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Section of Endocrinology, Children’s Mercy Hospital, University of Missouri-Kansas City School of Medicine and the University of Missouri Kansas City School of Pharmacy, Kansas City, Missouri 64108
Address all correspondence and requests for reprints to: Jill D. Jacobson, M.D., Professor, University of Missouri-Kansas City School of Medicine, 2401 Gillham Road, Kansas City, Missouri 64108. E-mail: jjacobson{at}cmh.edu.
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Abstract |
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Introduction |
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Many of the studies establishing effects of steroids on the immune system have been performed in vivo, in which alterations of gonadal steroids alter feedback on a variety of central nervous system peptides, many of which are now known to be immunomodulatory. Yet, historically, these feedback effects have not been taken into account. One hypothalamic hormone with immunostimulatory properties is GnRH (8). Thymic and splenic lymphocytes produce immunoreactive and bioactive GnRH and express GnRH receptors (9, 10, 11, 12). Studies in rodents demonstrate that GnRH exerts stimulatory influences on expression of the IL-2 receptor, on B and T lymphocyte proliferation, and on serum IgG levels (13, 14, 15, 16, 17). We have previously shown that GnRH receptor antagonists ameliorate murine systemic lupus erythematosus (SLE) and that GnRH receptor agonists exacerbate disease.
GnRH is exquisitely sensitive to feedback effects of androgens and estrogens. Rising estradiol increases GnRH production at the level of the hypothalamus and GnRH action at the level of the pituitary (18, 19, 20, 21, 22, 23), whereas androgens suppress both GnRH production and responsiveness at the levels of the hypothalamus and pituitary (24, 25, 26, 27, 28, 29, 30). Our primary hypothesis in this study is that the immunostimulatory effects of estrogens may relate to their positive feedback effects on GnRH production and/or action and that the immunosuppressive effects of androgens may relate to their negative feedback effects on GnRH production and/or action at the level of the immune system.
We used the GnRH-deficient HPG/Bm mouse in an effort to disentangle the immune effects of estrogens from the immune effects of GnRH. The HPG/Bm mouse is characterized by a truncating mutation in the GnRH gene, resulting in lack of production of GnRH as well as GnRH-associated peptide (GAP) (31). We quantitated GnRH receptor mRNA and GnRH receptor binding in lymphoid organs from GnRH-sufficient and GnRH-deficient mice. We administered gonadal steroids and GnRH to gonadectomized GnRH-deficient HPG/Bm mice and their GnRH-sufficient littermates and then assessed B and T lymphocyte proliferation as well as serial serum IgG levels.
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Materials and Methods |
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Genotyping
We genotyped HPG/Bm mice using a previously described technique (32). A 0.5- to 1-mm ear punch was placed in 200 µl of a PCR-compatible buffer with 10 µl proteinase K (10 mg/ml). This sample was incubated at 55 C for 1 h and then heated to 98 C for 10 min. The sample was centrifuged at 12,000 x g for 10 min. We used published primer sequences to distinguish homozygous HPG/Bm mice from heterozygous (HPG/Bm-GnRH hpg/+) mice. These primers recognize regions flanking the deletional mutation in HPG/Bm mice. PCR was performed in 50 µl, using a 28-cycle regimen (95 C, 2 min; 95 C, 45 sec; 60 C, 45 sec; 72 C, 90 sec x 20 cycles; 95 C, 45 sec; 60 C, 45 sec; 72 C, 2 min x 8 cycles).
Experimental design
Gonadectomized animals were randomized at 14–21 d of age and begun immediately in one of the following treatment groups: vehicle injections + placebo pellets, vehicle injections + estradiol pellets, vehicle injections + testosterone, dehydroepiandrosterone (DHEA), or dihydrotestosterone pellets, GnRH agonist injections + placebo pellets. Blood was obtained at weekly intervals for measurement of IgG.
Gonadectomy
For IgG studies males were gonadectomized via a single scrotal incision, and females were gonadectomized via bilateral flank incisions between 14 and 21 d of age under pentobarbital anesthesia. For proliferation experiments, animals were gonadectomized between 6 and 10 wk of age.
Pellets
Estrogen, in the form of 2.5 mg 17 ß-estradiol pellets, and androgen, in the forms of 25-mg testosterone pellets, 15 mg 5
-dihydrotestosterone pellets, and 25 mg DHEA pellets, were obtained from Innovative Research of America (Sarasota, FL). These pellet hormone delivery systems have been demonstrated to provide steady high physiologic levels of steroids for the duration of the implantation (33, 34). Either 21- or 90-d release pellets were used. Pellets were implanted following pentobarbital anesthesia on the day of gonadectomy. For the long-term experiments, 90-d release pellets were used.
Injections
Animals were injected sc in the nape of the neck six times weekly, in the morning, with 100 µg GnRH (native decapeptide; Sigma Chemical Co., St. Louis, MO) or vehicle in 100 µl volume consisting of 50% normal saline and 50% double-distilled water. We have previously established that this daily GnRH regimen induces a quadrupling of serum LH levels compared with vehicle treatment in gonadectomized mice (35).
Sera
Sera were collected from blood obtained every 3 wk by retroorbital puncture after isoflurane anesthesia. Sera for anti-DNA antibody measurements were stored at -20 C, and all samples from each time point were run in the same assay in an effort to avoid interassay variability.
IgG levels
Total IgG concentrations were measured by single radial immunodiffusion assay using immunodiffusion plates containing monospecific antiserum for IgG (ICN Biomedicals, Inc., Costa Mesa, CA).
Mononuclear cell preparation
Splenic mononuclear cells were prepared as follows: After rapid collection under sterile conditions, spleens were placed in cold RPMI 1640 containing 5% charcoal-stripped, delipidated fetal calf serum supplemented with antibiotics (100 IU penicillin and 100 µg streptomycin). Cells were passed through a 40-µm nylon mesh to remove clumps of cells and connective tissue. Mononuclear cells were isolated by density centrifugation.
Proliferation assays
Mononuclear cells were washed twice, and cell suspensions were prepared in RPMI 1640 with 10% fetal calf serum to yield a final concentration of 4 x 105 cells/well in 96-well microtiter plates (Falcon, Becton Dickinson, Lincoln Park, NJ). Blastogenic assays were carried out with 0.2-ml cell suspensions in quadruplicate. Cell viability was determined by the use of trypan blue. Lipopolysaccharide was used at a final concentration of 10 µg/ml. Concanavalin A was used at a final concentration of 2.5 µg/ml. Control wells contained no mitogen. Plates were incubated for 48 h at 37 C in 5% CO2. Thymidine (1 µCi/well; 25 Ci/mmol) was added before an additional 24 h of incubation. Labeled DNA was collected on fiber filters using an automatic cell harvester. Radioactivity was determined by liquid scintillation spectrophotometry. Data were expressed as a 
cpm, the difference between the counts per minute of experimental wells and a set of wells without mitogen and as a stimulation index, the ratio of experimental wells to control wells without mitogen.
RNA purification
RNA was isolated as follows: Mice were euthanized by CO2 narcosis. Whole organs were individually removed, placed in 1 ml TRI reagent (Molecular Research Center, Inc., Cincinnati, OH), homogenized, and placed on dry ice. RNA was extracted with chloroform and centrifuged at 12,000 x g for 15 min. RNA was precipitated with isopropanol, centrifuged at 12,000 x g for 15 min, washed in 80% ethanol, and air dried for 1 h. The pellet was then resolubilized in 0.05% diethyl pyrocarbonate-treated H2O. RNA concentration was determined by spectrophotometry.
Reverse transcription (RT)
RT was performed by a previously described technique as follows (36): 5 µg RNA in a total volume of 22.5 µl were added to each tube containing 10 µl of 5 x RT buffer, 0.5 µl each of dATP, dGTP, dCTP, and deoxythymidine triphosphate (dTTP), 0.5 µl RNAsin, 2.0 µl of 60 ng/µl random hexamers (PerkinElmer, Boston, MA), 2 µl mouse murine leukemia virus reverse transcriptase (Invitrogen, Carlsbad, CA), in a total volume of 50 µl. The mixtures were heated to 22 C for 10 min, 42 C for 30 min, and 95 C for 5 min. Resultant cDNA was diluted 1:10 in 0.05% diethyl pyrocarbonate-treated double-distilled deionized water (ddH2O) and reheated to 95 C for 10 min. Control reaction tubes were identical except for the omission of reverse transcriptase in one set and RNA in another set.
PCR amplification
Using the published sequence of the murine GnRH receptor mRNA (37), we synthesized the following oligonucleotide primers for the purpose of amplifying mRNA by competitive RT-PCR.
Murine GnRH receptor primers were as follows: sense, 5'-GCTCTCAAGGATGAAGGTGCTT-3'; and antisense, 5'-CCAGGCTAATCACCACCATCAT-3'.
Because quantitation is of importance, we performed competitive PCR using a modification of a previously described technique (38). We used a mimic construction (Clontech, Palo Alto, CA) to construct a nonhomologous DNA fragment to be used as a competitive template for each gene. Briefly, a composite primer pair (below) was designed to include the original primer sequence (underlined) and cDNA complementary to the irrelevant cDNA provided in the mimic construction kit. The composite primers were used in primary PCR according to the manufacturer’s directions. The resultant primary PCR product was then amplified in a secondary reaction using the original primers. The resultant nonhomologous PCR product (competitor) was then quantitated by densitometry. The competitor was added to the experimental PCR (described below) at a concentration of 1.0–1.3 fg/tube.
Murine GnRH receptor composite primers were as follows: sense, 5'-GCTCTCAAGGATGAAGGTGCTTCAAGTTTCGTGAGCTGATTG-3'; and antisense, 5'-CCAGGCTAATCACCACCATCATATTTGATTCTGGACCATGGC-3').
The competitive fragment corresponding to the GnRH receptor is 323 bp in length and is easily distinguishable from the gene product band (198 bp). The above sets of primers were used to amplify both the target gene and nonhomologous DNA fragment in the same reaction tube.
We plotted the log of the density of product band against the cycle number to confirm that the target and competitor sequences amplified with similar efficiencies (39). We compared the amplification of the competitor and target. The linear portions of the product and competitor curves were parallel, indicating that the amplification efficiencies of the targets and the competitors were equal.
We used commercially available primers to measure ß-actin mRNA expression as a control (Clontech).
PCR was performed under intermediate stringency in the linear phase of amplification, by mixing 5 µl of the cDNA with PCR buffer, 1.75–2 mM MgC12, 0.2 mM deoxynucleotide triphosphates, 2–3 ng/ml sense and antisense primers, 2–5 U Taq polymerase, various concentrations of competitor, 5 µl cDNA, and 27 µl RNAase and DNAase-free H2O in a total volume of 50 µl. For radiolabeled PCR, 0.5 µl 32P- or 0.1 µl 33P-labeled dCTP was added to the reaction mixture. PCR cycles were programed as follows: GnRH receptor mRNA: 95 C x 2 min, 1 cycle; 95 C x 1 min, 65 C x 1 min, 35 cycles; 72 C x 7 min, 1 cycle; 4 C soak; ß-actin: 95 C x 2 min, 1 cycle; 95 C x 1 min, 60 C x 1 min, 72 C x 1 min, 35 cycles; 72 C x 7 min, 1 cycle; 4 C soak.
An aliquot (5 µl) of each PCR was subjected to electrophoresis through a 5% polyacrylamide gel. Product and competitor bands were quantitated using densitometry. The concentration of the target cDNA in the samples was deduced by determining the concentration of competitor at which the target and competitor product concentrations were equal.
Tissue preparation for GnRH receptor assays
For receptor studies, thymus, spleen, and pituitary glands were removed carefully, immediately placed on ice, cleaned, and weighed. All subsequent manipulations were performed at 4 C, as described (40). Thymuses and spleen were individually homogenized (1:30, wt/vol) in ice-cold 0.25 M sucrose and 50 mM Tris-HCl (pH 7.6) in a Dounce homogenizer, centrifuged twice at 800 x g for 5 min before centrifugation of the collected supernatants at 10,000 x g for 30 min. The 10,000 x g pellet were resuspended in assay buffer (30 mg tissue wt/ml) and used directly in the binding assay. Protein concentration was determined by Lowry et al. (41) using BSA as a standard.
Iodination of GnRH
Iodinated LHRH was obtained from Amersham Biosciences (Piscataway, NJ) and reconstituted in water to a specific activity of 100 µCi/ml.
GnRH receptor assay
GnRH receptor assay was carried out as previously described (13), in a total volume of 500 µl, for 90 min at 0–4 C. Assays were performed in triplicate in polypropylene test tubes presoaked overnight in 1% albumin. The tubes contained 200 µl buffer (40 mM Tris-HCl, 0.1% BSA, pH 7.6), 100 µl iodinated GnRH (
150,000–180,000 cpm), 100 µl unlabeled GnRH (10 µg/tube) or buffer, and 100 µl splenic membrane preparation. Bound hormone was separated after washing with 3 ml ice-cold Tris-HCl buffer (pH 7.6), and by centrifugation at 15,000 x g. The supernatants were aspirated, and the radioactivity contained in the pellet was counted in a 
-counter at 70% efficiency.
Statistical analysis
Overall differences in proliferation among various doses were analyzed by ANOVA. Intragroup analyses were conducted by the Wilcoxon signed-rank test and unpaired Student’s t tests.
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Results |
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shows a representative autoradiograph of three different genotypes. The full-length gene is 712 bases. The truncated gene is 586 bases in length.
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). Testosterone, DHEA, and dihydrotestosterone were equally ineffective in suppressing serum IgG levels regardless of the presence of GnRH (data for testosterone are shown). Similar results were seen in prepubertally gonadectomized male HPG/Bm mice (data not shown).
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is a representative autoradiograph). GnRH-deficient mice display a greater ratio of target: competitor for GnRH receptor mRNA, compared with GnRH-sufficient mice (P = 0.001) and heterozygous mice (P = 0.02; n = 8–10; Fig. 3B).
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. GnRH-deficient mice display a greater maximum binding (Bma) compared with GnRH-sufficient mice (P = 0.016).
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demonstrates that GnRH significantly increased B lymphocyte proliferative responses to mitogen, compared with vehicle in females (P < 0.05) but only in GnRH-sufficient mice. In contrast, testosterone significantly decreased T lymphocyte proliferation, compared with vehicle but again only in GnRH-sufficient mice (P < 0.05). Male GnRH-deficient, HPG/Bm mice displayed proliferation patterns identical with female GnRH-deficient mice (data for females are shown). GnRH alone increased B lymphocyte proliferation in GnRH sufficient mice. Surprisingly, it was ineffective in increasing proliferation in GnRH-deficient mice. The combination of GnRH and estradiol in GnRH-sufficient mice was associated with a high mortality and with decreased cell viability in the 72-h proliferation assays, but only in GnRH-sufficient mice. Cell viability after 72 h was 52.0 ± 8.8% in vehicle-treated vs. 15.9 ± 6.5% in GnRH plus estradiol-treated, gonadectomized, wild-type mice (P = 0.017). The combination of GnRH and estradiol was not associated with a high mortality nor was it associated with toxicity in the GnRH-deficient mice. In fact, the combination of GnRH and estradiol was associated with enhanced B cell proliferation compared with either compound alone in these mice.
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demonstrates that in vivo exposure to estrogen or GnRH significantly increased T lymphocyte proliferative response mitogen, compared with vehicle. In contrast, testosterone significantly decreased T lymphocyte proliferation, compared with vehicle (P < 0.05). Similar proliferation results were seen in male and female GnRH-deficient HPG/Bm mice (data for females are shown). As with B lymphocyte proliferation, the combination of GnRH and estradiol was associated with significant toxicity (see previous section). The combination of GnRH and estradiol was not associated with a high mortality nor was it associated with toxicity in the GnRH-deficient mice. In fact, the combination of GnRH and estradiol was associated with enhanced T cell proliferation compared with GnRH alone.
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Discussion |
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Both B and T lymphocyte proliferation were markedly attenuated in GnRH-deficient mice compared with GnRH-sufficient mice regardless of hormonal treatment or gonadectomy. These findings are consistent with a 1988 study, in which abnormalities in many aspects of thymic development in HPG/Bm mice were characterized (42). Thymic weights, thymic cellularity, and double staining (CD8+, CD4+) thymocyte numbers were higher in hypogonadal mice than in normal male mice but lower in hypogonadal mice than in normal females. When brain grafts containing GnRH neurons were used to correct reproductive deficits in hypogonadal mice, lymphocytic abnormalities were normalized, regardless of whether or not the grafts corrected the gonadal steroid status of the recipients, in agreement with GnRH’s known direct effects on the immune system (42).
The finding that GnRH receptor mRNA and GnRH receptor binding were up-regulated in immune cells from GnRH-deficient mice suggests that a regulatory process is in place in immune cells to compensate for GnRH deficiency. We have previously demonstrated that chronic exposure to GnRH suppresses GnRH receptor expression in immune cells (17).
Although GnRH did augment B lymphocyte proliferation in GnRH-sufficient mice, our regimen of in vivo administration of GnRH over a 2-wk course was not sufficient to stimulate B cell proliferation in GnRH-deficient mice. We speculate that the 2-wk duration of treatment may have been insufficient to augment B lymphocyte proliferation in mice not previously exposed to GnRH. This concept is supported by our finding that serum IgG levels were not yet significantly increased at 3 wk of treatment but were significantly increased at 6 wk in GnRH-deficient mice. The effects of GnRH on the pituitary are known to be dependent on repetitive pulsatile administration. GnRH is known to exert an autopriming effect at the level of the pituitary; i.e. it augments its own action. We have observed similar self-priming effects of GnRH in immune cells. We have recently demonstrated that both estrogen and GnRH are able to induce GnRH’s primary signal transducers, namely, the homologous G proteins, Gq and G11, in immune cells in mice (17, 43). We speculate that GnRH-deficient mice, which are deficient in both estrogen and GnRH, may have a relative insufficiency of these stimulatory G proteins. In fact, the combination of estrogen and GnRH was more effective than either compound in increasing B cell proliferation by the 2-wk time point in GnRH-deficient mice.
In the present study, we were unable to document an effect of androgens on serum IgG levels regardless of the presence of GnRH. Testosterone, 5-dihydrotestosterone, as well as with DHEA were equally ineffective in reducing serum IgG levels in all mice, regardless of genotype. On the other hand, testosterone administration clearly suppressed B cell proliferation in GnRH-sufficient mice. Importantly, androgens exerted no effect in the GnRH-deficient mice. The finding that male GnRH-deficient mice, which are deficient in testosterone as well as in GnRH, exhibited diminished immune responses compared with their GnRH-sufficient littermates would further argue against the common notion that androgen plays purely suppressive roles in the immune system.
GnRH-deficient mice are deficient in gonadotropins, GAP, and gonadal steroids as well as in GnRH (31). Thus, the immunosuppression seen in deficient mice suggests that GnRH and/or gonadotropins, and/or gonadal steroids, may be important modulators of lymphocyte function. GnRH stimulated both B and T lymphocyte proliferation as well as Ig levels in gonadectomized mice, suggesting that GnRH exerts effects independently of gonadal steroids. The current study does not enable us to exclude a direct effect of gonadotropins. However, little evidence exists for immunological actions for the gonadotropins or for the presence of gonadotropin receptors at the level of the immune system. Nor can we exclude an effect of GAP, which is also absent in these mice. However, no studies to date have attributed any immune function to GAP. On the other hand, numerous in vitro and in vivo studies have shown that GnRH possesses direct immunostimulatory properties (14, 44).
In contrast to the results with B cell function, the divergent effects of androgens and estrogens on T cell function did not appear to be dependent on the presence of GnRH. Estrogens exerted stimulatory effects and androgens exerted suppressive effects on T cell proliferative responses to mitogen even in the absence of GnRH. Still, T cell proliferation was much more exuberant in GnRH-sufficient mice than in GnRH-deficient mice.
Our data suggest that GnRH may play a greater role in B cell immunity than in T cell immunity. We note that those diseases that display the greatest female predominance (e.g. SLE and autoimmune thyroid disease) tend to display prominent B cell or humoral components. SLE is characterized by polyclonal B cell activation and displays a 10:1 female to male predominance. Autoimmune thyroid disease displays a 9:1 female to male ratio and has been characterized as having a predominant T helper 2 or humoral cytokine profile (45). Type 1 diabetes, on the other hand, shows no gender predisposition and is generally considered to be a predominantly T helper 1- or T cell-mediated disease and involves CD8-positive lymphocytes. We have previously reported that GnRH antagonists prevent lupus in a lupus-prone mouse model (15). The effects were present even in the presence of high dose estrogen (15). On the other hand, we have also reported that GnRH antagonists exert no effect on CD8 counts and no effect on the incidence or timing of onset of diabetes in a rat model of diabetes, the diabetes-prone BB rat (46).
The notion that immune actions of gonadal steroids may be mediated, at least in part, indirectly via feedback effects on hypothalamic and/or pituitary hormones may have implications for future immunoendocrine studies. If even some of the immune effects of androgens and estrogens are mediated indirectly via GnRH, then the effects of gonadectomy on immune function cannot be attributed directly to alterations in gonadal steroids. Even in vitro studies of androgens and estrogens on immune functions must be interpreted with caution, as GnRH is ubiquitously expressed in all immune cells.
Taken together, our results indicate that GnRH may play an important role in the gender differences in immune function. Further studies examining the effects of androgens and estrogens on GnRH production and action at the level of the lymphocyte and on the effects of GnRH on cytokine profiles might shed further light on gender differences in immunity and autoimmunity.
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Footnotes |
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Abbreviations: DHEA, Dehydroepiandrosterone; GAP, GnRH-associated peptide; RT, reverse transcription; SLE, systemic lupus erythematosus.
Received April 23, 2003.
Accepted for publication August 21, 2003.
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References |
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T3–1 cells. Mol Cell Endocrinol 87:95–103[CrossRef][Medline]
(q/11) expression in lymphoid organs. Neuroendocrinology 78:147–153[CrossRef][Medline]
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