The use of human peripheral blood mononuclear cells (PBMC) as an in vitro system to predict in vivo toxicity was investigated. For 58 chemicals, the effect on cytokine secretion (IL-5, IFNgamma and TNFalpha) by phytohaemagglutinin-activated PBMC was measured, IC50 values were calculated and correlations of these endpoints with human LC50 values were determined. The best result was obtained with IFNgamma as an endpoint for which the calculated R(2) value was 0.58 which is comparable with the R(2) values for the classical neutral red uptake (NRU) assays using murine 3T3 cells and normal human keratinocytes (R(2)=0.56 and 0.59, respectively). When for each chemical the lowest IC50 value of the three endpoints was correlated with LC50 the calculated R(2) increased slightly to 0.63. A specific strength of our test is that it corrects several outliers (diazepam, digoxin, malathion and verapamil hydrochloride) which do not fit in the linear regression analysis for IC50 values obtained with the classical 3T3 NRU assay. Furthermore, 2,4-dichlorophenoxyacetic acid, cyclosporine A and pentachlorophenol had a 10 times lower IC50 value than the estimated human LC50 value and were identified as immunotoxic alerts. In conclusion, new endpoints investigated in this study contribute to the prediction of immunotoxic effects and correct outliers of classical cytotoxicity assays.

Abstract In the rat two major molecular variants of prolactin are recorded i.e. 23,000 M(r) and glycosylated 26,000 M(r). In order to further characterize the glycosylated 26,000 rat prolactin molecular variant, rat pituitary cell lysates were digested with several glycoen-zymes and the digestion products submitted to sodium dodecyl sulphate polyacrylamide gel electrophoresis and subsequent immunoblotting. The results were as follows: treatment with 1) neuraminidase, specific for sialic acid, yielded an M(r) decrease of the glycosidic variant from 26,000 to 24,500, 23,800, 23,000 and 22,000; 2) endo-alpha-N-acetylgalactosaminidase, which releases the disaccharide Gal (beta 1-3) GalNac from O-glycans, split 26,000 rat prolactin into a doublet of M(r) 26,000 to 25,500; and 3) mixed exoglycosidases from Turbo cornutus caused a gradual M(r) shift from 26,000 to 23,000. Affinity chromatography on wheat germ agglutinin Sepharose 6MB and soybean agglutinin agarose of rat pituitary homogenates and competitive inhibition tests showed that glycosylated rat prolactin has distinct affinity for these lectins. From the experimental data it is proposed that glycosylated rat prolactin is O-linked through threonine by the disaccharide Gal (beta 1-3) GalNac and possesses at least GalNac, and/or Gal and sialyl residues.

Abstract Prolactin cells derived from the anterior pituitaries of female rats were cultured in the presence of tunicamycin, swainsonine, castanospermine, beta-hydroxynorvaline and monensin in order to study their effect on the post-translational processing of the M(r) 17,000, 23,000 and 26,000 prolactin molecular forms. Sodium-dodecyl-sulphate polyacrylamide electrophoresis and subsequent immunoblotting revealed that: 1) tunicamycin, swainsonine and castanospermine, compounds that are essentially known as inhibitors of the N-glycosylation processus, had no effect on M(r) 17,000, 23,000 and 26,000 rat prolactin; 2) betahydroxynorvaline, which has been assumed to inhibit processing of pre-prolactin to mature 23,000 prolactin, did not increase the synthesis of 26,000 rat prolactin. In case of inhibition of the processing of a pre-prolactin to mature prolactin, one would expect an increase of the pre-prolactin; consequently, we could not establish the 26,000 rat prolactin, we revealed in immunoblotting, as a pre-prolactin; 3) monensin affected the post-translational processing of 17,000 and 26,000 rat prolactin, but left the 23,000 mature form intact. This is an important finding for the following reasons: monensin blocks the transport of secretory and membrane proteins, and this blockade prevents the cleavage of these molecules; indeed, production of 17,000 rat prolactin, a form of cleaved prolactin, was inhibited. Monensin also affects glycosylation and 26,000 rat prolactin has been identified as a presumably O-iinked glycosylated variant. The fact that its synthesis is inhibited by monensin treatment, but not by inhibitors of the N-linked process, particularly tunicamycin, and that 26,000 rat prolactin is susceptible to mild alkali and decomposition via beta-elimination are decisive arguments in favour of the O-linked glycosidic linkage.

Summary Background The contribution of IL-9 to human atopy is supported by genetic studies. However, IL-9 production in response to allergen in vitro has been reported only in children. Objective Study IL-9 induction by allergen in adults, compare it with IL-5 and IL-13 and evaluate its association with atopy. Methods Peripheral blood mononuclear cell (PBMC) from control adults and from atopic patients were cultured with various allergens or phytohaemagglutinin (PHA) and secreted IL-5, IL-9 and IL-13 were measured by ELISA. Results IL-9 was produced in response to Dermatophagoides pteronyssinus (Der p) by PBMC from Der p-hypersensitive adults at levels equivalent to those induced by PHA but with slower kinetics. The induction of IL-9 was allergen specific, reflecting donor RAST profile. In Der p-triggered reactions of non-atopic and atopic subjects, IL-9 showed the highest selectivity for atopics, IL-5 and IL-13 being produced more frequently in non-atopic donors. Significant correlations with specific IgE titres were found for IL-9 with all allergens tested (Der p and two peptides of Bet v 1 birch allergen). For IL-5 and IL-13, they were in the same range for Der p but more variable for birch allergens. Patterns of cytokine production by individual patients in response to allergen reflected these differences: for Der p, IL-5, IL-9 and IL-13 productions were strongly correlated but for birch IL-5 differed from the latter two. The in vitro production of IL-9 reflected clinical hypersensitivity profiles and was higher in individuals with asthma than in those with disease limited to rhinitis and/or conjunctivitis. Conclusions Allergen-triggered IL-9 production in vitro is an excellent marker for atopy in adults given its virtual absence in allergen-stimulated PBMC from non-atopic individuals and its correlation with allergen-specific IgE and asthma.

Beside its pivotal role in reproduction, the pituitary hormone prolactin (PRL) has been attributed an immunomodulatory function. Here we report that cAMP is an important stimulator of PRL transcription in primary human T lymphocytes. Inhibition of both protein kinase A (PKA) and p38 MAPK partially abrogated cAMP-induced PRL expression. In addition, cAMP-induced phosphorylation of p38 was shown to occur independently of PKA and could be mimicked by a methylated cAMP analogue which specifically activates the recently discovered cAMP receptor EPAC (exchange protein directly activated by cAMP). Our findings suggest that cAMP induces PRL expression in T lymphocytes via cooperation of at least two different signaling pathways: a PKA-dependent pathway leading to the phosphorylation of cAMP response element-binding protein, and a PKA-independent pathway leading to p38 phosphorylation.

Human lymphocyte subpopulations differ in their cellular responses to ionizing radiation. To shed light on the molecular basis of this effect, we characterized the transcriptional response to 1 Gy X-rays of CD4+ T lymphocytes. Of 18,433 genes tested, 102 were modulated more than 1.5-fold. The majority of the strongly activated genes were p53 targets involved in DNA repair and apoptosis. The expression of three of these genes was further tested by quantitative RT-PCR in lymphocyte subpopulations [CD4+ and CD8+ T, CD19+ B, CD56+ natural killer cells and peripheral blood lymphocytes (PBLs)] from ten adult donors. In contrast to DDB2, TNFRSF10B and BAX were differentially modulated among the subpopulations and the PBLs, being more activated in irradiated CD19+ B and CD8+ T lymphocytes. The level of BAX activation in the various subpopulations correlated with the sensitivity of the cells to radiation, suggesting its possible role in the differential radiosensitivity of hematopoietic cell subsets.

Besides its pivotal role in reproduction, the polypeptide hormone prolactin (PRL) has immunomodulatory properties. Whereas the bulk of circulating PRL is produced by the pituitary, PRL is also produced by the decidua, the myometrium, the mammary gland and leukocytes. Extrapituitary PRL expression is regulated differently from that in the pituitary, due to the use of an alternative promoter. Here we show for the first time that in T lymphocytes PRL expression is subject to regulation by cytokines. We established that both IL-2 and IL-4 reduced PRL mRNA levels in T lymphocytes to 25 and 28% of control values, respectively. PRL mRNA expression was inhibited to a lesser extent by IL-1beta, which decreased PRL mRNA levels to 58% of control values.

Besides its pivotal role in reproduction, the polypeptide hormone prolactin (PRL) has been attributed an immunomodulatory function. Extrapituitary PRL expression is regulated differently from that in the pituitary, due to the use of an alternative promoter. In leukocytes, cAMP is an important regulator of PRL expression. We report that in the human eosinophilic cell line Eol-1, cAMP-induced PRL expression is partially abrogated by two protein kinase A (PKA) inhibitors (H89, PKI) and by the p38 inhibitor SB203580. Phosphorylation of p38 was PKA-independent and could be stimulated by a methylated cAMP analogue, which specifically activates the exchange factor directly activated by cAMP (EPAC). Furthermore, cAMP induced a PKA-dependent phosphorylation of cAMP-responsive element binding protein (CREB). We postulate that cAMP induces PRL expression via two different signalling pathways: a PKA-dependent pathway leading to the phosphorylation of CREB, and a PKA-independent pathway leading to the phosphorylation of p38.

We report here the role of one of the less studied members of the family of suppressors of cytokine signaling (SOCS), namely SOCS-7, in cytokine signaling. We demonstrate that SOCS-7 inhibits prolactin (PRL), growth hormone (GH), or leptin (LEP) signaling mediated through STAT3 and STAT5 in a dose-dependent manner. SOCS-7 also attenuated STAT3 and STAT5 signaling induced by overexpression of JH1, the catalytic subdomain of JAK2. Since SOCS-7 interacted with phosphorylated STAT3 or STAT5, we assumed that SOCS-7 acts at the level of STAT proteins. Indeed, we showed that SOCS-7 inhibits PRL- and leptin-induced STAT5 and STAT3 phosphorylation and prevented the nuclear translocation of activated STAT3. Taken together, our results indicate that SOCS-7 is a physiological dysregulator of PRL, leptin, and probably also GH signaling and that its mode of action is a novel variation of SOCS protein inhibition of cytokine-inducible STAT-mediated signal transduction.

We previously reported that prolactin gene expression in the T-leukemic cell line Jurkat is stimulated by PGE(2) and that cAMP acts synergistically with Ca(2+) or protein kinase C on the activation of the upstream prolactin promoter. Using the transcription inhibitor actinomycin D, we now show that PGE(2)-induced prolactin expression requires de novo prolactin mRNA synthesis and that PGE(2) does not influence prolactin mRNA stability. Furthermore, PGE(2)-induced prolactin expression was inhibited by protein kinase inhibitor fragment 14-22 and BAPTA-AM, which respectively, inhibit protein kinase A- and Ca(2+)-mediated signaling cascades. Using specific PGE(2) receptor agonists and antagonists, we show that PGE(2) induces prolactin expression through engagement of E-prostanoid (EP) 3 and EP4 receptors. We also found that PGE(2) induces an increase in intracellular cAMP concentration as well as intracellular calcium concentration via EP4 and EP3 receptors, respectively. In transient transfections, 3000 bp flanking the leukocyte prolactin promoter conferred a weak induction of the luciferase reporter gene by PGE(2) and cAMP, whereas cAMP in synergy with ionomycin strongly activated the promoter. Mutation of a C/EBP responsive element at -214 partially abolished the response of the leukocyte prolactin promoter to PGE(2), cAMP, and ionomycin plus cAMP.