Insight into the Mechanism of Gender-specific Response to Trauma-hemorrhage

to Trauma-hemorrhage

M.A. Choudhry, K.I. Bland, and I.H. Chaudry

Introduction

Gender-based differences in patient response to injury/disease have long been recog- nized both in clinical and experimental settings [1, 2]. Despite this, some remain skeptical on the role of gender in the overall outcome of patients [1, 3]. From an analysis of more than 150,000 trauma patients, it was concluded that male patients are at higher risk of death as compared to female patients following blunt trauma [4]. Similarly, other studies have also indicated that females are more resistant to sepsis as compared to males [5, 6]. However, gender was not found to be a signifi- cant factor in the outcome of trauma patients in some other studies [7, 8]. Thus, the role of gender in the outcome of trauma patients remains somewhat controversial.

In contrast, the findings from experimental studies clearly indicate that gender plays a critical role in the host response to major injury [1, 2]. These studies have shown that immune and cardiovascular functions are suppressed following trauma-hemor- rhage in mature males, ovariectomized and aged females, while both immune and cardiac functions are maintained in proestrus females under those conditions [1, 3, 9, 10]. Similarly, liver functions following trauma-hemorrhage were depressed in males, but were maintained in proestrus females. Moreover, the survival rate of pro- estrus females subjected to sepsis after trauma-hemorrhage is significantly higher than age-matched males or ovariectomized females. In this chapter, we will review studies delineating the potential mechanisms by which male and female sex hor- mones influence immune and other organ functions following trauma-hemorrhage.

Gender and Immune Responses

A suppression of the immune response is apparent immediately after injury in males and persists for a prolonged period of time, despite fluid resuscitation [1, 3, 9 – 14].

Studies have shown that macrophage antigen presentation, T cell proliferation, and

cytokine production (Th1 cytokines, interleukin [IL]-2, and interferon [IFN] * ) are

decreased in male animals following trauma-hemorrhage. This is accompanied by

an augmented release of Th2 cytokines (IL-4 and IL-10) and increased mortality

from subsequent sepsis [1, 9, 15, 16]. In contrast to males, females in the proestrus

stage of the estrus cycle have normal/maintained immune responses following

trauma-hemorrhage [1, 9, 15, 16]. However, depletion of estrogen by ovariectomy

prior to trauma-hemorrhage resulted in suppressed immune responses similar to

that observed in male mice following trauma-hemorrhage [1, 9, 17]. Studies have

also demonstrated that treatment with the androgen receptor antagonist, flutamide,

Fig. 1. Influence of trauma-hemorrhage on immune cell function in various compartments. TNF: tumor necrosis factor; IL: interleukin; PBMC: peripheral blood mononuclear cell; IFN: interferon

following trauma-hemorrhage in male mice restored immune functions [9, 18].

Alternatively, the depletion of male sex hormones by castration prior to trauma- hemorrhage also prevented the suppression in immune functions [9, 18, 19]. The role of male sex steroids in suppressed immune response following trauma-hemor- rhage is further supported by findings suggesting that administration of 5 [ -dihyd- rotestosterone (DHT) in castrated males or in female mice prior to trauma-hemor- rhage resulted in the depression of splenic and peritoneal macrophage cytokine production as well as suppressed Th1 cytokines following trauma-hemorrhage [9, 18, 19]. In contrast, treatment of male or ovariectomized female mice with the female sex steroid, 17 q -estradiol, prevented the depression in splenic, peritoneal macrophage and Th1 cytokine production following trauma-hemorrhage [9, 18, 19]. These findings, therefore, indicate that male sex steroids cause immune depres- sion while female sex hormones maintain immune response following trauma-hem- orrhage.

In addition to suppressed immune responses, another significant observation in these studies is the finding of differences in immune cell effector responses in vari- ous tissue compartments of the body following trauma-hemorrhage [9, 20 – 23]. As shown in Figure 1, the cytokine production (IL-6, tumor necrosis factor [TNF]- [ ) capacity of peripheral blood mononuclear cells, splenic macrophages, and perito- neal macrophages was significantly decreased following trauma-hemorrhage. On the other hand, cytokine production by Kupffer cells and alveolar macrophages was sig- nificantly increased under the same experimental conditions [9, 20 – 23]. The precise mechanism by which such compartmentalized immune cell responses occur follow- ing trauma-hemorrhage in males, but not in proestrus females remains to be estab- lished.

Gender and Organ Function

Similar to immune response, other organs such as heart, liver, lung, and intestine,

are also severely affected following trauma-hemorrhage in males but not in proes-

trus females. Studies have shown that cardiac output, stroke volume, rate of pressure

development (+dP/dt), and total peripheral resistance were markedly depressed after trauma-hemorrhage in males despite fluid resuscitation [3]. Our results also indicate that these parameters of cardiac function are significantly depressed in estrus, metestrus, diestrus, and ovariectomized females following trauma-hemor- rhage and resuscitation. However, females in the proestrus stage of the estrus cycle maintained cardiac function following trauma-hemorrhage [3, 24].

Similarly, development of edema in lung, liver, and intestine tissue was observed within a few hours after trauma-hemorrhage and persisted for a prolonged period despite fluid resuscitation. However, edema following trauma-hemorrhage was not observed in proestrus females or in males treated with estrogen or other estrogenic agents (e.g., androstenediol). In a recent study, we have determined that the female reproductive cycle is an important variable in the regulation of lung injury following trauma-hemorrhage [25]. The findings from this study suggested that lung myelope- roxidase (MPO) activity (a marker for neutrophil infiltration) was significantly increased in the diestrus, estrus, and ovariectomized animals following trauma- hemorrhage. However, no difference in lung MPO activity following trauma-hemor- rhage was observed in proestrus females compared to shams. Furthermore, the increase in lung MPO activity in ovariectomized females was found to be higher compared with female rats during the diestrus, estrus, and metestrus phases of reproductive cycle. This was accompanied by increases in lung neutrophil chemoki- nes (e.g., cytokine-induced neutrophil chemoattractant [CINC]-1, CINC-3) and intercellular adhesion molecule (ICAM)-1 expression in the diestrus, estrus, and ovariectomized animals [25]. The proestrus females did not exhibit an increase in CINC-1, CINC-3, or ICAM-1 expression following trauma-hemorrhage compared to shams. Consistent with the MPO activity, the levels of CINC-1, CINC-3, and ICAM-1 were also significantly higher in ovariectomized females compared to female rats during the diestrus, estrus, and metestrus phases of reproductive cycle [25].

The maintenance of cardiac function and lung injury markers following trauma- hemorrhage in proestrus females was associated with the highest levels of estradiol, whereas all other stages of the estrus cycle had significantly lower plasma levels of estradiol [3, 24, 25]. Although estradiol levels were relatively higher in estrus and metestrus cycles compared to ovariectomized females, the findings of decreased heart function or increased lung injury markers in those animals suggest that the levels of estrogen in the estrus and metestrus cycle were not sufficient to prevent cardiac depression or lung injury following trauma-hemorrhage.

Additional findings suggest that administration of 17 q -estradiol in ovariecto-

mized females preventes the depression in cardiac function following trauma-hem-

orrhage [3]. Similar administration of 17 q -estradiol in males also prevented cardiac

and other organ dysfunction following trauma-hemorrhage [3, 10, 26]. On the other

hand, trauma-hemorrhage in pre-castrated animals did not influence cardiac func-

tion in males [3, 10, 26]. However, administration of the male sex hormone,

5 [ -DHT, in castrated males and in females suppressed cardiac function following

trauma-hemorrhage. These findings further support the suggestion that female hor-

mones maintain organ functions while male hormones adversely affect those func-

tions following trauma-hemorrhage.

Potential Mechanisms of Altered Immune and Organ Function Following Trauma-Hemorrhage

The mechanism of the beneficial effects of female sex steroids or harmful effect of male sex steroids after trauma-hemorrhage remains to be established. We have reviewed the studies suggesting the role of inflammatory mediators, heat shock proteins (HSP), and other genomic and non-genomic signaling mechanisms of estrogen-mediated beneficial effects following trauma-hemorrhage in experimen- tal settings.

Sex Hormone Receptors

There are two major subtypes of estrogen receptors (ER), ER- [ and ER- q . However, several isoforms of ER- [ and ER- q have been reported to-date. For example, ER- [ can further be classified into ER- [ A, ER- [ C, ER- [ E, and ER- [ F, and ER- q into ER-

q 1, ER- q 2, ER- q 4, and ER- q 5 [2, 3, 10, 27, 28]. It has been suggested that ER- [ and ER- q are encoded by different genes located on different chromosomes and thus do not represent splice variants [27]. Similarly, androgen receptors (AR) are also sug- gested to exist in more than one subtype; however, these subtypes have not been well studied, especially in the mammalian system. Moreover, it is also not clear whether they are isoforms or subtypes and whether these two subtypes/isoforms are derived from the same or distinct genes [29].

The distribution of AR and ER may vary from organ to organ. Similarly the dis- tribution of AR and ER subtypes is likely to be cell- or organ-specific. In a recent study, we found that heart is primarily rich in ER- q , intestine in both ER- [ and - q , lung has more ER- q than [ and liver was found to be rich in ER- [ [30, 31]. Studies have also shown that the distribution may further be affected by trauma-hemor- rhage [32]. Thus, although the effects of 17 q -estrogen on immune and other organ functions appear to be mediated via estrogen receptors, it is likely that these benefi- cial effects are mediated via organ-specific ERs. Recently our studies have utilized the ER- [ - and - q -specific agonists, propyl pyrazole triol (PPT) and diarylpropioni- trile (DPN), and examined the role of ER- [ and - q in different organs following trauma-hemorrhage [30, 31, 33]. The findings from these studies have shown that treatment of animals with the ER- [ agonist, PPT, prevented increased MPO activity in the liver following trauma-hemorrhage, while DPN normalized MPO activity in the lung [30, 31, 33].

Inflammatory Mediators

Trauma-hemorrhage results in increased production of pro-inflammatory cytokines,

which includes IL-1, IL-6, and TNF- [ , as well as the anti-inflammatory cytokine,

IL-10 [3, 9, 10, 21]. The elevation in IL-6 and TNF- [ has been correlated with poor

outcome following major injury including trauma-hemorrhage. Studies have

reported that IL-6, which controls multiple cell functions, may have a role in gender-

specific alterations in organ functions following trauma-hemorrhage, burn, and sep-

sis. In this regard, we found that plasma IL-6 levels were significantly elevated

within a few hours after trauma-hemorrhage and remained elevated at 24 h follow-

ing trauma-hemorrhage; however, administration of estradiol during resuscitation

downregulated the trauma-hemorrhage-induced increase in plasma IL-6 [3, 9, 10,

21]. Although our previous studies suggest that Kupffer cells are the primary source

of circulatory IL-6 levels, cardiomyocytes can also synthesize IL-6 and the local pro- duction of IL-6 is likely more critical in regulating cardiac function following trauma-hemorrhage [3, 9, 10, 21, 26].

TNF- [ is another pro-inflammatory cytokine that has been studied in great detail in animal models of trauma. The findings from these studies suggest that TNF- [ is increased within a few hours after injury and that treatment of animals with 17 q - estradiol prevents the increase in TNF- [ following trauma-hemorrhage [3, 9, 10, 20, 21, 34]. Thus, these findings suggest that 17 q -estradiol may mediate its salutary effect on cardiac function via modulation of pro-inflammatory cytokines, such as IL-6 and TNF- [ .

In addition to cytokines, 17 q -estradiol has also been shown to prevent the decrease in nitric oxide synthase (NOS) and to increase leukocyte infiltration [35].

Both diminished NOS activity and increased leukocyte infiltration potentially con- tribute to altered cardiac function following trauma-hemorrhage [25, 31, 33, 34, 36 – 38]. Thus, estradiol may also mediate its beneficial effects on organ function by modulating NOS and/or leukocyte infiltration following trauma-hemorrhage.

Nuclear Factor-kappa B

Nuclear factor-kappa B (NF-κB) is a pleiotropic transcription factor implicated in the regulation of diverse biological phenomena, including the cellular responses to stress, hypoxia, ischemia, and hemorrhagic shock [24, 39 – 41]. Studies have shown that NF-κB is activated in various heart diseases, such as myocarditis, congestive heart failure, dilated cardiomyopathy, and heart transplant rejection. NF- κB is also activated following burn, ischemia/reperfusion, and hypoxia. Moreover, studies have demonstrated that induction of IL-6 by hypoxia, a condition associated with trauma- hemorrhage, is mediated by NF- κB and NF-IL-6 in cardiomyocytes [24, 41]. NF-IL-6 and NF-κB are known to synergistically activate the transcription of inflammatory cytokines. Studies have also shown that estrogen represses IL-6 gene expression through inhibition of the DNA-binding activities of the transcription factors NF-IL-6 and NF-κB by the estrogen receptors [24, 41]. Thus, administration of estrogen fol- lowing trauma-hemorrhage may inhibit NF- κB binding activity and, thereby, sup- press IL-6 production. Altogether, there are multiple possibilities through which estrogen may mediate its beneficial effects on the heart and other organs following trauma-hemorrhage and resuscitation.

Heat Shock Proteins

Estrogen can induce the expression of HSP and several lines of evidence suggest that HSP upregulation plays a major role in the preservation of organ function after ischemic and low-flow conditions [25, 30, 42 – 45]. Multiple mechanisms have been proposed that HSP may utilize in mediating their protective effects. For example, HSP60 and HSP70 serve as molecular chaperones and maintain protein structures under stress conditions. HSP60 is localized in the mitochondria and is reported to be helpful in maintaining electron chain integrity [42].

HSP70, the most intensively studied member of the HSP family, similar to HSP60, is shown to assist the transfer of newly synthesized proteins into the mitochondria.

HSP70 also plays a role in maintaining overall mitochondrial integrity [42]. In addi-

tion, there is a growing body of evidence that HSP70 can block the pro-inflamma-

tory cascade via the suppression of NF-κB activation [42–44, 46].

HSP32, also referred to as heme oxygenase 1 (HO-1), has been shown to play a protective role following trauma-hemorrhage [25, 30, 43, 44]. While a definitive mechanism by which HSP32 mediates its beneficial effects remains to be established, studies have shown that it participates in heme elimination. The accumulation of free heme under hypoxic conditions becomes toxic and, therefore, elimination of free heme from the cellular milieu is necessary. Carbon monoxide which is a by- product of heme degradation can activate soluble guanylate cyclase and induce vasodilatation via cGMP. Another potential mechanism of HSP32-mediated tissue protection may be the carbon monoxide-mediated activation of Ca

-dependent potassium channels. Since the activation of Ca

-dependent potassium channels leads to hyperpolarization of the smooth muscle cells, their stimulation results in decreased vascular contractility. Bilirubin, another product of HSP32 enzyme activ- ity, has been shown to have potent antioxidant activity. A recent study from our lab- oratory has shown that HSP32 upregulation inhibits the expression of adhesion mol- ecules and prevents subsequent leukocyte-endothelial cell interactions [25, 30, 43, 44]. Furthermore, it has also been reported that HSP32 upregulation protects mito- chondrial function and prevents ATP-depletion after oxidative stress.

HSPs are also known to regulate the process of programmed cell death/apoptosis [42, 46]. One major pathway of apoptosis involves the release of cytochrome C from mitochondria. In turn, cytochrome C binds to a protein known as apoptotic pepti- dase activating factor (Apaf)-1 and triggers its oligomerization. This complex then attracts the inactive unprocessed pro-form of the proteolytic enzyme, caspase-9, which is then cleaved to its active form, thereby initiating apoptosis. HSPs have been shown to inhibit this process at various points such as preventing the binding of cytochrome C to Apaf-1. Furthermore, HSP70 prevents oilgomerized Apaf-1 from recruiting pro-caspase-9 [42, 46]. Studies have also suggested that over-expression of HSP60 inhibits myocardial apoptosis in response to ischemic injury. Furthermore, a recent study has shown that reducing HSP60 expression with antisense oligonucleo- tides is associated with an increase in Bax and a reduction in Bcl-2, which induces apoptosis of cardiomyocytes [42, 46]. In addition, HSP90 has been shown to bind to endothelial NOS (eNOS) and stimulate its activity [36, 42, 46]. Thus, HSPs protect cells via multiple mechanisms which target key cellular components and regulatory process.

The overall mechanism by which estradiol upregulates HSP after trauma-hemor- rhage remains to be established; however, studies have shown that HSP synthesis is controlled by a family of transcription factors, the heat shock factors (HSF).

Although four HSF have been identified, HSF-1 has been shown to regulate the

expression of HSP in response to ischemia, hypoxia, heat, stretch, or injury [36, 42,

46 – 48]. It is suggested that in the absence of ligand, the ER in the cytoplasm of

E2-target cells associate with the HSP (e.g., HSP90), which maintains the receptors

in an inactive state. Studies have shown that HSP90 also forms a complex with HSF-1

[42, 47, 48]. Interactions involving HSP90 and ER as well as the binding between

HSP90 and HSF thus represent an important element in the activation of HSF-1 by

E2 [42, 47, 48]. HSF-1, normally present in the cytoplasm in an inactive, monomeric

form, migrates to the nucleus upon activation where it binds to the heat shock ele-

ment (HSE). HSE is present in the promoter of the stress response gene and initiates

HSP transcription and synthesis.

Genomic Signaling

It is a widely held belief that steroid receptors (AR and ER) are mainly localized in the cytoplasm and nucleus of the cell. Thus, both androgens and estrogens mediate their actions by activating the transcription factors and accordingly are expected to alter signaling at the nuclear level (i.e., the genomic mechanism). This genomic effect of estrogen last from hours to days. As presented in Fig. 2, upon binding to estrogen, ER becomes activated, dimerized and the complex translocates to the nucleus. In the nucleus, it binds a specific target DNA sequence within estrogen- responsive genes called an estrogen response element (ERE). This leads to the enhancement of transcription. Studies have also indicated that unbound ER can also bind to ERE consensus sequences and activate transcription, but interaction of the receptor with estrogen stabilizes dimerization and enhances its interaction with tar- get sequences within estrogen-responsive genes. It has been suggested that several soluble growth factors, including epidermal growth factor and insulin-like growth factor-1 can activate ER and thus promote gene transactivation in the absence of estrogen [36, 42, 47, 48].

Non-genomic Signaling

In addition to the genomic action, there is evidence indicating that ERs are also localized on the plasma membrane and thus estrogen can also influence cell func- tion by inducing nongenomic effects via plasma membrane ERs. Studies have shown that these so-called nongenomic effects or cell membrane-initiated signals are acti- vated quickly within minutes (Fig. 2). Utilizing a cell-impermeable estrogen conju-

Fig. 2. Genomic and non-genomic actions of estrogen. E2: estrogen; R: receptor; iNOS: inducible nitric oxide synthase; PTK: protein tyrosine kinases; NO: nitric oxide; PI3K: phosphatidylinositol 3-kinase; PIP2:

phosphatidylinositol 4,5-bisphosphate; PLC: phospholipase C; IP3: inositol 3-phopshate; DAG: diacyl gryce-

rol; PKC: protein kinase C; MAPK: mitogen activated protein kinase

gated with bovine serum albumin (E2-BSA), studies have shown that many of the signaling pathways (e.g., PLC/PKC; p38/MAPK; PI3k/AKT, NO, Ca

) are turned on after stimulation of cells within E2-BSA [27, 36, 47, 49, 50].

The PI3K/AKT signaling pathway can also be activated by estrogen in different cell lines (e.g., vascular or epithelial cells). Engagement of membrane ERs results in rapid endothelial NO release through a PI3K/AKT-dependent pathway. The non- genomic effects of estrogen can regulate different cellular processes, such as prolifer- ation, survival, apoptosis, and differentiated functions in diverse cell types, includ- ing breast cancer cells [27, 36, 47, 49, 50]. The nature of the plasma membrane bind- ing sites for estrogens is currently under intense debate and investigation. However, to date, both classic ER- [ and ER- q and non-classical ER (e.g., GPR30) have been identified at the membrane. It has been shown that GPR30 binds only high concen- trations of 17 q -estradiol.

We have used E2-BSA to delineate the role of cell surface versus nuclear mem- brane receptors in estrogen-mediated restoration of organ function following trauma-hemorrhage. Our results indicate that male rats treated with E2-BSA display improvement in cardiac functions at 2 hours following trauma-hemorrhage. We fur- ther found that the biologic effects of E2-BSA on cardiac function are receptor- dependent since the administration of ICI 182,780, a selective ER antagonist, along with E2-BSA abolished the E2-BSA-induced cardioprotection in trauma-hemorrhage rats. However, it is to be noted that the restoration of cardiac function following E2- BSA treatment was not complete as compared to the rats treated with estrogen alone. Thus, it is likely that the activation of both surface and nuclear ERs is required for full restoration of cardiac function following trauma-hemorrhage. Our studies also demonstrated that PI3K/Akt pathway plays a major role in mediating the non-genomic effects of estrogen on cardiac function. Similar findings were obtained by other investigators indicating the role of PI3K/Akt signaling in non- genomic estrogen effects [27, 36, 47, 49, 50]. The activation of the PI3K pathway pro- tects organs or cells against ischemia-reperfusion injury and hypoxia through sup- pression of the cell death machinery. One of the downstream targets of PI3K path- way is Akt and studies have shown that an increase in Akt activity leads to improved left ventricular contractile recovery following transient ischemia. PI3K/Akt has been reported to play an important role in the cell survival pathway [27, 36, 47, 49, 50].

Studies have also reported that the PI3K/Akt signal has an anti-apoptotic activity

through different mechanisms, e.g., by phosphorylation of Akt, it induces BAD

phosphorylation and hence inhibits its translocation into the mitochondria and

binding to Bcl-2 [27, 36, 47, 49, 50]. While it is likely that E2-BSA-induced Akt phos-

phorylation prevents DNA fragmentation in cardiomyocytes and thus maintains car-

diac functions following trauma-hemorrhage, the role of other molecules in mediat-

ing the nongenomic effects of estrogen has not been ruled out. In view of this, addi-

tional studies are needed to delineate the role of both genomic and non-genomic

pathways in estrogen-mediated actions on immune and other organ functions fol-

lowing trauma-hemorrhage. However, it should be noted that although the genomic

and non-genomic actions of estrogen have been studied separately, these actions are

interdependent and should be considered as synergistically acting aspects of the

molecular response to estrogen, leading to the final physiologic outcomes such as

survival versus apoptosis, growth regulation and cell motility to name a few.

Conclusion

Clinical and experimental studies suggest that gender is an important factor which should be considered in the overall management of trauma patients. Experimental findings suggest that trauma-hemorrhage results in profound alterations in immune and other organ functions in males and ovariectomized females, but these functions are maintained in pre-castrated males and proestrus females. However, administra- tion of estrogen restored both immune and other organ functions in males and ovariectomized females following trauma-hemorrhage. While more studies are needed to fully understand the precise mechanism by which estrogen mediates its beneficial effects, the studies reviewed in this chapter suggest that estrogen modu- lates a number of factors including inflammatory mediators and HSPs, and thereby protects organ functions following trauma-hemorrhage.

Acknowledgement: This work was supported by the National Institutes of Health grants AA015979-01A1 (MAC), R37 GM39519 and R01 GM37127 (IHC). The authors also thank Ms. Bobbi Smith for editing the manuscript.

References

1. Choudhry MA, Bland KI, Chaudry IH (2006) Gender and susceptibility to sepsis following trauma. Endocr Metab Immune Disord Drug Targets 6:127 – 135

2. Orshal JM, Khalil RA (2004) Gender, sex hormones, and vascular tone. Am J Physiol Regul Integr Comp Physiol 286:R233-R249

3. Choudhry MA, Schwacha MG, Hubbard WJ, et al (2005) Gender differences in acute response to trauma-hemorrhage. Shock 24 (Suppl 1):101 – 106

4. George RL, McGwin G Jr, Windham ST, et al (2003) Age-related gender differential in out- come after blunt or penetrating trauma. Shock 19:28 – 32

5. Offner PJ, Moore EE, Biffl WL (1999) Male gender is a risk factor for major infections after surgery. Arch Surg 134:935 – 938

6. Schroder J, Kahlke V, Staubach KH, Zabel P, Stuber F (1998) Gender differences in human sepsis. Arch Surg 133:1200 – 1205

7. Croce MA, Fabian TC, Malhotra AK, Bee TK, Miller PR (2002) Does gender difference influ- ence outcome? J Trauma 53:889 – 894

8. Eachempati SR, Hydo L, Barie PS (1999) Gender-based differences in outcome in patients with sepsis. Arch Surg 134:1342 – 1347

9. Angele MK, Schwacha MG, Ayala A, Chaudry IH (2000) Effect of gender and sex hormones on immune responses following shock. Shock 14:81 – 90

10. Chaudry IH, Samy TS, Schwacha MG, Wang P, Rue LW III, Bland KI (2003) Endocrine targets in experimental shock. J Trauma 54:S118-S125

11. Angle N, Hoyt DB, Coimbra R, et al (1998) Hypertonic saline resuscitation diminishes lung injury by suppressing neutrophil activation after hemorrhagic shock. Shock 9:164 – 170 12. Kher A, Wang M, Tsai BM, et al (2005) Sex differences in the myocardial inflammatory

response to acute injury. Shock 23:1 – 10

13. Noel JG, Guo X, Wells-Byrum D, Schwemberger S, Caldwell CC, Ogle CK (2005) Effect of thermal injury on splenic myelopoiesis. Shock 23:115 – 122

14. Shelley O, Murphy T, Paterson H, Mannick JA, Lederer JA (2003) Interaction between the innate and adaptive immune systems is required to survive sepsis and control inflammation after injury. Shock 20:123 – 129

15. Angele MK, Knöferl MW, Ayala A, Bland KI, Chaudry IH (2001) Testosterone and estrogen differently effect th1 and th2 cytokine release following trauma-haemorrhage. Cytokine 16:22 – 30

16. Zellweger R, Wichmann MW, et al (1997) Females in proestrus state maintain splenic

immune functions and tolerate sepsis better than males. Crit Care Med 25:106 – 110

17. Knöferl MW, Jarrar D, Angele MK, et al (2001) 17beta-Estradiol normalizes immune responses in ovariectomized females after trauma-hemorrhage. Am J Physiol Cell Physiol 281:C1131-C1138

18. Wichmann MW, Angele MK, Ayala A, Cioffi WG, Chaudry IH (1997) Flutamide: a novel agent for restoring the depressed cell-mediated immunity following soft-tissue trauma and hemor- rhagic shock. Shock 8:242 – 248

19. Wichmann MW, Zellweger R, DeMaso CM, Ayala A, Chaudry IH (1996) Mechanism of immu- nosuppression in males following trauma-hemorrhage. Critical role of testosterone. Arch Surg 131:1186 – 1191

20. Ayala A, Wang P, Ba ZF, Perrin MM, Ertel W, Chaudry IH (1991) Differential alterations in plasma IL-6 and TNF levels after trauma and hemorrhage. Am J Physiol 260:R167-R171 21. Catania RA, Chaudry IH (1999) Immunological consequences of trauma and shock. Ann.

Acad Med Singapore 28:120 – 132

22. Hildebrand F, Hubbard WJ, Choudhry MA, et al (2006) Kupffer cells and their mediators: the culprits in producing distant organ damage after trauma-hemorrhage. Am J Pathol 169:784 – 794

23. Schneider CP, Schwacha MG, Chaudry IH (2006) Influence of gender and age on T-cell responses in a murine model of trauma-hemorrhage: differences between circulating and tis- sue-fixed cells. J Appl Physiol 100:826 – 833

24. Yang S, Choudhry MA, Hsieh YC, et al (2006) Estrus cycle: Influence on cardiac function fol- lowing trauma-hemorrhage. Am J Physiol Heart Circ Physiol 291:H2807 – 2815

25. Yu HP, Yang S, Hsieh YC, Choudhry MA, Bland KI, Chaudry IH (2006) Maintenance of lung myeloperoxidase activity in proestrus females after trauma-hemorrhage: upregulation of heme oxygenase-1. Am J Physiol Lung Cell Mol Physiol 291:L400-L406

26. Yang S, Zheng R, Hu S, et al (2004) Mechanism of cardiac depression after trauma-hemor- rhage: increased cardiomyocyte IL-6 and effect of sex steroids on IL-6 regulation and cardiac function. Am J Physiol Heart Circ Physiol 287:H2183-H2191

27. Acconcia F, Kumar R (2006) Signaling regulation of genomic and nongenomic functions of estrogen receptors. Cancer Lett 238:1 – 14

28. Kos M, Denger S, Reid G, Gannon F (2002) Upstream open reading frames regulate the trans- lation of the multiple mRNA variants of the estrogen receptor alpha. J Biol Chem 277:

37131 – 37138

29. Ikeuchi T, Todo T, Kobayashi T, Nagahama Y (1999) cDNA cloning of a novel androgen recep- tor subtype. J Biol Chem 274:25205 – 25209

30. Yu HP, Shimizu T, Choudhry MA, et al (2006) Mechanism of cardioprotection following trauma-hemorrhagic shock by a selective estrogen receptor-beta agonist: up-regulation of cardiac heat shock factor-1 and heat shock proteins. J Mol Cell Cardiol 40:185 – 194 31. Yu HP, Shimizu T, Hsieh YC, et al (2006) Tissue-specific expression of estrogen receptors and

their role in the regulation of neutrophil infiltration in various organs following trauma- hemorrhage. J Leukoc Biol 79:963 – 970

32. Samy TS, Schwacha MG, Cioffi WG, Bland KI, Chaudry IH (2000) Androgen and estrogen receptors in splenic T lymphocytes: effects of flutamide and trauma-hemorrhage. Shock 14:465 – 470

33. Yu HP, Hsieh YC, Suzuki T, et al (2006) Salutary effects of estrogen receptor-beta agonist on lung injury after trauma-hemorrhage. Am J Physiol Lung Cell Mol Physiol 290:L1004-L1009 34. Hildebrand F, Hubbard WJ, Choudhry MA, Thobe BM, Pape HC, Chaudry IH (2006) Effects

of 17{beta}-estradiol and flutamide on inflammatory response and distant organ damage fol- lowing trauma-hemorrhage in metestrus females. J Leukoc Biol 80:759 – 765

35. Angele MK, Fitzal F, Smail N, et al (2000) L-arginine attenuates trauma-hemorrhage-induced liver injury. Crit Care Med 28:3242 – 3248

36. Haynes MP, Russell KS, Bender JR (2000) Molecular mechanisms of estrogen actions on the vasculature. J Nucl Cardiol 7:500 – 508

37. Hierholzer C, Harbrecht B, Menezes JM, et al (1998) Essential role of induced nitric oxide in

the initiation of the inflammatory response after hemorrhagic shock. J Exp Med 187:917 – 928

38. Kerger H, Waschke KF, Ackern KV, Tsai AG, Intaglietta M (1999) Systemic and microcircula-

tory effects of autologous whole blood resuscitation in severe hemorrhagic shock. Am J Phy-

siol 276:H2035-H2043

39. Abraham E (2000) NF-kappaB activation. Crit Care Med 28:N100-N104

40. Liu SF, Malik AB (2006) NF-kappa B activation as a pathological mechanism of septic shock and inflammation. Am J Physiol Lung Cell Mol. Physiol 290:L622-L645

41. Matsusaka T, Fujikawa K, Nishio Y, et al (1993) Transcription factors NF-IL6 and NF-kappa B synergistically activate transcription of the inflammatory cytokines, interleukin 6 and interleukin 8. Proc Natl Acad Sci USA 90:10193 – 10197

42. Latchman DS (2001) Heat shock proteins and cardiac protection. Cardiovasc Res 51:637 – 646 43. Szalay L, Shimizu T, Suzuki T, et al (2006) Estradiol improves cardiac and hepatic function

after trauma-hemorrhage: role of enhanced heat shock protein expression. Am J Physiol Regul Integr Comp Physiol 290:R812-R818

44. Szalay L, Shimizu T, Schwacha MG, et al (2005) Mechanism of salutary effects of estradiol on organ function after trauma-hemorrhage: upregulation of heme oxygenase. Am J Physiol Heart Circ Physiol 289:H92-H98

45. Su F, Nguyen ND, Wang Z, Cai Y, Rogiers P, Vincent JL (2005) Fever control in septic shock:

beneficial or harmful? Shock 23:516 – 520

46. Meng X, Harken AH (2002) The interaction between Hsp70 and TNF-alpha expression: a novel mechanism for protection of the myocardium against post-injury depression. Shock 17:

345 – 353

47. Hall JM, Couse JF, Korach KS (2001) The multifaceted mechanisms of estradiol and estrogen receptor signaling. J Biol Chem 276:36869 – 36872

48. Knowlton AA, Sun L (2001) Heat-shock factor-1, steroid hormones, and regulation of heat- shock protein expression in the heart. Am J Physiol Heart Circ Physiol 280:H455-H464 49. Bjornstrom L, Sjoberg M (2005) Mechanisms of estrogen receptor signaling: convergence of

genomic and nongenomic actions on target genes. Mol Endocrinol 19:833 – 842

50. Driggers PH, Segars JH (2002) Estrogen action and cytoplasmic signaling pathways. Part II:

the role of growth factors and phosphorylation in estrogen signaling. Trends Endocrinol

Metab 13:422 – 427