Gabriele Baier-Bitterlich

Protein kinase C-theta isoenzyme selective stimulation of the transcription factor complex AP-1 in T lymphocytes.

T-lymphocyte stimulation requires activation of several protein kinases, including the major phorbol ester receptor protein kinase C (PKC), ultimately leading to induction of lymphokines, such as interleukin-2 (IL-2). The revelant PKC isoforms which are involved in the activation cascades of nuclear transcription factors involved in IL-2 production have not yet been clearly defined. We have examined the potential role of two representative PKC isoforms in the induction of the IL-2 gene, i.e., PKC-alpha and PKC-theta, the latter being expressed predominantly in hematopoietic cell lines, particularly T cells. Similar to that of PKC-alpha, PKC-theta overexpression in murine EL4 thymoma cells caused a significant increase in phorbol 12-myristate 13-acetate (PMA)-induced transcriptional activation of full-length IL-2-chloramphenicol acetyltransferase (CAT) and NF-AT-CAT but not of NF-IL2A-CAT or NF-kappaB promoter-CAT reporter gene constructs. Importantly, the critical AP-1 enhancer element was differentially modulated by these two distinct PKC isoenzymes, since only PKC-theta but not PKC-alpha overexpression resulted in an approximately 2.8-fold increase in AP-1-collagenase promoter CAT expression in comparison with the vector control. Deletion of the AP-1 enhancer site in the collagenase promoter rendered it unresponsive to PKC-theta. Expression of a constitutively active mutant PKC-theta A148E (but not PKC-alpha A25E) was sufficient to induce activation of AP-1 transcription factor complex in the absence of PMA stimulation. Conversely, a catalytically inactive PKC-theta K409R (but not PKC-alpha K368R) mutant abrogated endogenous PMA-mediated activation of AP-1 transcriptional complex. Dominant negative mutant Ha-RasS17N completely inhibited the PKC-O A148E-induced signal, PKC-O. Expression of a constitutively active mutant PKC-O A148E (but not PKC-alpha A25E) was sufficient to induce activation of AP-1 transcription factor complex in the absence of PMA stimulation. Conversely, a catalytically inactive PKC-O K409R (but not PKC-alpha K368R) mutant abrogated endogenous PMA-mediated activation of AP-1 transcriptional complex. Dominant negative mutant Ha-enRasS17N completely inhibited in the PKC-O A148E-induced signal, identifying PKC-theta as a specific constituent upstream of or parallel to Ras in the signaling cascade leading to AP transcriptional activation.

Soluble receptors for tumour necrosis factor in clinical laboratory diagnosis

Abstract:  Soluble tumour necrosis factor receptors (sTNF‐Rs) play a role as modulators of the biological function of tumour necrosis factor‐α (TNF‐α) in an agonist/antagonist pattern. In various pathologic states the production and release of sTNF‐Rs may mediate host response and determine the course and outcome of disease by interacting with TNF‐α and competing with cell surface receptors. The determination of sTNF‐Rs in body fluids such as plasma or serum is a new tool to gain information about immune processes and provides valuable insight into a variety of pathological conditions. Regarding its immediate clinical use, sTNF‐Rs levels show high accuracy in the follow‐up and prognosis of various diseases. In HIV infection and sepsis, sTNF‐Rs concentrations strongly correlate with the clinical stage and the progression of disease and can be of predictive value. Determination of sTNF‐Rs also gives useful information for monitoring cancer and autoimmune diseases. The information provided is often even superior to that obtained with classical disease markers, probably due to the direct involvement of the “TNF system” in the pathogenetic mechanisms in these patients. The available data imply that the measurement of sTNF‐Rs, especially of the sTNF‐R 75kD type, is a useful adjunct for quantification of the Th1‐type immune response, similar to other immune activation markers such as neopterin and β2‐microglobulin. Endogenous sTNF‐Rs concentrations appear to reflect the activation state of the TNF‐α/TNF receptor system.

Direct stimulation of Vav guanine nucleotide exchange activity for Ras by phorbol esters and diglycerides.

We recently identified Vav as a Ras-activating guanine nucleotide exchange factor (GEF) stimulated by a T-cell antigen receptor-coupled protein tyrosine kinase (PTK). Here, we describe a novel, protein kinase-independent alternative pathway of Vav activation. Phorbol ester, 1,2-diacylglycerol, or ceramide treatment of intact T cells, Vav immunoprecipitates, or partially purified Vav generated by in vitro translation or COS-1 cell transfection stimulated the Ras exchange activity of Vav in the absence of detectable tyrosine phosphorylation. GEF activity of gel-purified Vav was similarly stimulated by phorbol myristate acetate (PMA). Stimulation was resistant to PTK and protein kinase C inhibitors but was blocked by calphostin, a PMA and diacylglycerol antagonist. In vitro-translated Vav lacking its cysteine-rich domain, or mutated at a single cysteine residue within this domain (C528A), was not stimulated by PMA but was fully activated by p56lck. This correlated with increased binding of radiolabeled phorbol ester to COS-1 cells expressing wild-type, but not C528A-mutated, Vav. Thus, Vav itself is a PMA-binding and -activated Ras GEF. Recombinant interleukin-1 alpha stimulated Vav via this pathway, suggesting that diglyceride-mediated Vav activation may couple PTK-independent receptors which stimulate production of lipid second messengers to Ras in hematopoietic cells.

Decreased plasma tryptophan in pregnancy

Objective To examine levels of serum tryptophan and its degradation product kynurenine in uncomplicated pregnancy, according to the week of pregnancy and the concentrations of neopterin. Methods Plasma was analyzed from 45 healthy pregnant women (15 in each trimester), 15 healthy puerperas, and 20 nonpregnant controls. Tryptophan and kynurenine were measured by reverse-phase, high-performance liquid chromatography, and neopterin by radioimmunoassay. Results In healthy pregnant women, tryptophan values decreased (median first trimester: 72 μmol/L; second trimester: 51 μmol/L; third trimester: 46 μmol/L; P < .001) in a manner correlated with the duration of pregnancy (Spearman rank correlation coefficient rs = −0.771, P < .001) and normalized in the puerperium (median 60 14mol/L). No change in kynurenine, a tryptophan degradation product, was observed, but the ratio of kynurenine to tryptophan increased during pregnancy and correlated positively with gestational age (rs = 0.714, P < .001). In addition, an inverse correlation existed between neopterin and tryptophan concentrations (rs = −0.566, P < .001), as well as a positive one between neopterin and the kynurenine to tryptophan ratio (rs = 0.660, P < .001). Conclusion Tryptophan levels decrease during normal pregnancy and the decrease may be related to immune activation phenomena.

Protein kinase Cθ, a selective upstream regulator of JNK/SAPK and IL‐2 promoter activation in Jurkat T cells

The predominant expression of protein kinase C (PKC) θ in T cells (J. Biol. Chem.1993. 268: 4997– 5004), its isoenzyme‐specific ability to stimulate AP‐1 transcriptional activity (Mol. Cell. Biol.1996. 16: 1842 –1850) and the recent discovery of its selective and antigen‐dependent colocalization with the contact region between T cells and antigen‐presenting cells (Nature1997. 385: 83 – 89) suggest that, among the PKC family members, PKCθ plays a specialized role in T cell activation. By investigating the downstream effectors of PKCθ we now demonstrate a direct and isoenzyme‐specific contribution of PKCθ to c‐Jun‐N‐terminal kinase/stress‐activated protein kinase (JNK/SAPK) but not extracellular regulated kinase (ERK) activation. Expression of a constitutively active (CA) form of PKCθ (but not CA‐PKCα, ϵ and λ / ι) resulted in strong activation of JNK/SAPK and expression of a dominant‐negative form of PKCθ interfered with the endogenous activation signal for JNK/SAPK. Importantly, Ca2+ ionophore and CA‐PKCθ (but not CA‐PKCα, ϵ and λ / ι) caused synergistic activation of the IL‐2 promoter. Together, these data establish that PKCθ is required for activation of JNK/SAPK signaling leading to IL‐2 promoter transcription in T lymphocytes.

Enhanced Tryptophan Degradation in Systemic Lupus Erythematosus

In vitro and in vivo, tryptophan degradation was found to be associated with T cell functional loss and tolerance induction. In systemic lupus erythematosus (SLE) besides the Th2-type cytokine interleukin-10, Th1-type cytokines including interferon-gamma (IFN-gamma) are expressed especially during exacerbation of the disease. IFN-gamma stimulates the enzyme indoleamine (2,3)-dioxygenase (IDO) converting tryptophan to the metabolite kynurenine which in macrophages is subsequently degraded to other, partly neurotoxic compounds like quinolinic acid, and finally to nicrotinamides. We measured kynurenine and tryptophan concentrations in the sera of 55 SLE patients. In these patients, the concentrations of tryptophan (median, interquartile range: 53.9, 45.7-64.1 microM) were lower (p < 0.0001), and the kynurenine concentrations (2.45, 1.75-3.40 microM) were increased (p < 0.0005) compared to healthy blood donors (70.0, 63.8-80.6; 1.80, 1.45-2.27 microM, respectively). Also the kynurenine per tryptophan quotients (K/T), which allow to estimate IDO activity, were significantly higher in patients than in normals (0.043, 0.033-0.062 vs. 0.027, 0.021-0.030; p < 0.0001), indicating enhanced IDO-induced tryptophan degradation in SLE. There was no significant relationship between tryptophan, kynurenine and the SLEDAI, and also the correlation of K/T with SLEDAI was rather weak (rs = 0.243, p < 0.05). Higher K/T was found in patients presenting with serositis (p = 0.01), decrease of complement (c3, c4; p < 0.01) and blood count change (anemia, leucopenia, lymphopenia; p = 0.032) than in patients without such disease manifestations. The significant correlation found between K/T and neopterin (rs = 0.808, p < 0.001), a marker of immune activation, points to a role of immune activation to be responsible for tryptophan degradation in SLE patients.

Activation of p56lck by p72syk through physical association and N-terminal tyrosine phosphorylation.

The p56lck and p59fyn protein tyrosine kinases are important signal transmission elements in the activation of mature T lymphocytes by ligands to the T-cell antigen receptor (TCR)/CD3 complex. The lack of either kinase results in deficient early signaling events, and pharmacological agents that block tyrosine phosphorylation prevent T-cell activation altogether. After triggering of the TCR/CD3 complex, both kinases are moderately activated and begin to phosphorylate cellular substrates, but the molecular mechanisms responsible for these changes have remained unclear. We recently found that the p72syk protein tyrosine kinase is physically associated with the TCR/CD3 complex and is rapidly tyrosine phosphorylated and activated by receptor triggering also in T cells lacking p56lck. Here we examine the regulation of p72syk and its interaction with p56lck in transfected COS-1 cells. p72syk was catalytically active and heavily phosphorylated on its putative autophosphorylation site, Tyr-518/519. Mutation of these residues to phenylalanines abolished its activity in vitro and toward cellular substrates in vivo and reduced its tyrosine phosphorylation in intact cells by approximately 90%. Coexpression of lck did not alter the catalytic activity of p72syk, but the expressed p56lck was much more active in the presence of p72syk than when expressed alone. This activation was also seen as increased phosphorylation of cellular proteins. Concomitantly, p56lck was phosphorylated at Tyr-192 in its SH2 domain, and a Phe-192 mutant p56lck was no longer phosphorylated by p72syk. Phosphate was also detected in p56lck at Tyr-192 in lymphoid cells. These findings suggest that p56lck is positively regulated by the p72syk kinase.

Effect of neopterin and 7,8-dihydroneopterin on tumor necrosis factor-α induced programmed cell death

Tumor necrosis factor‐α and the formation of reactive oxygen intermediates are central mediators of apoptosis. Recent data indicated a role of neopterin and 7,8‐dihydroneopterin in oxygen radical mediated processes. We have therefore investigated the effect of neopterin‐derivatives on TNFα induced apoptosis of the monocyte‐like cell line U937. At an elevated concentration 7,8‐dihydroneopterin was found to superinduce TNFα mediated programmed cell death due to the formation of reactive oxygen intermediates. Our results imply that in combination with TNFα high concentrations of 7,8‐dihydroneopterin enhances apoptosis due to oxidative stress on cells.

Neopterin and 7,8-dihydroneopterin induce apoptosis in the rat alveolar epithelial cell line L2

The neopterin derivatives, neopterin and 7,8‐dihydroneopterin, modulate the cellular oxidant‐antioxidant balance as well as the expression of the inducible nitric oxide synthase (iNOS) gene. Since apoptosis can be induced by reactive oxygen intermediates and nitric oxide (NO) we investigated whether these neopterin derivatives induce apoptotic cell death. As model we selected the rat alveolar epithelial cell line L2. 24 h incubation of neopterin (1–1000 μM) as well as 7,8‐dihydroneopterin (1–1000 μM) resulted in a significant increase of percent apoptotic cells (measured by FACS analysis). Coincubation of both pteridines with the cytomix (interferon‐γ plus tumor necrosis factor‐α) led to a significantly higher apoptosis than the cytomix alone. In contrast to the cytomix, no iNOS gene expression and no NO release could be detected after incubation with neopterin or 7,8‐dihydroneopterin. We conclude that neopterin and 7,8‐dihydroneopterin are per se inducers of apoptosis which is not mediated by nitric oxide. This may be of importance in inflammatory pulmonary diseases associated with an activation of the cellular immune system.

Chronic immune stimulation, oxidative stress, and apoptosis in HIV infection☆

Infection with the human immunodeficiency virus (HIV) is accompanied by a decrease in CD4+ T cell numbers and the ultimate disruption of immunological functions. In sera of infected patients, elevated levels of interferon-gamma are detected, which is indicative of an activated TH1-type immune response. T-cell-derived interferon-gamma leads to the expression of various proinflammatory cytokines and enhanced macrophage capacity to secrete reactive oxygen intermediates. In addition, interferon-gamma is the major stimulator for the biosynthesis of neopterin and its reduced form, 7,8-dihydroneopterin. Neopterin is known as a sensitive immune activation marker in clinical laboratory diagnosis. Recent data implied a potential role of neopterin derivatives in oxygen free-radical-mediated processes, e.g. high concentrations of 7,8-dihydroneopterin were found to interfere with the oxidant-antioxidant balance, and may lead to apoptosis of human cells. In addition, 7,8-dihydroneopterin was found to be effective in the activation of redox-sensitive transcription factors and in the induction of HIV-1 gene expression. In this commentary, we describe our current view as to how neopterin derivatives, in concert with cytokines and reactive oxygen intermediates, may lead the way to the final destruction of the cellular immune system.