Endocrinology, doi:10.1210/en.2003-0512
Endocrinology Vol. 145, No. 1 337-342
Copyright © 2004 by The Endocrine Society
Modulation of Diabetes with Gonadotropin-Releasing Hormone Antagonists in the Nonobese Mouse Model of Autoimmune Diabetes
Mansoor A. Ansari,
Minati Dhar,
Sreelatha Spieker,
Noshaba Bakht,
Ayaz M. Rahman,
Wayne V. Moore and
Jill D. Jacobson
Section of Endocrinology, Children’s Mercy Hospital, Kansas City, Missouri 64108
Address all correspondence and requests for reprints to: Jill D. Jacobson, M.D, Section of Endocrinology, Children’s Mercy Hospital, 2401 Gillham Road, Kansas City, Missouri 64108. E-mail: jjacobson{at}cmh.edu.
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Abstract
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The nonobese mouse model of autoimmune diabetes (NOD mouse) exhibits a strain-dependent preponderance of disease in females. Castration of male NOD mice leads to an increased incidence of diabetes, suggesting that testosterone directly modulates the expression of diabetes in the NOD mouse. However, castration also modulates hypothalamic and pituitary hormone production via removal of the negative feedback effects of testosterone. One hypothalamic hormone with immunomodulatory properties whose expression is increased by castration is GnRH. To test whether the increased incidence of diabetes in castrated male NOD mice is related to an increase in GnRH activity, we treated castrated male NOD mice with Antide, a GnRH receptor antagonist, to determine the effect on the incidence and timing of onset of diabetes. The prevalence of diabetes at 40 wk of age in male NOD mice was 50% in sham-operated mice, compared with an 83% prevalence in castrated males. Antide administration prevented the increased incidence of diabetes in the castrated male mice. Antide reduced total serum IgG levels, IL-6 cytokine expression in cultured splenocytes, and the lymphocytic infiltration of islets. GnRH administration exerted reciprocal effects, leading to earlier timing of onset of diabetes and increases in serum total IgG levels. We conclude that GnRH modulates the expression of diabetes in the NOD mouse independently of gonadal steroids.
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Introduction
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SEVERAL AUTOIMMUNE DISEASES display a definite female gender bias. Although this does not appear to be the case with autoimmune diabetes in humans, females do display an increased incidence of autoimmune antibodies (1). An important animal model of autoimmune diabetes, the nonobese mouse model of autoimmune diabetes (NOD), displays a strong gender bias for overt diabetes that varies with the strain.
The exact roles of gonadal steroids in autoimmune diseases remain unclear, even though the gender bias suggests a prominent role. Several studies in experimental models have shown that gonadectomy modifies the expression of autoimmune diseases, including diabetes (2, 3, 4, 5). Historically, investigators have attributed the immune effects of gonadectomy to alterations in gonadal steroids. However, gonadectomy or administration of gonadal steroids also dramatically alters feedback effects on hypothalamic hormones. One hypothalamic hormone with immunomodulatory properties is GnRH. GnRH agonists prevent involution of the thymus that normally occurs with aging in the rat (6). GnRH agonist administration leads to increased B and T cell proliferative responses and increased numbers of T lymphocytes expressing the IL-2 receptor in rats (7, 8). Moreover, spleen and thymus preparations have been shown to contain mRNA for GnRH and produce an immunoreactive GnRH (9, 10). A recent study (11) demonstrated that lymphocytic GnRH production increases when T cells are activated by mitogen in vitro. Using a murine model of systemic lupus erythematosus (SLE), we previously demonstrated that various GnRH receptor antagonists ameliorate disease severity independently of gonadal steroids. Conversely, administration of native GnRH exacerbated lupus independently of gonadal steroids.
Based on the findings that 1) GnRH exacerbates autoimmunity in mice, 2) castration of male mice leads to an increase in GnRH action, and 3) castration of male NOD mice leads to increased incidence of diabetes, we determined the effect of GnRH agonists and antagonists on expression of autoimmune diabetes and parameters of immune responsiveness in castrated male NOD mice.
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Materials and Methods
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Mice
Male NOD/LtJ mice were purchased from the Jackson Laboratory (Bar Harbor, ME) and maintained in our animal facility. Overt diabetes occurs in about 40–60% of males and 90–100% of females mice by 30 wk in this strain (11, 12). All experiments were carried out in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals and the University of Missouri-Kansas City Animal Care and Use Committee.
Mice were gonadectomized via a single scrotal incision after sodium pentobarbital anesthesia (50 mg/kg body weight) as previously described (12). Sham-operated males underwent pentobarbital anesthesia, scrotal incisions, and suture placement.
Experimental design
Male NOD mice underwent gonadectomy or sham gonadectomy at 14–18 d of age. Gonadectomy was confirmed by measurements of serum testosterone at 4 wk of treatment, serum LH at 4-wk intervals and seeking residual gonadal tissue at necropsy. Gonadectomized animals were randomly placed into one of three treatment groups: GnRH, Antide, or vehicle on the day of gonadectomy at 14–18 d of age. A random table of numbers was used for randomization. Sham-operated mice were treated with vehicle.
Serum glucoses were checked weekly using a One Touch Fast Take meter (Lifescan, Milpitas, CA). Urine was tested for glucose weekly by urinalysis reagent strips (Miles, Inc., Elkhart, IN). Animals were considered diabetic if the serum glucose was more than 13.8 mmol/liter (>250 mg/dl), and the urine glucose was more than 27.7 mmol/liter (>500 mg/dl).
Sera collected during isoflurane anesthesia at 4-wk intervals were stored at -20 C. Samples from all time points were run in the same assay to avoid interassay variability.
In an additional set of animals, gonadectomized males were treated with either antagonist or vehicle. Five mice per treatment group were euthanized at 3, 5, 7, 9, and 11 wk of treatment, and the pancreas was removed for histological examination.
GnRH and Antide administration
GnRH (native decapeptide) was purchased from Bachem (Bubendorf, Switzerland). GnRH antagonist, Antide (acetyl-ß-[2-naphthyl]-D-Ala-D-p-chloro-phe-ß-[3-pyridyl]-D-ala-ser-N
-[nicotinoyl]-lys-N-[nicotinoyl]-D-lys-leu-N-[isopropyl]-lys-pro-D-ala-NH2, was supplied by Ares-Serono (Randolph, MA) or purchased from Bachem. Animals were injected sc in the nape of the neck six times weekly, in the morning, with 100 µg GnRH in 50% normal saline in distilled water or 100 µg GnRH antagonist in 50% propylene glycol in sterile distilled water. We previously demonstrated that this GnRH dosing regimen leads to an exacerbation of murine lupus. Two separate groups of gonadectomized, vehicle-treated mice were used to exclude effects of vehicle on disease severity: One group was treated with 50% normal saline in water, and one was treated with 50% propylene glycol in water as above. Sham-operated control mice received injections in water/propylene glycol. All injections were in 100-µl volumes.
Hormone, IgG, and cytokine measurements
Serum testosterone concentrations were measured by RIA using a commercial kit for rat testosterone (Coat-A-Count, Diagnostic Products Corp., Los Angeles, CA). The lower limits of detection were 0.17 nmol/liter. Serum LH and prolactin were measured by RIA using previously described methods (13, 14). The lower limit of detection was 0.07 ng/ml. LH levels are expressed in terms of NIADDK-rLH RP-2 standard.
Total IgG concentrations were measured by single radial immunodiffusion assay using immunodiffusion plates containing monospecific antiserum for IgG (ICN Biomedicals, Inc., Costa Mesa, CA).
Splenic mononuclear cells were isolated using Ficoll Hypaque density gradients and cocultured with vehicle, GnRH (10-7 M), or Antide (10-7 M) for 24 h. Cells were pelleted by centrifugation, and the supernatant was harvested for measurement of IL-6 using a standard enzyme immunoassay for mouse IL-6 (OptEIA, PharMingen, San Diego, CA) and interferon-
(IFN-) using an ELISA kit (Amersham Pharmacia Biotech, Piscataway, NJ).
Histology
Sections (5 µm) of pancreas imbedded in paraffin were stained with hematoxylin and eosin. The frequency and severity of the insulitis were determined by a blinded observer (W.V.M.) with experience in detecting and judging severity of the insulitis. Frequency was reported as the percentage of islets showing signs of insulitis in 10 fields at x200 magnification. Severity of the insulitis was graded according to the following scale: 0, no mononuclear cells present; 1, peripheral accumulation of mononuclear cells; 2, infiltration of islet parenchyma with mononuclear cells; 3, complete infiltration of the islet with mononuclear cells; and 4, contracted islet with residual glucagon-positive cells.
Statistics
Data are expressed as mean ± SE. Serum immunoglobulin measurements were compared by two-tailed Student’s paired t tests. The effect of Antide on prevention of diabetes was compared by survival curves. The statistical significance of the difference between survival curves was determined by the log rank test that is equivalent to the Mantel-Haenszel test (15).
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Results
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Incidence and timing of onset of diabetes
In sham-operated/vehicle-treated and gonadectomized/Antide-treated NOD male mice, about 50% of the mice developed diabetes by 60 wk of treatment (Fig. 1
). Antide treatment significantly decreased the incidence of diabetes, compared with vehicle in gonadectomized mice (P < 0.05). Antide treatment of gonadectomized mice led to diabetes-free survival similar to sham-operated controls. Sham-operated/vehicle-treated mice exhibited a significantly decreased incidence of diabetes, compared with gonadectomized/vehicle-treated mice. In comparison, all of the gonadectomized/GnRH-treated mice had developed diabetes by 37 wk of treatment (P < 0.05, compared with gonadectomized/vehicle-treated animals). There were no differences in the incidence or timing of onset of diabetes between the two vehicle-treated groups.
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FIG. 1. Effect of GnRH receptor agonist, vehicle, or GnRH receptor antagonist on the timing and incidence of diabetes in intact and gonadectomized male NOD mice (sham-operated/vehicle, n = 8; gonadectomized/vehicle, n = 26; gonadectomized/Antide, n = 20; gonadectomized/GnRH, n = 10). Antide treatment significantly decreased the incidence of diabetes, compared with vehicle in gonadectomized mice (P < 0.05). Antide treatment of gonadectomized mice led to diabetes-free survival similar to sham-operated controls. Sham-operated/vehicle-treated mice exhibited a significantly decreased incidence of diabetes, compared with gonadectomized/vehicle-treated mice. There was an increased incidence of diabetes with GnRH (P < 0.05). *, Significantly lower than gonadectomized/vehicle-treated mice. **, Significantly higher than gonadectomized/vehicle-treated mice.
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Confirmation of gonadectomy and hormonal effects of GnRH and Antide
The mean serum testosterone level was 11.6 ± 3.3 nmol/liter in sham-operated mice at 7 wk of treatment. All testosterone levels were less than 0.17 nmol/liter in gonadectomized mice at the same time point.
GnRH-treated, gonadectomized mice displayed a 4-fold increase in serum LH levels, compared with vehicle after 4 wk of treatment (4.6 ± 0.01 vs. 1.08 ± 0.64; P = 0.02). LH levels were undetectable in all except one of the Antide-treated mice (Fig. 2; P < 0.05 at all time points).
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FIG. 2. Serum LH levels measured by RIA reveal that gonadectomized/Antide-treated mice display significant reductions in LH levels, compared with gonadectomized/vehicle-treated mice at all time points tested. Results are mean ± SE (n = 3–4 mice/group; P < 0.05 at all time points). *, Significantly different from gonadectomized/vehicle-treated mice.
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Histology
In the separate set of animals used to study the histology of the insulitis, gonadectomized/Antide-treated mice exhibited a lower frequency of infiltrated islets, compared with gonadectomized/vehicle-treated mice. Figure 3, A and B, shows representative histologic sections. Figure 3C shows a graphic representation of percentages of islets infiltrated at various time points (P < 0.05; n = 10 sections/time point). Figure 3D shows a graphic representation of percentages of the islet histology score at various time points (P < 0.05; n = 10 sections/time point).
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FIG. 3. Effect of vehicle or GnRH receptor antagonist on islet cell lymphocytic infiltrates in gonadectomized male NOD mice. A, A representative histologic section from a gonadectomized/vehicle-treated mouse is shown. B, A representative histologic section from a gonadectomized/antagonist-treated mouse is shown. C, Graphic representation of the percentages of islets infiltrated at various time points. At least 10 tissue sections from at least three mice in each treatment were counted. Results are mean ± SE. *, Significantly different from gonadectomized/vehicle-treated mice (n = 10; P < 0.05). D, Graphic representation of the islet histology score at various time points. At least 10 tissue sections from at least three mice in each treatment were counted. Results are mean ± SE. *, Significantly different from gonadectomized/vehicle-treated mice (n = 10; P < 0.05).
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IL-6 production by cultured splenocytes
In vitro exposure of isolated splenic mononuclear cells to Antide significantly suppressed IL-6 levels in the supernatant (P = 0.018; Fig. 4). Supernatants of splenocytes exposed to GnRH displayed a trend toward increased IL-6 levels, but the increase was not statistically significant (data not shown).
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FIG. 4. Effect of in vitro exposure of spleen cells from male NOD mice to vehicle and GnRH receptor antagonists on IL-6 production. Results are mean ± SE. GnRH antagonist exposure significantly reduces IL-6 production in the supernatant (n = 6; P < 0.05). *, Significantly different from vehicle-exposed cells. **, Significantly different from male, vehicle-exposed cells.
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IFN- production by cultured splenocytes
In vitro exposure of isolated splenic mononuclear cells to Antide significantly suppressed IFN- levels in the supernatant (P = 0.018; Fig. 5).
Serum IgG concentrations
The serum total IgG levels were significantly increased in gonadectomized/GnRH-treated mice, compared with the gonadectomized/vehicle-treated mice at 8 wk of treatment (P < 0.05). Sham-operated mice displayed significantly lower IgG levels than gonadectomized mice (P < 0.05; Fig. 6). Gonadectomized/Antide-treated mice exhibited the lowest serum IgG concentrations (P < 0.05, compared with gonadectomized/vehicle-treated mice). Serum IgG levels remained elevated in the gonadectomized/GnRH-treated mice and suppressed in the gonadectomized/Antide-treated mice, even at 24 wk of treatment.
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FIG. 6. Effect of GnRH, vehicle, or GnRH receptor antagonist on serum IgG levels in intact and gonadectomized male NOD mice (sham-operated/vehicle, n = 8; gonadectomized/vehicle, n = 26; gonadectomized/Antide, n = 20). Results are mean ± SE. *, Significantly different from gonadectomized/vehicle-treated mice (P < 0.05).
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Discussion
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One aim of the present study was to determine whether a reduction in GnRH activity might prevent autoimmune diabetes in NOD mice. In fact, administration of the GnRH antagonist Antide was associated with both delayed timing of onset and reduced incidence of diabetes in gonadectomized mice. In contrast, GnRH agonist administration led to an accelerating timing and an increased incidence of diabetes, associated with a transient increase in serum LH levels.
A second aim was to determine whether GnRH receptor antagonism would eliminate the increased incidence of diabetes known to be associated with castration. Various autoimmune diseases including murine SLE and diabetes are associated with an exacerbation of disease severity in males after gonadectomy (2, 4, 5, 16). The exacerbation associated with castration has been attributed to loss of the immunosuppressive effects of androgen. However, studies in many animal models including the NOD mouse have failed to show a direct protective effect of testosterone on expression of disease (3, 12). We hypothesized that the immunoprotective effects of androgens may be mediated by negative feedback effects on GnRH production and action. This hypothesis is based on the observations that GnRH is immunostimulatory and that androgens negatively regulate both GnRH production and action (18, 19, 20). Indeed, in the present study, gonadectomized/Antide-treated mice displayed reduced incidence of disease, compared with gonadectomized controls. Thus, gonadectomy does not increase the incidence of diabetes when GnRH action is blocked. Our data support the hypothesis that increased GnRH, rather than loss of testosterone, contributes to the increased incidence of diabetes associated with castration in the NOD mouse.
In these experiments, gonadectomized mice were used to eliminate the actions of GnRH on gonadal steroid production and eliminate gonadal steroid feedback effects on GnRH release. This allowed us to assess a direct role of GnRH in modulating autoimmune diabetes in the NOD mouse. A limitation of the current study was that these experiments did not address the possible effect of gonadotropins (LH and FSH) on expression of diabetes. Although numerous lines of evidence demonstrate that prolactin exerts actions in immunity and autoimmunity (21, 22), there is little evidence for immunomodulatory effects of gonadotropins. Although immune cells express prolactin receptors (21), no studies have confirmed the presence of gonadotropin receptors in immune cells.
A potential limitation of the GnRH agonist treatment arm of the study is that GnRH agonists exert biphasic effects: Agonists and superagonists are known to cause an initial stimulation of the GnRH receptor followed by a down-regulation of the GnRH receptor at the level of the pituitary. We have previously demonstrated that GnRH agonists exert similar biphasic effects at the level of the immune system. Acute (24 h) exposure of splenic mononuclear cells to either GnRH leads to an increase in GnRH receptor (23). On the other hand, chronic in vivo exposure to GnRH leads to down-regulation of the GnRH receptor in both thymic and splenic mononuclear cells (24). Thus, the use of GnRH agonists in studies of immunity is potentially confounded by these biphasic effects because an initial stimulation of the immune system might set off a cascade of immunological events that is not reversible. We speculate that the initial stimulation achieved with GnRH agonist in this study was sufficient to trigger a cascade of events that culminated in ß-cell damage. In our previous studies using GnRH agonist in murine lupus, we demonstrated an early significant increase in disease severity (at 6–12 wk of treatment) followed by a later significant reduction in disease severity at 24 wk (24).
GnRH is known to exert an autopriming effect (i.e. it augments its own action at the level of the pituitary). We observed similar effects of GnRH agonists in the immune system. We recently demonstrated that GnRH induces its own signal transducer in immune cells in mice (24). This is one mechanism whereby GnRH agonist administration may induce a cascade of immunological events.
Possible mechanisms for the observed protective effects of GnRH antagonists include effects on cellular immunity, humoral immunity, or cytokine production by immune cells. GnRH is known to augment both B and T cell proliferation in vivo and in vitro. It increases serum IgG levels in mice and increases expression of the IL-2 receptor in rats (6, 7, 8, 12). Diabetes in the NOD mouse is thought to involve both B and T cell components. Herein we demonstrated that GnRH antagonists alter humoral immunity as demonstrated by decreased IgG levels and decreases in the T helper (TH2) cytokine IL-6. This is in agreement with our previous studies demonstrating that GnRH antagonists ameliorate murine lupus in association with a reduction in B cell numbers (12). GnRH antagonists also lowered the TH1 cytokine IFN-.
It is interesting to note that although GnRH antagonist exposure effectively reduced both IFN- (a TH1 cytokine) and IL-6 (a TH2 cytokine), IL-6 levels were strikingly different between the sexes even in the absence of GnRH, with female cells expressing more. This confirms a previous report that IL-6 levels display a sexual dimorphism in the NOD mouse (25). We note that IL-6 is thought to play an important role in autoimmune diseases that show a female predominance, including lupus and the NOD model of diabetes (26, 27, 28). We note that those autoimmune diseases that display prominent gender differences are characterized by a strong humoral or B cell component. The NOD mouse model displays a striking gender differences, and B cells have been shown to be essential for disease pathogenesis (29, 30). SLE is an autoimmune disease with one of the highest female to male ratios, and it is characterized by polyclonal B cell activation. Type 1 diabetes in humans, on the other hand, is characterized by a strong T cell component and displays no gender differences. Because GnRH production and action are augmented in females, compared with males, we speculate that GnRH may play a greater role in those autoimmune diseases with the larger gender differences. GnRH may exert its immune actions via TH2 cytokines such as IL-6.
Taken together, our data suggest that GnRH antagonists modulate the expression of murine diabetes independently of their effects on the gonads. These findings point to a need for further understanding of the roles of hypothalamic and pituitary hormones, especially in relationship to cytokine production. These studies raise the possibility of new therapeutic interventions for autoimmune diseases.
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Acknowledgments
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GnRH antagonist Antide was supplied in part for these studies by the Ares-Serono Group.
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Footnotes
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This work was supported by NIH Grants 1R29AR43152 and 1R01AI50878 and a grant from the Paul Henson Trust. The LH assay was supported by National Institute of Child Health and Human Development/NIH through a cooperative agreement (U54 HD28934) as part of the Specialized Cooperative Centers Program in Reproduction Research.
Abbreviations: IFN-, Interferon-; NOD, nonobese mouse model of autoimmune diabetes; SLE, systemic lupus erythematosus; TH, T helper.
Received April 23, 2003.
Accepted for publication August 21, 2003.
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Abstract
Introduction
