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E A Clark

Publications and source records attributed to E A Clark.

At least 91 records · Page 5Linked to original sources
PubMed1994

Cross-linking CD40 on B cells rapidly activates nuclear factor-kappa B.

The B cell-associated surface molecule CD40 functions to regulate B cell responses. Cross-linking CD40 on B cells can lead to homotypic cell adhesion, IL-6 production, and, in combination with cytokines, to Ig isotype switching. Tyrosine kinase activity is increased shortly after engagement of this receptor. Little is known about how the very early events induced by CD40 cross-linking link to cellular responses. In this study, we demonstrate that nuclear factor (NF)-kappa B and NF-kappa B-like transcription factors are activated after cross-linking CD40 on resting human tonsillar B cells and on B cell lines. The activation is rapid and is mediated through a tyrosine kinase-dependent pathway. The complexes detected in electrophoretic mobility shift assays contain p50, p65 (RelA), c-Rel, and most likely other components. By using transient transfection assays, we found that cross-linking CD40 supports NF-kappa B-dependent gene expression. Our results define the NF-kappa B system as an intermediate event in CD40 signaling and suggest that the CD40 pathway can influence the expression of B cell-associated genes with NF-kappa B consensus sites.

Animals↗
PubMed1994

Structurally distinct disintegrins contortrostatin and multisquamatin differentially regulate platelet tyrosine phosphorylation.

Tyrosine phosphorylation of multiple platelet proteins is regulated by the integrin alpha IIb beta 3. In order to further examine integrin-regulated tyrosine phosphorylation, we have used small Arg-Gly-Asp-containing snake venom proteins (termed disintegrins) that inhibit platelet aggregation to competitively block the agonist-induced binding of fibrinogen to alpha IIb beta 3. One structurally unique disintegrin, contortrostatin (which appears to be a disulfide-linked dimer of 13.5 kDa with two Arg-Gly-Asp sites), was found to trigger signaling events typically mediated by fibrinogen cross-linking of alpha IIb beta 3, as demonstrated by tyrosine phosphorylation of the tyrosine kinase pp72syk and a 140-kDa protein. Contortrostatin and another disintegrin, multisquamatin (a monomer of 5.7 kDa with a single Arg-Gly-Asp site), did not affect thrombin-induced platelet shape change, secretion, or integrin-independent tyrosine phosphorylation; however, they inhibited aggregation and aggregation-dependent tyrosine phosphorylation of numerous proteins, including the focal adhesion kinase pp125FAK. Our results suggest that structurally distinct disintegrins have varying effects on tyrosine phosphorylation; while monomeric multisquamatin and dimeric contortrostatin both inhibit aggregation-dependent tyrosine phosphorylation, contortrostatin also possesses a unique functional activity that allows it to activate an intracellular signaling pathway leading to tyrosine phosphorylation. This activity may be involved in the function of this snake venom protein on hemostasis.

Amino Acid Sequence↗
PubMed1994

Costimulation through CD28 enhances T cell-dependent B cell activation via CD40-CD40L interaction.

Changes in T cell helper function were analyzed when anti-CD3-activated T cells were costimulated with mAbs to the CD28 receptor (anti-CD28). T cell-dependent B cell growth and differentiation were consistently augmented if anti-CD3 stimulated-T cells were simultaneously activated with anti-CD28. Although anti-CD28 enhanced IL-2 and IL-4 production, it did not increase B cell responses solely by augmenting production of soluble lymphokines. Anti-CD28 costimulation induced increases on T cells of CD40 ligand (CD40L), known to promote B cell proliferation and Ig secretion. Because anti-CD28 promoted T cell helper functions and expression of CD40L, we examined the dependence for CD40L during T cell-dependent B cell responses. Although soluble CD40 fusion proteins only partially inhibited T cell-dependent B cell activation, we found a strict requirement for CD40L expression at initiating B cell responses. Both CD40L expression and T cell help were blocked by cyclosporin A after TCR cross-linking, and, unlike T cell proliferation, both remained cyclosporin A sensitive during CD28 costimulation. In addition, anti-CD28 could not compensate for the T cell helper deficiency of hyper IgM syndrome patients who lack functional CD40L. Thus, anti-CD28-induced T cell help is delivered via a CD40L-dependent process. The fact that cross-linking CD40 on B cells promotes expression of the B7/BB-1 ligand for CD28 suggest T and B interactions may have a reciprocal amplification mechanism.

Antigens, CD↗
PubMed1994

Differential expression of ZAP-70 and Syk protein tyrosine kinases, and the role of this family of protein tyrosine kinases in TCR signaling.

TCR stimulation results in the tyrosine phosphorylation of a number of cellular substrates. We have recently identified a 70-kDa protein tyrosine kinase, ZAP-70, which associates with the human TCR zeta-chain after TCR stimulation. We report here the isolation and sequence of a cDNA clone that encodes murine ZAP-70. Murine and human ZAP-70 share 93% amino acid identity and are homologous to the 72-kDa protein tyrosine kinase Syk. Syk has been implicated in the signal transduction pathways of the B cell membrane Ig and high affinity IgE receptors, Fc epsilon RI. In addition, we examined the tissue distribution of ZAP-70 and Syk in human and murine thymocyte subsets, B cells, and peripheral T cell subsets. ZAP-70 protein is expressed in all major thymocyte populations, with the level of expression being comparable to that found in both CD4+ and CD8+ peripheral T cells. Although Syk protein is also present in all thymocyte subsets, expression of Syk protein is down-regulated threefold to fourfold in peripheral T cells. In contrast to ZAP-70, expression of Syk is 12- to 15-fold higher in peripheral B cells when compared with peripheral T cells. In addition, whereas T cell stimulation results in down-regulation of Lck, no significant change in ZAP-70 or Syk protein is detected. Finally, we provide evidence that both ZAP-70 and Syk can associate with the TCR after TCR stimulation. With the use of a heterologous expression system, we show that, like ZAP-70, Syk is dependent upon a Src-family protein tyrosine kinase for association with the phosphorylated zeta-chain. Thus, the differential expression of these kinases suggests the possibility of different roles for ZAP-70 and Syk in TCR signaling and thymic development.

Amino Acid Sequence↗
PubMed1994

Molecular cloning of human Syk. A B cell protein-tyrosine kinase associated with the surface immunoglobulin M-B cell receptor complex.

B cell antigen receptors are multicomponent complexes consisting of the surface immunoglobulin and accessory molecules with associating protein-tyrosine kinases. A spleen tyrosine kinase, Syk, in porcine B cells and a 72-kDa protein-tyrosine kinase, PTK72, in murine B cells associate with the B cell antigen receptor. Herein, we report the isolation of a full-length cDNA encoding the human homologue of Syk. This cDNA predicted a polypeptide consisting of two NH2-terminal SH2 domains and a COOH-terminal tyrosine kinase domain. Syk is highly conserved between human and swine and is homologous to the T cell-associated protein-tyrosine kinase ZAP-70. Both Syk mRNA and protein were detected in cells derived from multiple hematopoietic lineages. Within the B cell compartment, Syk was expressed from pro-B cells to plasma cells. In vitro kinase assays conducted on the human Syk protein isolated from B cells revealed the presence of autophosphorylation activity on Syk tyrosine residues. Tyrosine phosphorylation of Syk associating with the B cell receptor complex in human was augmented rapidly after surface immunoglobulin cross-linking. The human SYK locus was mapped to chromosome 9 at band q22.

Amino Acid Sequence↗
PubMed1994

How B and T cells talk to each other.

The B cells of the immune system, which secrete antibodies against foreign antigens, are fully specific and effective only after maturation in lymph nodes and other lymphoid tissues. Immunocompetent T cells play a crucial part in this process, but the molecular details of the way in which the two cell types interact have only recently become apparent.

Animals↗
PubMed1994

CD40 molecules induce down-modulation and endocytosis of T cell surface T cell-B cell activating molecule/CD40-L. Potential role in regulating helper effector function.

The T-BAM/CD40-L molecule on CD4+ T cells interacts with B cell CD40 molecules to deliver contact-dependent signals that drive B cell activation and Ig secretion. Cell surface T-BAM/CD40-L expression is transient and may be closely regulated in order to limit the activation and clonal selection of noncognate B cells. We demonstrate that B cells, but not non-B cells, rapidly and specifically down-modulate surface T-BAM/CD40-L expression in a contact-dependent and temperature-sensitive manner that renders T cells unable to activate resting bystander B cells. Because the ability to down-modulate T-BAM/CD40-L correlated with CD40 expression, the role of CD40 molecules in down-modulating its ligand was directly assessed. Anti-CD40 mAb, but not control mAb, block B cell-induced T-BAM/CD40-L down-modulation. Furthermore, CD40+ nonlymphoid transfectants specifically down-modulate surface T-BAM/CD40-L expression. B cells induce T-BAM/CD40-L internalization into cytoplasmic compartments in a process that is inhibited by cytochalasin B. Pretreatment of activated T cells with lysosomotropic agents does not affect CD40-induced down-modulation of surface T-BAM/CD40-L but results in a marked accumulation of T-BAM/CD40-L in cytoplasmic vesicles. Together, these studies strongly suggest that CD40 induced T-BAM/CD40-L down-modulation occurs, in part, by receptor-mediated endocytosis followed by lysosomal degradation and may represent a mechanism to regulate CD4+ T cell helper effector functions.

Amino Acid Sequence↗
PubMed1994

Properties of mouse CD40: cellular distribution of CD40 and B cell activation by monoclonal anti-mouse CD40 antibodies.

We describe here the derivation of a rat monoclonal antibody (mAb) against mouse CD40 (designated 3/23), which stains 45-50% of spleen cells of adult mice, approximately 90% of which are B cells. Interestingly, some 5-10% of both CD4+ and CD8+ T cells in the spleens of (some, but not all) adult, unimmunized mice are also CD40+, whereas CD40+ cells were not detectable in the thymus, even following collagenase digestion. Some 35-40% of lymphoid cells in the bone marrow of adult mice are CD40+ and virtually all of these are B220+, and hence of the B cell lineage: triple-color flow cytometry showed that CD40 is expressed at low levels on some 30% of pre-B cells, at intermediate levels on 80% of immature B cells and on essentially all mature B cells in the bone marrow. These results, therefore, suggest that in the mouse CD40 is expressed relatively late during the process of B cell differentiation. The mAb induced marked up-regulation of major histocompatibility complex class II molecules, CD23 and B7.2 antigens on mature B cells. It also stimulated modest levels of DNA synthesis in mature B cells by itself: this was markedly enhanced by suboptimal concentrations of mitogenic (but not non-mitogenic) anti-mu and anti-delta mAb, and moderately enhanced by co-stimulation with interleukin-4. Hypercross-linking of CD40 (using biotinylated mAb and avidin) also enhanced the proliferative response to anti-CD40.

Animals↗
PubMed1994

CD40 and its ligand in the regulation of humoral immunity.

T cells provide help for B cell antibody production by cell-cell contact and by soluble factors. CD40L is the predominant protein induced on activated T cells which constitutes contact-dependent help, and lack of CD40L or blocking CD40-CD40L interactions leads to severely impaired antibody production. In addition to CD40-CD40L, B and T cells express costimulatory, accessory molecules which amplify T and B cell function and allow for reciprocal dialogues during antigen presentation. Interactions between costimulatory counter-receptors can determine lymphocyte activation or nonresponsiveness, and provides a means for regulating self-tolerance.

Animals↗
PubMed1994

Regulation of lymphocyte activation by the cell-surface molecule CD22.

Accessory molecules play an important role in the regulation of lymphocyte activation mediated by the B-cell antigen receptor (BCR). CD22 is one such accessory molecule expressed on B-lineage cells. Here, Che-Leung Law and colleagues review current knowledge on the structure-function relationship between CD22 and the BCR, discuss the role of CD22 as a cell-adhesion molecule and suggest models for potential in vivo functions of CD22.

Animals↗
PubMed1994

Cell-cell interactions that regulate the development of B-lineage cells.

Significant progress has been made recently in our understanding of the functions of lymphocyte-associated surface proteins. The latest developments involve the identification of ligands or co-receptors for many of these surface proteins. The signal transduction mechanisms utilized by these molecules are also beginning to be elucidated.

Animals↗
PubMed1994

Regulation of protein tyrosine kinases in platelets.

Platelet activation is accompanied by a dramatic increase in tyrosine phosphorylation of many cellular proteins. Phosphorylation of these proteins occurs in successive waves during the activation process, suggesting that several distinct mechanisms, occurring in a temporal order, regulate protein tyrosine kinases and/or phosphatases in activated platelets. Several tyrosine kinases, including Src family kinases, Syk and FAK, have been implicated in these phosphorylation events. These kinases are regulated by distinct receptor-mediated events involving activation of their catalytic activity and alterations in their cellular localization.

Blood Platelets↗
PubMed1994

The role of CD40 and CD80 accessory cell molecules in dendritic cell-dependent HIV-1 infection.

We investigated the role of blood dendritic cells (DCs) in transmission of HIV-1 from infected to uninfected CD4+ T cells, and the accessory molecules involved. DCs promoted transmission from infected to uninfected CD4+ cells, but DCs themselves were not infectable. DC-mediated transmission was blocked by MAb to CD4 and MHC class II, but strongly increased by MAb to CD40 on DCs or CD28 on T cells. The DC-dependent infection was inhibitable by anti-CD80 and a soluble fusion protein of the CD80 ligand, CTLA4; soluble CTLA4 immunoglobulin also blocked infection augmented by cross-linking CD40. These data suggest a linkage between CD40-CD40L and CD28-CD80 counterreceptors on DCs and T cells, and spread of HIV infection in vivo.

Antibodies, Monoclonal↗
PubMed1994

Immunologic markers in a longitudinal study of aging in pigtailed macaques (Macaca nemestrina).

Blood lymphocyte subsets, serum immunoglobulins (Ig), response of lymphocytes to mitogens, and natural killer (NK) cell activity were evaluated as potential biomarkers for primate aging. All were evaluated in a cross-sectional study of 60 pigtailed macaques (Macaca nemestrina) ranging from 2 to 32 years of age. Lymphocyte responses to mitogens were lower in older animals than in most younger ones. NK cell activity showed no clear relation to age cohort. In a longitudinal study of lymphocyte subsets and immunoglobulins, class I restricted T cells (CD8+bri) of the memory (CD18+bri) subtype increased with age, whereas those of the naive (CD18+dull) subtype decreased with age in females. A class II restricted T-cell subset (CD4+CD45RA-), which includes memory T cells, increased with age in females. Serum IgA increased. These results support the utility of memory and naive subsets of CD8+ T cells, CD4+ T cells, and serum IgA as biomarkers for longitudinal studies. Total lymphocytes, total T cells, CD4+ T cells, and CD8+ T cells may also be useful in this species.

Aging↗
PubMed1994

T-cell proliferation to subinfectious SIV correlates with lack of infection after challenge of macaques.

OBJECTIVES: To analyze correlates of protection in macaques exposed to SIV. METHODS: Peripheral blood mononuclear cells (PBMC) from macaques inoculated intrarectally with various dilutions of SIV were examined for their in vitro proliferative response to SIV envelope peptides and generation of SIV-specific antibodies. Some macaques previously exposed intravenously to subinfectious doses of SIV were subsequently challenged 16 months later with an infectious intrarectal dose of SIV. RESULTS: The viral-specific immune responses of macaques exposed to infectious doses of SIV were characterized by generation of antibodies and weak or undetectable T-cell-mediated responses. In contrast, macaques inoculated with doses of SIV below the threshold required for seroconversion and recovery of virus exhibited T-cell proliferation in response to SIV envelope synthetic peptides. The macaques that had previously been exposed to SIV resisted the subsequent virus challenge, whereas the naive macaques (never exposed to SIV) all became infected. CONCLUSIONS: The inability to productively infect macaques previously exposed to subinfectious doses of SIV suggests that a T-cell-mediated response may confer long-term protection against infection, and that AIDS vaccines should be designed to optimize the cellular arm of the immune response.

Amino Acid Sequence↗
PubMed1994

Intrarectal inoculation of macaques by the simian immunodeficiency virus, SIVmne E11S: CD4+ depletion and AIDS.

Macaca nemestrina and Macaca fascicularis were inoculated with various doses of a single-cell clone of SIVmne-infected HuT 78 cells (E11S) by both the intravenous and intrarectal routes. Animals inoculated intravenously at each dose seroconverted and virus was isolated from peripheral blood mononuclear cells, but only the high-dose intrarectally exposed macaques became viremic and seroconverted. However, some seronegative, virus isolation negative intrarectally inoculated macaques showed evidence of infection and disease.

Animals↗
PubMed1993

On the role of the platelet membrane skeleton in mediating signal transduction. Association of GP IIb-IIIa, pp60c-src, pp62c-yes, and the p21ras GTPase-activating protein with the membrane skeleton.

The platelet plasma membrane is lined with a membrane skeleton composed of short actin filaments, actin-binding protein, spectrin, vinculin, and other unidentified proteins. It is connected to the outside of the cell through association with the cytoplasmic domains of transmembrane receptors. In detergent-lysed platelets, cytoplasmic actin filaments are sedimented by centrifugation at 15,600 x g, but the sedimentation of membrane skeleton fragments requires higher g-forces (100,000 x g). In the present study, we show that the major platelet integrin, glycoprotein (GP) IIb-IIIa, sediments from detergent-lysed platelets at 100,000 x g together with fragments of the membrane skeleton that contain the cytoskeletal proteins spectrin, vinculin, and talin. In addition, this cell fraction contained the tyrosine kinases pp60c-src and pp62c-yes and the p21ras GTPase-activating protein (GAP). After thrombin-induced platelet aggregation mediated by fibrinogen binding to GP IIb-IIIa on adjacent platelets, we detected a redistribution of spectrin, talin, vinculin, pp60c-src, and pp62c-yes to the fraction that sediments at 15,600 x g. The redistribution of these proteins from the high-speed detergent-insoluble fraction to the low-speed fraction correlated with the extent of aggregation and was not detected in aggregation-defective thrombasthenic platelets (which lack the GP IIb-IIIa complex). In addition, many of the proteins phosphorylated on tyrosine in activated platelets were present in detergent-insoluble fractions. These results are consistent with the possibilities that 1) GP IIb-IIIa, pp60c-src, pp62c-yes, and GAP associate with a membrane skeleton fraction that contains spectrin, vinculin, and talin, 2) the association of GP IIb-IIIa with adhesive ligand in a platelet aggregate causes components of the membrane skeleton to undergo altered association with cytoplasmic actin filaments, and 3) many of the proteins phosphorylated on tyrosine residues in activated platelets are components of the cytoskeleton. The results imply that the membrane skeleton may play an important role in binding signaling molecules at sites of integrin-cytoskeleton interactions and in mediating signal transduction events in platelets. Further, GP IIb-IIIa-induced redistribution of components of the membrane skeleton and associated signaling molecules may represent an important step in regulating integrin-induced motile events in platelets.

Actins↗