Limited effects of placental and pituitary growth hormone on cytokine expression in vitro.

ARTICLE

INTRODUCTION

In vitro and in vivo observations in animals and man indicate that growth hormone (GH) and prolactin (PRL) may play a role in the development, the function and some diseases of the immune system (reviewed in [1-4]). Congenital deficiency in GH or PRL, in contrast, has only very limited effects in rodents and man [3, 5]. In particular, pituitary dwarfs (lacking GH) and Laron dwarfs (lacking functional GH-receptors (GH-R)) have no symptoms related to immune dysfunction [3, 4]. Several GH variants have been described, in particular the pituitary (GH-N) and the placental (GH-V) forms [2, 6-8]. GH-N signals through GH-R but also, in primates, through PRL-R, while GH-V binding capacities to PRL-R are more than 10 times lower [8]. No specific function for GH-V is known. Immunoregulatory properties could be different from those of GH-N and might contribute e.g. to tolerance of the fetus. The present studies were devised separately to evaluate possible effects of GH on human leukocytes. First, the production of several important cytokines was monitored in leukocytes grown in the presence of GH. Next, the effects of pituitary and placental GH on cytokine gene expression were compared. As both studies yielded mainly negative results, they are presented together.

MATERIALS AND METHODS

GH

Cell cultures were treated with recombinant human GH-N (Novo Nordisk, Denmark for mRNA studies and from Pharmacia, Sweden for cytokine production studies) or recombinant human GH-V (produced in the Laboratory of Biochemistry/Endocrinology, Liège). The bioactivity of each preparation was checked in the Nb2 bioassay.

Cells and culture

For cytokine production, buffy coats were obtained from the blood bank within 18 hours of collection. Peripheral blood mononuclear cells (PBMC) were isolated using Lymphoprep (Nycomed Pharma, Norway). Tonsils were obtained from patients with recurrent tonsillitis and/or obstructive respiratory disease and mechanically dissociated within 1 hour of removal. Cells were grown at a concentration of 3 x 10⁶ cells/ml in a serum-free medium (RPMI 1640 (Gibco, Scotland), supplemented with 0.2% bovine serum albumin, 12.5 mug/ml transferrin and 30 nM sodium selenite and 100 U/ml penicillin, 100 mug/ml streptomycin) [9]. GH (or PBS) was present from the start. Mitogens were: for PBMC, PHA (10 mug/ml); for tonsillar cells, PHA (4 mug/ml) + LPS (12 ng/ml). Supernatants were collected after 3 days and kept at ­ 20° C until use.

For the RNase protection assays (RPA), PBMC were prepared from human blood, as described [10]. Briefly, blood was diluted 1:1 in RPMI 1640 (Gibco), and centrifuged on a Ficoll (Pharmacia) gradient for 25 min at 830 g without braking. Cells were harvested, washed three times in RPMI 1640 and cultured (3 x 10⁶ cells/ml) for 4 or 16 hours in DMEM/Ham's F12 1:1 with antibiotics (150 U/ml Penicillin G, 150 mug/ml streptomycin, and 50 mug/ml gentamycin, Gibco) and fungizone (1.25 mug/ml) (Gibco) with or without mitogens (0.1 mug/ml PMA and 1 mug/ml ionomycin) and/or GH (100 ng/ml GH-N or GH-V).

Enzyme-linked immunosorbent assays (ELISA)

The ELISA were run in duplicate according to manufacturer's instructions (Biosource, Nivelles, Belgium, for IFN-gamma and TNF-alpha, and Pharmingen, San Diego, CA, for IL-5). In view of the known variability in cytokine production by PBMC from normal human donors (see legend to Table 1), results from hormone-treated cultures were expressed as percentage of control (only PBS-treated) cultures from the same donor. Data analysis was performed using the Wilcoxon signed rank test.

RNA extraction

Total RNA was extracted and prepared as previously described [11]. Briefly, cultured cells were harvested and RNA was extracted using Intapure kit (Eurogentec, Belgium), quantified by agarose gel densitometry and kept in RNase-free conditions at ­ 80° C in 75% ethanol ­ 25% water.

RNase protection assay (RPA)

The RiboQuant Multi-Probe RPA System was used with an hCK-1 template set (Pharmingen), targeting templates for IL-2, IL-4, IL-5, IL-9, IL-10, IL-13, IL-14, IL-15 and IFN-gamma mRNA, and for two housekeeping genes (hL32 and GAPDH), as internal standards. Briefly, cytokine mRNA-specific [alpha-³²P]UTP-labeled RNA probes were synthesized from a cDNA template set and purified. The probes were hybridized with RNA. Single-stranded RNA was digested with RNase (A + T1 mix) and hybridized fragments were purified and separated on a 5% acrylamide/bisacrylamide gel containing 8 M urea. Autoradiographs, obtained after different exposure times, were scanned with the HP ScanJet 6100C and band densitometry was performed using Gel-Pro Analyzer 3.0 (Media Cybernetics, MD). Band density values were plotted on the probe dilution standard curve and converted to cpm. Values were then expressed as percentages of each internal standard, hL32 and GAPDH, from the same donor. Comparisons between various conditions were performed using the Wilcoxon signed rank test.

RESULTS

Pituitary GH does not significantly modulate the secretion of IL-1beta, IL-5, IFN-gamma and TNF-alpha by cultured PBMC or tonsillar cells

The effect of GH on the production of IFN-gamma, IL-5, IL-1beta and TNF-alpha by PHA-stimulated PBMC (n = 8) was monitored using ELISA (Table 1). After 3 days of culture in serum-free conditions (to avoid interference with e.g. lactogenic hormones and insulin-like growth factors), there were no significant effects with the exception of a slight (10-15%) decrease in IL-5 production (in the presence of 1, 10 or 100 ng/ml GH) and a slight (15%) increase in IFN-gamma (with 10 and 100 ng GH/ml). The effect on IL-5 was possibly mediated by the PRL-R as it was also observed with PRL. When tonsillar cells (n = 4) were grown in the presence of mitogens, a slight increase in the production of IFN-gamma was also observed in GH-treated cultures but there was no consistent effect of GH on the production of IL-5 or TNF-alpha (results not shown).

Pituitary and placental GH do not significantly modulate the level of IL-2, IL-4, IL-5, IL-9, IL-10, IL-13, IL-14, IL-15 and IFN-gamma mRNA in PBMC

The effect of GH on the level of cytokine mRNA in unstimulated or PMA-ionomycin-stimulated PBMC (n = 3 to 6) was screened by RPA after 4 hours and 16 hours of culture and expressed as a percentage of GAPDH or L32 mRNA. In men, there were no significant effects on the levels of IL-2, IL-4, IL-5, IL-9, IL-10, IL-13, IL-14, IL-15 and IFN-gamma mRNA (results not shown). The only significant changes, out of around 800 comparisons, were seen in women (Table 2): after 16 hours, IL-9 mRNA was significantly increased in cells treated with mitogens and GH-V (p = 0.0142) versus cells given mitogens alone. After 4 hours, the amount of IL-4 mRNA was significantly higher in cells treated with mitogens plus GH-N in comparison to mitogens plus GH-V (p = 0.0136). Neither value, however, was significantly different from the controls without GH.

DISCUSSION

Receptors for GH are expressed on human B cells and monocytes and, at a lower level, on T lymphocytes [3, 12, 13]. In addition, GH, in particular GH-N, can act on leukocytes through the PRL-R, which has a similar distribution [2, 3, 14].

We have observed a modest increase in IL-9 mRNA (with GH-V, in women only) and a decrease in IL-5 secretion (with GH-N). As the latter effect was also observed with PRL, it could be mediated by the PRL-R. In addition, the results obtained with RPA do not show strong effects of GH-V on the mRNA synthesis of a wide range of cytokines, as was also the case with GH-N. Only in women did GH-N and GH-V have opposite effects on IL-4 expression, an observation that will be repeated in a larger number of donors.

In different systems, effects of GH-N on proliferation, cytokine production and functional activity of lymphocytes and monocytes have been reported [4, 9, 15-20]. Leukocytes secrete limited amounts of GH [2, 3], that may act in a paracrine manner and this could possibly mask effects of added GH in some systems. Although some effects of GH might be more striking in pure populations, we have used here mixed cell populations in order to also detect indirect effects due, for instance, to interactions between macrophages and T-cells [20]. In view of the known redundancy in the immune system, in particular in cytokine signaling pathways, we propose that effects of GH may only be manifest when the immune capacity has been impaired by some form of stress as a result of e.g. infection, cancer, hormonal imbalance (glucocorticoid treatment) or irradiation.

CONCLUSION

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