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. 2005 Oct;142(1):103–110. doi: 10.1111/j.1365-2249.2005.02904.x

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

F Tanriverdi *,†, D Gonzalez-Martinez †, Y Hu †, F Kelestimur *, PMG Bouloux †
PMCID: PMC1809497  PMID: 16178862

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 γ-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 γ-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-2Rγ mRNA in a dose dependent manner, with a significant increase of percentage 162·3 ± 14 of control at 10−5 M. In contrast, IL-2Rγ 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-2Rγ 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.

Keywords: GnRH-I, GnRH-II, IL-2R, mononuclear cell, IL-2

Introduction

Gonadotropin-releasing hormone type-I (GnRH-I) is a decapeptide primarily known for its role in reproductive function, through regulation of GnRH receptor (GnRHR) mediated FSH and LH secretion from pituitary [1]. GnRH-I and GnRHR-I expression are not however, limited to the central nervous system, and have been demonstrated in different tissues such as breast, ovary and prostate [2,3]. The presence of specific GnRH binding sites and expression of GnRH mRNA in the immune system has also been extensively reported, including in cultured porcine lymphocytes [4,5] as well as in rat/murine thymus and spleen [6–9].

Local production of GnRH-I has been demonstrated in human peripheral T-cells (CD4+, CD8+), in the leukaemic (Jurkat) cell line similar to T lymphocytes and human B lymphocytes [10–12]. Expression of GnRH-I and GnRHR-I mRNA in human peripheral lymphocytes suggest a potential autocrine and/or paracrine effect of GnRH in immune system regulation [13,14]. Previous animal studies have implied that GnRH-I possesses immune stimulatory actions and may have a potential role in prenatal and postnatal programming of immune cells [15–17]. For example, in an animal model of immunodeficiency, total IgG levels and CD4+ lymphocytes increased after native GnRH administration [18].

Until recently, GnRH-I was thought to be the only isoform present in humans. However GnRH does not represent a unique molecule. A form of GnRH originally isolated from chicken hypothalamus (cGnRH-II) [19] has been found to be universally present and conserved among vertebrates including mammals [20–22]. In the human, GnRH Type-II (GnRH-II), which has an amino acid sequence 70% homologous to GnRH-I but is encoded by a different gene, is expressed at significantly higher levels outside the brain especially in the kidney, bone marrow and prostate, though its function has not yet been elucidated [23]. Furthermore, a novel human GnRH-R Type-II homolog gene and wide tissue distribution of the antisense transcript, including lymph node, thymus and peripheral lymphocytes has been demonstrated [24]. We have recently demonstrated the expression of both GnRH-I and GnRH-II mRNA and their corresponding decapeptides in human peripheral blood mononuclear cells [12]. To our knowledge, regulation of human peripheral lymphocyte proliferation by GnRH-II and interaction of GnRH-I and GnRH-II at immune system level have not yet been studied.

IL-2 plays a pivotal role in lymphocyte activation and proliferation through IL-2R complex. The IL-2R is recognized as a heteromeric receptor complex composed of at least three distinct chains (α, β and γ) that mediate a variety of critical immune responses. The noncovalent combination of these three chains constitutes the high affinity receptor [25]. However, IL-2R α itself is not able to process lymphocyte activation and proliferation due to lack of signal transducing cytoplasmic domain. Because of this reason, IL-2R γ chain expression is recommended as another indicator of lymphocyte activation [13,25]. In several studies in human and rat, although the functional consequence was unknown, regulation of IL-2R α and IL-2R γ chain expression by GnRH-I has been demonstrated in peripheral lymphocytes [9,13]. After several experiments related to GnRH induction of IL-2R expression in rats, Batticane et al. [9] speculated that IL-2R expression might represent a crucial step in the mechanisms underlying GnRH action in immune cells. All these findings suggest that endogenous and exogenous GnRH-I and/or GnRH-II might have a regulatory effect on IL-2 proliferative response and IL-2R γ chain expression in human PMBCs.

Understanding the roles of GnRH-I and GnRH-II in immune function might have clinical relevance for several reasons. First, in recent years GnRH-I agonists have become widely used in a variety of disorders, even though little is known about their potential immunological effects. Second, the potential immune modulatory and programming effects of these neuropeptides might provide further insight into pathogenesis of autoimmune disorders and gender difference in immune response. Moreover novel future applications of these peptides might be possible in several immune system related disorders [26].

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 modulatory 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 γ chain expression in PMBCs.

Materials and methods

Isolation of peripheral mononuclear cells

The use of human mononuclear cells for research was approved by the Royal Free and University College Medical School Local Ethical Committee and Erciyes University Medical School Ethical Committee. Peripheral venous blood samples (10 ml) were obtained from six male healthy volunteers. Mean age of the volunteers was 27·75 ± 1·5 years (mean ± SD). The Ficoll-Hypaque method was used to isolate PMBC as previously described [13]. Blood samples were obtained from six males at the same time and separate PMBC cultures were prepared for each individual. After 1 week later we called the same six volunteers and repeated the cultures. Cells were resuspended in RPMI-1640 medium (Sigma-Aldrich, Dorset, UK) containing 10% FBS, 100 µg/ml streptomycin, 100 µg/ml ampicillin, 25 mmol/l HEPES, 2 mmol l-glutamine and incubated at 37 °C, with 5% CO2. The viability of lymphocytes was confirmed by a trypan blue exclusion test (> 95%).

Proliferation assay

Three different experimental designs were performed for proliferation assay. PMBC's from six males were plated separately for each individual in 96-well flat bottom tissue culture plates (Nunclon, NY, USA) at a concentration of 1 × 104 cells/100 µl in the culture medium described above. Just after the onset of culture, cells were treated in triplicate, firstly; with either human GnRH-I (pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2 (Sigma-Aldrich) 10−5 M and 10−9 M), IL-2 50 U/ml (Sigma-Aldrich), IL-2 (50 U/ml) + GnRH-I (10−9 M to 10−5 M), or GnRH-I antagonist (Cetrorelix; Ac-d-Nal-d-Cpa-D-Pal-Ser-Tyr-d-Cit-Leu-Arg-Pro-d-Ala-NH2 (ASTA medica, Frankfurt, Germany) 10−6 M) + IL-2 (50 U/ml) + hGnRH-I (10−5 M). Secondly; a similar experimental design with human GnRH-II (pGlu-His-Trp-Ser-His-Gly-Trp-Tyr-Pro-Gly-NH2 (kindly provided by Prof R. Millar, Edinburgh, UK) 10−5 M and 10−9 M), IL-2 50 U/ml, and IL-2 (50 U/ml) + GnRH-II (10−9 M to 10−5 M) was undertaken in PMBC’s. And finally; human GnRH-I + human GnRH-II (each in equal molar concentrations; 10−5 M and 10−9 M), IL-2 50 U/ml, and IL-2 (50 U/ml) + GnRH-I (10−9 M to 10−5 M) + GnRH-II (10−9 M to 10−5 M) were applied. To confirm the reproducibility of the assay, these three experimental designs were repeated on a separate occasion in the same six healthy volunteers.

Plates were incubated for 72 h at 37 °C with 5% CO2. Cell proliferation was estimated according to the manufacturer's instructions with the Cell Titer 96 AQueous nonradioactive cell proliferation assay using tetrazolium compounds (Promega, Southampton, UK). At the end of a 72 h incubation period, 20 µl of the MTS/PMS solution was added directly to the each well and incubated for 4 h. The absorbance (Mean Optic Density: mean OD) was measured at 492 nm using an ELISA plate reader (Anthos ht III). The blank comprised 100 µl of culture medium, and absorbances of samples were corrected by subtracting blank absorbance.

Culture of mononuclear cells with GnRH-I and GnRH-II

PMBC's were harvested from one of the randomly selected volunteers, rapidly plated onto 3 different 6 well plates (Corning, NY, USA) at a concentration of 3 × 106 cells/well and were incubated at 37 °C with 5% CO2 for 15–18 h. The culture medium used was described above. Then the cells were treated with single doses of human GnRH-I (10−9 M to 10−5 M), GnRH-I (10−5 M) + GnRH-I antagonist (Cetrorelix; 10−6 M), and human GnRH-II (10−9 M to 10−5 M) for 24 h. In addition, untreated cells were incubated at the same conditions as control groups.

Quantitative real-time RT-PCR

Total RNA was extracted from control and treated PMBC's using a commercially available kit (RNeasy midi kit, Qiagen, West Sussex, UK). RNA concentration was quantified by absorbance at 260 nm. Total RNA (2 µg) was reverse-transcribed using a First Strand cDNA Synthesis Kit (Amersham Pharmacia Biotech, Buckinghamshire, UK), according to the manufacturer's instructions. The integrity of the RNA samples was verified by examining the RT-PCR product of β-actin mRNA.

Quantitative real-time PCR was performed using the Rotor-Gene 2000 amplification system (Biogene, Kimbolton, UK). The resultant cDNAs from each control, GnRH-I and GnRH-II treated samples were submitted to PCR using intron-spanning primer pairs for IL-2R γ-chain (target gene) and beta-actin (reference gene). The sequences were: IL-2R (sense 5′-CCAGGACCCACGGGAACCCA-3′ and anti sense 5′-GGTGGGAATTCGGGGCATCG-3′) [13] and beta-actin (sense 5′-TCACCCACACTGTGCCCATCTACGA-3′ and anti sense 5′-CAGCGGAACCG CTCATTGCCAATGG −3′).

The template cDNAs (2 µl) were amplified in a 50 µl PCR reaction containing, 50 pmol of each primer, 25 µl OF QuantiTect SYBR Green Master Mix (Qiagen, West Sussex, UK) and RNase-free water. PCR reactions for both target and reference genes were carried out for 35 cycles in the following sequence; denaturation at 94 °C for 1 min, annealing at 61 °C for 40 s, extension at 72 °C for 1 min, final extension at 72 °C for 120 s after the last cycle. Fluorescent data were acquired at 585 nm during each 72 °C extension phase. Product specifity was examined by melt curve analysis and agarose gel electrophoresis after each real-time PCR run. For analysis of quantitative results, relative quantification method was performed by software provided with Rotor-Gene 2000.

For both target gene and reference gene, standard curves were generated by using 5 different concentrations (1 unit to 1/16 unit) of control (untreated) samples. The relative amounts of target and reference genes for each sample were calculated in units, by using the resulting threshold cycle (CT) of the samples and the corresponding standard curves. To correct for differences in both RNA quality and quantity between samples, data were normalized using the ratio of the IL-2R relative amount to that of beta-actin. By using the resultant normalized ratios, the target gene expression in treated samples were compared with control samples. To confirm the reproducibility of the assay, PCR was repeated twice on a separate occasion for each sample.

Sequencing of RT-PCR product

PCR products were sequenced by automatic DNA sequencing using dye terminator chemistry in an ABI 377 sequencer (MWG, Ebersberg, Germany). The sequencing results were matched with the available sequences using the BLAST (basic length alignment search tool) program provided by the National Centre for Biotechnology Information.

Statistical analysis

Data were expressed as mean and standard error of the mean (mean ± SEM) OD. Pooled data were compared by one-way anova followed by posthoc comparison of individual groups by Dunns's Test after Levene's Test had shown that variances were homogenous. P < 0·05 was considered statistically significant.

Results

Effects of GnRH-I, GnRH-II and GnRH-I + GnRH-II, with or without IL-2, on PMBC proliferation

To evaluate the peripheral lymphocyte proliferation with three different experimental designs described before, the MTS/PMS assay was performed in PMBCs from 6 healthy males.

Treatment of PMBCs with GnRH-I (10−5 M and 10−9 M) and with IL-2 (50 U/ml) resulted in a significant increase in proliferation compared with untreated control (P < 0·05; Table 1). Co-treatment with IL-2 and GnRH (10−9−10−5 M) increased proliferation significantly at all concentrations of GnRH when compared with control (P < 0·05). In all groups, PMBCs cotreated with IL-2 and GnRH-I demonstrated higher proliferative responses than IL-2 treatment alone, but the enhancement of GnRH-I on IL-2 response was significant only at 10−5 M GnRH concentration (P < 0·05). The GnRH-I antagonist (Cetrorelix; 10−6 M) coincubated with IL-2 + GnRH-I 10−5 M treated PMBCs, resulted in a significant decrease in proliferation when compared to IL-2 + GnRH-I 10−5 M alone (P < 0·05). Treatment effects and mean OD values in normal PMBCs with first experimental design are shown in Table 1.

Table 1. Effects of GnRH -I (10−9 M to 10−5 M) and/or IL-2 (50 U/ml), and GnRH-II (10−9 M to 10−5 M) and/or IL-2 (50 U/ml) on PMBC proliferation.

Mean optic densities (% of control)
Treatment groups GnRH-I treatment GnRH-II treatment
Control 100% 100%
10−9 M 133·5 ± 7·2%† 88·8 ± 8·5%
10−5M 135·9 ± 7·6%† 97·8 ± 9·5%
IL-2 (50 U/ml) 179·6 ± 11·1%† 188·4 ± 20·1%†
10−9 M +IL-2 191·1 ± 17·7%† 175·7 ± 19·6%†
10−8M + IL-2 194·6 ± 13·6%† 171·6 ± 21·8%†
10−7 M +IL-2 196·2 ± 18·1%† 159·3 ± 17·7%†
10−6 M +IL-2 194·1 ± 12·1%† 169·4 ± 21·1%†
10−5M + IL-2 230·3 ± 7·7%†‡ 157·6 ± 13·7%†
10−5M + IL-2 + ant* 170·2 ± 6·5%§ 163·3 ± 11·7%†
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The values represent the mean OD's of triplicates for two separate experiments in 6 healthy males. Levels of mean OD's are expressed as a percentage (mean ± SEM) of the level in control (without GnRH-I and GnRH-II treatment).

*

GnRH-I Antagonist 10−6 M;

†

P < 0·05 versus untreated control;

‡

P < 0·05 versus IL-2 treatment;

§

P < 0·05 versus IL-2 + GnRH-I 10−5 M treatment.

There were no significant proliferative responses to GnRH-II (10−5 M and 10−9 M) (P > 0·05), but IL-2 (50 U/ml) treatment was resulted in a significant increase in proliferation compared with untreated control (P < 0·05; Table 1). Co-treatment with IL-2 and GnRH-II (10−9−10−5 M) increased proliferation significantly at all concentrations of GnRH when compared with control (P < 0·05). In all groups, PMBCs cotreated with IL-2 and GnRH-II (10−9−10−5 M) demonstrated lower proliferative responses than IL-2 treatment alone, but the reduction of IL-2 response by GnRH-II was not significant (P > 0·05). In addition when GnRH-I antagonist (Cetrorelix; 10−6 M) was applied to IL-2 + GnRH-II 10−5 M treated PMBCs, there was not any significant change in the proliferative response when compared to IL-2 + GnRH-II 10−5 M alone (P > 0·05). Data with the second experimental design are shown in (Table 1).

Treatment of PMBCs with GnRH-I + GnRH-II (each in equal molar concentrations; 10−5 M and 10−9 M) did not show a significant increase in proliferation and there was no significant increase in mean OD (Levels of mean OD's are expressed as a percentage (mean ± SEM) of the level in untreated control) when compared with control (P > 0·05). IL-2 (50 U/ml) treatment and cotreatment of IL-2 (50 U/ml) + GnRH-I + GnRH-II (each in equal molar concentrations; 10−5 M to 10−9 M) were resulted in a significant increase in proliferation compared with untreated control (P < 0·05). Although there was a trend for a mean OD decrease in all IL-2 (50 U/ml) + GnRH-I + GnRH-II (10−5 M−10−9 M) cotreatment groups when compared with IL-2 treatment alone, this was not statistically significant (P > 0·05).

Regulation of IL-2R gamma chain mRNA by GnRH-I and GnRH-II

During in vitro cultures, different doses of GnRH-I and GnRH-II were added to the medium, and mononuclear cells were cultured for 24 h. After harvesting the cells quantitative real-time RT-PCR was performed using specific primers for IL-2R gamma chain (gene of interest) and beta actin (internal reference). The quality and predicted size of PCR products (481 bp for IL-2R and 295 bp for beta-actin) were assessed by 1·5% agarose gel electrophoresis (Figs 1b and 2b). Furthermore, resultant PCR products were sequenced and found to be identical sequence to human IL-2R and beta actin.

Fig. 1.

Fig. 1

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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.

Fig. 2

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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.

Normalized ratios of the samples, derived from relative amounts of target and reference genes, were used to compare the expression level of IL-2R in GnRH-I and GnRH-II treated mononuclear cells. Levels of mRNA were expressed as a percentage (mean ± SEM) of the level in untreated controls.

It was found that GnRH-I increased the expression of IL-2R mRNA with a significant increase of percentage 162·3 ± 14 of control at 10−5 M after 24 h GnRH-I treatment (P < 0·05; Fig. 1a). IL-2R mRNA level was significantly decreased at GnRH-I (10−5 M) + GnRH-I antagonist (Cetrorelix; 10−6 M) treated group when compared with GnRH-I (10−5 M) treatment (P < 0·05; Fig. 1a).

In contrast, IL-2R expression was significantly decreased in all concentrations of GnRH-II (10−9 M to10−5 M) (P < 0·05). The maximum decrease was detected at 10−5 M, with percentage 37·7 ± 6·6 of control (Fig. 2a).

Discussion

The present study clearly indicates the potential stimulatory effects of GnRH-I in PMBCs and reduction of these effects by GnRH-II. Because there is a gender difference in immune response, we have undertaken our study only in males [26–28]. Treatment of total lymphocytes with GnRH-I produced a significant proliferative response when compared with control, suggesting a potential regulatory role of GnRH-I in lymphocyte proliferation. This finding is consistent with previous studies demonstrating proliferative effect of GnRH-I in rat immune cells and human T lymphocytes [9,11]. Expression of GnRH-R Type-I in peripheral lymphocytes and its functional capacity have been demonstrated [13,14]. The mechanism of GnRH-I induced proliferation is unknown, but may be mediated by activation of PKC and a decrease in cAMP level [9,11].

Although GnRH-II administration alone did not effect the lymphocyte proliferation, the significant proliferative effect of GnRH-I was abolished by GnRH-II when added together in same concentrations. Chen et al. [29] have clearly demonstrated that increased laminin receptor expression with GnRH-II in T cells is unaffected by cetrorelix, suggesting a distinct and yet-uncharacterized receptor for GnRH-II. A specific GnRH-R Type-II cDNA, showing only 40% identity with the GnRH-R Type-I, was cloned from monkey but not from human [24,30].

The type II receptors are highly selective for GnRH-II in receptor binding assays [30,31], and there are distinct differences in signalling by the two receptors (marmoset type II receptor and human type I receptor), suggesting that there might be different effects where receptors are found in the same cells [32]. Although we could not demonstrate the molecular mechanisms of differential effect of GnRH-I and GnRH-II, and their interaction at immune system level, action through their distinct receptors on peripheral lymphocytes might be one possible explanation.

IL-2 induces proliferation and/or activation of T and B cells, and its receptor expression (IL-2Rα) is stimulated in rat thymocyte and splenocyte cultures incubated with native GnRH-I and its analogue [9]. Stimulation of IL-2Rã mRNA expression by GnRH-I and GnRH analogue has also been observed in human PMBC by using semiquantitative RT-PCR [13]. However, the functional consequence of increase in IL-2R expression by GnRH-I has not been mentioned. In present study, cotreatment of GnRH-I (only at 10−5 M GnRH-I concentration) and IL-2 demonstrated significant enhancement of proliferation when compared with IL-2 treatment alone. Although GnRH-I was significantly effective at high concentrations, physiological importance is not clear. It is known that concentration of hypothalamic GnRH extremely low in the systemic circulation. This makes it unlikely that hypothalamic GnRH could exert a significant action on extrapituitary sites. Many studies clearly demonstrate that immune cells locally produce GnRH-I and GnRH-II [23,26]. These recent findings suggest that GnRH may play a role in an autocrine and/or paracrine manner. The local concentrations of these neuropeptides are unknown and future studies will clarify the physiological importance of GnRH effect in terms of concentration. Consistent with previous studies [9,13] we observed that IL-2Rγ expression was stimulated by GnRH-I, suggesting the enhancement of IL-2 proliferative response is via up-regulation of IL-2R expression. However in the study by Chen et al. [13] IL-2Rγ expression responses to GnRH-I are slightly higher than our IL-2Rγ expression responses. One possible explanation could be the difference in gender between the subjects in both studies. Chen et al. have isolated PMBCs from female donors, and its known that there is a sexual dimorphism in immune response between males and females. Even though the mechanisms are unclear autoimmune disorders are typically more common in females, and they demonstrate greater humoral and/or cellular immune response than males [26,33]. Future in vitro and in vivo studies are warranted to clarify the impact of locally produced GnRH in sexual dimorphism in immune response.

On the other hand GnRHR Type-I antagonist (Cetrorelix) reversed the significant increases of GnRH-I + IL-2 proliferative response and IL-2 receptor expression, suggesting that these effects are via a functional GnRHR Type-I. Moreover in a recent study from our laboratory, B-Lymphoblastoid cells were treated IL-2 (50 U/ml) + GnRHR Type-I antagonist (Cetrorelix; 10−6 M). An apparent decrease in proliferation was observed compared to IL-2 treatment, implying that local tonic expression of GnRH contributes to the IL-2 response [12].

A further novel finding in this study was the demonstration of differential regulation of IL-2 proliferative response and IL-2R gamma expression by GnRH-II. Co-treatment of GnRH-II and IL-2 demonstrated substantial but not statistically significant reduction in proliferation at all concentrations when compared with IL-2 treatment alone. In addition we demonstrated that IL-2R gamma expression was significantly reduced by GnRH-II at all concentrations, implying the reduction of IL-2 proliferative response is via down-regulation of IL-2R expression. Furthermore the proliferative response profile was similar to GnRH-II response when IL-2 + GnRH-I + GnRH-II applied, suggesting the dominant effect of GnRH-II on IL-2 proliferative response at equivalent concentrations. Although the GnRH-II effect on immune function is not yet clarified, the evolutionary conservation of this decapeptide and its wide distribution outside the central nervous system; including immune system suggest a potential important physiological role [24,30,34]. Recently local production of GnRH-I and GnRH-II in T lymphocytes and GnRH-I and GnRH-II induced laminin receptor expression increase that is involved in cellular adhesion and migration have been demonstrated [29]. Based on our findings, its tempting to speculate that regulation of IL-2R expression may therefore be an important target for GnRH-I and GnRH-II in PMBCs in males. Moreover different local concentrations of these neuropeptides probably finely tune the IL-2R level and consequently may have an impact in immune response and lymphocyte subtype shift. Further functional studies are therefore necessary to explore the role of local GnRH-I and GnRH-II in lymphocytes and their interaction with sex steroids and cytokines at immune system level in both genders.

The clinical significance of GnRH-I/GnRH–II interaction at immune system level and their modulatory effect on IL-2 proliferative response and IL-2R expression in peripheral lymphocytes are unclear. Jacobson et al. [35] have suggested that exogenous or local GnRH-I may play a role in the exacerbation of autoimmune disorders. Treatment with a GnRH-I agonist and antagonist in intact and castrated, male and female, lupus-prone (SWR×NZB) F1 hybrid mice leads to increased severity of renal disease with the agonist and a protective effect of antagonist. The immune stimulatory effects of GnRH-I and the relation between IL-2 response and GnRH at immune system level may have potential clinical implications for GnRH-I and GnRH-I analogues, in immune deficiency conditions when immune reconstitution is crucial. Although selective GnRH-II therapeutics is not yet available, elucidation of the immune system effects of GnRH-II may provide important insights for potential applications [32].

In summary, present study clearly demonstrates the differential effects of GnRH-I and GnRH-II on PMBC proliferation, IL-2 proliferative response, and IL-2Rγ expression in males. To our knowledge, our observations provide the first evidence for the differential interactions of these local neuropeptides at lymphocyte level. Further experimental data in human are warranted to explore the clinical implications of these data.

Acknowledgments

We thank to Professor R.Millar for providing us GnRH-II.

References

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