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Endocrinology Vol. 144, No. 4 1496-1505
Copyright © 2003 by The Endocrine Society

ARTICLE

Gonadotropin-Releasing Hormone Attenuates Pregnancy-Associated Thymic Involution and Modulates the Expression of Antiproliferative Gene Product Prohibitin

Vishwa Deep Dixit, Rajagopala Sridaran, Megan A. Edmonsond, Dennis Taub and Winston E. Thompson

Department of Physiology (V.D.D., R.S., M.A.E.), Cooperative Reproductive Science Research Center (R.S., W.E.T.), Laboratory of Immunology (D.T.), National Institute on Aging, National Institutes of Health, Baltimore, Maryland 21224; and Department of Obstetrics and Gynecology (W.E.T.), Morehouse School of Medicine, Atlanta, Georgia 30310

Address all correspondence and requests for reprints to: Dr. Winston E. Thompson, Department of Obstetrics & Gynecology, Cooperative Reproductive Science Research Center, Morehouse School of Medicine, 720 Westview Drive Southwest, Atlanta, Georgia 30310. E-mail: thompsw{at}msm.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Thymic involution during pregnancy is believed to be a critical adaptive mechanism for regulation and control of the maternal immune system. These regulatory feedback mechanisms are important for the survival of the semiallogeneic fetus. In the present study, we examined the effects of GnRH on pregnancy-induced thymic involution by characterizing the expression patterns of prohibitin (PHB), an antiproliferative gene product, GnRH, and GnRH receptor (GnRH-R) proteins in the rat thymus and in mature splenic lymphocytes. GnRH agonist infusions in pregnant rats markedly attenuated pregnancy-induced thymic involution resulting in significant increases in thymic weight and thymocyte numbers. In addition, histological examination of the thymus revealed increase in cortical cellularity. Western blot analyses revealed a significant increase of total PHB protein content in thymi during pregnancy. Furthermore, distinct changes in PHB isoform expression were observed in the pregnant involuting thymi with greater expression of the basic PHB isoform. Basic isoform expression decreased in pregnant rats and was comparable with nonpregnant rat thymi upon GnRH agonist treatment. PHB is mainly expressed in mature cells of the thymic medulla, where it strongly colocalized with GnRH. We have observed GnRH-R immunoreactivity mainly in thymic medulla. Furthermore, as assessed by immunofluorescence double labeling with proliferating cell nuclear antigen, PHB was preferentially expressed in nonproliferating thymocytes. In this study, we demonstrated that GnRH, GnRH-R, and PHB show characteristic polarized expression in thymocytes. In addition, GnRH and PHB were coexpressed in mature splenic T cells. Our results suggest that PHB and GnRH are involved in thymic growth and may be important for maturation of T lymphocytes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE THYMUS PLAYS a crucial role in the development of T cells by providing an inductive microenvironment in which bone marrow-derived progenitor cells undergo proliferation, T cell receptor gene rearrangement, positive and negative selection, and thymocyte differentiation into CD4+- or CD8+-expressing cells (1). The earliest thymic precursor population, CD4-CD8- cells comprise approximately 3% of total thymocyte numbers. These cells proliferate extensively within the thymic cortex and differentiate into immature CD4+CD8+ T cells, which serve as targets for both positive and negative selection (2). The majority of these cells undergo apoptosis, whereas positively selected thymocytes enter the medulla before exiting to the periphery (3, 4, 5). A number of mediators and coreceptors present within the thymic microenvironment play a critical role in the selection process and are believed to facilitate the differentiation of immature double positive cells to mature naïve CD4+ and CD8+ T cells (3, 4).

Various hormones and cytokines are expressed within the thymic microenvironment and appear to play an important role in providing signals that regulate development of the thymocytes (5). GnRH and its receptor mRNAs are known to be expressed in lymphoid cells and tissues, including murine thymus (6). In addition to its role in controlling the hypothalamo-pituitary-gonadal axis, GnRH also appears to exert potent regulatory effects on immune function (7, 8) and has been reported to prevent age-associated thymic involution in mice (9). Cloning of cDNAs coding for GnRH receptors from various species has demonstrated that the GnRH receptors belong to a superfamily of seven transmembrane G protein-coupled receptors (GPCR) (10, 11). GnRH has been shown to induce the expression of the IL-2 receptor in lymphocytes (7) and cumulative evidence suggests that GnRH might mediate cross-talk between immune and reproductive systems. Moreover, GnRH has been also shown to function as a signal modulating proliferation in various cell types (7, 12), including ovarian cancer cells. Significantly, more than 80% of human ovarian cancers coexpress GnRH and its specific receptor as a part of an autocrine feedback mechanism to control cells growth (13, 14). It is thought that GnRH may serve as an autocrine or paracrine regulatory cytokine, controlling cell growth and differentiation.

We have previously shown that a GnRH agonist (GnRH-Ag) modulates the expression of PHB in rat ovaries (15, 16). PHB is a regulatory protein believed to play an important role in the control of cell differentiation and proliferation in various cell types. DNA sequence comparison studies indicate that the PHB gene is most closely related to the mammalian analog of the Drosophila Cc gene shown to be necessary for normal cell development (17). The PHB gene encodes a 30-kDa posttranslationally modified protein that is located primarily in the mitochondria in fibroblasts, in HeLa cells (18), and in rat ovarian granulosa cells (15, 16). PHB has also been shown to be coexpressed with membrane IgM receptors on B lymphocytes (19). PHB is a highly conserved protein that is thought to play a role in cell cycle control (20), differentiation (21), senescence (22), and antiproliferative activity (23). Microinjection of PHB mRNA into HeLa, normal fibroblasts cells, and chronic lymphocytic leukemia B cells has been reported to block DNA synthesis and entry of cells into S phase, whereas antisense PHB oligonucleotides stimulated entry into S phase (17, 21). However, the expression and function of PHB in thymus and T cell physiology is still unknown.

Pregnancy is characterized by specific changes in the maternal immune system to accommodate genetically disparate allogeneic fetus (24). One of the adaptive changes that occur during pregnancy is the inhibition of T cell development, as manifested by the involution of thymus in different species (25, 26). We have previously reported that GnRH-Ag infusion in d 8 pregnant rat model leads to termination of pregnancy (27). Interestingly, we consistently observed an increase in thymic size in the pregnant rats that aborted the concepti. Therefore, in the present study, we examined the effects of GnRH-Ag on pregnancy-induced thymic involution and characterized the expression pattern of the antiproliferative gene product PHB, GnRH, and GnRH receptor (GnRH-R) protein in the rat thymus and mature splenic lymphocytes. Our current data demonstrate that: 1) GnRH-Ag has strong thymotropic effects in pregnancy; 2) PHB expression is modulated during thymic involution and regeneration by GnRH-Ag; 3) PHB and GnRH proteins are expressed and colocalized in rat thymus and demonstrate polarized expression pattern in thymocytes and peripheral lymphocytes; and 4) PHB is preferentially expressed in nonproliferating thymocytes. These observations suggest that GnRH and PHB may play an important role in differentiation and maturation of T lymphocytes within the thymus and peripheral lymphatic tissues.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
Polyclonal antibody for GnRH was purchased from Peninsula Laboratories, Inc. (Belmont, CA). Monoclonal antibodies for proliferating cell nuclear antigen (PCNA) and GnRH-R were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), respectively. PHB monoclonal and polyclonal antibodies were obtained from Lab Vision Corp./NeoMarkers, Inc. (Fremont, CA). Anti CD4+ and CD8+ antibodies were purchased from R&D Biosystems (Minneapolis, MN). Goat antimouse IgG (H + L) conjugated to Alexa Fluor 488, goat antirabbit IgG conjugated to Alexa Fluor 594, and 4'6-diamidino-2-phenylindole dihydrochloride (DAPI) was purchased from Molecular Probes, Inc. (Eugene, OR).

Animals
Nonpregnant and timed-pregnant age matched (8- to 10-wk-old first time pregnant mothers) SASCO rats were purchased from Charles River Laboratories, Inc. (Wilmington, MA). They were housed at the animal facilities of Morehouse School of Medicine in a temperature (23–25 C) and light (14-h light, 10-h dark) controlled room. Purina rodent chow and tap water were freely accessible. The day of insemination, which was identified by a sperm plug, was designated as d 1 of pregnancy. Nonpregnant and d 8 pregnant rats were euthanized by Halothane and thymus tissues were excised and snap frozen for cryosectioning and protein extraction.

GnRH-Ag infusion
GnRH-Ag ([pyro]-Glu-His-Trp-Ser-Tyr-D-Trp-NMeLeu-Arg-Pro-ethylamide-LH-releasing hormone; Wyeth-40972) was a gift from Wyeth-Ayerst Laboratories (Philadelphia, PA). GnRH-Ag (5 µg/d) was administered continuously via osmotic pump (Model 1003D, Alza Corp., Palo Alto, CA) starting on the morning of d 8 of pregnancy for a period of 24 h as described previously (27). This dose was selected primarily because of its consistent and reproducible inhibitory effects on progesterone production (27). Briefly, d 8 of pregnancy was selected for infusion because thymus starts to involute early in pregnancy and changes in size and structure are clearly distinguishable around d 8 of pregnancy. In addition, GnRH-Ag infusion at d 8 induces strong antipregnancy effects and ultimately causes termination of pregnancy within 48 h of GnRH-Ag infusion, i.e. by d 10 of pregnancy (27).

Briefly, each osmotic minipump was loaded with GnRH-Ag or saline and then incubated in saline for 4 h at 37 C before implantation into d 8 pregnant rats. While under Metofane anesthesia, each rat was implanted sc with one osmotic minipump in the dorsal surface of the neck. The rats were euthanized with Halothane after 24 h of GnRH-Ag treatment, and each thymus was excised, weighed, and snap frozen in liquid nitrogen. 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 NIH guide for the care and use of laboratory animals.

Immunofluorescence staining and microscopy
Frozen thymi were embedded in Stephens Scientific Frozen Section Medium (Riverdale, NJ) and cut into 10-µm-thick cryostat sections using the Microm HM 500 OM microtome. At least three series were used for each staining. Tissue sections were then fixed with 3.7% paraformaldehyde in PBS for 30 min, followed by PBS wash and subsequent treatment with cold absolute methanol for 5 min at -20 C. Following treatment with 50 nM NH4Cl PBS for 10 min, the tissue sections were washed in PBS and then permeabilized with 0.2% Triton X-100 for 5 min. After blocking nonspecific binding using blocking buffer containing 10% calf serum, the slides were incubated overnight with either antirabbit GnRH (1:100), antimouse GnRH-R (1:100), antirabbit PHB (1:200), or antimouse PHB (1:200) at 4 C in blocking buffer. The cellular localization of GnRH, GnRH-R, and PHB were visualized with an Alexa Fluor 594-conjugated-goat antirabbit IgG, Alexa Fluor 488-conjugated-goat antimouse IgG at 2 µg/ml, respectively (Molecular Probes, Inc.). Thereafter, slides were washed and subsequently incubated in DAPI for nuclear staining at a concentration of 1 µg/ml for 15 min. After extensive washing, the sections were mounted in Vectashield mounting medium (Vector Laboratories, Burlingame, CA). Negative controls were performed omitting the primary antibody or using an isotype-matched control antibody derived from the same species. Mounted slides were examined using an Olympus laser scanning confocal microscope (Olympus America Inc., Melville, NY) imaging system equipped with a 15-mW krypton/argon laser emitting at 488 nm and 647 nm. Lymphocytes and thymocytes were also similarly triple stained for GnRH, GnRH-R, PHB, and DAPI on poly-L-lysine-coated slides. Alexa Fluor 488 has a laser absorption and emission spectrum profile similar to fluorescein, whereas Alexa Fluor 594 has a profile similar to Texas Red. Hematoxylin and 1% eosin staining were performed using standard methods, and thymic sections were analyzed under a x10 objective.

Preparation of splenic, thymocytes single cell suspension
Cells were isolated and washed in RPMI 1640 medium (Life Technologies, Inc., Gaithersburg, MD). Splenocytes were isolated from the excised spleens of rats using sterile techniques, and single-cell suspension was prepared using established cell isolation techniques. T cells were isolated using nylon wool columns. Isolated lymphocytes were placed on poly-L-lysine-coated slides for immunofluorescence staining.

Western blot analysis
The procedure used for Western blot analysis has been previously described (17). Briefly, 50 µg of protein extracts from thymi of nonpregnant, pregnant, and pregnant GnRH-Ag-treated rats were subjected to either one- or two-dimensional gel electrophoresis. The membranes were subsequently blotted with anti-PHB antibody (1:1000) followed by extensive washing and incubation with the appropriate secondary antibody for 2 h at room temperature. Antibody binding was detected by chemiluminescence (Amersham Pharmacia Biotech, Arlington Heights, IL).

Progesterone estimation
Progesterone in the serum was measured using RIA kit obtained from Diagnostic Laboratories, Inc. (Webster, TX). The sensitivity or the minimum detection limit of the kit was 0.12 ng/ml. Intraassay coefficient of variation was 7.2%.

Statistical analysis
Results were expressed as the mean ± SEM of three to five experiments. Quantification of the scanned images was performed using the NIH Image version 1.61 software program. Statistical analysis was subsequently carried out by one- or two-way ANOVA. Significant differences between treatment groups were determined by the Tukey test, which protects the significance tests of all combinations of pairs. Statistical significance was inferred at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
GnRH treatment attenuates pregnancy induced thymic involution
GnRH has been previously shown to reverse age-associated thymic involution leading to increased thymic cellularity and size (9). To assess whether GnRH-Ag infusion could reverse pregnancy-associated thymic involution, we examined the thymi from nonpregnant, d 8 pregnant, and GnRH-Ag-treated pregnant rats for thymic structure and function. Representative histologic sections of thymuses from each group are shown in Fig. 1Go. Similar to aging thymus, the normal cortico-medullary differentiation of the thymus was lost during pregnancy. In addition, pregnant thymus demonstrated an increase in eosin-stained adipose tissue. Continuous infusion of GnRH-Ag via osmotic minipumps over a period of 24 h led to a significant (P < 0.05) increase in thymic weight and thymocyte numbers. Furthermore, in contrast to pregnant sham rats, GnRH-Ag treatment resulted in a marked increase in cellularity within the cortical area with relative preservation of medullary structures. Similar to our previous studies (27, 28), the circulating progesterone concentration declined from 64.3 ± 6.9 in pregnant sham to 21.3 ± 5.2 pg/ml (n = 7) after 24 h continuous GnRH-Ag infusion in these rats, suggesting that drop in progesterone may disinhibit the regenerative process in thymus after GnRH-Ag infusion. To more fully understand the effect of GnRH-Ag on the maturation of thymocytes, we stained thymi of pregnant and GnRH-Ag-treated pregnant rats for the CD4+ and CD8+ cell surface markers. As shown in Fig. 1Go (lower panel), there was a marked increase in CD4+ and CD8+ expressing thymocytes in medulla compared with sham controls upon GnRH-Ag infusion, suggesting that GnRH-Ag treatment facilitated an increase in thymocyte maturation.



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  		Figure 1. GnRH-Ag administration attenuates pregnancy-associated thymic involution in rats. Upper left panel shows thymic weight, thymocyte numbers, and thymic size in pregnant sham and GnRH-Ag-infused pregnant rats (n = 7). Values are expressed as mean ± SEM; statistical significance was inferred at P < 0.05. Representative thymic sections (upper right panel) are shown (x10) after hematoxylin and eosin staining. A, Nonpregnant; B, pregnant; and C, pregnant rat infused with GnRH-Ag. Reduction and partial loss of cortico-medullary area, reduced cortical cellularity characterizes thymic involution. GnRH-Ag treatment is associated with an increase in CD4+ and CD8+ cells in the thymic medulla of pregnant rats. Immunofluorescence analysis of CD4+ cell staining is displayed in bottom left vertical panels (x10). D, Pregnant sham (control for GnRH-Ag-treated pregnant rats). F, GnRH-Ag-treated pregnant rats. Immunostaining of CD8+ thymocytes (bottom right panel). E, Pregnant sham and G, after GnRH-Ag treatment. m, Medulla; c, cortex.

 
GnRH-R and GnRH expression in thymus
Given the dramatic effects of GnRH in regeneration of thymus during pregnancy, we next sought to examine the expression pattern of the GnRH-R in the thymus. Using confocal microscopy, we demonstrated that GnRH-R is constitutively expressed in the thymus with greater expression in the medullary regions and lower expression in the cortex (Fig. 2Go). The GnRH infusion for 24 h had no effect on GnRH-R expression in different areas of thymus (data not shown). Furthermore, thymocytes derived from nonpregnant and pregnant rats demonstrated both GnRH and GnRH-R expression, with typical polarized patterns of coexpression (Fig. 2Go, lower panel). In addition, GnRH-R was found to be specifically colocalized with GnRH in some thymocytes but not all, suggesting a role for GnRH as a paracrine or autocrine immunomodulator in thymic physiology.



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  		Figure 2. Localization of GnRH-R in the thymus of pregnant rat. A, Negative control without the primary antibody, B, GnRH-R is highly expressed in the thymic (x10) medulla (M) compared with cortex (C). Isolated thymocytes were double labeled using specific antibodies GnRH-R (mouse monoclonal) and GnRH (rabbit polyclonal). GnRH-R- and GnRH-positive cells were visualized by incubating cells in secondary antibodies conjugated with Alexa Fluor 488 and Alexa Fluor 594. Cell nuclei were counterstained by DAPI. GnRH-R shows capped expression on thymocytes. Overlay of images demonstrates colocalization of GnRH and GnRH-R in some cells (x60).

 
PHB expression increases with thymic involution in pregnant rats
We have previously demonstrated that PHB expression is modulated with GnRH-Ag treatment in rat ovaries (15, 16). In the present study, we sought to determine whether PHB an antiproliferative gene product is expressed in the thymus, a major site of lymphocyte proliferation and differentiation. These studies were initiated to determine if the thymic involution process during pregnancy is associated with changes in PHB expression. In addition, we also examined the patterns of PHB expression during regrowth of the thymus after in vivo GnRH-Ag treatment. Total protein extracts were prepared from rat thymi of nonpregnant, pregnant (control, d 8) and GnRH-Ag-treated (24 h) d 8 pregnant rats and subjected to SDS-PAGE. The results (Fig. 3Go, A and B) demonstrate the presence of lower levels of PHB (30 kDa) in nonpregnant thymi and significantly increased expression (P < 0.05) in pregnant thymi. Interestingly, d 8 pregnant rats treated for 24 h with GnRH failed to demonstrate any significant change in total PHB expression upon comparison with pregnant sham age-matched rats.



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  		Figure 3. Western blot analyses of protein content for PHB in thymus from nonpregnant (lane 1), d 8 pregnant (control for GnRH-Ag-treated pregnant rats; lane 2) and GnRH-Ag-infused pregnant rats (lane 3). A, The PHB expression in thymi. Fifty micrograms of protein were applied to each lane and subjected to Western blot analyses. B, The bar graphs represent the mean ± SEM of results from three replicate experiments after normalization of data for PHB against cyclophilin. C, Two-dimensional gel electrophoresis for PHB in thymi. Protein samples were further focused for 16,000 V/h, with a mixture of pH 3–10 and pH 5–7 ampholyte and following the second dimension Western blotting procedure detected spots for PHB. Asterisk denotes significant difference at P < 0.05; arrowheads indicate acidic PHB isoform.

 
Because PHB is posttranslationally modified and expressed as two distinct isoforms (15), we next sought to determine if different PHB isoforms are also expressed in these various thymic preparations. Two-dimensional electrophoresis was performed to further delineate PHB isoforms expression in these rats. Immunoblot analysis revealed that two 30-kDa isoforms of this protein were expressed in the various thymic preparations. Day 8 pregnant (control) rats demonstrated a marked increase in the basic isoform of PHB when compared with age-matched nonpregnant and GnRH-Ag-treated rat thymi. Moreover, upon infusion of GnRH-Ag into rats using osmotic minipumps, we observed a significant increase in the more acidic PHB isoform, whereas basic PHB isoform was remarkably down-regulated compared with pregnant sham. Furthermore, expression of basic PHB isoform in GnRH-Ag- treated pregnant rats was reduced compared with pregnant shams and the level of expression was comparable to noninvoluting nonpregnant thymi. Interestingly, these studies identified a 37-kDa protein, which was expressed only in thymi of pregnant rats. This 37-kDa protein may constitute another uncharacterized up-regulated protein that is recognized by the PHB antibody.

Colocalization of GnRH and PHB in the rat thymus
As GnRH appears to regulate PHB isoform expression, we next investigated the expression pattern of these proteins within the thymus. To determine which cells within the thymi are expressing PHB, indirect immunofluorescence staining of thymic tissue sections was performed using either monoclonal or polyclonal antibodies specific for PHB to determine cell and organ associations. We used frozen thymic tissue sections; this approach prevents the detection of artifacts potentially associated with tissue processing allowing the examination of cells in their native states. The results in Fig. 4Go demonstrate that PHB is expressed at increased levels in the medulla compared with cortex. Moreover, additional staining of nonpregnant and pregnant thymi revealed that PHB and GnRH were highly coexpressed in thymic medulla, an area of thymus containing mature thymocytes. Isolated thymocytes from pregnant rats showed a characteristic polarized PHB expression, with strong colocalization with GnRH (Fig. 4Go, middle panel). Although it is unclear which specific isoforms of PHB are coexpressed with GnRH, the coexpression does suggest a possible conjoint role of these proteins in the homeostasis of the thymic microenvironment. Additional studies were performed to determine if GnRH and PHB are also coexpressed in mature lymphocytes. Splenic T lymphocytes derived from pregnant rats (Fig. 4Go, lower panel) reveal that PHB is indeed expressed within mature splenic T lymphocytes and exhibit a polarized capped expression pattern. In addition, GnRH also appears to be constitutively coexpressed in these cells. Together, these results demonstrate that PHB and GnRH signals might be important for immune cell differentiation and function.



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  		Figure 4. PHB and GnRH are coexpressed in thymic medulla and T cells. Immunofluorescence double labeling of GnRH was performed by polyclonal antibody, followed by antirabbit Alexa Fluor 594 conjugated secondary antibody, PHB was localized using monoclonal antibody, followed by antimouse Alexa Fluor 488-conjugated secondary antibody. Upper panel shows representative thymic sections (x40) of pregnant rat, GnRH is strongly coexpressed with PHB in medulla. Middle panel demonstrates (x100) polarized expression of (A) PHB and (B) GnRH from pregnant rat thymocytes. C, Nuclear DAPI stain and (D) overlay shows a strong colocalization of PHB and GnRH. Mature T cells were isolated from spleen lower panel (E) PHB, (F) GnRH, (G) DAPI, and (H) merge of E, F, and G shows a similar colocalization and polar expression of PHB and GnRH as seen in thymocytes.

 
PHB is preferentially expressed in nonproliferating thymocytes
As PHB has been shown to negatively regulate cell cycle (16, 20, 21, 22), we sought to determine if PHB is preferentially associated with nonproliferating thymocytes. To this end, the thymic tissue sections were double labeled with polyclonal anti-PHB antibody in combination with monoclonal anti-PCNA antibody that stains proliferating cell populations. The results in the Fig. 5Go demonstrate that the majority of the proliferating cells (PCNA+) failed to express any PHB protein, whereas nonproliferating thymocytes appeared to express significantly higher levels of PHB. Further, thymic medulla having fewer PCNA+ cells had higher PHB expression compared with thymic cortex where the majority of proliferating cells localized.



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  		Figure 5. PHB distribution in rat thymus relative to proliferating cell marker PCNA. PHB is mainly expressed in nonproliferating cells. Left vertical panel shows localization of PHB in thymic cortex (x100) (A) PHB, (B) PCNA, (C) overlay of A and B shows segregation of PHB from proliferating cells. Right vertical panel shows relative distribution of (D) PHB, (E) PCNA, (F) overlay of D and E in thymic medulla. Thymic medulla consist of few proliferating PCNA+ cells and has higher PHB expression compared with cortex.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
T cell development in the thymus is under tight control by various cytokines, hormones, and neurotransmitters. The efficiency of this homeostatic process depends on an array of proteins expressed in the thymus during differentiation of the thymocytes. In the present study, we used pregnant and nonpregnant rats to evaluate the effects of GnRH on the process of thymic involution in pregnancy. We also examined the expression patterns of PHB during pregnancy induced thymic involution. These studies are biologically significant because our data demonstrate that GnRH attenuates pregnancy-induced thymic involution and regulates the expression of PHB during thymic growth. We believe that PHB and GnRH may play an important role in T cell development and differentiation.

Immunologically, pregnancy can be considered as a chronic alloreactive state where the maternal immune system undergoes specific changes to accommodate the genetically disparate fetus (24). A number of regulatory factors play specific roles in this process including several hormones and cytokines (29). During pregnancy, the thymus has been shown to involute, a mechanism that is likely to dampen the T cell output and facilitate the survival of fetus by preventing the replenishment of clonally deleted paternally reactive T cells (24, 26). In the current pregnant rat model, we have consistently observed luteolysis and drop in circulating progesterone levels that subsequently lead to termination of pregnancy after 24-h-long continuous GnRH-Ag infusion (27, 28). In this study, we demonstrate the GnRH-Ag-induced termination of pregnancy is also associated with marked increase in thymic size and function. The marked increase in thymic size and thymocyte numbers in GnRH-Ag-treated pregnant thymus may be in response to direct signaling effect through G protein-coupled GnRH-R within the thymus, or may be due to a precipitous drop in circulating progesterone levels in these rats (27) or combination of both. Published studies have reported that removal of sex steroids by castration leads to stimulation of thymic size (30) and subsequent increase in thymus-associated GnRH levels (31). In addition, it should be noted that GnRH has previously been shown to reverse age-associated thymic involution (9). High serum estradiol and progesterone levels and presence of functional progesterone receptors are also critical to thymic involution during pregnancy (26). Therefore, we speculate that the decline in progesterone resulting from luteal demise might further facilitate the growth of pregnant thymus after GnRH-Ag treatment. However, further studies are required to delineate these mechanisms. These studies support our hypothesis that pregnancy induced thymic involution is an adaptive change and is perhaps one of many complex mechanisms geared for fetal survival. GnRH-Ag administration disrupts this homeostasis and consequently stimulates thymic function leading to termination of pregnancy in our pregnant rat model.

GnRH mRNA is expressed in the thymus and is thought to be important for thymic development (9, 31). In this study, we show that GnRH protein is expressed in the thymus of both pregnant and nonpregnant rats. However, thymus involutes despite of presence of GnRH and its receptor in pregnant rat thymus; the exact mechanism behind this process remains to be established. It should be noted that involution of thymus and attenuation of cell-mediated immune response in pregnancy is a reversible process and is merely a adaptive change as a result of complex interaction between endocrine and immune system to accommodate the genetically disparate fetus. After a successful pregnancy, involuted thymus regains its functions, which implies that all the regenerative mechanisms, including various hormones and growth factors like GnRH and GnRH-R, are probably intact in pregnant thymus but probably under override inhibitory control of high estradiol and progesterone, which are known to inhibit thymic function (26). Recently, it has been proposed that promiscuity of genes expressed in thymus is a vital mechanism for the maintenance of tolerance to these self-molecules (32, 33). Expression of highly tissue-restricted proteins like insulin (33) or GnRH in thymus might be necessary for tolerance to self-molecules and prevention of autoimmune disorders (34). However, we propose that GnRH have additional functional roles in thymic development because GnRH and its receptor are strongly colocalized and have a polarized expression pattern in thymocytes similar to GPCR chemokine receptors in lymphocytes (35). Interestingly, we observed that GnRH was strongly colocalized in PHB positive cells of the thymic medulla. Therefore, coexpression of PHB and GnRH in thymic medulla might provide inductive signals for T cell maturation and differentiation.

Total PHB expression is significantly increased in the thymus of pregnant rats compared with nonpregnant rats of the same age group. Recently, PHB was identified as a potential regulator of growth arrest and tumor suppression (36). Our results demonstrate that increased PHB expression within the regressing pregnant thymus may be one of the mechanisms regulating growth arrest and involution of thymus. Using two-dimensional gel electrophoresis, we observed a strong increase in the basic and acidic isoform of PHB in involuting pregnant rat thymus upon comparison with nonpregnant rats. Increase in thymic function after GnRH-Ag treatment was accompanied by a clear decrease in basic isoform. An increase in the acidic isoform of PHB was observed in GnRH-treated pregnant rat thymus compared with untreated pregnant and nonpregnant. These data suggest that PHB isoform expression in thymus is hormonally regulated perhaps via the posttranslational modification of the PHB protein. Whereas the exact functional relevance of this phenomenon warrants further detailed experimentation, the increase in the basic isoform of PHB in involuting pregnant thymus and its down-regulation during GnRH-mediated attenuation of thymic involution suggests that PHB may be playing an active role in thymic regeneration.

Spatial distribution of PHB was observed in the cortex, as well as the medulla; however, localization of PHB was more pronounced in the medullary regions of the thymus. The observed increase in PHB localization in the medulla indicates that PHB expression might be important in the maturation of developing T cells. These results were confirmed by double-labeling PHB with PCNA. PCNA is a 34-kDa intranuclear protein whose expression increases in the late G1 to S phases of the cell cycle immediately before DNA synthesis (37). Furthermore, PCNA functions as an auxiliary protein of DNA polymerase-{delta} and plays essential role in DNA replication and repair (38, 39, 40). Consistent with the functional role of PCNA, we observed a large number of PCNA+ cells in thymic cortex and markedly low PCNA labeling in thymic medulla. Interestingly, there was a reduced to undetectable localization of PHB in PCNA+ thymocytes, whereas higher PHB labeling in thymic medulla was associated with much fewer proliferating thymocytes. Our data on thymus support the current idea that PHB functions as an inhibitor of cell proliferation (20, 21, 22, 23). However, PHB was not totally excluded from PCNA+ cells as certain thymocytes demonstrated both PHB and PCNA colocalization. Coexpression of these markers may suggest that a mechanism to control cell cycle in some cells in late G1 phase exists as was demonstrated by recent studies showing that certain cell cycle proteins (e.g. P21, cyclin D, and Gadd45) bind to PCNA and regulate cell cycle progression and DNA replication (41, 42, 43). Additional studies revealed no preferential expression of PHB occurs in CD4+ or CD8+ cells (data not shown).

Recently, it has been proposed that PHB may function as a molecular chaperone (44). Because T cell maturation is associated with large increases in synthesis of proteins required to mount an effective immune response against foreign antigens, the presence of PHB in mature cells may be required for the stabilization of these proteins. We observed a characteristic polarized expression of PHB in thymocytes. Immune cells differ from most other cells because they show a typical pattern of polarization and aggregation of signaling proteins in the cholesterol-rich microdomains called lipid rafts (45). As immune cells are activated, they aggregate the proteins in lipid rafts to facilitate the signaling. The capping of receptors and signal molecules, such as chemokine and cytokine receptors in rafts, is a major focus of immunological signal transduction research. It has been proposed that proteins having lipid moieties at N terminus have preferential aggregation in cholesterol and spingolipid rich microdomains (46, 47). A plausible and currently untested hypothesis is because PHB is palmitoylated at its N terminus (48) and as a result is able to preferentially localize in lipid rafts. This hypothesis is further supported by a recent report describing a hydrophobic stretch and palmitoylation in PHB homology domain imparting raft association to Flotillin protein, a ubiquitous marker of lipid-raft domains considered important for insulin signaling and neuronal regeneration (48). This suggests and supports the role for PHB in cell signaling and as a molecular chaperone (44) responsible for preventing misfolding of wide repertoire of proteins aggregating in lipid rafts in immune cells.

Our data on rat thymocytes with increased expression in thymic medulla is in agreement with the previous report that showed increases in PHB expression being associated with ovarian follicular maturation, granulosa cell differentiation, as well as phorbol ester-treated chronic leukemic B-lymphocyte maturation (15, 21) To evaluate whether GnRH and PHB are important in the maturation of lymphocytes, we isolated mature T cells from spleen and demonstrated that, like mature thymic cells, PHB and GnRH are also colocalized in mature lymphocytes. PHB may also function in the maturation and differentiation of other immune cells. Recently, using microarray analyses, it was reported that differentiation of human naïve T cells into memory phenotype is associated with greater than 2-fold change in PHB gene (48). Similarly, monocytes induced to differentiate into dendritic cells also show more than 2-fold increase in PHB on the array; however, confirmation of microarray data at protein level was not performed in these studies and awaits further analysis (49).

In summary, we report that expression of PHB is increased in involuted pregnant thymi and undergoes posttranslational modifications during thymic regeneration by GnRH. Our current studies support an active role for PHB and GnRH in the thymus and T cell maturation and that these proteins might be a target for stimulation of thymic function.


   Acknowledgments
 
We thank Drs. Firyal Khan-Dawood, Melissa Green, Kelwyn Thomas, and Dzung H. Nguyen for their critique and valuable comments.


   Footnotes
 
This investigation was supported, in part, by the National Aeronautics and Space Administration (NASA) Grant NAG9-963 (to R.S.), NIH Grants GM-08248 (to R.S., W.E.T.), HD-41749 (to R.S. and W.E.T.), and NCI-P50-CA83591 SPORE in Ovarian Cancer (to W.E.T.). M.A.E., a student from the University of Houston, was supported for summer research at the Morehouse School of Medicine by NCC9-112 from NASA.

This work was presented at the 35th Annual Meeting of the Society for the Study of Reproduction in Baltimore, MD, July 27–31, 2002 (Abstracts 335, 336).

Abbreviations: DAPI, 4'6-Diamidino-2-phenylindole dihydrochloride; GnRH-Ag, GnRH agonist; GnRH-R, GnRH receptor; GPCR, G protein-coupled receptors; PCNA, proliferating cell nuclear antigen; PHB, prohibitin.

Received September 10, 2002.

Accepted for publication December 12, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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