Center for Surgical Research and Department of Surgery, Brown University School of Medicine and Rhode Island Hospital, Providence, Rhode Island 02903
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ABSTRACT |
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Studies indicate that macrophage immune responses in males are
depressed after trauma-hemorrhage, whereas they are enhanced in females
under such conditions. Nonetheless, the involvement of male and female
sex steroids in this gender-dependent dimorphic immune response after
trauma-hemorrhage remains unclear. To study this, male C3H/HeN mice
were castrated and treated with pellets containing either vehicle,
5-dihydrotestosterone (DHT), 17
-estradiol, or a combination of
both steroid hormones for 14 days before soft tissue trauma (i.e.,
laparotomy) and hemorrhagic shock (35 ± 5 mmHg for 90 min followed by
adequate fluid resuscitation) or a sham operation. Twenty-four hours
later the animals were killed, plasma was obtained, and Kupffer cell
and splenic and peritoneal macrophage cultures were established. For
DHT-treated mice, we observed significantly decreased releases of the
proinflammatory cytokines interleukin 1
(IL-1
) and IL-6 by
splenic macrophage (
50 and
57%, respectively) and
peritoneal macrophage (
51 and
52%, respectively)
cultures after trauma-hemorrhage compared with releases by cultures of
cells from mice subjected to a sham operation; in contrast, responses
of splenic and peritoneal macrophage cultures from other groups
subjected to trauma-hemorrhage did not change
significantly. In addition, only DHT-treated animals exhibited increased Kupffer cell IL-6 release (+634%). The release of
IL-10 in DHT-treated hemorrhaged animals was increased compared with
that in sham-operated animals but was decreased in estrogen-treated mice under such conditions. These results suggest that male and female
sex steroids exhibit divergent immunomodulatory properties with respect
to cell-mediated immune responses after trauma-hemorrhage.
gender; immune depression; Kupffer cells; shock
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INTRODUCTION |
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SEVERAL CLINICAL and experimental studies demonstrate gender differences in the susceptibility to and morbidity from sepsis and trauma (13, 22, 25, 38, 40). Female mice in the proestrus state tolerate sepsis better than male mice, as evidenced by a significantly lower mortality rate after polymicrobial sepsis (40). Furthermore, female mice exhibit enhanced immune responses, as opposed to depressed immune function in males, after trauma-hemorrhage (35). Studies indicate that this immunological gender dimorphism appears to be hormonally regulated and that the hormones involved originate primarily from the gonads and secondarily from the thymus and the hypothalamus-pituitary gland (18).
In this respect, studies examining the effects of depletion of male sex
steroids by castration of male mice before mycobacterium infection
showed that castration increased the host resistance to infection (38).
Moreover, castration of male animals before hemorrhagic shock prevented
the depression of immune responses typically observed in normal males
after this event (34). Similarly, the administration of an androgen
receptor blocker, e.g., flutamide, restored depressed immune responses
and increased the survival rate of hemorrhaged animals subjected to
subsequent sepsis (3, 33). In addition, the administration of
5-dihydrotestosterone (DHT) to female mice depressed the splenic and
peritoneal macrophage immune response after trauma-hemorrhage to levels
comparable to those in males under such conditions (1, 2).
In contrast to testosterone, female sex steroids appear to have immunoenhancing effects after infection or circulatory stress (16, 38). In this regard, estrogens have been shown to increase the resistance of host animals to infections, i.e., those caused by Streptococcus and Mycobacterium marinum (25, 38). Moreover, estrogens have been shown to stimulate macrophage functions, as evidenced by increased clearance of IgG-coated erythrocytes (16). Overall these findings suggest that female sex steroids should improve macrophage function in contrast to the immunodepressive effects of male sex steroids.
Despite the abundance of information demonstrating the effect of sex
steroids in modulating normal immune cell responsiveness, there is
limited knowledge concerning the capacities of these hormones to alter
the release of pro- and, in particular, anti-inflammatory cytokines by
macrophages after trauma-hemorrhage. Therefore the aim of the present
study was to investigate the cell-mediated immune response after
trauma-hemorrhage in animals that were depleted of sex steroids by
castration and then supplemented with known amounts of DHT and/or
17-estradiol. The release of pro- and anti-inflammatory cytokines by
Kupffer cells and splenic and peritoneal macrophages was measured to
determine the effect of sex steroids on macrophage populations
harvested from these different microenvironments.
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MATERIALS AND METHODS |
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Animals. Inbred male C3H/HeN mice (Charles River Laboratories, Wilmington, MA), 7 wk old (24-26 g body wt), were used in this study. Mice were castrated 2 wk before the experiment as previously described (34). All procedures were carried out in accordance with the guidelines set forth in the Animal Welfare Act and the Guide for the Care and Use of Laboratory Animals by the National Institutes of Health. This project was approved by the Institutional Animal Care and Use Committee of Rhode Island Hospital and Brown University.
Hormonal supplementation.
Twenty-one-day release pellets containing 7.5 mg
5-dihydrotestosterone (DHT), 0.5 mg 17
-estradiol, or vehicle
(Innovative Research of America, Sarasota, FL) were implanted
subcutaneously with a 10-gauge trocar (Innovative Research of America)
after castration, i.e., 2 wk before the initiation of the experiment.
Experimental groups.
Castrated male mice were randomized into eight groups:
groups 1 and
2 received vehicle;
groups 3 and
4 received DHT;
groups 5 and
6 were treated with 17-estradiol;
and groups 7 and
8 were treated with both DHT and
17
-estradiol. Each group consisted of seven animals.
Groups
1, 3,
5, and
7 contained animals subjected to sham
operations (sham-operated animals). The animals in
groups 2, 4,
6, and
8 were subjected to the
trauma-hemorrhage procedure. The experiments were performed seven
different times. On each experimental day, one animal from each group
was incorporated into the study.
Trauma-hemorrhage procedure. Mice in the 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 (soft tissue trauma) was performed. The laparotomy was then closed aseptically in two layers with 6-0 Ethilon sutures (Ethicon, Somerville, NJ). After this, both femoral arteries were aseptically cannulated with polyethylene 10 tubing (Clay-Adams, Parsippany, NJ) by a minimal-dissection technique. The animals were then allowed to awaken. Blood pressure (BP) was constantly monitored by attaching one of the catheters to a BP analyzer (Digi-Med; Micro-Med, Louisville, KY). On awakening, the animals were bled rapidly through the other catheter to a mean arterial BP of 35 ± 5 mmHg (mean arterial BP prehemorrhage was 95 ± 5 mmHg), which was maintained for 90 min. At the end of that period, four times the shed blood volume (the average shed blood volume was 0.95 ml, ~60% of the circulating blood volume) was infused in the form of lactated Ringer solution to provide adequate fluid resuscitation. Lidocaine was applied to the incision sites, the catheters were then removed, the vessels were ligated, and the groin incisions were closed. Sham-operated animals in groups 1, 3, 5, and 7 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 trauma-hemorrhage model within the first 24 h.
Blood, tissue, and cell harvesting procedure. The animals were killed by methoxyflurane overdose at 24 h after the completion of the experiment to obtain the spleen, the liver, peritoneal macrophages, and whole blood. The mice were sacrificed at the same time of the day to avoid fluctuations of plasma hormone levels due to the circadian rhythm.
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 (80°C) until assayed for DHT and
17
-estradiol.
RIAs for plasma DHT and 17-estradiol.
Plasma DHT concentration was determined with a commercially available
coated-tube RIA kit (Diagnostic Systems Laboratories, Webster, TX) in
which 100 µl of unextracted plasma were assayed in duplicate. The
sensitivity of the DHT RIA has been found to be 0.05 ng/ml. The average
coefficient of variation of the standard curve was 2.0%.
Preparation of peritoneal macrophage culture.
Resident peritoneal macrophages were obtained from mice by lavaging the
peritoneal cavity, and monolayers were established as previously
described (6, 39). The macrophage monolayers were stimulated (for 48 h
at 37°C, 5% CO2, and 90%
humidity) with 10 µg lipopolysaccharide (LPS)/ml
Click's medium containing 10% FCS (Biologos, Naperville, IL). At the
end of the incubation period, the culture supernatants were collected,
aliquoted, and stored at 80°C until assayed for
interleukin-1 (IL-1), IL-6, and IL-10. This protocol provided adherent
cells that were >95% positive, as determined by nonspecific esterase
staining, and that exhibited typical macrophage morphology.
Preparation of splenic macrophage culture.
The spleens were removed aseptically, placed in separate petri dishes
containing cold (4°C) PBS, and dissociated by grinding. The
splenocyte suspension was used to establish a macrophage culture as
previously described (39). The macrophage monolayers were stimulated
for 48 h (at 37°C, 5% CO2,
and 90% humidity) with 10 µg LPS/ml Click's medium containing 10%
FCS. At the end of the incubation period, the culture supernatants were
collected, aliquoted, and stored at 80°C until assayed for
IL-1, IL-6, and IL-10.
Preparation of Kupffer cell culture.
Kupffer cells were harvested as previously described (5). In brief,
retrograde perfusion of the liver was performed with 35 ml of ice-cold
Hanks' balanced salt solution (HBSS) through the portal vein. This was
immediately followed by perfusion with 10 ml of 0.05% collagenase IV
(Worthington Biochemical, Freehold, NJ) in HBSS at 37°C. The liver
was then transferred to a petri dish containing warm 0.05%
collagenase, minced finely, incubated at 37°C for 15 min, and
passed through a sterile 150-mesh stainless steel screen into a beaker
containing 10 ml of cold HBSS and 10% FCS. The cell suspension was
then layered over 16% Metrizamide (Accurate Chemical, Westbury, NY) in
HBSS and centrifuged at 3,000 g,
4°C, for 45 min to separate the Kupffer cells from the remaining parenchymal cells in the pellet. After removal of the nonparenchymal cells from the interface with a Pasteur pipette, the cells were washed
twice by centrifugation (800 g, 10 min, 4°C) with HBSS and resuspended in Click's medium containing
10% FCS. The cells were then transferred to a 24-well plate that was
precoated with 0.5 ml of 6 µg/ml Vitrogen 100 (Collagen Biomaterials,
Palo Alto, CA) and incubated for 3 h at 37°C (5%
CO2 and 90% humidity).
Nonadherent cells were removed by washing three times with Click's
medium. This protocol provides adherent cells that are >95% positive
as determined by nonspecific esterase staining and that exhibit typical macrophage morphology (39). The Kupffer cells (3 × 106 Kupffer
cells · ml1 · well
1)
were incubated for 24 h (37°C, 5%
CO2) with 10 mg LPS/ml Clicks's medium and 10% FCS, and the production of IL-6 and IL-10 was assessed.
Assessment of IL-6 release. IL-6 activities in culture supernatant of splenic and peritoneal macrophages and Kupffer cells were determined by the degree of proliferation of the IL-6-dependent murine B-cell hybridoma cell line 7TD1 (19). The 7TD1 cell line (gift from Dr. Jacques Van Snick) was maintained as previously described (20). Serial dilutions of macrophage supernatants were added to 4 × 104 7TD1 cells/ml, and the cells were incubated for 72 h at 37°C in 5% CO2. For the last 4 h of incubation, 20 µl of a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution (5 mg/ml in RPMI 1640; Sigma Chemical, St. Louis, MO) were added to each well (only viable cells incorporate MTT). The assay was stopped by aspiration of 100 µl of supernatant from each well, with subsequent replacement by 150 µl of 10% SDS solution in PBS (lauryl sulfate; Sigma Chemical) to dissolve the dark blue formazan crystals. An automated microplate reader (EL-311; Bio-Tek Instruments, Winooski, VT) was used to measure the light absorbance at 595 nm.
Assessment of IL-1 and IL-10 release.
IL-1
and IL-10 levels in the macrophage supernatants were determined
by the Sandwich ELISA technique described by Mossmann et al. (24). In
brief, Nunc-Immuno 96-well plates with MaxiSorp surfaces were coated
overnight with either 2 µg monoclonal hamster anti-mouse IL-1
capture antibody (Genzyme Diagnostics, Cambridge, MA)/ml 0.1 M
carbonate, pH 9.5, or 4 µg rat anti-mouse IL-10 capture antibody
(clone JES-5; Pharmingen, San Diego, CA)/ml 0.1 M
NaHCO3, pH 8.2. The plates were
washed three times with PBS containing 0.05% Tween 20 (Sigma Chemical)
and blocked with PBS containing 20% FCS for 2 h. After the plates were
washed, 100 µl of the samples and standard [1,000 pg/ml murine
IL-1
(Genzyme Diagnostics) or 10 ng/ml murine IL-10
(Pharmingen)] were added to the plates, and then they were
incubated overnight (4°C). After repeated washings the plates were
incubated for 1 h with 100 µl of either biotinylated polyclonal
rabbit anti-mouse IL-1
(Genzyme Diagnostics) at a concentration of
0.8 µg/ml at 37°C or biotinylated monoclonal rat anti-mouse IL-10
(clone SXC-1; Pharmingen) at a concentration of 2 µg/ml at room
temperature. Then the plates for IL-1
detection were
incubated with horseradish peroxidase-conjugated streptavidin (Genzyme
Diagnostics) for 15 min at 37°C. After multiple washings, 100 µl
of 3,3',5,5
-tetramethylbenzidine (TMB; Sigma Chemical) was added for 10 min at room temperature. After the addition of 100 µl of stop solution (0.5 M
H2SO4)
the optical density of each well at 450 nm was determined on a plate
reader (EL-311; Bio-Tek Instruments). For detection of IL-10 the plates
were washed and incubated at room temperature for 30 min with
avidin-peroxidase (diluted 1:400; Sigma Chemical). After the washing,
100 µl of 2,2'-azinobis(3-ethylbenzthiazolinesulfonic acid)
(ABTS)-H2O2 substrate buffer was added to each well to initiate color development. The optical density at 405 nm for each well was then determined on a
microplate reader. The concentrations of IL-1
and IL-10 present in
the samples were determined by interpolation from a standard curve
produced with murine IL-1
and IL-10, respectively.
Statistical analysis. The results are presented as means ± SE. One-way ANOVA, followed by the Student-Newman-Keuls test or Tukey's 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 to be significant.
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RESULTS |
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Plasma DHT and 17-estradiol levels.
In castrated animals receiving vehicle, plasma DHT levels (Fig.
1A)
were found to be markedly decreased compared with the physiological
plasma DHT levels previously reported for normal male mice (2, 21).
Plasma DHT levels in DHT-treated animals were significantly increased
compared with those in vehicle-treated castrated animals
(P < 0.05), and were comparable to
physiological plasma testosterone levels observed in normal male mice
(2, 21). The plasma DHT levels for sham-operated and trauma-hemorrhage groups were not significantly different.
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Effect of sex steroids on peritoneal macrophage cytokine release.
The release of IL-1, IL-6, and IL-10 (Fig.
2,
A-C)
from peritoneal macrophages in castrated animals receiving vehicle
after trauma-hemorrhage was similar to that in sham-operated animals. Castrated animals treated with DHT, however, showed significantly decreased IL-1 and IL-6 release after trauma-hemorrhage compared with sham-operated animals (
50.6% for IL-1
and
52.0%
for IL-6; P < 0.05). In contrast,
the release of the anti-inflammatory cytokine IL-10 was not
significantly altered by DHT treatment after trauma-hemorrhage. The
administration of estrogen to castrated male animals did not significantly affect IL-1
or IL-6 release from peritoneal
macrophages after trauma-hemorrhage (+56.2% compared with release from
peritoneal macrophages of 17
-estradiol-treated sham-operated
animals; P > 0.05).
However, IL-10 release from peritoneal macrophages was significantly
decreased in 17
-estradiol-treated animals after trauma-hemorrhage
(
40.9% compared with release from 17
-estradiol-treated sham-operated animals; P < 0.05).
Moreover, unlike DHT-treated mice subjected to trauma-hemorrhage,
animals receiving a combination of DHT and 17
-estradiol did not
display suppressed proinflammatory cytokine production or enhanced
IL-10 production.
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Effect of sex steroids on splenic macrophage cytokine release.
The release of IL-1, IL-6, and IL-10 in vehicle-treated castrated
male animals after trauma-hemorrhage was similar to that in
sham-operated animals receiving vehicle (Fig.
3,
A-C).
The administration of DHT did not change IL-1
and IL-6 releases in sham-operated animals compared with releases in sham-operated animals
receiving vehicle. In mice subjected to trauma-hemorrhage, however,
treatment with DHT resulted in significantly
(P < 0.05) decreased IL-1
(
49.6%) and IL-6 (
56.7%) releases from splenic macrophages compared with releases in DHT-treated, sham-operated animals. Furthermore, IL-10 release in sham-operated animals receiving DHT was significantly decreased (
76.9% compared with release in
vehicle-treated sham-operated animals;
P < 0.05). In contrast to what was
found for proinflammatory cytokines, the release of IL-10 significantly
increased (+433.2%; P < 0.05) after
trauma-hemorrhage compared with that in sham-operated animals receiving
DHT. The administration of DHT in the presence of 17
-estradiol
prevented the depression of IL-1
and IL-6 release from splenic
macrophages after trauma-hemorrhage. Moreover, splenic macrophage IL-10
release in sham-operated animals treated with DHT and 17
-estradiol
was significantly higher (+466%; P < 0.05) than the IL-10 release from DHT-treated sham-operated
animals.
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Effect of sex steroids on Kupffer cell cytokine release.
Kupffer cell IL-6 release was unchanged in vehicle-treated animals
after trauma-hemorrhage (Fig.
4A).
Although the administration of DHT did not change Kupffer cell IL-6
release in sham-operated animals, it resulted in significantly
increased IL-6 release after trauma-hemorrhage (+631% compared with
release from sham-operated animals treated with DHT;
P < 0.05). The results also indicate that Kupffer cell IL-10 release (Fig.
4B) significantly increased in
vehicle-treated and DHT-treated animals after traumahemorrhage (+77.1% in vehicle-treated and +80.8% in DHT-treated animals
compared with release from sham-operated animals receiving vehicle or
DHT, respectively; P < 0.05).
Furthermore, the administration of 17-estradiol significantly
increased (+42%; P < 0.05) the
Kupffer cell IL-10 release compared with that from DHT-treated,
sham-operated animals. Treatment of castrated male mice with DHT and
17
-estradiol prevented the increase of Kupffer cell IL-6 and IL-10
release after trauma-hemorrhage seen in mice treated with DHT only.
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DISCUSSION |
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Previous studies have reported gender differences in humoral as well as cell-mediated immune function (8, 9, 22, 26). These differences are believed to be responsible for the higher incidence of autoimmune diseases in females (26) as well as for the increased susceptibility to and morbidity from sepsis in males (8, 9, 22, 40). Similarly, differences in immune responsiveness with regard to gender have also been observed after trauma-hemorrhage. Proestrus female mice show enhanced immune function after trauma-hemorrhage compared with male mice, in which there is a depressed response (35).
Although several studies indicate that sex steroids play a central role
in the immune response under physiological and pathophysiological conditions (1, 12, 18, 31, 34, 37), the precise effect of male and
female sex steroids on macrophage function after trauma-hemorrhage remains unknown. In an attempt to elucidate the complex relationship of
male and female sex steroids on macrophage function after
trauma-hemorrhage, male mice were castrated and supplemented with
controlled amounts of DHT and/or 17-estradiol by implantation of
constant-release pellets. The plasma DHT and 17
-estradiol levels in
treated, castrated animals were found to be similar to physiological
levels seen in normal male (range 0.8-2.5 ng/ml) (1) and female
mice (range incorporating all states of the estrus cycle 45.6-220
pg/ml) (1), respectively (1, 29). Recent studies using extracted plasma further confirm that the levels of DHT in plasma obtained in the present study are physiological and that in normal mice the majority (~99%) of the circulating androgen is in the form of DHT rather than
free testosterone (21). Because only one dose of sex steroids was
administered in the present study, it remains unknown whether sub- or
supraphysiological plasma DHT or estradiol levels exhibit similar
immunomodulatory properties after trauma-hemorrhage. It should be noted
that trauma-hemorrhage did not alter plasma sex steroid levels.
Similarly, trauma-hemorrhage did not change plasma testosterone and
estradiol levels in normal male and female animals (2).
The results presented for vehicle-treated mice are consistent with previous findings in which the depletion of testosterone via castration normalized the cytokine release of peritoneal and splenic macrophages and Kupffer cells after trauma-hemorrhage (34). Thus it is our hypothesis that male sex steroids are responsible for producing the immunodepression in males after trauma-hemorrhage. Although intact male mice were not used in the present study, the findings further support this hypothesis by illustrating that by providing DHT to castrated animals, the depression of splenic and peritoneal macrophage proinflammatory cytokine release after trauma-hemorrhage was reestablished. Similarly, the administration of DHT to female mice decreased the release of splenic and peritoneal macrophages after trauma-hemorrhage; in contrast immune responses in untreated female mice were maintained (2). Interestingly, unlike splenic and peritoneal macrophages, Kupffer cells from castrated DHT-treated mice exhibited an enhanced ability to release IL-6 after trauma-hemorrhage. However, this is also in keeping with prior studies of normal male mice subjected to hemorrhage, which indicate that Kupffer cells are primed to release increased levels of proinflammatory cytokines, i.e., IL-6, under such conditions (5).
It should be pointed out that the administration of testosterone did not alter the cytokine release in sham-operated animals. In this regard, numerous studies suggest that androgens do not depress macrophage cytokine release in normal animals (1, 3, 10, 11). These results suggest that physiological concentrations of testosterone are only harmful in an immunologically compromised host, not in normal animals.
In contrast to DHT treatment, treatment of castrated mice with estrogen
did not depress the release of IL-1 and IL-6 from splenic and
peritoneal macrophages after trauma-hemorrhage. Because female mice in
the proestrus state have been shown to have an enhanced release of
IL-1
and IL-6, the data of the present study suggest that the
additive effects of other female sex hormones, e.g., progesterone,
might contribute to enhanced immune responses in proestrus female mice
after trauma-hemorrhage (35). In this regard, progesterone and
estrogens have been reported to have similar immunoenhancing effects on
rat macrophages (10, 14, 17, 23). It should be pointed out, however,
that 17
-estradiol treatment prevented the depression of splenic and
peritoneal macrophage proinflammatory cytokine release and normalized
Kupffer cell IL-6 release even in the presence of physiological
concentrations of testosterone. In this respect, several studies have
demonstrated the stimulatory effects of estrogen on various immune
cells (7, 11, 23, 30). The administration of estrogen in ovariectomized mice restored the depressed phagocytic activity of macrophages from
those mice (11). It should be noted that the effects of estrogens on
macrophage functions appear to be dose dependent. In this regard,
studies have shown that physiological levels of estrogen increase the
release of proinflammatory cytokines, whereas higher levels of estrogen
inhibit peritoneal macrophage functions (10, 15, 28). Thus the
immunostimulatory effects of estrogen might contribute to the
maintained immune responses in females after trauma-hemorrhage.
Kupffer cells have been suggested to be the primary source for circulating plasma IL-6 in males after trauma-hemorrhage (27). Although the increase of plasma IL-6 has been reported to be more pronounced in males than in females, IL-6 levels also increased in female mice at 4 h after trauma-hemorrhage (35). The release of IL-6 by Kupffer cells only increased in DHTtreated animals in the present study, suggesting that Kupffer cells from estradiol-treated mice might release increased IL-6 earlier after trauma-hemorrhage and resuscitation. Further support for this notion comes from studies that showed that after endotoxemia, female sex steroids produced the maximum increase of plasma IL-6 at 1 h after LPS injection compared with 3 h for vehicle-treated animals (42). Alternatively, other cells, e.g., keratinocytes, might contribute to increased IL-6 levels in females after trauma-hemorrhage. This, however, remains to be determined.
In contrast to the release of proinflammatory cytokines, the release of
the anti-inflammatory cytokine IL-10 by splenic and peritoneal
macrophages was increased in DHT-treated castrated animals after
trauma-hemorrhage compared with the release in sham-operated animals
receiving DHT. It should be pointed out that the releases of IL-10 from
splenic and peritoneal macrophages in DHT-treated sham-operated animals
were significantly decreased compared with those in vehicle- or
estrogen-treated sham-operated animals, which might represent
macrophage dysfunction in those sham-operated animals. In
vehicle-treated animals the release of IL-10 from splenic and
peritoneal macrophages was unchanged after trauma-hemorrhage, whereas
IL-10 release in 17-estradiol-treated mice decreased under such
conditions. In this regard, an increased release of IL-10 after
trauma-hemorrhage in normal male mice has been reported (4). Because
the release of IL-10 by splenic and peritoneal macrophages decreases
after trauma-hemorrhage, the decreased release of IL-10 in those
animals might contribute to the maintenance of macrophage responses
after trauma-hemorrhage. Similarly, Wilder (36) demonstrated enhanced
release of IL-10 and IL-4 in healthy women in the reproductive years
with high plasma estrogen levels compared with decreased
anti-inflammatory cytokine production in the postpartum period.
The underlying mechanisms by which sex hormones mediate their effects on macrophages after trauma-hemorrhage remain unclear at present. Studies have, however, demonstrated the presence of estrogen receptors on various immune cells, i.e., thymocytes, macrophages, and leukocytes (11, 26, 32). Although androgen receptors have not yet been identified on macrophages, receptors for male sex steroids have been observed on macrophage-like synovial cells, immature monocytic cells and T and B cells (26). Thus sex steroids may modulate immune responses directly via specific receptor-mediated processes. Further support for the hypothesis that sex steroids exert their effects through receptor-mediated processes comes from recent studies that indicate that blocking the effect of testosterone at the receptor in vivo with flutamide after trauma-hemorrhage restored macrophage and splenocyte functions (3, 33). Because sex steroids primarily exert their immunomodulating effects after trauma-hemorrhage, it is possible that increased receptor expression or changes in receptor affinity for these hormones occur because of trauma-hemorrhage. This, however, remains to be tested.
Alternatively, changes in the cytokine pattern might be due to the
indirect effects of these hormones on the macrophages. Sex steroids
might alter secondary mediators from lymphocytes (e.g.,
interferon-), endothelial cells, or other interactive cell
populations and thereby modulate the cytokine release of macrophages
after trauma-hemorrhage. However, which other mediators are involved in
the immunomodulation of macrophages by sex steroids after
trauma-hemorrhage remains to be determined. In this regard, studies
indicate that prolactin, which is known to have immune system-enhancing
effects, may potentially be a mediator for the immunoenhancing effects
of estrogen because estrogen can stimulate prolactin secretion (41).
In summary, the present study demonstrates that the female and male sex
steroids 17-estradiol and DHT differentially modulate macrophage
cytokine release after trauma-hemorrhage. DHT decreases the release of
proinflammatory cytokines, whereas estrogen decreases the release of
anti-inflammatory cytokines, thereby maintaining macrophage functions
even in the presence of testosterone after trauma-hemorrhage. These
findings further extend our previous observations that indicate that
testosterone might be the culprit for producing depression of
macrophage functions after trauma-hemorrhage in males. Furthermore,
17
-estradiol, a female sex steroid, appears to exhibit
immunoprotective effects on macrophage functions potentially by
down-regulating anti-inflammatory cytokine release. The exact mechanism, however, for the immunomodulatory properties of male and
female sex steroids after trauma-hemorrhage remains unknown. Nonetheless, the results of this study suggest that the administration of sex steroids or treatment with their specific blockers should be
considered as a novel and useful approach for modulating the release of
pro- and/or anti-inflammatory cytokines after trauma-hemorrhage.
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ACKNOWLEDGEMENTS |
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This investigation was supported by National Institute of General Medical Sciences Grant R01-GM-37127.
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FOOTNOTES |
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Present address of M. K. Angele: Ludwig-Maximilians Univ., Department of Surgery, Klinikum Grosshadern, Marchioninstrasse 15, 81377 Munich, Germany.
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: I. H. Chaudry, Center for Surgical Research, Brown Univ. School of Medicine and Rhode Island Hospital, Middle House II, 593 Eddy St., Providence, RI 02903 (E-mail: ichaudry{at}lifespan.org).
Received 17 December 1998; accepted in final form 28 March 1999.
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REFERENCES |
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1.
Angele, M. K.,
A. Ayala,
W. G. Cioffi,
K. I. Bland,
and
I. H. Chaudry.
Testosterone: the culprit for producing splenocyte immune depression following trauma-hemorrhage.
Am. J. Physiol.
274 (Cell Physiol. 43):
C1530-C1536,
1998
2.
Angele, M. K.,
A. Ayala,
B. A. Monfils,
W. G. Cioffi,
K. I. Bland,
and
I. H. Chaudry.
Testosterone and/or low estradiol: normally required but harmful immunologically for males after trauma-hemorrhage.
J. Trauma
44:
78-85,
1998[Medline].
3.
Angele, M. K.,
M. W. Wichmann,
A. Ayala,
W. G. Cioffi,
and
I. H. Chaudry.
Testosterone receptor blockade after hemorrhage in males: restoration of the depressed immune functions and improved survival following subsequent sepsis.
Arch. Surg.
132:
1207-1214,
1997[Abstract].
4.
Ayala, A.,
D. L. Lehman,
C. D. Herdon,
and
I. H. Chaudry.
Mechanism of enhanced susceptibility to sepsis following hemorrhage: interleukin (IL)-10 suppression of T-cell response is mediated by eicosanoid induced IL-4 release.
Arch. Surg.
129:
1172-1178,
1994[Abstract].
5.
Ayala, A.,
M. M. Perrin,
W. Ertel,
and
I. H. Chaudry.
Differential effects of haemorrhage on Kupffer cells: decreased antigen presentation despite increased inflammatory cytokine (IL-1, IL-6 and TNF) release.
Cytokine
4:
66-75,
1992[Medline].
6.
Ayala, A.,
M. M. Perrin,
M. A. Wagner,
and
I. H. Chaudry.
Enhanced susceptibility to sepsis following simple hemorrhage: depression of Fc and C3b receptor mediated phagocytosis.
Arch. Surg.
125:
70-75,
1990[Abstract].
7.
Baranao, R. I.,
A. Tenenbaum,
and
L. S. Rumi.
Effects of sexual steroid hormones on the functionality of murine peritoneal macrophages.
Steroids
56:
481-485,
1991[Medline].
8.
Bone, R. C.
Toward an epidemiology and natural history of SIRS (systemic inflammatory response syndrome).
JAMA
268:
3452-3455,
1992[Abstract].
9.
Centers for Disease Control.
Mortality patternsUnited States, 1989.
Morb. Mortal. Wkly. Rep.
41:
121-125,
1992[Medline].
10.
Chao, T. C.,
P. J. VanAlten,
J. A. Greager,
and
R. J. Walter.
Steroid sex hormones regulate the release of tumor necrosis factor by macrophages.
Cell. Immunol.
160:
43-49,
1995[Medline].
11.
Cutolo, M.,
A. Sulli,
B. Seriolo,
S. Accardo,
and
A. T. Masi.
Estrogens, the immune response and autoimmunity.
Clin. Exp. Rheumatol.
13:
217-226,
1995[Medline].
12.
Da Silva, J.,
and
G. M. Hall.
The effects of gender and sex hormones on outcome in rheumatoid arthritis.
Baillieres Clin. Rheumatol.
6:
193-219,
1992.
13.
Dinkel, R. H.,
and
U. Lebok.
A survey of nosocomial infections and their influence on hospital mortality rates.
J. Hosp. Infect.
28:
297-304,
1994[Medline].
14.
Dresser, D. W.
Specific inhibition of antibody production. Protein over-loading paralysis.
Immunology
5:
161-168,
1962.
15.
Forsberg, J. G.
Short-term and long-term effects of estrogen on lymphoid tissues and lymphoid cells with some remarks on the significance for carcinogenesis.
Arch. Toxicol.
55:
79-90,
1984[Medline].
16.
Friedman, D.,
F. Netti,
and
A. D. Schreiber.
Effect of estradiol and steroid analogues on the clearance of immunoglobulin G-coated erythrocytes.
J. Clin. Invest.
75:
162-167,
1985[Medline].
17.
Graff, R. J.,
M. A. Lappe,
and
G. D. Snell.
The influence of the gonads and adrenal glands on the immune response to skin grafts.
Transplantation
7:
105-111,
1969[Medline].
18.
Grossman, C. J.
Possible underlying mechanisms of sexual dimorphism in the immune response, fact and hypothesis.
J. Steroid Biochem.
34:
241-251,
1989[Medline].
19.
Hültner, L.,
H. Szöts,
M. Welle,
J. Van Snick,
J. Moeller,
and
P. Dörmer.
Mouse bone marrow-derived interleukin 3-dependent mast cells and autonomous sublines produce interleukin 6.
Immunology
67:
408-413,
1989[Medline].
20.
Ihle, J. N.,
J. Keller,
J. S. Greenberger,
L. Henderson,
R. A. Yetter,
and
H. C. Morse.
Phenotypic characteristics of cell lines requiring IL-3 for growth.
J. Immunol.
129:
1377-1383,
1982
21.
Kahlke, V., M. K. Angele, A. Ayala, M. G. Schwacha, W. G. Cioffi, K. I. Bland, and I. H. Chaudry. Immune dysfunction following trauma-hemorrhage:
influence of gender and age. Cytokine.
In press.
22.
McGowan, J. E.,
M. W. Barnes,
and
N. Finland.
Bacteremia at Boston City Hospital: occurrence and mortality during 12 selected years (1935-1972) with special reference to hospital-acquired cases.
J. Infect. Dis.
132:
316-335,
1975[Medline].
23.
Miller, L.,
and
J. S. Hunt.
Sex steroid hormones and macrophage function.
Life Sci.
59:
1-14,
1996[Medline].
24.
Mosmann, T. R.,
J. H. Schumacher,
D. F. Fiorentino,
J. Leverah,
K. W. Moore,
and
M. W. Bond.
Isolation of monoclonal antibodies specific for IL-4, IL-5, IL-6, and a new Th2-specific cytokine (IL-10), cytokine synthesis inhibitory factor, by using a solid phase radioimmunoadsorbent assay.
J. Immunol.
145:
2938-2945,
1990
25.
Nicol, T.,
D. L. J. Bilbey,
L. M. Charles,
J. L. Cordingle,
and
B. Vernon-Roberts.
Oestrogen: the natural stimulant of body defense.
J. Endocrinol.
30:
277-291,
1964.
26.
Olsen, N. J.,
and
W. J. Kovacs.
Gonadal steroids and immunity.
Endocr. Rev.
17:
369-384,
1996[Medline].
27.
O'Neill, P. J.,
A. Ayala,
P. Wang,
Z. F. Ba,
M. H. Morrison,
A. E. Schultze,
S. S. Reich,
and
I. H. Chaudry.
Role of Kupffer cells in interleukin-6 release following trauma-hemorrhage and resuscitation.
Shock
1:
43-47,
1994[Medline].
28.
Polan, M. L.,
A. Kuo,
J. Loukides,
and
K. Bottomly.
Cultured human luteal monocytes secrete increased levels of interleukin-1.
J. Clin. Endocrinol. Metab.
7:
480-484,
1990.
29.
Reburn, C. J.,
and
K. E. Wynne-Edwards.
Novel patterns of progesterone and prolactin in plasma during the estrus cycle in the Djungarian hamster (Phodopus campbelli) as determined by repeated sampling of individual females.
Biol. Reprod.
54:
819-825,
1996[Abstract].
30.
Roubinian, J. R.,
N. Talal,
J. S. Greenspan,
J. R. Goodman,
and
P. K. Siiteri.
Effect of castration and sex hormone treatment on survival, anti-nucleic acid antibodies, and glomerulonephritis in NZB/NZW F1 mice.
J. Exp. Med.
147:
1568-1583,
1978[Abstract].
31.
Sthoeger, Z. M.,
N. Chiorazzi,
and
R. G. Lahita.
Regulation of the immune response by sex hormones. I. In vitro effects of estradiol and testosterone on pokeweed mitogen-induced human B cell differentiation.
J. Immunol.
141:
91-98,
1988
32.
Viselli, S. M.,
N. J. Olsen,
K. Shults,
G. Steizer,
and
W. J. Kovacs.
Immunochemical and flow cytometric analysis of androgen receptor expression in thymocytes.
Mol. Cell. Endocrinol.
109:
19-26,
1995[Medline].
33.
Wichmann, M. W.,
M. K. Angele,
A. Ayala,
W. G. Cioffi,
and
I. H. Chaudry.
Flutamide: a novel agent for restoring the depressed cell-mediated immunity following soft-tissue trauma and hemorrhagic shock.
Shock
8:
242-248,
1997[Medline].
34.
Wichmann, M. W.,
A. Ayala,
and
I. H. Chaudry.
Male sex steroids are responsible for depressing macrophage immune function after trauma-hemorrhage.
Am. J. Physiol.
273 (Cell Physiol. 42):
C1335-C1340,
1997
35.
Wichmann, M. W.,
R. Zellweger,
C. M. DeMaso,
A. Ayala,
and
I. H. Chaudry.
Enhanced immune responses in females as opposed to decreased responses in males following hemorrhagic shock.
Cytokine
8:
853-863,
1996[Medline].
36.
Wilder, R. L.
Hormones, pregnancy, autoimmune diseases.
Ann. NY Acad. Sci.
840:
45-50,
1998
37.
Wira, C. R.,
and
R. M. Rossoll.
Antigen-presenting cells in the female reproductive tract: influence of sex hormones on antigen presentation in the vagina.
Immunology
84:
505-508,
1995[Medline].
38.
Yamamoto, Y.,
H. Saito,
T. Setogawa,
and
H. Tomioka.
Sex differences in host resistance to Mycobacterium marinum infection in mice.
Infect. Immun.
59:
4089-4096,
1991[Medline].
39.
Zellweger, R.,
A. Ayala,
C. M. DeMaso,
and
I. H. Chaudry.
Trauma-hemorrhage causes prolonged depression in cellular immunity.
Shock
4:
149-153,
1995[Medline].
40.
Zellweger, R.,
M. W. Wichmann,
A. Ayala,
S. Stein,
C. M. DeMaso,
and
I. H. Chaudry.
Females in proestrus state maintain splenic immune functions and tolerate sepsis better than males.
Crit. Care Med.
25:
106-110,
1997[Medline].
41.
Zellweger, R.,
X.-H. Zhu,
M. W. Wichmann,
A. Ayala,
C. M. DeMaso,
and
I. H. Chaudry.
Prolactin administration following hemorrhagic shock improves macrophage cytokine release capacity and decreases mortality from subsequent sepsis.
J. Immunol.
157:
5748-5754,
1996[Abstract].
42.
Zuckerman, S. H.,
S. E. Ahmari,
N. Bryan-Poole,
G. F. Evans,
L. Short,
and
A. L. Glasebrook.
Estriol: a potent regulator of TNF and IL-6 expression in a murine model of endotoxemia.
Inflammation
20:
581-597,
1996[Medline].