Negative regulation of T-cell adhesion and activation by CD43

CD43 is a cell-surface sialoglycoprotein expressed by a variety of haematopoietically derived cells, including T lymphocytes. Earlier observations of defective CD43 expression by T lymphocytes from boys with the X-chromosome-linked Wiskott-Aldrich syndrome suggested the importance of CD43 in lymphocyte function. Subsequent studies have suggested that CD43 facilitates leukocyte adhesion and has a co-stimulatory role during T-cell activation. To define the physiologically relevant function(s) of CD43, we have generated CD43-knockout mice. We report here that CD43-deficient T cells from such mice show a marked increase in their in vitro proliferative response to concanavalin A, anti-CD3, the superantigen SEB and allostimulation. Additionally, CD43-deficient T cells show a substantial enhancement in homotypic adhesion and in their ability to bind different ligands, including fibronectin and the intercellular adhesion molecule ICAM-1. Vaccinia-virus-infected CD43-knockout mice mounted an augmented anti-vaccinia cytotoxic T-cell response compared with their wild-type littermates, yet developed an increased virus load. We conclude that CD43 negatively regulates T-cell activation and adhesion and is important for viral clearance.     

Rho-kinase phosphorylates COOH-terminal threonines of ezrin/radixin/moesin (ERM) proteins and regulates their head-to-tail association

The ezrin/radixin/moesin (ERM) proteins are involved in actin filament/plasma membrane interaction that is regulated by Rho. We examined whether ERM proteins are directly phosphorylated by Rho-associated kinase (Rho-kinase), a direct target of Rho. Recombinant full-length and COOH-terminal half radixin were incubated with constitutively active catalytic domain of Rho-kinase, and approximately 30 and approximately 100% of these molecules, respectively, were phosphorylated mainly at the COOH-terminal threonine (T564). Next, to detect Rho-kinase-dependent phosphorylation of ERM proteins in vivo, we raised a mAb that recognized the T564-phosphorylated radixin as well as ezrin and moesin phosphorylated at the corresponding threonine residue (T567 and T558, respectively). Immunoblotting of serum-starved Swiss 3T3 cells with this mAb revealed that after LPA stimulation ERM proteins were rapidly phosphorylated at T567 (ezrin), T564 (radixin), and T558 (moesin) in a Rho-dependent manner and then dephosphorylated within 2 min. Furthermore, the T564 phosphorylation of recombinant COOH-terminal half radixin did not affect its ability to bind to actin filaments in vitro but significantly suppressed its direct interaction with the NH2-terminal half of radixin. These observations indicate that the Rho-kinase-dependent phosphorylation interferes with the intramolecular and/ or intermolecular head-to-tail association of ERM proteins, which is an important mechanism of regulation of their activity as actin filament/plasma membrane cross-linkers.     

Ligation of CD4 surface antigen induces rapid tyrosine phosphorylation of the cytoskeletal protein ezrin

Ezrin is a cytoskeletal protein which is tyrosine phosphorylated in human T lymphocytes upon stimulation through CD3 antigen (Egerton, M., Burgess, W., Chen, D., Druker, B. J., Bretscher, A., and Samelson, L. A., J. Immunol. 149, 1847, 1992). We found that tyrosine phosphorylation of ezrin was markedly enhanced by ligation of either CD3 or CD4 antigen and peaked between 1 and 2 min. Furthermore, stimulations through CD4 and CD3 antigens were additive. Using the cell line HUT 78 T transfected with either normal human CD4 or mutated CD4 molecules unable to associate with p56lck tyrosine kinase, we showed that this kinase plays a major role in the tyrosine phosphorylation of ezrin. Moreover, CD45R ligation studies provided evidence that the membrane-associated tyrosine phosphatase CD45 activity regulates ezrin tyrosine phosphorylation. Subcellular fractionation showed that although ezrin is mainly located in the cytosol of T cells, anti-CD4-induced ezrin phosphorylation involved the membrane fraction, with no concomitant translocation of the protein from the cytosol to the membrane.     

Expression of the CD8alpha beta-heterodimer on CD8(+) T lymphocytes in peripheral blood lymphocytes of human immunodeficiency virus- and human immunodeficiency virus+ individuals

CD8(+) T lymphocytes play a pivotal role in controlling human immunodeficiency virus (HIV)-1 replication in vivo. We have performed four-color flow cytometric analysis of CD8(+) peripheral blood lymphocytes (PBL) from 21 HIV-1 seronegative and 103 seropositive individuals to explore the phenotypic heterogeneity of CD8beta-chain expression on CD8(+) T lymphocytes and to clarify how its expression on CD8(+) T lymphocytes may relate to acquired immunodeficiency syndrome (AIDS) clinical progression. We showed that the single monoclonal antibody (MoAb) 2ST8-5H7, directed against the CD8alpha beta-heterodimer, identifies CD8(+) T lymphocytes as effectively as the conventional combination of anti-CD3 and anti-CD8alpha antibodies. However, we detected a significantly lower mean fluorescence (MF) of anti-CD8alpha beta staining on PBL from HIV-1 seropositive donors as compared with seronegative donors. In fact, CD8(+) T lymphocytes from HIV-1-infected individuals with the lowest CD4 counts showed the lowest levels of CD8alpha beta MF. To explore further this change in CD8alpha beta expression, we assessed the expression of 14 different cell surface molecules on CD8alpha beta+ T lymphocytes of PBL from 11 HIV-1 seronegative and 22 HIV-1 seropositive individuals. The MF of anti-CD8alpha beta staining was significantly reduced on CD8(+) T lymphocyte subsets that showed immunophenotypic evidence of activation. The subset of lymphocytes expressing low levels of CD8alpha beta expressed higher levels of activation, adhesion, and cytotoxic-associated molecules and was predominantly CD45RO+ and CD28(-). Finally, we monitored the expression of the CD8alpha beta-heterodimer on PBL of eight HIV-1-infected individuals over a 16-week period after the initiation of highly active antiretroviral therapy (HAART), including zidovudine (ZDV), lamivudine (3TC), and indinavir (IDV), and found a significant increase in the expression of the CD8alpha beta-heterodimer. These results suggest that antibodies recognizing the CD8alpha beta-heterodimer are useful tools to specifically identify CD8(+) T lymphocytes. Moreover, the quantitative monitoring of CD8alpha beta expression allows the detection of discrete CD8(+) T lymphocyte subsets and may be useful for assessing the immune status of individuals infected with HIV-1.     

Biomechanical analysis and clinical treatment of blunt renal trauma

Moesin, one of the ERM (ezrin; radixin; moesin) family members, is directly associated with the cytoplasmic domain of CD44, which is now thought to be related to the metastatic potential of tumor cells. Using immunohistochemistry we investigated the expression of moesin in normal epidermis and various kinds of epithelial skin tumors: squamous cell carcinoma, verrucous carcinoma, Bowen's disease, solar keratosis, keratoacanthoma, basal cell carcinoma, and extramammary Paget's disease. Normal skin showed positive epidermal staining for moesin with the exception of the stratum corneum. The expression of moesin varied with the type of skin tumor. In basal cell carcinoma, Bowen's disease, and extramammary Paget's disease, moesin expression was either faint or negative. In contrast to Bowen's disease, invasive squamous cell carcinoma showed more intense and heterogeneous staining of the cytoplasm and the cell membrane. Verrucous carcinoma was weakly positive, with a tendency for the moesin to be distributed in the cell membrane. The staining pattern of moesin varied among the different kinds of epithelial skin tumors, and its expression was generally similar to that of the standard form of CD44. These results suggest that moesin is closely inter-related with CD44 in epithelial skin cells as seen in other cellular systems, and that the variable pattern of moesin staining among the skin tumor cells could reflect expression disorders associated with the transformation.     

Identification and functional analysis of the ezrin-binding site in the hyaluronan receptor, CD44

ERM (ezrin, radixin and moesin) proteins function as linkers between the actin cytoskeleton and the plasma membrane. In addition to this structural role, these proteins are highly regulatable making them ideal candidates to mediate important physiological events such as adhesion and membrane morphology and to control formation and breakdown of membrane-cytoskeletal junctions. Recently, a direct interaction in vitro has been demonstrated between ERM proteins and the hyaluronan receptor, CD44. We have mapped the ezrin-binding site to two clusters of basic amino acids in a membrane-proximal 9 amino-acid region within the CD44 cytoplasmic domain. To investigate the functional importance of this interaction in vivo, we created a number of mutations within full-length CD44 and expressed these mutants in human melanoma cells. We demonstrate here that mutations within the ezrin-binding site do not disrupt the plasma membrane localization of CD44 and, in addition, that this region is not required to mediate efficient hyaluronan binding. These studies suggest that ERM proteins mediate the outside-in, rather than inside-out, signalling of adhesion receptors.     

POP66, a paraneoplastic encephalomyelitis-related antigen, is a marker of adult oligodendrocytes

L-selectin on lymphocytes reacts with glycosylated ligands on high endothelial venule walls in lymphoid organs. Through this carbohydrate-dependent interaction, rolling and initial attachment of lymphocytes to endothelium is mediated. Here we have studied an earlier described L-selectin-induced homotypic aggregation, to further elucidate the events that occur after engagement of L-selectin. It was found that the interaction of L-selectin with fucoidan, but not with other carbohydrates, or with monoclonal antibodies directed against the carbohydrate recognition domain of L-selectin, resulted in homotypic aggregation among both B- or T lymphocytes. Importantly, this aggregation was shown to be both lymphocyte function-associated antigen-1 (LFA-1) and calcium-independent. Furthermore, for aggregation metabolic energy was required, and signalling via protein tyrosine kinase appeared to be involved. Neither de novo protein synthesis, protein kinase C mediated signalling, Gi-protein mediated signal transduction, nor calcium mobilization were required for aggregation. During aggregation, L-selectin was not shed from the lymphocyte's cell surface. Finally, it was found that the lymphocyte binding capacity to high endothelial venules on cryostat sections was not altered upon triggering these lymphocytes via L-selectin. Interestingly, L-selectin-triggered cells showed increased binding to paracortical areas in peripheral lymph nodes. Our data suggest that signals via L-selectin, might lead to altered expression of cell surface molecules, important in interactions other than the first stage of lymphocyte rolling.     

Interleukin-15 induces adhesion receptor redistribution in T lymphocytes

Chemotactic factors such as cytokines and chemokines direct the migration of leukocytes into inflammatory sites. Chemokines play a role regulating both the expression and adhesive properties of leukocyte integrins. We have recently described an additional function of chemokines in the induction of cell polarization and adhesion receptor redistribution during the initial step of leukocyte locomotion. We herein report that interleukin (IL)-15, a newly described cytokine with chemotactic properties, is able to induce uropod formation on T lymphoblasts to which intercellular adhesion molecule (ICAM)-3, a leukocyte-restricted counter-receptor for the lymphocyte function-associated antigen (LFA)-1 integrin, is redistributed. Other adhesion molecules, such as ICAM-1, ICAM-2, CD43 and CD44, also redistributed to the uropod, although in a lower proportion of the cells. The induction of uropod formation by IL-15 was observed on T lymphoblasts adhering to the integrin ligands fibronectin, vascular cell adhesion molecule (VCAM)-1 and ICAM-1, but not to bovine serum albumin or poly-L-lysine. The effect of IL-15 was dose dependent and specifically inhibited by a monoclonal antibody (mAb) against this cytokine. Blocking experiments with anti-IL-2 receptor beta chain mAb showed an inhibitory effect on IL-15-mediated redistribution of ICAM-3, whereas no effect was observed in the presence of anti-IL-2 receptor alpha chain mAb. The uropod induced by IL-15 is enriched in many different adhesion receptors and, being well exposed to the external milieu, is likely to modulate the adhesive properties of lymphocytes.     

The expression of carbohydrate antigens in activated T cells and in autoimmune diseases

Cell surface carbohydrate antigens have been implicated in cell differentiation and maturation and may play a role in immunoregulation. The expression of carbohydrates in peripheral blood lymphocytes (PBL) was studied by double immunofluorescence flow cytometry, using MoAbs CT1 and CT2 but only a small proportion of cells bound these MoAbs. MoAbs CT1, CT2 and the lectin vicia villosa (VV) which share specificity for Gal NAc were then used to examine lymphocytes from patients with systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), Behcet's disease (BD) and IgA nephropathy. A significant increase in MoAbs CT1 CT2 and VV binding CD4 or CD8 cells was found only with lymphocytes from patients with SLE. However, MoAbs CT or VV binding lymphocytes from healthy subjects were significantly up-regulated by activation with a mitogen (PHA), cross-linked anti-CD3 MoAb or a common antigen (65kDa heat shock protein), suggesting that an increased proportion of T cells expressing these carbohydrates results from any of the three types of lymphocyte activating agents. Inhibition studies were then carried out to determine the relationship between the MoAbs CT1 and CT2, VV and GalNAc. Indeed, VV binding to T cells was significantly inhibited by either MoAbs CT1 or CT2, or GalNAc but not GlucNAc, suggesting that VV shares a common binding site with MoAb CT and that GalNAc may constitute one of the sugar receptors. Investigations of lymphocytes from adult peripheral blood in health and disease suggest that carbohydrate antigens may play a role in activation and immunoregulation.     

Direct interaction of the Rho GDP dissociation inhibitor with ezrin/radixin/moesin initiates the activation of the Rho small G protein

The Rho GDP dissociation inhibitor (GDI) forms a complex with the GDP-bound form of the Rho family small G proteins and inhibits their activation. The GDP-bound form complexed with Rho GDI is not activated by the GDP/GTP exchange factor for the Rho family members, suggesting the presence of another factor necessary for this activation. We have reported that the Rho subfamily members regulate the ezrin/radixin/moesin (ERM)-CD44 system, implicated in reorganization of actin filaments. Here we report that Rho GDI directly interacts with ERM, initiating the activation of the Rho subfamily members by reducing the Rho GDI activity. These results suggest that ERM as well as Rho GDI and the Rho GDP/GTP exchange factor are involved in the activation of the Rho subfamily members, which then regulate reorganization of actin filaments through the ERM system.     

Immunolocalization of CD44 and the ERM family in bone cells of mouse tibiae

We studied the immunohistochemical localization of CD44, hyaluronate receptor, and the ezrin-radixin-moesin (ERM) family, actin binding proteins, in bone cells using confocal laser scanning microscopy and transmission electron microscopy to clarify the mechanism of the organization of their cytoskeletons. In osteoclasts, intense immunoreactivity to CD44 could be detected on their basolateral plasma membranes. There was less reactivity observed in the area of the plasma membrane in direct contact with the bone surface. The immunogold electron-microscopical method revealed that CD44 was mainly localized on the microvilli of the basolateral plasma membrane. The plasma membrane of the clear zone and the ruffled border were not immunolabeled with CD44. As for the ERM family, the basolateral plasma membrane of osteoclasts was stained with antimoesin monoclonal antibody, but not with ezrin or radixin. In osteoblasts attached to the bone surface, immunoreactivity to CD44 was restricted to their cytoplasmic processes. They showed immunoreactivities to radixin and moesin on the cytoplasmic side of their plasma membrane when in contact with each other. However, although osteocytes in the bone matrix demonstrate an intense immunolabeling with CD44 on their plasma membrane, they scarcely show immunoreactivity to the ERM family. These findings suggest that: (1) the CD44-moesin-actin filament system is involved in the organization of cytoskeletons in the basolateral plasma membrane of osteoclasts; and (2) other mechanisms, rather than the CD44 and the ERM family, may be involved in the cells of osteoblast lineage.     

Tobacco use among multi-ethnic Latino populations

The equine homologue of the leucocyte integrin LFA-1 (CD11a/CD18) has been characterized using a panel of four monoclonal antibodies (mAbs). The antibodies labelled almost all leukocytes, thymocytes and lymph node cells from normal horses, and immunoprecipitated two noncovalently associated polypeptides with molecular weights of 180 kDa and 100 kDa, respectively. The antigen recognized by one mAb could be precipitated by another in this cluster in a sequential immunoprecipitation assay. The mAbs, however, did not block the activities on lymphocyte function of one another. A mAb to the beta subunit of human LFA-1 cross-reacted with equine LFA-1, but an antibody to its alpha subunit did not, suggesting that the beta subunit of the leukocyte integrin may be more highly-conserved. Functionally, H20A and a human CD18 antibody (MHM23) inhibited phorbol ester-mediated homotypic lymphocyte aggregation, whereas mAb CZ3.2 induced rather than inhibited the homotypic cell aggregation. The formation of lymphocyte aggregates induced by CZ3.2 was not blocked by the inhibitory antibodies H20A or MHM23. CZ3.1 seemed to have little inducible or inhibitory effects on homotypic cell aggregation. The mAb CZ3.1 defined a unique LFA-1 determinant present on granulocytes, but absent on lymphocytes in members of an extended horse family, in contrast to the other antibodies which labelled both granulocytes and lymphocytes from these animals. In all other horses tested, no differences in reactivity of CZ3.1 and the other LFA-1 antibodies were observed when the antibodies were tested on lymphocytes or granulocytes. Our results indicate that common epitopes are shared' between human and equine LFA-1, and that the described panel of monoclonal antibodies identifies distinct determinants present on the equine LFA-1 molecule. The following monoclonal antibodies used in this study were given official workshop designations at the Second International Workshop on Equine Leukocyte Antigens (Lunn et al., 1998) CZ3.1 (Cor) = W45; CZ3.2 (Cor) = W77.     

Ligation of CD31/PECAM-1 modulates the function of lymphocytes, monocytes and neutrophils

CD31 or platelet/endothelial cell adhesion molecule (PECAM-1) is a 130-kDa glycoprotein expressed on endothelial cells, granulocytes, a subset of lymphocytes and platelets. In this study, we examined the ability of four monoclonal antibodies (mAb) against different domains of CD31 to modulate the function of T lymphocytes, monocytes and neutrophils. Engagement of CD31 on T lymphocytes results in co-stimulation of T lymphocyte proliferation to suboptimal doses of anti-CD31 mAb. This proliferation is accompanied by secretion of numerous cytokines and chemokines, up-regulation of CD25 and an increase in cell size. Purification of T lymphocytes into CD45RO and CD45RA subsets showed that only naive CD45RA T lymphocytes are co-stimulated by anti-CD31 mAb. Further studies on neutrophils show that engagement of CD31 results in down-regulation of CD62L and up-regulation of CD11b/CD18 as well as oxidative burst, as assessed by superoxide release. In addition, ligation of CD31 on monocytes results in TNF-alpha secretion, and studies with various cell signaling inhibitors indicate that tyrosine kinases and cAMP-dependent kinases are involved in monocyte activation via CD31. Of the four mAb used in this study, only two activated human leukocytes. These mAb were PECAM-1.3 and hec7, which bind to domains 1 and 2 of CD31. We conclude that engagement of domains 1 and 2 of CD31 results in outside-in signaling in leukocytes.     

Cell-type specificity of anti-CD45 autoantibodies in systemic lupus erythematosus

OBJECTIVE: To determine the specificity of anti-CD45 autoantibodies in systemic lupus erythematosus (SLE) for native CD45 and for CD45 expressed by T cells and B cells, and at different stages of cellular activation. METHODS: CD45 purified from different types of lymphocytes was examined by immunoblotting with sera from patients with SLE. Indirect immunofluorescence experiments were performed with purified anti-CD45 autoantibodies. RESULTS: IgM anti-CD45 autoantibodies in SLE recognize native CD45 expressed on the surface membrane of viable lymphocytes and CD45 purified from activated peripheral T cells and certain T cell lines, but not CD45 purified from B cells or resting peripheral T cells. The presence or absence of reactivity is independent of the individual isoforms expressed. CONCLUSION: Recognition of CD45 by IgM antilymphocyte autoantibodies in SLE varies with the lineage and state of activation of the lymphocyte target. This pattern of reactivity is consistent with autoantibody specificities involving CD45 glycoforms, rather than CD45 isoforms.     

Identification of EBP50: A PDZ-containing phosphoprotein that associates with members of the ezrin-radixin-moesin family

Members of the ezrin-radixin-moesin (ERM) family of membrane-cytoskeletal linking proteins have NH2- and COOH-terminal domains that associate with the plasma membrane and the actin cytoskeleton, respectively. To search for ERM binding partners potentially involved in membrane association, tissue lysates were subjected to affinity chromatography on the immobilized NH2-terminal domains of ezrin and moesin, which comprise the ezrin-radixin-moesin-association domain (N-ERMAD). A collection of polypeptides at 50-53 kD from human placenta and at 58-59 kD from bovine brain bound directly to both N-ERMADs. The 50-53-kD placental proteins migrated as a major 50-kD species after phosphatase treatment, indicating that the heterogeneity is due to different phosphorylation states. We refer to these polypeptides as ERM-binding phosphoprotein 50 (EBP50). Sequence analysis of human EBP50 was used to identify an approximately 2-kb human cDNA that encodes a 357-residue polypeptide. Recombinant EBP50 binds tightly to the N-ERMADs of ezrin and moesin. Peptide sequences from the brain candidate indicated that it is closely related to EBP50. EBP50 has two PSD-95/DlgA/ZO-1-like (PDZ) domains and is most likely a homologue of rabbit protein cofactor, which is involved in the protein kinase A regulation of the renal brush border Na+/H+ exchanger. EBP50 is widely distributed in tissues, and is particularly enriched in those containing polarized epithelia. Immunofluorescence microscopy of cultured cells and tissues revealed that EBP50 colocalizes with actin and ezrin in the apical microvilli of epithelial cells, and immunoelectron microscopy demonstrated that it is specifically associated with the microvilli of the placental syncytiotrophoblast. Moreover, EBP50 and ezrin can be coimmunoprecipitated as a complex from isolated human placental microvilli. These findings show that EBP50 is a physiologically relevant ezrin binding protein. Since PDZ domains are known to mediate associations with integral membrane proteins, one mode of membrane attachment of ezrin is likely to be mediated through EBP50.     

T cell activation through the CD43 molecule leads to Vav tyrosine phosphorylation and mitogen-activated protein kinase pathway activation

CD43, the most abundant membrane protein of T lymphocytes, is able to initiate signals that lead to Ca2+ mobilization and interleukin-2 production, yet the molecular events involved in signal transduction pathway of the CD43 molecule are only beginning to be understood. We have shown recently that cross-linking CD43 on the cell surface of human T lymphocytes with the anti-CD43 monoclonal antibody L10 leads to CD43-Fyn kinase interactions and to Fyn phosphorylation on tyrosine residues. This interaction seems to be mediated by the SH3 domain of Fyn and a proline-rich sequence located in the cytoplasmic domain of CD43. Here we show that CD43-specific activation of human T lymphocytes induced tyrosine phosphorylation of the adaptor protein Shc and of the guanine exchange factor Vav, as well as the formation of a macromolecular complex that comprises Shc, GRB2, and Vav. CD43 ligation resulted in enhanced formation of Vav.SLP-76 complexes and in the activation and nuclear translocation of ERK2. Cross-linking of the CD43 molecule in 3T3-CD43(+) cells induced luciferase activity from a construct under the control of the Fos serum responsive element. Altogether, these data suggest that the mitogen-activated protein kinase pathway is involved in CD43-dependent interleukin-2 gene expression.     

CD30 expression identifies a functional alloreactive human T-lymphocyte subset

BACKGROUND: CD30 is a member of the tumor necrosis factor/nerve growth factor receptor family and has been proposed as a marker of specific cytokine-producing subsets in humans. Previous studies have examined the expression of CD30 on established T helper type 1 and T helper type 2 cell clones and the function of CD30+ cells after mitogenic stimulation. In this study, we examined the development and function of CD30+ T cells generated in response to alloantigen. METHODS: Primary one-way mixed lymphocyte reactions were established, and the expression of CD30 on T lymphocytes was determined by immunofluorescence and flow cytometry. Fluorescence-activated cell sorting was utilized to define the cytokine profile of alloactivated CD30+ cells after restimulation with anti-CD3 monoclonal antibodies or alloantigen. The effect of cyclosporine on the development of CD30+ cells, and on cytokines produced by CD30+ T lymphocytes, in response to alloantigen was determined. RESULTS: CD30+ T lymphocytes could be detected on day 2 of mixed lymphocyte reactions and continued to increase in number and proportion through day 6. Both CD4 and CD8 T cells expressed CD30 after primary alloantigenic stimulation. CD30+ T cells are a subset of alloactivated T cells and are the major source of interferon-gamma and interleukin-5 produced in response to alloantigen. Cyclosporine partially, but not completely, inhibits the development of CD30+ cells, and has a greater effect on interferon-gamma production than on interleukin-5 production. CONCLUSIONS: CD30+ T lymphocytes may constitute an important immunoregulatory subset in human allograft rejection.     

Specific regulation of VLA-4 and alpha 4 beta 7 integrin expression on human activated T lymphocytes

Modulation of VLA integrins was studied in several human T cell clones upon specific and nonspecific cellular activation. Human activated T lymphocytes down-regulated both alpha 4 beta 1 and alpha 4 beta 7 integrins upon specific recognition of alloantigens (cytotoxic T cells) or in the presence of Staphylococcus enterotoxin B (superantigen recognizing noncytotoxic T cells). In contrast, the expression of other membrane integrins, such as VLA-1 and VLA-5 integrins, was not modified. Down-regulation of alpha 4 beta 1 and alpha 4 beta 7 integrins was observed as early as 3 h after stimulation, lasted later than 72 h and was partially inhibited by cytochalasin D. Interestingly, neither target cells nor NK cells modulated CD49d expression after interaction with T cells of K562, respectively, suggesting that CD49d expression was linked to specific T cell activation. The down-regulation of the CD49d chain in T cell clones stimulated with immobilized anti-CD3 mAbs confirmed the role of TCR-mediated activation in CD49d regulation. However, the CD3-independent cellular aggregation induced by soluble anti-CD43 mAb was also able to strongly down-regulate alpha 4 beta 1 and alpha 4 beta 7. The present work shows the first evidence that CD49d subunit-bearing integrin expression is distinctly regulated from other integrins after Ag or superantigen recognition by human activated T cells. CD49d modulation may be relevant for the traffic and tissue localization of locally activated T cells during immune responses.     

Adenosine diphosphate (ADP)-ribosylation of the guanosine triphosphatase (GTPase) rho in resting peripheral blood human T lymphocytes results in pseudopodial extension and the inhibition of T cell activation

Scrape loading Clostridium botulinum C3 exoenzyme into primary peripheral blood human T lymphocytes (PB T cells) efficiently adenosine diphosphate (ADP)-ribosylates and thus inactivates the guanosine triphosphatase (GTPase) Rho. Basal adhesion of PB T cells to the beta1 integrin substrate fibronectin (Fn) was not inhibited by inactivation of Rho, nor was upregulation of adhesion using phorbol myristate acetate (PMA; 10 ng/ml) or Mn++ (1 mM) affected. Whereas untreated PB T cells adherent to Fn remain spherical, C3-treated PB T cells extend F-actin-containing pseudopodia. Inactivation of Rho delayed the kinetics of PMA-dependent PB T cell homotypic aggregation, a process involving integrin alphaLbeta2. Although C3 treatment of PB T cells did not prevent adhesion to the beta1 integrin substrate Fn, it did inhibit beta1 integrin/CD3-mediated costimulation of proliferation. Analysis of intracellular cytokine production at the single cell level demonstrated that ADP-ribosylation of Rho inhibited beta1 integrin/ CD3 and CD28/CD3 costimulation of IL-2 production within 6 h of activation. Strikingly, IL-2 production induced by PMA and ionomycin was unaffected by C3 treatment. Thus, the GTPase Rho is a novel regulator of T lymphocyte cytoarchitecture, and functional Rho is required for very early events regulating costimulation of IL-2 production in PB T cells.