Immunological detection of cell surface components related with aggregation of Chinese hamster and chick embryonic cells.

Previous studies suggested that Chinese hamster V79 cells possess two mechanisms for their mutual adhesion, Ca²⁺-dependent and Ca²⁺-independent ones. We could prepare cells with only the Ca²⁺-dependent mechanism intact by dispersing cell monolayers with trypsin (0.01%) containing Ca²⁺. In the present study, we found that cells dispersed with a very low concentration of trypsin (0.0001%) in the absence of Ca²⁺ retain only the Ca²⁺-independent mechanism intact. Fab fragments of antibodies directed against surface antigens of V79 cells inhibited the aggregation of V79 cells by the Ca²⁺-independent mechanism, but did not inhibit the aggregation of these cells by the Ca²⁺-dependent mechanism. These results suggest that the two mechanisms of cell adhesion are based on different cellular components. Molecules responsible for the Ca²⁺-independent adhesion mechanism are probably cell surface components, because they were released from cells by the treatment with 0.01% trypsin without losing their specific antigenicity. The presence of adhesion mechanisms similar to those in V79 cells was shown in neural retinal cells of chick embryos. It was assumed, therefore, that these mechanisms of cell adhesion are generally present among a variety of cell types.     

Cleavage stage mouse embryos share a common cell adhesion system with teratocarcinoma cells

We examined similarities in adhesive properties of mouse cleaving embryos at one- to eight-cell stages and of teratocarcinoma cells by aggregation studies. Teratocarcinoma cells and fibroblastic cells have a Ca²⁺-dependent cell-cell adhesion site (CDS), which is resistant to trypsin in the presence of Ca²⁺ but sensitive in the absence of Ca²⁺. When several embryos treated with trypsin in the presence of Ca²⁺ (TC) were kept in contact with each other, they fused into a single aggregate in the medium with Ca²⁺ but not without Ca²⁺. Embryos treated with trypsin in the absence of Ca²⁺ (TE) did not show such Ca²⁺-dependent aggregation. Aggregation of TC-treated embryos was inhibited by Fab fragments of antibody raised against TC-treated teratocarcinoma F9 cells. The aggregation-inhibitory effect of the Fab was removed by absorption with TC-treated teratocarcinoma cells, but not with TE-treated teratocarcinoma cells. This effect was not removed by absorption with fibroblasts and some other tissue cells. TC-treated embryos adhered to TC-treated teratocarcinoma cells, but not to TC-treated fibroblastic cells. These results suggest that early mouse embryos share a common CDS molecule with teratocarcinoma cells but not with fibroblastic cells.     

Two distinct adhesion mechanisms in embryonic neural retina cells. II. An immunological analysis

Antibodies were raised against neural retina cells prepared by dissociation in EGTA alone (E cells, Ca²⁺-independent aggregation), in trypsin + Ca²⁺ (TC cells, Ca²⁺-dependent aggregation), or in trypsin + EGTA (TE cells, nonadhesive). Anti-E-cell Fab selectively inhibited Ca²⁺-independent aggregation, anti-TC-cell Fab selectively inhibited Ca²⁺-dependent aggregation, and anti-TE-cell Fab inhibited neither. Fab from a fourth preparation, also raised against E cells, inhibited both Ca²⁺-independent and Ca²⁺-dependent aggregation but was separated by immunoadsorption into two fractions, one specific for each mode of aggregation. In cells which utilize both modes simultaneously (LTC cells), each was inhibited exclusively by the appropriate Fab. The immunological data presented here demonstrate the existence in the same cells of two distinct and functionally independent adhesion mechanisms, each responsible for one of the two modes of aggregation. The differing adhesive properties of retinal cells prepared by different procedures are explained by the presence, absence, or degree of activity of these two mechanisms, qualities regulated by the concentrations of trypsin and Ca²⁺ used in the tissue dissociation.     

Experimental manipulation of cell surface to affect cellular recognition mechanisms.

The mechanism of selective cell adhesion was studied using Chinese hamster V79 and chick embryonic neural retinal cells. Both of these cell types have been shown to have two experimentally separable mechanisms of adhesion; Ca²⁺-dependent and Ca²⁺-independent. Cells can be dispersed so that either or both of the mechanisms remain intact by use of different treatments. A method of labeling cells with FITC was devised to identify one of the two types of cells in a binary cell population. When cells with one of the two adhesion mechanisms were mixed with cells with the other mechanism, they segregated completely, forming independent aggregates, not only in the heterotypic combination of these cell types but also in the homotypic combination of each cell type. In contrast, when cells were mixed with others with the same adhesion mechanism, either Ca²⁺-dependent or -independent, they formed chimeric aggregates, even in the heterotypic cell combination. These results suggest that the specificity in each of those two mechanisms of cell adhesion plays an important role in cellular recognition processes.     

Cell to Cell Adhesion Systems in Xenopus laevis, the South African Clawed Frog I. Detection of Ca2+ Dependent and Independent Adhesion Systems in Adult and Embryonic Cells

The reaggregation kinetics of frog ( Xenopus laevis ) cells prepared using several different dissociation procedures were monitored. Two distinct modes of cell adhesion were revealed, one mediated by Ca²⁺ dependent cell-cell adhesion system (CDS) and the other mediated by Ca²⁺ independent one (CIDS).
CDS was detected in frog embryonic (two-cell stage to neurula) cells and in cells of epithelial cell lines (A6 and A8), while CIDS was detected only in A6 cells. Frog CDS was resistant to 0.01% tryptic digestion in the presence of 1 mM Ca²⁺ and CIDS was resistant to dilute (0.0001%) tryptic digestion. Cells with both mechanisms were prepared by dissociation with 1 mM EDTA and both mechanisms were absent in cells dissociated with 0.01% trypsin and 1 mM EDTA. Frog CDS and CIDS functioned in a temperature dependent and independent manner, respectively. Properties of frog CDS and CIDS revealed in this study were the same as those of mammalian or avian CDS and CIDS, respectively. Frog cells with CDS cross-adhered to mammalian epithelial (F9) cells but not to fibroblastic (V79) cells Ca²⁺ dependently. Monoclonal antibody ECCD-1 raised against mouse epithelial CDS blocked aggregation of frog A8 cells.
These results suggest the similarity between frog CDS and mammalian epithelial type CDS.     

Rearrangement of the PH-20 protein on the surface of macaque spermatozoa following exposure to anti-PH-20 antibodies or binding to zona pellucida

Capacitated cynomolgus macaque sperm have a surface hyaluronidase (PH-20) that is evenly distributed over the entire head and can be visualized at the ultrastructural level using a secondary antibody labeled with colloidal gold . Exposure of sperm to mono-specific, bivalent polyclonal antibodies to PH-20 causes a rapid clustering of PH-20 . The predominant morphological consequence of PH-20 redistribution is its aggregation along the lateral edge of the sperm head. Monovalent Fab fragments of the anti-PH-20 antibody bound to the sperm head but did not induce a change in PH-20 distribution. PH-20 aggregation was observed in almost all sperm following treatment with the polyclonal antibody, but only about 20% of the sperm had morphological acrosome reactions, regardless of the time of exposure or the concentration of antibody. There was morphological evidence of swelling of the acrosomal matrix in over 50% of the sperm following exposure to anti-PH-20 antibodies. Anti-PH-20 Fab fragments did not induce the acrosome reaction or acrosomal matrix swelling. Sperm bound to macaque zona pellucida also showed aggregation of the PH-20 protein as soon as 30 sec after sperm-zona interaction. This aggregation was not observed when macaque sperm were bound to hamster zona pellucida. When macaque sperm were surface-labeled with biotin and then incubated with anti-PH-20 antibodies or macaque zona pellucida, there was no evidence of a global surface protein rearrangement, although PH-20 protein was aggregated on the surface of the same sperm cells. An increase in levels of internal sperm Ca⁺⁺ was measured in association with the antibody-induced PH-20 aggregation. Fab fragments did not increase Ca⁺⁺ levels, but when they were crosslinked with anti-Fab antibody there was a significant Ca⁺⁺ increase and induction of acrosome reactions. Anti-PH-20 Fab fragments did not block macaque sperm binding to macaque zona pellucida or the zona-induced acrosome reaction. We conclude that PH-20 on the sperm surface is involved in sperm-zona pellucida interaction and the zona-induced acrosome reaction. Mol. Reprod. Dev. 50:207–220, 1998. © 1998 Wiley-Liss, Inc.     

Selective adhesion of embryonal carcinoma cells and differentiated cells by Ca2+-dependent sites

The specificity of adhesion between embryonal carcinoma cells and fibroblastic cells of various origins was studied. Embryonal carcinoma cells have intercellular adhesion sites requiring Ca²⁺ (CDS). These sites were found to be sensitive to proteases but resistant to them in the presence of Ca²⁺. CDS with a similar protease sensitivity is present in fibroblastic cells. When embryonal carcinoma cells of different lines were mixed, they adhered to each other nonselectively by CDS. Nonselective adhesion by CDS occurred also between fibroblastic cells of various lines. When embryonal carcinoma and fibroblastic cells were mixed, they preferentially adhered to homotypic cells. Fab fragments of antibodies raised against F9 cells (a nullipotent line of embryonal carcinoma) inhibited the adhesion between embryonal carcinoma cells but not between fibroblastic cells. This inhibitory activity of Fab was absorbed with embryonal carcinoma cells with CDS, but not with fibroblastic cells with CDS or embryonal carcinoma cells from which CDS was experimentally removed. SDS-polyacrylamide gel electrophoresis of radioiodinated cell surface proteins showed that the presence of a 140K-dalton component correlated with the presence of CDS in embryonal carcinoma cells, while the presence of a 150K-dalton component correlated with the presence of CDS in fibroblastic cells. These results suggest that CDS in embryonal carcinoma and fibroblastic cells comprise distinct molecules.     

Teratocarcinoma cell adhesion: Identification of a cell-surface protein involved in calcium-dependent cell aggregation

Teratocarcinoma cells have a Ca²⁺-dependent cell-cell adhesion site (t-CDS) that is unique in being inactivated with trypsin in the absence of Ca²⁺ but not in the presence of Ca²⁺. Fab fragments of antibodies raised against teratocarcinoma F9 cells dissociated by treatment with trypsin and calcium (anti-TC-F9) inhibit the aggregation of teratocarcinoma cells mediated by t-CDS. This inhibitory effect of Fab is removed when anti-TC-F9 is absorbed with F9 cells treated with trypsin and calcium (TC-F9), but not when it is absorbed with F9 cells treated with trypsin and EGTA (TE-F9). Comparisons of cell-surface antigens reactive to anti-TC-F9 in TC-F9 cells with those in TE-F9 cells reveal that only one component, with an approximate molecular weight of 140,000 (p140), is detected specifically on the surface of TC-F9 cells. When TC-F9 cells are retrypsinized in the absence of Ca²⁺, a substance with an approximate molecular weight of 34,000 (p34) is released that can neutralize the aggregation-inhibitory effect of the Fab. This p34 interferes with the immunoprecipitation of p140 with anti-TC-F9, suggesting that p34 is a tryptic fragment of p140. Anti-TC-F9 Fab causes the dissociation of the monolayers of teratocarcinoma cells. This effect is removed by absorption of the Fab with p34 as well as with TC-F9 cells, but not with TE-F9 cells. These results suggest that p140 is essential for the function of t-CDS, and that this is an actual cell-adhesion molecule active in the establishment of monolayers of teratocarcinoma cells.     

Two distinct adhesion mechanisms in embryonic neural retina cells. I. A kinetic analysis

The reaggregation kinetics of embryonic chick neural retina cells prepared using several different dissociation procedures were monitored through decreases in the small-angle light scattering of aggregating samples. Two distinct modes of aggregation were revealed, one Ca²⁺ independent, the other Ca²⁺ dependent, suggesting the existence of two separate adhesion mechanisms. By varying the concentrations of Ca²⁺ and trypsin in the dissociation medium, we obtained cells which exhibited both, either, or neither mode of aggregation. The Ca²⁺-independent adhesiveness is active in the absence of proteolysis, is resistant to low levels of trypsin (0.001%), but is readily inactivated at higher trypsin concentrations in either the presence or absence of Ca²⁺. It is relatively temperature independent. By contrast, the Ca²⁺-dependent adhesiveness is not detected before exposure of the cells to proteolysis. It is expressed after tryptic proteolysis in the presence of Ca²⁺ and is then highly temperature dependent. It is resistant to further digestion by trypsin in the continued presence of Ca²⁺ but is lost when Ca²⁺ is subsequently removed, apparently through the expression of tryptic cleavage incurred earlier. We suggest that its increased activity may result at least in part from the clustering of surface components into adhesive patches. A provisional model is presented correlating these data.     

Identification of platelet membrane thrombospondin binding molecules using an anti-thrombospondin antibody

A rat monoclonal IgG_2a antibody, 5G11, was raised against native human platelet thrombospondin (TSP). Western blot analysis revealed that 5G11 bound (i) to TSP before and after disulfide reduction, and (ii) to a 15-kDa fragment released after prolonged trypsin digestion. Crossed immunoelectrophoresis confirmed that the binding epitope was expressed in the presence of Ca²⁺ and after treatment of TSP with EDTA. Since 5G11 had no effect on platelet aggregation, the antibody was used to immunoprecipitate Ca²⁺-dependent and Ca²⁺-independent TSP-binding molecules on the surface of thrombin-activated surface-labeled ¹²⁵I-platelets. The experimental basis was that ligand-receptor interactions are of high affinity and that anti-ligand antibodies should precipitate the ligand-receptor complex. With platelets activated in the presence of EDTA, 5G11 predominantly precipitated a ¹²⁵I-labeled band of M_r 88 000, identified as glycoprotein (GP) IV. In contrast, in the presence of 2 mM Ca²⁺ and 1 mM Mg²⁺, 5G11 precipitated a complex of five radiolabeled proteins, among which GPIIb, GPIIIa and GPIV were the most prominent.     

In vitro reaggregation of dissociated mouse cerebellar cells : I. Demonstration of different aggregation mechanisms

Reaggregation of mechanically dissociated mouse cerebellar cells (M cells) was compared with cells that received an additional trypsinization either before (T cells) or after (MT cells) the dissociation step. Reaggregation behaviour was followed by measuring the number and size distribution of particles with a Coulter counter. Aggregation rates which were calculated as percentage of decrease of particles could be measured reproducibly. Since the percentage of very large particles (> 100 cells) formed during aggregation varied considerably from one experiment to the next, size distribution curves of particles were used more to distinguish qualitative differences in a less quantitative way.Whereas aggregation rates and size distribution of particles with M cells were almost identical when aggregation occurred in medium of high (1.1 mM) or low (0.1 mM) Ca²⁺ concentrations, T and MT cells aggregated better at high Ca²⁺ concentration. Their aggregation rates were reduced by approx. 50% at low Ca²⁺ concentrations and larger aggregates were hardly formed under these conditions. The aggregation rates of T and MT cells showed a clear dependence on Ca²⁺ concentration, being half maximal at approx. 0.1 mM Ca²⁺.The ability of M cells to aggregate at low or high Ca²⁺ concentrations was influenced by subsequent trypsinization to produce MT cells. When the trypsin concentration was changed from 0.001 to 0.1% during this procedure the aggregation rates at high Ca²⁺ concentration were reduced to approx. 80% of the maximal value, whereas those at low Ca²⁺ concentrations were reduced to 35%. Variation of the Ca²⁺ concentration between 1.1 and 0.1 mM during the trypsinization step (0.015% trypsin) revealed no difference on the aggregation rates.We propose that M cells aggregate mainly or exclusively by a Ca²⁺-independent binding mechanism, whereas T or MT cells aggregate using a Ca²⁺-dependent one which may be functionally silent in M cells.     

Increases of T-type Ca2+ current in heart cells of the cardiomyopathic hamster

In the present study, the whole-cell voltage clamp technique was used in order to record the T- and L-type Ca²⁺ currents in single heart cells of newborn and young normal and hereditary cardiomyopathic hamsters. Our results showed that the I/V relationship curve as well as the kinetics of the L-type Ca²⁺ currents (I_Ca(L)) in both normal and cardiomyopathic heart cells were the same. However, the proportion of myocytes from normal heart hamster that showed L-type I_Ca was less than that of heart cells from cardiomyopathic hamster. The I/V relationship curve of the T-type I_Ca (I_Ca(T)) was the same in myocytes of both normal and cardiomyopathic hamsters. The main differences between I_Ca(T) of cardiomyopathic and normal hamster are a larger window current and the proportion of ventricular myocytes that showed this type of current in cardiomyopathic hamster. The high density of I_Ca(T) as well as the large window current and proportion of myocytes showing I_Ca(T) may explain in part Ca²⁺ overload observed in cardiomyopathic heart cells of the hamster.     

The Macrophage Galactose-Type Lectin Can Function as an Attachment and Entry Receptor for Influenza Virus

Specific protein receptors that mediate internalization and entry of influenza A virus (IAV) have not been identified for any cell type. Sialic acid (SIA), the primary attachment factor for IAV hemagglutinin, is expressed by numerous cell surface glycoproteins and glycolipids, confounding efforts to identify specific receptors involved in virus infection. Lec1 Chinese hamster ovary (CHO) epithelial cells express cell surface SIA and bind IAV yet are largely resistant to infection. Here, we demonstrate that expression of the murine macrophage galactose-type lectin 1 (MGL1) by Lec1 cells enhanced Ca²⁺-dependent IAV binding and restored permissivity to infection. Lec1 cells expressing MGL1 were infected in the presence or absence of cell surface SIA, indicating that MGL1 can act as a primary receptor or as a coreceptor with SIA. Lec1 cells expressing endocytosis-deficient MGL1 mediated Ca²⁺-dependent IAV binding but were less sensitive to IAV infection, indicating that direct internalization via MGL1 can result in cellular infection. Together, these studies identify MGL1 as a cell surface glycoprotein that can act as an authentic receptor for both attachment and infectious entry of IAV.     

An antibody that inhibits fibronectin-independent adhesion of fibroblasts to extracellular matrix material

Chinese hamster ovary (CHO) fibroblasts adhere to the extracellular matrix by both fibronectin-dependent and -independent mechanisms (Harper and Juliano, 1981a,b). Previous studies have suggested that a trypsin-sensitive, 265,000-dalton membrane glycoprotein (gp265) is involved in the fibronectin-independent adhesion process. Using a polyclonal antibody against soluble products obtained from trypsin-treated CHO cells, we have been able to further analyze this involvement. This antibody immunoprecipitates a trypsin-sensitive 265,000-dalton protein from detergent-solubilized cells. Incubation of AdvF11, a variant cell line that does not utilize fibronectin for adhesion, with this antibody blocks their adhesion to extracellular matrix material (ECM). The immunoglobulin fraction will also partially block adhesion of the parental cell line to ECM particularly when the ECM is first treated with an antifibronectin antibody. Taken together these results add support for the involvement of gp265 in fibronectin-independent adhesion and provide a methodology for further characterization.     

Interaction of lentil lectin with human platelets. Evidence against glycoprotein II as aggregation mediator

Human platelets bind on an average of 5 × 10⁵ molecules of lentil lectin/cell with an apparent dissociation constant of 3 × 10^?7 M. The lectin binds mainly to surface glycoprotein II with an apparent molecular weight of 125,000. Lentil lectin neither caused aggregation nor did it inhibit platelet aggregation by other agents. It had no influence on the binding of thrombin to platelets or on thrombin-induced clot retraction. The hypothesis that glycoprotein II mediates platelet aggregation needs reevaluation.     

Early human T cell activation events with engagement of surface MHC class II

Major histocompatibility complex (MHC) class II are expressed on most activated human lymphocytes. They direct antigen presentation events in dendritic cells and B cells (collectively called antigen presenting cells), but the role for MHC class II in human T cells is not well understood. To understand the role of surface MHC class II and to identify the molecules involved in signaling, we have defined the early activation sequence in T cells when MHC class II are engaged by a specific antibody. Specifically, we have characterized the involvement of phosphotyrosine kinases, phospholipase C (PLC), and Ca²⁺ mobilization. With the engagement by either whole anti-class II antibody or its Fab fragments, the enzymatic activity of p56^lck and ZAP-70 increased, but there was no increase in p59^fyn activity. In addition, the intracellular free Ca²⁺ increased, which was due to enhanced influx and not to the mobilization of intracytoplasmic Ca²⁺. These events did not require cross-linking because they were not significantly augmented by the addition of antispecies antibody. The coimmunoprecipitation of tyrosine phosphorylated PLC-γ1 with surface MHC class II suggested that PLC-γ1 could be recruited to MHC class II after engagement. These results show the complexities of the early signals transduced by the engagement of surface MHC class II on T cells. J. Cell. Biochem. 70:346–353, 1998. © 1998 Wiley-Liss, Inc.     

Calcium-dependent self-aggregation of toposome,a sea urchin embryo cell adhesion molecule

Summary— Sea urchin embryos can be easily dissociated into single cells by exposure to Ca²⁺- and Mg²⁺-free seawater. When transferred back to normal seawater, isolated cells spontaneously form aggregates capable of development. Here, the Ca²⁺-dependent self-aggregation of toposome, a 22S glycoprotein complex which mediates cell-cell adhesion in sea urchin embryos, has been investigated using the purified molecule. Results show that the 22S complex is completely converted to 15S particles by sedimentation on sucrose isokinetic gradients in the presence of EDTA. Reconstitution of the 22S complex is achieved by readdition of Ca²⁺. We propose that the 15S particle constitutes the toposome functional unit on the cell surface.     

A comparison of the interaction of Chinese hamster fibroblasts with human and bovine transferrins, and conalbumin (chick-egg transferrin)

The iron requirement of a cell line of Chinese hamster fibroblasts is met more efficiently by human transferrin than by bovine transferrin or conalbumin. One possible explanation is that the binding of these transferrins to the Chinese hamster V79 cells may differ. Binding studies now show that the affinity of V79 cells for human transferrin is about 40 times greater than for bovine transferrin. Conalbumin has no detectable affinity for the human transferrin binding sites. Human apotransferrin has approximately one-sixth the affinity for the transferrin binding sites. The binding constant for the relation of human transferrin with the V79 cell is about 2.3·10⁶1· mole⁻¹, and the approximate number of binding sites per cell is 9 · 10⁵.     

Functional correlation between cell adhesive properties and some cell surface proteins

The adhesive properties of Chinese hamster V79 cells were analyzed and characterized by various cell dissociation treatments. The comparisons of aggregability among cells dissociated with EDTA, trypsin + Ca2+, and trypsin + EDTA, revealed that these cells have two adhesion mechanisms, a Ca2+-independent and a Ca2+-dependent one. The former did not depend on temperature, whereas the latter occurred only at physiological temperatures. Both mechanisms were trypsin sensitive, but the Ca2+- dependent one was protected by Ca2+ against trypsinization. In morphological studies, the Ca2+-independent adhesion appeared to be a simple agglutination or flocculation of cells, whereas the Ca2+- dependent adhesion seemed to be more physiological, being accompanied by cell deformation resulting in the increase of contact area between adjacent cells. Lactoperoxidase-catalyzed iodination of cell surface proteins revealed that several proteins are more intensely labeled in cells with Ca2+-independent adhesiveness than in cells without that property. It was also found that a cell surface protein with a molecular weight of approximately 150,000 is present only in cells with Ca2+-dependent adhesiveness. The iodination and trypsinization of this protein were protected by Ca2+, suggesting its reactivity to Ca2+. Possible mechanisms for each adhesion property are discussed, taking into account the correlation of these proteins with cell adhesiveness.     

Stabilin-2 mediates homophilic cell-cell interactions via its FAS1 domains

Stabilin-2 was recently shown to mediate a heterophilic interaction with integrin alphaMbeta2 via its FAS1 domain. Here, we demonstrate that stabilin-2 also mediates homophilic cell-cell interactions. L cells expressing stabilin-2 mediate a significant level of cell aggregation, and this aggregation is significantly inhibited by anti-stabilin-2 antibody. Stabilin-2-mediated aggregation is mediated by homophilic interactions and enhanced in the presence of Ca²⁺ and Mg²⁺. Interestingly, exogenous addition of FAS1 domains but not EGF-like domains enhances stabilin-2-mediated cell aggregation, suggesting that exogenous FAS1 domains may form polymeric structure with FAS1 domains of stabilin-2. Together, these data show the participation of stabilin-2 in homophilic cell adhesion and role of FAS1 domains.