Download Mechanism for the maintenance of splenic T lymphocyte functions in

Articles in PresS. Am J Physiol Cell Physiol (March 26, 2003). 10.1152/ajpcell.00058.2003
Mechanism for the maintenance of splenic T lymphocyte functions in proestrus females
following trauma-hemorrhage: Enhanced local synthesis of 17β-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, AL 35294.
Running title: 17β-estradiol synthesis and cytokine release by T cells
Please address correspondence, proofs and reprint request to:
Dr. Irshad H. Chaudry
Center for Surgical Research
University of Alabama School of Medicine
G094, Volker Hall
1670 University Boulevard
Birmingham, AL 35294
Tel: 205-975-2195
Fax: 205-975-9719
E-mail: [email protected]
Key words: Inflammation, immune suppression, steroid synthesis, T lymphocytes,
cytokines.
Copyright (c) 2003 by the American Physiological Society.
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Abstract
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 following 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 17β-estradiol was assessed in the splenic T lymphocytes
of proestrus and ovariectomized mice by using appropriate substrates following TH. Analysis
for aromatase and 17β-hydroxysteroid dehydrogenases indicated increased 17β-estradiol
synthesis and low conversion into estrone in T lymphocytes of proestrus but not of
ovariectomized mice. The effect of 17β-estradiol on T lymphocyte cytokine release was reliant
upon ER expressions. This was apparent in the differences of ER expression, especially that of
ER-β, and an association between increased 17β-estradiol synthesis and sustained release of IL-2
and IL-6 in T lymphocytes of proestrus females following TH. Since 17β-estradiol is able to
regulate cytokine genes and the splenic T lymphocyte cytokine releases is altered following TH,
continued synthesis of 17β-estradiol in proestrus females appears to be responsible for the
maintenance of T lymphocyte cytokine release associated with the protection of immune
functions following TH.
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Introduction.
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 to men and are
more prone to autoimmune diseases (9,11,20). 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 prolong period, which may lead to subsequent sepsis with high mortality rates
(43,56). The immune depression is pronounced in males and ovariectomized females (OVX)
following TH compared to proestrus females (54,55). Moreover, immune functions in males and
OVX females can be restored by the administration of 17β-estradiol (E2) following TH (16-18).
Thus, gender dimorphism is obvious in the loss of immune functions following TH implicating a
major role for sex steroid hormones (2,3).
Steroid hormones regulate immune functions in vivo and the mechanisms involve not
only the control of cytokine gene transcription by the classical steroid hormone-receptor
complex, but also the tissue specific metabolism of sex steroids (12,21,24,29,33,36,47). Among
the sex steroids, estrogen is demonstrated to protect immune functions following TH because
proestrus females are not immunodepressed compared to male and OVX mice. Furthermore, the
depressed immune functions in males and OVX females following TH can be normalized by
parenteral E2 administration (16-18). Ovary is the primary site of estrogen synthesis in females
and, in spite of that, the enzymes involved in estrogen metabolism are also present in peripheral
tissues, including spleen and the T lymphocytes (21,36). 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) and the production of cytokines as needed. E2 is a highly potent
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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 following TH. In this regard, the
expression and analysis of enzymes involved in steroid metabolism is meaningful compared to
active steroid quantification, since regulatory steroids have a short half-life and quantification of
steroid at sub-picomole 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
using relevant substrates. Since 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 α and β expressions, and IL-2 and IL-6 release (the cytokines whose
release are altered following TH) in proestrus and OVX mice following TH in the same cell
preparations. The results indicated 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 probably one of the reasons why proestrus females are not immunodepressed following
TH.
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Material and Methods
Chemicals. Analytical grade reagents were used in all experiments. Androstene-4-ene-3,17dione[1,2,6,7-3H], specific activity 74 Ci/mmol; androstene-4-ene-3,17 dione[4-14C], specific
activity 54 Ci/mmol; testosterone[4-14C], 5α-dihydrotestosterone [4-14C], specific activity 57
Ci/mmol; 17β-estradiol[4-14C], specific activity 54 Ci/mmol; and estrone[4-14C], 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, Inc. (Gaithersburg, MD).
Mice. Inbred C3H/HeN female mice, 6-8 week 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 two weeks after
ovariectomy the animals were used in experiments. Animals were assigned to the following four
groups (n=8 per group): female shams; females undergoing TH; OVX female shams; 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, softtissue trauma was induced in mice by performing a 2-cm ventral midline laparotomy, which was
6
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 4x the volume of shed blood 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
sacrificed 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 (37,39). 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
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 5x106 for bioassays.
Enzyme assays. The modified assay procedures for 5α-reductase and for 17β-hydroxysteroid
dehydrogenase oxidative and reductive activities have been described in detail previously
(1,36,48). The assay mixtures after the enzyme reaction were extracted 5 times with methylene
chloride and the steroids in the organic phase were analyzed by thin layer chromatography using
the mobile phase of chloroform-ethyl acetate (3:1, v/v).
The radioactivity of the separated
steroids in the chromatographic plates was measured using InstantImager (Packard, Downers
Grove, IL) and steroids were identified by comparison to 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
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assay mixture. After centrifugation at 10000 x g for 10 min, the radioactivity in 500 µl of
supernatant was measured after addition of 5 ml 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, v/v) 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 unit/µl) for 30 min at 37o C.
PolyA+ mRNA preparation and reverse-transcriptase (RT)-PCR reactions were carried out using
the Access RT-PCR kit (Promega, Access RT-PCR System Kit, Madison, WI.). The primers
used in PCR analysis (Table I) were chosen from the cDNA sequences of GeneBank and the
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 gradient Mastercycler (Eppendorf,
Westbury, NY). The first cycle of reverse transcriptase reaction was carried out at 48o C for 45
min. The PCR cycle for amplification consisted of 30s denaturation at 94o C, followed by
annealing at 60o C for 1 min and 2 min extension at 68o C. The final products were extended for
7 min at 68o 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 expressions between the sham and TH for each enzyme was made at the cycle
where expression was nearly 50%. β-actin expression was used as the internal control. The PCR
products were analyzed by electrophoresis on 1.5% agarose gels in 1x TAE buffer and visualized
8
by ethidium bromide staining under UV illumination. The intensity of cDNA bands was
measured in the 500 Fluorescence Chemilimager (San Leandro, CA).
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 of
anti-CD3 (BD Biosciences, San Jose, CA) in complete Click’s medium at 37oC 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 (1x105 cells/ml) were added. The cultures were incubated for 48 h at 37oC
with 5% CO2. At the end of this time, 1µCi of 3H-thymidine (specific activity 6.7 Ci/mmol, New
England Nuclear, Boston, MA) 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 after during processed for liquid scintillation
counting on Model 1205 Betaplate (Pharmacia/LKB Nuclear, Gaithersburg, MD). For the IL-6
assay, to the serial dilutions of the lymphocyte culture supernatant were added 100 µl of 7TD1
cells (5x105 cells/ml) and incubated for 72 h at 37oC in 5% CO2. At the end 20 µl of 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, 5 mg/ml in RPMI1640 media)
was added to each well and the plate was incubated for additional 4h. 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 Bio-Tek automated microplate reader (Bio-Tek Instruments,
Winooski, VT). Relative units of cytokine activity was computed by comparison of the curves
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produced from dilution of the experimental samples to that generated by dilution of recombinant
mouse IL-2 or IL-6 standards (R&D, Minneapolis, MN).
Protein content. The protein content was determined by micro Bradford method (BioRad,
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. Sigma Plot 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, San Rafael, CA) 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 by Student’s t-test. Significance was achieved when p< 0.05.
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Results
17β-estardiol 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 following TH (Figure 1A). Nonetheless, the increase in aromatase activity following
TH in the tissues was not observed in OVX mice except in the adipose tissue where a significant
increase in the activity was observed (Figure 1B).
These results are in accordance with our
earlier observation (36).
E2 is also synthesized from androstenedione with E1 as the intermediate in the reaction.
Two enzymes participate in this catalysis: aromatase for conversion of androstenedione to E1
and 17β-HSD for reduction of E1 to E2. The aromatase activity associated with androstenedione
conversion to E1 in T lymphocytes of proestrus and OVX animals following TH is shown in
Figures 1C and 1D. Aromatase activity in all the tissues was low with androstenedione as a
substrate, compared to testosterone as the substrate, suggesting low conversion to E1 in all the
tissues. The reduction of E1 to E2 is catalyzed by 17β-HSD and the activity of this enzyme in T
lymphocytes is shown in Figures 2A and 2B. The 17β-HSD activities for conversion of E1 to E2
did not alter in the tissues and in T lymphocytes, following TH, in both the proestrus and OVX
animals.
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 II. The catalytic efficiency of the enzymes, Vmax/Km, indicated that the production of
E2 is from androstenedione through testosterone and not E1. Moreover, the analysis showed the
presence of relatively low 5α-reductase activity, indicating less 5α-dihydrotestosterone (DHT)
syntheses in the T lymphocytes of proestrus female mice.
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Testosterone metabolism. Testosterone is synthesized from androstenedione by reductive
catalysis. The activity of 17β-HSD associated in this catalysis in tissues of proestrus and OVX
mice, following TH, is shown in Figures 2C and 2D. In proestrus females, significant increase in
the activity was observed only in the ovary, spleen and T lymphocytes following TH (Figure
2C). The enzyme activity in adipose tissue was lower in OVX mice compared to proestrus
females, and the enzyme activity did not change in this tissue in either group following TH
(Figure 2D).
5α-reductase converts testosterone into DHT, which is a highly active androgen. There
was no change in 5α-reductase activity of T lymphocytes or other tissues of both proestrus and
OVX mice following TH (Figures 3A and 3B).
Conversion to E1. E1 is an inactive estrogen because of low binding affinity to ER. The 17βHSD 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, spleen and T lymphocytes from proestrus
females (Figures 3C and 3D). Trauma-hemorrhage and resuscitation 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 5α-reductase, aromatase and oxidative
isomers II, IV and V of 17β-HSD in T lymphocytes of sham and trauma-hemorrhaged female
mice is shown in Figure 4. The expression of aromatase did not change significantly following
TH in proestrus females or in OVX mice. Likewise, the expression of the threE17β-HSD isomers
was similar in both sham and trauma-hemorrhaged proestrus females but their expression was
reduced in OVX females following TH. 5α-reductase expression was not different in proestrus
or OVX mice following TH.
12
ER-α and ER-β expressions. The expression of ERs, α and β, in the splenic T lymphocytes from
proestrus and OVX female mice, following TH are shown in Figure 5. The ER-β expression was
low in OVX animals compared to ER-α expression. There was no change in the ER-α
expression in the T lymphocytes of proestrus and OVX animals following TH. In contrast, in
proestrus female ER-β expression decreased significantly following TH, whereas its expression
significantly increased in OVX females under those same conditions.
Cytokine expression and release. The expression of IL-2 and IL-6 in T lymphocytes of proestrus
and OVX mice, following TH, is shown in Figure 6. The IL-2 expression was low in OVX
females compared to proestrus females and TH did not alter IL-2 expression in either groups.
Stimulation of T lymphocytes with Con A, however, resulted in a significant reduction in the IL2 release in OVX animals but not in proestrus females following TH. In contrast, IL-6 expression
and release were different. Significant increase in IL-6 expression was observed in proestrus
mice following TH, however, the expression decreased significantly in OVX animals.
Moreover, Con A stimulation of T lymphocytes did not alter the release of IL-6 in T
lymphocytes from proestrus females following TH, whereas, a three decrease in the IL-6 release
was observed in OVX females under such conditions.
13
Discussion
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 since its role also documented in many autoimmune disorders and
in the outcome following TH (7,14,16,17,23,55). The majority of the estrogen effects are
mediated by two distinct intracellular receptors, ER-α and ER-β, each encoded by unique genes
(14,20,34). However, studies have also suggested that E2 interaction with other cell surface
receptors including growth factor or dopamine receptors (13,19,30,47). Non-genomically
estrogen is capable of regulating Ca++i mobilization and iNOS release 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
following TH. The divergent immune responses following TH in males and proestrus females is
also manifested by the altered release of cytokines, IL-2 and IL-6, by the splenic T lymphocytes
(2,3,16,54,55). Since 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. 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
following TH.
Enzyme kinetics shows that synthesis of E2 from androstenedione is via the formation of
testosterone and not via E1. This study indicates a correlation between increased endogenous
synthesis of E2, low conversion to E1 (Figure 7), and the persistent release of IL-2 and IL-6 in
14
the lymphocytes of proestrus female following TH.
This is substantiated by: (a) 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, (b) increased
production of testosterone from catalytic reduction of androstenedione by 17β-HSD in proestrus
animals following 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
(c) the comparatively low oxidative catalysis by 17β-HSD in both proestrus and OVX animals
suggesting little or no conversion of E2 into E1. The expressions of 17β-HSD isomers were
analyzed by routine RT-PCR analysis, which is not quantitative. Our aim was to determine
whether different forms of the 17β-HSD isomers are expressed in T lymphocytes and if they are
expressed, whether their expression is altered following TH. The enzyme expressions were
therefore evaluated in the same T lymphocyte preparation that was used for enzyme assays,
estrogen receptor expression as well as IL-2 and IL-6 expression and release. The results show
changes in the expression of 17β-HSD isomers after ovariectomy and following TH. It is,
however, necessary to quantify the 17β-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 5α-reductase activity,
either after ovariectomy or following TH in proestrus and OVX females was evident indicating
little change in the production of DHT in T lymphocytes. Since DHT is considered as an
inhibitor of aromatase activity (6,41), an increase in its activity would have lowered E2
production.
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. This, however, is
15
not surprising since E2 formation from testosterone is the result of a coupled reaction involving
aromatase P450 and a flavoprotein NADPH-cytochrome P450 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:NADH ratio in tissues following 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-α and ER-β, and our study shows that both the subtypes are present in the splenic
T lymphocytes of female mice. However, their expression in response to TH is different. ER-α
expression did not change after ovariectomy or following TH, whereas ER-β expression
decreases significantly following ovariectomy. Moreover, expression of ER-β is significantly
different in the proestrus and OVX animals after TH; its expression is decreased in proestrus and
increased in OVX animals. The increased production of E2, attenuated expression of ER-β in the
proestrus and the opposite effects in the OVX females following TH suggest that down
regulation of ER-β may be a factor associated with the change in the cytokine releases by T
lymphocytes. Since the T lymphocyte populations used in the experiments consisted of both
CD4+ and CD8+ phenotypes (37), 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 since: (i) the alterations in the release of these cytokines is an indication
16
of the proinflammatory condition, i.e., Th1 to Th2 shift, and (ii) our earlier studies have
demonstrated marked alterations in the release of these cytokines in OVX females but not in
proestrus females following TH (16,55). Furthermore, the release of these cytokines can by
restored by administration of E2 in OVX females during or immediately resuscitation (16). In the
present study, we observed the expression and release of the pro inflammatory 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 animals when compared to proestrus, 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 following
TH. The release of the cytokines, determined by bioassay in response to antiCD3 stimulation of
T lymphocytes, was also different. The use of antiCD3 as a stimulant for T lymphocytes
cytokine releases is evocative since Con A is primarily a mitogen associated with cell
proliferation whereas antiCD3 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 following 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 following TH appears to be
associated with sustained release of IL-2 and IL-6 in those animals since loss in E2 production is
reflected by decreased release of these cytokines in the OVX females following TH. The
cytokine releases in this study were determined in T lymphocyte preparation that contained
CD4+ and CD8+ subsets. Analysis of cytokines expression and release in each T lymphocyte
subsets is important for any meaningful correlation.
17
Substantial emphasis has been focused recently on the regulation of extra-gonadal
biosynthesis of sex steroids. The local synthesis of active steroids in T lymphocytes is essential
for carrying out of their specific functions, especially, the release of cytokines. The rate of
formation of each steroid depends upon the level of expression of the specific androgen- and
estrogen-synthesizing enzymes in the tissue. Moreover, local synthesis of active steroids is
meaningful compared to availability in circulation, since 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
following TH as reflected in the decreased release of IL-2 and IL-6 by T lymphocytes (58). In
this study, we demonstrate enhanced synthesis of E2, which promotes the maintenance IL-2 and
IL-6 release by T lymphocytes in proestrus mice following TH. Thus, both of our studies
suggest an important role for steroid metabolizing enzymes in the release of cytokines by T
lymphocyte following TH. Among the sex steroid metabolizing enzymes, the activities of 17βHSD isomers appears to be critical since they catalyzes 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 5androstene-3β,17β-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 following TH restores immune functions in male mice and the effects appear to be
mediated via the ER since 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,
17β-HSD isomers play a unique role in the sex-steroid sensitive physiological functions.
18
Clinical trauma is a pathological condition that produces an inflammatory response and
our recent retrospective study reveals female patients in pre-menopausal 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 following 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 the basis
of the pathophysiology of TH and help in the development of improved therapy to
prevent/decrease morbidity and mortality following TH.
19
Acknowledgments.
This work was supported by a grant from the National Institutes of Health, R01 GM-37127.
20
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Footnotes.
Abbreviations: Trauma-hemorrhage and resuscitation, TH; Ovariectomized, OVX; E1, estrone;
E2, 17β-Estradiol; DHT, 5α-Dihydrotestosterone; DHEA, Dehydroepiandrosterone; ER-α,
Estrogen receptor α; ER-β, Estrogen receptor β; 17β-HSD, 17β-Hydroxysteroid dehydrogenase.
28
Table 1. Primers used in PCR analysis.
Target mRNA
Primer sequences
Location
5α-Reductase type II (22)
5’CAGTGGTACTAAGCACAGAAACTCAC3’
5’AGCTACACCTGAAGATTTACTTCACC3’
5’CTTTCAGCCTTTTGGCTTTG3’
5’ TTTCTTCACTGGTCCCCAAC3’
5’ TCCTGGCCATAGTTCTCTCCTG3’
5’CAAGGCGTTTCTGCCTCTACTT3’
5’ GGATTTTCTGCAAGGCATGT3’
5’ CCCTTCAGAGCTTGGCATAG3’
5’ TTCTGACTTCCATGGGCATT3’
5’ TAGACGCACTGTCTGCTGCT3’
5’GAAGCTAGTGCTCAGCTCTCTA3’
5’TAGAGTCTCTGTACAGCCAAGG3’
5’ATGGCATTCTACAGTCCTGCTGTGATGAAC3’
5’TCACTGAGACTGGAGGTTCTG3’
5’ACAGGTTCCCCGGGCAGCATCTCTA3’
5’TCAGCATCTCATCCCAGCCCAAGCA3’
5’TGATGCTGGTGACAACCACGGCCTTC3’
5’AGCCACTCCTTCCTGTGACTCCAGCTT3’
3793-3819
4245-4271
1324-1343
1754-1773
697-718
971-990
1621-1640
2105-2224
63-84
345-365
590-610
1146-1168
1162-1184
1375-1497
100-121
336-357
34-59
647-671
Aromatase (44)
17β-HSD type II (26)
17β-HSD type IV (28)
17β-HSD type V (8)
ER-α (53)
ER-β (46)
IL-2 (15)
IL-6 (53)
29
Table 2. Kinetics of aromatase, 5α-reductase and 17β-HSD of splenic T lymphocytes with
different substrates
Activity
Aromatase
Substrate
Testosterone
Androstenedione
Testosterone
5α-Reductase
Androstenedione
17β-HSD
Estrone
(reductive)
*pmoles/mg protein/min
Product
Vmax*
Km
Vmax/ Km
17β-estradiol
Estrone
5α-dihydrotestosterone
Testosterone
17β-estradiol
200
186
150
150
120
7.5
25.8
24
16.7
48.3
26.6
7.2
6.25
9.0
2.5
30
Figure Legends
Figure 1. Aromatase activity in different tissues isolated from proestrus and ovariectomized
(OVX) mice following trauma-hemorrhage, with testosterone (A and B) and 4-androstenedione
(C and D) as substrates. The data are expressed as mean + SD of 8 experiments for each group.
S, sham. *p<0.05 vs. sham.
Figure 2. The reductase activity of 17β-hydroxysteroid dehydrogenase (17β-HSD) in different
tissues isolated from proestrus and OVX mice following trauma-hemorrhage, with estrone and
(A and B) and 4-androstenedione (C and D) as substrates. The data are expressed as mean + SD
of 8 experiments for each group. S, sham. *p<0.05 vs. sham.
Figure 3. The activity of 5α-reductase with testosterone (A and B) as the substrate and 17βhydroxysteroid dehydrogenase (17β-HSD), oxidative, with 17β-estradiol (C and D) as the
substrate in different tissues isolated from proestrus and OVX mice following TH. The data are
expressed as mean + SD of experiments for each group. S, sham. *p<0.05 vs. sham.
Figure 4: The expression of 5α-reductase (5AR, 479 bp); aromatase (ARO, 450 bp); 17βhydroxysteroid dehydrogenase oxidative isomers (17β-HSD type II 294 bp, type IV 605 bp and
type V 303 bp), analyzed by RT-PCR assay, in splenic T lymphocytes of proestrus and OVX
mice following TH. S, sham. The data shown is a representative of four separate experiments.
Figure 5: The effect of trauma-hemorrhage on ER-α and ER-β expression in T lymphocytes of
proestrus (P) and OVX mice. S, sham. The data is representative of four separate experiments.
Figure 6: The effect of trauma-hemorrhage on the expression and release of IL-2 and IL-6 by T
lymphocytes from proestrus and OVX mice. The IL-2 and IL-6 expression in lymphocytes were
by RT-PCR analysis and the data is a representative of 4 separate experiments. The relative
intensity of the band (receptor expression from 4-6 analyses) is shown in the histogram. For
31
cytokine release, the lymphocytes were stimulated with 10 µg per ml of anti-CD3 in Click’s
medium at 37oC for 36 h. S, sham. Data are expressed as mean + SD of 8 experiments for each
group. *p<0.05 vs. sham.
Figure 7: Metabolism of 17β-estradiol in the T lymphocytes. 5-AR, 5α-reductase; 17β-HSD,
17β-hydrosysteroid dehydrogenase; R, reduction; O, oxidation.
32
Samy et al Figure 1
Aromatase activity
17β-estradiol formed
pmoles/mg protein/min
80
80
A
Proestrus
*
60
TH
S
40
*
20
*
*
*
*
20
0
0
Adrenal
gland
Ovary Adrenal Spleen T cells Adipose
gland
Aromatase activity
estrone formed
pmoles/mg protein/min
TH
S
60
40
10
OVX
B
C
Proestrus
S
TH
5
10
Spleen T cells
Adipose
OVX
D
S
5
0
0
Ovary
Adrenal Spleen
gland
T cells
Adipose
Adrenal
gland
Spleen T cells
Adipose
TH
33
17β-HSD activity (reductive)
17β-estradiol formed
pmoles/mg protein/min
Samy et al Figure 2
Proestrus
A
10
S
5
10
OVX
B
S
TH
5
0
0
Adrenal
gland
17 β-HSD activity (reductive)
testosterone formed
pmoles/mg protein/min
TH
Ovary
Spleen
C
Adrenal
gland
Adipose
T cells
D
Proestrus
50
S
*
*
25
TH
Spleen T cells
50
Adipose
OVX
S
*
25
0
0
Adrenal
gland
Ovary
Spleen
T cells
Adipose
Adrenal
Spleen
gland
T cells Adipose
TH
34
Samy et al Figure 3
5α-Reductase activity
5α-dihydrotestosterone formed
pmoles/mg protein/min
60
50
60
A
Proestrus
S
40
40
30
30
20
20
10
10
0
17β-HSD activity, (oxidative)
estrone formed
pmoles/mg protein/min
S
TH
0
Adrenal
gland
Ovary Adrenal Spleen T cells Adipose
gland
30
OVX
B
50
TH
C
15
TH
T cells
D
Proestrus
S
Spleen
Adipose
OVX
S
30
15
0
0
Adrenal
gland
Ovary
Spleen
T cells
Adipose
Adrenal
gland
Spleen
T cells
Adipose
TH
35
Samy et al Figure 4
17β-HSD
5AR
ARO
II
IV
V
600 bp
400 bp
300 bp
S TH S TH S TH S TH S TH
Proestrus
17β-HSD
5AR
II
ARO
IV
V
600 bp
400 bp
S TH S TH S TH S TH S TH
OVX
36
Samy et al Figure 5
ER-α
600 bp
600 bp
ER-β
S
TH
S
Proestrus (P)
TH
OVX
Relative band intensity
100
S
TH
80
60
*
40
*
20
0
P OVX
ER-α
P OVX
ER-β
37
Samy et al Figure 6
600 bp
TH
IL-2 expression
Relative band intensity
Proestrus
S
TH
OVX
100
S
TH
80
60
40
20
0
S TH
Proestrus
TH
IL-6 expression
Relative band intensity
100
80
TH
OVX
S
TH
60
40
0
*
S TH
Proestrus
S TH
OVX
*
100
0
Proestrus
S
*
20
200
OVX
IL-6
S
S
TH
S TH
600 bp
Proestrus
300
S TH
IL-6 release, units/mg protein
S
IL-2 release, units/mg protein
IL-2
S TH
OVX
S
TH
300
*
150
0
S TH
Proestrus
S
TH
OVX
38
Samy et al Figure 7
Metabolism of 17β-estradiol in T lymphocytes
Aromatase
4-Androstenedione
Estrone
17β-HSD R
O
O
R 17β-HSD
Testosterone
17β-Estradiol
Aromatase
5AR
5α-Dihydrotestosterone