Induction of hyperphosphorylation and activation of the p56lck protein tyrosine kinase by phenylarsine oxide, a phosphotyrosine phosphatase inhibitor.

The T cell protein tyrosine kinase p56lck is implicated in thymic development and mitogenic activation of T lymphocytes, and is itself regulated by reversible tyrosine phosphorylation. When phenylarsine oxide (PAO), a membrane-permeable inhibitor of phosphotyrosine phosphatases, was added to Jurkat T leukemia or LSTRA thymoma cells, the phosphate content of p56lck increased rapidly. The sites of increased phosphorylation were mapped to Tyr-192, Tyr-394 and Tyr-505. Hyperphosphorylated p56lck displayed retarded mobility on SDS gels, unaltered or marginally increased cytoskeletal association, and its catalytic activity changed in a biphasic manner; during the first 10-20 min of PAO-treatment the activity increased and then it declined to very low values within 1-2 hr. Our data suggest that p56lck contains both positive and negative regulatory sites which are constantly dephosphorylated at an unexpectedly high rate by cellular phosphotyrosine phosphatases.

Inhibition of CD3-induced Ca2+ signals in Jurkat T-cells by myristic acid.

Stimulation of T lymphocytes with antibodies against the T cell receptor/CD3 complex induces within seconds a rise in the concentration of intracellular free Ca2+. Here we show that treatment with 20 microM free myristic acid completely inhibits this Ca2+ signal and the cellular proliferation in Jurkat T cells. Also lauric acid inhibited cell growth while its blocking effect on the Ca2+ signal was weaker than that of myristic acid. Other saturated free fatty acids were inactive. The inhibitory effect of myristic acid could be reversed by the addition of fatty acid free albumin, which will bind the fatty acid. Myristic acid, but not its methyl ester, inhibited both the anti-CD3-induced Ca2+ influx across the cell membrane and Ca2+ release from intracellular stores, but not the formation of inositol phosphates. In contrast, thapsigargin-induced release of Ca2+ from the same intracellular stores was unaffected by myristic acid. Thus, myristic acid specifically blocks T cell antigen receptor-CD3 induced Ca2+ mobilization in T cells.

Signalling initiated with CD4-TCR or TCR-TCR interactions: comparison of tyrosine phosphorylation patterns and CD45 effects.

Antigen-triggered response in T cells is mediated by the T cell receptor (TCR)/CD3-complex. This signalling, however, is modulated by a number of other surface molecules. Among the most important of these is the CD4/CD8 molecule which associates with the TCR/CD3-complex and binds to the MHC complex. The molecular mechanisms involved in interactions between TCR-TCR and TCR-CD4 are not fully understood. We have earlier described an experimental model that allows us to dissect signals involving CD4-TCR interactions and those involving TCR-TCR interactions using a mouse CD4-CD8- T cell hybridoma cell-line transfected either with the TCR from a mouse T-helper 2 cell-line (D10) alone or with both the TCR and the CD4 molecule. To further characterize these two different modes of signalling in T lymphocytes we have studied the tyrosine phosphorylation patterns resulting from these interactions. In addition, we have studied the modulatory effect of the CD45 molecule on these interactions. In contrast to some earlier reports, we found that both the patterns of induced tyrosine phosphorylation and the effects of CD45 modulation were essentially similar in the CD4-TCR and the TCR-TCR signal transduction cascades. The results are consistent with a purely synergistically amplifying function for CD4 on the TCR-mediated signalling.

Upregulation of major histocompatibility complex class II antigens in hepatocytes in Doberman hepatitis.

Major histocompatibility complex (MHC) class II antigen expression in hepatocytes and its correlation with mononuclear cell infiltration into the liver were studied using immunohistochemical techniques in 38 Dobermans with Doberman hepatitis (DH). Liver biopsy samples were obtained from 18 dogs at the subclinical stage. Autopsy samples were taken from 6 DH dogs euthanized for a reason other than DH, from 14 dogs euthanized because of advanced liver failure and from 6 control Dobermans. Upon examination of the control liver samples, no expression of MHC class II antigens was detected in hepatocytes. By contrast, in 15 of the 18 DH biopsies (83%) and in all 20 DH autopsy liver samples, hepatocytes expressed MHC class II molecules. MHC class II expression was either cytoplasmic or membranous and occurred in conjunction with lymphocyte infiltration. A correlation between the inflammatory reaction and the expression of MHC class II in hepatocytes suggests that the aberrant expression of MHC class II in hepatocytes is induced by cytokines. Hepatocytes presenting a putative MHC class II molecule-associated autoantigen could thus become the target of an immune attack mediated by CD4+ T cells. In addition, corticosteroid treatment was observed to significantly decrease MHC class II expression in DH hepatocytes. Inappropriate MHC class II expression in hepatocytes and mononuclear cell infiltration are suggesting an autoimmune nature for chronic hepatitis in Dobermans.

Colostral growth factors. Possible role in bovine udder epithelial cell regeneration.

Growth of the secretory epithelium during prepartum time, and for a short period after calving, is under hormonal control by estrogen, progesterone and prolactin. The mechanism(s) by which these hormones act is not known but colostrum and milk have been shown to contain different growth promoting substances. In an attempt to unravel these relationships the effect of bovine colostrum on cellular proliferation in vitro have been characterized. Colostral thermostable factors not present in milk nor associated with fat, potently induce the proliferation associated enzyme, ornithine decarboxylase, in fibroblast cell lines. However, mammary epithelial cells appear to proliferate in response to different colostral heat sensitive factor(s) that await further characterization.

The sulfhydryl reagent N-ethylmaleimide induces hyperphosphorylation on tyrosine residues in the Jurkat T-cell line.

Tyrosine protein kinases have been shown to be functionally involved in regulation of cellular signalling, proliferation and transformation. The activity of tyrosine protein kinases is counterbalanced by phospho tyrosine phosphatases that maintain constitutively low levels of protein phosphotyrosine in most cells. In this study the effect of N-ethylmaleimide on the protein tyrosine phosphorylation was tested in Jurkat T-cells. Treatment of intact cells for 5-10 mins with 50-100 microM N-ethylmaleimide resulted in a dramatic increase in phosphorylation on tyrosine residues. Phosphoaminoacid analysis revealed an up to ten-fold increase in the content of phosphotyrosine. N-ethylmaleimide blocked the phospho tyrosine phosphatases activity of immunoprecipitated CD45 while in a kinase assay N-ethylmaleimide did not affect the 32P-gamma-ATP phosphorylation of substrates. The N-ethylmaleimide-induced hyperphosphorylation was reversed by treatment with 2 mM dithiotreitol. It is concluded that N-ethylmaleimide offers a novel useful tool for identification of substrates for tyrosine protein kinases and for studies on phosphotyrosine-dependent protein interactions.

Generation of inositol phosphates during triggering of cytotoxicity in human natural killer and lymphokine-activated killer cells.

We investigated early molecular mechanisms involved in the triggering of cytolytic responses in natural killer (NK) and lymphokine-activated (LAK) cells. When NK or LAK cells were conjugated to the sensitive target cells K562, an increased formation of both inositol monophosphate (IP1) and inositol trisphosphate (IP3) was detected. Target cells like Raji or Jok-1, which form conjugates with NK cells but are insensitive to NK lysis, did not elicit IP1 formation. Treatment of NK cells with interleukin 2 increased the basal turnover of inositol phosphates and enhanced the phosphatidyl inositol breakdown upon confrontation with sensitive targets. These finding indicate that hydrolysis of phosphatidyl inositols is associated with the signal which triggers the cytolytic response in NK and LAK cells. These events therefore constitute an early marker of the cytolytic activation.

Role of CD16 (Fc receptor III) and interleukin-2 in the reactivation of natural killer cells after inhibitory target cell contact.

Contact with natural killer (NK)-resistant monolayer targets is an inhibitory signal to NK cells. In this study, we have analyzed the effect of such effector/target cell interactions on the CD16 (FcRIII) expression on lymphocytes and the role of CD16 and interleukin-2 (IL-2) in the reactivation of their cytolytic machinery. Coculturing peripheral blood mononuclear cells with NK-resistant monolayer cells did not change the percentage of CD 16-positive effector cells, although this treatment effectively inhibited their cytotoxicity against NK-sensitive targets. The inhibited effector cells partially regained their activity by incubating for 24 h in medium supplemented with 10% fetal calf serum (FCS), whereas human albumin-, newborn calf serum- or human AB serum-supplemented media had no reactivating effect. Monoclonal class IgG1, IgG2a and IgM anti-CD16 antibodies [Abs; 3G8, CLB-CD16 (CLB-FcR gr1) and Leu 11b], and normal rabbit IgG (NR-IgG) prevented the FCS-mediated reactivation of cytotoxicity, whereas nonreactive control Abs significantly enhanced it. The detection of the CD16 antigen by the monoclonal anti-CD16 Abs Leu 11a and Leu 11c was blocked by the above anti-CD16 Abs and NR-IgG, while the expression of other NK cell-associated surface molecules (CD2, CD56) remained unchanged. Mere blocking of CD16, using a short-term incubation with anti-CD16 Abs, had an insignificant effect on endogenous NK activity, suggesting that CD16 is involved in NK cell (re)activation rather than in the killing process itself. In the presence of IL-2, inactivated effector cells also regained their killing activity. The IL-2-induced reactivation was not inhibited by anti-CD16 Abs. The results suggest that FCS-derived factors and soluble nonreactive immunoglobulins enhance the NK activity of down-regulated effector cells via CD16, and that CD16 and IL-2 receptors represent alternative independent pathways of NK cell reactivation.

Activation of natural killer cells via the Fc gamma RIII (CD16) requires initial tyrosine phosphorylation.

Triggering of the Fc gamma RIII (CD16) on natural killer (NK) cells by monoclonal antibodies or antibody-coated target cells stimulates a rapid phospholipase C (PLC)-mediated hydrolysis of inositol phospholipids and results in subsequent delivery of the lytic hit. The role of initial tyrosine phosphorylation in these events was investigated with a tyrosine protein kinase (TPK) inhibitor, genistein. At doses that inhibited CD16-triggered tyrosine phosphorylation of substrates in intact cells, genistein did not influence serine/threonine phosphorylation or target cell binding but prevented PLC activation, cell-mediated cytotoxicity and antibody-dependent cellular cytotoxicity. These findings indicate that tyrosine phosphorylation is an early and critical event during receptor-mediated activation of the lytic machinery.

Rapid Ca2+ mobilization in single LGL cells upon interaction with K562 target cells--role of the CD18 and CD16 molecules.

Changes in the intracellular Ca2+ levels of human large granular lymphocytes (LGL), loaded with the fluorescent Ca2+ indicator fura-2, have been studied upon addition of human chronic myelogenous leukemia K562 cells. The measurements, analyzed at the single-cell level using image analysis, indicate a rapid Ca2+ mobilization in the effector cell upon interaction with its target cell. This mobilization appeared to be localized to an area within the effector cell that was in physical contact with target cells. The LGL responded with different kinetics in a transient manner and about 19% of them could undergo two or more responses. Data obtained from experiments performed with anti-CD16- and anti-CD18-pretreated LGL in the presence of target cells indicate that the CD16 and CD18 molecules are not likely to be the triggers of the Ca2+ response, although they might participate in the recognition of the target cell.

Transduction of mitogenic signals in T lymphocytes. Role of inositol phospholipids for the rapid activation of ornithine decarboxylase.

Treatment of human T lymphocytes with mitogenic ligands, such as concanavalin A (Con A), induces a rapid activation of the enzyme ornithine decarboxylase (ODC). This activation occurs within minutes and is completely inhibited when the cells are treated with 1 mM Li+ (in an inositol-free medium) prior to stimulation with Con A. In the presence of 1 mM myo-inositol Li+ has no effect on the Con A-induced activation of ODC. To elucidate why inositol is needed for the mitogen-induced activation of ODC in T lymphocytes, we tested the ability of different inositol metabolites to reverse the inhibitory effect of Li+. Here we report that inositol phospholipids, in addition to inositol, reverse the Li+-induced inhibition of ODC activation, while all other inositol derivatives tested were ineffective. This indicates that Li+ does not block the activation of ODC by inhibiting the generation of inositol phosphates, but rather by a mechanism which is circumvented if inositol phospholipids are added. The molecular mechanisms involved in the rapid activation of ODC by mitogens in human T lymphocytes apparently require inositol phospholipids, but are not directly mediated by inositol-1,4,5-trisphosphate (IP3) alone, diacylglycerol alone, or other inositol phosphates.

Triggering of human natural killer cells through CD16 induces tyrosine phosphorylation of the p72syk kinase.

Activation of protein tyrosine kinases (PTK) is an initial and obligatory event for the triggering of human natural killer (NK) cells to cytotoxicity. Of the different PTK detected in NK cells, only p56lck has previously been shown to participate in NK cell activation. Here we present evidence that another PTK, p72syk, is involved in activation of NK cells. Stimulation with a monoclonal antibody to to the Fc gamma RIII receptor (CD16) induced an increased tyrosine phosphorylation of p72syk. This phosphorylation correlated with an increased tyrosine kinase activity of p72syk towards a synthetic peptide substrate. A severalfold increase in the catalytic activity of p72syk was also seen after treatment of NK cells with an inhibitor of phosphotyrosine phosphatases, pervanadate. We conclude that triggering of the cytotoxic response in NK cells is associated with activation of p72syk.

Phorbol 12-myristate 13-acetate (TPA) blocks CD3-mediated Ca2+ mobilization in Jurkat T cells independently of protein kinase C activation.

Stimulation of Jurkat T cells with antibodies against the T cell receptor/CD3 complex induces a rise in the intracellular concentration of Ca2+ within seconds. The inositol phosphate-dependent Ca2+ mobilization induced by OKT3 was completely abrogated when Jurkat cells were pretreated for 1 min with the phorbol 12-myristate 13-acetate TPA (10nM), a concentration which activates protein kinase C (PKC). The effects of TPA on the Ca2+ fluxes were insensitive to treatment of the cells with known PKC inhibitors (H-7 and staurosporin) under conditions where the PKC-mediated phosphorylation was blocked. Furthermore, another activator of PKC, mezerein, inhibited the Ca2+ signal induced by OKT3. This inhibition, however, could completely be reversed by pretreatment with H-7 or staurosporine. We conclude that the TPA-mediated inhibition of Ca2+ fluxes in Jurkat T cells largely acts through a PKC-independent pathway.

p72syk protein tyrosine kinase: an early transducer of sIgG-triggered apoptotic signalling in human follicular lymphoma cells.

Cross-linking of B cell antigen receptor (sIg) elicits different biological responses, including cell activation, proliferation, differentiation, anergy and cell death depending on the maturational stage of the cell. We established the tumor cell lines HF-1.3.4 and HF-4-9 from two patients with follicular lymphoma. Both cell lines carry the characteristic t(14;18) chromosomal translocation and display constitutively overexpressed Bcl-2. HF-1.3.4 represents a mature B cell with sIgG and several somatic hypermutations in its Ig genes, while HF-4-9 is a less mature B cell, expressing sIgM and only a few mutations in its Ig genes. Cross-linking of sIg with antibodies leads to apoptosis in HF-1.3.4 cells but not in HF-4-9 cells. Triggering of sIg induced, within seconds, identical tyrosine phosphorylation of p53/56lyn protein tyrosine kinase (PTK) and p55blk PTK in both of the cell lines; however, a prominent tyrosine phosphorylation and activation of p72syk PTK only in HF-1.3.4 cells. We conclude that p72syk PTK is of importance in relaying apoptotic signalling upon sIg cross-linking in the HF-1.3.4 cell line. Given the mature phenotype of the HF-1.3.4 cell line it serves as a model for the late negative selection during B cell ontogeny. Moreover, our results question the current concept that a constitutive overexpression of BcI-2 confers resistance to sIg ligation-induced apoptosis in lymphoma cells.

Biodegradation of the copolymeric polylactide stent. Long-term follow-up in a rabbit aorta model.

The behavior of biodegradable polylactide as a stent material has not yet been fully established in small vessels such as arteries with a diameter <3 mm. The aim of this study was to investigate the long-term effect of a copolymeric polylactide (PLA96) stent. Appropriately sized spiral PLA96 stents were implanted into the infrarenal aortas of 20 rabbits. Intraoperative systemic heparinization (150 IU/kg), perioperative subcutaneous enoxaheparin sodium (10 mg), ticlopidine (250 mg/day) for 1 month, and acetosalicylic acid (12.5 mg/day) were continuously administered. Animals were euthanized according to a fixed timetable for up to 34 months for histologic and scanning-electron-microscopic assessment. Endothelialization was complete within 1 month. In 2 of the 3 aortas sampled 3 months after implantation, a mild inflammatory reaction was visible, with no sign of granulomatous or foreign-body reaction in the vessel wall. Instead, in 1 sample examined at the same time point, neointimal chondroid metaplasia was detected. After 6 months, inflammatory reaction declined in the vessel wall. Hydrolyzation of the stent was histologically evident at 12 months, with mild foreign-body reaction detectable in 2 of 5 aortas sampled at this time point. The stent disintegrated without fragmentation by 24 months, as it was gradually replaced by fibrosis. The vessel lumen remained patent at all time points. We conclude that the PLA96 stent degraded with minimal tissue response within 24 months. PLA96 may thus be a promising stent core material for small vessels in the future, although further investigation is needed to establish its final biocompatibility.