Development of an allergy test model: activation of human mast cells with potentially allergenic substances.

Due to the permanent increase of newly developed and already existing allergies, simple, quick, and reliable test models for detecting potentially allergenic substances are still required. Here, we describe the development of a new in vitro allergy test based on isolated primary mast cells (MC) of non-allergic patients from lung tissue and foreskin specimens, respectively. To establish the specificity of the test model we used primary MC stimulated with immunoglobulin E (IgE), human recombinant stem cell factor (hrSCF), and anti-IgE antibodies to release significant amounts of histamine indicating the ability of MC to cause a hypersensitivity reaction of the immediate type. The general applicability of this test model for detecting allergenic substances could be confirmed by histamine release of primary MC stimulated with sera of patients suffering from house dust allergy, and the corresponding antigen Dermatophagoides pteronyssinus. The results of the present work suggest that this newly developed human in vitro model provides the opportunity of testing substances for their allergenic potential within days and at low costs. This could also be of particular interest for newly produced compounds.

Regulation by rho family GTPases of IL-1 receptor induced signaling: C3-like chimeric toxin and Clostridium difficile toxin B inhibit signaling pathways involved in IL-2 gene expression.

In this study the participation of Rho family GTPases in the regulation of IL-1-activated protein kinase cascades controlling IL-2 synthesis was investigated in murine EL-4 thymoma cells. The recombinant C3-like chimeric toxin, which consists of the C3 toxin of Clostridium limosum and the N-terminal part of Clostridium botulinum C2 toxin (C2IN-C3) interacting with the C2II binding subunit to facilitate uptake into cells, and selectively inactivates Rho A by ADP-ribosylation, prevented IL-1-stimulated activation of Jun-NH2-terminal-kinases (JNK) and p38 mitogen-activated-protein kinases (MAPK). UDP-monoglucosylation and concomitant inactivation of Rho A and of Rac-2 by Clostridium difficile toxin B also inhibited IL-1-induced activation of JNK and p38 MAPK, but additionally inhibited activation of the extracellular-regulated-kinase pathway and DNA binding of the transcription factor NFkappaB. Accordingly, pre-treatment of cells with C21N-C3 fusion toxin only decreased IL-1-stimulated IL-2 synthesis by 50%, while in C. difficile toxin B-treated cells IL-1-induced IL-2 secretion was reduced by 90%. These results imply that together with Rho A an additional member of the Rho family G proteins, i.e. Rac-2, is critically involved as an upstream regulator in IL-1-induced activation of different MAPK, stress-activated protein kinases, and in NFkappaB activation controlling IL-2 gene expression in response to IL-1, acting in close proximity to the IL-1-receptor complex.

Comparative quantification of IL-1beta, IL-10, IL-10r, TNFalpha and IL-7 mRNA levels in UV-irradiated human skin in vivo.

OBJECTIVE AND DESIGN: Ultraviolet (UV) exposure induces local immunosuppression and inflammation in human skin. Cytokines are, in part, responsible for these responses. To investigate the effects of UV-induced gene expression at the molecular level we established a sensitive in vivo/ex vivo method for a comparative quantification of cytokines and receptors involved in the local skin immune reactions. MATERIAL AND METHODS: Specific mRNA levels of human UV-irradiated skin were determined by real time quantification (TaqMan RT-PCR). Highly efficient PCR-reaction conditions were obtained by designing very short PCR-templates (72-87 bp). The most sensitive PCR-conditions were obtained by optimisation of primer and Mn(OAc)2-concentrations, which led to significant PCR signals (C(T)-value) of less than 36 cycles. A strong correlation between PCR efficiency of the internal control (GAPDH) compared to targets (IL-1beta, IL-10, IL-10r, TNFalpha, IL-7) allowed the use of deltadelta C(T)-method to quantify comparable mRNA levels. RESULTS: Interleukin-1beta (IL-1beta), Interleukin-10 (IL-10), and tumour necrosis factor alpha (TNFalpha) mRNA levels were increased in a time- and dose-dependent manner. Interleukin-1beta induction reached a maximum (approx. 44-fold) 6 h after a UV-dose equivalent to 3 times the minimal erythemal doses just perceptible (MEDjp). Maximal TNFalpha mRNA expression (approx. 14-fold) was also detected 6 h after UV exposure. Interleukin-10 mRNA induction reached a maximum of approximately 14-fold 24 h after UV-irradiation of 3 MEDjp. Time- and dose-dependent changes in Interleukin-7 and Interleukin-10 receptor mRNA levels did not occur after UV-irradiation. CONCLUSIONS: Time-distinct gene induction of IL-1beta, TNFalpha and IL-1beta is involved in UV-induced immune reactions, but no considerable changes were found for IL-10r or IL-7.

The Hsp90-specific inhibitor geldanamycin selectively disrupts kinase-mediated signaling events of T-lymphocyte activation.

The 90-kDa heat shock protein (Hsp90) is the most abundant molecular chaperone of eukaryotic cells. Its chaperone function in folding nascent proteins seems to be restricted to a subset of proteins including major components of signal transduction pathways (eg, nuclear hormone receptors, transcription factors, and protein kinases). Improper function of these proteins can be induced by selective disruption of their complexes with Hsp90 using the benzoquinonoid ansamycin geldanamycin. In this study, we demonstrate that geldanamycin treatment blocks interleukin (IL)-2 secretion, IL-2 receptor expression, and proliferation of stimulated T-lymphocytes. Moreover, geldanamycin decreases the amount and phosphorylation of Lck and Raf-1 kinases and prevents activation of the extracellular signal regulated kinase (ERK)-2 kinase. Geldanamycin also disrupts the T-cell receptor-mediated activation of nuclear factor of activated T-cells (NF-AT). Treatment with geldanamycin, however, does not affect the activation of lysophosphatide acyltransferase, which is a plasma membrane enzyme coupled to the T-cell receptor after T-cell stimulation. Through demonstrating the selective inhibition of kinase-related T-lymphocyte responses by geldanamycin, our results emphasize the substantial role of Hsp90-kinase complexes in T-cell activation.

Molecular mechanisms of T lymphocyte activation: convergence of T cell antigen receptor and IL-1 receptor-induced signaling at the level of IL-2 gene transcription.

Co-stimulation of murine EL-4 thymoma cells-carrying high numbers of TCR and type I IL-1 receptors (IL-1R)-with anti-CD3 antibodies and IL-1 resulted in synergistic enhancement of IL-2 synthesis. While the extracellular signal-regulated kinase (ERK) cascade was activated by both receptors, IL-1 preferentially stimulated Jun-N-terminal kinases (JNK) and p38 mitogen-activated kinase or microtubule-associated protein kinase (MAPK). Interruption of TCR- or IL-1R-stimulated ERK cascade by PD-98059, a specific inhibitor of MAP/ERK kinase (MEK), resulted in partial suppression of nuclear factor of activated T cells activation and in complete inhibition of IL-1-stimulated NFkappaB activation. Suppression of activation of both MEK and p38 MAPK resulted in significant inhibition of IL-2 gene expression. The results show that maximal activation of the IL-2 gene requires activation of at least two different protein kinase cascades, i.e. of the ERK and p38 pathways but presumably also that of JNK which converge at the level of the IL-2 promoter resulting in enhancement of its transcriptional activity.

The Hsp90-specific inhibitor, geldanamycin, blocks CD28-mediated activation of human T lymphocytes.

The 90 kDa heat shock protein (Hsp90) is a molecular chaperone aiding the folding of nuclear hormone receptors and protein kinases. Hsp90-mediated folding can be disrupted by the Hsp90-specific drug, geldanamycin. Here we provide evidence for the inhibition of the CD28-specific BW 828 antibody-mediated activation of human T lymphocyte proliferation, IL-2 secretion and IL-2 receptor expression by geldanamycin. Our results suggest that the major cytoplasmic chaperone, Hsp90, plays an important role in CD28-mediated T lymphocyte activation.

Stress-activated protein kinase/Jun N-terminal kinase is required for interleukin (IL)-1-induced IL-6 and IL-8 gene expression in the human epidermal carcinoma cell line KB.

The cytokine interleukin-1 (IL-1) is a major inflammatory hormone which activates a broad range of genes during inflammation. The signaling mechanisms triggered by IL-1 include activation of several distinct protein kinase systems. The stress-activated protein kinase (SAPK), also termed Jun N-terminal kinase (JNK), is activated particularly strongly by the cytokine. In an attempt to delineate its role in activation of gene expression by IL-1, we inhibited the IL-1-induced SAPK/JNK activity by stable overexpression of either a catalytically inactive mutant of SAPKbeta (SAPKbeta(K-R)) or antisense RNA to SAPKbeta in human epidermal carcinoma cells. A detailed analysis of signal transduction in those cells showed that activation of neither NFkappaB nor p38 mitogen-activated protein kinase was affected, suggesting that we achieved specific blockade of the SAPK/JNK. In untransfected and vector-transfected KB cells, IL-1 induced a strong increase in expression of IL-6 and IL-8 mRNA, along with the synthesis of high amounts of the proteins. In two KB cell clones stably overexpressing the mutant SAPKbeta(K-R), and three clones stably overexpressing antisense RNA to SAPKbeta, expression of IL-6 and IL-8 in response to IL-1 was strongly reduced at both the mRNA and protein level. These data indicate that the SAPK/JNK pathway provides an indispensable signal for IL-1-induced expression of IL-6 and IL-8.

Complex effects of long-term 50 Hz magnetic field exposure in vivo on immune functions in female Sprague-Dawley rats depend on duration of exposure.

In previous studies we have demonstrated that 50 Hz, 100 microT magnetic field (MF) exposure of female Sprague-Dawley rats for 13 weeks significantly enhances the development and growth of mammary tumors in a breast cancer model. The present study was designed to test the hypothesis that, at least in part, the tumor (co)promoting effect of MF exposure is due to MF effects on the immune surveillance system, which is of critical importance in protecting an organism against the development and growth of tumors. For this purpose, female Sprague-Dawley rats of the same age as in the mammary tumor experiments were continuously exposed for different periods (2, 4, 8, and 13 weeks) to a 50 Hz, 100 microT MF. Control groups were sham-exposed simultaneously. Following the different exposure periods, splenic lymphocytes were cultured and the proliferative responses to the T-cell-selective mitogen concanavalin A (Con A) and the B-cell-selective pokeweed mitogen (PWM) were determined. Furthermore, the production of interleukin-1 (IL-1) was determined in the splenocyte cultures. The mitogenic responsiveness of T cells was markedly enhanced after 2 weeks of MF exposure, suggesting a co-mitogenic action of MF. A significant, but less marked increase in T-cell mitogenesis was seen after 4 weeks of MF exposure, whereas no difference from sham controls was determined after 8 weeks, indicating adaptation or tolerance to this effect of MF exposure. Following 13 weeks of MF exposure, a significant decrease in the mitogenic responsiveness of lymphocytes to Con A was obtained. This triphasic alteration in T-cell function (i.e., activation, tolerance, and suppression) during prolonged MF exposure resembles alterations observed during chronic administration of mild stressors, substantiating the hypothesis that cells respond to MF in the same way as they do to other environmental stresses. In contrast to T cells, the mitogenic responsiveness of B cells and IL-1 production of PWM-stimulated cells were not altered during MF exposure. The data demonstrate that MF in vivo exposure of female rats induces complex effects on the mitogenic responsiveness of T cells, which may lead to impaired immune surveillance after long-term exposure.

Different protein kinase C isoenzymes regulate IL-2 receptor expression or IL-2 synthesis in human lymphocytes stimulated via the TCR.

Stimulation of purified human PBL with mAbs raised against the T cell receptor resulted in an immediate and transient activation of protein kinase C-alpha (PKC-alpha) and PKC-theta, peaking at 10 min, whereas PKC-beta, -delta, and -epsilon were translocated with a delay of >90 min and remained activated for up to 2 h. To characterize specific functions of distinct PKC isoenzymes, Abs against different PKC isoenzymes were introduced by means of electropermeabilization. Neutralization of PKC-alpha and -theta resulted in the complete inhibition of IL-2R expression, whereas anti-PKC-beta, -delta, and -epsilon Abs inhibited IL-2 synthesis. Extensive control experiments have shown that neither electropermeabilization nor control Ig influenced PKC activity and cellular functions. Our data thus clearly show that specific PKC isoenzymes regulate different cellular functions in stimulated human lymphocytes.

T cell antigen receptor dependent signalling in human lymphocytes: cholera toxin inhibits interleukin-2 receptor expression but not interleukin-2 synthesis by preventing activation of a protein kinase C isotype, PKC-alpha.

Activation and translocation of protein kinases C is a key event in the regulation of T lymphocyte activation, proliferation and function. Stimulation of human peripheral blood lymphocytes with the monoclonal antibody BMA 031 raised against the T cell antigen receptor led to a bimodal activation of protein kinases C. The immediate activation and translocation of the protein kinase C isoform PKC-alpha was followed by activation and translocation of the protein kinase C-beta isoenzyme after 90 min of stimulation. Pretreatment of the cells with cholera toxin for 90 min completely abolished activation of protein kinase C-alpha. In sharp contrast, activation and translocation of protein kinase C-beta was not influenced by the bacterial toxin, suggesting that activation and translocation of different protein kinase C isoenzymes are regulated by distinct mechanisms of transmembrane signalling coupled to the T cell antigen receptor/CD3 complex. The expression of high affinity IL-2 receptors was completely inhibited by cholera toxin, while IL-2 synthesis and secretion were not influenced in BMA 031-stimulated human lymphocytes. Extensive control experiments have shown that the effects of cholera toxin were not mediated by its B subunit, and were independent of elevation of intracellular cAMP concentration, suggesting that cholera toxin interfered with a signalling pathway leading to activation of protein kinase C-alpha, which could be responsible for the inhibition of IL-2 receptor expression. This hypothesis was substantiated by the finding that upon introduction of antibodies against protein kinase C-alpha, IL-2 receptor gene expression was completely suppressed. The results suggest, that protein kinase C-alpha might be the major protein kinase C isoenzyme of a signal transduction cascade regulating IL-2 receptor expression in stimulated human lymphocytes.

Interleukin-1 induced signalling: biphasic activation of mitogen-activated protein kinase kinase and mitogen-activated protein kinases in HeLa cells. Involvement of phosphoprotein phosphatases.

Results of this study document a biphasic activation of protein kinases of the MAP kinase cascade-MEK and MAP kinases-upon interleukin-1 stimulation in human HeLa cells. The specific activities of both MEK and MAP kinases were increased within 1 min, declined rapidly to control levels and increased again after 15 min of interleukin-1 stimulation. Inhibition by okadaic acid of serine/threonine specific phosphatases resulted in a marked increase in interleukin-1 stimulated MEK and MAP kinase activities. Elevation by interleukin-1 of the specific activities of MEK and MAP kinases correlated with suppression of serine/threonine phosphatases in the late phase of stimulation. The data indicate, that enhanced phosphorylation of cellular proteins by enzymes of the MAP kinase cascade might represent a fine balance between activated protein kinases and repressed phosphoprotein phosphatase 2A in interleukin-1 stimulated HeLa cells.

Interleukin 1 activates jun N-terminal kinases JNK1 and JNK2 but not extracellular regulated MAP kinase (ERK) in human glomerular mesangial cells.

Interleukin 1 (IL-1) potently activates human glomerular mesangial cells (HMC). In cytosolic extracts of IL-1-stimulated HMC or in anion exchange chromatography fractions we could not find any change in phosphorylation of myelin basic protein (MBP), a good substrate for extracellular regulated kinase (ERK). In contrast, IL-1 stimulated GST-jun kinase activity at least 10-fold. The jun kinase activity could be characterised as JNK1 and JNK2 at the protein and mRNA level. IL-1, TNF, UV light and osmotic stress, but not PMA, LPS, IL-3, IL-4, IL-6, IL-8, IL-10, IL-13, GM-CSF, PDGF, bFGF, TGF-beta and interferon-gamma were able to stimulate jun kinase activity in HMC, suggesting that jun kinase is selectively mediating signal transduction of the proinflammatory cytokines IL-1 and TNF as well as of cellular stress in HMC.

Exposure of DMBA-treated female rats in a 50-Hz, 50 microTesla magnetic field: effects on mammary tumor growth, melatonin levels, and T lymphocyte activation.

There is growing public concern about the possible health risks, particularly increased cancer risks of exposure to magnetic fields (MF) associated with power distribution systems. Recently, we have started a series of animal studies to investigate this issue, using the DMBA (7,12-dimethylbenz[a]anthracene) model of breast cancer in female rats. In the present study, female rats were chronically exposed to a 50-Hz, 50 microTesla (microT) MF with or without DMBA treatment. Because alterations in circulating levels of the pineal hormone melatonin and impaired immune system functions have been involved in breast cancer growth, and both melatonin and immune system are thought to be targets for MF-effects, serum melatonin and the proliferative capacity of splenic lymphocytes were determined in MF-exposed and sham-exposed rats. For this purpose, 216 female Sprague-Dawley rats were divided into four groups. Two of the groups (with 99 animals each) received oral applications of DMBA and were either sham-exposed or exposed in a 50-Hz, 50 microT MF for 24 h/day 7 days/week for a period of 91 days. The other two groups (9 animals each) were either sham-exposed or MF-exposed without DMBA treatment. The exposure chambers and all other environmental factors were identical for MF-exposed and sham-exposed animals. The DMBA-treated animals were palpated once weekly to assess the development of mammary tumors. At the end of the three-month period of MF exposure, the number and size of mammary tumors was determined by autopsy. In controls, DMBA induced tumors in approximately 55% of the animals within the 3 month period of sham-exposure. Already 8 weeks after DMBA application, the MF-exposed group exhibited significantly more tumors than sham-exposed animals. At time of autopsy, significantly more MF-exposed DMBA-treated rats exhibited macroscopically visible mammary tumors than DMBA-treated controls, thus indicating that MF exposure enhances the development and growth of cancers in this model. Comparison of the data from 50 microT with recent data from other flux densities indicated that long-term MF exposure of DMBA-treated rats increases the incidence of palpable and/or macroscopically visible mammary tumors in a highly dose-related fashion. Determination of nocturnal serum melatonin after 9 and 12 weeks of exposure at 50 microT did not yield significant differences between MF-exposed rats and sham-exposed controls, whereas a marked suppression of T cell proliferative capacity was seen in MF exposed rats. The data add further evidence to the hypothesis that hormone-dependent tissues such as breast might be particularly sensitive to MF-effects and indicate that immune system depression is involved in the increased breast cancer growth observed in MF exposed rats.

Differential signal transduction pathways regulating interleukin-2 synthesis and interleukin-2 receptor expression in stimulated human lymphocytes.

In human peripheral blood lymphocytes stimulated via the T-cell antigen receptor/CD3 complex IL-2 synthesis and cellular proliferation were effectively inhibited by a concentration of ouabain as low as 50 nM, whilst the expression of high affinity IL-2 receptors was not influenced. Binding of the monoclonal antibody, BMA 031 to the T-cell antigen receptor/CD3 complex resulted in a bimodal activation of protein kinase C. The activation of protein kinase C-alpha in the early phase of T-lymphocyte activation was not affected by 50 nM ouabain, in contrast sustained activation of protein kinase C-beta, between 90-240 min of stimulation was completely abolished by the cardiac glycoside. When protein kinase C was directly activated by PMA + ionomycin, 50 nM ouabain was ineffective in inhibiting protein kinase C activation, as well as subsequent IL-2 synthesis, suggesting that the glycoside interfered with signal transducing mechanism(s) upstream of the activation of protein kinase C. Ouabain had no influence on the elevation of intracellular calcium concentration in BMA 031 stimulated lymphocytes, ruling out the possibility that it interfered with the T-cell antigen receptor dependent phosphatidylinositol response. In contrast, lysophosphatide acyltransferase catalysed elevated incorporation of polyunsaturated fatty acids was effectively inhibited by low concentrations of ouabain in BMA 031-stimulated T-lymphocytes, whereas stimulation with PMA + ionomycin had no influence on the plasma membrane phospholipid fatty acid metabolism. These results suggest, that differential signal transduction pathways are involved in the activation of protein kinases C-alpha and -beta. They implicate that elevated incorporation of polyunsaturated fatty acids into plasma membrane phospholipids might contribute to sustained activation of protein kinase C-beta, and establish a link between activation of protein kinase C-beta and induction of IL-2 synthesis in human lymphocytes.

T-cell antigen receptor-induced signal-transduction pathways--activation and function of protein kinases C in T lymphocytes.

CONTENTS. T-cell activation--Structure of the T-cell antigen receptor--Modular organisation of the T-cell antigen receptor--T-cell antigen receptor-coupled signaling pathways: Activation of protein-tyrosine kinase by the T-cell antigen receptor; Signal transduction in lymphoid cells involves several protein-tyrosine kinases in parallel; Regulation of T-cell antigen receptor signaling by the phosphoprotein phosphatase CD45--Consequences of T-cell antigen receptor-induced tyrosine phosphorylation: Activation of phosphoinositol-lipid-turnover pathways--Activation of phospholipase C-gamma-1: p59fyn or p56lck?--G-protein motif of CD3-gamma: relevance for signal transduction--Association of lipid kinase with the T-cell antigen receptor--Intracellular signaling by phospholipid metabolites and calcium: activation of protein kinase C--Protein kinase C isoenzymes--Heterogenity of protein kinase C and mode of activation--Phospholipid-derived mediators in activation of protein kinase C in T-cells--Role of phospholipase D metabolites in activation of protein kinase C--Polyunsaturated fatty acids and lysophosphatidylcholine as activators of protein kinase C--Potein kinase C and p21ras function in interdependent and distinct signaling pathways during T-cell activation--Raf-1 kinase: regulator or target of protein kinase C?--Summary and perspectives.

The NF kappa B heterodimer/homodimer balance and IL-1-stimulated IL-2 production in murine T lymphocytes.

Activation of NF kappa B is considered one way IL-1 exerts its costimulatory effect on T cell activation and IL-2 production. However, T helper 2 cells do not synthesize IL-2 upon IL-1 stimulation, although they depend on IL-1 for proliferation. The involvement of NF kappa B in IL-2 production was therefore addressed in two different murine cell lines: D10N, a T helper 2 cell line that does not synthesize IL-2, and EL4 6.1., a thymoma cell line producing IL-2 when stimulated with IL-1. In both cell types IL-1 activated the DNA binding activity of the NF kappa B heterodimers p50/65 and p50/c-rel via the IL-1 type I receptor. In D10N cells, however, the p50/p50 homodimer is present in large amounts in unstimulated cells, whereas the heterodimers were only activated by IL-1-treatment. In contrast, only marginal amounts of the p50/p50 homodimer were found in EL4 6.1. cells, but a strong activation of heterodimers was induced by IL-1. The findings are compatible with the concept of p50 homodimer being inhibitory by replacing the p50/p65 or p50/c-rel heterodimer from nuclear kappa B binding sites. The inability of D10N cells to respond to IL-1 exposure with IL-2 formation might therefore be due to their high constitutive levels of p50 homodimers.

The nonapeptide leucinostatin A acts as a weak ionophore and as an immunosuppressant on T lymphocytes.

Earlier studies have disclosed that leucinostatin A, a hydrophobic nonapeptide antibiotic, assumes an alpha-helical secondary structure in nonpolar environments. The present report demonstrates that the peptide acts as a weak ionophore facilitating the transport of mono-and divalent cations through the plasma membrane of T lymphocytes and through artificial membranes. Leucinostatin A does not change the thymidine uptake of both resting mouse thymocytes and peripheral blood lymphocytes but dose-dependently prevents the activation of T lymphocytes by tetradecanoyl-phorbol-acetate and by anti-T cell receptor antibody.

Cyclosporin A inhibits T cell receptor-induced interleukin-2 synthesis of human T lymphocytes by selectively preventing a transmembrane signal transduction pathway leading to sustained activation of a protein kinase C isoenzyme, protein kinase C-beta.

Stimulation of human peripheral blood lymphocytes via T cell receptor/CD3 complex resulted in a bimodal activation of protein kinase(s) C (PKC). Within 10 min of stimulation PKC-alpha was translocated to, and thus activated in, the plasma membranes of human lymphocytes, followed by a fast dissociation of this isotype from the plasma membrane. This short term activation and translocation PKC-alpha proved to be cyclosporin A (CsA) insensitive. After 90 min of stimulation PKC-beta was translocated to and remained bound to the plasma membranes for up to 4 h. Preincubation of human lymphocytes with 200 ng/ml CsA specifically and completely abolished the sustained activation of PKC-beta. Neither the phorbol ester-induced direct activation of PKC nor the specific activity of the plasma membrane-bound enzyme was influenced by CsA, suggesting that a signal transduction pathway leading to sustained activation of PKC-beta was influenced by the immunosuppressive agent. In fact, CsA inhibited, in a concentration-dependent manner, the activation of lysophosphatid acyltransferase-catalyzed elevated incorporation of cis-polyunsaturated fatty acids into plasma membrane phospholipids. While interleukin-2 (IL-2) synthesis and cellular proliferation were completely inhibited by 200 ng/ml CsA in BMA 030- or BMA 031-stimulated cells, expression of high-affinity IL-2 receptors was not influenced by the immunosuppressive drug. These results suggest that synthesis and expression of high-affinity IL-2 receptors might be regulated by a signal-transducing pathway involving activation and translocation of PKC-alpha. Lysophosphatid acyltransferase-catalyzed incorporation of cis-polyunsaturated fatty acids might represent another mechanism of signal transduction implicated in the activation and translocation of PKC-beta, which is specifically inhibited by CsA. Neutralization of PKC-beta by introducing anti-PKC-beta antibodies prevented IL-2 synthesis and proliferation in stimulated human lymphocytes. The results suggest a possible link between activation of PKC-beta and regulation of IL-2 synthesis in activated human lymphocytes. Thus, inhibition of the activation and translocation of PKC-beta by CsA may result in inhibition of IL-2 gene expression in human lymphocytes.