| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
ARTICLES |
Center for Surgical Research and Department of Surgery, University of Alabama, Birmingham, Alabama 35294
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: irshad.chaudry{at}ccc.uab.edu
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-reductase, aromatase, and 3ß- and 17ß-
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-reductase activity in male T
lymphocytes is immunosuppressive because of increased
5-dihydrotestosterone synthesis, whereas in females increased
aromatase activity triggering 17ß-estradiol synthesis is
immunoprotective. This study also demonstrates the importance of
17ß-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. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Testosterone and 17ß-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, 9, 10). These receptors function as transcription factors, which regulate several cytokine genes (11, 12, 13, 14, 15, 16, 17, 18). Alterations in the release of pro- and antiinflammatory cytokines have been demonstrated in males after trauma-hemorrhage. 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, 3ß-hydroxysteroid dehydrogenase
(3ßHSD), and 17ß-hydroxysteroid dehydrogenase (17ßHSD), that
participate in the biosynthesis of testosterone, its active metabolite
5-dihydrotestosterone, and 17ß-estradiol in peripheral tissues
besides the gonads and adrenal glands (19, 20, 21, 22, 23, 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 17ßHSD
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.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-dihydrotestosterone (SA, 57
Ci/mmol), [4-14C]17ß-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 [1,2,6,7-3H]progesterone (SA, 110 mCi/mmol)
were obtained from NEN Life Science Products (Boston, MA).
The unlabeled steroids were obtained from Sigma (St.
Louis, MO). ß-Actin amplimers were purchased from CLONTECH Laboratories, Inc. (Palo Alto, CA).
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 x
g for 15 min at 4 C. The supernatant was centrifuged at
100,000 x g for 30 min at 4 C. The microsomal pellet
was suspended in the homogenization buffer at 25 µg/10 µl and
stored at -70 C. The reaction mixture for the 5-reductase assay
consisted of a 20-µl aliquot of cell homogenate (50 µg protein) in
0.5 ml 100 mM potassium phosphate buffer, pH 6.6,
and testosterone (1 µCi 14C-labeled steroid and
50 µM unlabeled steroid dissolved in 5 µl
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 µl 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
The aromatase activity was assayed by the procedure of Thompson
and Siiteri (31). Each tissue was homogenized in 3 vol 50
mM Tris-maleate buffer, pH 7.4, containing 1 mM
ß-mercaptoethanol, 40 mM niacinamide, and 250
mM sucrose. The homogenate was centrifuged at 5,000 x
g for 15 min, and the supernatant was centrifuged at
150,000 x 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 µl microsomal preparation (50
µg protein) and steroids (0.5 µCi radiolabeled and 5 µmol
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 µl 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 x g for 10 min, the radioactivity in 500
µl supernatant was measured after the addition of 5 ml liquid
scintillation cocktail in the scintillation counter (Wallac, Inc., Gaithersburg, MD). For estimating
[14C]17ß-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 µl 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.
3ßHSD and 17ßHSD activities
The procedure of Sturgeon et al. (32)
was followed for the assay of 3ßHSD and 17ßHSD 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 x g for 15 min at 4 C to remove the cell debris
and then at 105,000 x 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 3ßHSD and 17ßHSD
activities.
The 3ßHSD activity was assayed in 500 µl 50 mM NaH2PO4 buffer, pH 7.4, containing 20% glycerol and 10 mM EDTA. A 20-µl aliquot of the microsomal preparation (50 µg 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 µl 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 17ßHSD assay was carried out in 500 µl 100 mM potassium phosphate buffer, pH 7.4, containing 50 µg protein (20 µl microsomal preparation), 1 mM NADPH, 5 µM androstenedione or estrone, and 0.5 µCi [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 17ßHSD oxidative functions
The spleen homogenate preparation used for reductive activity
was also used to assay the oxidative reaction of 17ßHSD. The assay
was carried out at pH 7.4 and 8.6. The reaction mixture (500 µl)
consisted of 100 mM
NaH2P04, 0.5 mM
dithiothreitol, 5 µM 17ß-estradiol, 0.1 µCi
[14C]17ß-estradiol, and a 20-µl aliquot (50
µg 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 µl 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 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.
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/µl) 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 µl) in 1 x 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 deoxy-NTP mix, 1 mM
MgSO4, 0.1 U AMV reverse transcriptase, 0.1 U
TfI DNA polymerase, and 1 µM 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 ß-actin was used as the internal control.
The PCR products were analyzed by electrophoresis on 1.5% agarose gels
in Tris-acetate-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).
|
0.05. |
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-Reductase, aromatase, 3ßHSD, and 17ßHSD activities in
mouse tissues. 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 activity of 3ßHSD, 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 3ßHSD was observed in the tissues of males and females.
The activity of 17ßHSD, assessed for the reductive conversion of androstenedione to testosterone, was present in the adrenal glands, gonads, and spleen. Adrenal glands and gonads expressed higher 17ßHSD activity than spleen. No significant activity of 17ßHSD 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, 3ßHSD, and 17ßHSD 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 3ßHSD was very low in B lymphocytes.
Moreover, there were no 17ßHSD activities in the B lymphocytes. Most
of the activities of the splenic steroidogenic enzymes (>85%) were
localized in the T lymphocytes.
|
-reductase, aromatase, 3ßHSD, and 17ßHSD
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.
|
|
Trauma-hemorrhage did not alter 3ßHSD activities in tissues of either
males or females (Fig. 2
). Although there was no change in 17ßHSD
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),
3ßHSD, and type II, IV, and V isoforms of 17ßHSD in T lymphocytes
from male and female mice after trauma- hemorrhage 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 3ßHSD expression. Of the
17ßHSD isomers, type IV showed decreased expression after
trauma-hemorrhage.
|
), the expressions
of aromatase and 3ßHSD did not change after trauma-hemorrhage. Of the
17ßHSD 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.
|
-reductase, aromatase, 3ßHSD, and 17ßHSD 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 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 3ßHSD or 17ßHSD (reductive) in any
tissue (Fig. 6). A significant observation, however, was the difference
in the reductive function of 17ßHSD in spleen and T lymphocytes of
proestrous mice and mice ovariectomized after trauma-hemorrhage. The
activity of aromatase and 17ßHSD (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).
|
|
-reductase, aromatase, 3ßHSD, and 17ßHSD from
spleen-reductase,
aromatase, 3ßHSD, and 17ßHSD 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 maximum velocity (Vmax) and
Km (Table 4). The
Vmax and Km of
5-reductase with testosterone as the substrate were 150 pmol/mg
protein·min and 24 nM, respectively. The
Vmax and Km of aromatase
differed for testosterone and androstenedione: 200 pmol/mg
protein·min and 7.5 nM vs. 186 pmol/mg
protein·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 17ß-estradiol was greater than that of
androstenedione to estrone. The Vmax and
Km of 3ßHSD for reductive catalysis of
dehydroepiandrosterone and pregnenolone were also different: 51 pmol/mg
protein·min and 35 nM vs. 21 pmol/mg
protein·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 17ßHSD
for reductive conversion of androstenedione to testosterone were 150
pmol/mg protein·min and 16.7 nM, and those for
conversion of estrone to 17ß-estradiol were 120 pmol/mg protein·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 17ß-estradiol.
|
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 animals 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 17ßHSD isomer types II, IV, and V in T
lymphocytes. Trauma- hemorrhage 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).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Studies have shown the requirement of ß-OH function on carbon-17 of
testosterone, 5-dihydrotestosterone, and 17ß-estradiol for
receptor binding (40, 41). 5-Reductase, aromatase,
3ßHSD, and 17ßHSD 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).
|
-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 6-fold higher than that of testosterone,
and its transcriptional activity is also greater and more prolonged
(43, 44, 45, 46). In male mice, the activity of 5-reductase
increased significantly (>2-fold) 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 17ßHSD (oxidative) into
androsterone (24), a metabolite that is inactive because
of its inability to bind to the AR due to the lack of ß-hydroxyl
function on carbon 17. Our data show the expression of 17ßHSD 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 trauma-
hemorrhage 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
17ßHSD reductive activities in proestrous females after
trauma-hemorrhage demonstrate increased synthesis of 17ß-
estradiol. This would suggest that 17ß-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
17ßHSD (reductive) with different steroid substrates suggest that the
synthesis of 17ß-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 17ßHSD
oxidative functions or in the expression of oxidant type IV isomer
suggests less conversion of 17ß-estradiol to estrone (which does not
bind to ER). Moreover, the very low activity of aromatase and 17ßHSD
(reductive) in spleen and T lymphocytes of ovariectomized females
compared with proestrous female mice after trauma hemorrhage is
consistent with lower 17ß-estradiol production in those animals.
Thus, the decreased local production of 17ß-estradiol in
ovariectomized females appears to be the reason why these animals are
immunosuppressed after trauma-hemorrhage (4).
The activity of 3ßHSD was not altered in either males or proestrous females after trauma-hemorrhage or gonadectomy. The relative efficiencies (Vmax/Km) of the reactions catalyzed by 3ßHSD indicate that the formation of androstenedione from dehydroepiandrosterone was greater than that of progesterone from pregnenolone. The lack of change in splenic 3ßHSD 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, 3ß,17ß-androstenediol (50, 51).
This study implies a key role for 17ßHSD 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 17ßHSD, on the other hand,
will lead to the production of inactive steroids, androsterone from
5-dihydrotestosterone and estrone from 17ß-estradiol. Therefore,
alterations in the ratio of 17ßHSD 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 17ßHSD isotypes (36, 37, 38, 41, 52, 53, 54, 55, 56, 57, 58). Each 17ßHSD 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 17ßHSD
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 17ßHSD 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 17ß-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
17ß-estradiol increased in T lymphocytes of proestrous female mice
after trauma-hemorrhage, but not in males. Thus, trauma-hemorrhage led
to enhancement of the activity of different steroidogenic enzymes 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
17ß-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.
|
Footnotes |
|---|
1 Present address: Department of Trauma-Surgery, University of Ulm,
Steinhövel Strasse 9, 89075 Ulm, Germany. 
Abbreviations: 3ßHSD, 3ß-Hydroxysteroid dehydrogenase; 17ßHSD, 17ß-hydroxysteroid dehydrogenase.
Received February 13, 2001.
Accepted for publication April 11, 2001.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
B activity through maintenance of IkB levels contributes to
dihydrotestosterone-mediated repression of interleukin-6 promoter.
J Biol Chem 271:26267–26275
promoter. J Immunol 146: 4362–4367
5-4
isomerase (3ß-HSD) 17ß-HSD, 5-reductase and aromatase activities
in the rhesus monkey. Mol Cell Endocrinol 104:103–111[CrossRef][Medline]
-reductase
and aromatase in brain differentiation and functions. J Steroid Biochem
Mol Biol 58:445–466
-hydroxysteroid
dehydrogenases from rat and human prostate. J Biol Chem 272:15959–15966-reductase, an enzyme essential
for male sexual differentiation. J Biol Chem 264:16249–16255-reductase gene. Endocrinology 131:1571–1573-reductase. Rev Reprod. 4:179–183
-androstene-3ß,17ß-diol.
Endocrinology 92:941–946This article has been cited by other articles:
 |
|
 |
|
  R. Raju and I. H. Chaudry Sex Steroids/Receptor Antagonist: Their Use as Adjuncts After Trauma-Hemorrhage for Improving Immune/Cardiovascular Responses and for Decreasing Mortality from Subsequent Sepsis Anesth. Analg., July 1, 2008; 107(1): 159 - 166. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. H. Haider, D. T. Efron, E. R. Haut, D. C. Chang, C. N. Paidas, and E. E. Cornwell III Mortality in Adolescent Girls vs Boys Following Traumatic Shock: An Analysis of the National Pediatric Trauma Registry Arch Surg, September 1, 2007; 142(9): 875 - 880. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K Radojevic, N Arsenovic-Ranin, D Kosec, V Pesic, I Pilipovic, M Perisic, B Plecas-Solarovic, and G Leposavic Neonatal castration affects intrathymic kinetics of T-cell differentiation and the spleen T-cell level J. Endocrinol., March 1, 2007; 192(3): 669 - 682. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. D. Salazar, M. R. Miller, J. B. Barnett, and R. Schafer Evidence for a Novel Endocrine Disruptor: The Pesticide Propanil Requires the Ovaries and Steroid Synthesis to Enhance Humoral Immunity Toxicol. Sci., September 1, 2006; 93(1): 62 - 74. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Mayr, C. R. Walz, P. Angele, T. Hernandez-Richter, I. H. Chaudry, F. Loehe, K. W. Jauch, and M. K. Angele Castration prevents suppression of MHC class II (Ia) expression on macrophages after trauma-hemorrhage J Appl Physiol, August 1, 2006; 101(2): 448 - 453. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y.-C. Hsieh, S. Yang, M. A. Choudhry, H.-P. Yu, K. I. Bland, M. G. Schwacha, and I. H. Chaudry Flutamide restores cardiac function after trauma-hemorrhage via an estrogen-dependent pathway through upregulation of PGC-1 Am J Physiol Heart Circ Physiol, January 1, 2006; 290(1): H416 - H423. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Hammer, D. G. Drescher, S. B. Schneider, M. Quinkler, P. M. Stewart, B. Allolio, and W. Arlt Sex Steroid Metabolism in Human Peripheral Blood Mononuclear Cells Changes with Aging J. Clin. Endocrinol. Metab., November 1, 2005; 90(11): 6283 - 6289. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. L. Tosi, B. D. Boyan, and A. L. Boskey Does Sex Matter in Musculoskeletal Health? The Influence of Sex and Gender on Musculoskeletal Health J. Bone Joint Surg. Am., July 1, 2005; 87(7): 1631 - 1647. [Full Text] [PDF]
|
|
|
|
 |
|
 T. Matsutani, T. S. A. Samy, L. W. Rue III, K. I. Bland, and I. H. Chaudry Transgenic prolactin-/- mice: effect of trauma-hemorrhage on splenocyte functions Am J Physiol Cell Physiol, May 1, 2005; 288(5): C1109 - C1116. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. C. Roden, M. T. Moser, S. D. Tri, M. Mercader, S. M. Kuntz, H. Dong, A. A. Hurwitz, D. J. McKean, E. Celis, B. C. Leibovich, et al. Augmentation of T Cell Levels and Responses Induced by Androgen Deprivation J. Immunol., November 15, 2004; 173(10): 6098 - 6108. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. M. Nitsch, F. Wittmann, P. Angele, M. W. Wichmann, R. Hatz, T. Hernandez-Richter, I. H. Chaudry, K. W. Jauch, and M. K. Angele Physiological Levels of 5{alpha}-Dihydrotestosterone Depress Wound Immune Function and Impair Wound Healing Following Trauma-Hemorrhage Arch Surg, February 1, 2004; 139(2): 157 - 163. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. J. Molloy, A. J. O'Neill, J. J. Grantham, M. Sheridan-Pereira, J. M. Fitzpatrick, D. W. Webb, and R. W. G. Watson Sex-specific alterations in neutrophil apoptosis: the role of estradiol and progesterone Blood, October 1, 2003; 102(7): 2653 - 2659. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. P. Schneider, M. G. Schwacha, T. S. A. Samy, K. I. Bland, and I. H. Chaudry Androgen-mediated modulation of macrophage function after trauma-hemorrhage: central role of 5{alpha}-dihydrotestosterone J Appl Physiol, July 1, 2003; 95(1): 104 - 112. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. K. Angele, S. Nitsch, M. W. Knoferl, A. Ayala, P. Angele, F. W. Schildberg, K. W. Jauch, and I. H. Chaudry Sex-specific p38 MAP kinase activation following trauma-hemorrhage: involvement of testosterone and estradiol Am J Physiol Endocrinol Metab, July 1, 2003; 285(1): E189 - E196. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Ueyama, N. Shirasawa, M. Numazawa, K. Yamada, M. Shelangouski, T. Ito, and Y. Tsuruo Gastric Parietal Cells: Potent Endocrine Role in Secreting Estrogen as a Possible Regulator of Gastro-Hepatic Axis Endocrinology, August 1, 2002; 143(8): 3162 - 3170. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
, Sham; 
, trauma-hemorrhage. n = 8.
Data are expressed as the mean ± SD. *,
P < 0.05 vs. sham control.
