cells requires high densities of adsorbed sulfatide. In the presence of laminin, however, specific adhesion of some cell types to sulfatide is strongly stimulated and requires only low densities of adsorbed lipid, suggesting that laminin is mediating adhesion by crosslinking receptors on the cell surface to sulfatide adsorbed on the plastic. Although thrombospondin also binds to sulfatides and to melanoma cells, it does not enhance but rather inhibits direct and laminin-dependent melanoma cell adhesion to sulfatide, presumably because it is unable to bind simultaneously to ligands on opposing surfaces. Thus, sulfated glycolipids can participate in both laminin- and thrombospondin-mediated cell adhesion, but their mechanisms of interaction are different.     

Multiple carbohydrate receptors on lymphocytes revealed by adhesion to immobilized polysaccharides

Phosphomannan polysaccharides and fucoidan, a polymer of fucose 4-sulfate, have been demonstrated to inhibit adhesion of lymphocytes to tissue sections that contain high endothelial venules (Stoolman, L. M., T. S. Tenforde, and S. D. Rosen, 1984, J. Cell Biol., 99:1535-1540). We have investigated the potential cell surface carbohydrate receptors involved by quantitating adhesion of rat cervical lymph node lymphocytes to purified polysaccharides immobilized on otherwise inert polyacrylamide gels. One-sixth of the lymphocytes adhered specifically to surfaces derivatized with PPME (a phosphomannan polysaccharide prepared from Hansenula holstii yeast), whereas up to half of the cells adhered to surfaces derivatized with fucoidan. Several lines of evidence demonstrated that two distinct receptors were involved. Adhesion to PPME-derivatized gels was labile at 37 degrees C (decreasing to background levels within 120 min) whereas adhesion to fucoidan-derivatized gels was stable. Soluble PPME and other phosphomannans blocked adhesion only to PPME-derivatized gels; fucoidan and a structurally related fucan blocked adhesion to fucoidan-derivatized gels. Other highly charged anionic polysaccharides, such as heparin, did not block adhesion to either polysaccharide-derivatized gel. Adhesion to PPME-derivatized gels was dependent on divalent cations, whereas that to fucoidan-derivatized gels was not. The PPME-adherent lymphocytes were shown to be a subpopulation of the fucoidan-adhesive lymphocytes which contained both saccharide receptors. These data reveal that at least two distinct carbohydrate receptors can be found on peripheral lymphocytes.     

Interactions between lymphocytes and dermal fibroblasts: an in vitro model of cutaneous lymphocyte trafficking.

Cultures of dermal fibroblasts were established from skin biopsies of CBA mice and used to study the interactions with murine T-lymphocytes. Electron microscopy showed that zones of contact developed between the fibroblasts and the T-cells, particularly after mitogenic activation. The adhesion of the lymphocytes was temperature-dependent, and many more lymphoblasts than resting cells attached to the fibroblast monolayers. Flow cytometry analysis of the adherent population showed that the most prominent type of resting lymphocyte was of the CD4 phenotype, which was also observed using a T-helper lymphoid cell line. However, neither the CD4 nor the CD8 (T-cytotoxic) antigens were involved in the binding process, and while the fibroblasts expressed Class I MHC molecules (but not Class II), these also had no role in mediating lymphocyte adhesion. Although the fibroblasts did not express the ligand Mala-2, the murine homologue of human ICAM-1, a monoclonal antibody against LFA-1, its cognate receptor on the lymphocytes, nevertheless effectively inhibited binding. T-cell attachment was also partially prevented by antibody against the lymphocyte CD2 antigen and by RGDS, a protein epitope known to mediate a number of receptor-integrin interactions. Moreover, this peptide also rapidly and preferentially detached T-lymphocytes which had previously adhered to the fibroblast monolayers. Lymphocyte binding was substantially elevated following treatment of the fibroblasts with cytokines such as tumor necrosis factor-alpha and interferon-gamma, but not interleukin-1 alpha. This increase in adhesiveness was, however, almost completely abolished by monoclonal antibodies specific for LFA-1 or for Mala-2. The results of this study show that while lymphocytes recognize fibroblasts normally via a number of constitutively expressed receptor-integrin interactions, their adhesion can also be modulated by cytokine-induced changes in the expression of other surface ligands.     

Cellular distribution of p68, a new calcium-binding protein from lymphocytes.

A Ca2+-binding protein of mol. wt. 68 000 ( p68 ) is a major component of a Nonidet P-40 insoluble fraction of human and pig lymphocyte plasma membrane. An affinity-purified rabbit antibody has been produced against p68 and used to study its cellular distribution. The antibody stained fixed and permeabilised human B lymphoblastoid cells, peripheral blood lymphocytes and sections of human tonsil. Whole cells, however, were not stained, indicating that the protein was not represented at the cell surface. This assignment was consistent with the detection of p68 in immunoprecipitates from biosynthetically- but not surface-labelled cells. It is concluded that p68 is located on the cytoplasmic face of the plasma membrane. Subcellular fractionation experiments confirmed that p68 was largely membrane-bound in lymphocytes, although a small soluble fraction (approximately 10% of the total) was detected. Sub-fractionation of lymphocyte membranes revealed that p68 was associated not only with the plasma membrane but also with other endomembrane systems. As judged by immunoprecipitation, p68 was present in a variety of cultured cell lines of both lymphoid and non-lymphoid origin. p68 demonstrated a diffuse distribution in fixed and permeabilised fibroblasts which did not correspond to the distribution of either microfilaments or intermediate filaments. However, in detergent-extracted cells the protein was localised in a lamina-like network. A similar immunofluorescent staining pattern has recently been observed for spectrin-related proteins in the detergent-resistant cytoskeleton of fibroblasts. It is suggested that p68 is part of a sub-membranous cytoskeletal complex not only in lymphocytes but also in other cell types.     

Receptors involved in lymphocyte homing: relationship between a carbohydrate-binding receptor and the MEL-14 antigen

Blood-borne lymphocytes extravasate in large numbers within peripheral lymph nodes (PN) and other secondary lymphoid organs. It has been proposed that the initiation of extravasation is based upon a family of cell adhesion molecules (homing receptors) that mediate lymphocyte attachment to specialized high endothelial venules (HEV) within the lymphoid tissues. A putative homing receptor has been identified by the monoclonal antibody, MEL-14, which recognizes an 80-90-kD glycoprotein on the surface of mouse lymphocytes and blocks the attachment of lymphocytes to PN HEV. In a companion study we characterize a carbohydrate-binding receptor on the surface of mouse lymphocytes that also appears to be involved in the interaction of lymphocytes with PN HEV. This receptor selectively binds to fluorescent beads derivatized with PPME, a polysaccharide rich in mannose-6-phosphate. In this report we examine the relationship between this carbohydrate-binding receptor and the putative homing receptor identified by the MEL-14 antibody. We found that: MEL-14 completely and selectively blocks the activity of the carbohydrate-binding receptor on mouse lymphocytes; the ability of six lymphoma cell lines to bind PPME beads correlates with cell-surface expression of the MEL-14 antigen, as well as PN HEV-binding activity; selection of lymphoma cell line variants for PPME-bead binding by fluorescence-activated cell sorting (FACS) produces highly correlated (r = 0.974, P less than 0.001) and selective changes in MEL-14 antigen expression. These results show that the carbohydrate-binding receptor on lymphocytes and the MEL-14 antigen, which have been independently implicated as receptors involved in PN-specific HEV attachment, are very closely related, if not identical, molecules.     

Relevance of drug uptake,cellular distribution and cell membrane fluidity to the enhanced sensitivity of Down's syndrome fibroblasts to anticancer antibiotic-mitoxantrone

Sensitivity of human fibroblasts derived from Down's syndrome (DS) individuals (S-240, T-158, T-74, T-164) and normal donors (S-126, WA-1) to anticancer antibiotic-mitoxantrone (1,4-dihydroxy-5,8-bis((2-((2-hydroxy-ethyl)amino)ethyl)amino)-9,10-anthracenedione dihydrochloride; MIT) and its relationship to the transport rate, cellular distribution and interaction with cell membrane were studied. The survival assay showed that MIT was more toxic to trisomic fibroblast lines than to normal cells. Studies of transport kinetics indicated that the amount of drug taken up and extruded by DS cells was diminished, compared to control cells. In contrast, the cellular level of MIT associated with DNA was greater in trisomic than in normal cells. The fluorescence anisotropy measurements of TMA-DPH and 12-AS demonstrated that the fluidity of the polar region of the outer lipid monolayer of DS cell membrane was decreased in comparison with normal cells. MIT treatment decreased fluidity of the inner hydrophobic region of plasma membrane, but only slightly influenced the fluidity of the outer surface of the cell membrane. Finally, we concluded that lowered membrane fluidity, diminished amount of MIT extruded by cells and the enhanced level of the drug associated with DNA could be responsible for the enhanced sensitivity of DS fibroblasts to the MIT treatment.     

Effect of insulin receptor down regulation on insulin-stimulated thymidine incorporation in cultured human fibroblasts and tumor cell lines.

Insulin receptors in transformed tissue are relatively resistant to down regulation by insulin, and although receptor downregulation reduces rapid onset biologic responses to insulin in normal tissue, this is not observed in tumor cells. The present study compares longterm insulin responses (thymidine incorporation and cell growth) in normal human fibroblasts with responses in human tumor cell lines (MCF-7, T-47D and HCT-8) to determine whether these responses are also resistant to the effects of receptor down regulation. Thymidine incorporation into fibroblasts was more responsive to insulin than was incorporation into tumor cells, although stimulation of uptake into fibroblasts was not paralleled by changes in cell replication. In contrast, physiological insulin concentrations inhibited, and high concentrations of insulin stimulated, thymidine incorporation and cell replication in MCF-7 and T-47D cells. All insulin concentrations inhibited thymidine incorporation in HCT-8 cells without affecting cell replication. The responsiveness of fibroblasts, MCF-7 and HCT-8 cells to insulin was unaltered by down regulation of insulin receptors prior to measuring thymidine incorporation, whereas receptor down regulation paradoxically increased the responsiveness of T-47D cells to insulin. Exposure of fibroblasts to 5 x 10(-8) M dexamethasone for 24h increased their responsiveness to insulin but did not influence the response of MCF-7 or HCT-8 cells, whereas insulin-stimulated incorporation of thymidine in T-47D cells was inhibited. Thus, receptor down regulation does not influence the longterm biologic response to insulin in normal cells, and paradoxically increases responsiveness in one of three tumor cell lines. These changes may contribute to the well-described stimulatory effects of insulin on tumor cell growth and inhibition of this response with dexamethasone may be relevant to cancer treatment programs.     

Demonstration of a surface antigen on Epstein-Barr virus genome-carrying lymphoid cells: distinction from the virus-determined membrane antigen.

A surface antigen (SA) was detected on Epstein-Barr virus (EBV)-carrying lymphoid cell lines by indirect membrane immunofluorescence with an antiserum from a rabbit immunized with Raji cells; the antiserum had been extensively absorbed with normal human blood and tonsil cells. The SA was not detected on normal human umbilical cord and adult peripheral blood lymphocytes or EBV-negative cell lines. Incidences of the SA and EBV-determined membrane antigen (MA) on certain EBV-carrying cell lines were not compatible. Antibody against SA was differentially absorbed by the SA-positive MA-negative cell lines whereas MA antibody was absorbed by MA-positive SA-negative cell lines. The results of cross-absorption tests of antiserum against Raji cells or P3HR-1 cells suggested that SA may contain more than one antigenic determinant.     

Rat natural killer cells synthesize fibronectin. Possible involvement in the cytotoxic function

The ability of NK cells to synthesize and secrete fibronectin (FN), an extracellular matrix glycoprotein which plays a key role in many biologic processes including cellular adhesion, morphology, cytoskeletal organization, cell migration, and invasiveness, was studied. By using affinity-purified polyclonal antibodies directed against human cellular or plasma FN, the presence of FN was evidentiated on Percoll-purified rat large granular lymphocyte or on a large granular lymphocyte tumor cell line (CRC) by flow cytometry and immunoelectron microscopy. Its expression increased after NK cell activation by poly I:C administration. Biochemical analysis by immunoprecipitation and SDS-PAGE indicated that FN was associated to cell surface and secreted in the supernatant in a molecular form similar to that of FN from L929 fibroblasts. In an attempt to understand the role of FN in the NK cell function, we found that an antibody against human plasma FN and its F(ab')2 fragment inhibited NK cytotoxicity against YAC-1 target at the effector cell level. Inhibition occurred at the postbinding level, because F(ab')2 anti-FN inhibited induction of phosphatidylinositol hydrolysis by YAC-1 target cells, whereas binding to target cells was not affected. The possible role of FN in the NK cytotoxic function is suggested.     

Anionic sites,fucose residues and class I human leukocyte antigen fate during interaction of Toxoplasma gondii with endothelial cells

Toxoplasma gondii invades and proliferates in human umbilical vein endothelial cells where it resides in a parasitophorous vacuole. In order to analyze which components of the endothelial cell plasma membrane are internalized and become part of the parasitophorous vacuole membrane, the culture of endothelial cells was labeled with cationized ferritin or UEA I lectin or anti Class I human leukocyte antigen (HLA) before or after infection with T. gondii. The results showed no cationized ferritin and UEA I lectin in any parasitophorous vacuole membrane, however, the Class I HLA molecule labeling was observed in some endocytic vacuoles containing parasite until 1 h of interaction with T. gondii. After 24 h parasite-host cell interaction, the labeling was absent on the vacuolar membrane, but presents only in small vesicles near parasitophorous vacuole. These results suggest the anionic site and fucose residues are excluded at the time of parasitophorous vacuole formation while Class I HLA molecules are present only on a minority of Toxoplasma-containing vacuoles.     

Lymphocyte recognition of high endothelium: antibodies to distinct epitopes of an 85-95-kD glycoprotein antigen differentially inhibit lymphocyte binding to lymph node, mucosal, or synovial endothelial cells

The tissue-specific homing of lymphocytes is directed by specialized high endothelial venules (HEV). At least three functionally independent lymphocyte/HEV recognition systems exist, controlling the extravasation of circulating lymphocytes into peripheral lymph nodes, mucosal lymphoid tissues (Peyer's patches or appendix), and the synovium of inflamed joints. We report here that antibodies capable of inhibiting human lymphocyte binding to one or more HEV types recognize a common 85-95-kD lymphocyte surface glycoprotein antigen, defined by the non-blocking monoclonal antibody, Hermes-1. We demonstrate that MEL-14, a monoclonal antibody against putative lymph node "homing receptors" in the mouse, functionally inhibits human lymphocyte binding to lymph node HEV but not to mucosal or synovial HEV, and cross-reacts with the 85-95-kD Hermes-1 antigen. Furthermore, we show that Hermes-3, a novel antibody produced by immunization with Hermes-1 antigen isolated from a mucosal HEV-specific cell line, selectively blocks lymphocyte binding to mucosal HEV. Such tissue specificity of inhibition suggests that MEL-14 and Hermes-3 block the function of specific lymphocyte recognition elements for lymph node and mucosal HEV, respectively. Recognition of synovial HEV also involves the 85-95-kD Hermes-1 antigen, in that a polyclonal antiserum produced against the isolated antigen blocks all three classes of lymphocyte-HEV interaction. From these studies, it is likely that the Hermes-1-defined 85-95-kD glycoprotein class either comprises a family of related but functionally independent receptors for HEV, or associates both physically and functionally with such receptors. The findings imply that related molecular mechanisms are involved in several functionally independent cell-cell recognition events that direct lymphocyte traffic.     

Fibronectin-plasma membrane interaction in the adhesion of hemopoietic cells

Many hemopoietic cell lines were examined for their ability to adhere to culture dishes coated with extracellular matrix proteins. Adhesion assay was performed with murine and human leukemic cell lines representative of different stages of differentiation along both erythroid and myeloid lineages. All the hemopoietic cell lines tested adhered to fibronectin but not to laminin, types I, III, and IV collagen, serum-spreading factor, and cartilage proteoglycans. In addition to immortalized cell lines, immature erythroid and myeloid mouse bone marrow cells adhered to fibronectin. To define the fibronectin region involved in hemopoietic cell adhesion, proteolytic fragments, monoclonal antibodies, and synthetic peptides were used. Among different fibronectin fragments tested, only a 110-kD polypeptide, corresponding to the fibroblast attachment domain, was active in promoting adhesion. Moreover, a monoclonal antibody to the cell binding site located within this domain prevented hemopoietic cell adhesion. Finally, the tetrapeptide Arg-Gly-Asp-Ser, which corresponds to the fibronectin sequence recognized by fibroblastic cells, specifically and competitively inhibited attachment of hemopoietic cells to this molecule. The cell surface molecule involved in the interaction of mouse hemopoietic cells with fibronectin was identified as a 145,000-D membrane glycoprotein by adhesion-blocking antibodies. This glycoprotein was found to be antigenically and functionally related to the GP135 membrane glycoprotein involved in the adhesion of fibroblasts to fibronectin (Giancotti, F. G., P. M. Comoglio, and G. Tarone, 1986, Exp. Cell Res., 163:47-62). On the basis of these data, we conclude that interaction of hemopoietic cells with fibronectin involves a specific fibronectin sequence and a 145,000-D cell surface glycoprotein. We speculate that this property might be relevant for the interaction of hemopoietic cells with the bone marrow stroma, which represents the natural site of hemopoiesis.     

Identification of three major target molecules of IgM antilymphocyte autoantibodies in systemic lupus erythematosus

Three cell lymphocyte antigens of m.w. 55,000, 70,000, and 105,000 to 110,000 were identified by Western blotting as targets of IgM autoantibodies in serum from a group of 49 patients with systemic lupus erythematosus. The 55- and 70-kDa antigens were well expressed on unstimulated peripheral T cells, whereas the 105- to 110-kDa target was demonstrable only on mitogen-activated T cells and lymphoblastoid T cell lines. Localization of these molecules to the plasma membrane was established by cytoabsorption experiments in which IgM antibody staining of blotted antigens was specifically absorbed from systemic lupus erythematosus serum during 4 degrees C incubations with intact lymphocytes, and by their detection in purified lymphocyte plasma membranes. While the identity of these target antigens vis a vis known surface determinants was not defined, their expression on peripheral T cells from multiple donors and on cell lines of both undifferentiated (HSB-2) and phenotypically mature (Jurkat; HUT 78) types excluded alloantigens, major histocompatibility complex-encoded determinants, and most T cell differentiation antigens as candidates in this regard. Expression of the IgM autoantibody targets on HSB-2 cells argues against discrete T subset specificities as well. IgM reactivity with the 55-, 70-, and 105- to 110-kDa antigens by blotting was highly correlated with antilymphocyte antibody activity in complement-dependent cytotoxicity assays (Fisher's p less than 0.001), and paralleled flow microfluorimetric and microcytotoxicity quantitation of IgM antibody activity in serial observations of individual patients studied during different phases of disease activity. Taken together, these data suggest that IgM lymphocytotoxic antibodies in systemic lupus erythematosus are directed predominantly against a limited number of non-T cell subset-specific antigens.     

Attachment of Madin-Darby canine kidney cells to extracellular matrix: role of a laminin binding protein related to the 37/67 kDa laminin receptor in the development of plasma membrane polarization.

Madin-Darby canine kidney (MDCK) cells have been extensively used as a model for the study of epithelial polarization. The contacts between the cell and extra-cellular matrix (ECM) provide a signal for the polarization of apical membrane markers. In order to study the molecular basis of these contacts, MDCK cells extracts in Triton X-100 were affinity-purified on laminin, yielding polypeptides of 100-110 and 36 kDa, but only the second one could be enzymatically iodinated from the cell surface. This protein was also recognized by an antibody against the 37/67-kDa laminin/elastin family of proteins. Different polypeptides were purified by the same method on type I collagen. An antibody developed against the polypeptides purified on laminin recognized also a 67-kDa protein, blocked 125I-laminin binding to a population of high affinity (1.5 nM KD) binding sites and caused a significant decrease in cell attachment and spreading to laminin or endogenous ECM. This antibody did not interfere with MDCK cell attachment to fibronectin or collagen matrices, but still impaired cell spreading. An apical MDCK plasma membrane protein (184 kDa), fully polarized in untreated cells, was partially mispolarized after treatment with anti-36 kDa antibody. These results are consistent with a model of various ECM receptors operating together in these cells, and show an important role of a non-integrin 36-kDa laminin binding protein related to the 67-kDa laminin receptor family in cell attachment, spreading and polarization.     

Identification of multiple cell adhesion receptors for collagen and fibronectin in human fibrosarcoma cells possessing unique alpha and common beta subunits

Using monoclonal antibody technology and affinity chromatography we have identified four distinct classes of cell surface receptors for native collagen on a cultured human fibrosarcoma cell line, HT-1080. Two classes of monoclonal antibodies prepared against HT-1080 cells inhibited adhesion to extracellular matrix components. Class I antibodies inhibited cell adhesion to collagen, fibronectin, and laminin. These antibodies immunoprecipitated two noncovalently linked proteins (subunits) with molecular masses of 147 and 125 kD, termed alpha and beta, respectively. Class II antibodies inhibited cell adhesion to native collagen only and not fibronectin or laminin. Class II antibodies immunoprecipitated a single cell surface protein containing two noncovalently linked subunits with molecular masses of 145 and 125 kD, termed alpha and beta, respectively. The two classes of antibodies did not cross-react with the same cell surface protein and recognized epitopes present on the alpha subunits. Pulse-chase labeling studies with [35S]methionine indicated that neither class I nor II antigen was a metabolic precursor of the other. Comparison of the alpha and beta subunits of the class I and II antigens by peptide mapping indicated that the beta subunits were identical while the alpha subunits were distinct. In affinity chromatography experiments HT-1080 cells were extracted with Triton X-100 or octylglucoside detergents and chromatographed on insoluble fibronectin or native type I or VI collagens. A single membrane protein with the biochemical characteristics of the class I antigen was isolated on fibronectin-Sepharose and could be immunoprecipitated with the class I monoclonal antibody. The class I antigen also specifically bound to type I and VI collagens, consistent with the observation that the class I antibodies inhibit cell adhesion to types VI and I collagen and fibronectin. The class II antigen, however, did not bind to collagen (or fibronectin) even though class II monoclonal antibodies completely inhibited adhesion of HT-1080 cells to types I and III-VI collagen. The class I beta and II beta subunits were structurally related to the beta subunit of the fibronectin receptor described by others. However, none of these receptors shared the same alpha subunits. Additional membrane glycoprotein(s) with molecular mass ranges of 80-90 and 35-45 kD, termed the class III and IV receptors, respectively, bound to types I and VI collagen but not to fibronectin. Monoclonal antibodies prepared against the class III receptor had no consistent effect on cell attachment or spreading, suggesting that it is not directly involved in adhesion to collagen-coated substrates.(ABSTRACT TRUNCATED AT 400 WORDS)     

Glycoconjugates and cell adhesion: the adhesive proteins laminin, thrombospondin and von Willebrand's factor bind specifically to sulfated glycolipids

The adhesive glycoproteins laminin, thrombospondin and von Willebrand's factor bind specifically and with high affinity to sulfated glycolipids, and it is this binding that probably accounts for their ability to agglutinate glutaraldehyde-fixed erythrocytes. The 3 proteins differ, however, in the effect of sulfated polysaccharides on their binding to sulfatides. Fucoidan strongly inhibits binding of both laminin and thrombospondin, but not of von Willebrand's factor, suggesting the involvement of laminin or thrombospondin or other unknown sulfatide-binding proteins in specific cell interactions that are also inhibited by fucoidan. Thrombospondin adsorbed onto plastic promotes the attachment and spreading of G361 melanoma cells. Interestingly, fucoidan and an antibody directed against the sulfatide-binding domain of thrombospondin selectively inhibit spreading but not attachment. Sulfatides, but not neutral glycolipids or gangliosides, when adsorbed onto plastic also promote attachment and spreading of G361 melanoma cells. Direct adhesion of G361 cells requires high densities of sulfatide. In the presence of laminin, however, specific adhesion of G361 cells to sulfatide is strongly stimulated and requires only low densities of adsorbed lipid, suggesting that laminin mediates adhesion by cross-linking receptors on the melanoma cell surface to sulfatide adsorbed onto the plastic. Although thrombospondin binds to sulfatide and to G361 cells, it does not enhance but rather inhibits direct and laminin-dependent G361 cell adhesion to sulfatide, presumably because it is unable to bind simultaneously to ligands on opposing surfaces. Thus, sulfated glycoconjugates participate in both laminin- and thrombospondin-mediated cell adhesion, but their mechanisms of interaction are different.     

Immune complexes and immunoconglutinin interactions associated with altered lymphocyte activity in Plasmodium chabaudi infections

Fresh plasma from rats infected with Plasmodium chabaudi, incubated with splenic lymphocytes from rats immunized 5 days previously with sheep blood cells, suppressed the capacity of the spleen cells to produce antibody against the sheep cells as was indicated by reductions in the numbers of hemolytic Jerne plaques formed by the treated cells. The effect was maximal in plasma of rats drawn on the 7th day of infection at a time the rats experienced a hemolytic crisis. Serologic studies indicated that the active plasma contained elevated titers of antibody against fibrinogen products, antibody against the soluble serum antigens elaborated during blood infections and antibody against the third component of fixed complement (C3) or immunoconglutinin. Titers of lytic complement were reduced and amounts of soluble immune complex precipitated with polyethylene glycol 6000 were elevated. The active plasma may have affected the antibody producing cells by one or both of two mechanisms. Soluble antigen-antibody complexes could have interacted with Fc receptors of activated lymphocytes to alter their function. Alternatively, the complexes may have fixed complement and interacted with receptors for fixed C3 on the lymphocyte membrane. Such cells, being coated with the antigen for immunoconglutinin, could be altered by immunoconglutination. Inasmuch as the immune complexes in the active plasma were generated in vivo, it would seem unlikely that the plasma would contain significant amounts of complex that had not fixed complement. With immunoconglutinin present in the plasma, alteration of the cells by immunoconglutination seems a more likely possibility.     

Identification,isolation, and characterization of a human T cell population by a monoclonal antibody

The hybridization of spleen cells from mice immunized with mononuclear leukocytes with the HAT-sensitive nonsecreting myeloma, NS1, resulted in the production of hybrid cell lines secreting monoclonal antibodies to lymphocyte surface antigens. One of these, anti-Ta, was shown by fluorescence-activated cell sorter analysis to be specific for a subpopulation of peripheral human T cells. Anti-Ta did not react with peripheral human B cells. Immunoprecipitation followed by two-dimensional gel analysis demonstrated that the T cell subpopulation-specific antigen recognized by this monoclonal antibody is part of, or firmly associated with, a protein of the plasma membrane.