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Center for Surgical Research and Department of Surgery, University of Alabama at Birmingham School of Medicine, Birmingham, Alabama 35294
Submitted 30 December 2002 ; accepted in final form 7 April 2003
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ABSTRACT |
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 TOP ABSTRACT METHODS AND MATERIALSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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, IL-10 release, and splenic
M
IL-1
, IL-6, IL-10, and IL-12 release were determined 24 h after
TH. Plasma IL-6 levels were significantly increased in vehicle-treated
females, and DHEA administration markedly attenuated this response. In
vehicle-treated females, splenocyte proliferation, IL-2, IL-3, and IFN-
release, and splenic M IL-1, IL-6, and IL-12 release were
maintained or slightly enhanced after TH. In DHEA-treated females, however,
these immune functional parameters were either unaltered compared with
vehicle-treated animals or even further enhanced, but surprisingly were not
depressed. Moreover, DHEA reduced splenocyte and splenic M
anti-inflammatory cytokine (i.e., IL-10) production after TH compared with
vehicle-treated females. Because DHEA further enhances the immune
responsiveness in proestrus females after TH, this hormone might be a useful
adjunct even in females for further enhancing immune responses and decreasing
the mortality rate after trauma and severe blood loss. inflammation; cytokines; gender
It is well known that dehydroepiandrosterone (DHEA), the most abundant
steroid hormone in humans
(27), is an intermediate in
the pathway for the synthesis of both androgen and estrogen. In this regard,
estrogen administration has been shown to have immunomodulatory effects under
various physiological and pathological conditions
(15). Recently, studies in
animal models have shown that DHEA is effective in restoring immune functions
after thermal injury as well as sepsis
(4,
5,
22,
23). Furthermore, DHEA
administration normalized splenocyte apoptosis and lymphocyte migration
(23) after hemorrhagic shock,
restored the depressed cell-mediated immune responses after trauma-hemorrhage,
and significantly reduced the mortality rates from a subsequent septic
challenge (3,
8). It is of interest to note
that in those studies male animals were used to examine the effects of
exogenous hormone administration. However, depending on the hormonal milieu,
both androgenic and estrogenic effects of DHEA have been reported
(9). Studies have also shown
that administration of 17-estradiol in male animals after
trauma-hemorrhage had beneficial immunomodulatory effects
(14). If DHEA is converted to
estrogens in male animals, the increased concentrations of estrogen would be
expected to further enhance/improve immune responses after trauma-hemorrhage.
Conversely, in female animals, the conversion of DHEA to testosterone should
have immunodepressive effects. This notion is supported by data from studies
that showed that pretreatment of proestrus female animals with testosterone
before the onset of trauma-hemorrhage led to marked immune depression
(1). In view of this, it could
be postulated that the administration of DHEA in female animals after
trauma-hemorrhage will have deleterious effects on immune responses since DHEA
will be metabolized to testosterone, a hormone with immunosuppressive
properties. The aim of the present study, therefore, was to determine whether
administration of DHEA in female animals in the proestrus state of the estrus
cycle has any deleterious effects on immune functional parameters after
trauma-hemorrhage.
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METHODS AND MATERIALS |
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TOPABSTRACTMETHODS AND MATERIALSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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Experimental groups. The state of the estrus cycle was determined daily in each female animal by light microscopic examination of vaginal smear cytology for at least 1 day before the experiment. In the morning of the experiment, female mice were selected in the proestrus state of the estrus cycle by typical cytology and then randomly assigned to the treatment or vehicle groups. Proestrus mice underwent trauma-hemorrhage or sham operation. After sham operation, the mice received a subcutaneous injection of vehicle (propylene glycol, Sigma Chemical, St. Louis, MO). Immediately before fluid resuscitation, the mice in the trauma-hemorrhage group received a subcutaneous injection of DHEA (4 mg/kg body wt, Sigma Chemical) or vehicle. This dose and route of DHEA administration have been previously found to be effective in restoring immune functional parameters after trauma-hemorrhage (3, 8).
Trauma-hemorrhage procedure. Mice in the trauma-hemorrhage groups were lightly anesthetized with methoxyflurane (Metofane, Pitman Moore, Mundelein, IL) and restrained in a supine position, and a 2.5-cm midline laparotomy (i.e., soft tissue trauma induced) was performed, which was then closed aseptically in two layers by using 6-0 Ethilon sutures (Ethicon, Somerville, NJ). Both femoral arteries were then aseptically cannulated with polyethylene 10 tubing (Clay-Adams, Parsippany, NJ) by using a minimal dissection technique, and the animals were allowed to awaken. Blood pressure was constantly monitored by attaching one of the catheters to a blood pressure analyzer (Micro-Med, Louisville, KY). Lidocaine was applied to the incision sites to provide analgesia during the study period. On awakening, the animals were bled rapidly through the other catheter to a mean arterial blood pressure of 35 ± 5 mmHg (mean arterial blood pressure prehemorrhage was 95 ± 5 mmHg), which was maintained for 90 min. At the end of that procedure, the animals were resuscitated with four times the shed blood volume in the form of lactated Ringer solution. The catheters were then removed, the vessels ligated, and the groin incisions closed. Sham-operated animals underwent the same surgical procedure, which included ligation of both femoral arteries, but neither hemorrhage nor fluid resuscitation was carried out. There was no mortality observed in this model of trauma-hemorrhage (26).
Blood, tissue, and cell harvesting procedure. The animals were killed by methoxyflurane overdose at 24 h after trauma-hemorrhage to obtain the spleen and whole blood.
Plasma collection and storage. Whole blood was obtained by cardiac puncture and placed in microcentrifuge tubes (Microtainer, Becton Dickinson, Rutherford, NJ). The tubes were then centrifuged at 16,000 g for 15 min at 4°C. Plasma was separated, placed in pyrogen-free microcentrifuge tubes, immediately frozen, and stored at -80°C until assayed.
Cell line maintenance. The IL-2-dependent CTLL-2 cells and the
IFN--dependent RAW 264.7 cells were obtained from the American Type
Culture Collection and maintained according to their directions. The
IL-3-dependent FDC-P1 cells (a gift from Dr. Charles Janeway, Yale University,
New Haven, CN) were maintained as previously described
(10). The IL-6-sensitive
murine B-cell hybridoma (7TD1) (a gift from Dr. Jacques Van Snick, The Ludwig
Institute for Cancer Research, Brussels, Belgium) was maintained as previously
described (10).
Preparation of splenocyte culture. At 24 h after trauma-hemorrhage
or sham operation, the spleens were removed aseptically, and splenocytes were
isolated as previously described in detail
(29). In brief, the spleens
were gently ground between frosted microscope slides to produce a single-cell
suspension. This suspension was centrifuged at 300 g for 15 min.
After resuspension, the erythrocytes were lysed hypotonically, and the
remaining cells were washed with PBS by centrifugation (300 g for 15
min). Viability was tested by using trypan blue exclusion and found to be
95% irrespective of the group assessed. The splenocytes were then
resuspended in RPMI/1640 (GIBCO-BRL, Grand Islands, NY) containing 10%
heat-inactivated fetal bovine serum (GIBCO-BRL) to yield a final concentration
of 1 x 106 cells/ml. The ability of the splenocyte cultures
to produce lymphokines in response to a mitogenic challenge, was assessed by
incubation for 48 h (at 37°C, 5% CO2, and 90% humidity) in the
presence of 2.5 µ/ml Concanavalin A (Pharmacia/LKB Biotech, Piscataway,
NJ). After incubation, the cell suspension was centrifuged at 300 g
for 15 min, and the supernatants were harvested and stored at -80°C until
assayed.
Splenocyte proliferation. The cells' ability to proliferate in response to mitogenic stimulation with 0 (negative control) or 2.5 µ/ml Concanavalin A was measured by 3[H]thymidine incorporation technique as previously described (19, 26).
Preparation of splenic macrophage culture. Splenic macrophage cultures were established as previously described in detail (30). The monolayers of splenic macrophages (1 x 106 cells/ml) were stimulated with 10 µ of lipopolysaccharide W (from Escherichia coli 055:B5, Difco Laboratories, Detroit, MI) per milliliter of Click's medium containing 10% heat-inactivated fetal bovine serum (GIBCO-BRL) for 48 h at 37°C, 5% CO2, and 90% humidity to assess the cells' ability to release cytokines. At the end of the incubation period, the culture supernatants were removed, centrifuged at 300 g for 15 min, divided into aliquots, and stored at -80°C until assayed for cytokine concentrations.
Assessment of cytokine release. The capacity of the mixed
splenocyte culture to produce IL-2 (CTLL-2), IL-3 (FDC-P1), and IFN-
(RAW 264.7) was assessed by determining the amount of respective lymphokines
in the collected culture supernatant by using specific bioassays as previously
described in detail (16,
20). IL-6 activity was
determined by assessing the 72-h proliferation of the IL-6-dependent murine
hybridoma 7TD1 cells stimulated by serial dilutions of plasma or supernatants
as described in detail elsewhere
(6,
21). Concentrations of
IL-1 (DuoSet, Genzyme, Cambridge, MA), IL-10, and IL-12 (BD OptEIA ELISA
Set, BD Pharmingen, San Diego, CA) in macrophage and splenocyte supernatants
were determined by using a sandwich-enzyme-linked immunosorbent assay
technique (ELISA) according to the manufacturer's recommendations.
Determination of plasma hormone concentrations. Concentrations of
17-estradiol, testosterone, and DHEA were determined by using
commercially available radioimmunoassays (ICN Biomedicals, Costa Mesa, CA), as
described by the manufacturer.
Statistical analysis. The results are presented as means ± SE. One-way ANOVA followed by the Student-Newman-Keuls test as a post hoc test for multiple comparisons was used to determine the significance of the differences between experimental means. A P value of <0.05 was considered significant.
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RESULTS |
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TOPABSTRACTMETHODS AND MATERIALSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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Plasma hormone levels. At 24 h after administration of DHEA, no
significant effects on circulating concentrations of DHEA were detectable
(Fig. 2A). Plasma
concentrations of 17-estradiol decreased after traumahemorrhage, and
administration of DHEA had no significant effects on 17-estradiol levels
(Fig. 2B). Likewise,
plasma testosterone concentrations were decreased in animals that underwent
trauma-hemorrhage, and administration of DHEA did not increase plasma levels
of testosterone (Fig.
2C).
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Splenocyte proliferation. At 24 h after trauma-hemorrhage, splenocyte proliferative capacity was unaltered in proestrus females, irrespective of whether the animals received vehicle (64.964 ± 16.431 counts/min) or DHEA (59.195 ± 16.339 counts/min) during resuscitation (sham group: 65.754 ± 12.868 counts/min).
Splenocyte T helper 1 and 2 cytokine release. Although splenocyte
IL-2 release was slightly elevated in vehicle-treated female mice after
trauma-hemorrhage, DHEA administration led to a further increase in IL-2
release that did not reach statistical significance
(Fig. 3A). Comparable
to IL-2, the release of IL-3 was increased (P > 0.05) in
vehicle-treated female mice after trauma-hemorrhage, and treatment with DHEA
led to a significantly increased splenocyte IL-3 release in proestrus female
mice (P < 0.05; Fig.
3B). In proestrus female mice, trauma-hemorrhage as well
as DHEA administration had no effect on IFN- release. DHEA
administration significantly reduced anti-inflammatory, i.e., IL-10, release
in proestrus female mice after trauma-hemorrhage (P < 0.05;
Fig. 4A).
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Splenic macrophage responses. In vehicle-treated proestrus female
mice, there was a trend toward increased splenic macrophage release of
IL-1, IL-6, and IL-12, which was even more pronounced in DHEA-treated
female mice (P > 0.05; Fig.
5). In contrast to proinflammatory cytokine release, splenic
macrophage anti-inflammatory cytokine (i.e., IL-10) release decreased in
female mice that received DHEA during resuscitation. This decrease, however,
did not reach statistical significance
(Fig. 4B).
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DISCUSSION |
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TOPABSTRACTMETHODS AND MATERIALSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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-dihydrotesterone, and
estrogen, and, depending on the hormonal milieu, both androgenic and
estrogenic effects of DHEA have been reported
(9). In this respect,
testosterone has been shown to exhibit immunosuppressive properties in male
and female mice (1,
2). Thus we postulated that the
prevailing hormonal milieu in female mice might lead to conversion of DHEA
into testosterone, thereby exerting deleterious effects on immune responses.
To test this hypothesis, female mice in the proestrus state of the estrus
cycle were treated with DHEA after trauma-hemorrhage. Because our laboratory's
previous studies (28) have
shown that female mice in this state exhibit maintained/enhanced immune
responses after trauma-hemorrhage, DHEA could be immunosuppressive under those
conditions. Our results indicate that plasma concentrations of IL-6 significantly increased in vehicle-treated proestrus female mice after trauma-hemorrhage. Administration of DHEA significantly reduced the increase in circulating IL-6, suggesting an attenuation of the inflammatory response to trauma-hemorrhage. However, in view of the fact that stimulated in vitro IL-6 production by splenic macrophages was further increased in DHEA-treated female mice, it appears that cell populations other than splenic macrophages are susceptible to functional modulation by DHEA. In this regard, the effects of DHEA on Kupffer cell function should be further investigated, because these liver tissue-fixed macrophages have been previously shown to play a pivotal role in the elaboration of systemic cytokine levels after trauma-hemorrhage (24).
The data further indicate that vehicle-treated proestrus female mice showed no depression in splenocyte immune functions after trauma-hemorrhage. Interestingly, administration of DHEA further enhanced splenocyte responses after trauma-hemorrhage. These findings are in sharp contrast to our hypothesis, which was that in female mice DHEA would be converted to androgenic intermediates and cause deleterious effects on immune responses. Comparable to splenocytes, splenic macrophage function was unaltered in vehicle-treated proestrus female mice after trauma-hemorrhage, and administration of DHEA had no adverse effects on splenic macrophage function. In both splenocytes and splenic macrophages, DHEA administration led to decreased anti-inflammatory cytokine (i.e., IL-10) production after trauma-hemorrhage. In this regard, an increased IL-10 release in male mice has been shown to contribute to the immunosuppression after trauma-hemorrhage (7). The decreased release of anti-inflammatory cytokines in DHEA-treated mice after trauma-hemorrhage might thus contribute to the enhanced immune responses in those animals. Whether the improved immune response in DHEA-treated female mice after trauma-hemorrhage is associated with an increased survival rate after the induction of subsequent sepsis remains to be determined.
The analysis of circulating hormone concentrations at 24 h after
trauma-hemorrhage or sham operation revealed no significant changes in DHEA
plasma levels between animals receiving vehicle or DHEA. Furthermore, plasma
concentrations of 17-estradiol and testosterone were decreased in female
mice that underwent trauma-hemorrhage compared with sham-operated female mice,
and DHEA administration had no significant effect on hormonal status at that
time point studied. It could therefore be suggested that dilutionary effects
due to resuscitation with Ringer lactate after trauma-hemorrhage masked the
increase in circulating DHEA in proestrus female mice. It is also possible
that DHEA concentrations were significantly elevated at an earlier time after
trauma-hemorrhage. The findings that neither 17-estradiol nor
testosterone levels were significantly increased in DHEA-treated mice compared
with animals receiving vehicle are in line with the results of Jarrar et al.
(11). In that study, a higher
dose of DHEA (30 mg/kg body wt) was used compared with the dose used in this
study, and the authors found an 17- to 19-fold increase in the plasma
levels of DHEA at 24 h after its administration with no effect on
17-estradiol or testosterone concentrations. In view of this, it could
be postulated that DHEA modulates immune functions primarily by direct
effects. Support for the notion that DHEA has actions independent of the
conversion to androgens comes from studies that have shown that DHEA produces
salutary effects on immune functions in androgen-unresponsive mice
(27). However, because DHEA
can be metabolized at the cellular level in peripheral tissues
(18), one cannot rule out
alterations in the site-specific hormonal micro-milieu under those conditions.
It appears, however, that, in female mice, the conversion of DHEA to
androgenic steroids plays a minor role in regulating posttrauma immune
functions. Alternatively, the female sex hormones present in the proestrus
state of the estrus cycle might be sufficient to preserve immune responses
despite newly developed testosterone. In addition, it is possible that in
proestrus females DHEA acts synergistically with other hormones to further
stimulate/prevent immune responses after trauma-hemorrhage. In this regard,
female rodents in the proestrus state of the estrus cycle have elevated
concentrations of the immunomodulatory hormones such as prolactin, estrogen,
and progesterone (25). If DHEA
acts via estrogen receptors, it is possible that upregulation of estrogen
receptors because of elevated estrogen levels during the proestrus phase may
also contribute to the immunomodulatory effects of DHEA. In this regard,
recent studies have suggested that DHEA can also directly bind to and activate
the estrogen receptor (8,
11). Support for an important
role of estrogen receptors comes from studies by Catania et al.
(8). The in vitro stimulatory
effects of DHEA on splenocyte proliferative capacity were blocked in the
presence of the estrogen-receptor antagonist tamoxifen; however, addition of
the androgen-receptor antagonist flutamide had no inhibitory effects
(8). Furthermore, it has been
shown that the salutary effects of DHEA administration on cardiovascular and
hepatocellular function after trauma-hemorrhage were abolished in the presence
of the estrogen receptor-antagonist ICI-182,780
(11). Thus it appears that the
beneficial effects of DHEA on immune, cardiac, and hepatocellular functions in
male rodents after trauma-hemorrhage are, in part, mediated via the estrogen
receptor. Support for the pivotal role of estrogen receptors for
immunoprotection after trauma-hemorrhage comes from studies that indicate that
in vivo administration of a selective estrogen-receptor antagonist in
proestrus female mice significantly depressed immune responsiveness
(17). Additional studies are
required to further elucidate the precise mechanisms involved in mediating the
beneficial immunomodulatory effects of DHEA in proestrus female mice after
trauma-hemorrhage.
Although our findings and previous data indicate that immune responses are
not compromised in young proestrus female mice, it has been shown that
ovariectomized female mice as well as aged female mice with decreased
circulating levels of female sex steroids have depressed immune functional
parameters after traumahemorrhage
(12,
13). However, administration
of 17-estradiol after trauma-hemorrhage normalized cell-mediated immune
responses in ovariectomized female mice
(15). Because the adverse
immune response to trauma is closely related to the hormonal environment in
the host, the use of immunomodulatory hormones in female mice with lowered
hormone levels or hormonal deficiencies appears a valuable therapeutic
strategy. In view of the known salutary immunomodulatory properties of DHEA in
male animals and of our findings that exogenous DHEA had stimulatory effects
on immune functions even in proestrus female mice after trauma-hemorrhage, it
could be proposed that this hormone should be considered as a potential
therapeutic adjunct for use in patients with known hormonal deficiency, such
as postmenopausal or postovariectomy patients.
In summary, the data presented indicate that administration of DHEA in proestrus female mice after trauma-hemorrhage further improved splenocyte and splenic macrophage function compared with vehicle-treated animals. Therefore, it appears that DHEA has beneficial immunomodulatory effects in female mice despite the possibility of its potential metabolism toward immunosuppressive androgen. Thus DHEA represents a safe steroid hormone in males and females for improving the depressed immune responses after trauma-hemorrhage and for decreasing the mortality rates from subsequent sepsis.
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DISCLOSURES |
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TOPABSTRACTMETHODS AND MATERIALSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Present address of M. W. Knöferl: University of Ulm, Dept. of Trauma-Surgery, Steinhövelstr. 9, 89075 Ulm, Germany.
Present address of M. K. Angele: Dept. of Surgery, Klinikum Grosshadern, Marchioninstr. 15, 81377 Munich, Germany.
Present address of R. A. Catania and M. D. Diodato: Center for Surgical Research and Department of Surgery, Brown University School of Medicine and Rhode Island Hospital, Providence, RI 02903.
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REFERENCES |
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TOPABSTRACTMETHODS AND MATERIALSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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