17beta -Estradiol normalizes immune responses in ovariectomized females after trauma-hemorrhage

Markus W. Knöferl1, Doraid Jarrar1, Martin K. Angele1, Alfred Ayala1, Martin G. Schwacha2, Kirby I. Bland2, and Irshad H. Chaudry2

1 Center for Surgical Research and Department of Surgery, Brown University School of Medicine and Rhode Island Hospital, Providence, Rhode Island 02903; and 2 Center for Surgical Research and Department of Surgery, University of Alabama at Birmingham, Birmingham, Alabama 35394


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Recent studies indicate that immune responses in proestrus females are maintained after trauma-hemorrhage but markedly depressed in ovariectomized females under such conditions. The current study tested the hypothesis that the decreased estrogen levels after ovariectomy are responsible for this immune depression. To study this hypothesis, ovariectomized female CBA/J mice were subjected to laparotomy (i.e., soft tissue trauma) and hemorrhagic shock (35 ± 5 mmHg for 90 min, then resuscitated) or sham operation. The mice received either 17beta -estradiol (E2; 100 µg/25 g body wt) or vehicle subcutaneously during resuscitation. Immune cells were isolated 24 h thereafter. Splenocyte proliferation and interferon-gamma , interleukin (IL)-2, and IL-3 release were significantly depressed after trauma-hemorrhage in vehicle-treated mice, whereas these functions were maintained in E2-treated mice. Peritoneal macrophage IL-1beta and IL-6 release and splenic macrophage IL-6 and IL-12 release were also significantly depressed in vehicle-treated mice after trauma-hemorrhage, and release of these cytokines was restored by E2 treatment. In summary our findings indicate that the depressed splenic and peritoneal immune responses after trauma-hemorrhage can be normalized by a single dose of E2. Thus estrogen appears to be the causative factor in the maintenance of immunocompetence in females after trauma-hemorrhage, and its administration to ovariectomized or postmenopausal females should be helpful in preventing immune depression under such conditions.

T lymphocyte; macrophage; inflammation; immunosuppression; gender


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

A NUMBER OF STUDIES HAVE SHOWN that immune functions are markedly depressed in males after trauma-hemorrhage and that these changes persist for several days despite adequate fluid resuscitation (11, 44, 45). There is increasing evidence that hormonal mechanisms are involved in regulating immune responses under those conditions. In this regard, recent studies indicate that testosterone plays a crucial role in producing immunosuppression in males after trauma-hemorrhage. For instance, it has been shown that depletion of testosterone by castration of male mice before the insult prevented the depression of immune functions after trauma-hemorrhage (2, 42). Furthermore, treatment of male mice with a specific testosterone receptor antagonist after trauma-hemorrhage restored the depressed immune functions and decreased the mortality rate of animals subjected to subsequent sepsis (3, 41).

In contrast to the immunosuppression observed in males, females in the proestrus state of the estrus cycle have enhanced immune functions after trauma-hemorrhage (23, 43). The maintenance of immunocompetence in females was associated with a significantly higher survival rate after a subsequent septic challenge than that seen in males (12). Because systemic concentrations of female sex hormones are increased in the proestrus state (36), we speculated that those hormones are involved in maintaining immunocompetence in females. In this regard, recent studies have shown that in ovariectomized females, with reduced systemic concentrations of female sex hormones, trauma-hemorrhage resulted in significantly depressed macrophage functions (23). This depression in macrophage function after trauma-hemorrhage in ovariectomized females was associated with a significantly increased mortality rate from subsequent sepsis when compared with females in the proestrus state of the estrus cycle (23). Because ovariectomy produces complex changes in the hormonal milieu of the host, it is not possible to postulate whether the immunosuppressive effects after trauma-hemorrhage are attributable to a single hormone. Nonetheless, studies have shown that administration of the female sex hormone 17beta -estradiol (E2) in males after trauma-hemorrhage significantly improved splenocyte, splenic macrophage (sMphi ), and peritoneal macrophage (pMphi ) immune functions compared with vehicle-treated males (24). Although immunoprotective effects of E2 have been reported in males, it remains unknown whether decreased systemic levels of this steroid are responsible for the immunosuppression encountered in ovariectomized females after trauma-hemorrhage. The aim of the present study, therefore, was to determine whether administration of E2 to ovariectomized females has any beneficial or deleterious effects on splenocyte, sMphi , and pMphi immune functions after trauma-hemorrhage.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals. Inbred female CBA/J mice (Jackson Laboratories, Bar Harbor, ME), 8-9 wk old (24-26 g body wt), were used in this study. 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.

Experimental groups. Ovariectomy was performed in female CBA/J mice 2 wk before trauma-hemorrhage using two dorsolateral incisions. In preliminary studies it was found that, during this period, plasma concentrations of E2 and uterine wet weight as a sensitive parameter of systemic estrogen exposure significantly decreased in ovariectomized females compared with females in the proestrus state (data not shown). Two weeks after ovariectomy, the animals were divided into four groups. Groups 1 and 2 consisted of sham-operated ovariectomized females, which were neither hemorrhaged nor resuscitated. Animals in groups 3 and 4 consisted of ovariectomized females, which were subjected to the trauma-hemorrhage procedure. Immediately before initiation of fluid resuscitation, animals in groups 1 and 3 received a subcutaneous injection of vehicle (200 µl corn oil), and animals in groups 2 and 4 were treated with E2 (100 µg/25 g body wt dissolved in 200 µl corn oil). Each group consisted of 7-8 animals.

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 using 6-0 Ethilon sutures (Ethicon, Somerville, NJ). Both femoral arteries were then aseptically cannulated with polyethylene-10 tubing (Clay-Adams, Parsippany, NJ) 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. Upon awakening, the animals were bled 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 were ligated, and the groin incisions were 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.

Blood, tissue, and cell harvesting procedure. The animals were killed by methoxyflurane overdose at 24 h after trauma-hemorrhage and resuscitation to obtain the spleen, pMphi , uteri, 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 (-80°C) until assayed.

Cell line maintenance. The interleukin (IL)-2-dependent CTLL-2 cells were obtained from 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, CT) were maintained as previously described (20). 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 (20).

Preparation of splenocyte culture. At 24 h after sham operation or trauma-hemorrhage and resuscitation, the spleens were removed aseptically and placed in separate petri dishes containing 4°C phosphate-buffered saline (PBS) solution. Splenocytes were isolated as previously described in detail (45). Briefly, the organs 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, 15 min). Viability was tested using trypan blue exclusion and found to be ~95% regardless of the group assessed. The splenocytes were then resuspended in RPMI 1640 (GIBCO-BRL, Grand Island, NY) containing 10% heat-inactivated fetal bovine serum (FBS; GIBCO-BRL) to yield a final concentration of 1 × 107 cells/ml. The ability of the splenocyte cultures to produce cytokines 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 µg/ml concanavalin A (Con 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 for interferon-gamma (IFN-gamma ), IL-2, IL-3, and IL-10.

Splenocyte proliferation. A second portion of the splenocyte suspension was placed into a 96-well microtiter plate (Corning Glass, Corning, NY) in aliquots of 100 µl. The cells' ability to proliferate in response to mitogenic stimulation with 0 (negative control) or 2.5 µg/ml Con A was measured by [3H]thymidine incorporation technique as previously described (38). Briefly, after incubation for 48 h at 37°C, 5% CO2, and 90% humidity, 1 µCi of the radionucleotide (sp act 6.7 Ci/mmol; NEN, Wilmington, DE) was added to each well, and the cultures were incubated for another 16 h. The cells were then harvested onto glass fiber filter mats, and the beta decay was detected by liquid scintillation counting, as previously described (27).

Preparation of sMphi and pMphi culture. Spleens were harvested aseptically, and sMphi cultures were established as previously described in detail (46). Resident pMphi were harvested by peritoneal lavage at 24 h after sham operation or trauma-hemorrhage and resuscitation, and monolayers were established as previously described (6). The monolayers of sMphi and pMphi (1 × 106 cells/ml) were stimulated with 10 µg of lipopolysaccharide W/ml Click's medium containing 10% heat-inactivated FBS for 48 h at 37°C, 5% CO2, and 90% humidity to assess the cells' ability to release cytokines. (Lipopolysaccharide W was from Escherichia coli 055:B5, Difco Laboratories, Detroit, MI.) 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 or IL-3 was assessed by determining the amount of IL-2 or IL-3 in the collected culture supernatant. Serial dilutions of the supernatants and standards were added to CTLL-2 cells (1 × 105 cells/ml) or to FDC-P1 cells (2.5 × 105 cells/ml) and incubated for 24 h (FDC-P1) or 48 h (CTLL-2) at 37°C, 5% CO2, and 90% humidity. At the end of this period, 1 µCi of [3H]thymidine (sp act 6.7 Ci/mmol, NEN) was added to each well, and the cultures were incubated for an additional 16 h. The cells were then harvested onto glass-fiber mats, and the beta decay was detected by liquid scintillation radiography as previously described (27).

IL-10 concentrations in macrophage and splenocyte supernatants and IFN-gamma levels in the splenocyte supernatants were determined using sandwich-enzyme-linked immunosorbent assay technique (ELISA) according to the manufacturer's recommendations (BD OptEIA ELISA set; BD Pharmingen, San Diego, CA).

IL-6 activity was determined by assessing the 72-h proliferation of the IL-6-dependent murine B cell hybridoma 7TD1 stimulated by serial dilutions of sMphi and pMphi supernatants as previously described (7). For the last 3 h of incubation, 20 µl of a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide solution (5 mg/ml in RPMI 1640; Sigma Chemical, St. Louis, MO) was added to each well. The amount of dark blue formazan crystal formation was then measured spectrophotometrically. The units of IL-6 activity were determined by comparison of curves produced from dilutions of a recombinant mouse IL-6 standard (200 U/ml; Genzyme, Cambridge, MA) according to the methods of Mizel (30). IL-1beta levels in pMphi supernatants were determined by ELISA according to the manufacturer's recommendations (Genzyme).

Determination of plasma E2 concentration. E2 concentration was determined using a commercially available radioimmunoassay (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 < 0.05 was considered significant.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Biological effect of E2 treatment. At 24 h after vehicle administration, plasma concentrations of E2 were 16.6 ± 5.3 pg/ml in ovariectomized females that were sham operated and 15.7 ± 8.3 pg/ml in females that underwent trauma-hemorrhage. Administration of E2 in ovariectomized females resulted in significantly increased plasma concentrations of E2 in sham-operated as well as hemorrhaged animals (P < 0.05; Fig. 1A) compared with corresponding vehicle-treated ovariectomized females. In addition, uterine wet weight, a sensitive parameter of systemic estrogen exposure, significantly increased in E2-treated ovariectomized females that were sham operated or hemorrhaged (P < 0.05 vs. corresponding vehicle-treated ovariectomized females; Fig. 1B).


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Fig. 1.   Plasma 17beta -estradiol concentrations (A) and uterine wet weight (B) of female CBA/J mice that were subjected to trauma-hemorrhage and resuscitation or to sham operation at 2 wk after ovariectomy. At the end of trauma-hemorrhage or sham operation, the ovariectomized females were either treated with corn oil vehicle or 100 µg 17beta -estradiol/25 g body wt. Plasma 17beta -estradiol concentrations were measured by radioimmunoassay. Values are means ± SE of 7-8 animals in each group. ANOVA: * P < 0.05 vs. sham vehicle, # P < 0.05 vs. trauma-hemorrhage vehicle.

Splenocyte proliferation. At 24 h after trauma-hemorrhage, splenocyte proliferative capacity was significantly depressed in ovariectomized females that received vehicle at the beginning of resuscitation compared with sham-operated females receiving vehicle (P < 0.05; Fig. 2). In ovariectomized females treated with E2 after trauma-hemorrhage, however, no depression of splenocyte proliferative capacity was observed.


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Fig. 2.   Proliferative capacity of splenocytes harvested from ovariectomized female CBA/J mice at 24 h after sham operation or trauma-hemorrhage and treatment with corn oil vehicle or 100 µg 17beta -estradiol/25 g body wt. All female mice were ovariectomized 2 wk before the experiment. Splenocytes were stimulated with 2.5 µg/ml concanavalin A for 48 h, and proliferation was measured by [3H]thymidine incorporation technique. Values are means ± SE of 7-8 animals in each group. ANOVA: * P < 0.05 vs. sham vehicle, # P < 0.05 vs. trauma-hemorrhage vehicle.

Splenocyte cytokine production. After trauma-hemorrhage, splenocyte IFN-gamma release was significantly depressed in vehicle-treated ovariectomized females compared with sham-operated animals (P < 0.05; Fig. 3A). In contrast, no depression of IFN-gamma production was observed in splenocytes harvested from ovariectomized females treated with E2 after trauma-hemorrhage. Trauma-hemorrhage resulted in a significantly depressed production of IL-2 by splenocytes harvested from vehicle-treated ovariectomized females (P < 0.05; Fig. 3B). Treatment with E2 restored IL-2 productive capacity. IL-3 production was significantly suppressed after trauma-hemorrhage in splenocytes harvested from vehicle-treated ovariectomized females (P < 0.05; Fig. 3C); however, treatment of ovariectomized females with E2 at the beginning of resuscitation significantly improved splenocyte IL-3 release capacity after trauma-hemorrhage toward sham levels (P < 0.05; Fig. 3C). In contrast to the production of IFN-gamma , IL-2, and IL-3, the release of IL-10 was maintained after trauma-hemorrhage in ovariectomized females receiving vehicle (Fig. 3D). However, treatment with E2 led to significantly reduced IL-10 production after trauma-hemorrhage compared with E2-treated sham-operated animals (P < 0.05, Fig. 3D).


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Fig. 3.   Interferon-gamma (IFN-gamma ; A), interleukin-2 (IL-2; B), IL-3 (C), and IL-10 (D) release of splenocytes harvested from ovariectomized female CBA/J mice at 24 h after sham operation or trauma-hemorrhage and treatment with corn oil vehicle or 100 µg 17beta -estradiol/25 g body wt. All female mice were ovariectomized 2 wk before the experiment. Splenocytes were stimulated with 2.5 µg/ml concanavalin A for 48 h. IL-2 levels in splenocyte supernatants were measured using a bioassay specific for IL-2 (CTLL-2), IL-3 levels were determined by a specific bioassay for IL-3 (FDC-P1), and IFN-gamma and IL-10 concentrations were measured by sandwich-enzyme-linked immunosorbent assay technique. Values are means ± SE of 7-8 animals in each group. ANOVA: * P < 0.05 vs. sham vehicle; # P < 0.05 vs. trauma-hemorrhage vehicle, + P < 0.05 vs. sham estradiol.

Macrophage cytokine production. sMphi IL-6 release was significantly depressed in vehicle-treated ovariectomized females after trauma-hemorrhage (P < 0.05; Fig. 4A). Administration of E2 in ovariectomized females during resuscitation did not influence IL-6 release under these conditions. IL-10 production by sMphi was not affected by trauma-hemorrhage in vehicle-treated mice; however, E2 treatment of such animals significantly reduced IL-10 release (P < 0.05; Fig. 4B). sMphi IL-12 production was significantly depressed after trauma-hemorrhage in vehicle-treated ovariectomized females compared with their corresponding sham-operated animals (P < 0.05; Fig. 4C). Treatment with E2 prevented the depression in sMphi IL-12 production after trauma-hemorrhage.


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Fig. 4.   Release of IL-6 (A), IL-10 (B), and IL-12 (C) by splenic macrophages (sMphi ) harvested from ovariectomized female CBA/J mice at 24 h after sham operation or trauma-hemorrhage and treatment with corn oil vehicle or 100 µg 17beta -estradiol/25 g body wt. All female mice were ovariectomized 2 wk before the experiment. sMphi were cultured in the presence of 10 µg/ml lipopolysaccharide W for 48 h. IL-6 levels in macrophage supernatants were measured using a specific bioassay (7TD1); IL-10 and IL-12 concentrations were determined by sandwich-enzyme-linked immunosorbent assay technique. Values are means ± SE of 7-8 animals in each group. ANOVA: * P < 0.05 vs. sham vehicle, # P < 0.05 vs. trauma-hemorrhage vehicle.

At 24 h after trauma-hemorrhage, pMphi IL-1beta release was significantly depressed in ovariectomized females receiving vehicle compared with vehicle-treated sham-operated animals (P < 0.05; Fig. 5A). Treatment with E2 led to a partial restoration of pMphi IL-1beta release. Similar to sMphi , pMphi IL-6 production was significantly depressed in ovariectomized females that underwent trauma-hemorrhage and received vehicle (P < 0.05, Fig. 5B). In contrast to sMphi , IL-6 production by pMphi was maintained at sham levels in mice that were treated with E2 at the beginning of resuscitation. pMphi IL-10 production was not affected by either trauma-hemorrhage or E2 treatment (Fig. 5C).


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Fig. 5.   Release of IL-1beta (A), IL-6 (B), and IL-10 (C) by peritoneal macrophages (pMphi ) harvested from ovariectomized female CBA/J mice at 24 h after sham operation or trauma-hemorrhage and treatment with corn oil vehicle or 100 µg 17beta -estradiol/25 g body wt. All female mice were ovariectomized 2 wk before the experiment. pMphi were cultured in the presence of 10 µg/ml lipopolysaccharide W for 48 h. IL-6 levels in macrophage supernatants were measured by specific bioassay (7TD1); IL-1beta and IL-10 were measured by sandwich-enzyme-linked immunosorbent assay technique. Values are means ± SE of 7-8 animals in each group. ANOVA: * P < 0.05 vs. sham vehicle, # P < 0.05 vs. trauma-hemorrhage vehicle.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The aim of the present study was to determine whether administration of E2 in ovariectomized females has any salutary effects on splenocyte, sMphi , or pMphi immune functions after trauma-hemorrhage. The results presented here indicate that at 24 h after sham operation or trauma-hemorrhage and administration of 100 µg E2/25 g body wt, plasma concentrations of E2 were significantly increased compared with vehicle-treated ovariectomized females. Furthermore, uterine wet weight, a sensitive parameter of systemic estrogen exposure, significantly increased in ovariectomized females that received E2 compared with vehicle-treated ovariectomized females. In all experiments discussed here, ovariectomized females were used at 2 wk after ovariectomy. This time point was selected because preliminary studies have shown that during this period, plasma concentrations of E2 and uterine wet weight significantly decreased in ovariectomized females compared with females in the proestrus state of the estrus cycle. Thus the findings that E2 administration significantly increased plasma levels of this hormone as well as uterine wet weight in ovariectomized females indicate the biological effectiveness of the hormone treatment used.

Administration of E2 in ovariectomized females after trauma-hemorrhage led to profound changes in immune functions compared with ovariectomized females receiving vehicle. Comparable with the findings from previous studies (23), our results indicate that in ovariectomized females, sMphi and pMphi proinflammatory cytokine production (IL-1beta , IL-6, and IL-12) was significantly depressed after trauma-hemorrhage. However, administration of a single dose of E2 in ovariectomized females at the beginning of fluid resuscitation resulted in partial normalization of sMphi and pMphi proinflammatory cytokine release capacity under those conditions. In this regard, it has previously been shown that normal females in the proestrus state of the estrus cycle also maintain sMphi and pMphi function after trauma-hemorrhage (23). Thus the findings that ovariectomy leads to a depression of sMphi and pMphi proinflammatory cytokine release after trauma-hemorrhage indicate that physiological levels of female sex steroids and, in particular, E2 are involved in maintaining macrophage proinflammatory cytokine release in the splenic and peritoneal compartment. Furthermore, the present finding that administration of E2 in ovariectomized females partially restored sMphi and pMphi proinflammatory cytokine release suggests that this female sex steroid plays a critical role in regulating macrophage functions. In this regard, it should be noted that the pattern of immune depression observed in ovariectomized females after trauma-hemorrhage is comparable to the depression of immune functions seen in male mice after trauma-hemorrhage (45). Thus the observation that high concentrations of testosterone as well as low levels of estradiol are associated with immunosuppression after trauma-hemorrhage could lead to the hypothesis that the ratio of male to female sex hormones is essential to the course of the immune response to a traumatic insult.

Comparable to the depression observed in sMphi and pMphi function, trauma-hemorrhage resulted in a significantly depressed splenocyte proliferative capacity and splenocyte cytokine production in ovariectomized females receiving vehicle. Administration of E2 at the end of trauma-hemorrhage, however, restored splenocyte proliferation to values comparable with sham-operated animals. Furthermore, E2 treatment normalized the depressed production of IFN-gamma , IL-2, and IL-3 in ovariectomized females that underwent trauma-hemorrhage. IL-3 is a growth factor that influences growth of specific T lymphocyte subsets as well as proliferation of early T cell precursors (32, 35). With regard to our finding, the suppression in splenocyte proliferation correlated with suppressed IL-3 production after trauma-hemorrhage, and the restoration of IL-3 production by E2 paralleled restored splenocyte proliferative responses. Previous studies have shown that suppressed IL-3 production correlates with lower immune functional capacity in aged mice (17, 21, 29). These findings suggest that E2 is involved in the regulation of splenocyte immune responses and that the decreased levels of E2 in ovariectomized females contribute to the depression of splenocyte immune functions after trauma-hemorrhage. Support for the notion that E2 has stimulatory effects on posthemorrhage splenocyte function comes from studies that have shown that administration of E2 in males restored splenocyte immune responses after trauma-hemorrhage (24). It should be noted, however, that these findings do not allow us to distinguish whether the stimulatory effects of E2 on splenocyte immune functions are due to the direct actions of this hormone on splenocytes or are also being mediated via indirect mechanisms, such as macrophage/splenocyte interactions. In this regard, our results indicate that administration of E2 restored sMphi IL-12 production in ovariectomized females that underwent trauma-hemorrhage. Because macrophages are present in splenocyte cultures, it is possible that macrophage-derived IL-12, a well-characterized stimulant of splenocyte immune functions (40), contributes to the beneficial effects of E2 treatment. Whether indirect mechanisms other than sMphi IL-12 production are involved in restoring splenocyte immune functions in E2-treated ovariectomized females remains to be determined. The precise mechanism of action of E2 on immune cell function remains to be fully elucidated. A possible explanation for why E2 normalized many immune parameters in ovariectomized mice after trauma-hemorrhage is suppressed apoptosis. E2 has been shown to suppress apoptosis (13, 15, 18) due to upregulation of Bcl-2, an antiapoptotic protein (9, 13). Additionally, decreased estrogen levels in ovariectomized females might also result in increased apoptosis. Further support for the potential role of apoptosis in the effect of E2 is provided in studies by Angele et al. (4), who demonstrated that thymocytes from proestrus females, which have elevated circulating estrogen levels, are resistant to apoptosis after trauma-hemorrhage compared with cells from male mice.

In contrast to the depression of sMphi proinflammatory cytokine release and splenocyte cytokine production observed in vehicle-treated ovariectomized females after trauma-hemorrhage, anti-inflammatory cytokine, i.e., IL-10, production by sMphi and splenocytes was maintained under those conditions. The maintained production of IL-10 by sMphi and splenocytes after trauma-hemorrhage was associated with a significantly depressed T lymphocyte proliferative capacity. It can be speculated that normal IL-10 production may play a role in the regulation of the inflammatory response by limiting T lymphocyte proliferation and the proinflammatory response. In ovariectomized females treated with E2, however, IL-10 production decreased after trauma-hemorrhage compared with sham-operated animals, and this was associated with maintained proinflammatory cytokine production. The anti-inflammatory cytokine IL-10 has previously been described as an important immunosuppressant of cell-mediated immunity (19) and has been implicated in the suppression of splenocyte immune functions after hemorrhage (5). Therefore, our findings suggest that E2 decreases anti-inflammatory cytokine production by sMphi and splenocytes after trauma-hemorrhage and that the maintained production of IL-10 observed in ovariectomized females receiving vehicle might contribute to the depression of immune functions under those conditions. Nonetheless, multiple mechanisms exist by which splenic T lymphocyte proliferation can be suppressed. These mechanisms include elevated levels of prostaglandins, nitric oxide and/or IL-6, and suppression of antigen-presenting cell function. With regard to IL-6, previous studies have suggested an anti-inflammatory role for this multifunctional cytokine. IL-6 can induce the expression of multiple factors with anti-inflammatory properties, including IL-1R antagonist, soluble tumor necrosis factor receptors, IL-10, glucocorticoids, and suppressors of cytokine signaling (8, 25, 33, 37, 39). IL-6 may play an important role in a negative-feedback loop that suppresses inflammation similar to IL-10. With regard to this concept, Ahmed and Ivashkiv (1) have recently shown that IL-6 and IL-10 both activate the JAK-STAT signaling pathway that has pro- and anti-inflammatory actions. Our findings that E2 can modulate IL-6 and IL-10 production may be related to regulation of this signal transduction pathway.

Although our findings suggest several mechanisms by which E2 administration might contribute to maintaining immune functions in ovariectomized females after trauma-hemorrhage, the exact target level of immunoendocrine interactions remains unclear. Because estrogen receptors have been identified on macrophages (16) and splenic T lymphocytes (34), it is possible that these cells may be susceptible to functional modulation by the exogenous estrogens administered after trauma-hemorrhage. Additional studies should, therefore, be directed at determining 1) whether the salutary effects of E2 on immune responsiveness after trauma-hemorrhage are not only receptor mediated and 2) which immune cell populations are primarily affected by this treatment. Alternatively, these salutary effects of E2 treatment on immune functions seen after trauma-hemorrhage might, in part, be the indirect result of effects on other organ systems. In this regard, recent findings in cardiovascular disease research provide data that E2 can act both via rapid nongenomic, as well as long-term genomic, mechanisms (28). Therefore, it is possible that the nongenomic effects of pharmacological doses of E2, such as vasodilatation due to changes in ion-channel function (14, 22) as well as increased endothelial nitric oxide production (10, 26), might contribute to the beneficial effects on immune functions by improving microcirculation after trauma-hemorrhage. Nonetheless, support for this notion comes from recent studies that have shown that the depressed cardiovascular responses after trauma-hemorrhage in males were normalized by treatment with E2 after trauma-hemorrhage (31).

In summary, the data presented here demonstrate that in vehicle-treated ovariectomized females, splenocyte cytokine production as well as sMphi and pMphi proinflammatory cytokine production were significantly depressed after trauma-hemorrhage. Administration of a single dose of the female sex steroid E2 after trauma-hemorrhage restored splenocyte as well as sMphi and pMphi immune functions under those conditions. Furthermore, E2 administration reduced the elevated production of the anti-inflammatory cytokine IL-10 by splenocytes and sMphi observed in vehicle-treated ovariectomized females after trauma-hemorrhage. These findings indicate that the reduction of female sex steroids in ovariectomized females is associated with adverse effects on immune functions after trauma-hemorrhage. Because administration of the female sex steroid E2 after trauma-hemorrhage normalized immune responses in ovariectomized females, this hormone appears to play a critical role in maintaining immunocompetence in females under those conditions. Thus E2 administration in females with lowered estrogen levels (due to ovariectomy or postmenopausal) appears to be a safe and novel approach for the prevention of immunosuppression after trauma-hemorrhage.


    ACKNOWLEDGEMENTS

The experiments contained in this study were conducted at Rhode Island Hospital, Providence, RI 02903.


    FOOTNOTES

This research was supported by National Institute of General Medical Sciences Grant R01-GM-37127 and by Deutsche Forschungsgemeinschaft Fellowship KN-475/1-1 (awarded to M. W. Knöferl).

Present address of M. W. Knöferl: Univ. 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.

Address for reprint requests and other correspondence: I. H. Chaudry, The Univ. of Alabama at Birmingham, Center for Surgical Research, G094 Volker Hall, 1670 Univ. Blvd., Birmingham, AL 35294-0019 (E-mail: Irshad.Chaudry{at}ccc.uab.edu).

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.

Received 26 February 2001; accepted in final form 5 June 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Cell Physiol 281(4):C1131-C1138
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