Trauma-hemorrhage produces profound immunosuppression in males but not in proestrus females. Prior castration or flutamide treatment of males following trauma-hemorrhage prevents immunosuppression, implicating 5α-dihydrotestosterone for the immunosuppressive effects. 5α-Dihydrotestosterone, a high-affinity androgen receptor-binding steroid, is synthesized in tissues as needed and seldom accumulates. The presence of steroidogenic enzymes in T lymphocytes suggests both synthesis and catabolism of 5α-dihydrotestosterone. We hypothesized, therefore, that the basis for high 5α-dihydrotestosterone activity in T lymphocytes of males following trauma-hemorrhage is due to decreased catabolism. Accordingly, catabolism of 5α-dihydrotestosterone was assessed in splenic T lymphocytes by examining the activity and expression of enzymes involved. Analysis showed increased synthesis and decreased catabolism of 5α-dihydrotestosterone in intact male T lymphocytes following trauma-hemorrhage. In contrast, reduced 5α-reductase activity and increased expression of 17β-hydroxysteroid dehydrogenase oxidative isomers suggest inactivation of 5α-dihydrotestosterone in precastrated males. Thus our study suggests increased synthesis and decreased catabolism of 5α-dihydrotestosterone as a reason for loss of T lymphocyte functions in intact males following trauma-hemorrhage, as evidenced by decreased release of interleukin-2 and -6.
- gonadal steroids
- androgen metabolism
suppression of immune functions is severe and prolonged following trauma-hemorrhage, and release of proinflammatory cytokines appears to be one of the outcomes of immune suppression (8, 37, 38). Studies have demonstrated gender dimorphism in immune responses following trauma-hemorrhage (3). The immune responses are markedly suppressed in male mice following trauma-hemorrhage, whereas they are enhanced/unaltered in proestrus females under such conditions, thus linking gonadal steroids to the change in immune functions (2, 10, 11, 35,36). Immune suppression in males is evidenced by 1) loss of the ability of splenocytes to proliferate and 2) alterations in release of cytokines by splenic T lymphocytes (37,38). Furthermore, studies have demonstrated that immune suppression in males can be prevented by castration of the animals before trauma-hemorrhage or by pharmacological blockade of the androgen receptors with flutamide after trauma-hemorrhage (34, 35). Thus the studies suggest an androgen-dependent mechanism for the immune suppression in males following trauma-hemorrhage.
T lymphocytes are targets for sex steroids because they have receptors for both the androgen and estrogen (19, 25). The sex steroid receptors function as transcription factors for cytokines synthesis in lymphoid cells (4, 9, 31). Thus the activity of steroid hormone receptor for the release of cytokines by T lymphocytes is dependent on the presence of an active ligand. This appears particularly logical for 5α-dihydrotestosterone because of its higher affinity for the androgen receptor as well as increased transcriptional activity of the 5α-dihydrotestosterone-bound receptor compared with testosterone (21, 27, 39). Furthermore, our recent study demonstrates the presence of enzymes that contribute to the metabolism of androgen and estrogen in splenic T lymphocytes (23). The study also shows that the activity of 5α-reductase, which synthesizes 5α-dihydrotestosterone from testosterone, increases in lymphocytes from males following trauma-hemorrhage (23).1
5α-Dihydrotestosterone is a highly potent androgen that is rapidly metabolized in tissues. Thus investigation of its catabolism in T lymphocytes following trauma-hemorrhage is important because such analysis will provide information on the availability of steroid in the receptor-active form for T lymphocyte functions. Because of rapid tissue turnover of 5α-dihydrotestosterone and the difficulties in quantification of steroids at subpicomole levels in lymphocytes, evaluation for enzyme activities appears more appropriate. Accordingly, our aim was to determine the catabolism of 5α-dihydrotestosterone in T lymphocytes of male mice following trauma-hemorrhage. This was accomplished by assessing the activity and expression of catabolic enzymes under those conditions.
MATERIALS AND METHODS
Analytical grade reagents were used in all the experiments. [1,2,6,7-3H]androstene-4-ene-3,17-dione (specific activity 74 Ci/mmol), [4-14C]androstene-4-ene-3,17-dione (specific activity 54 Ci/mmol), [4-14C]testosterone, 5α-[4-14C]dihydrotestosterone (specific activity 57 Ci/mmol), and 5α-[3-3H]androstane-3α,17β-diol (specific activity 53 Ci/mmol) were purchased from NEN Life Science Products (Boston, MA). The unlabeled steroids were from Sigma (St. Louis, MO). The oligonucleotide primers for PCR assay were synthesized at BRL Life Technologies (Gaithersburg, MD).
Inbred C3H/HeN male mice 6–8 wk old weighing 20–25 g were obtained from Charles River Laboratories (Wilmington, MA). The CTLL-2 cell line (TIB-214) for interleukin (IL)-2 assay and the hybrid cell line 7TD1 (CRL-1851) for IL-6 assay were obtained from the American Type Culture Collection (ATCC; Rockville, MD), and cells were maintained in culture according to ATCC directions. The animal studies were conducted according to the guidelines established by the National Institutes of Health, and the protocols were approved by the University of Alabama at Birmingham Institutional Animal Care and Use Committee.
Animals were assigned to the following four groups (n = 8 animals/group): male shams, males undergoing trauma-hemorrhage, precastrated male shams, and precastrated males undergoing trauma-hemorrhage. The protocol described by Waynforth (32) was followed for castration of male mice. Two weeks after castration, the animals were used in experiments.
The procedure for inducing trauma (laparotomy)-hemorrhage is described in detail in earlier publications (22, 38). Briefly, after overnight fast, soft-tissue trauma was induced in mice by performing a 2-cm ventral midline laparotomy, which was closed in two layers. Both femoral arteries were then catheterized, and the animals were allowed to awaken. Upon awakening, the animals were bled rapidly to a mean arterial pressure of 30 mmHg, maintained at that pressure for 90 min, and then resuscitated with four times the volume of blood drawn with Ringer's lactate solution. Sham-operated mice underwent the same anesthetic and surgical procedures, but neither hemorrhage nor resuscitation was carried out. The animals were killed 2 h after resuscitation, and the spleen was removed for analysis.
Preparation of T lymphocytes.
The procedures for the preparation of splenocytes and enrichment of T lymphocytes are described in an earlier publication (22). T lymphocyte enrichment was carried out by passage of the splenocyte suspension through the nylon wool column. The enriched T lymphocytes were >95% pure and consisted of both CD4+ and CD8+ subsets (24).
The procedure by Andersson et al. (1) was used for the assay of 5α-reductase activity and the procedure by Turgeon et al. (30) for the assay of 17β-hydroxysteroid dehydrogenase (17β-HSD) reductive and oxidative activities, as described previously (23). NADPH was used as a cofactor for reductive catalysis and NAD+ as a cofactor for the oxidative catalysis in these assays. The procedure of Biswas and Russell (5) was followed for measurement of 3α-hydroxysteroid dehydrogenase (3α-HSD) activities. Briefly, the cells were homogenized in 10 mM phosphate buffer, pH 7.0, containing 150 mM KCl and 1 mM EDTA with a Brinkman Polytron. The 800 g supernatant was used for the assay. The assay mixture (0.5 ml, pH 7.5) consisted of 100 mM sodium phosphate, 150 mM KCl, 1 mM EDTA, and 1.5 mM NADPH and was incubated at 37°C with 2 μM steroids containing 0.1 μCi 3H- or14C-labeled steroid. The reaction mixtures were extracted five times with methylene chloride, and the steroids in the organic phases were analyzed by thin-layer chromatography using the mobile phase of chloroform-ethyl acetate (3:1). The radioactivity of the separated steroids in the chromatographic plates in the enzyme assays was measured by using the InstantImager (Packard, Downers Grove, IL), and the steroids were identified by comparison to the retardation factor (R f) values of standards.
The RNA was prepared from T lymphocytes by using the Atlas total RNA kit (Clontech, Palo Alto, CA) and purified by DNase treatment (1 U/μl) for 30 min at 37°C. Poly(A+) mRNA preparation and RT-PCR reactions were carried out using the Access RT-PCR system kit (Promega, Madison, WI.). The RT-PCR reaction mixture (50 μl) in 1× buffer (100 mM KCl, 0.1 mM EDTA, 1 mM dithiothreitol, 20 mM Tris · HCl, pH 8.0, 50% glycerol, 0.5% Nonidet P-40, and 0.5% Tween 20) contained 200 μM dNTP mix, 1 mM MgSO4, 0.1 unit of avian myeloblastosis virus reverse transcriptase, 0.1 unit of Tfi DNA polymerase, and 1 nM each of the primers (Table1). Cloned cDNA sequences of the enzymes were taken from GenBank, and software (www.genome.wi.mit.edu/genome_software/other/primer3.html) was used for the selection of primers. The PCR reactions were carried out in a gradient Mastercycler (Eppendorf, Westbury, NY). The first cycle of the RT reaction was carried out at 48°C for 45 min. The PCR cycle for amplification consisted of 30 s of denaturation at 94°C, followed by annealing at 60°C for 1 min and 2 min of extension at 68°C. The final products were extended for 7 min at 68°C. Each enzyme was analyzed for amplification between 5 and 40 cycles. The enzyme amplifications were examined together with the internal standard β-actin. Because the level of amplification was dependent on the reaction cycle, comparison of the enzyme expression between the sham and the trauma-hemorrhage was made at the cycle where ∼50% expression was apparent. The PCR products were analyzed by electrophoresis of cDNA on 1.5% agarose gels in 1× TAE (Tris, acetic acid, EDTA) buffer and visualized by ethidium bromide staining under ultraviolet illumination. The intensity of cDNA bands was measured in the 500 Fluorescence ChemiImager (San Leandro, CA).
IL-2 and IL-6 activities.
The bioassay procedures for estimation of IL-2 and IL-6 release in the T lymphocyte culture supernatants have been previously described (36, 38). The lymphocytes were stimulated with 2.5 μg/ml concanavalin A (Sigma) in complete Click's medium at 37°C for 36 h before culture supernatants were assayed for the cytokine releases.
The protein content was determined by the micro Bradford method (Bio-Rad, Hercules, CA). BSA was used as standard.
Kinetic constants for steroid substrates were determined by conventional Lineweaver-Burk analysis. All assays were carried out in triplicate by using microsomal preparations of tissue homogenates. Ten concentrations of substrates between 1 and 200 μM were used for each steroid. SigmaPlot software (version 2.0; Jandel Scientific, San Rafael, CA) was used to generate hyperbolic functions and nonlinear regression plots.
SigmaStat software (version 2.0; Jandel Scientific) was used in all nonlinear regression analysis. All data were analyzed by separate one-way ANOVA. When a significant F value was obtained, the effects were differentiated by using Tukey's test. Tests between effects were performed by Student's t-test. Significance was achieved when P ≤ 0.05.
5α-Reductase, 3α-HSD, and 17β-HSD activities in intact males.
The activity of the enzymes in splenic T lymphocytes from intact male mice following trauma-hemorrhage is shown in Fig.1. Trauma-hemorrhage led to significant increase (>2-fold) in the activities of 5α-reductase (Fig.1 A) and 3α-HSD (Fig. 1 B). There was no change in the 17β-HSD reductive activities when testosterone was used as the substrate, i.e., conversion of testosterone into 4Δ-androsteronedione (Fig. 1 C). However, when 5α-androstane-5α,17β-diol was used as a substrate, the 17β-HSD activity significantly decreased, suggesting a reduction in the oxidative conversion of 5α-androstane-5α,17β-diol into androsterone following trauma-hemorrhage. The kinetic parameters of the enzymes in T lymphocytes with relevant steroid substrates are provided in Table2.
The mRNA expression of these enzymes in the T lymphocytes following trauma-hemorrhage is shown in Fig. 2. Increases in the expression of 5α-reductase (55%) and 3α-HSD (84%) and decreases in the expression of aromatase (80%) and 17β-HSD isomer IV (37%) were observed following trauma-hemorrhage. The increase in 5α-reductase expression correlated with the increase in enzyme activity following trauma-hemorrhage. There was, however, no change in the expressions of 3β-HSD or the 17β-HSD isomers II and V following trauma-hemorrhage. It should be noted, however, that the RT-PCR analysis carried out in our study is at most semiquantitative.
Cis-retinoic acid dehydrogenase (CRAD2) is a bifunctional enzyme that catalyzes the activity of 3α-HSD as well as the oxidative function of 17β-HSD type VI (19, 21). Figure3 shows increased expression of a 1,059-bp fragment of CRAD2 mRNA in the T lymphocytes following trauma-hemorrhage. According to Tomita et al. (29), amplification of this mRNA fragment reflects amplification of mouse CRAD2 mRNA, which includes the sequences of both 3α-HSD and 17β-HSD type VI.
Enzyme expressions in castrated males.
Because prior castration of males prevents them from being immunosuppressed following trauma-hemorrhage (36), we evaluated the expression of the enzymes following trauma-hemorrhage in T lymphocytes from castrated mice. The results show that expression of 5α-reductase was lacking in both sham and trauma-hemorrhaged mice (Fig. 4). Furthermore, there were no changes in the expressions of 3α-HSD and aromatase in castrated males after trauma-hemorrhage. Among the 17β-HSD isomers, unlike in noncastrated males, the expression of isomer types II (39%) and V (45%) was significantly decreased following trauma-hemorrhage compared with sham control.
IL-2 and IL-6 release in T lymphocytes.
The in vitro release of cytokines by T lymphocytes following concanavalin A stimulation was evaluated (Fig.5). Trauma-hemorrhage caused a significant reduction in the releases of both IL-2 (85%) and IL-6 (80%). Castration of animals before trauma-hemorrhage, however, maintained normal release of both the cytokines.
Studies demonstrate that male mice are immunosuppressed following trauma-hemorrhage, whereas proestrus females do not show any signs of immunosuppression under those conditions (2, 3,35). These studies also implicate 5α-dihydrotestosterone in producing immunosuppression in males (34, 36) and 17β-estradiol for protection from immunosuppression following trauma-hemorrhage (10, 11, 35). The characteristic effect of immunosuppression following trauma-hemorrhage is the alteration in the release of cytokines by splenic T lymphocytes. Because T lymphocytes have receptors for androgen and estrogen and the enzymes involved in sex steroids metabolism (23, 25), we investigated whether a correlation exists between the steroid metabolism and cytokine release. 5α-Dihydrotestosterone is the main sex steroid, which is bound to the androgen receptor in the nucleus. Its availability for T lymphocyte functions, which also includes regulated release of cytokines, is largely dependant on its local metabolism. Our previous studies have indicated increased 5α-dihydrotestosterone synthesis in T lymphocytes from intact males following trauma-hemorrhage (23). In the present study, we assessed the catabolism of 5α-dihydrotestosterone in T lymphocytes by analysis of the enzymes involved. Such enzymatic assessment is meaningful because of the high intracellular steroid turnover and the limitations of steroid quantification in the T lymphocytes.
The pathway of testosterone metabolism in T lymphocytes and the enzymes involved are shown in Fig. 6. The enzymes engaged in 5α-dihydrotestosterone catabolism are 3α-HSD in the conversion of 5α-dihydrotestosterone into 5α-androstane-3α,17β-diol and 17β-HSD (oxidative form) in the formation of androsterone from 5α-androstane-3α,17β-diol. Androsterone is an inactive steroid because of the lack of C-17 hydroxyl function essential for androgen receptor binding (12,15, 33). Our results indicate that T lymphocytes of intact males following trauma-hemorrhage, when assayed with appropriate substrates, show enhanced activity of 3α-HSD and reduction in the activity of 17β-HSD. Furthermore, the changes observed in the enzyme activities agree with changes in enzyme expression by PCR analysis. Our previous study (23) as well as the present one shows increased 5α-reductase activity in T lymphocytes of intact males following trauma-hemorrhage. Thus 1) increased activity of 5α-reductase (Fig. 1 A) suggests enhanced synthesis of 5α-dihydrotestosterone, and 2) low activity of the 17β-HSD (oxidative) suggests decreased conversion of 5α-dihydrotestosterone to androsterone. The increase in 3α-HSD activity suggests that 5α-dihydrotestosterone synthesized in T lymphocytes, following trauma-hemorrhage, readily converts into 5α-androstane-3α,17β-diol and is present at equilibrium concentration with 5α-androstane-3α,17β-diol in the T lymphocytes. 5α-Androstane-3α,17β-diol is a weak androgen, and the reversible catalysis of 3α-HSD allows for continued presence of androgen in the receptor-active form in the T lymphocytes.
17β-HSD is an oxidoreductase that catalyzes the interconversion of 17-keto and 17β-hydroxy steroids. Because both androgens and estrogens exhibit the highest receptor-binding activity in the 17β-hydroxy form, this enzyme plays an important role in the production of active steroids for T lymphocyte functions. In this regard, several isoforms of 17β-HSD that display tissue, substrate, and reaction specificities have been identified (13, 20). Among the isomers, types II, IV, and VI catalyze the oxidation of 17-hydroxy steroids into inactive 17-keto steroids. Our results indicate alteration in type IV, but not type II, expression in T lymphocytes from intact males following trauma-hemorrhage. The other isomer with oxidative function is type VI, which is established as a catalytic component of CRAD2, a retinal dehydrogenase that catalyzes 3α-HSD reductive activity as well as 17β-HSD oxidative reactions (5, 29). Our results also show that the CRAD2 expression increases in T lymphocytes of intact males following trauma-hemorrhage. This increased expression appears to be associated with 3α-HSD reductive and not with the 17β-HSD type VI oxidative function because less androsterone was formed in T lymphocytes following trauma-hemorrhage when 5α-androstane-3α,17β-diol was used as the substrate (Fig. 1 D in the enzyme assay). Because CRAD2 is involved in the synthesis as well as the catabolism of 5α-dihydrotestosterone in vivo, increased expression of the enzyme in T lymphocytes following trauma-hemorrhage emphasizes the importance of the enzyme in the regulation of the active steroid in T lymphocyte functions.
Our earlier studies implicate 17β-estradiol for protection of immune functions in proestrus females following trauma-hemorrhage (3,35). 17β-Estradiol is also synthesized from testosterone by aromatase, and the immunoprotective role of 17β-estradiol was confirmed in our recent studies that showed the restoration of immune functions in intact males as well as ovariectomized females following trauma-hemorrhage by the administration of 17β-estradiol (10,11). It could be argued that administration of 17β-estradiol in males following trauma-hemorrhage should not be beneficial because 5α-dihydrotestosterone, which is synthesized more in T lymphocytes,1) is unlike testosterone, resistant to aromatization, and2) inhibits aromatase activity (6, 26). However, we observed a decreased expression of aromatase in T lymphocytes of intact males following trauma-hemorrhage. Thus the results collectively suggest that the increased synthesis of 5α-dihydrotestosterone together with decreased conversion of 5α-dihydrotestosterone into androsterone and the decreased synthesis of 17β-estradiol appear to contribute to the suppression of T lymphocyte functions in intact males following trauma-hemorrhage. In the present study, suppression of T lymphocyte function was noticed as a result of the lowered release of IL-2 and IL-6 by T lymphocytes following trauma-hemorrhage.
Our previous studies have shown that castration of males before trauma-hemorrhage prevents immune suppression following trauma-hemorrhage (36); however, the precise mechanism responsible remains unknown. The present study suggests that the synthesis of 5α-dihydrotestosterone was not high enough in T lymphocytes of precastrated animals to interfere with cytokine production due to the lack of 5α-reductase activity in the lymphocytes (23). The absence of 5α-reductase expression in T lymphocytes of castrated males was expected because sex steroids have been shown to regulate the 5α-reductase gene differently in the androgen-sensitive and androgen-insensitive tissues. Moreover, precastration also resulted in increased expression of oxidative isomer types II and V, whose expressions were not altered following trauma-hemorrhage in noncastrated mice. This finding again emphasizes the critical role of 17β-HSD in the regulation of active steroid for T lymphocyte functions. Thus, on the basis of the enzyme activities and the release of IL-2 and IL-6 by T lymphocytes following trauma-hemorrhage, it is reasonable to conclude that increased synthesis and decreased catabolism of 5α-dihydrotestosterone are the likely reasons for the immune suppression in males following trauma-hemorrhage. The presence of androgen receptor in the lymphocytes allows 5α-dihydrotestosterone to interact with the receptor and regulate the release of cytokines, thus contributing to the loss of splenocyte functions following trauma-hemorrhage. However, the precise mechanism by which this occurs remains to be determined.
Testosterone is metabolized into 5α-dihydrotestosterone or 17β-estradiol in vivo to serve important immunoregulatory functions. Our studies demonstrate the presence of enzymes required for 5α-dihydrotestosterone synthesis and catabolism, as well the presence of androgen and estrogen receptors in T lymphocytes where regulated release of cytokines occurs (23, 25). The mechanisms involved in the cytokine release thus include not only the classic steroid-hormone-receptor complex regulation of gene transcription but also tissue-specific end-organ metabolism of steroid hormones. The end-organ metabolism by enzymatic processes provides a biochemical means to create a unique transcriptional regulatory microenvironment within the specific lymphoid tissue. Thus these studies point to the likely regulation of cytokine release in T lymphocytes by changes in the activities of enzymes involved in sex steroid metabolism.
This investigation was supported by National Institute of General Medical Sciences Grant R01-GM-37127.
Present address of C. P. Schneider: Dept. of Surgery, Klinikum Grosshadern, Marchionenstr, 80933 Munich, Germany.
Address for reprint requests and other correspondence: I. H. Chaudry, Center for Surgical Research, Univ. of Alabama School of Medicine, G094, Volker Hall, 1670 Univ. Boulevard, Birmingham, AL 35294-0018 (E-mail:).
↵1 Steroid nomenclature: androstenedione, 4-androstene-3,17-dione; androsterone, 3α-hydroxy-5α-androstan-17-one; 3α-androstanediol, 5α-androstane-3α,17β-diol; 5α-dihydrotestosterone, 17β-hydroxy-5α-androstan-3-one; 17β-estradiol, 1,3,5(10)-estratriene-3,17β-diol; estrone, 3-hydroxy-1,3,5(10)-estratriene-17-one. 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. Section 1734 soley to indicate this fact.
First published February 6, 2002;10.1152/ajpcell.00560.2001
- Copyright © 2002 the American Physiological Society