Robert Hooghe

Effects of selected herbicides on cytokine production in vitro

To evaluate possible deleterious effects of commonly used herbicides on leukocytes, cytokine production was selected as a sensitive indicator. After in vitro exposure of human peripheral blood mononuclear cells from normal donors, the production of all 3 cytokines tested--interferon-gamma (a type 1 cytokine), interleukin-5 (a type 2 cytokine) and tumor necrosis factor-alpha (an inflammatory cytokine)--was impaired by up to 70, 50 and 70% respectively in a concentration-dependent manner in cultures exposed to atrazine (0.03-3 microM in 1% dimethylsulfoxide, DMSO). The effect paralleled that seen with dexamethasone, a known immunosuppressive agent. Other pesticides also dissolved in DMSO--mecoprop, simazine or MCPA (each up to 1 microM)--or dissolved in phosphate-buffered saline--diuron (up to 1 microM), isoproturon (up to 3 microM), metoxuron (up to 8 microM) or metamitron (up to 80 microM)--showed no concentration-related effects on cytokine production. There was, however, an inhibition of IFN-gamma and TNF-alpha production by simazine, metoxuron and mecoprop and of all three cytokines tested by diuron. MCPA (0.01 and 0.1 microM) stimulated the production of TNF-alpha. Thus, exposure to herbicides leading to plasma levels in the micromolar range induces imbalance in cytokine production.

Cytokine-like effects of prolactin in human mononuclear and polymorphonuclear leukocytes

Some biochemical events following the binding of prolactin (PRL) to its receptor in normal human leukocytes were investigated. PRL enhanced JAK2 phosphorylation in peripheral blood mononuclear cells (PBMC) but not in granulocytes. PRL also induced phosphorylation of Stat-5 in PBMC and Stat-1 in granulocytes. Subsequent binding of Stat-5- and of Stat-1-like molecules to a GAS responsive element from the beta-casein promoter was detected by EMSA. p38 MAPK (but not p42/p44 MAPK) was activated by PRL in both leukocyte populations. PRL induced iNOS and CIS mRNA expression in granulocytes. Increased expression of IRF-1 and SOCS-2 was observed in granulocytes and of SOCS-3 and iNOS in PBMC. Similar effects were obtained with ovine and human PRL. Antiserum to PRL reduced iNOS and IRF-1 expression induced by PRL in granulocytes and reduced iNOS expression in PBMC. Also, pretreatment of granulocytes with a p38 MAPK inhibitor (SB 203580) prevented in part PRL-induced iNOS and IRF-1 expression. In PBMC, the p38 inhibitor decreased PRL-induced iNOS gene expression. These results indicate that PRL-induced gene regulation in leukocytes requires the activation of at least two different pathways: the Stat and the MAP kinase pathways. Moreover, although PRL activates Stat in both leukocyte types, signal transduction is different in granulocytes and in PBMC. Most importantly, PRL modulates the expression of genes crucial to leukocyte function. The present findings reinforce the concept that PRL has cytokine-like activity in human leukocytes.

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.

A role for growth hormone and prolactin in leukaemia and lymphoma

Growth hormone (GH) and prolactin (PRL) qualify as lymphohaemopoietic growth and differentiation factors, and so does insulin-like growth factor (IGF)-I, which mediates many of GH activities. Although there is only limited evidence that endocrine, paracrine or autocrine GH or PRL play a role in human leukaemia and lymphoma, the expression of these factors or their receptors may have diagnostic or therapeutic implications. Indeed, the participation of GH, PRL or IGF-I in the development or progression of certain haematological malignancies or to the antitumour immune response has been documented. Examples discussed in this review include a rat lymphoma in which the PRL receptor acts as an oncogene; the rat Nb2 lymphoma, which is dependent on PRL for growth; and experiments showing that PRL stimulates natural killer cell activity and the development of lymphokine-activated killer cells.

Modification of blood-borne arrest properties of lymphoma cells by inhibitors of protein glycosylation suggests the existence of endogenous lectins

The requirement for intact carbohydrates of glycoproteins at the cell surface was investigated after treatment of lymphoma cells with compounds which interfere at different steps in N-linked glycosylation: swainsonine and 1-deoxynojirimycin act at different levels during the processing, so that complex oligosaccharides cannot be formed; 2-deoxyglucose, beta-hydroxynorvaline, and tunicamycin completely prevent the formation of N-linked (high-mannose as well as complex) oligosaccharides. The role of sialic acid was investigated by treating the cells with neuraminidase. These treatments resulted in altered patterns of surface-labelled glycoproteins after SDS-polyacrylamide gel electrophoresis. Blood-borne arrest of lymphoma cells in the spleen was sensitive to neuraminidase and to treatments interfering with the processing of complex N-linked oligosaccharides. It is suggested that carbohydrates are signals for cellular interactions involved in the recirculation and homing behaviour of lymphoid cells and probably interact with endogenous lectins at their site of homing.

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.

Effect of inhibitors of glycosylation and carbohydrate processing on invasion of malignant mouse MO4 cells in organ culture.

Inhibitors of glycosylation and carbohydrate processing were used to investigate the role of carbohydrates exposed at the cell surface in invasion. Malignant mouse MO4 cells were confronted with embryonic chick heart in organ culture, an assay shown to be relevant for a number of aspects of invasionin vivo. Tunicamycin (1·0μg/ml), 2-deoxy-d-glucose (100mm),β-OH-norvaline (1·0mm), and Monensin (0·1μg/ml) reversibly inhibited the invasion of MO4 cells. At these concentrations the drugs also inhibited the growth of MO4 cells. 1-Deoxynojirimycin (10mm), swainsonine (0·4μg/ml), and Marcellomycin (0·1 μg/ml) permitted invasion. Marcellomycin also reversibly inhibited the growth of MO4 cells. These results show that drugs known to interfere with the glycosylation or processing of carbohydrate chains of glycoproteins in different ways have different effects on the invasion of MO4 cellsin vitro.

In Vitro Effects of Prolactin on the Lympho-Hemopoietic System

Functional receptors for prolactin (PRL-R) are expressed on many cell types in various tissues, including leukocytes. The PRL-R belongs to the hemopoietin-cytokine receptor family. PRL itself is also expressed in the lympho-hemopoietic system (LHS) where it induces discrete effects. PRL thus qualifies as a cytokine. Effects on the LHS are modest in rodents and man and, though dispensable, PRL is clearly able to affect leukocyte physiology. The present contribution summarizes mainly recent in vitro studies on the role of PRL in the hemopoietic and in the immune systems.

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.

Phenotype of thymic lymphomas in the mouse

The MP2 cell line was established from a murine leukemia virus-induced thymic lymphoma. Half of the cells were consistently L3T4 positive and less than 5% of the cells were Lyt-2 positive. Single cell cloning on the basis of the presence or absence of Lyt-2 allowed the isolation of four clones with stable phenotypes: (1) Lyt-2-, L3T4-; (2) Lyt-2+, L3T4+; (3) Lyt-2-, L3T4+; (4) Lyt2+, L3T4-. These data are discussed in relation to tumour cell heterogeneity and to normal T-cell differentiation pathways.