Mechanism for normal splenic T lymphocyte functions in proestrus females after trauma: enhanced local synthesis of 17{beta}-estradiol

T. S. Anantha Samy, Rui Zheng, Takeshi Matsutani, Loring W. Rue, III, Kirby I. Bland, and Irshad H. Chaudry

Center for Surgical Research and Department of Surgery, University of Alabama School of Medicine, Birmingham, Alabama 35294

Submitted 11 February 2003 ; accepted in final form 17 March 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Trauma-hemorrhage and resuscitation (TH) produces profound immunodepression and enhances susceptibility to sepsis in males but not in proestrus females, suggesting gender dimorphism in the immune responses. However, the mechanism responsible for the maintenance of immune functions in proestrus females after TH is unclear. Splenic T lymphocytes express receptors for estrogen (ER), contain enzymes involved in estrogen metabolism, and are the major source of cytokine production; the metabolism of 17{beta}-estradiol was assessed in the splenic T lymphocytes of proestrus and ovariectomized mice by using appropriate substrates after TH. Analysis for aromatase and 17{beta}-hydroxysteroid dehydrogenases indicated increased 17{beta}-estradiol synthesis and low conversion into estrone in T lymphocytes of proestrus but not of ovariectomized mice. The effect of 17{beta}-estradiol on T lymphocyte cytokine release was reliant on ER expressions. This was apparent in the differences of ER expression, especially that of ER-{beta}, and an association between increased 17{beta}-estradiol synthesis and sustained release of IL-2 and IL-6 in T lymphocytes of proestrus females after TH. Because 17{beta}-estradiol is able to regulate cytokine genes, and the splenic T lymphocyte cytokine releases is altered after TH, continued synthesis of 17{beta}-estradiol in proestrus females appears to be responsible for the maintenance of T lymphocyte cytokine release associated with the protection of immune functions after TH.

inflammation; immune suppression; steroid synthesis; T lymphocytes; cytokines


THE INFLUENCE OF GENDER on immune functions has been recognized for many years, and in general, women are known to develop enhanced humoral responses compared with men and are more prone to autoimmune diseases (9, 12, 20). Trauma-hemorrhage and resuscitation (TH) produce severe impairment of both immune and cardiovascular functions (51, 56, 57). Although depression of cellular immunity occurs very early following TH, the loss in immune functions persists for a prolonged period, which may lead to subsequent sepsis with high mortality rates (43, 56). The immune depression is pronounced in males and ovariectomized females (OVX) after TH compared with proestrus females (54, 55). Moreover, immune functions in males and OVX females can be restored by the administration of 17{beta}-estradiol (E2) after TH (16, 17, 18). Thus gender dimorphism is obvious in the loss of immune functions after TH, implicating a major role for sex steroid hormones (2, 4).

Steroid hormones regulate immune functions in vivo, and the mechanisms involve not only the control of cytokine gene transcription by the classic steroid hormone-receptor complex but also the tissue-specific metabolism of sex steroids (11, 21, 24, 29, 33, 36, 47). Among the sex steroids, estrogen is demonstrated to protect immune functions after TH because proestrus females are not immunodepressed compared with male and OVX mice. Furthermore, the depressed immune functions in males and OVX females after TH can be normalized by parenteral E2 administration (16, 17, 18). The ovary is the primary site of estrogen synthesis in females, and despite that, the enzymes involved in estrogen metabolism are also present in peripheral tissues, including spleen and the T lymphocytes (21, 37). The presence of steroidogenic enzymes, especially in the T lymphocytes, suggests a role for local synthesis of E2 for interaction with the estrogen receptor (ER) present in the cells (39) and the production of cytokines as needed. E2 is a highly potent regulatory sex steroid involved in a variety of metabolic functions. Because of its regulatory role, E2 seldom accumulates in the cells, and its synthesis is dependent on the tissue requirement. Thus understanding the E2 metabolism in the T lymphocytes is desirable for determining the basis for change in the cytokine releases by these cells after TH. In this regard, the expression and analysis of enzymes involved in steroid metabolism is meaningful compared with active steroid quantification, because regulatory steroids have a short half-life and quantification of steroid at subpicomole levels in the tissues or cells is ambivalent. We therefore measured the activity and expression of the enzymes involved in E2 metabolism in splenic T lymphocytes by using relevant substrates. Because the promoter regions of the cytokine genes have response elements for ER binding (24, 32, 33, 35), E2 synthesis in T lymphocytes was evaluated in conjunction with ER-{alpha} and ER-{beta} expression and IL-2 and IL-6 release (the cytokines whose release are altered after TH) in proestrus and OVX mice after TH in the same cell preparations. The results indicate that continued synthesis of E2 in splenic T lymphocytes of proestrus females appears to be responsible for the maintenance of IL-2 and IL-6 release in those cells and is probably one of the reasons why proestrus females are not immunodepressed after TH.


    MATERIAL AND METHODS
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 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
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Chemicals. Analytical grade reagents were used in all 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, {alpha}-[4-14C]dihydrotestosterone, specific activity 57 Ci/mmol; 17{beta}-[4-14C]estradiol, specific activity 54 Ci/mmol; and [4-14C]estrone, specific activity 56 Ci/mmol, were bought 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).

Mice. Inbred C3H/HeN female mice, 6–8 wk old weighing 20–25 g, were obtained from Charles River Laboratories (Wilmington, MA). The animal experiments were conducted according to the guidelines established by the National Institutes of Health and the protocols approved by the University of Alabama at Birmingham Institutional Animal Care and Use Committee.

Experimental groups. Proestrus female mice that showed a large number of nucleated epithelial cells and few cornified cells in the vaginal smear were used in the experiments. The procedure described by Waynforth et al. (52) was followed for ovariectomy, and 2 wk after ovariectomy the animals were used in experiments. Animals were assigned to the following four groups (n = 8 mice per group): female shams, females undergoing TH, OVX female shams, and OVX females undergoing TH.

Trauma-hemorrhage. The procedure for inducing trauma (i.e., midline laparotomy)-hemorrhage has been described in detail in our earlier publications (56, 57). 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. The animals were then bled rapidly to a mean arterial pressure of 30 mmHg, maintained at that pressure for 90 min, and resuscitated with four times the volume of shed blood with Ringer lactate solution. Sham-operated mice underwent the same anesthetic and surgical procedures, but neither hemorrhage nor resuscitation was carried out. The animals were killed at 2 h after resuscitation, and the spleens were removed for analysis.

Preparation of T lymphocytes. The procedures for the preparation of splenocytes and enrichment of T lymphocytes have been described in an earlier publication (36, 38). The purity of enriched lymphocytes was >95% and consisted of both CD4+ and CD8+ subsets. All analyses were carried out in the same population of T lymphocytes prepared from one mouse in each group or from a pooled population of lymphocytes prepared from two mice in each group. Approximately 109 lymphocytes were used for preparation of homogenate in enzyme assays, 106 lymphocytes for mRNA expression by PCR analysis, and 5 x 106 for bioassays.

Enzyme assays. The modified assay procedures for 5{alpha}-reductase and for 17{beta}-hydroxysteroid dehydrogenase (17{beta}-HSD) oxidative and reductive activities have been described in detail previously (1, 37, 48). The assay mixtures after the enzyme reaction were extracted five times with methylene chloride, and the steroids in the organic phase were analyzed by thin-layer chromatography (TLC) using the mobile phase of chloroform-ethyl acetate (3:1, vol/vol). The radioactivity of the separated steroids in the chromatographic plates was measured by using InstantImager (Packard, Downers Grove, IL), and steroids were identified by comparison with the Rf values of standards.

The aromatase activity was assayed by the procedure of Thompson and Siiteri (45). [3H]androstenedione and [14C]-testosterone were used as substrates in these assays. For estimation of 3H20 release, 1 ml of 10% activated charcoal with 1% dextran-T70 was added to the assay mixture. After centrifugation at 10,000 g for 10 min, the radioactivity in 500 µl of supernatant was measured after the addition of 5 ml of liquid scintillation cocktail in the scintillation counter (Wallac, Gaithersburg, MD). For estimating [14C]E2 conversion from [14C]testosterone, the reaction mixture was extracted twice with two volumes of dichloromethane. After removal of the organic solvent, the residue was dissolved in 100 µl of 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 InstantImager.

RT-PCR analysis. The RNA was prepared from T lymphocytes 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 kit (Promega, Madison, WI). The primers used in PCR analysis (Table 1) were chosen from the cDNA sequences of the GenBank, and Primer3 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 1 min of annealing at 60°C 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 38 cycles. The number of amplification cycles for measuring expression differed considerably for each enzyme. Comparison of expression between the sham and TH for each enzyme was made at the cycle where expression was nearly 50%. {beta}-Actin expression was used as the internal control. The PCR products were analyzed by electrophoresis on 1.5% agarose gels in 1x TAE (Tris-acetate-EDTA) buffer and visualized by ethidium bromide staining under UV illumination. The intensity of cDNA bands was measured in the 500 Fluorescence Chemilimager (San Leandro, CA).


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Table 1. Primers used in PCR analysis

 

Cytokine assays. The CTLL-2 cell line (TIB-214) for IL-2 assay and the hybrid cell line 7TD1 (CRL-1851) for IL-6 assay were obtained from the American Type Culture Collection (Rockville, MD). The bioassay procedures for IL-2 and IL-6 release in T cell culture supernatants have been described previously (57). The cells were stimulated with 10 µg/ml anti-CD3 (BD Biosciences, San Jose, CA) in complete Click's medium at 37°C for 36 h before the culture supernatants were assayed for the cytokine release. IL-2 activity in the T lymphocyte culture supernatants was determined by making serial dilutions of the supernatant (in 500 µl) to which CTLL-2 cells (1 x 105 cells/ml) were added. The cultures were incubated for 48 h at 37°C with 5% CO2. At the end of this time, 1 µCi of [3H]thymidine (specific activity 6.7 Ci/mmol; NEN) was added to each well and cultures were further incubated for 16 h. The cultures were then harvested with a multiple automated sample harvester (Skatron AS, Trombay, Norway) onto a glass fiber-filter mat and processed for liquid scintillation counting on a Betaplate (model 1205; Pharmacia/LKB Nuclear, Gaithersburg, MD). For the IL-6 assay, 100 µl of 7TD1 cells (5 x 105 cells/ml) were added to the serial dilutions of the lymphocyte culture supernatant and incubated for 72 h at 37°C in 5% CO2. At the end, 20 µl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; 5 mg/ml in RPMI 1640 medium) were added to each well, and the plate was incubated for an additional 4 h. The MTT crystals that incorporated into the viable cells were dissolved by aspiration of the supernatant from each well and addition of 100 µl of isopropanol containing 0.04 M HCl. The absorbance of fluids in each well was measured at 620 nm using an automated microplate reader (Bio-Tek Instruments, Winooski, VT). Relative units of cytokine activity were computed by comparison of the curves produced from dilution of the experimental samples to that generated by dilution of recombinant mouse IL-2 or IL-6 standards (R&D Systems, Minneapolis, MN).

Protein content. The protein content was determined by the micro Bradford method (Bio-Rad, Hercules, CA) with BSA as standard.

Enzyme kinetics. Kinetic constants for steroid substrates were determined by Lineweaver-Burk analysis. Assays were carried out in triplicate using microsomal preparations of tissue homogenates. Ten concentrations of substrates between 1 and 200 µM were used for each steroid. SigmaPlot software (vers. 2.0; Jandel Scientific, San Rafael, CA) was used to generate hyperbolic functions and nonlinear regression plots.

Statistical analysis. SigmaStat (vers. 2.0; Jandel Scientific) was used in nonlinear regression analysis. Data were analyzed by separate one-way ANOVA. When a significant F value was obtained, the effects were differentiated using Tukey's test. Tests between effects were performed with Student's t-test. Significance was achieved when P <= 0.05.


    RESULTS
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 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
17{beta}-Estradiol synthesis. E2 is primarily synthesized from testosterone by aromatase. The activity of aromatase increased significantly in all the tissues and splenic T lymphocytes in proestrus females after TH (Fig. 1A). Nonetheless, the increase in aromatase activity after TH in the tissues was not observed in OVX mice except in the adipose tissue, where a significant increase in the activity was observed (Fig. 1B). These results are in accordance with our earlier observation (37).



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Fig. 1. Aromatase activity in different tissues isolated from proestrus (A and C) and ovariectomized (OVX) mice (B and D) after trauma-hemorrhage (TH), with testosterone (A and B) and 4-androstenedione (C and D) as substrates. Data are expressed as means ± SD of 8 experiments for each group. *P < 0.05 vs. sham (S).

 

E2 is also synthesized from androstenedione with estrone (E1) as the intermediate in the reaction. Two enzymes participate in this catalysis: aromatase for conversion of androstenedione to E1 and 17{beta}-HSD for reduction of E1 to E2. The aromatase activity associated with androstenedione conversion to E1 in T lymphocytes of proestrus and OVX animals after TH is shown in Fig. 1, C and D. Aromatase activity in all the tissues was low with androstenedione as a substrate, compared with testosterone as the substrate, suggesting low conversion to E1 in all the tissues. The reduction of E1 to E2 is catalyzed by 17{beta}-HSD, and the activity of this enzyme in T lymphocytes is shown in Fig. 2, A and B. The 17{beta}-HSD activities for conversion of E1 to E2 did not alter in the tissues and in T lymphocytes, after TH, in both the proestrus and OVX animals.



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Fig. 2. Reductase activity of 17{beta}-hydroxysteroid dehydrogenase (17{beta}-HSD) in different tissues isolated from proestrus (A and C) and OVX mice (B and D) after TH, with estrone (A and B) and 4-androstenedione (C and D) as substrates. Data are expressed as means ± SD of 8 experiments for each group. *P < 0.05 vs. sham.

 

Enzyme kinetics in T lymphocytes. The activity of enzymes involved in the metabolism of testosterone and E2 in the T lymphocytes of proestrus females, with pertinent substrates, is given in Table 2. The catalytic efficiency of the enzymes (Vmax/Km) indicates that the production of E2 is from androstenedione through testosterone, and not E1. Moreover, the analysis showed the presence of relatively low 5{alpha}-reductase activity, indicating less 5{alpha}-dihydrotestosterone (DHT) synthesis in the T lymphocytes of proestrus female mice.


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Table 2. Kinetics of aromatase, 5{alpha}-reductase, and 17{beta}-HSD of splenic T lymphocytes with different substrates

 

Testosterone metabolism. Testosterone is synthesized from androstenedione by reductive catalysis. The activity of 17{beta}-HSD associated in this catalysis in tissues of proestrus and OVX mice, after TH, is shown in Fig. 2, C and D. In proestrus females, significant increase in the activity was observed only in the ovary, spleen, and T lymphocytes after TH (Fig. 2C). The enzyme activity in adipose tissue was lower in OVX mice compared with proestrus females, and the enzyme activity did not change in this tissue in either group after TH (Fig. 2D).

5{alpha}-Reductase converts testosterone into DHT, which is a highly active androgen. There was no change in 5{alpha}-reductase activity of T lymphocytes or other tissues of both proestrus and OVX mice after TH (Fig. 3, A and B).



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Fig. 3. Activity of 5{alpha}-reductase with testosterone (A and B) as substrate and activity of 17{beta}-HSD (oxidative) with 17{beta}-estradiol (C and D) as substrate in different tissues isolated from proestrus (A and C) and OVX mice (B and D) after TH. Data are expressed as means ± SD of experiments for each group. *P < 0.05 vs. sham.

 

Conversion to E1. E1 is an inactive estrogen because of low binding affinity to ER. The 17{beta}-HSD converts E2 into E1 by oxidative catalysis. Similar to reductase, the oxidation of E2 by this enzyme was low in the adrenal gland, ovary, adipose tissue, spleen, and T lymphocytes from proestrus females (Fig. 3, C and D). TH did not alter the oxidative activity of the enzyme in any of the tissues, including T lymphocytes from the proestrus or OVX animals.

Enzyme expression in T lymphocytes. The expression of 5{alpha}-reductase, aromatase, and oxidative isomers II, IV, and V of 17{beta}-HSD in T lymphocytes of sham and TH female mice is shown in Fig. 4. The expression of aromatase did not change significantly after TH in proestrus females or in OVX mice. Likewise, the expression of the 17{beta}-HSD isomers was similar in both sham and TH proestrus females, but expression was reduced after TH in OVX females. 5{alpha}-Reductase expression was not different after TH in proestrus or OVX mice.



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Fig. 4. Expression of 5{alpha}-reductase (479 bp), aromatase (450 bp), and 17{beta}-HSD oxidative isomers (type II, 294 bp; type IV, 605 bp; and type V, 303 bp), analyzed by RT-PCR assay, in splenic T lymphocytes of proestrus (A) and OVX mice (B) after TH. 5AR, 5{alpha}-reductase; ARO aromatase. Data shown are representative of 4 separate experiments.

 

ER-{alpha} and ER-{beta} expressions. The expression of ER-{alpha} and ER-{beta}, in the splenic T lymphocytes from proestrus and OVX female mice, after TH, is shown in Fig. 5. The ER-{beta} expression was low in OVX animals compared with ER-{alpha} expression. There was no change in the ER-{alpha} expression in the T lymphocytes of proestrus and OVX animals after TH. In contrast, in proestrus females ER-{beta} expression decreased significantly after TH, whereas its expression significantly increased in OVX females under those same conditions.



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Fig. 5. Effect of TH on estrogen receptor (ER)-{alpha} and ER-{beta} expression in T lymphocytes of proestrus and OVX mice. Data are representative of 4 separate experiments.

 

Cytokine expression and release. The expression of IL-2 and IL-6 in T lymphocytes of proestrus and OVX mice, after TH, is shown in Fig. 6. The IL-2 expression was low in OVX females compared with proestrus females, and TH did not alter IL-2 expression in either group. Stimulation of T lymphocytes with anti-CD3, however, resulted in a significant reduction in the IL-2 release in OVX animals but not in proestrus females after TH. In contrast, IL-6 expression and release were different. Significant increase in IL-6 expression was observed in proestrus mice after TH; however, the expression decreased significantly in OVX animals. Moreover, anti-CD3 stimulation of T lymphocytes did not alter the release of IL-6 in T lymphocytes from proestrus females after TH, whereas a threefold decrease in the IL-6 release was observed in OVX females under such conditions.



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Fig. 6. Effect of TH on the expression and release of IL-2 (A) and IL-6 (B)byT lymphocytes from proestrus and OVX mice. The IL-2 and IL-6 expression in lymphocytes was determined by RT-PCR analysis. Data are representative of 4 separate experiments. The relative intensity of the band (top, receptor expression from 4–6 analyses) is shown in the histogram at left. For cytokine release (right), the lymphocytes were stimulated with 10 µg/ml anti-CD3 in Click's medium at 37°C for 36 h. Data are expressed as means ± SD of 8 experiments for each group. *P < 0.05 vs. sham.

 


    DISCUSSION
 TOP
 ABSTRACT
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Estrogen is a key regulator of cell growth, differentiation, and function in a wide variety of tissues. It plays an important role during pregnancy in the modulation of the maternal immune system to prevent rejection of the fetus. Estrogen modulation of the immune system is not restricted to pregnancy alone, because its role is also documented in many autoimmune disorders and in the outcome after TH (7, 14, 17, 18, 23, 55). The majority of the estrogen effects are mediated by two distinct intracellular receptors, ER-{alpha} and ER-{beta}, each encoded by unique genes (14, 20, 34). However, studies have also suggested that E2 interacts with other cell surface receptors including growth factor or dopamine receptors (13, 19, 30, 47). Nongenomically, estrogen is capable of regulating intracellular Ca2+ mobilization and release of inducible nitric oxide synthase in the cells (19, 34).

The major consequence of TH, besides impairment of the cardiovascular system, is severe depression of immune functions (51, 56, 57). The depression is profound in males but is not observed in proestrus females, indicating sexual dimorphism in the immune response after TH. The divergent immune responses after TH in males and proestrus females are also manifested by the altered release of cytokines IL-2 and IL-6 by the splenic T lymphocytes (2, 4, 18, 54, 55). Because T lymphocytes express receptors for E2 and are also capable of synthesizing E2 locally, the assessment of local active steroid synthesis in the release of cytokines by T lymphocytes becomes significant (37, 39). Hence, the metabolism of E2 and its effect on the release of IL-2 and IL-6 was assessed in T lymphocytes of proestrus and OVX mice after TH.

Enzyme kinetics show that synthesis of E2 from androstenedione is via the formation of testosterone and not via E1. This study indicates a correlation among increased endogenous synthesis of E2, low conversion to E1 (Fig. 7), and the persistent release of IL-2 and IL-6 in the lymphocytes of proestrus female after TH. This is substantiated by 1) the enhancement of aromatase activity, which leads to E2 synthesis in T lymphocytes after TH, unlike reduction in the enzyme activity in OVX females under the same conditions; 2) increased production of testosterone from catalytic reduction of androstenedione by 17{beta}-HSD in proestrus animals after TH, whereas this enzyme activity was unchanged in OVX animals, indicating sustained availability of testosterone for conversion to E2 by aromatase in proestrus animals; and 3) the comparatively low oxidative catalysis by 17{beta}-HSD in both proestrus and OVX animals, suggesting little or no conversion of E2 into E1. The expression of 17{beta}-HSD isomers was analyzed by routine RT-PCR analysis, which is not quantitative. Our aim was to determine whether different forms of the 17{beta}-HSD isomers are expressed in T lymphocytes and, if they are expressed, whether their expression is altered after TH. The enzyme expressions were therefore evaluated in the same T lymphocyte preparation that was used for enzyme assays and ER expression as well as IL-2 and IL-6 expression and release. The results show changes in the expression of 17{beta}-HSD isomers after ovariectomy and after TH. It is, however, necessary to quantify the 17{beta}-HSD isomer expressions by a quantitative PCR procedure for meaningful association of the different isomers in the E2 metabolism in T lymphocytes. Testosterone is also the precursor of DHT. No change in the 5{alpha}-reductase activity, either after ovariectomy or after TH, in proestrus and OVX females was evident, indicating little change in the production of DHT in T lymphocytes. Because DHT is considered to be an inhibitor of aromatase activity (6, 41), an increase in its activity would have lowered E2 production.



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Fig. 7. Metabolism of 17{beta}-estradiol in the T lymphocytes. R, reduction; O, oxidation.

 

A significant observation of our study is the lack of correlation of aromatase expression in T lymphocytes with the enzyme activity in both proestrus and OVX animals. However, this is not surprising because E2 formation from testosterone is the result of a coupled reaction involving aromatase P-450 and a flavoprotein NADPH-cytochrome P-450 reductase (42), and the expression of aromatase alone was assessed in this study. Furthermore, this enzyme reaction requires NADPH as a cofactor. In this regard, our previous studies have indicated decreased splenocyte ATP levels and NAD-to-NADH ratios in tissues after hemorrhagic shock (25). This suggests that not only the expression but also the cofactor requirements are important for the assessment of aromatase activity.

The predominant biological effects of E2 are mediated through two intracellular receptors, ER-{alpha} and ER-{beta}, and our study shows that both these subtypes are present in the splenic T lymphocytes of female mice. However, their expression in response to TH is different. ER-{alpha} expression did not change after ovariectomy or after TH, whereas ER-{beta} expression decreased significantly after ovariectomy. Moreover, expression of ER-{beta} was significantly different in the proestrus and OVX animals after TH; its expression was decreased in proestrus and increased in OVX animals. The increased production of E2-attenuated expression of ER-{beta} in the proestrus and the opposite effects in the OVX females after TH suggest that down-regulation of ER-{beta} may be a factor associated with the change in the cytokine releases by T lymphocytes. Because the T lymphocyte populations used in the experiments consisted of both CD4+ and CD8+ phenotypes (38), analysis of each phenotype for receptor expressions is needed for correlating the changes in receptor subtype expression to lymphocyte differentiation or functional changes, such as a particular cytokine release.

The present study compared E2 synthesis with the in vitro stimulated release of IL-2 and IL-6 in the same cell preparations obtained from different groups. We selected the release of IL-2 and IL-6 in these studies because 1) the alterations in the release of these cytokines are indications of the proinflammatory condition, i.e., Th1 to Th2 shift, and 2) our earlier studies demonstrated marked alterations in the release of these cytokines in OVX females but not in proestrus females after TH (18, 55). Furthermore, the release of these cytokines can be restored by administration of E2 in OVX females during or immediately resuscitation (18). In the present study, we observed that the expression and release of the proinflammatory cytokines IL-2 and IL-6 in T lymphocytes are also different in the proestrus and OVX animals and in response to TH. IL-2 expression, although low in OVX compared with proestrus animals, did not change after TH in either group. In contrast, IL-6 expression was similar in both proestrus and OVX mice, but it was augmented in proestrus and markedly decreased in OVX mice after TH. The release of the cytokines, determined by bioassay in response to anti-CD3 stimulation of T lymphocytes, was also different. The use of anti-CD3 as a stimulant for T lymphocytes cytokine releases is evocative because Con A is primarily a mitogen associated with cell proliferation, whereas anti-CD3 is associated with the T lymphocyte functions. The release of IL-2 and IL-6 was similar in proestrus and OVX females in sham controls, but significantly decreased release of both cytokines was observed only in the OVX animals after TH. A distinct association between E2 synthesis and cytokine release in different groups is evident in this study. Increased E2 synthesis in T lymphocytes proestrus females after TH appears to be associated with sustained release of IL-2 and IL-6 in those animals, because loss in E2 production is reflected by decreased release of these cytokines in the OVX females after TH. The cytokine releases in this study were determined in T lymphocyte preparation that contained CD4+ and CD8+ subsets. Analysis of cytokine expression and release in each T lymphocyte subset is important for any meaningful correlation.

Substantial emphasis has been focused recently on the regulation of extragonadal biosynthesis of sex steroids. The local synthesis of active steroids in T lymphocytes is essential for carrying out their specific functions, especially the release of cytokines. The rate of formation of each steroid depends on the level of expression of the specific androgen- and estrogen-synthesizing enzymes in the tissue. Moreover, local synthesis of active steroids is meaningful compared with availability in circulation, because the steroid can be synthesized as needed and catabolized immediately after fulfillment of tissue function. This is especially true for any regulatory molecule, of which E2 is one. Our recent studies have shown augmented synthesis and decreased catabolism of DHT as the likely cause for loss of T lymphocyte functions in males after TH as reflected in the decreased release of IL-2 and IL-6 by T lymphocytes (58). In this study, we have demonstrated enhanced synthesis of E2, which promotes the maintenance of IL-2 and IL-6 release by T lymphocytes in proestrus mice after TH. Thus both of our studies suggest an important role for steroid-metabolizing enzymes in the release of cytokines by T lymphocytes after TH. Among the sex steroid-metabolizing enzymes, the activities of 17{beta}-HSD isomers appear to be critical because they catalyze both the oxidative and reductive reactions that are required for the synthesis of testosterone, DHT, and E2 as well as their catabolism into inactive steroids (21, 31). This enzyme is also involved in the formation of 5-androstene-3{beta},17{beta}-diol from dehydroepiandrosterone (DHEA), which has been shown to bind to the ER (21, 27, 40). In this regard, our previous studies have demonstrated that DHEA administration after TH restores immune functions in male mice, and the effects appear to be mediated via the ER because tamoxifen blocked the salutary effects of this adrenal steroid (4, 5). Thus, being at the final steps of the formation and inactivation of active estrogens and androgens, 17{beta}-HSD isomers play a unique role in the sex steroid-sensitive physiological functions.

Clinical trauma is a pathological condition that produces an inflammatory response, and our recent retrospective study reveals female patients in premenopausal age range tolerating blunt trauma far better than the males (10). Our experimental results point to sex hormones significantly influencing the immune system in males and females after TH. Thus gender and the hormonal status of the host appear to be critical in the outcome of TH, and estrogen appears to be beneficial in the favorable outcome. Estrogen functions in different tissues and cells by distinct mechanisms, either by regulation of gene activity or by regulation of signal transduction processes (13, 19, 47, 50). Our studies show that the local synthesis of the active steroid appears to be important at least for the T lymphocyte cytokine releases. Thus a thorough understanding of the mechanisms of action of estrogen in different tissues as well as in different immune cells is important. Such studies are expected to lead to further understanding of the basis of the pathophysiology of TH and to help in the development of improved therapy to prevent/decrease morbidity and mortality after TH.


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


    FOOTNOTES
 

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 (E-mail: Irshad.Chaudry{at}ccc.uab.edu).

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 solely to indicate this fact.


    REFERENCES
 TOP
 ABSTRACT
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
1. Andersson S, Bishop RW, and Russell DW. Expression, cloning and regulation of steroid 5{alpha}-reductase, an enzyme essential for male sexual differentiation. J Biol Chem 264: 16249–16255, 1989.[Abstract/Free Full Text]

2. Angele MK, Ayala A, Monfils BA, Cioffi WG, Bland KI, and Chaudry IH. Testosterone and/or low estradiol: normally required but harmful immunologically for males after trauma-hemorrhage. J Trauma 44: 78–85, 1998.[ISI][Medline]

3. Angele MK, Catania RA, Ayala A, Cioffi WG, Bland KI, and Chaudry IH. Dehydroepiandrosterone: an inexpensive steroid hormone that decreases the mortality from sepsis following trauma-induced hemorrhage. Arch Surg 133: 1281–1288, 1998.[Abstract/Free Full Text]

4. Angele MK, Schwacha MG, Ayala A, and Chaudry IH. Effect of gender and sex hormones on immune responses following shock. Shock 14: 81–90, 2000.[ISI][Medline]

5. Catania RA, Angele MK, Ayala A, Cioffi WG, Bland KI, and Chaudry IH. Dehydroepiandrosterone restores immune function following trauma-hemorrhage by a direct effect on T lymphocytes. Cytokine 11: 443–450, 1999.[ISI][Medline]

6. Chan WK and Tan CH. FSH-induced aromatase activity in porcine granulose cells: non-competitive inhibition by non-aromatizable androgens. J Endocrinol 108: 335–341, 1986.[Abstract]

7. Cutolo M, Sulli A, Seriolo B, Accardo S, and Masi AT. Estrogens, the immune response and autoimmunity. Clin Exp Rheumatol 13: 217–226, 1995.[ISI][Medline]

8. Dufort I, Rheault XF, Huang P, Soucy T, and Luu-The V. Characteristics of a highly labile human type V 17{beta}-hydroxysteroid dehydrogenase. Endocrinology 140: 568–575, 1999.[Abstract/Free Full Text]

9. Eidinger D and Garrett TJ. Studies of the regulatory effects of the sex hormones on antibody formation and stem cell differentiation. J Exp Med 136: 1098–1116, 1972.[ISI][Medline]

10. George RL, McGwin G Jr, Windham ST, Melton SM, Metzger J, Chaudry IH, and Rue LW III. Age related gender differential in outcome following blunt or penetrating trauma. Shock 19: 28–32, 2003.[ISI][Medline]

11. Grossman CJ. Regulation of the immune system by sex steroids. Endocr Rev 5: 435–455, 1994.

12. Grossman CJ, Roselle GA, and Mendenhall CL. Sex steroid regulation of autoimmunity. J Steroid Biochem Mol Biol 40: 649–659, 1991.[ISI][Medline]

13. Hall JM, Couse JF, and Korach KS. The multifaceted mechanisms of estradiol and estrogen receptor signaling. J Biol Chem 276: 36869–36872, 2001.[Free Full Text]

14. Jansson L and Holmdahl R. Estrogen-mediated immunosuppression in autoimmune diseases. Inflamm Res 47: 290–301, 1998.[ISI][Medline]

15. Kashima N, Nishi-Takaoka C, Fujita T, Taki S, Hamuro J, and Taniguchi T. Unique structure of murine interleukin-2 as deduced from cloned cDNAs. Nature 313: 402–404, 1985.[ISI][Medline]

16. Knöferl MW, Angele MK, Diodato MD, Schwacha MG, Ayala A, Cioffi WG, Bland KI, and Chaudry IH. Female sex hormones regulate macrophage function following trauma-hemorrhage and prevent increased mortality from subsequent sepsis. Ann Surg 235: 105–112, 2002.[ISI][Medline]

17. Knöferl MW, Diadoto MD, Angele MK, Ayala A, Cioffi WG, Bland KI, and Chaudry IH. Do female sex steroids adversely or beneficially affect the depressed immune responses in males after trauma-hemorrhage? Arch Surg 135: 425–433, 2000.[Abstract/Free Full Text]

18. Knöferl MW, Jarrar D, Angele MK, Ayala A, Schwacha MG, Bland KI, and Chaudry IH. 17{beta}-Estradiol normalizes immune responses in ovariectomized females after trauma-hemorrhage. Am J Physiol Cell Physiol 281: C1131–C1138, 2001.[Abstract/Free Full Text]

19. Kousteni S, Bellido T, Plotkin LT, O'Brien CA, Bodenner DL, Han L, Han K, DiGregorio GB, Katzenellenbogen JA, Katzenellenbogen BS, Roberson PK, Weinstein RS, Jilka RL, and Manolagas SC. Nongenotropic, sex-nonspecific signaling through the estrogen and androgen receptors: dissociation from transcriptional activity. Cell 104: 719–730, 2001.[ISI][Medline]

20. Krzych U, Strausser HR, Bressler JP, and Goldstein AL. Effects of sex hormones on some T and B cell functions, evidenced by differential immune expression between male and female mice by cyclic pattern of immune responsiveness during the estrous cycle in female mice. Am J Reprod Immunol 1: 73–77, 1981.[ISI][Medline]

21. Labrie F, Luu-The V, Lin SX, Simard J, Labrie C, El-Alfy M, Pelletier G, and Bélanger A. Intracrinology: role of the family of 17{beta}-hydroxysteroid dehydrogenases in human physiology and disease. J Mol Endocrinol 25: 1–16, 2000.[Abstract/Free Full Text]

22. Labrie F, Sugimoto Y, Luu-The V, Simard J, Lachance Y, Bachvarov D, Leblanc G, Durocher F, and Paquet N. Structure of human type II 5 alpha-reductase gene. Endocrinology 131: 1571–1573, 1992.[Abstract]

23. Lahita RJ. The role of sex hormone in systemic lupus erythematosus. Curr Opin Rheumatol 11: 352–356, 1999.[Medline]

24. McDonnell DP. Definition of the molecular action of tissueselective oestrogen-receptor molecules. Biochem Soc Trans 26: 54–60, 1998.[ISI][Medline]

25. Meldrum DR, Ayala A, Wang P, Ertel W, and Chaudry IH. Association between decreased splenic ATP levels and immunodepression: amelioration with ATP-MgCl2. Am J Physiol Regul Integr Comp Physiol 261: R351–R357, 1991.[Abstract/Free Full Text]

26. Mustonen MVJ, Poutanen MH, Isomaa VV, Vihko PT, and Vihko RK. Cloning of mouse 17{beta}-hydroxysteroid-dehydrogenase type 2 and analyzing expressions of the mRNAs for types 1, 2, 3, 4 and 5 in mouse embryos and adult tissues. Biochem J 325: 199–205, 1997.[ISI][Medline]

27. Nephew KP, Sheeler CQ, Dudley MD, Gordon S, Nayfield SG, and Khan SA. Studies of dehydroepiandrosterone (DHEA) with the human estrogen receptor in yeast. Mol Cell Endocrinol 143: 133–142, 1998.[ISI][Medline]

28. Normand T, Husen B, Leenders F, Pelczar H, Baert JL, Begue A, Flourens AC, Adamski J, and de Launoit Y. Molecular characterization of mouse {beta}-hydroxysteroid dehydrogenase IV. J Steroid Biochem Mol Biol 55: 541–548, 1995.[ISI][Medline]

29. Olsen NJ and Kovacs WJ. Gonadal steroids and immunity. Endocr Rev 17: 369–384, 1994.

30. Patterson K and Gustafsson JA. Role of estrogen receptor beta in estrogen action. Annu Rev Physiol 63: 165–192, 2001.[ISI][Medline]

31. Peltoketo H, Vihko P, and Vihko R. Regulation of estrogen action: role of 17{beta}-hydroxysteroid dehydrogenases. Vitam Horm 55: 353–398, 1999.[ISI][Medline]

32. Pottratz ST, Bellido T, Mocharla M, Crabb D, and Manolagas SC. 17{beta}-estradiol inhibits expression of human interleukin-6 promoter-receptor constructs by a receptor-dependent mechanism. J Clin Invest 93: 944–950, 1994.[ISI][Medline]

33. Ray A, Prefontaine KE, and Ray P. Down-modulation of interleukin-6 gene expression by 17{beta}-estradiol in the absence of high affinity DNA binding by the estrogen receptor. J Biol Chem 269: 12940–12946, 1994.[Abstract/Free Full Text]

34. Rosenfeld CR, White RE, Roy T, and Cox BE. Calcium-activated potassium channels and nitric oxide coregulate estrogen-induced vasodilation. Am J Physiol Heart Circ Physiol 279: H319–H328, 2000.[Abstract/Free Full Text]

35. Ruh MF, Bi Y, D'Alonzo R, and Bellone CJ. Effect of estrogens on IL-1{beta} promoter activity. J Steroid Biochem Mol Biol 66: 203–210, 1998.[ISI][Medline]

36. Samy TSA, Catania RA, Ayala A, and Chaudry IH. Trauma-hemorrhage activates signal transduction pathways in mouse splenic T lymphocytes. Shock 9: 443–450, 1998.[ISI][Medline]

37. Samy TSA, Knöferl MW, Zheng R, Schwacha MG, Bland KI, and Chaudry IH. Divergent immune responses in male and female mice following trauma-hemorrhage: dimorphic alterations in T lymphocyte steroidogenic enzyme activities. Endocrinology 42: 3519–3529, 2001.

38. Samy TSA, Schwacha MG, Chung SC, Cioffi WG, Bland KI, and Chaudry IH. Proteasome participates in the alteration of signal transduction in T and B lymphocytes following trauma-hemorrhage. Biochim Biophys Acta 1453: 92–104, 1999.[ISI][Medline]

39. Samy TSA, Schwacha MG, Cioffi WG, Bland KI, and Chaudry IH. Androgen and estrogen receptors in splenic T lymphocytes: the effects of flutamide and trauma-hemorrhage. Shock 14: 465–470, 2000.[ISI][Medline]

40. Schmidt M, Kreutz M, Loffler G, Scholmerich J, and Straub RH. Conversion of dehydroepiandrosterone to down stream steroid hormones in macrophages. J Endocrinol 164: 161–169, 2000.[Abstract/Free Full Text]

41. Shilling AD and Williams DE. The non-aromatizable androgen, dihydrotestosterone, induces antiestrogenic responses in the rainbow trout. J Steroid Biochem Mol Biol 74: 187–194, 2000.[ISI][Medline]

42. Simpson ER, Mahendroo MS, Means GD, Kilgore MW, Hinshelwood MM, Graham-Lorence S, Amernesh B, Ito Y, Fisher CR, Michael MD, Mendelson CR, and Bulun SE. Aromatase cytochrome P450, the enzyme responsible for estrogen biosynthesis. Endocr Rev 15: 342–355, 1994.[ISI][Medline]

43. Stephan RN, Kupper TS, Geha AS, Baue AE, and Chaudry IH. Hemorrhage without tissue trauma produces immunosuppression and enhances susceptibility to sepsis. Arch Surg 122: 62–68, 1987.[Abstract]

44. Terashima M, Toda K, Kawamoto T, Kuribayashi I, Ogawa Y, Maeda T, and Shizuta Y. Isolation of a full-length cDNA encoding mouse aromatase P450. Arch Biochem Biophys 285: 231–237, 1991.[ISI][Medline]

45. Thompson EA and Siiteri PK. Utilization of oxygen and reduced nicotinamide adenine dinucleotide phosphate by human placental microsomes during aromatization of androstenedione. J Biol Chem 279: 5364–5372, 1974.

46. Tremblay GB, Tremblay A, Copeland NG, Gilbert DJ, Jenkins NA, Labrie F, and Giguere V. Cloning, chromosomal localization, and functional analysis of the murine estrogen receptor beta. Mol Endocrinol 11: 353–365, 1997.[Abstract/Free Full Text]

47. Tsai MJ and O'Malley BW. Molecular mechanisms of action of steroid/thyroid receptor superfamily members. Annu Rev Biochem 63: 451–486, 1994.[ISI][Medline]

48. Turgeon C, Gingras S, Carrière MC, Blais Y, Labrie F, and Simard J. Regulation of sex steroid formation by interleukin-4 and interleukin-6 in breast cancer cells. J Steroid Biochem Mol Biol 65: 151–162, 1998.[ISI][Medline]

49. Van Snick J, Cayphas S, Szikora Renauld JC, Van Roost Boon T, and Simpson RJ. cDNA cloning of murine interleukin-HP1: homology with human interleukin 6. Eur J Immunol 18: 193–197, 1988.[ISI][Medline]

50. Wade CB, Robinson S, Shapiro RA, and Dorsa DM. Estrogen receptor (ER){alpha} and ER{beta} exhibit unique pharmacologic properties when coupled to activation of the mitogen-activated protein kinase pathway. Endocrinology 142: 2336–2342, 2001.[Abstract/Free Full Text]

51. Wang P, Ba ZF, Burkhardt J, and Chaudry IH. Trauma-hemorrhage and resuscitation in the mouse: effects on cardiac output and organ blood flow. Am J Physiol Heart Circ Physiol 264: H1166–H1173, 1993.[Abstract/Free Full Text]

52. Waynforth HB. Experimental and Surgical Techniques in the Rat. London: Academic, 1980.

53. White R, Lees JA, Needham M, Ham J, and Parker M. Structural organization and expression of the mouse estrogen receptor. Mol Endocrinol 1: 735–744, 1987.[Abstract]

54. Wichmann MW, Angele MK, Ayala A, Cioffi WG, Chaudry IH. Flutamide: a novel agent for restoring the depressed cellmediated immunity following soft-tissue trauma and hemorrhagic shock. Shock 8: 242–248, 1997.[ISI][Medline]

55. Wichmann MW, Zellweger R, DeMaso CM, Ayala A, and Chaudry IH. Enhanced immune responses in females, as opposed to decreased responses in males following hemorrhagic shock and resuscitation. Cytokine 8: 853–863, 1996.[ISI][Medline]

56. Xu YX, Ayala A, and Chaudry IH. Prolonged immunodepression following trauma and hemorrhagic shock. J Trauma 44: 335–341, 1998.[ISI][Medline]

57. Zellweger R, Ayala A, DeMaso CM, and Chaudry IH. Trauma-hemorrhage causes prolonged depression in cellular immunity. Shock 4: 149–153, 1995.[ISI][Medline]

58. Zheng R, Samy TSA, Schneider CP, Rue III LW, Bland KI, and Chaudry IH. Decreased 5{alpha}-dihydrotestosterone catabolism suppress T lymphocyte functions in males after trauma-hemorrhage. Am J Physiol Cell Physiol 282: C1332–C1338, 2002.[Abstract/Free Full Text]