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Endocrinology 142(8):3519 –3529 Copyright © 2001 by The Endocrine Society

Divergent Immune Responses in Male and Female Mice after Trauma-Hemorrhage: Dimorphic Alterations in T Lymphocyte Steroidogenic Enzyme Activities ¨ FERL*, RUI ZHENG, T. S. ANANTHA SAMY, MARKUS W. KNO MARTIN G. SCHWACHA, KIRBY I. BLAND, AND IRSHAD H. CHAUDRY Center for Surgical Research and Department of Surgery, University of Alabama, Birmingham, Alabama 35294 Immune responses are suppressed in males, but not in proestrous females, after trauma-hemorrhage. Testosterone and 17␤-estradiol appear to be responsible for divergent immune effects. There is considerable evidence to suggest sex steroid hormone involvement in immune functions. As formation of active steroid depends on the activity of androgen- and estrogen-synthesizing enzymes, expression and activity of 5␣reductase, aromatase, and 3␤- and 17␤- hydroxysteroid dehydrogenases were determined in spleen and T lymphocytes of male and proestrous female mice after trauma-hemorrhage. All of the enzymes were present in spleen, specifically in T lymphocytes. 5␣-Reductase expression and activity increased in male T lymphocytes, whereas aromatase activity, but not expression, increased in female T lymphocytes. Increased 5␣-

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ELL-MEDIATED IMMUNE responses are markedly suppressed in male mice after trauma-hemorrhage, whereas they are enhanced in proestrous females under such conditions (1, 2). Castration of males before trauma-hemorrhage (2) or administration of flutamide, an androgen receptor antagonist, in males after trauma-hemorrhage (3) prevented immunosuppression and decreased mortality rates from subsequent sepsis. These findings implicated testosterone as a causative factor for the immunosuppression observed in males under such conditions. Although immune functions are enhanced in proestrous female mice after trauma-hemorrhage, they are significantly suppressed in ovariectomized females (4). Furthermore, administration of 17␤estradiol after trauma-hemorrhage in male mice restores immune functions (5). Thus, the primary gonadal steroids, testosterone and 17␤-estradiol, appear to elicit divergent immune responses in males and females after trauma-hemorrhagic shock. Testosterone and 17␤-estradiol, primarily synthesized in the gonads and to a lesser extent in the adrenal glands (⬍1% of gonads), exert a large array of biological effects. They regulate cellular functions through interaction with cognate receptors (6, 7), and their effects are found in many tissues, including those populated with immune cells. Earlier studies have shown that immune cells express receptors for androgen and estrogen (8 –10). These receptors function as transcription factors, which regulate several cytokine genes (11– Abbreviations: 3␤HSD, 3␤-Hydroxysteroid dehydrogenase; 17␤HSD, 17␤-hydroxysteroid dehydrogenase.

reductase activity in male T lymphocytes is immunosuppressive because of increased 5␣-dihydrotestosterone synthesis, whereas in females increased aromatase activity triggering 17␤-estradiol synthesis is immunoprotective. This study also demonstrates the importance of 17␤-hydroxysteroid dehydrogenase oxidative and reductive functions. The immunoprotection of proestrous females is associated with enhanced reductase function of the enzyme. In males, decreased expression of oxidative isomer type IV, which impairs catabolism of 5␣-dihydrotestosterone, probably augments immunosuppression. This study provides evidence for the involvement of intracrine sex steroid synthesis in gender dimorphic immune responses after trauma-hemorrhage. (Endocrinology 142: 3519 –3529, 2001)

18). Alterations in the release of pro- and antiinflammatory cytokines have been demonstrated in males after traumahemorrhage. Although studies show that sex steroids play a significant role in T lymphocyte functions (8, 9), it is unclear whether their endogenous synthesis in T lymphocytes is needed for the regulation of immune functions. Our objective, therefore, was to ascertain whether active steroids needed for receptor binding and activation are synthesized locally in the spleen and T lymphocytes and their metabolism altered after trauma-hemorrhage. A number of studies have shown the presence of steroidogenic enzymes, 5␣-reductase, aromatase, 3␤-hydroxysteroid dehydrogenase (3␤HSD), and 17␤-hydroxysteroid dehydrogenase (17␤HSD), that participate in the biosynthesis of testosterone, its active metabolite 5␣-dihydrotestosterone, and 17␤-estradiol in peripheral tissues besides the gonads and adrenal glands (19 –24). Our results show the presence of these enzymes in the mouse spleen, specifically in T lymphocytes. Furthermore, trauma-hemorrhage altered the expression and activity of 5␣-reductase, aromatase, and 17␤HSD in T lymphocytes in a gender-dimorphic manner. As T lymphocytes express receptors for androgen and estrogen (8, 9), alterations in the activity of these enzymes after trauma-hemorrhage affect the availability of active steroids needed for receptor binding and activation. Thus, investigation of the expression and activity of androgen- and estrogen-metabolizing enzymes is important for determining the mechanism of alteration in T lymphocyte functions after trauma-hemorrhage.

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Endocrinology, August 2001, 142(8):3519 –3529

Materials and Methods Chemicals Analytical grade reagents were used in all the experiments. [1,2,6,7H]Androstene-4-ene-3,17-dione (SA, 74 Ci/mmol), [4-14C]androstene4-ene-3,17-dione (SA, 54 Ci/mmol), [4-14C]testosterone, [4-14C]5␣-dihydrotestosterone (SA, 57 Ci/mmol), [4-14C]17␤-estradiol (SA, 54 Ci/mmol), [4-14C]estrone (SA, 56 Ci/mmol), [4-14C]dehydroepiandrosterone (SA, 55 Ci/mmol), [7-3H]pregnenolone (SA, 25 Ci/mmol), and [1,2,6,73 H]progesterone (SA, 110 mCi/mmol) were obtained from NEN Life Science Products (Boston, MA). The unlabeled steroids were obtained from Sigma (St. Louis, MO). ␤-Actin amplimers were purchased from CLONTECH Laboratories, Inc. (Palo Alto, CA). 3

Samy et al. • Alterations in T Lymphocyte Steroidogenic Enzymes

steroid and 50 ␮m unlabeled steroid dissolved in 5 ␮l ethanol). The reaction was initiated by the addition of NADPH at a final concentration of 5 mm. Incubations were carried out for 1 h at 37 C and were terminated by addition of 1 ml dichloromethane. The organic phase was collected by centrifugation, and the aqueous phase was extracted twice with 1 ml dichloromethane. The pooled organic phases were evaporated under nitrogen; the residue was dissolved in 100 ␮l methanol and subjected to TLC on Silica gel 60A plates (Whatman, Clifton, NJ). The mobile phase used was ethyl acetate/chloroform (3:1, vol/vol). The radioactivity of the separated steroids in the chromatographic plates was measured using the InstantImager (Packard, Downers Grove, IL). The steroids were identified by comparison to the Rf values of standards.

Aromatase activity Experimental animals Inbred C3H/HeN 6- to 8-wk-old male and female mice, weighing 20 –25 g, were obtained from Charles River Laboratories, Inc. (Wilmington, MA). The animal studies were conducted according to the guidelines set by the NIH and the protocol approved by the University of Alabama institutional animal care and use committee.

Experimental groups Animals were assigned to the following groups (n ⫽ 12/group): male sham, male undergoing trauma-hemorrhage, proestrous female sham, proestrous female undergoing trauma-hemorrhage, castrated male sham, castrated male undergoing trauma-hemorrhage, ovariectomized female sham, and ovariectomized female undergoing trauma-hemorrhage.

Castration and ovariectomy The protocols described by Waynforth (25) were followed for castration of male and ovariectomy of female mice. Two weeks after castration or ovariectomy, the animals were used in experiments.

Trauma-hemorrhage The procedure for inducing trauma (laparotomy)-hemorrhage was described in detail previously (26, 27). Briefly, 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 mm Hg, maintained at that pressure for 90 min and then resuscitated with 4 times the volume of blood drawn with Ringer’s lactate solution. Sham-operated mice underwent the same anesthetic and surgical procedures, but hemorrhage and resuscitation were not carried out. The animals were killed 2 h after resuscitation, and blood, spleen, adrenal glands, testes, ovaries, and brown adipose tissue were removed for analysis.

Preparation of splenocytes and enrichment of T and B lymphocytes The procedure for the preparation of splenocytes was described in our earlier publication (27). T lymphocyte enrichment was accomplished by passage of the splenocyte suspension through a nylon wool column packed to 10 ml in 20-ml syringes. The resultant cells were more than 90% T lymphocytes (27, 28). Petri dishes coated with antimouse Ig antibodies were used for panning and isolation of B lymphocytes from the splenocyte suspension (28, 29).

5␣-Reductase activity Steroid 5␣-reductase activity was assayed by the procedure of Andersson et al. (30). Each tissue was homogenized in 10 vol 10 mm potassium phosphate buffer, pH 7.0, containing 150 mm KCl and 1 mm EDTA and centrifuged at 3,000 ⫻ g for 15 min at 4 C. The supernatant was centrifuged at 100,000 ⫻ g for 30 min at 4 C. The microsomal pellet was suspended in the homogenization buffer at 25 ␮g/10 ␮l and stored at ⫺70 C. The reaction mixture for the 5␣-reductase assay consisted of a 20-␮l aliquot of cell homogenate (50 ␮g protein) in 0.5 ml 100 mm potassium phosphate buffer, pH 6.6, and testosterone (1 ␮Ci 14C-labeled

The aromatase activity was assayed by the procedure of Thompson and Siiteri (31). Each tissue was homogenized in 3 vol 50 mm Trismaleate buffer, pH 7.4, containing 1 mm ␤-mercaptoethanol, 40 mm niacinamide, and 250 mm sucrose. The homogenate was centrifuged at 5,000 ⫻ g for 15 min, and the supernatant was centrifuged at 150,000 ⫻ g for 30 min. The microsomal pellet was washed three times with the same buffer. The last washing consisted of centrifugation in Tris-maleate buffer without sucrose. The microsomal pellet was suspended in the homogenization buffer supplemented with 20% glycerol and stored at ⫺70 C. The reaction mixture for the aromatase activity consisted of 100 ␮l microsomal preparation (50 ␮g protein) and steroids (0.5 ␮Ci radiolabeled and 5 ␮mol unlabeled in ethanol) in 1.5 ml 50 mm Tris-maleate buffer, pH 7.4, containing 0.5 mm dithiothreitol, 40 mm niacinamide, and 250 mm sucrose. [3H]Androstenedione or [14C]testosterone was used as substrate in these assays. The assay mixture was preincubated for 10 min at 37 C, and the reaction was started by the addition of 150 ␮l of a solution consisting of 10 mm NADPH, 50 mm glucose-6-phosphate, and 62.5 U glucose-6-phosphate dehydrogenase. The 1-h reaction at 37 C was terminated by the addition of 2 vol chloroform. For 3H20 release, 1 ml 10% activated charcoal with 1% dextran T70 was added. After centrifugation at 10,000 ⫻ g for 10 min, the radioactivity in 500 ␮l supernatant was measured after the addition of 5 ml liquid scintillation cocktail in the scintillation counter (Wallac, Inc., Gaithersburg, MD). For estimating [14C]17␤-estradiol conversion from [14C]testosterone, the reaction mixture was extracted twice with 2 vol dichloromethane. After removal of the organic solvent, the residue was dissolved in 100 ␮l methanol and subjected to TLC on silica gel plates with chloroform/ethyl acetate (3:1, vol/vol) as the mobile phase. The separated steroids in the chromatographic plates were measured for radioactivity with the InstantImager.

3␤HSD and 17␤HSD activities The procedure of Sturgeon et al. (32) was followed for the assay of 3␤HSD and 17␤HSD activities. The tissue was homogenized in 100 mm potassium phosphate buffer, pH 7.4, containing 20% glycerol and 10 mm EDTA. The homogenate was centrifuged at 3,000 ⫻ g for 15 min at 4 C to remove the cell debris and then at 105,000 ⫻ g at 4 C for 45 min. The microsomal pellet was dissolved in 100 mm phosphate buffer, pH 7.4, containing 10 mm EDTA, but without glycerol, and was stored at ⫺70 C until analysis. This microsomal preparation was used for assay of both 3␤HSD and 17␤HSD activities. The 3␤HSD activity was assayed in 500 ␮l 50 mm NaH2PO4 buffer, pH 7.4, containing 20% glycerol and 10 mm EDTA. A 20-␮l aliquot of the microsomal preparation (50 ␮g protein) was added, and the reaction mixture was incubated at 37 C in the presence of [14C]dehydroepiandrosterone or [3H]pregnenolone and 1 mm NADPH for 1 h. The reaction was stopped by the addition of 2 vol chloroform. The steroids were extracted with diethyl ether, and the organic solvent was evaporated to dryness. The residue was dissolved in 100 ␮l methanol and chromatographed on silica gel-coated TLC plates with toluene/acetone (80:20, vol/vol) as the mobile phase. The 14C radioactivity of separated steroids was quantified by the InstantImager, and 3H-associated radioactivity was quantified in the scintillation counter after the addition of 5 ml scintillation fluid. The identity of separated steroids was established by comparison with pure samples. The 17␤HSD assay was carried out in 500 ␮l 100 mm potassium phosphate buffer, pH 7.4, containing 50 ␮g protein (20 ␮l microsomal preparation), 1 mm NADPH, 5 ␮m androstenedione or estrone, and 0.5


␮Ci [14C]androstenedione or [14C]estrone. The reaction was carried out at 37 C for 1 h and was terminated with the addition of methanol. The steroids in the reaction mixture were extracted twice with 3 vol dichloromethane. The steroid residue was recovered by evaporation of the organic solvent under N2 and separated on thin layer silica gel chromatographic plates using toluene/acetone (4:1, vol/vol) as the mobile phase. The steroid-associated radioactivity in the silica gel TLC plates were measured by InstantImager, and steroids were identified by comparison with authentic samples.

Assay of 17␤HSD oxidative functions The spleen homogenate preparation used for reductive activity was also used to assay the oxidative reaction of 17␤HSD. The assay was carried out at pH 7.4 and 8.6. The reaction mixture (500 ␮l) consisted of 100 mm NaH2P04, 0.5 mm dithiothreitol, 5 ␮m 17␤-estradiol, 0.1 ␮Ci [14C]17␤-estradiol, and a 20-␮l aliquot (50 ␮g protein) of the microsomal preparation. After incubation of the assay mixture for 10 min at 37 C, the reaction was started by the addition of a NAD⫹ or NADP⫹ (1 mm). There was no difference in the enzyme oxidative activity between the two cofactors. After additional incubation at 37 C for 1 h, the reaction was stopped by the addition of 2 vol chloroform and was extracted twice in chloroform. The steroids in the pooled organic phase were evaporated to dryness. The residue was dissolved in 100 ␮l methanol and separated by TLC on silica gel as described above. The radioactivity of the separated steroids was measured with the InstantImager.

Protein content The protein content of the microsomal preparations was determined by the micro Bradford method (Bio-Rad Laboratories, Inc., Hercules, CA). BSA was used as a standard.

Enzyme kinetics Kinetic constants for steroid substrates were determined by conventional Lineweaver-Burk analysis. All assays were carried out in triplicate using microsomal preparations of tissue homogenates. Ten concentrations of substrates between 1–200 ␮m 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.

PCR amplification of reverse transcribed mRNA The RNA was prepared from the purified T lymphocytes using the Atlas pure total RNA kit (CLONTECH Laboratories, Inc., Palo Alto, CA) and was purified by treatment with deoxyribonuclease (1 U/␮l) for 30 min at 37 C. Poly(A)⫹ mRNA preparation and RT-PCR reaction were carried out using the Access RT-PCR kit (Promega Corp., Madison, WI). The RT-PCR reaction mixture (50 ␮l) 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 ␮m deoxy-NTP mix, 1 mm MgSO4, 0.1 U AMV reverse transcriptase, 0.1 U TfI DNA polymerase, and 1 ␮m of each of the primers (Table 1). The enzyme gene sequences were chosen from the GenBank database, and primers were selected using the software, www.genome.wi.mit.edu/genome_ software/


other/primer3.html. The oligonucleotide primers were synthesized by BRL Life Technologies, Inc. (Gaithersburg, MD). The PCR reaction was carried out in the Mastercycler gradient (Eppendorf, Westbury, NY). To optimize reaction conditions, the amplification was carried out initially at 8 different cycle points, from 5– 40 with increments of five cycles. PCR products begin to appear after 15 cycles, and PCR expression at the 25th cycle was used in the comparative evaluations. The first cycle of reverse transcriptase reaction was carried out at 48 C for 45 min, and 25 cycles of amplification were performed sequentially at 94 C for 30 s, 60 C for 1 min, 68 C for 2 min, and final extension at 68 C for 7 min. The amplification of ␤-actin was used as the internal control. The PCR products were analyzed by electrophoresis on 1.5% agarose gels in Trisacetate-EDTA buffer and were visualized by ethidium bromide staining under UV illumination. The intensity of cDNA bands was measured in the 500 Fluorescence ChemiImager (San Leandro, CA).

Statistical analysis SigmaStat software version 2.0 (Jandel Scientific) was used in all nonlinear regression analysis. All data were analyzed by separate oneway ANOVA. When a significant F value was obtained, the effects were differentiated using Tukey’s test. Tests between effects were performed by Student’s t test. Significance was achieved at P ⱕ 0.05.

Results 5␣-Reductase, aromatase, 3␤HSD, and 17␤HSD activities in mouse tissues

The activities of the above enzymes in the adrenal gland, gonads, and spleen of male and proestrous female mice are shown in Table 2. The 5␣-reductase activity, assessed for the conversion of testosterone to 5␣-dihydrotestosterone, was present in the adrenal glands, gonads, and spleen of both male and female mice. In the adrenals, 5␣-reductase activity was more than 2-fold higher in males compared with females. In testes, 5␣-reductase activity was 100-fold higher than that in the ovaries. Spleen from males showed 2-fold higher reductase activity compared with spleen from females. Ovaries and the adipose tissue exhibited very low 5␣-reductase activity. The aromatase activity, assessed for conversion of testosterone to 17␤-estradiol, was higher in the ovaries and adipose tissue compared with the adrenal glands and spleen. This enzyme activity was barely detectable in the testes. The activity of 3␤HSD, assessed for the conversion of dehydroepiandrosterone to androstenedione, was higher in the adrenal glands and gonads compared with the other tissues. No significant difference in the activities of 3␤HSD was observed in the tissues of males and females. The activity of 17␤HSD, assessed for the reductive conver-

TABLE 1. PCR primers used in mRNA phenotype analysis of steroidogenic enzymes Target mRNA (33–38)

5␣-Reductase type II (33) Aromatase P450 CYP19 (34) 3␤HSD (35) 17␤HSD type II (36) 17␤HSD type IV (37) 17␤HSD type V (38)

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Primer sequences

Location

5⬘-CAGTGGTACTAAGCACAGAAACTCAC-3⬘ 5⬘-AGCTACACCTGAAGATTTACTTCACC-3⬘ 5⬘-CTTTCAGCCTTTTGGCTTTG-3⬘ 5⬘-TTTCTTCACTGGTCCCCAAC-3⬘ 5⬘-TACCTTGCTTTCCTGCTATCTCTACT-3⬘ 5⬘-GACTTCACTTTAGTGCTGTCTTTCTG-3⬘ 5⬘-TCCTGGCCATAGTTCTCTCCTG-3⬘ 5⬘-CAAGGCGTTTCTGCCTCTACTT-3⬘ 5⬘-GGATTTTCTGCAAGGCATGT-3⬘ 5⬘-CCCTTCAGAGCTTGGCATAG-3⬘ 5⬘-TTCTGACTTCCATGGGCATT-3⬘ 5⬘-TAGACGCACTGTCTGCTGCT-3⬘

3793–3819 4245– 4271 1324 –1343 1754 –1773 1086 –1111 1560 –1585 697–718 971–990 1621–1640 2105–2224 63– 84 345–365

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sion of androstenedione to testosterone, was present in the adrenal glands, gonads, and spleen. Adrenal glands and gonads expressed higher 17␤HSD activity than spleen. No significant activity of 17␤HSD was detected in the adipose tissue. Steroidogenic enzyme activity in T and B lymphocytes

The T and B lymphocytes from male and female spleens were assayed for 5␣-reductase, aromatase, 3␤HSD, and 17␤HSD activity, and the results are presented in Table 3. All four enzyme activities were present in T lymphocytes. The activity of 5␣-reductase, aromatase, and 3␤HSD was very low in B lymphocytes. Moreover, there were no 17␤HSD activities in the B lymphocytes. Most of the activities of the splenic steroidogenic enzymes (⬎85%) were localized in the T lymphocytes. Effect of trauma-hemorrhage on the steroidogenic enzyme activity

The activities of 5␣-reductase, aromatase, 3␤HSD, and 17␤HSD in tissues and T lymphocytes of male and female mice after trauma-hemorrhage are compared with those of sham controls in Figs. 1 and 2. Trauma-hemorrhage resulted in increased 5␣-reductase activity in male tissues and increased aromatase activity in female tissues (Fig. 1). A significant increase in 5␣-reductase activity was observed in testes, spleen, and T lymphocytes compared with adrenal glands in males after trauma-hemorrhage; the increase in enzyme activity was more than 2-fold higher in spleen and T lymphocytes (Fig. 1). Trauma-hemorrhage did not affect 5␣-reductase activity in tissues from proestrous female mice. A significant (⬎2-fold) increase in aromatase activity was seen in all tissues of female mice after trauma-hemorrhage. The increase was more than 3-fold higher in spleen and T lymphocytes. In contrast, no significant change in aromatase activity was observed in male mice after trauma-hemorrhage. Trauma-hemorrhage did not alter 3␤HSD activities in tissues of either males or females (Fig. 2). Although there was

no change in 17␤HSD reductive activities in male tissues after trauma-hemorrhage, a significant (⬎5-fold) increase in the reductive activity occurred in the spleen and T lymphocytes of female mice after trauma-hemorrhagic shock. Enzyme expression in T lymphocytes after trauma-hemorrhage

The expressions of 5␣-reductase, aromatase (P450 CYP19), 3␤HSD, and type II, IV, and V isoforms of 17␤HSD in T lymphocytes from male and female mice after traumahemorrhage were assayed by RT-PCR analysis. In the male mice (Fig. 3), the enhanced expression of 5␣-reductase type II was consistent with the increased enzyme activity after trauma-hemorrhage. There was, however, a decrease in the expression of aromatase and no alteration in 3␤HSD expression. Of the 17␤HSD isomers, type IV showed decreased expression after trauma-hemorrhage. In female mice (Fig. 4), the expressions of aromatase and 3␤HSD did not change after trauma-hemorrhage. Of the 17␤HSD isomers, only the type II isomer (294 bp) showed a slight increase in expression after trauma-hemorrhage, whereas the expression of types IV and V isomers did not change. 5␣-Reductase II expression was not present in female T lymphocytes. It should be noted, however, that the enzyme expressions observed in the T lymphocytes are comparative and semiquantitative at best, and thus, quantitative expression of mRNA is needed for establishing differences in gene transcriptions after trauma-injury. Effect of gonadectomy on the activities of enzymes in male and female mice

The effect of castration and ovariectomy on the activities of 5␣-reductase, aromatase, 3␤HSD, and 17␤HSD in the tissues of sham and trauma-hemorrhaged mice are shown in Figs. 5 and 6. The castration of males increased 5␣-reductase activity only in the adrenal gland (Fig. 5). Furthermore, no

TABLE 2. Activities of 5␣-reductase, aromatase, 3␤HSD, and 17␤HSD (reductive) in mouse tissues 5␣-Reductasea (pmol/mg protein䡠min)

Adrenal gland Testes Ovary Spleen Adipose tissue

Male

Female

185.6 ⫾ 37.0 120.0 ⫾ 5.7

85.5 ⫾ 28.5

53.7 ⫾ 15.2 2.5 ⫾ 0.9

1.25 ⫾ 0.5 26.5 ⫾ 7.9 1.5 ⫾ 0.2

Aromataseb (pmol/mg protein䡠min) Male

5.8 ⫾ 2.3 0.9 ⫾ 0.1 6.5 ⫾ 1.5 10.5 ⫾ 2.3

Female

7.4 ⫾ 1.1 39.1 ⫾ 5.1 5.7 ⫾ 1.2 13.6 ⫾ 1.8

3␤HSDc (pmol/mg protein䡠min)

17␤HSDd (pmol/mg protein䡠min)

Male

Female

Male

Female

25.5 ⫾ 1.8 36. ⫾ 5.8

27.3 ⫾ 2.8

9.8 ⫾ 0.8 11.4 ⫾ 1.0

8.8 ⫾ 0.5

8.8 ⫾ 0.8 ND

47.6 ⫾ 9.5 7.9 ⫾ 0.4 ND

4.8 ⫾ 0.5 ND

7.8 ⫾ 0.8 5.0 ⫾ 0.4 ND

Conversion of testosterone to 5␣-dihydrotestosterone (a), testosterone to 17␤-estradiol (b), dehydroepiandrosterone to androstenedione (c), and androstenedione to testosterone (d). Values (mean ⫾ SD) were obtained from five different experiments performed in triplicate. ND, Not detected. TABLE 3. Activities of 5␣-reductase, aromatase, 3␤HSD, and 17␤HSD (reductive) in mouse splenic T and B lymphocytes

T Lymphocytes B Lymphocytes

5␣-Reductasea (pmol/mg protein䡠min)

Aromataseb (pmol/mg protein䡠min)

3␤HSDc (pmol/mg protein䡠min)

17␤HSDd (pmol/mg protein䡠min)

Male

Female

Male

Female

Male

Female

Male

Female

47.8 ⫾ 13.3 1.4 ⫾ 0.6

23.5 ⫾ 4.9 0.9 ⫾ .6

3.8 ⫾ 1.2 0.5 ⫾ 0.1

4.3 ⫾ 1.2 1.9 ⫾ 0.7

8.3 ⫾ 0.8 1.0 ⫾ 0.2

8.1 ⫾ 0.5 1.2 ⫾ 0.4

5.3 ⫾ 1.1 ND

6.1 ⫾ 1.2 ND

Conversion of testosterone to 5␣-dihydrotestosterone (a), testosterone to 17␤-estradiol (b), dehydroepiandrosterone to androstenedione (c), and androstenedione to testosterone (d). Values (mean ⫾ SD) were obtained from five different experiments performed in triplicates. ND, Not detected.



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FIG. 1. The effect of trauma-hemorrhage on the activity of 5␣-reductase and aromatase in different tissues of male and female mice. Testosterone was the substrate for both 5␣-reductase and aromatase activities. u, Sham; f, trauma-hemorrhage. n ⫽ 8. Data are expressed as the mean ⫾ SD. *, P ⬍ 0.05 vs. sham control.

change in 5␣-reductase activity was observed in ovariectomized females. A significant increase in aromatase activity was observed only in the adipose tissue of ovariectomized females and not in the ovary or spleen after trauma-hemorrhage. Castration had no effect on the aromatase activity of tissues from sham or trauma-hemorrhaged male mice (Fig. 5). Similarly, gonadectomy of male and female mice before trauma-hemorrhage did not change the activity of 3␤HSD or 17␤HSD (reductive) in any tissue (Fig. 6). A significant observation, however, was the difference in the reductive function of 17␤HSD in spleen and T lymphocytes of proestrous mice and mice ovariectomized after trauma-hemorrhage. The activity of aromatase and 17␤HSD (reductive) were severalfold higher in trauma-hemorrhaged proestrous female mice than in ovariectomized females that underwent the same insult (compare Figs. 1 and 5, and Figs. 2 and 6). Properties of 5␣-reductase, aromatase, 3␤HSD, and 17␤HSD from spleen

As our experiments showed the presence of 5␣-reductase, aromatase, 3␤HSD, and 17␤HSD in the spleen and T lymphocytes, and their activities were altered in the tissues after trauma-hemorrhage, the kinetic properties of the enzymes were determined. The microsomal enzyme preparations from male spleens were used for the determination of max-

imum velocity (Vmax) and Km (Table 4). The Vmax and Km of 5␣-reductase with testosterone as the substrate were 150 pmol/mg protein䡠min and 24 nm, respectively. The Vmax and Km of aromatase differed for testosterone and androstenedione: 200 pmol/mg protein䡠min and 7.5 nm vs. 186 pmol/mg protein䡠min and 25.8 mm. The reaction catalytic efficiencies, calculated as Vmax/Km, were 26.6 and 7.2, respectively, suggesting that the catalytic conversion of testosterone to 17␤estradiol was greater than that of androstenedione to estrone. The Vmax and Km of 3␤HSD for reductive catalysis of dehydroepiandrosterone and pregnenolone were also different: 51 pmol/mg protein䡠min and 35 nm vs. 21 pmol/mg protein䡠min and 25 nm, respectively. The reactive catalytic efficiencies, Vmax/Km, for dehydroepiandrosterone and pregnenolone were 1.4 and 0.8, respectively, suggesting a favored catalysis of androstenedione from dehydroepiandrosterone rather than of progesterone from pregnenolone. The Vmax and Km of 17␤HSD for reductive conversion of androstenedione to testosterone were 150 pmol/mg protein䡠min and 16.7 nm, and those for conversion of estrone to 17␤-estradiol were 120 pmol/mg protein䡠min and 48.3 nm, respectively. The reactive catalytic efficiencies, Vmax/Km, were 9.0 and 2.5, respectively, for androstenedione and estrone. These findings suggest that reductive catalysis of androstenedione to testosterone was more than that of estrone to 17␤-estradiol.

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FIG. 2. The effect of trauma hemorrhage on the activities of 3␤HSD and 17␤HSD in different tissues of male and female mice. Dehydroepiandrosterone was the substrate for 3␤HSD activity, and androstenedione was the substrate for reductive activity. u, Sham; f, traumahemorrhage. n ⫽ 8. Data are expressed as the mean ⫾ SD. *, P ⬍ 0.05 vs. sham control.

FIG. 3. The mRNA expression of steroidogenic enzymes in splenic T lymphocytes of male mice after trauma-hemorrhage. 5AR, 5␣-Reductase (479 bp); ARO, aromatase (450 bp); 17␤HSD type II, 294 bp; type IV, 605 bp; type V, 303 bp; 3␤HSD, 500 bp; S, sham; TH, traumahemorrhage.

17␤HSD oxidative activity

17␤HSD is an oxido-reductase that catalyzes the interconversion of 17␤-estradiol and estrone. The oxidative activity of the enzyme was, therefore, determined in spleen and T lymphocytes of male and female mice after trauma-hemorrhage to determine whether trauma-hemorrhagic shock has any effect on the reverse reaction (i.e. conversion of 17␤estradiol to estrone). The oxidative activity of 17␤HSD in spleen and T lymphocytes was low compared with its reductive activity. The data presented in Fig. 7 show no change in the oxidative activity of the enzyme at pH 7.8 in the spleen and T lymphocytes of sham and trauma-hemorrhaged ani-

FIG. 4. The mRNA expression of steroidogenic enzymes in splenic T lymphocytes of female mice after trauma-hemorrhage. 5AR, 5␣Reductase (479 bp); ARO, aromatase (450 bp); 17␤HSD type II, 294 bp; type IV, 605 bp; type V, 303 bp; 3␤HSD, 500 bp; S, sham; TH, trauma-hemorrhage.

mals of both genders. As the oxidative activity may be dependent on pH, assays were also carried out in phosphate buffer at pH 8.6. Although the oxidative activity was 20% higher at pH 8.6, there was still no significant difference in the activities between the sham controls and the experimental groups of both genders (data not shown). RT-PCR analysis was carried out to measure the expression of 17␤HSD isomer types II, IV, and V in T lymphocytes. Traumahemorrhage led to decreased expression of type IV isomer only in male T lymphocytes, whereas the expression of the three isotypes did not differ in female lymphocytes (Figs. 3 and 4).



3525

FIG. 5. The effect of trauma-hemorrhage on the activities of 5␣-reductase and aromatase in different tissues of castrated male (C) and ovariectomized female (Ovx) mice. Testosterone was the substrate for both 5␣-reductase and aromatase activities. u, Sham; f, traumahemorrhage. n ⫽ 8. Data are expressed as the mean ⫾ SD. *, P ⬍ 0.05 vs. sham control.

Discussion

Previous studies from our laboratory have demonstrated significant differences in the immune responses between males and proestrous female mice after trauma-hemorrhage (1, 2). Furthermore, our studies have shown divergent effects of testosterone and 17␤-estradiol on immune functions under those conditions (3, 5, 39). Immune cells express receptors for androgen and estrogen (7–9), which function as transcription factors for the regulation of several cytokine genes (10 –17). The presence of androgen- and estrogen-synthesizing enzymes in the peripheral tissues suggest active synthesis of steroids at the site of action. The rate of formation of active steroid is dependent on the level of expression and activity of the androgen- and estrogen-metabolizing enzymes in the tissue and reduces the need for the measurement of tissue steroid levels in fentomole quantities. In addition, any value obtained by quantitative analysis is likely to be meaningless due to high tissue or intracellular steroid turnover. Furthermore, as the activities of many of the steroidogenic enzymes are the outcome of coupled reactions, measurement of enzyme activity is more predictive of active steroid synthesis than is measurement of enzyme expression. Therefore, analysis of the expression and activity of androgen- and estrogenmetabolizing enzymes in T lymphocytes of male and female mice will indicate changes in the active steroid metabolism after trauma-hemorrhage. Such studies will also provide a

basis for the sex dimorphic immune responses in males and females. Studies have shown the requirement of ␤-OH function on carbon-17 of testosterone, 5␣-dihydrotestosterone, and 17␤estradiol for receptor binding (40, 41). 5␣-Reductase, aromatase, 3␤HSD, and 17␤HSD catalyze the reactions in the synthesis of these carbon-17-hydroxysteroids (Fig. 8). The data from this study demonstrate the presence of all four enzymes in the spleen and T lymphocytes as well as the adrenal glands and gonads. The activities of these enzymes, however, were tissue and gender dependent. The enhanced expression and activity of 5␣-reductase in male T lymphocytes and of aromatase in female T lymphocytes suggest an important role for these enzymes in the gender dimorphic immune responses after trauma-hemorrhage. Our studies also demonstrate that female T lymphocytes do not express 5␣-reductase type II. This is understandable, as recent studies by Mahendroo and Russell (42) have shown the presence of two isomers, I and II, each with gender specificity in reproductive functions. The 5␣-reductase activity detected in female T lymphocytes is probably from the enzyme type I isomer (42). 5␣-Dihydrotestosterone and testosterone, which are immunosuppressive in male mice after trauma-hemorrhage, are two androgens that are established ligands for the AR. The receptor binding affinity of 5␣-dihydrotestosterone is

3526



FIG. 6. The effect of trauma hemorrhage on the activity of 3␤HSD and 17␤HSD (reductive) in different tissues of castrated male (C) and ovariectomized female (Ovx) mice. Dehydroepiandrosterone was the substrate for 3␤HSD activity, and androstenedione was the substrate for the 17␤HSD reductive activity. u, Sham; f, trauma-hemorrhage. n ⫽ 8. Data are expressed as the mean ⫾ SD. *, P ⬍ 0.05 vs. sham control. TABLE 4. Kinetic constants of splenic 5␣-reductase, aromatase, 3␤HSD, and 17␤HSD with different steroid substrates Enzyme

Substrate

Product

Vmax (pmol/mg protein䡠min)

Km

Vmax/Km

5␣-Reductase Aromatase

Testosterone Testosterone Androstenedione Dehydroepiandrosterone Pregnenolone Androstenedione Estrone

5␣-Dihydrotestosterone 17␤-Estradiol Estrone Androstenedione Progesterone Testosterone 17␤-Estradiol

150 200 186 51 21 150 120

24 7.5 25.8 35 25 16.7 48.3

6.25 26.6 7.2 1.4 0.8 9.0 2.5

3␤HSDa 17␤HSDa a

Reductive activity.

6-fold higher than that of testosterone, and its transcriptional activity is also greater and more prolonged (43– 46). In male mice, the activity of 5␣-reductase increased significantly (⬎2fold) in the spleen and T lymphocytes after trauma-hemorrhage, demonstrating the increased synthesis of 5␣-dihydrotestosterone by these cells (Fig. 8). Increased production of the enzyme is evidenced by its enhanced expression in T lymphocytes after trauma-hemorrhage. Studies have shown that 5␣-dihydrotestosterone can be further metabolized by 3␣-hydroxysteroid dehydrogenase and 17␤HSD (oxidative) into androsterone (24), a metabolite that is inactive because of its inability to bind to the AR due to the lack of ␤-hydroxyl function on carbon 17. Our data show the expression of 17␤HSD type IV, which generates a decrease in oxidative function after trauma-hemorrhage. Thus, our finding suggests the predisposition of male T lymphocytes to increased levels of 5␣-dihydrotestosterone after trauma-hemorrhage.

5␣-Dihydrotestosterone appears to be a key factor for immune suppression in males after trauma-hemorrhage, as 1) the salutary effect of flutamide is due to its competition with 5␣-dihydrotestosterone for AR binding (46); 2) the absence of immunosuppression in castrated males after traumahemorrhage is related to decreased 5␣-dihydrotestosterone synthesis due to lowered 5␣-reductase activity (1, 3); and 3) this study provides evidence for the impaired catabolism of 5␣-dihydrotestosterone in T lymphocytes. Although 5␣-dihydrotestosterone levels are low in proestrous females, this steroid cannot induce immunosuppression, because 5␣-reductase type II is not expressed in female T lymphocytes and the activity of the enzyme in the spleen and T lymphocytes is low. In contrast, a significant increase in aromatase activity together with increased 17␤HSD reductive activities in proestrous females after trauma-hemorrhage demonstrate increased synthesis of 17␤-


FIG. 7. The effect of trauma hemorrhage on the oxidative activity of 17␤HSD in spleen and T lymphocytes of male and female mice. ␤Estradiol was the substrate. u, Sham; f, trauma-hemorrhage. n ⫽ 8. Data are expressed as the mean ⫾ SD.

FIG. 8. Biosynthesis of gonadal steroids.

estradiol. This would suggest that 17␤-estradiol is immunoprotective. Nevertheless, the lack of alteration in aromatase expression after trauma-hemorrhage is not consistent with the increased activity of the enzyme observed in T lymphocytes under such conditions. This was predictable, because aromatase reaction involves three separate steps, and the enzyme complex is composed of two proteins, aromatase P450 and a flavoprotein NADPH-cytochrome P450 reductase (47). The aromatase activity, assessed by the tritiated water method followed in our experiments, is determined by the coupled reaction of aromatase and NADPH-cytochrome P450 reductase. Therefore, the discrepancy observed in aromatase mRNA expression and activity may be attributed to the relative ratio of the two enzymes after trauma-hemorrhage, as revealed in another study (48). In spleen, the comparative catalytic reaction efficiencies (determined by Vmax/ Km) of aromatase and 17␤HSD (reductive) with different steroid substrates suggest that the synthesis of 17␤-estradiol from androstenedione was through the intermediate testosterone and not estrone. Interestingly, increases in the relative aromatase activities of spleen and T lymphocytes were greater than those in adrenal glands and ovary, further supporting the importance of local steroid conversion. The lack of change in 17␤HSD oxidative functions or in the expression of oxidant type IV isomer suggests less conversion of 17␤estradiol to estrone (which does not bind to ER). Moreover, the very low activity of aromatase and 17␤HSD (reductive) in spleen and T lymphocytes of ovariectomized females com-


3527

pared with proestrous female mice after trauma hemorrhage is consistent with lower 17␤-estradiol production in those animals. Thus, the decreased local production of 17␤-estradiol in ovariectomized females appears to be the reason why these animals are immunosuppressed after trauma-hemorrhage (4). The activity of 3␤HSD was not altered in either males or proestrous females after trauma-hemorrhage or gonadectomy. The relative efficiencies (Vmax/Km) of the reactions catalyzed by 3␤HSD indicate that the formation of androstenedione from dehydroepiandrosterone was greater than that of progesterone from pregnenolone. The lack of change in splenic 3␤HSD activity after trauma-hemorrhage suggests that dehydroepiandrosterone conversion to androstenedione was also minimal. It appears, therefore, that the previously observed salutary effects of dehydroepiandrosterone on immune functions in males after trauma-hemorrhage (49) are probably due to its conversion into a potent estrogen, 3␤,17␤-androstenediol (50, 51). This study implies a key role for 17␤HSD oxidative and reductive functions in the immune responses of males and females after trauma-hemorrhage. The reductive function of this enzyme appears to be an obligatory step in the biosynthesis of an active androgen or an active estrogen. The oxidative activity of 17␤HSD, on the other hand, will lead to the production of inactive steroids, androsterone from 5␣-dihydrotestosterone and estrone from 17␤-estradiol. Therefore, alterations in the ratio of 17␤HSD oxidative and reductive functions are critical for the regulation of splenic T lymphocyte function after trauma-hemorrhagic injury. A number of studies have shown the presence of several human and mouse 17␤HSD isotypes (36 –38, 41, 52–58). Each 17␤HSD isotype demonstrates a favored substrate and reaction direction and a unique tissue distribution (36, 41, 53, 56). Our recent study has demonstrated lowered release of IL-6 by splenic T lymphocytes of males after trauma-hemorrhage (10). We have evaluated the expression of three 17␤HSD isotypes, types II, IV, and V, in this study. There is a need to determine the expression of other enzyme isotypes, especially isotype I, because of their substrate, reaction, and tissue specificities. The characterization of the isotype(s) present in the splenic T lymphocytes as well as correlation to the 17␤HSD oxidative and reductive functions in relation to cytokine release by T lymphocyte subsets are probably essential for further deciphering the mechanisms involved in the gender dimorphic immune response to trauma-hemorrhagic shock. The significance of our study is that key enzymes involved in the synthesis of biologically potent sex steroids 5␣-dihydrotestosterone, testosterone, and 17␤-estradiol are present in the spleen, specifically in T lymphocytes. Furthermore, the activities of these enzymes are markedly altered after trauma-hemorrhage. The 5␣-reductase activity that is essential for conversion of testosterone to 5␣-dihydrotestosterone increased in male T lymphocytes, but not in females after trauma-hemorrhage. The aromatase activity that is required for the conversion of testosterone to 17␤-estradiol increased in T lymphocytes of proestrous female mice after traumahemorrhage, but not in males. Thus, trauma-hemorrhage led to enhancement of the activity of different steroidogenic en-

3528 Endocrinology, August 2001, 142(8):3519 –3529

zymes in T lymphocytes of males and females and provided an explanation for the divergent immune responses. Although sex steroids bind to their cognate receptors to activate cytokine genes, it remains to be elucidated which cytokine gene(s) is regulated by 5␣-dihydrotestosterone or 17␤-estradiol to produce different immune effects in males and females after trauma-hemorrhage. The precise delineation of the molecular events associated with T lymphocyte function and hormone-sensitive control sites will further establish the basis for sexual dimorphism in immune responses after trauma-hemorrhage. Acknowledgments Received February 13, 2001. Accepted April 11, 2001. Address all correspondence and requests for reprints to: Irshad H. Chaudry, Ph.D., Center for Surgical Research, Department of Surgery, University of Alabama, Volker Hall G094, 1670 University Boulevard, Birmingham, Alabama 35294. E-mail: [email protected]. This work was supported by USPHS Grant GM-37127. * Present address: Department of Trauma-Surgery, University of Ulm, Steinho¨vel Strasse 9, 89075 Ulm, Germany.

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