GnRH-I and GnRH-II have differential modulatory effects on human peripheral blood mononuclear cell proliferation and interleukin-2 receptor gamma-chain mRNA expression in healthy males

Abstract

GnRH-I and its receptor (GnRHR-I) have previously been demonstrated and shown to be biologically active in the immune system, notably within peripheral lymphocytes. Recently however, a second form of GnRH (GnRH-II) has been described in the human. The functions of both these neuropeptides in PMBCs have not been understood yet. The present study was therefore designed to investigate the effects of GnRH-I and/or GnRH-II on human PMBC proliferation in males. Secondly, the effects of GnRH-I and GnRH-II on IL-2 dependent lymphocyte proliferation were examined. Finally, we analysed the role of GnRH-I and GnRH-II in IL-2R gamma-chain expression. Peripheral venous blood samples were obtained from six male healthy volunteers (Mean age 27.75 +/- 1.5). Non-radioactive cell proliferation assay was used for proliferation studies and we used quantitative real-time RT-PCR to examine the role of GnRH-I and GnRH-II on IL-2R gamma-chain expression in PMBCs. Treatment of PMBCs with GnRH-I (10(-9) M and 10(-5) M) and with interleukin-2 (IL-2) (50 U/ml) resulted in a significant increase in cell proliferation compared with the untreated control. PMBCs cotreated with IL-2 and GnRH-I demonstrated higher proliferative responses than IL-2 treatment alone, the enhancement of GnRH-I on IL-2 response being significant only at GnRH-I concentration of 10(-5) M. Co-incubation of IL-2+ GnRH 10(-5) M with a GnRH antagonist (Cetrorelix; 10(-6) M) significantly decreased the proliferation. GnRH-II did not affect the proliferation of PMBCs alone, and did not alter the proliferative response to IL-2. The proliferative responses to GnRH-I (alone and with IL-2) were significantly attenuated by GnRH-II coincubation (each in equal molar concentrations; 10(-9) M to 10(-5) M). It was found that GnRH-I increased the expression of IL-2Rgamma mRNA in a dose dependent manner, with a significant increase of percentage 162.3 +/- 14 of control at 10(-5) M. In contrast, IL-2Rgamma expression was significantly decreased in all concentrations of GnRH-II (10(-9) M to10(-5) M), and the maximum decrease was detected at 10(-5) M, with percentage 37.7 +/- 6.6 of control. All these findings strongly suggest that regulation of IL-2R expression may therefore be an important target for GnRH-I and GnRH-II in PMBCs in males. In summary, present study clearly demonstrates the differential effects of GnRH-I and GnRH-II on PMBC proliferation, IL-2 proliferative response, and IL-2Rgamma expression in PMBCs in males. To our knowledge, our observations provide the first evidence for the interactions of these local neuropeptides at lymphocyte level. Further experimental data in human are warranted to explore the clinical implications of these data.

Fig. 1

Demonstration of the effect of GnRH-I (10⁻⁹ M to 10⁻⁵ M) and GnRH-I (10⁻⁵ M) + GnRH-I antagonist (Cetrorelix; 10⁻⁶ M) on IL-2Rγ mRNA levels in PMBCs. Total RNA was extracted after 24 h incubation and, Quantitative real-time RT-PCR (relative quantification method) was performed as detailed in Material and Methods. (a) The bars represent the mean normalized ratios (relative amounts of target gene/reference gene) of the samples derived from two individual experiments. Levels of mRNA were expressed as a percentage (mean ± SEM) of the level in untreated controls. The treatment groups are; control (100%), 10⁻⁹ M (92·0 ± 9·2%), 10⁻⁸ M (96·1 ± 4·1%), 10⁻⁷ M (121·5 ± 9·8%), 10⁻⁶M (128·2 ± 9·0%), 10⁻⁵ M (162·3 ± 14·1%), GnRH-I Antagonist 10⁻⁶ M +10⁻⁵ M (110·1 ± 8·8%), respectively. ^aP < 0·05 versus untreated control; ^bP < 0·05 versus GnRH-I 10⁻⁵ M treatment. (b) Ethidium bromide staining of the experiment shown in (a). Target gene: IL-2Rγ; Reference gene: beta-actin.

Fig. 2

Demonstration of the effect of GnRH-II (10⁻⁹ M to 10⁻⁵ M) on IL-2Rγ mRNA levels in PMBCs. Total RNA was extracted after 24 h incubation and, Quantitative real-time RT-PCR (relative quantification method) was performed as detailed in Material and methods. (a) The bars represent the mean normalized ratios (relative amounts of target gene/reference gene) of the samples derived from two individual experiments. Levels of mRNA were expressed as a percentage (mean ± SEM) of the level in untreated controls. The treatment groups are; control (100%), 10⁻⁹ M (58·4 ± 7·5%), 10⁻⁸ M (58·7 ± 7·2%), 10⁻⁷ M (51·7 ± 8·3%), 10⁻⁶ M (49·1 ± 3·1%), 10⁻⁵ M (37·7 ± 6·6), respectively. ^aP < 0·05 versus untreated control. (b) Ethidium bromide staining of the experiment shown in (a). Target gene: IL-2Rγ; Reference gene: beta-actin.