Summary Our earlier studies have demonstrated that gonadotropin-releasing hormone (GnRH) agonists suppress immune system function in female mice.
Immunology and Cell Biology (1996) 74, 134-143
Gonadotropin-releasing hormone agonist influences absolute levels of lymphocyte subsets in vivo in male mice LV RAO,' RP CLEVELAND,2 RJ KIMMEL' and KM ATAYA" Departments of' Obstetrics and Gynecology and ^Pathology. MetroHealth Medical Center, Case Western Reserve University. Cleveland. Ohio. USA Summary Our earlier studies have demonstrated that gonadotropin-releasing hormone (GnRH) agonists suppress immune system function in female mice. No systematic studies regarding the effect of gender on immune system function following GnRH agonist treatment, however, have been reported. This study, therefore, investigated sequential changes in lymphocyte subsets in 3- and 10-week-oId male mice following agonist or placebo administration. Changes in immunophenotypic expression of lymphocytes from thymus, bone marrow, spleen, and blood were analysed at periodic intervals. Upon agonist administration, plasma testosterone levels were significantly increased in pre-pubertal mice, but were significantly decreased in post-pubertal males. Absolute thymic weights, thymocytes and T subsets were significantly increased from the third week regardless of gonadal status. Blood lymphocyte subsets showed a decreasing trend after agonist administration in pre-pubertal males, whereas no differences were observed in post-pubertal males. No significant differences were observed in spleen cells after agonist administration. These studies, together with earlier observations in female mice indicate that GnRH agonist effects on the immune system, are independent of steroid hormone levels. In contrast to suppressive effects in females, GnRH agonists induce no change or ultimately enhanced lymphocyte counts in males, indicating differential effects on the immune system between males and females. This may have important implications for the treatment of various diseases. Key words: immune system, Lupron depot, lymphocyte subsets, sexual dimorphism.
Introduction In recent years, gonadotropin-releasing hormone (GnRH) agonists have been increasingly used to down-regulate the pituitary and induce a state of hypogonadism. This form of endocrine manipulation has therapeutic value in a wide range of hormone-dependent diseases such as endometriosis and uterine fibroids. Administration of GnRH agonists has also resulted in the inhibition of growth of hormone-dependent tumours. These agents are indicated for the palliative treatment of many other conditions including advanced prostate cancer' and fibrocystic breast disease.^ Recent studies from our laboratory''"^ and others^ have indicated that GnRH agonists significantly affect immune system function both in vitro and in vivo. GnRH agonist treatment in vivo induces significant reductions in lymphocyte subsets in various lymphoid tissues of both pre- and post-pubertal female mice. In vivo cellmediated immune function was also suppressed under similar conditions in these animals. These observations raise concems regarding the use of these substances as therapeutic agents. While the mechanisms of the effectiveness of GnRH agonists in these conditions may partly be due to effects on the immune system, there may also be harmful effects. The precise mechanisms of action of Correspondence : Dr LV Rao, Division of Clinical Chemistry, Department of Pathology, John Sealy Hospital, University of Texas Medical Branch, Galveston, Texas 77555-0551, USA. Received 4 May 1995; accepted 25 October 1995.
GnRH agonists on the immune system, however, are unclear. Studies from our laboratory using female mice indicated a general suppression of leucocyte maturation upon GnRH agonist treatment in vivo and suggested a potential effect at an early stem cell stage during leucocyte development. No systematic studies on the effect of gender on GnRH agonist treatment in vivo on immune system functions, however, have been reported. There is abundant evidence indicating that females have enhanced immunologic responses over males.^-^ Gender and sex hormones have clear-cut effects on various hetero and autoimmune responses, but the mechanisms of action are still unknown.*^"" Recent observations indicated that GnRH might alter the severity of autoimmune diseases through mechanisms independent of their effects on gonadal hormones.'-' As an extension of our previous studies in female mice, the present study investigated sequential changes in functional lymphocyte subpopulations in both primary and secondary lymphoid tissues following a single i.m. injection of GnRH agonist in depot formulation in both pre- and post-pubertal male mice.
Materials and methods
Animals Three-week-old pre-pubertal {n = 50) and 10 week old postpubertal {n = 50) male BALB/c mice were obtained from Charles River
GnRH agonist and lymphocyte subsets in male mice
Laboratories (Wilmington, MA. USA). They were fed and housed at the Animal Resource Center of Case Western Reserve University at MetroHealth Medical Center under controlled temperature (20-22 °C) and light (12 h light/12 h dark). Mice of each age group were randomly divided into two subgroups of 25 mice each. One subgroup received a single 50 ng i.m. injection of GnRH agonist (Lupron depot, Leuprolide acetate. TAP Pharmaceuticals, Chicago, IL, USA). The depot formulation is a microcapsule injection which contains r>Iactic acid-glycolic acid as a biodegradable copolymer. The average daily release rate of the GnRH agonist was 2.5% of the administered dose over a period of 4 weeks and this release is primarily controlled by the hydrolysis of water.'^ This dose corresponds on a weight basis to that used for treatment of human malignancies. Microcapsules without the GnRH agonist were simultaneously administered to the placebo group. Both placebo (n= 5) and Lupron-trealed (n = 5) groups were killed by an overdose of Ketamine at 1, 2, 3, 4 and 6 week intervals. The period between 4 and 6 weeks represents a recovery phase in the injected animals. Peripheral blood was collected via cardiac puncture and anti-coagulated with EDTA. Blood samples were monitored for total while blood cells (WBC) counts, absolute numbers of lymphocytes, granulocytes and monocytes as well as plasma oestradiol levels. The thymus, femurs, spleen, tcstes and seminal vesicles were collected and the organs were weighed.
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para formaldehyde and stored at 4'C before examination by flow cytometry. The percentage of positive cells in each cell preparation was determined using a flow cytometer {FACScan, Becton Dickinson Immunocytometry Systems, San Jose, CA, USA) in standard manufacturer provided configuration. Autocomp software using Calibrite beads (Becton Dickinson) was used to standardize instrument electronics prior to each analysis. All analyses were performed with a Consort 30 computer (HP9000, Model 310, Hewlett Packard Co., North Hollywood. CA, USA) and appropriate software. The fluorescence data were expressed as dual parameter histograms of green (FITC) versus orange (PE) fluorescence. Four quadrant analyses, with markers set on the isotype controls, were used to determine the percentage of positive cells for each set of mAb.
Testosterone assays Plasma testosterone levels were determined by RIA using a kit supplied by ICN (Irvine, CA, USA). All samples were assayed in duplicate in one assay and the minimum detection limit for testosterone was 10 pg/mL.
Statistical analyses Preparation ofthymic. bone marrow, splenic and peripheral blood lymphocytes The thymus, spleen and two femurs were removed from each animal at the designated time and lymphocytes were obtained as earlier described.^ Liberated cells from all tissues were resuspended in HBSS containing 2% FCS at a concentration of one million cells per 50 ^L. Lymphocyte subset analyses of blood lymphocytes were performed on unseparated blood anticoagulated with EDTA. A 50 fiL whole blood sample from each mouse was incubated with individual mouse mAb for 30 min at room temperature in the dark. The cells were suspended in 2 mL of IX FACS lysing solution (Becton-Dickinson, Sanjose, CA, USA) to lyse erythrocytes and incubated at room temperature in the dark for 10 min. The samples were centrifuged, washed in PBS, fixed with 0.5% para form aldehyde and stored at 4'C prior to analyses.
Immitnophenotypic analyses by two-colour immunofluorescence and flow cytometry Immunofluorescent analyses of lymphocyte subpopulations were carried out using different mouse mAb and flow cytometry.^•'' Monoclonal antibodies conjugated with FITC or phycoerythrin (PE) were obtained from Pharmingen (San Diego, CA, USA). Irrelevant mouse monoclonal immunoglobulins conjugated with the fluorochrome were used as isotypic controls. The primary mouse mAb used to identify the different lymphocyte subsets were FITC-Thyl.2 (reacts primarily with T cells). F1TC-CD4 (T-helper), PE-CD45R (reacts with both pre-B and B cells) and PE-CD8 (T-suppressor/cytotoxic cells). Fifty microlitres of each cell preparation (IxlO*" cells) was incubated with individual FITC- or PE-labelled antibodies and controls for 30 min at room temperature in the dark. Combinations of FITC- and PEconjugated antibodies were used to produce double staining. The tubes were then centrifuged and washed in PBS, fixed in 0.5%
Differences between different treatment groups were evaluated by two way analysis of variance and Fisher's Protected Least Significant DifTerence (PLSD) method for multiple comparisons as appropriate.
Results Fffect of GnRH agonist on testis and seminal vesicle weights and plasma testosterone levels Figure I shows the changes in mean testis and seminal vesicle weights and testosterone levels at weekly intervals following a single i.m. injection of GnRH agonist or placebo in both pre- and post-pubertal male mice. In prepubertal mice both the mean testis and seminal vesicle weights showed no significant alterations following agonist administration. In post-pubertal mice, however, both of these measures showed a decreasing trend in the agonist-treated mice throughout the study period. The mean testis weight was significantly decreased in the second week and the seminal vesicle weight was significantly decreased in the fourth week. The mean plasma testosterone levels in post-pubertal male mice were significantly decreased from the second through the fourth week following GnRH agonist treatment. In contrast, plasma testosterone levels in pre-pubertal male mice were significantly elevated during the initial 2 weeks following agonist treatment but did not differ from the controls for the remainder of the study period.
Effect of GnRH agonist on thymus and thymic lymphocyte subpopulations Figure 2 shows the changes in mean absolute thymus weight and thymocyte counts at weekly intervals af\er a
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Post-pubertai
Pre-pubertal
Testosterone
Testosterone
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Testis weight
Testis weight 0.1C
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o.u 0.12 0.17 0.10 .J
Seminal vesicles weight
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Figure 1 Effect of GnRH agonist on sequential changes in plasma testosterone, testis and seminal vesicle weights in both pre- and postpubertal male mice. Mice were injected with either 50 )ig of placebo (—O—) or 50 Hg of Lupron depot {-•-) i.m. (week 0) and killed at 1, 2. 3. 4 and 6 week intervals. The values are represented as mean±s.e.m.,
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