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BOR - Papers in Press, published online ahead of print January 22, 2003.
Biol Reprod 2003, 10.1095/biolreprod.102.012211
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BIOLOGY OF REPRODUCTION 68, 2215–2221 (2003)
DOI: 10.1095/biolreprod.102.012211
© 2003 by the Society for the Study of Reproduction, Inc.

Immunology

Gonadotropin-Releasing Hormone Alters the T Helper Cytokine Balance in the Pregnant Rat1

Vishwa Deep Dixit3, Hyunwon Yang3, Venkatachalam Udhayakumar4, and Rajagopala Sridaran2,3

Department of Physiology,3 Morehouse School of Medicine, Atlanta, Georgia 30310-1495 Division of Parasitic Diseases,4 National Center for Infectious Diseases, Centers for Disease Control and Prevention, Public Health Service, U.S. Department of Health and Human Services, Atlanta, Georgia 30341-3724


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The interactions between immune-endocrine and reproductive systems are heightened during pregnancy as an adaptive mechanism, and are regulated by a complex array of hormones and cytokines that control the survival of a semiallogeneic conceptus. GnRH can exert direct effects on the immune system via its receptor (GnRH-R) on lymphoid cells. In the present study, we employed in vitro, ex vivo, and in vivo approaches to investigate the role of GnRH in the modulation of T helper cytokines in pregnant rats undergoing termination of pregnancy. Day 8 pregnant rats were infused with a GnRH agonist (GnRH-Ag) for 24 h using an osmotic minipump. Sham control rats were infused with the vehicle, saline. Lymphocytes were isolated from sham and treated rats and polyclonally stimulated with immobilized anti-CD3 antibody. The levels of the signature T helper 1 (Th-1) cytokines (interferon-{gamma} [IFN-{gamma}] and interleukin-2 [IL-2]) and Th-2 cytokines (IL-4 and IL-10) were measured in culture supernatants. Using immunoflourescence confocal microscopy, we demonstrated for the first time the spatial localization of GnRH-R protein on the surface of lymphocytes. We observed a marked increase in IFN-{gamma} and inhibition of IL-4 production from lymphocytes of pregnant rats treated in vitro with different doses of GnRH-Ag. Further, the responsiveness of lymphocytes to produce IFN-{gamma} was markedly increased in cells cultured ex vivo from GnRH-Ag infused rats, whereas the capacity of lymphocytes to produce IL-4 was significantly inhibited. In addition, GnRH-Ag infusion in pregnant rats induced a shift toward Th-1 cytokines in the serum. We did not observe any significant difference in IL-2 and IL-10 production in response to GnRH-Ag. Our results suggest an additional function for GnRH as a Th-1 inducer and Th-2 inhibitor. GnRH can thus skew the cytokine balance to predominantly Th-1 type in pregnancy, leading to the termination of pregnancy in rats.

cytokines, GnRH, GnRH-receptor, immunology, pregnancy


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Immunologically, pregnancy can be considered as a chronic alloreactive state in which the maternal immune system undergoes specific changes to accommodate a genetically disparate fetus [1]. The immunological inertness of the mother toward the fetus is currently believed to be mediated by a complex interplay of hormones, cytokines, and other regulatory signals with immunomodulatory properties [2]. Despite the presence of maternal T cells specific for paternally inherited histocompatibility antigens, a semiallogeneic fetus survives during pregnancy. Further, down-regulation of major histocompatibility complex (MHC) class 1 and expression of Fas ligand on placenta are not the critical events for fetal survival [3]. It has been argued that maternal T cells acquire a transient state of tolerance specific for paternal alloantigens [4]. Because the survival of a species is critically dependent on successful pregnancy, multiple mechanisms are involved in the modulation of immunity during pregnancy and geared for dampening of adverse maternal immune response toward the fetus [5].

It has been reported that successful outcome of pregnancy is associated with an increase in production of T helper-2 (Th-2) cytokines from lymphocytes [6–9]. The CD4+ helper T cells can be divided into two subsets based on the pattern of cytokine expression and their subsequent roles in immune responses [10]. T helper 1 (Th-1) cells, as important regulators of cell-mediated immune response, secrete interleukin 2 (IL-2) and gamma interferon (IFN-{gamma}), whereas Th-2 cells, mediating predominantly humoral immune responses, secrete IL-4, IL-5, and IL-10 [8]. The balance between predominantly inflammatory Th-1 and counterregulatory Th-2 cytokines is critical for an effective immune response [11]. Currently, it is believed that increase in inflammatory Th-1 cytokines during pregnancy leads to loss of conceptus and termination of pregnancy. The role of Th-1/Th-2 cytokines in immunomodulation during pregnancy is further supported by clinical observations that Th-1 associated autoimmune inflammatory diseases such as rheumatoid arthritis show a marked improvement in pregnant women [12]. The maternal inflammatory and immune system undergoes alterations at both local as well as systemic levels as evident from a decline in T cell proliferative responses [13]. This gave rise to the speculation that the maternal immune system is specifically suppressed during pregnancy to prevent rejection of the fetus. This hypothesis is further supported by observations that during pregnancy, infections by intracellular pathogens such as Listeria monocytogenes and Toxoplasma gondii, which require a robust Th-1 immune response to control, were exacerbated [14].

Cumulative evidence has proved the involvement of hormones, cytokines, and neuromodulators in bidirectional communication and integration of immune and endocrine systems [15]. A number of peptide and steroid hormones are involved in the modulation of T helper cytokine balance. GnRH, a hypothalamic decapeptide, apart from regulating the hypothalamo-pituitary-gonadal axis, may also exert strong immunomodulatory effects on the immune system [16]. Messenger RNA for the GnRH receptor (GnRH-R), a G-protein coupled seven-transmembrane receptor, is known to be expressed in immune cells [17] and in lymphoid organs [18]. GnRH agonist (GnRH-Ag) administration is associated with an increase in T cell proliferation [19, 20] and natural killer cell activity [21], and can reverse thymic involution in aging mice [20] and increase the helper T cells during immunodeficiency [22]. Furthermore, GnRH has been shown to stimulate IFN-{gamma} production from human peripheral blood mononuclear cells (PBMCs) [23]. These findings suggest that GnRH might play an active role in immunity.

In a rat model, we have previously reported that continuous administration of a GnRH-Ag over a period of 24 h in Day 8 pregnant rats leads to termination of pregnancy [24–26]. In an effort to further our understanding of modulation of immune response during GnRH-induced abortion in rats, we tested the hypothesis that GnRH causes a shift in T helper cytokine balance, leading to termination of pregnancy.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals

Timed-pregnant SASCO rats were purchased from Charles River Laboratories (Wilmington, MA). They were housed at the animal facilities of Morehouse School of Medicine in a room with controlled temperature (23–25°C) and light (14L:10D). Purina (St. Louis, MO) rodent chow and tap water were freely accessible. The day of insemination, which was identified by a sperm plug, was designated as Day 1 of pregnancy.

GnRH-Agonist Treatment

GnRH-Ag ([pyro]-Glu-His-Trp-Ser-Tyr-D-Trp-NMeLeu-Arg-Pro-ethylamide-LHRH; Wyeth-40972) was a gift from Wyeth-Ayerst Laboratories (Philadelphia, PA). GnRH-Ag (5 µg/day) was administered continuously via osmotic minipump (model 1003D; Alza Corp., Palo Alto, CA) starting on the morning of Day 8 of pregnancy for a period of 24 h. Briefly, each osmotic minipump filled with GnRH-Ag was incubated in saline for 2–3 h at 37°C before implanting into Day 8 pregnant rats (n = 6). While under Metofane anesthesia, each rat was laparotomized to confirm pregnancy and then implanted s.c. with one osmotic minipump on the dorsal surface of the neck. Sham Day 8 pregnant rats (n = 6) were similarly handled and were infused with saline alone. On Day 9, the rats were killed with halothane after 24 h of GnRH-Ag treatment. Blood was collected from the abdominal aorta and serum was stored at -20°C pending analysis. At autopsy, spleens were removed. All procedures involving animals were approved by the Institutional Animal Care and Use Committee at the Morehouse School of Medicine and in accordance with the principles and procedures of the U.S. National Institutes of Health Guide to the Care and Use of Laboratory Animals.

Lymphocyte Culture

Spleens were excised from the killed rats (n = 12, 6 rats per treatment group) using sterile techniques and immediately placed in sterile Petri dishes containing RPMI-1640 (Gibco BRL; Gaithersburg, MD) media supplemented with 20 mM HEPES and 2% fetal calf serum (FCS; Hyclone Laboratories, Logan, UT). Cells were dispersed by teasing the spleen through fine wire mesh into the Petri dish, and then cells were drawn up into a syringe fitted with a 25-gauge needle to break up clumps of cells, and then transferred to 15-ml sterile falcon tubes. The cell suspension was centrifuged at 200 x g for 10 min. Contaminating red blood cells were lysed by hypotonic shock in 0.84% NH4Cl and the cell pellet was washed twice with RPMI-1640. Thereafter, the cell number was adjusted to 5 x 106/ml in complete RPMI-1640 supplemented with 10% FCS, glutamine (2 mM), penicillin (50 units/ml), streptomycin (50 µg/ml), 20 mM HEPES, nonessential amino acids (1x) and sodium pyruvate (1 mM) (Sigma, St. Louis, MO). Live cell counts using Trypan blue-exclusion was always greater than 95%.

Lymphocytes were plated and cultured in 24-well plates at 37°C, 5% CO2:95% air under humid conditions. T cells were stimulated using plate-bound rat anti-CD3 monoclonal antibody (BD Pharmingen, San Diego, CA) at an optimal concentration of 500 ng/well, which showed a robust proliferative response. Cells were stimulated with five different concentrations of GnRH-Ag (0.1 to 1000 nM) with each treatment repeated six times. For IL-2 measurement, lymphocytes were incubated for a period of 48 h, and for other cytokine detection, lymphocytes were incubated for a 72-h period. Supernatants from the lymphocyte culture were stored at -20°C pending analysis.

Cytokine Measurements

Culture supernatants were analyzed for IFN-{gamma}, IL-2, IL-4, and IL-10 using rat Opt EIA ELISA sets (BD Pharmingen) according to the manufacturer's instructions.

Immunoflourescence and Confocal Microscopy

Lymphocytes from sham and GnRH-Ag-treated pregnant rats were cultured for 72 h with plate-bound anti-CD3 antibody. After the culture, cells were fixed with 3.7% paraformaldehyde and permeabilized using 0.2% Triton X-100. Thereafter, cells were allowed to adhere to poly-L-lysine coated slides. Nonspecific binding sites were blocked with 5% FCS and washed thrice with wash buffer (0.45 M NaCl, 0.24 M Na2HPO4, 0.24 M NaH2PO4, 0.3% Triton X-100). Slides were incubated with primary antibody, GnRH-R mouse monoclonal immunoglobulin G (IgG; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), at a concentration of 1:100 in blocking buffer overnight at 4°C. Slides were washed twice with wash buffer and incubated with biotinylated goat anti-mouse IgG (Molecular Probes, Eugene, OR) at a concentration of 1:100 for 1 h at 37°C. Thereafter, streptavidin-conjugated Oregon green-480 was used to label the cells at a concentration of 10 µg/ml for 45 min in the dark at 37°C. Propidium iodide (Molecular Probes) was used to counterstain the nuclei. Cells were mounted in Vectashield (Vector Laboratories, Inc., Burlingame, CA). Negative controls were performed omitting the primary antibody or using an isotype control antibody from the same species. Mounted slides were examined using a Multiprobe 2001 laser scanning confocal microscope (Molecular Dynamics, Sunnyvale, CA) imaging system equipped with a 15 mW krypton/argon laser emitting at 488 nm and 647 nm.

Statistical Analysis

Results are expressed as means ± SEM. Differences between means and effects of treatments were determined by one-way ANOVA using the Tukey test, which protects the significance tests of all combinations of pairs.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Presence of GnRH Receptors on Lymphocytes

As a first step to elucidate the functional role of GnRH in immunomodulation, we demonstrated the expression of GnRH-R on the surface of lymphocytes. Using confocal immunofluorescence microscopy, we demonstrated, for the first time, the presence of GnRH-R protein on the lymphocytes in Day 9 pregnant rats (Fig. 1). GnRH-R is expressed constitutively in the freshly isolated lymphocytes. After stimulation of lymphocytes with immobilized anti-CD3 antibody, however, GnRH-R showed polarized expression similar to other classical G protein-coupled chemokine receptors.



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  		FIG. 1. Localization of GnRH receptors on cultured rat lymphocytes derived from Day 9 pregnant rats. A) Cells were incubated by omitting the primary antibody with isotype-matched IgG control. B) Nuclear staining with propidium iodide (PI). C) Merge of (A) and (B). D) GnRH-Rs were visualized by specific monoclonal antibody cross-linked with biotin and labeled with streptavidin Oregon green-488 (absorbance and fluorescence emission maxima of 496 and 524 nm). E) Nuclei were counterstained with PI (absorption 535, emission maxima 617 nm) and cells were visualized with a krypton-argon ion laser. F) Merge of (D) and (E) showing a polarized expression pattern of GnRH-R in lymphocytes. Magnification, x600

GnRH-Ag Modulates Production of Th-1 Cytokines from Lymphocytes

Lymphocytes isolated from pregnant rats were stimulated via T cell receptor-specific ligation with ant-CD3 antibody (500 ng/ml) in the presence and absence of different concentrations of GnRH-Ag. The levels of the signature Th-1 cytokines IL-2 and IFN-{gamma} were assayed in lymphocyte culture supernatants collected after 48 h and 72 h of culture, respectively. At the dose of 0.1 nM and 1 nM, GnRH-Ag-stimulated T cells produced IFN-{gamma}, but it was not statistically significant from that of the control group (Fig. 2). However, at higher doses of 10 nM, 100 nM, and 1 µM, GnRH-Ag stimulated IFN-{gamma} production from lymphocytes was higher compared with that of the control group. IL-2 production from lymphocytes was measured after 48 h in the cell culture supernatants exposed to five different doses of GnRH-Ag. IL-2 production after GnRH-Ag treatment showed an increasing trend. However, they were not statistically different from those of the control group.



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  		FIG. 2. Effects of GnRH-Ag treatment on the production of Th-1 cytokines. Lymphocytes were derived from Day 9 pregnant rats and polyclonally stimulated by immobilized anti-CD3 antibody (500 ng/ml) in the absence (0) and presence of different concentrations of GnRH-Ag. GnRH-Ag treatment in vitro stimulates IFN-{gamma} production, without significantly affecting of IL-2 production. Data are presented as means ± SEM (n = 6), *represents a significant difference (P < 0.05) compared with 0

GnRH-Ag Modulates Production of Th-2 Cytokines from Lymphocytes

We studied the production of classical Th-2 cytokines, IL-4 and IL-10, by lymphocytes from the pregnant rats stimulated with immobilized anti-CD3 antibody. GnRH-Ag treatment at a concentration of 1 nM or at higher concentrations led to the inhibition of IL-4 production. IL-10 production was not modulated by GnRH-Ag (Fig. 3).



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  		FIG. 3. Effects of GnRH-Ag treatment on the production of Th-2 cytokines. See Figure 2 for other details. GnRH-Ag treatment in vitro inhibits IL-4 production. In vitro GnRH-Ag treatment has no effect on the production of IL-10. Data are presented as means ± SEM (n = 6), *represents a significant difference (P < 0.05) compared with 0

Effects of In Vivo GnRH-Ag Infusion on Responsiveness of Lymphocytes to Produce Th-1 Cytokines

In our pregnant rat model, 24-h-long continuous infusion of GnRH-Ag via the osmotic minipump leads to termination of pregnancy [24–26]. Considering the strong in vitro effects of GnRH-Ag in shifting the balance of cytokines to primarily an inflammatory Th-1 type, we next examined the effect of in vivo GnRH infusion in pregnant rats on the capacity of lymphocytes to secrete Th-1 and Th-2 cytokines. Lymphocytes derived from Day 9 pregnant sham and GnRH-Ag-treated rats were cultured ex vivo with and without plate-bound anti-CD3 antibody to assess the responsiveness of T cells to produce IFN-{gamma} and IL-2. The IFN-{gamma} production in lymphocyte cultures of pregnant sham rats showed a 2-fold increase in the anti-CD3 antibody treated group compared with that of the unstimulated culture. On the other hand, we observed a 6.5-fold increase in IFN-{gamma} production from lymphocytes after T cell receptor (TCR) ligation in GnRH-Ag infused pregnant rats compared with that of unstimulated controls (Fig. 4 upper panel). Thus, GnRH-Ag infusion up-regulates in vivo serum levels of IFN-{gamma}.



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  		FIG. 4. Responsiveness of lymphocytes to produce Th-1 cytokines after GnRH-Ag infusion in pregnant rats. Lymphocytes derived from Day 9 pregnant sham (open bars) and after GnRH-Ag infusion (closed bars) (5 µg/day) were cultured ex vivo in the presence and absence of immobilized anti-CD3 antibody to assess the responsiveness of cells to produce cytokines. Upper panel, GnRH-Ag infusion up-regulates IFN-{gamma} production. Lower panel, GnRH-Ag infusion does not affect the responsiveness of lymphocytes to secrete IL-2. Data are presented as means ± SEM (n = 6), *represents a significant difference (P < 0.05) compared with controls

Similar to our in vitro data, the responsiveness of lymphocytes to secrete IL-2 after GnRH-Ag infusion did not show any significant difference (Fig. 4, lower panel). Unstimulated lymphocytes that produced IL-2 increased significantly after stimulation with plate-bound anti-CD3 antibody. Unstimulated lymphocytes derived from pregnant rats treated with GnRH-Ag showed a similar responsiveness to secrete IL-2 after TCR ligation as in sham rats when treated with GnRH-Ag.

Effects of In Vivo GnRH-Ag Infusion on Responsiveness of Lymphocytes to Produce Th-2 Cytokines

To confirm our in vitro findings on inhibition of Th-2 cytokines in rats, we assessed the ability of lymphocytes to secrete IL-4 and IL-10 after in vivo GnRH-Ag infusion in pregnant rats. The IL-4 production by unstimulated lymphocytes from sham pregnant rats increased 4-fold when stimulated with plate-bound anti-CD3 antibody. In contrast, lymphocytes derived from GnRH-Ag-treated rats stimulated with plate-bound anti-CD3 antibody did not show any significant increase in IL-4 production (Fig. 5, upper panel). This finding suggests that GnRH-Ag infusion markedly inhibits the ability of lymphocytes to produce IL-4.



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  		FIG. 5. Responsiveness of lymphocytes to produce Th-2 cytokines after GnRH-Ag infusion in pregnant rats. See Figure 4 for other details. Upper panel shows GnRH-Ag infusion (closed bars) markedly inhibits the capacity of lymphocytes to produce IL-4 compared with Day 9 pregnant sham controls (open bars). Lower panel, GnRH-Ag does not modulate the IL-10 production from lymphocytes. Data are presented as means ± SEM (n = 6), *represents a significant difference (P < 0.05) compared with controls

Unstimulated lymphocytes derived from sham control pregnant rats produced modest level of IL-10, which increased slightly after stimulation with immobilized anti-CD 3 antibody (Fig. 5, lower panel). Lymphocytes from GnRH-Ag infused rats produced slightly lower levels of IL-10 (in both unstimulated and anti-CD3 treated groups) as compared with that of sham control animals, however, the difference was not statistically significant. This finding suggests that GnRH-Ag infusion does not modulate IL-10 production from lymphocytes.

GnRH-Ag Infusion Modulates Serum Levels of T Helper Cytokines

To further assess the T helper cytokine balance in Day 8 pregnant rats infused with GnRH-Ag, we measured the circulating levels of Th-1 and Th-2 cytokines in serum 24 h after the treatment. The IFN-{gamma} levels increased significantly after GnRH-Ag infusion compared with that of pregnant sham control rats (Fig. 6). The IL-2 levels were not measurable in the serum. Further, we found that serum Th-2 cytokine IL-4 levels decreased significantly after GnRH-Ag infusion compared with pregnant sham control rats. The IL-10 concentrations did not show any significant difference between sham and GnRH-Ag infused rats.



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  		FIG. 6. Effects of GnRH-Ag treatment on serum levels of T helper cytokines on Day 9 of pregnancy. Data are presented as means ± SEM (n = 6), *represents a significant difference (P < 0.05) compared with controls. IL-2 levels were not measurable in serum


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study using in vitro, ex vivo, and in vivo experiments in a rat model, we demonstrated, for the first time, that GnRH induces a shift toward Th-1 cytokine production during pregnancy. Interactions between immune, endocrine, and reproductive systems are heightened during pregnancy, in which complex interactions between different cytokines and hormones ensure the survival of both mother and allogeneic conceptus [1–9]. Accumulating evidence suggests that GnRH, apart from regulating the hypothalamo-pituitary-gonadal axis, also exerts potent effects on the immune system [16, 23]. This is further evident from the current study, which demonstrated that GnRH-R, a G protein-coupled receptor with seven transmembrane domains [17, 18], is expressed on lymphocytes from Day 9 pregnant rats. The modulation of GnRH receptor was also evident following activation of lymphocytes with anti-CD3 receptor (Fig. 1).

Previously, we have reported that continuous administration of a GnRH-Ag over a period of 24 h during early pregnancy leads to suppressed serum progesterone levels and fetal allograft rejection by the mother [24–26]. GnRH-R mRNA is also expressed in immune cells and it has been reported that GnRH might exert immunomodulatory effects through its seven transmembrane G protein-coupled receptors [17, 18]. Considering the vital role played by T helper cytokines in immune regulation and the successful outcome of pregnancy, we studied the role of GnRH in controlling these cytokines in pregnant rats. We demonstrated, for the first time, that GnRH-R protein is expressed and shows a polarized expression on activation in lymphocytes. Our data demonstrate that GnRH stimulates IFN-{gamma} production from TCR ligated T cells in pregnant rats. Furthermore, responsiveness of T cells to secrete this Th-1 cytokine was markedly up-regulated in GnRH-Ag infused pregnant rats. We observed a marked increase in serum IFN-{gamma} levels after 24 h of continuous GnRH-Ag infusion in pregnant rats. This observation is consistent with a recent study in which an i.v. bolus injection of LHRH in normoprolactinemic women led to an increase in production of IFN-{gamma} from PBMCs and high circulating levels of this Th-1 cytokine [23]. It has earlier been reported that GnRH can stimulate proliferation of T cells [17], classically IL-2, a Th-1 cytokine, produced by lymphocytes, and functions as a critical growth factor and induces cell proliferation. It has been reported that GnRH up-regulates IL-2 receptor expression in lymphocytes [19]. However, GnRH-Ag treatment did not significantly alter IL-2 levels, suggesting that this hormone may not have any direct effect on IL-2 production. Alternatively, it is also a possibility that IL-2 might have been consumed by activated T cells, and we may have underestimated its level. To rule out this possibility, it may be necessary to measure mRNA levels for this cytokine in future studies.

IL-4 is regarded as a classical Th-2 type cytokine, and increased secretion of IL-4 from lymphocytes during pregnancy is considered vital for the survival of a semiallogeneic conceptus [10, 11]. Our results clearly demonstrated that GnRH-Ag caused a suppression of IL-4 production from lymphocytes in pregnant rats, without significantly affecting IL-10 levels. This finding suggests that the IL-4 production pathway, but not the IL-10 production pathway, is sensitive to GnRH treatment. Further studies on the specific effects of GnRH on an immune cell subset might shed more light on the mechanisms responsible for the inhibition of the IL-4 response.

Continuous administration of GnRH-Ag in Day 8 pregnant rats for 24 h using the osmotic minipump leads to inhibition of progesterone synthesis and secretion [24–26]. It has been reported in previous studies that higher levels of progesterone are associated with high levels of Th-2 cytokines such as IL-4 and IL-10, with reciprocal inhibition of Th-1 cytokines [27, 28]. It has been further shown that progesterone mediates this effect by inducing lymphocytes to release an immunomodulatory protein that enhances IL-3, IL-4, and IL-10 production [28]. We speculate that the down-regulation of IL-4 response after GnRH-Ag infusion might also be due to attenuation of progesterone levels in these rats. However, we cannot rule out the possibility that GnRH might have affected IL-4 production by other alternate mechanisms because we observed only the down-regulation of IL-4 and not IL-10. It remains to be determined whether up-regulation of IFN-{gamma} following GnRH infusion is due to a direct effect on its production pathway or through an indirect mechanism involving progesterone or other endogenous mediators. However, incubation of lymphocytes in vitro with GnRH-Ag derived from pregnant rats primed with high progesterone levels still caused an increase in IFN-{gamma} and a decrease in IL-4, and clearly suggests a role for GnRH in directly altering as-yet-unknown specific downstream signaling pathways.

Signature Th-2 cytokines, IL-4 and IL-10, are responsible for controlling the humoral immune response with increasing antibody production [10], and are effective in clearing extracellular microbial infections. A Th-2 response counterregulates the effects of IFN-{gamma} on macrophages through the actions of IL-4 and IL-10. The net result of cytokine-mediated self-amplification and regulation is that once a T cell-mediated immune response begins to develop along one pathway; namely Th-1 or Th-2, it tends to become progressively polarized in that direction [11]. Accumulating evidence suggests that reproductive success is dependent on a Th-2 response, permitting pregnancy to proceed [3–9]. Induction of a Th-2 environment by pregnancy can inhibit resolution of infections requiring a Th-1 response [14]. Furthermore, a potent Th-1 response during pregnancy is associated with a detrimental effect on survival of an allogeneic conceptus [29].

This is further supported from the studies in which injection of IFN-{gamma} and IL-2 in mice led to abortions [30], whereas IL-10, a Th-2 cytokine, protected against fetal loss in a murine spontaneous abortion model [31]. Recently, it was noted that women with normal fecundity exhibited a cytokine profile that drives T cells toward a Th-2 phenotype [32]. Furthermore, women with a stronger Th-1 cytokine profile optimized against the survival from infections, the chances of having successful pregnancies were less likely [32]. In addition, apart from systemic changes in the maternal immune system, local immunomodulation at the feto-maternal interface via wide array of hormones and cytokines, and immune effector cells also play a critical role in maintaining the balance of a desirable immune response [33, 34]. Accordingly, we hypothesize that GnRH-induced termination of pregnancy in rats might be due to increases in inflammatory type 1 cytokine-like IFN-{gamma} and decreased levels of type 2 cytokine, such as IL-4. Furthermore, the thymus is known to involute during pregnancy as an adaptive response for a successful pregnancy [35], and GnRH-Ag exerts strong thymotrophic effects [20]. This is supported by our recent observation that GnRH-Ag markedly increases the thymic size and function in these pregnant rats, which finally undergo termination of pregnancy [36]. Altogether, these observations suggest that GnRH plays an important immunoregulatory role, and further studies are necessary to better understand this function.

Currently, GnRH-agonists and antagonists are widely used for therapeutic management of endometriosis [37], leiomyomas [38], and cancer [39]. GnRH has been reported to exacerbate the inflammatory responses in systemic lupus erythematosus [40], and leads to massive leukocytic infiltration in uterine leiomyomas [38] and prostate tumors [41]. Further, decreased production of GnRH leads to hypogonadotropic hypogonadism, a condition observed in human immunodeficiency virus infections and chronic sickness, in which it might contribute to immune dysregulation [42]. During the preparation of this manuscript, it was reported that GnRH is produced by human T cells and drives the lymphocytes into de novo gene transcription and additionally leads to chemotaxis and homing of T cells into specific organs [43]. Therefore, a GnRH-induced increase in Th-1 cytokine IFN-{gamma}, and associated stimulation of cell-mediated immunity might be a target for beneficial immune effects in a variety of ailments. Our novel finding that GnRH-Ag may specifically stimulate Th-1 and inhibit Th-2 cytokines provides additional insight into its immunomodulatory role; therefore, implications regarding the current use of GnRH-Ag need to be revisited.


   ACKNOWLEDGMENTS
 
We thank Dr. Alan Corbin, Wyeth-Ayerst Laboratories (Philadelphia, PA), for the gift of GnRH-Ag (Wyeth-40972); Lionel M. Elder and Donna M. Floyd for animal care; and Andrew Shah for assistance in using the confocal microscope.


   FOOTNOTES
 
1 This study was supported by grants GM08248 and HD41749 from the National Institutes of Health (NIH) and grant NAG9-963 from the National Aeronautics and Space Administration (NASA) to R.S. Core facilities were supported by grants NCC9-53 from NASA and RR03034 from NIH. Back

2 Correspondence: Rajagopala Sridaran, Department of Physiology, Morehouse School of Medicine, 720 Westview Drive, S.W., Atlanta, GA 30310-1495. FAX: 404 752 1045; sridaran{at}msm.edu Back

Received: 11 October 2002.

First decision: 30 October 2002.

Accepted: 21 January 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Medawar PB. Some immunological and endocrinological problems raised by the evolution of viviparity in vertebrates. Symp Soc Exp Biol 1953 7:320-338
  2. Dixit VD, Parvizi N. Pregnancy stimulates production of adrenocorticotropin and nitric oxide from peripheral bovine lymphocytes. Biol Reprod 2001 64:242-248[Abstract/Free Full Text]
  3. Rogers AM, Boime I, Connoly J, Cook JR, Russel JH. Maternal fetal tolerance is maintained despite transgene-driven trophoblast expression of MHC class 1 and defects in Fas and its ligand. Eur J Immunol 1998 28:3479-3487[CrossRef][Medline]
  4. Tafuri A, Alferink J, Moller P, Hammerling GJ, Arnold B. T cell awareness of paternal alloantigens during pregnancy. Science 1995 270:630-633[Abstract/Free Full Text]
  5. Jiang SP, Vacchio MS. Multiple mechanism of peripheral T cell tolerance to the fetal "allograft". J Immunol 1998 160:3086-3090[Abstract/Free Full Text]
  6. Weetman P. The immunology of pregnancy. Thyroid 1999 9:643-666[Medline]
  7. Wegmann TG, Lin H, Guilbert L, Mosmann TR. Bidirectional cytokine interactions in the maternal-fetal relationship: is successful pregnancy a TH-2 phenomenon?. Immunol Today 1993 14:353-356[CrossRef][Medline]
  8. Reinhard G, Noll A, Schlebush H, Mallmann P, Ruecker AV. Shifts in the TH-1/TH-2 balance during normal human pregnancy correlate with apoptotic changes. Biochem Biophys Res Commun 1998 245:933-938[CrossRef][Medline]
  9. Piccinni MP, Beloni L, Livi C, Maggi E, Scarselli G, Romagnani S. Defective production of both leukemia inhibitory factors and type 2 T-helper cytokines by decidual T cells in unexplained recurrent abortions. Nat Med 1998 4:1020-1024[CrossRef][Medline]
  10. Abbas AK, Murphy KM, Sher A. Functional diversity of helper T lymphocytes. Nature 1996 383:787-793[CrossRef][Medline]
  11. Paul WE, Seder RA. Lymphocyte responses and cytokines. Cell 1994 76:241-251[CrossRef][Medline]
  12. Wilder RL. Hormones, pregnancy and autoimmune diseases. Ann N Y Acad Sci 1998 840:45-50[Abstract/Free Full Text]
  13. Vacchio MS, Jiang SP. The fetus and the maternal immune system: pregnancy as a model to study peripheral T-cell tolerance. Crit Rev Immunol 1999 19:461-480[Medline]
  14. Luft BJ, Remington JS. Effect of pregnancy on resistance to Listeria monocytogenes and Toxoplasma gondii infections in mice. Infect Immunol 1982 38:1164-1171[Abstract/Free Full Text]
  15. Besedovsky HO, Rey AD. Immune-neuro-endocrine interaction: facts and hypotheses. Endocr Rev 1996 17:64-102[CrossRef][Medline]
  16. Jacobson JD. Gonadotropin-releasing hormone and G proteins: potential roles in autoimmunity. Ann N Y Acad Sci 2000 917:809-818[Abstract/Free Full Text]
  17. Chen HF, Jeung EB, Stephenson M, Leung PC. Human peripheral blood mononuclear cells express gonadotropin releasing hormone (GnRH), GnRH receptor and interleukin 2 receptor gamma chain messenger ribonucleic acid that are regulated by GnRH in vitro. J Clin Endocrinol Metab 1999 84:743-750[Abstract/Free Full Text]
  18. Jacobson JD, Crofford LJ, Sun L, Wilder RL. Cyclical expression of GnRH and GnRH receptor mRNA in lymphoid organs. Neuroendocrinology 1998 67:117-125[CrossRef][Medline]
  19. Batticane N, Morale MC, Gallo F, Farinella Z, Marchetti B. Luteinizing hormone-releasing hormone signalling at the lymphocyte involves stimulation of interleukin-2 receptor expression. Endocrinology 1991 129:277-286[Abstract]
  20. Marchetti B, Guarcello V, Morale MC, Bartolini G, Raiti F, Palumbo G, Garinella Z, Cordaro S, Scapagnini U. Luteinizing hormone releasing hormone (LHRH) agonist restoration of age associated decline of thymus weight, thymic LHRH receptors and thymocyte proliferative capacity. Endocrinology 1989 125:1037-1045[Abstract]
  21. Umesaki N, Tanaka T, Miyama M, Mizumo K, Kawamura N, Ogita S. Increased natural killer cell activities in patients with gonadotropin releasing hormone agonist. Gynecol Obstet Invest 1999 48:66-68[CrossRef][Medline]
  22. Jacobson JD, Ansari MA, Mansfeild ME, McArthur CP, Clement LT. Gonadotropin-releasing hormone increases CD4 T-lymphocyte numbers in an animal model of immunodeficiency. J Allergy Clin Immunol 1999 104:653-658[CrossRef][Medline]
  23. Grasso G, Massai L, De Leo V, Muscettola M. The effect of LHRH and TRH on human interferon-gamma production in vivo and in vitro. Life Sci 1998 62:2005-2014[CrossRef][Medline]
  24. Sridaran R, Ghose M, Mahesh VB. Inhibitory effects of a gonadotropin-releasing hormone agonist on the luteal synthesis of progesterone, estradiol receptors, and prolactin surges during early pregnancy. Endocrinology 1988 123:1740-1746[Abstract]
  25. Sridaran R, Hisheh S, Dharmarajan AM. Induction of apoptosis by a gonadotropin-releasing hormone agonist during early pregnancy in the rat. Apoptosis 1998 3:51-57
  26. Sridaran R, Philip GH, Li H, Culty M, Liu Z, Stocco DM, Papadopoulos V. GnRH agonist treatment decreases progesterone synthesis, luteal peripheral benzodiazepine receptor mRNA, ligand binding and steroidogenic acute regulatory protein expression during pregnancy. J Mol Endocrinol 1999 22:45-54[Abstract]
  27. Piccinni MP, Giudizi MG, Biagiotti R, Beloni L, Giannarini L, Sampognaro S, Parronchi P, Manetti R, Annunziato F, Livi C. Progesterone favors the development of human T helper cells producing Th2-type cytokines and promotes both IL-4 production and membrane CD30 expression in established Th1 clones. J Immunol 1995 155:128-133[Abstract]
  28. Szekeres-Bartho J, Wegmann TG. A progesterone-dependent immunomodulatory protein alters the Th1/Th2 balance. J Reprod Immunol 1996 31:81-95[CrossRef][Medline]
  29. Dresser DW. The potentiating effect of pregnancy on humoral immune responses of mice. J Reprod Immunol 1991 20:253-266[CrossRef][Medline]
  30. Chaout G, Meliani AA, Clark DA, Dy M, Minkowski M, Wegmann TG. Control of fetal survival in CBAxDBA/2 mice by lymphokine therapy. J Reprod Fertil 1990 89:447-452[Abstract]
  31. Chaout G, Meliani AA, Martal J, Raghupathy R, Elliot J, Mosmann T, Wegmann TG. IL-10 prevents naturally occurring fetal loss in the CBAxDBA/2 mating combination, and local defect in IL-10 production in this abortion prone combination is corrected by in vivo injection of IFN-{iota}. J Immunol 1995 154:4621-4630
  32. Westendorp RGJ, van Dunne FM, Kirkwood TBL, Helmerhorst FM, Huizinga TW. Optimizing human fertility and survival. Nat Med 2001 7:873[CrossRef][Medline]
  33. Vives A, Balasch J, Yague J, Quinto L, Ordi J, Vanrell JA. Type-1 and type-2 cytokines in human decidual tissue and trophoblasts from normal and abnormal pregnancies detected by reverse transcriptase polymerase chain reaction (RT-PCR). Am J Reprod Immunol 1999 42:361-368
  34. Moffet-King A. Natural killer cells and pregnancy. Nat Rev Immunol 2002 2:656-663[CrossRef][Medline]
  35. Kendall MD, Clarke AG. The thymus in pregnancy: the interplay of neural, endocrine and immune influences. Immunol Today 1994 15:545-551[CrossRef][Medline]
  36. Dixit VD, Sridaran R, Edmonsond MA, Taub D, Thompson WE. Gonadotropin releasing hormone (GnRH) attenuates pregnancy-associated thymic involution and modulates the expression of antiproliferative gene product prohibitin. Endocrinology 2003 144:1496-1505[Abstract/Free Full Text]
  37. Hsu CC, Lin YS, Wang ST, Huang KE. Immunomodulation in women with endometriosis receiving GnRH agonists. Obstet Gynecol 1997 89:993-998[Abstract]
  38. Bardsley V, Cooper P, Peat DS. Massive lymphocytic infiltration of uterine leiomyomas associated with GnRH agonist treatment. Histopathology 1998 33:80-82[CrossRef][Medline]
  39. Grundker C, Gunthert AR, Westphalen S, Emons G. Biology of the gonadotropin-releasing hormone system in gynecological cancers. Eur J Endocrinol 2002 146:1-14[Abstract]
  40. Jacobson JD, Ansari MA, Kinealy M, Muthukrishnan V. Gender specific exacerbation of murine lupus by GnRH: potential role of G{alpha}q/11. Endocrinology 1999 140:3429-3437[Abstract/Free Full Text]
  41. Tieva A, Stattin P, Wikstrom P, Bergh A, Damber JE. Gonadotropin releasing hormone receptor expression in human prostate. Prostate 2001 47:276-284[CrossRef][Medline]
  42. Schurmeyer TH, Muller V, von zur Muhlen A, Schmidt RE. Endocrine testicular function in HIV-infected outpatients. Eur J Med Res 1997 2:275-281[Medline]
  43. Chen A, Ganor Y, Rahimipour S, Ben-Aroya N, Koch Y, Levite M. The neuropeptide GnRH-II and GnRH-I are produced by human T cell and trigger laminin receptor gene expression, adhesion, chemotaxis and homing to specific organs. Nat Med 2002 12:1421-1426




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