Sex steroids regulate pro- and anti-inflammatory cytokine release by macrophages after trauma-hemorrhage

Martin K. Angele, Markus W. Knöferl, Martin G. Schwacha, Alfred Ayala, William G. Cioffi, Kirby I. Bland, and Irshad H. Chaudry

Center for Surgical Research and Department of Surgery, Brown University School of Medicine and Rhode Island Hospital, Providence, Rhode Island 02903


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Studies indicate that macrophage immune responses in males are depressed after trauma-hemorrhage, whereas they are enhanced in females under such conditions. Nonetheless, the involvement of male and female sex steroids in this gender-dependent dimorphic immune response after trauma-hemorrhage remains unclear. To study this, male C3H/HeN mice were castrated and treated with pellets containing either vehicle, 5alpha -dihydrotestosterone (DHT), 17beta -estradiol, or a combination of both steroid hormones for 14 days before soft tissue trauma (i.e., laparotomy) and hemorrhagic shock (35 ± 5 mmHg for 90 min followed by adequate fluid resuscitation) or a sham operation. Twenty-four hours later the animals were killed, plasma was obtained, and Kupffer cell and splenic and peritoneal macrophage cultures were established. For DHT-treated mice, we observed significantly decreased releases of the proinflammatory cytokines interleukin 1beta (IL-1beta ) and IL-6 by splenic macrophage (-50 and -57%, respectively) and peritoneal macrophage (-51 and -52%, respectively) cultures after trauma-hemorrhage compared with releases by cultures of cells from mice subjected to a sham operation; in contrast, responses of splenic and peritoneal macrophage cultures from other groups subjected to trauma-hemorrhage did not change significantly. In addition, only DHT-treated animals exhibited increased Kupffer cell IL-6 release (+634%). The release of IL-10 in DHT-treated hemorrhaged animals was increased compared with that in sham-operated animals but was decreased in estrogen-treated mice under such conditions. These results suggest that male and female sex steroids exhibit divergent immunomodulatory properties with respect to cell-mediated immune responses after trauma-hemorrhage.

gender; immune depression; Kupffer cells; shock


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

SEVERAL CLINICAL and experimental studies demonstrate gender differences in the susceptibility to and morbidity from sepsis and trauma (13, 22, 25, 38, 40). Female mice in the proestrus state tolerate sepsis better than male mice, as evidenced by a significantly lower mortality rate after polymicrobial sepsis (40). Furthermore, female mice exhibit enhanced immune responses, as opposed to depressed immune function in males, after trauma-hemorrhage (35). Studies indicate that this immunological gender dimorphism appears to be hormonally regulated and that the hormones involved originate primarily from the gonads and secondarily from the thymus and the hypothalamus-pituitary gland (18).

In this respect, studies examining the effects of depletion of male sex steroids by castration of male mice before mycobacterium infection showed that castration increased the host resistance to infection (38). Moreover, castration of male animals before hemorrhagic shock prevented the depression of immune responses typically observed in normal males after this event (34). Similarly, the administration of an androgen receptor blocker, e.g., flutamide, restored depressed immune responses and increased the survival rate of hemorrhaged animals subjected to subsequent sepsis (3, 33). In addition, the administration of 5alpha -dihydrotestosterone (DHT) to female mice depressed the splenic and peritoneal macrophage immune response after trauma-hemorrhage to levels comparable to those in males under such conditions (1, 2).

In contrast to testosterone, female sex steroids appear to have immunoenhancing effects after infection or circulatory stress (16, 38). In this regard, estrogens have been shown to increase the resistance of host animals to infections, i.e., those caused by Streptococcus and Mycobacterium marinum (25, 38). Moreover, estrogens have been shown to stimulate macrophage functions, as evidenced by increased clearance of IgG-coated erythrocytes (16). Overall these findings suggest that female sex steroids should improve macrophage function in contrast to the immunodepressive effects of male sex steroids.

Despite the abundance of information demonstrating the effect of sex steroids in modulating normal immune cell responsiveness, there is limited knowledge concerning the capacities of these hormones to alter the release of pro- and, in particular, anti-inflammatory cytokines by macrophages after trauma-hemorrhage. Therefore the aim of the present study was to investigate the cell-mediated immune response after trauma-hemorrhage in animals that were depleted of sex steroids by castration and then supplemented with known amounts of DHT and/or 17beta -estradiol. The release of pro- and anti-inflammatory cytokines by Kupffer cells and splenic and peritoneal macrophages was measured to determine the effect of sex steroids on macrophage populations harvested from these different microenvironments.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals. Inbred male C3H/HeN mice (Charles River Laboratories, Wilmington, MA), 7 wk old (24-26 g body wt), were used in this study. Mice were castrated 2 wk before the experiment as previously described (34). 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 project was approved by the Institutional Animal Care and Use Committee of Rhode Island Hospital and Brown University.

Hormonal supplementation. Twenty-one-day release pellets containing 7.5 mg 5alpha -dihydrotestosterone (DHT), 0.5 mg 17beta -estradiol, or vehicle (Innovative Research of America, Sarasota, FL) were implanted subcutaneously with a 10-gauge trocar (Innovative Research of America) after castration, i.e., 2 wk before the initiation of the experiment.

Experimental groups. Castrated male mice were randomized into eight groups: groups 1 and 2 received vehicle; groups 3 and 4 received DHT; groups 5 and 6 were treated with 17beta -estradiol; and groups 7 and 8 were treated with both DHT and 17beta -estradiol. Each group consisted of seven animals. Groups 1, 3, 5, and 7 contained animals subjected to sham operations (sham-operated animals). The animals in groups 2, 4, 6, and 8 were subjected to the trauma-hemorrhage procedure. The experiments were performed seven different times. On each experimental day, one animal from each group was incorporated into the study.

Trauma-hemorrhage procedure. Mice in the hemorrhage groups were lightly anesthetized with methoxyflurane (Metofane; Pitman-Moore, Mundelein, IL) and restrained in a supine position, and a 2.5-cm midline laparotomy (soft tissue trauma) was performed. The laparotomy was then closed aseptically in two layers with 6-0 Ethilon sutures (Ethicon, Somerville, NJ). After this, both femoral arteries were aseptically cannulated with polyethylene 10 tubing (Clay-Adams, Parsippany, NJ) by a minimal-dissection technique. The animals were then allowed to awaken. Blood pressure (BP) was constantly monitored by attaching one of the catheters to a BP analyzer (Digi-Med; Micro-Med, Louisville, KY). On awakening, the animals were bled rapidly through the other catheter to a mean arterial BP of 35 ± 5 mmHg (mean arterial BP prehemorrhage was 95 ± 5 mmHg), which was maintained for 90 min. At the end of that period, four times the shed blood volume (the average shed blood volume was 0.95 ml, ~60% of the circulating blood volume) was infused in the form of lactated Ringer solution to provide adequate fluid resuscitation. Lidocaine was applied to the incision sites, the catheters were then removed, the vessels were ligated, and the groin incisions were closed. Sham-operated animals in groups 1, 3, 5, and 7 underwent the same surgical procedure, which included ligation of both femoral arteries, but neither hemorrhage nor fluid resuscitation was carried out. There was no mortality observed in this trauma-hemorrhage model within the first 24 h.

Blood, tissue, and cell harvesting procedure. The animals were killed by methoxyflurane overdose at 24 h after the completion of the experiment to obtain the spleen, the liver, peritoneal macrophages, and whole blood. The mice were sacrificed at the same time of the day to avoid fluctuations of plasma hormone levels due to the circadian rhythm.

Plasma collection and storage. Whole blood was obtained by cardiac puncture and placed in microcentrifuge tubes (Microtainer; Becton Dickinson, Rutherford, NJ). The tubes were then centrifuged at 16,000 g for 15 min at 4°C. Plasma was separated, placed in pyrogen-free microcentrifuge tubes, immediately frozen, and stored (-80°C) until assayed for DHT and 17beta -estradiol.

RIAs for plasma DHT and 17beta -estradiol. Plasma DHT concentration was determined with a commercially available coated-tube RIA kit (Diagnostic Systems Laboratories, Webster, TX) in which 100 µl of unextracted plasma were assayed in duplicate. The sensitivity of the DHT RIA has been found to be 0.05 ng/ml. The average coefficient of variation of the standard curve was 2.0%.

Plasma 17beta -estradiol concentration was determined with a commercially available RIA kit (ICN Biomedicals, Costa Mesa, CA) in which 50-µl plasma samples were assayed in duplicate. The sensitivity of the 17beta -estradiol RIA has been found to be 5 pg/ml. The average coefficient of variation of the standard curve was 2.3%. The reactivity of both RIAs was 100% for their respective steroids.

Preparation of peritoneal macrophage culture. Resident peritoneal macrophages were obtained from mice by lavaging the peritoneal cavity, and monolayers were established as previously described (6, 39). The macrophage monolayers were stimulated (for 48 h at 37°C, 5% CO2, and 90% humidity) with 10 µg lipopolysaccharide (LPS)/ml Click's medium containing 10% FCS (Biologos, Naperville, IL). At the end of the incubation period, the culture supernatants were collected, aliquoted, and stored at -80°C until assayed for interleukin-1 (IL-1), IL-6, and IL-10. This protocol provided adherent cells that were >95% positive, as determined by nonspecific esterase staining, and that exhibited typical macrophage morphology.

Preparation of splenic macrophage culture. The spleens were removed aseptically, placed in separate petri dishes containing cold (4°C) PBS, and dissociated by grinding. The splenocyte suspension was used to establish a macrophage culture as previously described (39). The macrophage monolayers were stimulated for 48 h (at 37°C, 5% CO2, and 90% humidity) with 10 µg LPS/ml Click's medium containing 10% FCS. At the end of the incubation period, the culture supernatants were collected, aliquoted, and stored at -80°C until assayed for IL-1, IL-6, and IL-10.

Preparation of Kupffer cell culture. Kupffer cells were harvested as previously described (5). In brief, retrograde perfusion of the liver was performed with 35 ml of ice-cold Hanks' balanced salt solution (HBSS) through the portal vein. This was immediately followed by perfusion with 10 ml of 0.05% collagenase IV (Worthington Biochemical, Freehold, NJ) in HBSS at 37°C. The liver was then transferred to a petri dish containing warm 0.05% collagenase, minced finely, incubated at 37°C for 15 min, and passed through a sterile 150-mesh stainless steel screen into a beaker containing 10 ml of cold HBSS and 10% FCS. The cell suspension was then layered over 16% Metrizamide (Accurate Chemical, Westbury, NY) in HBSS and centrifuged at 3,000 g, 4°C, for 45 min to separate the Kupffer cells from the remaining parenchymal cells in the pellet. After removal of the nonparenchymal cells from the interface with a Pasteur pipette, the cells were washed twice by centrifugation (800 g, 10 min, 4°C) with HBSS and resuspended in Click's medium containing 10% FCS. The cells were then transferred to a 24-well plate that was precoated with 0.5 ml of 6 µg/ml Vitrogen 100 (Collagen Biomaterials, Palo Alto, CA) and incubated for 3 h at 37°C (5% CO2 and 90% humidity). Nonadherent cells were removed by washing three times with Click's medium. This protocol provides adherent cells that are >95% positive as determined by nonspecific esterase staining and that exhibit typical macrophage morphology (39). The Kupffer cells (3 × 106 Kupffer cells · ml-1 · well-1) were incubated for 24 h (37°C, 5% CO2) with 10 mg LPS/ml Clicks's medium and 10% FCS, and the production of IL-6 and IL-10 was assessed.

Assessment of IL-6 release. IL-6 activities in culture supernatant of splenic and peritoneal macrophages and Kupffer cells were determined by the degree of proliferation of the IL-6-dependent murine B-cell hybridoma cell line 7TD1 (19). The 7TD1 cell line (gift from Dr. Jacques Van Snick) was maintained as previously described (20). Serial dilutions of macrophage supernatants were added to 4 × 104 7TD1 cells/ml, and the cells were incubated for 72 h at 37°C in 5% CO2. For the last 4 h of incubation, 20 µl of a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution (5 mg/ml in RPMI 1640; Sigma Chemical, St. Louis, MO) were added to each well (only viable cells incorporate MTT). The assay was stopped by aspiration of 100 µl of supernatant from each well, with subsequent replacement by 150 µl of 10% SDS solution in PBS (lauryl sulfate; Sigma Chemical) to dissolve the dark blue formazan crystals. An automated microplate reader (EL-311; Bio-Tek Instruments, Winooski, VT) was used to measure the light absorbance at 595 nm.

Assessment of IL-1beta and IL-10 release. IL-1beta and IL-10 levels in the macrophage supernatants were determined by the Sandwich ELISA technique described by Mossmann et al. (24). In brief, Nunc-Immuno 96-well plates with MaxiSorp surfaces were coated overnight with either 2 µg monoclonal hamster anti-mouse IL-1beta capture antibody (Genzyme Diagnostics, Cambridge, MA)/ml 0.1 M carbonate, pH 9.5, or 4 µg rat anti-mouse IL-10 capture antibody (clone JES-5; Pharmingen, San Diego, CA)/ml 0.1 M NaHCO3, pH 8.2. The plates were washed three times with PBS containing 0.05% Tween 20 (Sigma Chemical) and blocked with PBS containing 20% FCS for 2 h. After the plates were washed, 100 µl of the samples and standard [1,000 pg/ml murine IL-1beta (Genzyme Diagnostics) or 10 ng/ml murine IL-10 (Pharmingen)] were added to the plates, and then they were incubated overnight (4°C). After repeated washings the plates were incubated for 1 h with 100 µl of either biotinylated polyclonal rabbit anti-mouse IL-1beta (Genzyme Diagnostics) at a concentration of 0.8 µg/ml at 37°C or biotinylated monoclonal rat anti-mouse IL-10 (clone SXC-1; Pharmingen) at a concentration of 2 µg/ml at room temperature. Then the plates for IL-1beta detection were incubated with horseradish peroxidase-conjugated streptavidin (Genzyme Diagnostics) for 15 min at 37°C. After multiple washings, 100 µl of 3,3',5,5''-tetramethylbenzidine (TMB; Sigma Chemical) was added for 10 min at room temperature. After the addition of 100 µl of stop solution (0.5 M H2SO4) the optical density of each well at 450 nm was determined on a plate reader (EL-311; Bio-Tek Instruments). For detection of IL-10 the plates were washed and incubated at room temperature for 30 min with avidin-peroxidase (diluted 1:400; Sigma Chemical). After the washing, 100 µl of 2,2'-azinobis(3-ethylbenzthiazolinesulfonic acid) (ABTS)-H2O2 substrate buffer was added to each well to initiate color development. The optical density at 405 nm for each well was then determined on a microplate reader. The concentrations of IL-1beta and IL-10 present in the samples were determined by interpolation from a standard curve produced with murine IL-1beta and IL-10, respectively.

Statistical analysis. The results are presented as means ± SE. One-way ANOVA, followed by the Student-Newman-Keuls test or Tukey's test as a post hoc test for multiple comparisons, was used to determine the significance of the differences between experimental means. A P value of <0.05 was considered to be significant.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Plasma DHT and 17beta -estradiol levels. In castrated animals receiving vehicle, plasma DHT levels (Fig. 1A) were found to be markedly decreased compared with the physiological plasma DHT levels previously reported for normal male mice (2, 21). Plasma DHT levels in DHT-treated animals were significantly increased compared with those in vehicle-treated castrated animals (P < 0.05), and were comparable to physiological plasma testosterone levels observed in normal male mice (2, 21). The plasma DHT levels for sham-operated and trauma-hemorrhage groups were not significantly different.


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Fig. 1.   Plasma 5alpha -dihydrotestosterone (DHT; A) and 17beta -estradiol (B) levels in male mice (C3H/HeN; n = 7/group) that were castrated and treated with either vehicle (VEH), DHT, 17beta -estradiol (estradiol), or DHT and 17beta -estradiol (DHT/estradiol) 2 wk before trauma-hemorrhage or sham operation. At 24 h after trauma-hemorrhage or sham operation mice were killed. Plasma DHT and 17beta -estradiol levels were measured by specific RIA. Values are means ± SE. Data were analyzed by ANOVA. * P < 0.05 vs. sham operation after vehicle treatment.

Plasma 17beta -estradiol levels (Fig. 1B) in castrated animals receiving vehicle or DHT alone were found to be similar to physiological plasma estradiol levels observed in normal male mice (2). The implantation of 17beta -estradiol release pellets resulted in significantly increased plasma 17beta -estradiol levels (P < 0.05), which were comparable to reported plasma estradiol levels in proestrus female mice (29). Trauma-hemorrhage did not alter plasma 17beta -estradiol levels compared with those in the corresponding sham-operated animals. The administration of DHT and 17beta -estradiol resulted in significantly increased levels of both sex steroids in plasma (P < 0.05; Fig. 1, A and B).

Effect of sex steroids on peritoneal macrophage cytokine release. The release of IL-1, IL-6, and IL-10 (Fig. 2, A-C) from peritoneal macrophages in castrated animals receiving vehicle after trauma-hemorrhage was similar to that in sham-operated animals. Castrated animals treated with DHT, however, showed significantly decreased IL-1beta and IL-6 release after trauma-hemorrhage compared with sham-operated animals (-50.6% for IL-1beta and -52.0% for IL-6; P < 0.05). In contrast, the release of the anti-inflammatory cytokine IL-10 was not significantly altered by DHT treatment after trauma-hemorrhage. The administration of estrogen to castrated male animals did not significantly affect IL-1beta or IL-6 release from peritoneal macrophages after trauma-hemorrhage (+56.2% compared with release from peritoneal macrophages of 17beta -estradiol-treated sham-operated animals; P > 0.05). However, IL-10 release from peritoneal macrophages was significantly decreased in 17beta -estradiol-treated animals after trauma-hemorrhage (-40.9% compared with release from 17beta -estradiol-treated sham-operated animals; P < 0.05). Moreover, unlike DHT-treated mice subjected to trauma-hemorrhage, animals receiving a combination of DHT and 17beta -estradiol did not display suppressed proinflammatory cytokine production or enhanced IL-10 production.


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Fig. 2.   Peritoneal macrophages were harvested 24 h after trauma-hemorrhage or sham operation from male C3H/HeN mice that were castrated and treated with vehicle (VEH), DHT, 17beta -estradiol (estradiol), or DHT and 17beta -estradiol (DHT/estradiol) 2 wk before experiment. Peritoneal macrophages were cultured for 48 h in presence of 10 µg/ml lipopolysaccharide (LPS), and releases of interleukin-1beta (IL-1beta ; A), IL-6 (B), and IL-10 (C) were determined in supernatants. Values are means ± SE. Data were analyzed by ANOVA. * P < 0.05 vs. sham operation after vehicle treatment; dagger  P < 0.05 vs. sham operation after DHT treatment; ddager  P < 0.05 vs. sham operation after 17beta -estradiol treatment.

Effect of sex steroids on splenic macrophage cytokine release. The release of IL-1beta , IL-6, and IL-10 in vehicle-treated castrated male animals after trauma-hemorrhage was similar to that in sham-operated animals receiving vehicle (Fig. 3, A-C). The administration of DHT did not change IL-1beta and IL-6 releases in sham-operated animals compared with releases in sham-operated animals receiving vehicle. In mice subjected to trauma-hemorrhage, however, treatment with DHT resulted in significantly (P < 0.05) decreased IL-1beta (-49.6%) and IL-6 (-56.7%) releases from splenic macrophages compared with releases in DHT-treated, sham-operated animals. Furthermore, IL-10 release in sham-operated animals receiving DHT was significantly decreased (-76.9% compared with release in vehicle-treated sham-operated animals; P < 0.05). In contrast to what was found for proinflammatory cytokines, the release of IL-10 significantly increased (+433.2%; P < 0.05) after trauma-hemorrhage compared with that in sham-operated animals receiving DHT. The administration of DHT in the presence of 17beta -estradiol prevented the depression of IL-1beta and IL-6 release from splenic macrophages after trauma-hemorrhage. Moreover, splenic macrophage IL-10 release in sham-operated animals treated with DHT and 17beta -estradiol was significantly higher (+466%; P < 0.05) than the IL-10 release from DHT-treated sham-operated animals.


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Fig. 3.   Splenic macrophages were harvested 24 h after trauma-hemorrhage or sham operation from male C3H/HeN mice that were castrated and treated with vehicle (VEH), DHT, 17beta -estradiol (estradiol), or DHT and 17beta -estradiol (DHT/estradiol) 2 wk before experiment. Splenic macrophages were cultured for 48 h in presence of 10 µg/ml LPS, and releases of IL-1beta (A), IL-6 (B), and IL-10 (C) were determined in supernatants. Values are means ± SE. Data were analyzed by ANOVA. * P < 0.05 vs. sham operation after vehicle treatment. dagger  P < 0.05 vs. sham operation after DHT treatment. ddager  P < 0.05 vs. sham operation after 17beta -estradiol treatment.

Effect of sex steroids on Kupffer cell cytokine release. Kupffer cell IL-6 release was unchanged in vehicle-treated animals after trauma-hemorrhage (Fig. 4A). Although the administration of DHT did not change Kupffer cell IL-6 release in sham-operated animals, it resulted in significantly increased IL-6 release after trauma-hemorrhage (+631% compared with release from sham-operated animals treated with DHT; P < 0.05). The results also indicate that Kupffer cell IL-10 release (Fig. 4B) significantly increased in vehicle-treated and DHT-treated animals after traumahemorrhage (+77.1% in vehicle-treated and +80.8% in DHT-treated animals compared with release from sham-operated animals receiving vehicle or DHT, respectively; P < 0.05). Furthermore, the administration of 17beta -estradiol significantly increased (+42%; P < 0.05) the Kupffer cell IL-10 release compared with that from DHT-treated, sham-operated animals. Treatment of castrated male mice with DHT and 17beta -estradiol prevented the increase of Kupffer cell IL-6 and IL-10 release after trauma-hemorrhage seen in mice treated with DHT only.


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Fig. 4.   Kupffer cells were harvested 24 h after trauma-hemorrhage or sham operation from male C3H/HeN mice that were castrated and treated with vehicle (VEH), DHT, 17beta -estradiol (estradiol), or DHT and 17beta -estradiol (DHT/estradiol) 2 wk before experiment. Kupffer cells were cultured for 24 h in presence of 10 µg/ml LPS, and releases of IL-6 (A) and IL-10 (B) were determined in supernatants. Values are means ± SE. Data were analyzed by ANOVA. * P < 0.05 vs. sham operation after vehicle treatment; dagger  P < 0.05 vs. sham operation after DHT treatment.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Previous studies have reported gender differences in humoral as well as cell-mediated immune function (8, 9, 22, 26). These differences are believed to be responsible for the higher incidence of autoimmune diseases in females (26) as well as for the increased susceptibility to and morbidity from sepsis in males (8, 9, 22, 40). Similarly, differences in immune responsiveness with regard to gender have also been observed after trauma-hemorrhage. Proestrus female mice show enhanced immune function after trauma-hemorrhage compared with male mice, in which there is a depressed response (35).

Although several studies indicate that sex steroids play a central role in the immune response under physiological and pathophysiological conditions (1, 12, 18, 31, 34, 37), the precise effect of male and female sex steroids on macrophage function after trauma-hemorrhage remains unknown. In an attempt to elucidate the complex relationship of male and female sex steroids on macrophage function after trauma-hemorrhage, male mice were castrated and supplemented with controlled amounts of DHT and/or 17beta -estradiol by implantation of constant-release pellets. The plasma DHT and 17beta -estradiol levels in treated, castrated animals were found to be similar to physiological levels seen in normal male (range 0.8-2.5 ng/ml) (1) and female mice (range incorporating all states of the estrus cycle 45.6-220 pg/ml) (1), respectively (1, 29). Recent studies using extracted plasma further confirm that the levels of DHT in plasma obtained in the present study are physiological and that in normal mice the majority (~99%) of the circulating androgen is in the form of DHT rather than free testosterone (21). Because only one dose of sex steroids was administered in the present study, it remains unknown whether sub- or supraphysiological plasma DHT or estradiol levels exhibit similar immunomodulatory properties after trauma-hemorrhage. It should be noted that trauma-hemorrhage did not alter plasma sex steroid levels. Similarly, trauma-hemorrhage did not change plasma testosterone and estradiol levels in normal male and female animals (2).

The results presented for vehicle-treated mice are consistent with previous findings in which the depletion of testosterone via castration normalized the cytokine release of peritoneal and splenic macrophages and Kupffer cells after trauma-hemorrhage (34). Thus it is our hypothesis that male sex steroids are responsible for producing the immunodepression in males after trauma-hemorrhage. Although intact male mice were not used in the present study, the findings further support this hypothesis by illustrating that by providing DHT to castrated animals, the depression of splenic and peritoneal macrophage proinflammatory cytokine release after trauma-hemorrhage was reestablished. Similarly, the administration of DHT to female mice decreased the release of splenic and peritoneal macrophages after trauma-hemorrhage; in contrast immune responses in untreated female mice were maintained (2). Interestingly, unlike splenic and peritoneal macrophages, Kupffer cells from castrated DHT-treated mice exhibited an enhanced ability to release IL-6 after trauma-hemorrhage. However, this is also in keeping with prior studies of normal male mice subjected to hemorrhage, which indicate that Kupffer cells are primed to release increased levels of proinflammatory cytokines, i.e., IL-6, under such conditions (5).

It should be pointed out that the administration of testosterone did not alter the cytokine release in sham-operated animals. In this regard, numerous studies suggest that androgens do not depress macrophage cytokine release in normal animals (1, 3, 10, 11). These results suggest that physiological concentrations of testosterone are only harmful in an immunologically compromised host, not in normal animals.

In contrast to DHT treatment, treatment of castrated mice with estrogen did not depress the release of IL-1beta and IL-6 from splenic and peritoneal macrophages after trauma-hemorrhage. Because female mice in the proestrus state have been shown to have an enhanced release of IL-1beta and IL-6, the data of the present study suggest that the additive effects of other female sex hormones, e.g., progesterone, might contribute to enhanced immune responses in proestrus female mice after trauma-hemorrhage (35). In this regard, progesterone and estrogens have been reported to have similar immunoenhancing effects on rat macrophages (10, 14, 17, 23). It should be pointed out, however, that 17beta -estradiol treatment prevented the depression of splenic and peritoneal macrophage proinflammatory cytokine release and normalized Kupffer cell IL-6 release even in the presence of physiological concentrations of testosterone. In this respect, several studies have demonstrated the stimulatory effects of estrogen on various immune cells (7, 11, 23, 30). The administration of estrogen in ovariectomized mice restored the depressed phagocytic activity of macrophages from those mice (11). It should be noted that the effects of estrogens on macrophage functions appear to be dose dependent. In this regard, studies have shown that physiological levels of estrogen increase the release of proinflammatory cytokines, whereas higher levels of estrogen inhibit peritoneal macrophage functions (10, 15, 28). Thus the immunostimulatory effects of estrogen might contribute to the maintained immune responses in females after trauma-hemorrhage.

Kupffer cells have been suggested to be the primary source for circulating plasma IL-6 in males after trauma-hemorrhage (27). Although the increase of plasma IL-6 has been reported to be more pronounced in males than in females, IL-6 levels also increased in female mice at 4 h after trauma-hemorrhage (35). The release of IL-6 by Kupffer cells only increased in DHTtreated animals in the present study, suggesting that Kupffer cells from estradiol-treated mice might release increased IL-6 earlier after trauma-hemorrhage and resuscitation. Further support for this notion comes from studies that showed that after endotoxemia, female sex steroids produced the maximum increase of plasma IL-6 at 1 h after LPS injection compared with 3 h for vehicle-treated animals (42). Alternatively, other cells, e.g., keratinocytes, might contribute to increased IL-6 levels in females after trauma-hemorrhage. This, however, remains to be determined.

In contrast to the release of proinflammatory cytokines, the release of the anti-inflammatory cytokine IL-10 by splenic and peritoneal macrophages was increased in DHT-treated castrated animals after trauma-hemorrhage compared with the release in sham-operated animals receiving DHT. It should be pointed out that the releases of IL-10 from splenic and peritoneal macrophages in DHT-treated sham-operated animals were significantly decreased compared with those in vehicle- or estrogen-treated sham-operated animals, which might represent macrophage dysfunction in those sham-operated animals. In vehicle-treated animals the release of IL-10 from splenic and peritoneal macrophages was unchanged after trauma-hemorrhage, whereas IL-10 release in 17beta -estradiol-treated mice decreased under such conditions. In this regard, an increased release of IL-10 after trauma-hemorrhage in normal male mice has been reported (4). Because the release of IL-10 by splenic and peritoneal macrophages decreases after trauma-hemorrhage, the decreased release of IL-10 in those animals might contribute to the maintenance of macrophage responses after trauma-hemorrhage. Similarly, Wilder (36) demonstrated enhanced release of IL-10 and IL-4 in healthy women in the reproductive years with high plasma estrogen levels compared with decreased anti-inflammatory cytokine production in the postpartum period.

The underlying mechanisms by which sex hormones mediate their effects on macrophages after trauma-hemorrhage remain unclear at present. Studies have, however, demonstrated the presence of estrogen receptors on various immune cells, i.e., thymocytes, macrophages, and leukocytes (11, 26, 32). Although androgen receptors have not yet been identified on macrophages, receptors for male sex steroids have been observed on macrophage-like synovial cells, immature monocytic cells and T and B cells (26). Thus sex steroids may modulate immune responses directly via specific receptor-mediated processes. Further support for the hypothesis that sex steroids exert their effects through receptor-mediated processes comes from recent studies that indicate that blocking the effect of testosterone at the receptor in vivo with flutamide after trauma-hemorrhage restored macrophage and splenocyte functions (3, 33). Because sex steroids primarily exert their immunomodulating effects after trauma-hemorrhage, it is possible that increased receptor expression or changes in receptor affinity for these hormones occur because of trauma-hemorrhage. This, however, remains to be tested.

Alternatively, changes in the cytokine pattern might be due to the indirect effects of these hormones on the macrophages. Sex steroids might alter secondary mediators from lymphocytes (e.g., interferon-gamma ), endothelial cells, or other interactive cell populations and thereby modulate the cytokine release of macrophages after trauma-hemorrhage. However, which other mediators are involved in the immunomodulation of macrophages by sex steroids after trauma-hemorrhage remains to be determined. In this regard, studies indicate that prolactin, which is known to have immune system-enhancing effects, may potentially be a mediator for the immunoenhancing effects of estrogen because estrogen can stimulate prolactin secretion (41).

In summary, the present study demonstrates that the female and male sex steroids 17beta -estradiol and DHT differentially modulate macrophage cytokine release after trauma-hemorrhage. DHT decreases the release of proinflammatory cytokines, whereas estrogen decreases the release of anti-inflammatory cytokines, thereby maintaining macrophage functions even in the presence of testosterone after trauma-hemorrhage. These findings further extend our previous observations that indicate that testosterone might be the culprit for producing depression of macrophage functions after trauma-hemorrhage in males. Furthermore, 17beta -estradiol, a female sex steroid, appears to exhibit immunoprotective effects on macrophage functions potentially by down-regulating anti-inflammatory cytokine release. The exact mechanism, however, for the immunomodulatory properties of male and female sex steroids after trauma-hemorrhage remains unknown. Nonetheless, the results of this study suggest that the administration of sex steroids or treatment with their specific blockers should be considered as a novel and useful approach for modulating the release of pro- and/or anti-inflammatory cytokines after trauma-hemorrhage.


    ACKNOWLEDGEMENTS

This investigation was supported by National Institute of General Medical Sciences Grant R01-GM-37127.


    FOOTNOTES

Present address of M. K. Angele: Ludwig-Maximilians Univ., Department of Surgery, Klinikum Grosshadern, Marchioninstrasse 15, 81377 Munich, Germany.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: I. H. Chaudry, Center for Surgical Research, Brown Univ. School of Medicine and Rhode Island Hospital, Middle House II, 593 Eddy St., Providence, RI 02903 (E-mail: ichaudry{at}lifespan.org).

Received 17 December 1998; accepted in final form 28 March 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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