Published online before print March 10, 2006
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or ER-ß?
* Center for Surgical Research and Department of Surgery, University of Alabama at Birmingham; and
Trauma Department, Hannover Medical School, Germany
2Correspondence: Center for Surgical Research, University of Alabama at Birmingham, GO94 Volker Hall, 1670 University Boulevard, Birmingham, AL 35294-0019. E-mail: Irshad.chaudry{at}ccc.uab.edu
ABSTRACT
The depression in cell-mediated immune function following trauma-hemorrhage is shown to be restored by 17ß-estradiol (E2) administration. However, it remains unknown which of the two estrogen-receptors, (ER)-
or ER-ß, plays the predominant role in mediating the beneficial effects of E2. Female B57BL/J6 ER-ß–/– transgenic mice [knockout (KO)] and corresponding ovariectomized wild-type (WT) mice were subjected to laparotomy and hemorrhagic shock (35.0±5.0 mmHg for 90 min) and treated with E2 (50 µg/25 g) or ER- agonist propyl pyrazole triol (PPT; 50 µg/25 g) following trauma-hemorrhage. Four hours after resuscitation, systemic cytokine concentrations and cytokine release by splenocytes and splenic macrophages were determined by cytometric bead array. Trauma-hemorrhage resulted in a significant increase in plasma tumor necrosis factor (TNF-), interleukin (IL)-6, and IL-10. In contrast, the release of these cytokines by splenic macrophages was decreased significantly in WT and KO animals. Administration of E2 or PPT following trauma-hemorrhage produced a significant reduction in systemic TNF- and IL-6 concentrations in WT and KO mice. Although the suppression in the productive capacity of these cytokines following trauma-hemorrhage by macrophages and splenocyte was also prevented in E2- and PPT-treated WT mice, the release of cytokines by macrophages and splenocytes in E2- and PPT-treated KO mice was not restored to the levels observed in sham animals. These findings collectively suggest that both receptors appear to play a significant role in mediating the immunoprotective effects of E2 in different tissue compartments following trauma-hemorrhage.
Key Words: shock • immune response • injury • sex hormones • propyl pyrazole triol • knockout
INTRODUCTION
Trauma is the leading cause of death in patients under 40 years of age. Most of these patients die as a result of severe infections and the development of subsequent multiple organ dysfunction [1 2 3 4 5 6 7 ]. In this regard, alterations in the immune cell function have been implicated in the overall decrease in host resistance and subsequent susceptibility to sepsis after injury [8 9 10 11 12 13 14 15 16 ].
The spleen represents a significant source of T lymphocytes as well as tissue-fixed macrophages inside the body and is supposed to play an important role in the immune response after trauma and sepsis [17 18 19 ]. Kang et al. [17 ] reported that total abolition of splenic immune functions by removal of the spleen induces a series of important alterations in the immune cell functions in various tissue compartments (lung, liver, blood) leading to increased susceptibility to infection. In addition, an impairment of bactericidal activity of macrophages in different organs (lung, peritoneum, liver) after splenectomy has been described [20 21 22 ].
There is convincing evidence that the immune response to injury [23 ] exhibits a gender dimorphic pattern. Experimental studies demonstrated that immune depression is severe in aged and ovariectomized females and adult males. In contrast, females in the proestrus stage maintained immune functions after trauma-hemorrhage [9 , 17 , 24 ]. Furthermore, the survival rate of proestrus females subjected to sepsis after trauma-hemorrhage was observed to be significantly higher than age-matched males or ovariectomized females. As the levels of female sex hormones, including 17ß-estradiol (E2), are elevated significantly during the proestrus state of the estrus cycle of female rodents, it appears that these sex hormones are involved in preserving immune function following trauma-hemorrhage. This hypothesis is supported by many studies demonstrating that administration of E2 in ovariectomized females and males after trauma-hemorrhage restored immune functions [24 25 26 ].
There are two major estrogen-receptors, subtypes (ER)- and ER-ß, which are expressed on immune cells including T lymphocytes and macrophages, and it is not clear which of the two subtypes is predominant in mediating the beneficial effects of E2 following trauma-hemorrhage. The post-traumatic administration of a selective ER agonist might represent a therapeutic option for the modulation of the immune system following trauma-hemorrhage. Therefore, to profile which ER (ER- or -ß) mediates the salutary effects of E2 on splenocytes and splenic macrophages, in vitro cytokine release capacity of these cells after in vivo treatment with E2 or a specific ER- agonist [propyl pyrazole triol (PPT)] was evaluated in wild-type (WT) and ER-ß knockout (KO; ER-ß–/–) mice.
MATERIALS AND METHODS
Animals
Female B57BL/J6 ER-ß–/– transgenic mice (129Sve) [27
] and corresponding WT animals (12 months, 28–32 g body weight) were included in this study to determine whether ER- plays a role in the regulation of splenic immune function. These animals were ovariectomized at 6 weeks of age and were a gift from Wyeth Research (Collegeville, PA). All animal studies were carried out in accordance with the guidelines of the National Institutes of Health (NIH; Bethesda, MD) and were approved by the Institutional Animal Care and Use Committee, University of Alabama at Birmingham.
Trauma-hemorrhage procedure
Mice were anesthetized with isoflurane (Minrad, Bethlehem, PA) and restrained in a supine position [25
]. A midline laparotomy (2 cm) was performed, which was closed in two layers with sutures (Ethilon 6/0, Ethicon, Somerville, NJ). Femoral arteries and the right femoral vein were cannulated with polyethylene tubing (Becton Dickinson, Sparks, MD). Blood pressure was measured via one of the arterial lines using a blood pressure analyzer (Micro-Med, Louisville, KY). Within 10 min after awakening, animals were bled through the other arterial catheter to a mean arterial blood pressure of 35.0 ± 5.0 mmHg, which was maintained for 90 min. At the end of the procedure, the animals were resuscitated via the venous line with four times the shed blood volume using Ringer’s lactate. After removing the catheters, the incisions were closed. Sham-operated animals underwent the same surgical procedures, but were neither hemorrhaged nor resuscitated.
Treatment of animals with E2 or with ER- agonist PPT
Subcutaneous (s.c.) administration of the vehicle [dimethyl sulfoxide (DMSO)] was performed after completion of the sham operation. In trauma-hemorrhage groups, E2 (50 µg/25 g), PPT (50 µg/25 g), or vehicle (DMSO) was injected s.c. immediately before onset of fluid resuscitation.
Harvesting procedures
Four hours following the completion of resuscitation in the trauma-hemorrhage groups, the animals were anesthetized again with isoflurane and were killed. Blood was obtained via cardiac puncture using a syringe coated with EDTA (Sigma Chemical Co., St. Louis, MO). Blood was centrifuged (10,000 rpm, 10 min, 4°C), and the plasma was stored at –80°C. The spleen was removed aseptically and processed as follows.
Preparation of splenocyte cultures
Splenocytes were isolated as described previously [17
]. Spleens were gently ground between frosted microscope slides to produce a single cell suspension, which was centrifuged at 300 g for 10 min at 4°C. The erythrocytes were lysed with lysis buffer, and the remaining cells were washed with phosphate-buffered saline (PBS) by centrifugation (300 g, 15 min, 4°C). After centrifugation, cells were resuspended in RPMI 1640 (Gibco, Grand Island, NY) containing 10% heat-inactivated fetal bovine serum and antibiotics (50 U/ml penicillin, 50 µg/ml streptomycin, and 5 µg/ml gentamycin, all from Gibco) to get a final concentration of 1 x 106 cells/ml. The splenocytes were then cultured in the presence of 2.5 µg/ml Concanavalin A (Con A; Pharmacia/LKB Biotech, Piscataway, NJ) at 37°C, 95% humidity, and 5% CO2 for 24 h. After incubation, the cell-free suspension was collected and stored at –80°C until further analysis.
Splenic macrophages
The splenocyte suspension was used to establish splenic macrophage cultures as described previously [17
]. In brief, the splenocyte suspension was plated in a 12-well plate (1x106 cells/ml), and after 2 h of incubation (37°C, 95% humidity, and 5% CO2), nonadherent cells were removed by washing with PBS (Gibco). Splenic macrophages in complete RPMI-1640 medium were stimulated with 10 µg lipopolysaccharide (LPS; Sigma Chemical Co.) for 24 h at 37°C, 95% humidity, and 5% CO2. At the end of the incubation period, the supernatants were removed and stored at –80°C until analyzed.
Flow cytometry
Cytokine concentrations in plasma and cell-free supernatants were determined with cytokine Bead Array inflammatory kits using flow cytometry according to the manufacturer’s instructions (BD PharMingen, San Diego, CA).
Statistics
Statistical analysis was performed using Sigma-Stat computer software (SPSS, Chicago, IL). Statistical significance was assumed, where probability values of less than 0.05 were obtained. Comparisons between groups were performed using one-way ANOVA followed by the Student’s t-test or the rank-sum test (Mann-Whitney U-test). Results are expressed as mean ± SEM of six to eight animals in each group with the exception of KO, in which the numbers were three to four in each group.
RESULTS
Plasma cytokine concentrations in WT and ER-ß–/– mice
Trauma-hemorrhage led to a significant increase in plasma tumor necrosis factor (TNF-), interleukin (IL)-6, and IL-10 levels compared with the respective sham groups in WT and KO animals. Administration of E2 and PPT following trauma-hemorrhage resulted in a significant decrease in plasma TNF- concentrations in WT and KO animals compared with trauma-hemorrhage vehicle (P<0.05; Fig. 1A
). For IL-6 levels, there was a significant decrease in E2- and PPT-treated KO animals (P<0.05). In WT groups, E2 and PPT caused a significant reduction in systemic IL-6 levels; however, the decrease after E2 application failed to reach statistical significance (P=0.08; Fig. 1B
). No significant differences were found between respective WT and KO groups. IL-10 levels, conversely, were also reduced substantially following the treatment of trauma-hemorrhage animals with E2 and PPT in WT and KO animals. The reduction in KO animals following PPT treatment was not found to be significant compared with the vehicle-treated, trauma-hemorrhage group (Fig. 1C)
.
 View larger version (20K): [in a new window] |
Figure 1. Plasma TNF- (A), IL-6 (B), and IL-10 (C) concentrations in WT and ER-ß KO mice treated with E2 or PPT. *, P < 0.05, versus all other groups; #, P > 0.05, versus trauma-hemorrhage (T-H) vehicle.
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(IFN-) concentrations, but the levels were below 20 pg/ml in all groups and were not affected by administration of E2 or PPT.
In vitro cytokine secretion of splenic macrophages of WT and ER-ß–/– mice
In vitro TNF- secretion after LPS stimulation
Trauma-hemorrhage resulted in a significant (P<0.05) reduction of TNF- secretion in WT and in KO groups compared with sham groups. The administration of E2 and PPT led to a significant increase of TNF- secretion in the WT group (P<0.05). Although a similar administration of E2 and PPT prevented the decrease in TNF- levels in KO animals, TNF- levels remained significantly lower compared with those observed in sham animals. No significant differences were observed between respective KO and WT groups (Fig. 2A
).
 View larger version (32K): [in a new window] |
Figure 2. TNF- (A) and IL-6 (B) productive capacity of splenic macrophages in response to stimulation with LPS in WT and ER-ß KO mice treated with E2 or PPT. *, P < 0.05, versus all; #, P > 0.05, versus T-H vehicle.
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, the production of IL-6 by splenic macrophages after trauma-hemorrhage was also reduced significantly in WT and KO groups compared with their respective sham groups (P<0.05). However, in contrast to TNF-, the treatment of animals with PPT and E2 prevented the decrease in IL-6 secretion in WT and KO animals (P>0.05). No significant differences were observed between respective KO and WT groups (Fig. 2B)
.
In vitro IL-10 secretion after LPS stimulation
No significant differences in IL-10 secretion by splenic macrophages were observed between trauma-hemorrhage groups (all values are in pg/ml; WT: 80.6±12; KO: 125.4±21.3) and respective sham animals (WT: 101.3±11.4; KO: 160.1±11.3; P>0.05). However, administration of E2 was associated with increased IL-10 secretion (P>0.05) compared with vehicle-treated, trauma-hemorrhage groups (WT: 124.1±19.6; KO: 170.8±32.9) and PPT (WT: 144.9±48.1; KO: 170.6±8.3). No significant differences were observed between respective KO and WT groups (P>0.05).
In vitro cytokine secretion of splenocytes of WT and ER-ß–/– mice
In vitro TNF- secretion after Con A stimulation
In WT and KO groups, trauma-hemorrhage resulted in a significant reduction of TNF- secretion compared with the respective sham group (P<0.05). The treatment of animals with E2 prevented the decrease in TNF- secretion (P<0.05) in WT and KO; however, administration of PPT did not prevent the decrease in TNF- secretion (P>0.05). No significant differences were observed between respective KO and WT groups (Fig. 3A
).
 View larger version (26K): [in a new window] |
Figure 3. TNF- (A), IL-6 (B), and IFN- (C) productive capacity of splenocytes in response to stimulation with Con A in WT and ER-ß KO mice treated with E2 or PPT. *, P < 0.05, versus all; #, P > 0.05, versus T-H vehicle.
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secretion (P>0.05). No significant differences between respective KO and WT groups were observed (P>0.05; Fig. 3B
).
In vitro IL-10 secretion after Con A stimulation
No significant differences in IL-10 secretion by splenocytes were observed between trauma-hemorrhage groups (WT: 99.6±16.3 pg/ml; KO: 116.4±5.4 pg/ml) and respective sham groups (WT: 168.7±75.5 pg/ml; KO: 165.1±26.5 pg/ml; P>0.05). Furthermore, the administration of E2 (WT: 177.4±15.1 pg/ml; KO: 168.2±22.9 pg/ml) and PPT (WT: 139.0±19.0 pg/ml; KO: 146.1±14.5 pg/ml) resulted in a slight increase in IL-10 secretion (P>0.05) by splenocytes following trauma-hemorrhage. No significant differences between respective KO and WT animals were observed (P>0.05).
In vitro IFN- secretion after Con A stimulation
Trauma-hemorrhage resulted in a significant decrease in IFN- secretion compared with the respective sham groups (Fig. 3C)
. The treatment of animals with E2 prevented the decrease in IFN- secretion (P<0.05) in WT and KO, and the administration of PPT did not prevent the decrease in IFN- secretion (P>0.05) by splenocytes following trauma-hemorrhage. No significant differences were observed between respective sham groups (P>0.05).
DISCUSSION
We examined whether the protective effects of E2 on splenic macrophages and splenocytes following trauma-hemorrhage are mediated via ER- or ER-ß. Our results indicate that administration of E2 and PPT following trauma-hemorrhage was associated with a significant reduction in systemic TNF- and IL-6 concentrations in WT and KO animals. Therefore, the effects of E2 on systemic cytokine levels are likely mediated via ER-. Conversely, the cytokine productive capacity by splenic macrophages and splenocytes was decreased following trauma-hemorrhage. However, treatment of animals with E2 but not PPT restored the cytokine production capacity to sham levels. These data suggest that the increased secretion of TNF- and IL-6 by splenic macrophages and splenocytes after E2 treatment in vivo is mediated primarily by ER-ß.
Estrogen exerts its effects on target organs by interacting with specific ER, such as ER- and -ß [28
, 29
]. Splenocytes and splenic macrophages express both of these receptors [28
29
30
], so in this study, using ER-specific agonists, we determined whether E2-mediated beneficial effects are mediated via ER- and/or ER-ß. Our findings suggest that the use of the ER- agonist per se following trauma-hemorrhage was equally as effective as E2 in decreasing the systemic proinflammatory cytokine concentrations following trauma-hemorrhage. Furthermore, the use of ER--selective agonist PPT was effective, as was E2 in the ER-ß–/– mice in decreasing plasma concentrations of proinflammatory cytokines following trauma-hemorrhage. Our results are thus in accordance with previous studies, which indicated that ER- clearly has the potential to function as a specific inhibitor of inflammatory pathways of Kupffer cells [31
, 32
]. In this regard, the Kupffer cells represent the major source of inflammatory cytokine production and thus systemic concentrations of proinflammatory mediators [33
].
Our results suggest that the role of ER- and ER-ß in mediating the salutary effects of E2 appears to be cell-specific. In splenic macrophages, the increased secretion of proinflammatory cytokines following E2 treatment seems to be primarily mediated by ER-ß. In these cells, the decreased TNF- and IL-6 production following trauma-hemorrhage was improved significantly following treatment of WT mice with PPT. Although, a similar administration of E2 and PPT in ER-ß KO animals also prevented the decrease in splenocytes and macrophages cytokines, the levels of cytokines remained significantly lower than those observed in sham animals. To the best of our knowledge, no other study has examined whether the effects of E2 on splenic macrophages are mediated via ER- or ER-ß. Nonetheless, experimental studies demonstrated that in macrophages of different organs (brain, blood, peritoneum, liver), the effects of E2 may vary depending on the distribution of ER [28
, 34
]. For example, in peritoneal macrophages and in Kupffer cells, no expression of ER-ß was found [28
], and thus, ER- seems to be of major importance for the interaction of E2 with macrophages in these compartments.
For the splenic compartment, however, the relevance of ER-ß for the maintenance of immune function after trauma-hemorrhage appears to be much greater. Administration of E2 in WT mice resulted in a significantly higher IL-6 secretion of splenocytes compared with PPT application. These results thus lead us to conclude that the expression of the ER-ß might be associated with the modulation of the post-traumatic cytokine synthesis by splenocytes. However, according to our results, the interaction of E2 with the ER- might also be involved in the maintenance of the immune function of splenocytes, as the capacity of TNF- synthesis in WT animals did not reveal any significant differences between administration of E2 or PPT following trauma-hemorrhage. Experimental studies have also shown an E2-associated modulation of cytokine synthesis by splenocytes, which was primarily mediated via ER- [29
, 35
, 36
]. However, in those studies the interaction between E2 and ER- resulted in controversial changes in the splenocyte cytokine synthesis. Liu et al. [35
] reported reduced cytokine synthesis in a model of an autoimmune encephalomyelitis, whereas in other experimental studies, an increased cytokine production was observed [29
, 35
36
37
]. It might be concluded that the importance of the different ERs as well as the E2-mediated effect on the capacity of cytokine synthesis of splenocytes are modified by the experimental insult. It is interesting that the results of an experimental study indicated that the post-traumatic expression of ER-ß in splenocytes is increased significantly, whereas no increase in ER- expression was found [10
]. In contrast Lambert et al. [29
] reported that in splenic CD4+ T cells, which were incubated in vitro with E2 without a prior in vivo insult, only a significant expression of ER- was observed. There are also studies suggesting that in ER-ß–/– animals, the function of the ER-ß might be taken over by the ER- [38
]. In light of this, the data with the ER-ß–/– animals should be interpreted carefully. Furthermore, in ER KO animals, some remaining activity of the respective receptor has been demonstrated [39
]. However, we believe that our results were not influenced by the described interaction between the different ERs, as the administration of the highly selective agonist of the ER- in WT mice confirmed the results of the ER-ß–/– animals.
In could be argued that for the sake of completeness, we should have used not only ER-ß but also ER- KO mice. Although this is true, the fact remains that plasma TNF- and IL-6 levels were comparable by using E2 or PPT in WT and also the ER-ß–/– mice following trauma-hemorrhage.
The post-traumatic administration of a selective ER agonist might represent a therapeutic option for the modulation of the immune system after trauma-hemorrhage. A selective agonist might exert the well-known, protective effects of E2 without the potentially negative side-effects. As systemic cytokine concentrations were reduced in an ER--dependent manner, it might be speculated that the use of a selective ER- agonist is a useful, therapeutic option for the prevention of remote organ damage after trauma-hemorrhage. However, the results of the present and other experimental studies indicate that in the splenic compartment, ER-ß might play a significant role [40
]. Therefore, it can be postulated that the selective application of an agonist of the ER- or the ER-ß after trauma-hemorrhage might not be sufficient to protect all of the different organ systems. Nonetheless, additional studies are needed to elucidate how far the selective administration of ER agonists is helpful in decreasing the incidence of infectious complications after trauma-hemorrhage and increasing the survival rate following those conditions.
ACKNOWLEDGEMENTS
This work was supported by NIH Grant R01 GM37127. The authors thank Wyeth Research for providing KO mice and PPT for these studies and also Dr. Heather Harris for valuable suggestions about the manuscript.
FOOTNOTES
1 Current address: Trauma Department, Hannover Medical School, Carl Neuberg STR. 1, 30625, Hannover, Germany. 
Received January 14, 2006; accepted February 9, 2006.
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