Androgen-mediated modulation of macrophage function following trauma-hemorrhage: The central role of 5α-dihydrotestosterone

Androgens have been implicated as the causative factor for the post-injury immune dysfunction in males; however, it remains unknown whether androgens directly affect macrophages. To study this, male mice were sham-operated or subjected to trauma (i.e., midline laparotomy) and hemorrhagic shock (MAP 30 ± 5 mmHg for 90 min and then resuscitated). The mice received the 5α-reductase inhibitor 4-hydroxyandrostenedione (4-OHA) prior to resuscitation. Plasma TNF-α, IL-6 and IL-10 levels were elevated following trauma-hemorrhage and normalized by 4-OHA. TNF-α and IL-6 production by splenic macrophages (sMφ) was decreased after injury, whereas Kupffer cell (KC) production of these mediators was enhanced. 4-OHA normalized cytokine production. Androgens suppressed cytokine production by sMφ from hemorrhaged mice, whereas it enhanced TNF-α and IL-6 production by KC. Addition of 4-OHA, in vitro, normalized cytokine production by cells treated with testosterone, but had no effect on dihydrotestosterone (DHT)-treated cells. These results indicate that androgens directly affect macrophage function in males following trauma and hemorrhagic shock and that the intracellular conversion of testosterone to DHT is of particular importance in mediating the androgen-induced effects. 2 by 10.0.33.2 on N ovem er 1, 2017 http://jaysiology.org/ D ow nladed fom Schneider et al. / DHT-INDUCED IMMUNOSUPPRESSION INTRODUCTION Numerous clinical and epidemiological studies indicate that males are more susceptible to the lethal effects of sepsis as compared to females (29; 31). In addition, experimental studies indicate that proestrus females maintain immune function following trauma-hemorrhage, whereas males do not (3). The maintained immune functional capacity of females under such conditions also correlates with a decreased mortality from subsequent sepsis (16). Studies also indicate that following trauma-hemorrhage the immunoprotected state in proestrus females is estrogen-mediated, whereas the immunosuppressed state in males is androgen-mediated (3). Thus, gender dimorphism in the immune response to trauma, hemorrhage and sepsis, which is supported by both clinical and experimental findings, appears to be mediated by sex steroids. Sex steroid-mediated regulation of immune function is also supported by other studies (39). In particular, the most potent androgen, 5α-dihydrotestosterone (DHT), has been shown to suppress cytokine release by murine T cells and alter their cytokine profile (4; 14; 24; 30; 52). While several studies have demonstrated the presence of androgen receptors in human and murine immune cells (8; 14; 28), recent findings also indicate that immune cells have the ability to directly metabolize sex steroids (44; 51). A number of steroidogenic enzymes, 5α-reductase, aromatase, 3β-hydroxysteroid dehydrogenase, and 17β-hydroxysteroid dehydrogenase participate in the biosynthesis of testosterone, its active metabolite DHT, and 17β-estradiol in tissue other than the gonads and adrenal gland (23; 25; 26; 58). Studies have shown that 4-hydroxyandrostenedione (4-OHA) is a potent inhibitor of 5α-reductase activity which is the enzyme responsible for the conversion of testosterone to its active metabolite, DHT (35; 59) . Furthermore, recent studies by Samy et al. (42) indicate that trauma-hemorrhage increases 5α-reductase activity in the spleen and splenic T cells, which is immunosuppressive due to the increased intracrine 3 by 10.0.33.2 on N ovem er 1, 2017 http://jaysiology.org/ D ow nladed fom Schneider et al. / DHT-INDUCED IMMUNOSUPPRESSION synthesis of DHT. Macrophages represent the first line of defense in host resistance and play a central role in the regulation of the immune system. Macrophage functional capacity is significantly altered following traumatic injuries and hemorrhagic shock (49). Previous findings indicate that macrophage functions are; regulated by male sex steroids (2; 12; 33); that these immune cells express androgen receptors (10; 27; 40) and can metabolize steroid hormones (45); and that gender dimorphism in their responsiveness exists after traumahemorrhage (3). The present study was designed to elucidate the mechanism(s) by which androgens modulate macrophage function following trauma-hemorrhage. 4 by 10.0.33.2 on N ovem er 1, 2017 http://jaysiology.org/ D ow nladed fom Schneider et al. / DHT-INDUCED IMMUNOSUPPRESSION MATERIALS AND METHODS Animals. Inbred male C3H/HeN mice (Charles River Laboratories, Wilmington, Ma), 8-10 weeks old (23-26 g BW) were used for this study. All procedures were carried out in accordance with the guidelines set forth in the Animal Welfare Act and the Guide for the Care and Use of Laboratory Animals by the National Institutes of Health. This study was conducted at Rhode Island Hospital and Brown University and approved by their Institutional Animals Care and Use Committee. Male mice were randomized into four groups (7-8 mice/ group). Groups 1 and 2 mice underwent sham operation, while groups 3 and 4 mice were subjected to trauma-hemorrhage. Groups 1 and 3 received a 0.1ml subcutaneous (sac.) corn oil injection (hereafter referred to as vehicle) after the experimental procedure. Groups 2 and 4 received a subcutaneous injection of 4hydroxyandrostenedione (4-OHA; 4-androsten-4-ol-3, 17-dione, Steraloids Inc., Newport, RI) dissolved in corn oil (5 mg/ kg body weight) just prior to resuscitation. The dosage regime of 4OHA employed was based on previous in vitro studies, the treatment regime for patients with prostatic and breast cancer and previous work by our laboratory (17; 32; 46). No deleterious side effects of subcutaneous 4-OHA administrations at this dose were observed in the present study. Trauma-hemorrhage procedure. Mice were lightly anesthetized with methoxyflurane (Metofane, Pitman-Moore, Mundelein, IL) and restrained in a supine position. A 2.5-cm midline laparotomy (i.e., induction of soft tissue trauma) was performed, and the abdominal incision was closed aseptically in 2 layers using 6-0 sutures (Ethilon, Ethicon Inc., Somerville, NJ). Both femoral arteries were then aseptically cannulated with polyethylene 10 tubing (Clay-Adams, Parsippany, NJ) using a minimal dissection technique. Heparin (porcine intestine heparin, ElkinsSinn Inc., Cherry Hill, NJ), 2U/25g body weight, was then administrated and the mice were 5 by 10.0.33.2 on N ovem er 1, 2017 http://jaysiology.org/ D ow nladed fom Schneider et al. / DHT-INDUCED IMMUNOSUPPRESSION allowed to awaken. Blood pressure was constantly monitored by attaching one of the catheters to a blood pressure analyzer (Digi-Med BPA-190, Micro-Med Inc., Louisville, KY). Upon awakening, the animals were bled through the other catheter to a mean arterial blood pressure (BP) of 30±5 mm Hg (BP pre-hemorrhage was 90±5 mm Hg), which was maintained for 90 min. At the end of that period, animals were resuscitated with lactated Ringer solution (4 times the shed blood volume for 30 min). Lidocaine hydrochloride was applied to the groin incision sites, the catheters were removed, the vessels ligated and the groin incisions were closed. Sham operated animals underwent the same groin dissection, which included ligation of both femoral arteries; however, neither hemorrhage nor fluid resuscitation was performed. Assessment of plasma sex steroid and cytokine levels. Whole blood was obtained by cardiac puncture at 2 hr after sham operation or trauma-hemorrhage. Plasma was separated by centrifugation (Microtainer, Becton Dickinson and Co., Rutherford, NJ) and stored at -80°C until assayed. Androstenedione, 17β-estradiol and testosterone plasma levels were determined by radioimmunoassay (RIA; ICN Biomedicals Inc., Costa Mesa, CA) according to the manufacturers recommendation. Plasma 5α-dihydrotestosterone levels were also measured by RIA (Diagnostic Systems Laboratories Inc., Webster, TX), however, prior to assaying, the samples were subjected to an oxidation/extraction procedure to remove the testosterone according to the manufacturers recommendation. Plasma TNF-α, IL-6 and IL-10 plasma levels were determined by ELISA technique according to the manufacturers recommendation (Pharmingen). Macrophage isolation and culture. The animals were killed with methoxyflurane overdose 2 hr after the completion of the resuscitation. Spleens were removed aseptically and splenic macrophages were isolated as previously described (46). Briefly, the spleen was gently 6 by 10.0.33.2 on N ovem er 1, 2017 http://jaysiology.org/ D ow nladed fom Schneider et al. / DHT-INDUCED IMMUNOSUPPRESSION ground to produce a single cell suspension and the erythrocytes lysed by hypotonic shock. The splenocytes were then washed twice, resuspended in RPMI 1640 containing 10% fetal bovine serum (FCS), gentamycin (1μg/ml), penicillin-G 50 U/ml and streptomycin 50 μg/mL (Gibco, Grand Island, NY) and plated at 10 cells/ml onto 12-well plates. After incubation for 2 hr (37°C, 95% humidity and 5% CO2) nonadherent cells were removed by washing with warm PBS. Kupffer cells were obtained as previously described by Ayala et al. [18]. In brief, the peritoneal cavity was opened aseptically, the portal vein exposed, catheterized with a 27-gauge needle attached and vena cava inferior was nicked. The liver was then blanched to remove blood by perfusion of 20 ml ice cold HBSS without Ca/Mg (Gibco). This was immediately followed by perfusion of 10 ml of 0.075% collagenase class IV (Sigma Chemicals Co., St. Louis, MO) in HBSS without Ca/Mg at 37°C. The liver was removed (en bloc) and transferred to a petri dish containing ice-cold 0.075% collagenase in HBSS. After mincing and incubation at 37°C for 10 min cell suspension was then passed through a sterile 150-mesh stainless steel screen into a 150ml beaker containing 10 ml ice cold HBSS and the cell suspension was washed twice by centrifugation at 400 x g for 15 min with HBSS. The cells were then resuspended in Click’s medium containing 10% fetal bovine serum, gentamycin (1μg/ml), Penicillin-G 50 U/mL and Streptomycin 50 μg/mL (Gibco) and layered over 16% Metrizamide (Accurate Chem. Corp., Westbury, NY) in HBSS and centrifuged at 1500 x g for 45 min. After removing the nonparenchymal cells from the interface, the cells were washed twice again by centrifugation at 400 x g for 15 min with complete Click’s and plated in a 12-well plate at a cell density of 10 cells/ml. After 4 hr incubation (37°C, 95% humidity and 5% CO2) nonadherent cells were removed by gentle washing with complete Click’s medium. 7 by 10.0.33.2 on N ovem er 1, 2017 http://jaysiology.org/ D ow nladed fom Schneider et al. / DHT-INDUCED IMMUNOSUPPRESSION Viability of splenic macrophages and Kupffer cells was consistently >95% as determined by trypan blue exclusion, regardless of whether of not the cells were derived from hemorrhage or sham animals. Previous studies from our laboratory demonstrated that these protocols provide adherent cell populations that were greater than 95% positive by nonspecific esterase staining and exhibit typical macrophage morphology (6). Splenic macrophages and Kupffer cells were isolated from mice treated in vivo with either 4-OHA or vehicle 2 hr following trauma-hemorrhage and resuscitation or sham operation. Cells were resuspended in complete RPMI 1640 and stimulated with 10 μg/mL LPS (Sigma Chemicals Co.) for 24 hr and supernatants were collected as described above. In vitro treatment with testosterone, 5α-dihydrotestosterone and 4-hydroxyandrostnenedione. Testosterone and 5α-dihydrotestosterone (DHT) were purchased from Sigma Chemicals Co. (St. Louis, MO) and 4-hydroxyandrostenedione (4-OHA) was purchased from Steraloids Inc. (Newport, RI). A stock solution (1mM) of testosterone and DHT was prepared in ethanol and diluted to appropriate molarity in complete Phenol Red-free RPMI 1640 media containing 10% charcoal-dextran treated FCS, gentamycin (1μg/ml), Penicillin-G 50 U/mL and Streptomycin 50 μg/ml (Gibco). The 1 mM stock solution of 4-OHA was prepared by dissolving 4-OHA in DMSO and diluted to appropriate molarity in complete Phenol Red-free RPMI 1640 media. The amounts of ethanol and DMSO did not exceed 0.025% in the media. Splenic macrophages and Kupffer cells were isolated from untreated animals directly following traumahemorrhage and resuscitation or sham operation, resuspended in complete Phenol Red-free RPMI 1640 media and incubated with either 250 nM testosterone or DHT with or without 4OHA (100 nM) for 2 hr (37°C, 95% humidity and 5% CO2). A concentration of 250 nM testosterone or DHT was chosen based on previously reported plasma testosterone and 5α8 by 10.0.33.2 on N ovem er 1, 2017 http://jaysiology.org/ D ow nladed fom Schneider et al. / DHT-INDUCED IMMUNOSUPPRESSION dihydrotestosterone levels in the literature (28). Two hours thereafter, 10 μg/ml lipopolysaccharide (LPS; Sigma Chemicals Co.) was added to the cultures and all cell cultures was incubated for a further 24 hr period (37°C, 95% humidity and 5% CO2). At the end of the incubation cell free supernatants were harvested and stored at –80 °C until assayed. Assessment of macrophage cytokine productive capacity. TNF-α, IL-6 and IL-10 levels in cell-free splenic macrophage and Kupffer cell supernatants were determined by ELISA technique according to the manufacturers recommendation (Pharmingen). In certain experiments, IL-6 levels in the cell-free supernatants prepared from macrophages treated with vehicle or 4-OHA in vivo was determined by assessing the proliferation of the IL-6 dependent murine hybridoma 7TD1 as previously described (54). In brief, 7TD1 cells were cultured with serial dilutions of plasma for 72 hr. During the last 3 hr of culture, 20 μl of a 3-(4,5dimethythiazol-2-L)-2,5-diphenyltetrazolium-bromide solution (MTT; 5 mg/mL in RPMI-1640, Sigma Chemical Co.) was added to each well. The amount of dark blue formazan crystal formation was then measured spectrophotometrically. The units of IL-6 activity were determined by comparison of curves produced from dilutions of a recombinant mouse IL-6 standard (200 U/ml, Genzyme, Cambridge, Mass). RT-PCR analysis of cytokine gene expression. Total RNA was prepared from splenic macrophages and Kupffer cells using denaturing solution (Clontech, Palo Alto, CA) followed by 3 rounds of phenol/chloroform and chloroform/isoamyl alcohol (Sigma) extraction (9). RNA was precipitated with isopropanol and washed with 80% ethanol. After air-drying, the pellet was resuspended in DEPC-treated water. Total RNA concentration and purification was determined by measuring the absorbance at 260 and 280 nm. 9 by 10.0.33.2 on N ovem er 1, 2017 http://jaysiology.org/ D ow nladed fom Schneider et al. / DHT-INDUCED IMMUNOSUPPRESSION One microgram of RNA was reverse transcribed (RT) in 20-μl reaction volume containing 1 X RT reaction buffer (50 mM Tris-HCl, pH 8.3; 75 mM KCl; 3 mM MgCl2), 0.5 mM dNTP, 20 U RNase inhibitor, 20 pM oligo (dT)18 primer, and 200 U reverse transcriptase (Clonetech). The RT reaction was incubated at 42°C for 1 hr, followed by heating at 94°C for 5 min. The cDNA was diluted with 80 μL DEPC-treated water and stored at -80°C before undergoing PCR. Primers for mouse IL-6 and IL-10 were purchased from Clonetech (Palo Alto, CA) and the primer for TNF-α was purchased from Ambion (Austin, TX). Ten percent portions of cDNA were amplified with 0.4 μM each of 3’ and 5’ primers or G3PDH primers (Clonetech), in 50 μL of PCR mixture containing 1 X buffer (50 mM KCl; 10 mM Tris-HCl and 1.5 mM MgCl2), 0.2 mM dNTP mix, 2.5 U AmpliTaq DNA polymerase (Clonetech). Thirty-six cycles of amplification were performed using a “Robocycler” (Stratagene, Torrey Pines, CA) in thin walled 0.2 ml PCR tubes (Stratagene) according to the following protocol: first cycle of 95°C (3 min), 60°C (2 min), and 72°C (3 min) followed by 35 cycles of 95°C (40 sec), 60°C (1 min) and 72°C (1 min). A final 5 min incubation at 72°C was used to “polish” the DNA termini. Resulting amplicons (TNF-α 239bp, IL-6 638bp, IL-10 455bp and G3PDH 983bp) were resolved on ethidium bromide-stained (0.5 μg/ml) 1.8% TBE-agarose gels and photographed using a gel documentation system (ChemiImager, Alpha Inotech Corporation, San Leandro, CA). The amount of PCR products generated by the target gene and the control gene (G3PDH) were measured using a ChemiImager System (Alpha Inotech Corp, Palo Alto CA), and the amount of the target in percent of the control gene was calculated. The results of typical experiments, repeated a minimum of three times, are