Published online before print October 15, 2007
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Center for Surgical Research and Department of Surgery, University of Alabama at Birmingham, Birmingham, Alabama, USA
1Correspondence: Center for Surgical Research, The University of Alabama at Birmingham, Volker Hall G094, 1670 University Blvd., Birmingham, AL 35294-0019, USA. E-mail: irshad.chaudry{at}ccc.uab.edu
ABSTRACT
17β-Estradiol (i.e., estrogen or E2) is a female sex steroid, which plays an essential role in female reproductive physiology. However, several lines of evidence indicate that in addition to its role in reproductive physiology, E2 is critical for maintaining many other organ functions in stress conditions. These include immune, cardiovascular, and neuronal functions, as well as regulation of skin, bone, and lipid metabolism. Studies have examined the role of E2 as an adjunct in post-trauma responses, and this article will review whether E2 as an adjunct to fluid resuscitation following trauma-hemorrhage plays any role in improving/restoring immune and cardiovascular functions.
Key Words: ER-a • ER-β • cytokines • shock • cell signaling • Kupffer cells
INTRODUCTION
Trauma remains the leading cause of death in young adults, especially during the first three decades of life. Studies have shown that a simultaneous activation of numerous interacting inflammatory pathways is initiated following trauma. This is characterized by abnormal production of cytokines and chemokines and the recruitment of inflammatory cells to the site of injury and other vital organs [1
2
3
4
5
]. As a result, the immune cell effector responses and other organ functions are severely compromised [2
, 6
7
8
9
]. Organ dysfunction and failure remain the major causes of death in patients who survive the initial insult. Several lines of evidence indicate that males and females respond differently to trauma. Findings from clinical studies suggest that premenopausal women have a lower incidence of infection, pneumonia, sepsis, Intensive Care Unit visits, and multiple organ failure than men after trauma [10
, 11
]. In our retrospective analysis of blunt trauma patients [12
, 13
], we found that male patients had a significantly higher risk of death compared with premenopausal female patients. A few studies, however, have failed to establish the relationship between gender and the outcome in trauma patients [2
]. The reason for the different results may be a result of the fact that the hormonal status of the victim was not accounted for at the time of injury, and thus, females in different phases of the menstrual cycle [i.e., with different circulating 17β-estradiol (E2) levels] were included. This may explain why similar findings are not observed in different studies. Thus, more clinical studies that include hormone measurements at the time of injury are needed to understand the role of gender in post-trauma pathogenesis. Nonetheless, there is evidence that females in their reproductive years have more vigorous immune responses than males. Furthermore, E2 at physiological levels was found to stimulate humoral and cell-mediated immune responses [1
, 2
]. In contrast, the male hormone, 5
-dihydrotestosterone (DHT), adversely affects these responses [1
, 2
]. Sex hormones can also modulate immune response, including clonal expansion, phagocytosis, apoptosis, and antigen presentation. Thus, sex hormone levels at the time of injury rather than gender per se appear to be critical in shaping the immune response following trauma. The conflicting clinical studies that have been reported are therefore likely a result of the differences in hormonal levels at the time of injury. To understand the role of sex hormones in post-trauma pathogenesis, it is important to measure their levels at the time of injury and correlate those levels with the complications/outcome of trauma patients.
E2 AND POST-TRAUMA IMMUNE FUNCTION
Trauma results in a profound suppression of immune response despite fluid resuscitation [1
, 2
, 6
, 14
]. The suppression is visible within hours after injury, and it persists for a prolonged period of time. The suppression in immune response is characterized by a decrease in macrophage (M
) and dendritic cell antigen presentation, T cell proliferation, and cytokine production by peritoneal and splenic M (Th1 cytokines—IL-2 and IFN-
). This is accompanied with an augmented release of Th2 cytokines (IL-4 and IL-10) and increased mortality from subsequent sepsis [1
, 15
, 16
]. Studies have indicated that the suppression of immune response is more pronounced in males and also apparent in nonproestrus females. However, the suppression in immune response following trauma-hemorrhage is not evident in proestrus females [1
]. The estrus cycle of the mouse and rat can be divided into four stages: diestrus, proestrus, estrus, and post-estrus (metestrus). As E2 levels are the highest in the proestrus cycle, it was thought that it is the E2 that protects immune cell function in proestrus females, and studies were thus carried out to determine the role of E2 in post-trauma immune responses. Those studies used approaches from depleting E2 levels by ovariectomy to the reconstitution of E2 in E2-depleted animals, as well as administration of E2 in males. The findings of those studies demonstrate that depletion of E2 leads to a suppression of immune responses, and reconstitution of E2 protects immune cell functions following trauma-hemorrhage [1
, 17
].
Studies have also shown that male sex hormones are responsible for suppressing the immune cell functions following trauma-hemorrhage. Further proof for this notion came from studies which demonstrated that administration of the androgen receptor antagonist (i.e., flutamide) following trauma-hemorrhage in male mice restored immune functions [1
, 18
]. Furthermore, trauma-hemorrhage in castrated animals (e.g., depleted of male sex hormones) did not produce the suppression in immune functions [1
]. The immunosuppressive role of the male sex steroid following trauma-hemorrhage is supported further by the studies that demonstrated that the administration of the male sex steroid DHT in castrated males or in female mice prior to trauma-hemorrhage resulted in the depression of splenic and peritoneal M cytokine production as well as suppressed Th1 cytokines following trauma-hemorrhage [1
]. In contrast, treatment of male or ovariectomized female mice with the female sex steroid E2 prevented the depression in immune cell effector responses following trauma-hemorrhage [1
, 18
]. Another interesting observation made was that trauma-hemorrhage resulted in compartmentalized alterations in immune responses following trauma-hemorrhage [1
, 19
, 20
]. For example, IL-6 and TNF- production capacity of PBMC, splenic and peritoneal M, and bone marrow cells was decreased significantly following trauma-hemorrhage. In contrast, the production of these cytokines by M harvested from liver (i.e., Kupffer cells) and lung (i.e., alveolar M) was elevated significantly under the same experimental conditions [1
, 19
, 20
]. Administration of E2 following trauma-hemorrhage, however, prevented those alterations in various tissue compartments. These findings suggest collectively that E2 protects immune cell effector responses, not only in the splenic compartment but also globally following trauma-hemorrhage.
E2 AND ORGAN FUNCTION
In addition to suppressed immune responses, the functions of other organs such as heart, liver, lung, and intestine are compromised following trauma-hemorrhage in males but not in proestrus females. Studies have shown that cardiac functions, as determined by cardiac output, stroke volume, contractility, and total peripheral resistance, were markedly depressed after trauma-hemorrhage in males and females in estrus, metestrus, diestrus phase, and ovariectomized females, despite fluid resuscitation. However, these parameters are maintained in proestrus females following trauma-hemorrhage [2 , 21 ]. Furthermore, administration of E2 in males and ovariectomized females protected cardiac function following trauma-hemorrhage. Similarly, trauma-hemorrhage produces lung, liver, and intestinal tissue edema within a few hours after injury, and like cardiac functions, tissue edema was not observed in proestrus females or in males treated with E2 following trauma-hemorrhage. Our findings further indicate that an increase in chemokines and neutrophil infiltration is likely to play a role in increased lung, liver, and intestine edema [22 , 23 ]. Consistent with cardiac functions, an increase in lung myeloperoxidase (MPO) activity, neutrophil chemokines [e.g., cytokine-induced neutrophil chemoattractant 1 (CINC-1), CINC-3], and adhesion molecule ICAM-1 expression was observed in the diestrus, estrus, and ovariectomized animals [24 ]. However, these parameters were not elevated in proestrus females following trauma-hemorrhage [24 ]. The maintenance of cardiac function and lung inflammatory markers following trauma-hemorrhage in proestrus females was associated with the highest levels of E2, whereas all other stages of the estrus cycle had significantly lower plasma E2 levels [2 , 21 , 24 ]. Although E2 levels were relatively higher in estrus and metestrus cycles compared with ovariectomized females, the findings of decreased cardiac functions or increase in lung injury markers in those animals suggest that E2 levels in the estrus and metestrus cycle were not sufficient to protect cardiac depression or lung injury following trauma-hemorrhage. Thus, female hormones are responsible for improving/maintaining organ functions following trauma-hemorrhage.
MECHANISM OF ACTION
Two major subtypes of estrogen receptor (ER), i.e., ER- and ER-β, exist in the cells through which E2 mediates its action [25
, 26
]. Furthermore, several isoforms exist within each ER subtype. ER- isoforms are ER-A, ER-C, ER-E, and ER-F. Similarly ER-β isoforms are ER-β1, ER-β2, ER-β4, and ER-β5 [26
]. These receptors are present in almost all cells, but their distribution was found to be organ-specific. For example, liver was found to be rich in ER- and lung in ER-β. Intestine, conversely, has ER- and ER-β. The overall distribution of these receptors in various organs following trauma-hemorrhage remains to be established. Recent studies have determined the role of ERs in estrogen-mediated protection of various organ functions following trauma-hemorrhage. These studies used ER-- and -β-specific agonists, propyl pyrazole triol (PPT), and diarylpropionitrile (DPN). PPT, the best-characterized selective agonist for the ER- subtype, has a 410-fold higher affinity for ER- than ER-β [27
]. DPN, which is a specific agonist for ER-β, has 70-fold higher binding affinity for ER-β over ER-. Furthermore, DPN has a 170-fold higher relative estrogenic potency in transcription assays with ER-β than ER- [28
]. Our results provide evidence that following trauma-hemorrhage, estrogen-induced reduction of MPO activity (an index of neutrophil infiltration) is mediated via ER- activation in the liver, via ER-β activation in the lung, and via ER- and ER-β in the small intestine [23
]. Those findings are consistent with ER expression in the liver, small intestine, and lung (i.e., ER- mRNA expression is highest in the liver, and ER-β mRNA expression is greatest in the lung) [23
, 29
30
31
]. Thus, it is possible that the differences between the ER subtypes in tissue distribution may contribute to the selective action of ER agonists in different tissues.
It has long been thought that sex hormone receptors, including ERs, are localized in the cytoplasm and nucleus of the cell, and they thus mediate their actions by activating the signaling at the nuclear level (i.e., the genomic mechanism). However, there is evidence indicating that ERs are localized in mitochondria, and they enhance the levels of mitochondrial DNA (mtDNA)-encoded transcripts directly [32
]. A recent study has shown that E2 enhances the mitochondrial levels of ERs and increases the transcript levels of several mtDNA-encoded genes required for mitochondrial respiratory complex (MRC) proteins and MRC activity [32
]. These observations suggest that mtDNA-encoded MRC could be a direct target for E2 action in the mtERs. We examined the role of mitochondria in E2-mediated protection of cardiac function following trauma-hemorrhage [33
]. Rats received PPT (ER- agonist), DPN (ER-β agonist), or E2 following trauma-hemorrhage, and the effects of these treatments were examined on mtER-, mtER-β, mitochondrial estrogen response element (mtERE)-binding activity, and mtDNA-encoded genes for MRC-I and MRC-IV proteins [33
]. To determine the role of MRC-IV in DPN-mediated cardioprotection, a group of DPN-treated rats was cotreated with MRC-IV inhibitor sodium cyanide (SCN). We found that E2 or DPN treatment after trauma-hemorrhage normalized cardiac mtER-β expression and increased mtER-β DNA binding activity. This was accompanied by an increase in MRC-IV gene expressions and activity, and MRC-I gene expression remained unchanged. Inhibition of MRC-IV in DPN-treated trauma-hemorrhage rats by SCN abolished the DPN-mediated cardioprotection, ATP production, mitochondrial cytochrome c release, caspase-3 cleavage, and apoptosis [33
]. Thus, E2- and ER-β-mediated cardioprotection following trauma-hemorrhage appears to be mediated via mtER-β-dependent MRC-IV activity and inhibition of mitochondrial apoptotic signaling pathways.
As shown in Figure 1
, ER upon binding to E2 becomes activated and dimerized. The complex then translocates to the nucleus, where it binds to a specific target DNA sequence within the estrogen-responsive genes, ERE [26
, 34
35
36
]. Studies have indicated that ERs, which are not bound to E2, can also bind to ERE consensus sequences and activate transcription. However, an interaction of the receptor with E2 stabilizes dimerization and enhances its interaction with target sequences. Other growth factors, including epidermal growth factor and insulin-like growth factor-1, can also activate ERs and thus, promote gene activation in the absence of E2 [37
]. The role of nongenomic pathways is also explored in E2 actions in several studies [38
39
40
]. Although the findings from these studies remained somewhat controversial, investigations in the past decade do indicate existence of plasma membrane-associated ERs and their involvement in E2 action [38
, 40
]. Furthermore, a role for plasma membrane-associated ER- and ER-β in the activation of phosphatidylinositol 3-kinase (PI3K)/Akt and the rapid release of NO in endothelial cells has been reported [38
39
40
]. In addition to ER, studies have indicated a role for G-protein coupled receptors (GPRs) in nongenomic actions of E2 (see Fig. 1
). This GPR was termed GPR30 and was shown to respond to the classic ER antagonist ICI 182,780 in an agonist manner [38
39
40
]. GPR30 is located on the cell membrane and presents an alternative to the classical ERs. Thus, as shown in the Figure 1
, E2 can mediate its effect independently of ERs [41
]. Estrogen binds directly to GPR30 and rapidly activates the signaling pathway in the cells. Studies have shown that GPR30 overexpression in breast cancer cells deficient in ER- and ER-β restores the activation of adenyl cyclase by E2. Moreover, silencing GPR30 expression with small interfering RNA prevents E2-mediated cAMP-dependent signaling in keratinocytes and in SKBR3 breast cancer cells that lack ER- and ER-β. These findings therefore indicate that G-protein coupling itself may not mediate, but it enables membrane ERs to initiate signal transduction at the cell surface in different cell types [38
]. Furthermore, it is likely that ERs form a complex with G-protein, and that complex facilitates the action of E2. Studies have also indicated that multiple isoforms of G-protein form complexes, and those complexes may mediate E2 action in various cell types. Recent findings from our laboratory have also shown that E2 uses GPR30 in activating the PKA pathway in isolated hepatocytes [42
]. However, more studies are needed to fully understand the role of GPR30 in E2-mediated effects on immune cell functions following trauma-hemorrhage.
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Figure 1. E2-mediated genomic and nongenomic signaling. R, Receptor; GPR30, G-protein-coupled receptor 30; PTK, protein tyrosine kinases; PIP2, phosphatidylinositol 4,5-bisphosphate; PLC, phospholipase C; IP3, inositol 3-phopshate; DAG, diacylglycerol; PKC, protein kinase C; PI3K, phosphatidylinositol 3-kinase, MAPK, Mitogen activated protein kinase.
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Heat shock proteins (HSP) are also implicated in mediating E2 actions following trauma-hemorrhage [24 , 29 , 37 ]. In a recent study, we investigated the role of HSP32 (also referred to as heme oxygenase-1) in E2-mediated organ protection following trauma-hemorrhage. We found that E2 administration up-regulates HSP32 following trauma-hemorrhage. Although the mechanism by which HSP32 protects organ function following trauma-hemorrhage remains to be established, studies have indicated that HSP32 participates in the elimination of heme, which is accumulated as a result of excessive blood loss or hypoxic insult. We observed that HSP32 up-regulation inhibits the expression of adhesion molecules and prevents subsequent leukocyte-endothelial cell interactions under those conditions [24 , 29 ]. Others have shown that an up-regulation in HSP32 protects mitochondrial function and prevents ATP depletion after oxidative stress. Similarly, other HSPs such as HSP60 and HSP70 are considered as molecular chaperones and maintain protein structures under stress conditions [37 ]. Nonetheless, more studies are needed to delineate the mechanism by which E2 regulates HSP32 following trauma-hemorrhage.
CONCLUSION
The findings reviewed in this article indicate that E2, in addition to its role in reproductive physiology, is also critical in maintaining other organ function in stress conditions. Studies have examined the role of E2 in post-trauma pathogenesis. The findings indicate that trauma-hemorrhage causes a severe depression in immune cell functions and also impairs the functions of other organs. Administration of E2 following trauma-hemorrhage as an adjunct to fluid resuscitation normalizes immune and organ functions. The findings also indicate that there are two major receptors, ER- and ER-β, which mediate E2 actions. These receptors are widely located in the cytoplasm and on the nuclear membrane, but there is evidence that these receptors are also found on the plasma membrane. In addition, G-protein, particularly GPR30, is implicated in mediating E2 actions. However, more studies are needed to better understand the role of these receptors in mediating the beneficial effect of estrogen following trauma-hemorrhage. Furthermore, most of the data about the role of estrogens in trauma is coming from an experimental setting, and data from the clinical setting are limited; therefore, more studies are needed in the clinical setting. Those studies should be performed in such a way that other confounding factors such as preclinical manifestation and age should be minimized. The information gained from these studies will be extremely helpful in designing innovative, therapeutic approaches for the treatment of trauma patients.
ACKNOWLEDGEMENTS
This work was supported by National Institutes of Health grants R01 AA015731-01A2 (M. A. C.), R37 GM39519, and R01 GM37127 (I. H. C.).
Received June 9, 2007; revised August 30, 2007; accepted September 19, 2007.
REFERENCES
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