Inhibition of in vitro cytokine production by human peripheral blood mononuclear cells treated with xenobiotics: implications for the prediction of general toxicity and immunotoxicity.

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.

Modulation of prolactin expression in human T lymphocytes by cytokines.

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.

Multiple, PKA-dependent and PKA-independent, signals are involved in cAMP-induced PRL expression in the eosinophilic cell line Eol-1.

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.

Suppressor of cytokine signaling 7 inhibits prolactin, growth hormone, and leptin signaling by interacting with STAT5 or STAT3 and attenuating their nuclear translocation.

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.

Mechanism of prostaglandin (PG)E2-induced prolactin expression in human T cells: cooperation of two PGE2 receptor subtypes, E-prostanoid (EP) 3 and EP4, via calcium- and cyclic adenosine 5'-monophosphate-mediated signaling pathways.

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.

Limited effect of selected organic pollutants on cytokine production by peripheral blood leukocytes.

To test the hypothesis that some persistent organic pollutants contribute to the increased prevalence of allergic disease, the effects of selected compounds on cytokine production by PBMC from control and allergic donors were evaluated. Cells were cultured for six days in the presence of a xenobiotic (PCB 153, hexachlorobenzene, pentachlorobenzene, pentachlorophenol, lindane, atrazine or DMSO vehicle) with phytohemagglutinin (PHA) or Dermatophagoides pteronyssinus extract, then for one day in the presence of PHA + phorbol 12-myristate 13-acetate. PCB 153 reduced the levels of IL-10, IFN-gamma and TNF-alpha. Hexachlorobenzene reduced the levels of IL-5, IL-10 and IFN-gamma. Pentachlorobenzene reduced IL-6 levels. Pentachlorophenol reduced IL-5 levels. Lindane and atrazine reduced both IL-5 and IFN-gamma. In addition, lindane reduced TNF-alpha levels. As these compounds had similar effects on cells from allergic and non-allergic donors, and as these effects were, in all cases, very limited indeed, the potential deleterious impact of the xenobiotics tested on the allergic response seems unlikely.

The suppressor of cytokine signaling (SOCS)-7 interacts with the actin cytoskeleton through vinexin.

To understand the function of the suppressor of cytokine signaling (SOCS)-7, we have looked for proteins interacting with SOCS-7 in a stringent yeast two-hybrid screen of a human leukocyte cDNA-library. We identified the cytoskeletal molecule vinexin as a partner interacting with SOCS-7. Tests with deletion mutants of SOCS-7 demonstrated that a central region of the molecule containing several proline-rich regions, N-terminal to the SH2 domain, was responsible for the binding to vinexin. It is thus likely that one of the SH3 domains of vinexin interacts with a poly-proline region of SOCS-7. The interaction with vinexin was confirmed biochemically as vinexin-alpha was co-precipitated with SOCS-7. Confocal laser-scanning microscopy in HEK293T, MCF-7, and 3T3-L1 cells showed that part of the transfected SOCS-7-green fluorescent protein (GFP) molecules merged with vinexin and with actin. Taken together, our data indicate that SOCS-7 interacts with vinexin and the actin cytoskeleton.

IGF-I stimulates IL-8 production in the promyelocytic cell line HL-60 through activation of extracellular signal-regulated protein kinase.

Interleukin (IL)-8 serves as a major chemoattractant for neutrophils and has also been proposed to affect cancer progression. In the present study, we show that IGF-I stimulates IL-8 mRNA expression and IL-8 secretion in the leukemic cell line HL-60. Stimulation of IL-8 expression was completely attenuated by two inhibitors of mitogen-activated protein kinase (MAPK) kinase (MEK), which phosphorylates the MAPKs extracellular-regulated kinase (ERK)1 and ERK2, and by the c-Jun NH2-terminal kinase (JNK) inhibitor SP600125. In contrast, inhibitors of p38 MAPK and phosphatidylinositol-3 kinase (PI3K) did not abrogate the effect of IGF-I. We also show that IGF-I stimulates the activation of ERK1 and ERK2, but we could not detect any effect of IGF-I on the phosphorylation of p38, JNK(p46) or JNK(p54). Collectively, our results suggest that basal JNK activity and activation of the MEK-ERK pathway are required for upregulation of IL-8 by IGF-I in HL-60 cells.

Clinical and biological characterization of macroprolactinemia with and without prolactin-IgG complexes.

OBJECTIVE: Macroprolactinemia, which can be detected by a polyethylene glycol (PEG) precipitation test, is a clinically and biologically heterogeneous condition. In this study, we analyzed whether the clinical presentation, the hormonal findings and the in vitro lactogenic activity differed between macroprolactinemic patients with and without circulating prolactin (PRL)-IgG complexes. DESIGN: Clinical data were reviewed and additional hormonal studies were performed in 50 hyperprolactinemic patients with macroprolactinemia. METHODS: Macroprolactinemia was identified by a PRL recovery after PEG precipitation of <50%, as measured by an automated commercial immunoassay system and circulating PRL-IgG complexes by an abnormal PRL binding to anti-IgG agarose. RESULTS: PRL-IgG complexes were found in 46 patients. The origin of hyperprolactinemia in these 46 patients was idiopathic in 33 patients, while a pituitary lesion or stalk magnetic resonance imaging or computed tomography scan was detected in 13 patients found compression. Galactorrhea was found in 11 of these 46 patients, while this condition was present in three of the four patients without circulating PRL-IgG complexes. The median free PRL concentration was significantly lower in patients with PRL-IgG complexes than in the group without complexes (243 vs 969 mIU/l; P<0.005), whereas median total PRL immunoreactivity and median PRL bioactivity in the Nb2 assay were not significantly different. In patients with circulating PRL-IgG complexes, Nb2 bioassay results correlated significantly with total PRL immunoreactivity (r=0.64; P<0.0001), but not with free PRL results (r=0.24; P<0.17). CONCLUSIONS: These results indicate that PRL-IgG complexes (i) account for most cases of macroprolactinemia--as identified by PEG precipitation--in hyperprolactinemic patients presenting with a variety of diagnoses, (ii) are not associated with a specific clinical presentation, (iii) can be found in patients with diverse pituitary pathologies, and (iv) possess an in vitro lactogenic activity in the Nb2 bioassay in relation to their immunoreactivity.

Effects of prolactin on cloned human T-lymphocytes.

To evaluate the possible role of prolactin (PRL) in T-lymphocytes, we monitored gene induction in one cytotoxic T-lymphocyte (CTL) clone derived from a patient with hemochromatosis and in several T-helper clones generated from a normal donor and a patient with multiple sclerosis. The CTL clone expressed conventional PRL receptor (PRLR), and PRL induced the expression of suppressor of cytokine signaling-3 (SOCS-3) and increased the expression of SOCS-2 and cytokine-inducible src homology-2 containing protein (CIS, another member of the SOCS family). As is the case in granulocytes, expression of a conventional receptor for PRL could not be shown by polymerase chain reaction analysis on three helper clones. In addition, as in granulocytes, PRL modulated the expression of genes such as the interferon-regulatory factor-1, inducible nitric oxide synthase, CIS, and SOCS-2. These effects were also elicited with ovine PRL and could be prevented by anti-PRL antibodies. Thus, the use of clones allowed the detection of direct effects of PRL on T-cells, even when these have few or no detectable PRLR, confirming that human T-lymphocytes are targets for PRL.

Inhibition of cytokine production by the herbicide atrazine. Search for nuclear receptor targets.

The hematological toxicity of the commonly used triazine herbicides is a cause for concern. In a search for molecular targets of these compounds, as their effects paralleled those seen with dexamethasone (DEX), we first looked for interaction with the glucocorticoid receptor. In contrast to the effects on proliferation and cytokine production of DEX, those induced by atrazine were not prevented by the glucocorticoid antagonist RU486. Also, whereas DEX was able to inhibit the promoter activity of genes regulated by NF-kappaB, atrazine failed to do so. We next looked for interaction with members of the peroxisome proliferator-activated receptor (PPAR) family. No peroxisome proliferation was observed in the liver or kidneys of mice treated with atrazine. Moreover, no PPAR-mediated induction of promoter activity was seen on targets of PPARalpha, PPARgamma, or PPARdelta. Similarly, neither atrazine nor simazine were able to stimulate RORalpha-mediated promoter activity. Finally, no binding of atrazine to the AR was observed. In conclusion, the effects of atrazine-type herbicides most probably do not result from interaction with the above-mentioned nuclear receptors.

Igf-I inhibits spontaneous apoptosis in human granulocytes.

Granulocytes are key cells in inflammatory processes that are recruited to sites of inflammation by chemoattractants such as IL-8 produced by neutrophils and monocytes. Programmed cell death (apoptosis) of granulocytes and subsequent recognition and phagocytosis by macrophages is a crucial mechanism for resolution of inflammation. Because IGF-I is a potent antiapoptotic factor, we addressed the effects of IGF-I on in vitro apoptosis of human peripheral blood granulocytes. We detected 1390 +/- 467 IGF-I receptors with a dissociation constant of 2.3 +/- 0.9 nM on purified granulocytes. Using microscopical analysis, annexin V binding assays to detect relocation of phosphatidylserine to the cell surface, and DNA fragmentation assays, we showed that IGF-I inhibits spontaneous apoptosis of granulocytes in serum-free culture by 32-45%. IGF-I did not modulate the secretion of IL-6, TNF alpha, and IL-8 by granulocytes, but IL-8 secretion by peripheral blood mononuclear cells was enhanced by 40%. These observations indicate that IGF-I may promote granulocyte functions by increasing granulocyte longevity.