Center for Surgical Research and Department of Surgery, University of Alabama School of Medicine, Birmingham, Alabama 35294
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ABSTRACT |
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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
5-dihydrotestosterone for the immunosuppressive effects. 5
-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 5
-dihydrotestosterone. We hypothesized, therefore, that the basis for high 5
-dihydrotestosterone activity in
T lymphocytes of males following trauma-hemorrhage is due to decreased
catabolism. Accordingly, catabolism of 5
-dihydrotestosterone was
assessed in splenic T lymphocytes by examining the activity and
expression of enzymes involved. Analysis showed increased synthesis and
decreased catabolism of 5
-dihydrotestosterone in intact male T
lymphocytes following trauma-hemorrhage. In contrast, reduced
5
-reductase activity and increased expression of
17
-hydroxysteroid dehydrogenase oxidative isomers suggest
inactivation of 5
-dihydrotestosterone in precastrated males. Thus
our study suggests increased synthesis and decreased catabolism of
5
-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; cytokines
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INTRODUCTION |
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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 5-dihydrotestosterone because of
its higher affinity for the androgen receptor as well as increased transcriptional activity of the 5
-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
5
-reductase, which synthesizes 5
-dihydrotestosterone from
testosterone, increases in lymphocytes from males
following trauma-hemorrhage
(23).1
5-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 5
-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 5
-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.
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MATERIALS AND METHODS |
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Chemicals.
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,
5-[4-14C]dihydrotestosterone (specific activity 57 Ci/mmol), and 5
-[3-3H]androstane-3
,17
-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).
Animals. 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.
Experimental groups. 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.
Trauma-hemorrhage. 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).
Enzyme assays.
The procedure by Andersson et al. (1) was used for the
assay of 5-reductase activity and the procedure by Turgeon et al. (30) for the assay of 17
-hydroxysteroid dehydrogenase
(17
-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 3
-hydroxysteroid dehydrogenase
(3
-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- or
14C-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 (Rf) values of standards.
RT-PCR analysis.
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 (Table 1). 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
-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).
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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.
Protein content. The protein content was determined by the micro Bradford method (Bio-Rad, Hercules, CA). BSA was used as standard.
Enzyme kinetics. 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.
Statistical analysis.
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.
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RESULTS |
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5-Reductase, 3
-HSD, and 17
-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 5
-reductase (Fig.
1A) and 3
-HSD (Fig. 1B). There was no change
in the 17
-HSD reductive activities when testosterone was used as the
substrate, i.e., conversion of testosterone into 4
-androsteronedione
(Fig. 1C). However, when 5
-androstane-5
,17
-diol was
used as a substrate, the 17
-HSD activity significantly decreased, suggesting a reduction in the oxidative conversion of
5
-androstane-5
,17
-diol into androsterone following
trauma-hemorrhage. The kinetic parameters of the enzymes in T
lymphocytes with relevant steroid substrates are provided in Table
2.
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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 5-reductase was lacking in both sham
and trauma-hemorrhaged mice (Fig. 4).
Furthermore, there were no changes in the expressions of 3
-HSD and
aromatase in castrated males after trauma-hemorrhage. Among the
17
-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.
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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.
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DISCUSSION |
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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 5-dihydrotestosterone in producing immunosuppression in males (34, 36) and
17
-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. 5
-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
5
-dihydrotestosterone synthesis in T lymphocytes from intact males
following trauma-hemorrhage (23). In the present study, we
assessed the catabolism of 5
-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 5-dihydrotestosterone catabolism are 3
-HSD in the
conversion of 5
-dihydrotestosterone into
5
-androstane-3
,17
-diol and 17
-HSD (oxidative form) in the
formation of androsterone from 5
-androstane-3
,17
-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 3
-HSD and reduction in the activity of
17
-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
5
-reductase activity in T lymphocytes of intact males following
trauma-hemorrhage. Thus 1) increased activity of
5
-reductase (Fig. 1A) suggests enhanced synthesis of
5
-dihydrotestosterone, and 2) low activity of the
17
-HSD (oxidative) suggests decreased conversion of
5
-dihydrotestosterone to androsterone. The increase in 3
-HSD
activity suggests that 5
-dihydrotestosterone synthesized in T
lymphocytes, following trauma-hemorrhage, readily converts into
5
-androstane-3
,17
-diol and is present at equilibrium
concentration with 5
-androstane-3
,17
-diol in the T
lymphocytes. 5
-Androstane-3
,17
-diol is a weak androgen, and
the reversible catalysis of 3
-HSD allows for continued presence of
androgen in the receptor-active form in the T lymphocytes.
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17-HSD is an oxidoreductase that catalyzes the interconversion of
17-keto and 17
-hydroxy steroids. Because both androgens and
estrogens exhibit the highest receptor-binding activity in the
17
-hydroxy form, this enzyme plays an important role in the production of active steroids for T lymphocyte functions. In this regard, several isoforms of 17
-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
3
-HSD reductive activity as well as 17
-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 3
-HSD reductive and not with the 17
-HSD type VI oxidative function because
less androsterone was formed in T lymphocytes following trauma-hemorrhage when 5
-androstane-3
,17
-diol was used as the substrate (Fig. 1D in the enzyme assay). Because CRAD2 is
involved in the synthesis as well as the catabolism of
5
-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 17-estradiol for protection of immune
functions in proestrus females following trauma-hemorrhage (3,
35). 17
-Estradiol is also synthesized from testosterone by
aromatase, and the immunoprotective role of 17
-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 17
-estradiol (10,
11). It could be argued that administration of 17
-estradiol in males following trauma-hemorrhage should not be beneficial because
5
-dihydrotestosterone, which is synthesized more in T lymphocytes,
1) is unlike testosterone, resistant to aromatization, and
2) 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
5
-dihydrotestosterone together with decreased conversion of
5
-dihydrotestosterone into androsterone and the decreased synthesis
of 17
-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 5-dihydrotestosterone was not high enough in T
lymphocytes of precastrated animals to interfere with cytokine
production due to the lack of 5
-reductase activity in the
lymphocytes (23). The absence of 5
-reductase expression
in T lymphocytes of castrated males was expected because sex steroids
have been shown to regulate the 5
-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 17
-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 5
-dihydrotestosterone are the
likely reasons for the immune suppression in males following trauma-hemorrhage. The presence of androgen receptor in the lymphocytes allows 5
-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 5-dihydrotestosterone or
17
-estradiol in vivo to serve important immunoregulatory functions. Our studies demonstrate the presence of enzymes required for
5
-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.
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ACKNOWLEDGEMENTS |
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This investigation was supported by National Institute of General Medical Sciences Grant R01-GM-37127.
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FOOTNOTES |
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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: irshad.chaudry{at}ccc.uab.edu).
1
Steroid nomenclature: androstenedione,
4-androstene-3,17-dione; androsterone,
3-hydroxy-5
-androstan-17-one; 3
-androstanediol, 5
-androstane-3
,17
-diol; 5
-dihydrotestosterone,
17
-hydroxy-5
-androstan-3-one; 17
-estradiol,
1,3,5(10)-estratriene-3,17
-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
Received 20 November 2001; accepted in final form 24 January 2002.
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