Estradiol β Receptor Expression on Human B-lymphocytes in Response to

1. Estradiol β Receptor Expression on Human B-lymphocytes in Response to Acute Heavy Resistance Exercise Maren S. Fragala1,2, William J. Kraemer 2, Megan R. Wolf 2, Andrea M. Mastro3, Craig R. Denegar2, Jeff S. Volek2, Jay R. Hoffman 1 and Carl M. Maresh2 1 Human Performance Laboratory, University of Central Florida, Orlando, FL, 2 Human Performance Laboratory, University of Connecticut, Storrs, CT, 3 Department of Microbiology and Cell Biology, The Pennsylvania State University, University Park, PA ABSTRACT RESULTS RESULTS (CONT.) RESULTS (CONT.) METHODS Background: Estradiol has previously been attributed for mediating gender differences in physiological responses to tissue damage. Since the neuro-endocrine and immune systems coordinate the body’s response to such stress, we hypothesized that estradiol β-receptors (ER) on b-lymphocytes may coordinate such communications and explain the protective responses previously reported in women. Purpose: The purpose of this investigation was to examine ER expression on circulating B-lymphocytes in response to an acute bout of heavy resistance exercise in men and women. Methods:Using a within subject design, fifteen resistance trained women (n=7; age= 22±3 y) and men (n=8; age= 25±5 y) performed a heavy resistance exercise squat protocol (6 sets of 5 reps at 90% 1RM) (EX) and a control test (CON) in a balanced, randomized order. Blood samples were collected before, during and after the exercise and control trials. ER expression on circulating lymphocytes was evaluated with flow cytometryand serum estradiol was assayed by ELISA. Results:Serum estradiol did not significantly differ between men (CON=48.0±22.3 pg•ml-1) and women (CON=86.5 ± 60.5 pg•ml-1) nor change during recovery to the exercise stress in men (6 HR POST-EX = 49.7±13.6 pg•ml-1; 24 HR POST-EX=60.1 ± 15.1 pg•ml-1) or women (6 HR POST-EX = 73.9±35.8 pg•ml-1; 24 HR POST-EX=105.4 ± 66.9 pg•ml-1) . ER on b-lymphocytes showed large inter-individual variations before exercise (relative fluorescence = 22.3 - 28.8) and trends for differences at 6-hours post-exercise (relative fluorescence = 32.1 - 54.4). However, no significant gender differences or changes in response to the exercise protocol were observed. Conclusion:Present findings reveal that estradiol-β receptors on B-lymphocytes unlikely explain gender differences in physiological responses to tissue damage elicited by acute heavy resistance exercise. It is possible that interactions may occur beyond the recovery period measured in the present study or it may be that ER on muscle tissue, rather than B-lymphocytes, dictate the protective effects of estradiol previously reported in women. Figure 4.Circulating Estradiol (pg·mL-1) responses to heavy resistance exercise (squat protocol) (solid lines) and a control condition (dashed lines) in women (n=7) (◊) and men (n=8) (□). Data are presented as means + SEM. • Serum estradiol did not significantly differ between men (CON=48.0±22.3 pg•ml-1) and women (CON=86.5 ± 60.5 pg•ml-1) • Serum estradiol did not significantly change during recovery to the exercise stress in men (6 HR POST-EX = 49.7±13.6 pg•ml-1; 24 HR POST-EX=60.1 ± 15.1 pg•ml-1) or women (6 HR POST-EX = 73.9±35.8 pg•ml-1; 24 HR POST-EX=105.4 ± 66.9 pg•ml-1) . Study Protocol CONTROL RECOVERY or (1 week) 1- RM pre-ex 1 hr 6 hr 24 hr mid-ex post-ex Blood sample collection post-exercise 6 sets x 5 reps @ 90% of 1-RM • ER-βon B-lymphocytes showed large inter-individual variations before exercise (relative fluorescence = 22.3 – 28.8) and trends for differences at 6-hours post-exercise (relative fluorescence = 32.1 – 54.4). • No significant gender differences or changes in response to the exercise protocol were observed in ER-β on B-lymphocytes. • Resistance trained men (n=8) and women (n=7) completed two trials (exercise and control) under similar conditions in a matched, balanced and randomized order • 1-repetition maximum (1-RM) was determined with a Smith-machine barbell back squat • The exercise protocol was 6 sets of 5 repetitions at 90% of 1-RM with 3 min of rest • Women were eumenorrheic and were tested in the follicular phase of the menstrual cycle • Blood samples were obtained immediately before (pre), at the mid-point (mid), and immediately after the test (post), and at 1, 6, and 24-hrs into recovery. SUMMARY & CONCLUSIONS Figure 5.B-lymphocytes (CD19+) expressing estradiol receptors in response to heavy resistance exercise (squat protocol) (solid bars) and a control condition (dashed bars) in women (n=7) (red) and men (n=8) (blue). Data are percentages of cells positively stained for CD19+ and ER+. • Muscle damaging exercise protocols trigger an inflammatory response where chemo-attractive factors are released to recruit leukocytes to both “clean up” the damaged tissue and to activate satellite cells to repair the tissue • Despite the hypothesized protective influence of estradiol in tissue disruption, this study did not show any clear patterns or gender differences in estradiol-β receptor expression on B-lymphocytes in response to heavy resistance exercise. • Present findings reveal that estradiol-β receptors on B-lymphocytes unlikely explain gender differences in physiological responses to tissue damage elicited by acute heavy resistance exercise. Table 1.Participant Characteristics INTRODUCTION • Previous research has shown reduced tissue disruption and inflammatory responses in women as compared to men following acute strenuous exercise. • Gender differences have been attributed to the ‘protective’ effects of estradiol as an antioxidant and membrane stabilizer. ‡ ‡ Blood processing for analysis • The mechanisms of estradiol’s protective effects are not known, but may involve reduced inflammatory responses through its interaction with immune cells. • B-lymphocytes contain estradiol β receptors and estradiol is known to influence critical events in B lymphopoiesis • It is unknown how estradiol β receptor presence on immune cells responds to acute resistance exercise stress. • It is possible that interactions may occur beyond the recovery period measured in the present study or it may be that ER on muscle tissue, rather than B-lymphocytes, dictate the protective effects of estradiol previously reported in women. • It is also possible that the protective effects of estradiol on muscle damage are mediated predominantly by estradiol –α receptors, rather than the β receptors measured in the present study. • Figure 1. Neuro-endocrine-immune interactions occur via shared hormones, cytokines, and receptors *p <0.05 between men and women • Measures • Estradiol β receptors on circulating leukocytes were evaluated with flow cytometry • B-lymphocytes were labeled with a CD19 antibody conjugated to APC • Circulating 17 β- estradiol concentrations were assayed via enzyme-linked immunosorbent assay (ELISA) at pre-, +6HR, and +24HR • Although α and β receptors differ among tissue types with the β often attributed for immune modulation, recent results from animal models reveal that the α receptor is important following exercise-induced muscle damage • Despite the null findings, results have implications for understanding mechanisms underlying recovery, tissue damage and repair models, and other immune challenges and disease risks. ‡ Indicates statistical significance (p < 0.05) from corresponding control condition value. Figure 6.Estradiol β receptor expression (relative fluorescence) in response to heavy resistance exercise (squat protocol) on B-lymphocytes • Such information will contribute to our understanding of the potential gender differences in communication between the endocrine and immune systems in tissue damage and repair models, and other immune challenges and disease risks. Blood sample collection during the control trial Figure 3. Leukocytes were gated based on forward and side scatter characteristics of cells. • Figure 2. Leukocytes were labeled with a PeCy7 conjugated anti- ER β- antibody and analyzed via flow cytometry REFERENCES Carter, A., Dobridge, J., & Hackney, A. C. (2001). Influence of estrogen on markers of muscle tissue damage following eccentric exercise.FiziologiiaCheloveka, 27(5), 133-137. Clarkson, P. M., & Hubal, M. J. (2001). Are women less susceptible to exercise-induced muscle damage? Current Opinion in Clinical Nutrition and Metabolic Care, 4(6), 527-531. Stupka, N., Lowther, S., Chorneyko, K., Bourgeois, J. M., Hogben, C., & Tarnopolsky, M. A. (2000). Gender differences in muscle inflammation after eccentric exercise. Journal of Applied Physiology, 89(6), 2325-2332. Tiidus, P. M. (2001). Oestrogen and sex influence on muscle damage and inflammation: Evidence from animal models. Current Opinion in Clinical Nutrition and Metabolic Care, 4(6), 509-513. Tiidus, P. M. (2003). Influence of estrogen on skeletal muscle damage, inflammation, and repair. Exercise and Sport Sciences Reviews, 31(1), 40-44. PURPOSE Granulocytes Forward Scatter Light Detector Neuro-endocrine Cells Cell Injector Tip Immune Cells To examine whether estradiol β receptors on B-lymphocytes change in response to acute exercise stress To examine whether gender differences in estradiol β receptors expressed on B-lymphocytes exist in response to acute exercise stress are apparent. Lymphocytes Focusing Sheath Fluid Monocytes Side Scatter & Fluorescent Light Detector Cells Source of Laser Light Resistance exercise protocol