Concentration-dependent effects of insulin on Ca2+ influx in vascular smooth muscle cells of normotensive and spontaneously hypertensive rats

The levels of CD26 expression, their capacity to induce protein tyrosine phosphorylation and their functional implication in natural killer (NK) cytolysis have been studied. It was found that only a small fraction (12-15%) of peripheral NK cells expresses CD26 compared with the high expression (99%) found in NK clones. The protein tyrosine phosphorylation mediated by means of CD26 activation was studied in NK cells treated with the anti-CD26 MoAb 134-2C2, and two new proteins of 50 and 21 kDa appeared phosphorylated in tyrosine residues. To study the influence of CD26 antigen in NK lysis, we analysed the lytic capacity of NK cells stimulated with different anti-CD26 MoAbs or after separation into CD26+ and CD26- subsets and using K562 as target cells. Under these conditions, no differences were found in the chromium release by the target cells. Redirected lysis through CD16 was also measured by arming the effector cells (CD26+ and CD26-) with anti-CD16 antibody and using K562 as target cells. It was found that CD26- cells showed significantly less CD16-dependent lysis than CD26+ cells. These results indicate that CD26 is related to the CD16-dependent lysis but not to NK cytolysis which may be caused by mediation of protein tyrosine phosphorylation.     

Human NK cell and ADCC reactivity against xenogeneic porcine target cells including fetal porcine islet cells

In vitro studies of human NK cell-mediated cytotoxicity and ADCC against porcine target cells were performed. Stimulation of human PBMC responder cells with either allogeneic or xenogeneic porcine cells led to a marked increase in NK cell reactivity. Maximum reactivity was reached following 3-6 days of in vitro culture. The sensitivity of target cells ranked as follows: K562 > porcine PHA-induced lymphoblasts > resting porcine PBMC. Limiting dilution analysis showed that allo- and xeno-stimulation in vitro led to differentiation of similar frequencies of effector NK cells. Split culture experiments showed that single NK effector cells were cytotoxic against both K562 and porcine lymphoblasts, demonstrating that individual NK cells lack species specificity. NK effector cell generation stimulated by xenogeneic cells was cyclosporin A (CsA) sensitive and dependent on the presence of autologous responder T lymphocytes, a dependence that was completely reconstituted by the sole addition of human IL-2. Xenostimulation of enriched CD3+ cells also led to a preferential appearance of CD16+ or CD56+ lymphoblasts. Natural xenoreactive human anti-porcine antibodies are mainly of IgM and IgG2 subclasses, but antibodies in xenoimmunised patients reactive against porcine lymphocytes and fetal porcine islet cells were also of IgG1 and IgG3 subclasses. The same subclass distribution was found among antibodies specific for gal(alpha)1,3 gal epitopes as shown by tests performed with alpha1,3 galactosyltransferase-transfected Raji cells (human Burkitt lymphoma cells). Natural antibodies did not mediate ADCC, whereas gal(alpha)1,3 gal-specific antibodies in sera from xenoimmunised patients did. Fetal porcine islet cells were sensitive to human NK cell-mediated cytotoxicity and to ADCC mediated by xenoimmune sera.     

Normal Syk protein level but abnormal tyrosine phosphorylation in B-CLL cells

One characteristic of B cells that accumulate during chronic lymphocytic leukemia (CLL) is their highly heterogeneous functional responses to B cell receptor (BCR) stimulation. Leukemic B cells with very poor responses have defective rapid tyrosine phosphorylation of numerous substrates, especially phospholipase C (PLC)gamma, as well as a defective calcium elevation on BCR stimulation. This points to a defect in BCR-associated protein tyrosine kinase (PTK). We investigated whether a defect in Syk, a PTK that is pivotal in coupling BCR to downstream signaling events, could account for these alterations. Syk tyrosine phosphorylation triggered by BCR ligation was severely impaired in B-CLL cells with low calcium responses to anti-mu stimulation. Syk associations were also defective, as concomitant tyrosine phosphorylation of a Syk-associated 145 kDa protein comigrating with PLCgamma-2 was only detected in responding B-CLL cells. By contrast, we found similar expression of the kinase regardless of B-CLL cell responsiveness. These results are consistent with the possibility that very proximal BCR signaling elements in some B-CLL cells are unable to connect with downstream biochemical events dominated by tyrosine phosphorylation and the potential docking function of Syk PTK.     

Physical and functional association of cortactin with Syk in human leukemic cell line K562

Human leukemic cell line K562 is induced to differentiate into the megakaryocytic lineage by stimulation with 12-O-tetradecanoylphorbol-13-acetate (TPA). We demonstrate here that TPA stimulation increases tyrosine phosphorylation of an 80-kDa protein at an early stage of megakaryocytic differentiation and that this 80-kDa protein is identical with cortactin. Since tyrosine kinase Syk was activated by TPA stimulation, we examined the possibility that cortactin is a potential substrate of Syk in K562 cells. TPA-induced tyrosine phosphorylation of cortactin was decreased profoundly by overexpression of dominant-negative Syk. Furthermore, cortactin was associated with Syk even before TPA stimulation. Since cortactin was previously referred as an 80/85-kilodalton pp60src substrate, we examined the association between Src and cortactin, whereas its association could not be detected. These data suggest that Syk phosphorylates cortactin in K562 cells upon TPA treatment.     

Constitutive tyrosine phosphorylation of the T-cell receptor (TCR) zeta subunit: regulation of TCR-associated protein tyrosine kinase activity by TCR zeta

The T-cell receptor (TCR) zeta subunit is an important component of the TCR complex, involved in signal transduction events following TCR engagement. In this study, we showed that the TCR zeta chain is constitutively tyrosine phosphorylated to similar extents in thymocytes and lymph node T cells. Approximately 35% of the tyrosine-phosphorylated TCR zeta (phospho zeta) precipitated from total cell lysates appeared to be surface associated. Furthermore, constitutive phosphorylation of TCR zeta in T cells occurred independently of antigen stimulation and did not require CD4 or CD8 coreceptor expression. In lymph node T cells that constitutively express tyrosine-phosphorylated TCR zeta, there was a direct correlation between surface TCR-associated protein tyrosine kinase (PTK) activity and expression of phospho zeta. TCR stimulation of these cells resulted in an increase in PTK activity that coprecipitated with the surface TCR complex and a corresponding increase in the levels of phospho zeta. TCR ligations also contributed to the detection of several additional phosphoproteins that coprecipitated with surface TCR complexes, including a 72-kDa tyrosine-phosphorylated protein. The presence of TCR-associated PTK activity also correlated with the binding of a 72-kDa protein, which became tyrosine phosphorylated in vitro kinase assays, to tyrosine phosphorylated TCR zeta. The cytoplasmic region of the TCR zeta chain was synthesized, tyrosine phosphorylated, and conjugated to Sepharose beads. Only tyrosine-phosphorylated, not nonphosphorylated, TCR zeta beads were capable of immunoprecipitating the 72-kDa protein from total cell lysates. This 72-kDa protein is likely the murine equivalent of human PTK ZAP-70, which has been shown to associate specifically with phospho zeta. These results suggest that TCR-associated PTK activity is regulated, at least in part, by the tyrosine phosphorylation status of TCR zeta.     

Dependence of both spontaneous and antibody-dependent, granule exocytosis-mediated NK cell cytotoxicity on extracellular signal-regulated kinases

Extracellular signal-regulated kinases (ERK, also known as mitogen-activated protein kinases) are serine-threonine kinases transducing signals elicited upon ligand binding to several tyrosine kinase-associated receptors. We have reported that ERK2 phosphorylation and activation follows engagement of the low affinity receptor for the Fc portion of IgG (CD16) on NK cells, and is necessary for CD16-induced TNF-alpha mRNA expression. Here, we analyzed the involvement of ERK in NK cell-mediated cytotoxicity and IFN-gamma expression induced upon stimulation with targets cells, coated or not with Abs. Our data indicate that, as with immune complexes, ERK2 phosphorylation occurs in human primary NK cells upon interaction with target cells sensitive to granule exocytosis-mediated spontaneous cytotoxicity, and that this regulates both target cell- and immune complex-induced cytotoxicity and IFN-gamma mRNA expression. A specific inhibitor of mitogen-activated protein kinase kinase reduced both spontaneous and Ab-dependent cytotoxicity in a dose-dependent manner involving, at least in part, inhibition of granule exocytosis without affecting effector/target cell interaction and rearrangement of the cytoskeleton proteins actin and tubulin. Involvement of ERK in the regulation of Ca2+-dependent cell-mediated cytotoxicity was confirmed, using a genetic approach, in primary NK cells infected with a recombinant vaccinia virus encoding an ERK inactive mutant. These data indicate that the biochemical pathways elicited in NK cells upon engagement of receptors responsible for either spontaneous or Ab-dependent recognition of target cells, although distinct, utilize ERK as one of their downstream molecules to regulate effector functions.     

The presence of CD5LOW+NK cells in normal controls and patients with pulmonary tuberculosis

CD5 antigen is present on all normal alpha beta T cells and some B cells. Human NK cells do not usually express CD5 antigen, but we found a subset of CD5LOW+ (low density of CD5) NK cells in some patients with pulmonary tuberculosis. Unlike CD5-NK cells, most CD5LOW+NK cells had HLA-DR. We observed few CD5LOW+NK cells in the normal controls and some in the large granular lymphocyte (LGL) population purified by Percoll density centrifugation. Sorted CD5LOW+NK populations were LGL. The CD5LOW+NK cells had high lytic activity on K562 cells in a 4-h 51chromium release assay. Our results indicate that there is a previously unidentified subset of NK cells.     

Contribution of tyrosine kinases to the selective orientation of human CD4+ bifunctional cloned T cells toward proliferation or cytolytic function

We show that T cell activation of human CD4+ cloned T cells through the CD2 molecule can induce either autocrine proliferation or cytolysis, depending on the pair of anti-CD2 mAbs used for stimulation, that is, D66/T11(1) or GT2/T11(1), respectively. As the earliest biochemical event after CD2 stimulation is likely the induction of tyrosine phosphorylation of various proteins, we investigated whether differential activation of protein tyrosine kinases (PTKs) could contribute to the selective induction of each function. Results show that herbimycin A, a potent PTK inhibitor, markedly decreased the induction of both proliferation and cytolysis. This implies a regulatory role for tyrosine phosphorylation in the induction of each function by CD2. However, that PTKs are differentially activated upon induction of proliferation by D66/T11(1) or cytotoxic function by GT2/T11(1) emanated from two different approaches. First, immunoblotting total cellular extracts with an anti-phosphotyrosine mAb showed different patterns of tyrosine phosphorylation depending on the pair of CD2 mAbs used for stimulation. Second, a differential activation of p56lck, a src-related PTK, was observed after stimulation with D66/T11, and GT2/T11(1). Although induction of proliferation by D66/T11(1) was correlated with increased Lck activity, this was not observed when cells were triggered to lyse by GT2/T11(1). Thus, by providing striking correlative evidences linking differences in PTK activation with induction of different functions in bifunctional cloned T cells, our results strongly suggest that PTKs may contribute to the selective orientation of T cell functions at a single-cell level.     

Induction of the 2B9 antigen/dipeptidyl peptidase IV/CD26 on human natural killer cells by IL-2, IL-12 or IL-15

Activation of human natural killer (NK) cells involves sequential events including cytokine production and induction of cell surface molecules, resulting in the enhancement of cytolytic activity. To delineate the activation process of NK cells, we generated murine monoclonal antibodies (mAbs) against YT, a human large granular lymphocyte/natural killer (LGL/NK) cell line. Among the mAbs reactive with YT cells, one mAb, termed 2B9, was noted because of the lack of reactivity with most of the human T- and B-cell lines tested. In fresh peripheral blood mononuclear cells (PBMC), however, the majority of cells expressing this antigen (Ag) were T cells but not CD16+ nor CD56+ NK cells. Since YT cells showed an activated phenotype expressing interleukin-2 (IL-2) receptor alpha chain, we examined whether 2B9 Ag could be induced on normal human peripheral blood NK cells by cytokines known to activate NK cells. The 2B9 Ag was induced on NK cells by IL-2, IL-12 or IL-15 while no induction was observed by interferon-gamma (IFN-gamma). Biochemical analysis showed that anti-2B9 mAb recognized a 115 kDa molecule in YT cells. A cDNA clone encoding the 2B9 Ag was isolated from a cDNA expression library of YT cells and its sequence was identical to CD26 cDNA although it was not of full length. Transient expression of the 2B9 cDNA on COS-7 cells revealed that this cDNA encodes the antigenic epitope(s) recognized by anti-2B9 mAb as well as Ta1, an anti-CD26 mAb. These results showed that the 2B9 Ag is identical to CD26, and demonstrated that CD26 is an activation antigen on CD16+ CD56+ NK cells inducible by IL-2, IL-12 or IL-15.     

Cooperation between CD44 and LFA-1/CD11a adhesion receptors in lymphokine-activated killer cell cytotoxicity

IL-2-activated NK cells exhibit cytotoxic activity against a wide variety of tumor cells in a non-MHC-restricted fashion and in the absence of prior sensitization. The molecular mechanisms that regulate the cytotoxicity and attachment of activated killer cells to tumor target cells are not known. We provide genetic evidence in CD44(-/-) and LFA-1(-/-) mice that the cell adhesion receptors LFA-1 and CD44 regulate the cytotoxic activity of IL-2-activated NK cells against a variety of different tumor cells. This defect in cytotoxicity was significantly enhanced in mice that carried a double mutation of both CD44 and LFA-1. In vitro differentiation, TNF-alpha and IFN-gamma production, and expression of the cytolytic effector molecules perforin and Fas-L were comparable among IL-2-activated NK cells from LFA-1(-/-), CD44(-/-), CD44(-/-)LFA-1(-/-), and control mice. However, CD44(-/-), LFA-1(-/-), and CD44(-/-)LFA-1(-/-) IL-2-activated NK cells showed impaired binding and conjugate formation with target cells. We also show that hyaluronic acid is the principal ligand on tumor cells for CD44-mediated cytotoxicity of IL-2-activated NK cells. These results provide the first genetic evidence of the role of adhesion receptors in IL-2-activated NK killing. These data also indicate that distinct adhesion receptors cooperate to mediate binding between effector and target cells required for the initiation of "natural" cytotoxicity.     

Increase in tumor necrosis factor-alpha mRNA but not perforin mRNA expression in response to two newly characterized anti-LFA-1 monoclonal antibodies

We have generated two monoclonal antibodies (mAb), designated anti-1B11 and anti-4F9, directed to the human lymphocyte-function-associated antigen-1 (LFA-1). Indirect immunofluorescence with both mAb showed a bimodal distribution of antigen on the surface of T, natural killer (NK), and lymphokine-activated killer (LAK) cells. Neither mAb reacted with the epitopes recognized by TA1 and Mo-1 mAb on the alpha-chain of the heterodimer. Anti-1B11 and anti-4F9 immunoprecipitated polypeptide chains with molecular weights of 177 and 95 kD. Both mAb inhibited cytolytic T lymphocytes (CTL), NK, and LAK cell-mediated cytotoxicity without affecting antibody-dependent cellular cytotoxicity (ADCC). The proliferative responses of T cells to allogeneic cells were inhibited by anti-1B11 and anti-4F9, whereas the responses to phytohemagglutinin P and concanavalin A were not affected. Anti-1B11 and anti-4F9 blocked effector cell (EC)-target cell (TC) conjugate formation by 50%. Only anti-4F9 cross-reacted with LFA-1 on porcine peripheral blood lymphocytes and inhibited porcine NK, LAK, and ADCC activities. Because LFA-1 also functions at the level of signal transduction during T cell activation and we previously showed that CTL rapidly degraded perforin and tumor necrosis factor-alpha (TNF alpha) mRNA after interaction with sensitive TC, we examined the effects of the mAb on the messages for perforin and TNF alpha. Treatment of CTL with anti-1B11 and anti-4F9 induced TNF alpha message and protein levels of TNF alpha, but did not alter perforin mRNA levels.     

CD50 (ICAM-3) is phosphorylated on tyrosine and is associated with tyrosine kinase activity in human neutrophils

CD50 (ICAM-3) is expressed at a high level on resting blood granulocytes, monocytes, and lymphocytes. The constitutive high expression of CD50 on resting leukocytes suggests that it is an important LFA-1 ligand in the initiation of the immune/inflammatory response. Using a radiolabeling technique initially designed to detect ecto-protein kinase activity, we found that CD50 mAbs immunoprecipitated a approximately 125- to 170-kDa phosphoprotein from human neutrophils. Phosphorylation was increased after stimulation with the chemotactic agent FMLP, platelet-activating factor, 12-O-tetradecanoyl-phorbol-13-acetate, and the calcium ionophore A23187. This increase in phosphorylation was transient with the maximal phosphorylation, being observed by 1 min. Phosphoamino acid analysis revealed that CD50 contained predominantly phosphotyrosine. Although this assay system was designed initially to detect ecto-protein kinase activity, subsequent studies have shown that membrane proteins can be phosphorylated on the cytoplasmic domain under these conditions. When CD50 was immunoprecipitated from solubilized neutrophils, protein tyrosine kinase activity associated with CD50 was detected in the immunoprecipitate. The data suggest that phosphorylation of CD50 on tyrosine by an associated tyrosine kinase plays a role in the function of CD50.     

Focal adhesion kinase-related fakB is regulated by the integrin LFA-1 and interacts with the SH3 domain of phospholipase C gamma 1

Signal transduction through integrin molecules expressed on platelets and nonlymphoid cells involves activation of the intracellular focal adhesion kinase ppI25FAK (FAK) to phosphorylate substrate proteins on tyrosine residues. Similar mechanisms are also functional in T-lymphocytes through the beta 1-integrin VLA-4. A putative FAK-related phosphoprotein (fakB) was identified that is responsive to intracellular signals induced through ligation of antigen receptors on both T- and B-lymphocytes, and whose induced tyrosine phosphorylation is augmented by TCR costimulation through the adhesion/costimulatory receptors CD2 and CD4. In this report, fakB is shown to respond to extracellular signals through the beta 2-integrin LFA-1 in the absence of primary signals through the TCR. Protein-protein complex formation was observed involving an association between fakB, phospholipase C gamma 1 (PLC gamma 1), and the tyrosine phosphoprotein pp35-36. Evidence is provided here that fakB interacts with PLC gamma 1 through its SH3 domain. The association between fakB and PLC gamma 1 does not appear to require T-cell activation, whereas the induced tyrosine phosphorylation of the protein complex components occurs following engagement of LFA-1. These data indicate that the beta2-integrin LFA-1 expressed on T-lymphocytes stimulates a novel, FAK-related molecule that may function in the interplay between adhesion receptors and intracellular signaling enzymes responsible for downstream second messenger generation.     

T cell receptor- and beta 1 integrin-mediated signals synergize to induce tyrosine phosphorylation of focal adhesion kinase (pp125FAK) in human T cells

The beta 1 subfamily of integrins is thought to play an important role in both the adhesion/migration and proliferation/differentiation of T cells. beta 1 integrins can provide T cell costimulation through interaction of very late antigen (VLA) 4 (VLA-4) (alpha 4 beta 1) and VLA-5 (alpha 5 beta 1) with the extracellular matrix protein fibronectin (FN), or by VLA-4 binding to its cell surface ligand, vascular cell adhesion molecule (VCAM) 1. The mechanism by which beta 1 integrin members transduce T cell-costimulatory signals is poorly understood. Studies in non-T cells have demonstrated regulation of the tyrosine focal adhesion kinase pp125FAK by beta 1 integrin engagement and, most recently, indicate a role for pp125FAK in linking integrin-mediated signal transduction to the Ras pathway (Schaller, M. D., and J. T. Parsons, 1994, Curr. Opin. Cell. Biol. 6: 705-710; Schlaepfer, D. D., S. K. Hanks, T. Hunter, and P. Van der Geer. 1994. Nature (Lond.), 372:786-790). Although pp125FAK kinase occurs in T cells, there are no reports on its regulation in this cell type. The studies described in this article characterize novel regulation of pp125FAK by the T cell receptor (TCR)-CD3 antigen complex and beta 1 integrins, and provide the first account, in any cell type, of integrin alpha 4 beta 1-mediated pp125FAK tyrosine phosphorylation. We demonstrate a rapid and sustained synergistic increase in tyrosine phosphorylation of human pp125FAK in Jurkat T cells after simultaneous (a) triggering of the TCR-CD3 complex, and (b) alpha 4 beta 1 and alpha 5 beta 1 integrin-mediated binding of these cells to immobilized FN or alpha 4 beta 1 integrin-mediated binding to immobilized VCAM-1. Studies with normal peripheral blood-derived CD4+ human T blasts confirm the synergistic action of a TCR-CD3 complex-mediated costimulus with a FN- or VCAM-1-dependent signal in the induction of T cell pp125FAK tyrosine phosphorylation. In vitro kinase assays performed on pp125FAK immunoprecipitates isolated from Jurkat cells and normal CD4+ T cells identified a coprecipitating 57-kD tyrosine-phosphorylated protein (pp57), distinct from pp59fyn or pp56lck. These results indicate, for the first time, the involvement of a specific kinase, pp125FAK, in alpha 4 beta 1- and alpha 5 beta 1-mediated T cell-costimulatory signaling pathways. In addition, the data demonstrate novel regulation of pp125FAK tyrosine phosphorylation by the TCR-CD3 complex.     

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.     

Pyk2 is differentially regulated by beta1 integrin- and CD28-mediated co-stimulation in human CD4+ T lymphocytes

Beta1 integrins can provide T cell co-stimulation, but little is known concerning their downstream signaling pathways. We found that Pyk2, a focal adhesion kinase-related tyrosine kinase, is regulated by beta1 integrin signaling in human T cells. Stimulation of Jurkat T cells with the alpha4beta1 integrin ligand VCAM-1 results in Pyk2 tyrosine phosphorylation, and combined stimulation with VCAM-1 and anti-CD3 mAb induces rapid and sustained synergistic Pyk2 phosphorylation. Studies with mAb suggest that in synergistic CD3- and alpha4beta1 integrin-mediated Pyk2 tyrosine phosphorylation, a major contribution of CD3-derived signals is independent of their effects on regulating integrin adhesion. Analysis of resting human CD4+ T cells confirmed the ability of CD3-derived signals to synergize with beta1 integrin-dependent signals in the induction of Pyk2 tyrosine phosphorylation. In addition, although CD28-mediated co-stimulatory signals were able to synergize with CD3-mediated signals in inducing ERK and JNK activation and secretion of IL-2 in the primary T cells, they did not contribute to the induction of Pyk2 phosphorylation. Taken together, these results indicate a potential role for Pyk2 in T cell co-stimulation mediated specifically by beta1 integrins.     

Activation of endothelin ETA receptors induces phosphorylation of alpha1b-adrenoreceptors in Rat-1 fibroblasts

The effect of endothelin-1 on the phosphorylation of alpha1b-adrenoreceptors, transfected into rat-1 fibroblasts, was studied. Basal alpha1b-adrenoreceptor phosphorylation was markedly increased by endothelin-1, norepinephrine, and phorbol esters. The effect of endothelin-1 was dose dependent (EC50 approximately 1 nM), reached its maximum 5 min after stimulation, and was inhibited by BQ-123, an antagonist selective for ETA receptors. Endothelin-1-induced alpha1b-adrenoreceptor phosphorylation was attenuated by staurosporine or genistein and essentially abolished when both inhibitors were used together. The effect of norepinephrine was not modified by either staurosporine or genistein alone, and it was only partially inhibited when both were used together. These data suggest the participation of protein kinase C and tyrosine kinase(s) in endothelin-1-induced receptor phosphorylation. However, phosphoaminoacid analysis revealed the presence of phosphoserine and traces of phosphothreonine, but not of phosphotyrosine, suggesting that the putative tyrosine kinase(s), activated by endothelin, could act in a step previous to receptor phosphorylation. The effect of endothelin-1 on alpha1b-adrenoreceptor phosphorylation was not mediated through pertussis toxin-sensitive G proteins. Calcium mobilization induced by norepinephrine was diminished by endothelin-1. Norepinephrine and endothelin-1 increased [35S]GTPgammaS binding to control membranes. The effect of norepinephrine was abolished in membranes obtained from cells pretreated with endothelin-1. Interestingly, genistein plus staurosporine inhibited this effect of the endothelial peptide. Endothelin-1 did not induce alpha1b-adrenoreceptor internalization. Our data indicate that activation of ETA receptors by endothelin-1 induces alpha1b-adrenoreceptor phosphorylation and alters G protein coupling.     

Differential expression of natural killer cell markers: human versus baboon

The use of baboons as a model for the study of allo- and xenotransplantation has become increasingly important, but there are few studies on the basic immunological responses in baboons that might be relevant for a rejection reaction. In present study, the cell-surface phenotype, cytokine-induced activation and growth, and cytotoxicity of baboon and human natural killer (NK) and lymphokine-activated killer (LAK) cells were compared. A panel of murine monoclonal antibodies specific for human cell-surface markers expressed on lymphocytes was used to compare relevant baboon and human peripheral blood lymphocytes (PBL). Baboon PBL were 52.1+/-2.9% CD8+, 18.5+/-2.2% CD16+, 3.0+/-0.5% CD25+, and 5.5+/-1.8% CD69+. The corresponding proportions in humans were 23.8+/-7.1%, 12.8+/-3.2%, 4.5+/-1.0%, and 2.3+/-1.1%. In contrast to human PBL, less than 1% of baboon lymphocytes expressed CD56, CD57, and CD122 (interleukin [IL]-2Rbeta). Baboon lymphocytes showed NK cytotoxic activity against the human K562 and CEM cell lines, which was comparable to human NK activity. Depletion of baboon CD16+ or CD8+ cells led to dramatic decreases in NK cytotoxicity, and removal of both subsets completely abrogated NK activity. Incubation of baboon lymphocytes with human recombinant IL-2 for 1 week led to the appearance of CD56+ cells (11.3+/-2.8%). Most of the baboon CD56+ cells induced in culture were in S and G2 phases of cell cycle. Both baboon and human IL-2-activated lymphocytes were highly cytotoxic against the human LAK-sensitive cell line Daudi. Depletion of baboon CD8+ but not CD56+ cells significantly decreased LAK activity. These studies revealed differences in the NK system of humans and baboons that should be taken into consideration when analyzing immune responses to allo- and xenotransplantation in baboons.