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Am J Physiol Heart Circ Physiol 291: H1456-H1465, 2006. First published April 14, 2006; doi:10.1152/ajpheart.00694.2005
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Neutrophil activation is modulated by sex hormones after trauma-hemorrhagic shock and burn injuries

Edwin A. Deitch, Preya Ananthakrishnan, David B. Cohen, Da Zhong Xu, Eleonora Feketeova, and Carl J. Hauser

Department of Surgery, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, New Jersey

Submitted 24 June 2005 ; accepted in final form 11 April 2006


   ABSTRACT
TOP
 ABSTRACT
METHODS AND MATERIALS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Recent literature indicates that females are more resistant to shock-, trauma-, and sepsis-induced immune dysfunction and organ injury than are males. Consequently, using trauma-hemorrhagic shock (T/HS) and burn models, we tested whether the neutrophil response to trauma occurred in a sexually dimorphic fashion and, if so, the role of sex hormones. Neutrophil activation, as reflected by CD11b expression and respiratory burst activity, was increased to a greater extent in male rats than in female rats after T/HS or burn injury. Testosterone appeared to potentiate neutrophil activation, because castration reduced neutrophil activation, whereas ovariectomy had little effect. Mechanistically, this sexually dimorphic neutrophil response appeared to be due to both cellular and humoral factors. Evidence for a cellular difference between male and female neutrophils is based on the observation that naive female neutrophils were more resistant to activation by burn or T/HS plasma and lymph than naive male neutrophils and that this resistance varied over the estrus cycle. Additionally, the humoral environment was more neutrophil activating in male rats, because burn and T/HS plasma and lymph from male rats activated naive male neutrophils to a greater extent than comparable samples from females. Last, on the basis of in vitro experiments examining the effects of estrogen on calcium signaling, it appears that estrogen limits trauma-induced neutrophil activation, at least in part, by limiting the entry of calcium into the cell via store-operated calcium entry mechanisms. In conclusion, there is a striking sexual dimorphism in neutrophil responses after trauma, and these changes reflect both cellular resistance to activation as well as a less activating humoral environment.

polymorphonuclear neutrophils

SHOCK-, TRAUMA-, AND BURN-INDUCED neutrophil activation has been implicated in the pathogenesis of the adult respiratory distress syndrome and as a contributory factor in the development of the multiple organ dysfunction syndrome (7, 11). Thus studies directed at identifying the source and nature of injury- and shock-induced neutrophil-activating factors as well as the mechanisms by which these mediators activate neutrophils have been major areas of investigation. Although the exact factors responsible for neutrophil activation remain to be fully determined, recent studies (1, 7, 8, 12, 37) investigating the mechanisms of acute neutrophil activation after shock, thermal, or mechanical trauma have identified the ischemic gut as a major source of activating factors. Although most of the studies in this area have been performed with male animals, as recently reviewed (3–5), there is an increasing body of literature indicating that females are more resistant to shock-, trauma-, and sepsis-induced immune dysfunction and organ injury than are males. Consequently, the goal of this study was to investigate whether sex hormones act as modulators of acute shock and injury-induced neutrophil activation and, if so, to begin to investigate the mechanisms involved in that modulation. Because most polymorphonuclear neutrophil (PMN) inflammatory responses depend on prolonged increases in cytosolic calcium concentration ([Ca2+]i), and such prolonged elevations of neutrophil [Ca2+]i depend on calcium entry from the environment (11, 18, 32), we also studied the ability of estrogen and testosterone to modulate calcium signaling. Additionally, because we had previously shown that attenuation of PMN [Ca2+]i by inhibiting Ca2+ entry through store-operated calcium entry (SOCE) pathways decreased inflammatory PMN responses to trauma and hemorrhagic shock (26), we investigated the effects of estrogen and testosterone on this pathway.


   METHODS AND MATERIALS
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 ABSTRACT
 METHODS AND MATERIALS
RESULTS
 DISCUSSION
 GRANTS
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Animals. Adult female and male pathogen-free Sprague-Dawley rats (Taconic Farms, Germantown, NY) weighing 250–350 g were used in all experiments after a minimal acclimation period of 5 days. The castration and ovariectomy procedures were done a minimum of 2 wk before the rats were subjected to sham or actual injury. Animals had free access to food [Teklad 22/5 Rodent Diet (W) 8640, Harlan Teklad, Madison, WI] and water and were maintained under barrier-sustained conditions with 12:12-h light-dark cycles. Female estrus cycle stage was determined by examining the cytology of vaginal smears as described by Baker (6), with only proestrus females being used in this study. All animals were maintained in compliance with the recommendations of the New Jersey Medical School Animal Use and Care Committee, as described in Guide for the Care and Use of Laboratory Animals (National Institutes of Health Publication No. 85-23, Revised 1996). All experiments were approved by the Animal Use and Care Committee.

Experimental design. The primary goal of this study was to test the hypothesis that sex hormones modulate the acute neutrophil response after a major injury. This hypothesis was tested utilizing both a trauma-hemorrhagic shock (T/HS) and a thermal injury model. Two distinct models were used to more rigorously test this hypothesis. To isolate the effects of sex hormones from other sex-related factors, neutrophil activation in castrated, and ovariectomized rats were compared with normal male and proestrus female rats in these two model systems. The T/HS model consisted of a laparotomy (trauma) and hemorrhage (30 mmHg x 90 min). The burn model consisted of a 40% total body surface area third-degree scald burn. These models were chosen for several reasons. First, each model is associated with neutrophil activation, and there is evidence in male rats that T/HS-induced lung injury (27) and burn-induced acute lung injury (28) are mediated by factors liberated from the intestine. Secondly, we have recently documented that female rats are protected from T/HS-induced lung and gut injury (2, 9).

To test the role of sex hormones as modulators of the neutrophil response to injury, in the first group of experiments, the effect of T/HS on neutrophil activation was measured in male and female rats who had and had not undergone gonadectomy. In addition, the ability of plasma from these rats to activate naive neutrophils was tested. In these experiments, four groups of male and four groups of female rats were subjected to T/HS or trauma-sham shock (T/SS). These groups included male rats subjected to T/HS or T/SS, castrated male rats subjected to T/HS or T/SS, proestrus female rats subjected to T/HS or T/SS, and ovariectomized female rats subjected to T/HS or T/SS. To evaluate neutrophil activation, small aliquots of blood (0.5 ml) were obtained for measuring CD11b expression at three time points: before the induction of T/HS or T/SS and at 1 and 3 h after the end of the 90-min shock or sham shock period. Additionally, to evaluate the ability of postinjury plasma to activate naive neutrophils, at 3 h after resuscitation from T/HS or T/SS, plasma samples were obtained and tested for their ability to activate naive neutrophils from donor rats as reflected by upregulation of neutrophil adhesion molecule expression (CD11b) and augmentation of the respiratory burst.

In the second group of experiments, similar groups of male and female rats were subjected to a burn or sham burn injury. As described above, blood samples for in vivo neutrophil CD11b expression were obtained at two time points (preburn and 3 h postburn) and plasma was obtained at 3 h postburn for in vitro testing.

To test the hypothesis that differences in neutrophil activation between male and female rats were related to a sex-related differential resistance to T/HS-induced gut injury and the production of biologically active mesenteric lymph, mesenteric lymph was collected from male and proestrus female rats subjected to T/HS or T/SS, and the neutrophil-activating capacity of these lymph samples were tested on naive donor neutrophils. Thus, in this set of experiments, nongonadectomized male and proestrus female rats were subjected to T/SS or T/HS, and postnodal mesenteric lymph was collected as previously described (27). This group of experiments was considered necessary because proestrus female rats are relatively resistant to T/HS-induced gut injury (2, 9), and thus the neutrophil-activating potential of female T/HS lymph may be much less than male T/HS lymph and could help explain any observed differences in the in vivo neutrophil activation between the male and female rats.

Last, to begin to test the hypothesis that sex hormones can modulate normal neutrophil activation, neutrophils collected from naive donor rats were stimulated in the presence or absence of estrogen or testosterone. In this experiment, PMN were isolated from naive male or proestrus female rats. Male PMN were evaluated for their ability to mobilize [Ca2+]i in response to depletion of microsomal Ca2+ stores by the calcium-ATPase inhibitor thapsigargin (TG) in the presence of the synthetic estrogen diethylstilbestrol. The role of estrogens in modulating calcium influx through specific and nonspecific channel mechanisms was assessed by comparing the inhibition of Ca2+ influx by diethylstilbestrol to the inhibition of strontium (Sr2+) influx (20). Next, the effects of the major naturally occurring estrogen 17beta-estradiol (17beta-E2) on male and proestrus female PMN were assessed by using the area under the curve for calcium influx as our measure of SOCE over time. Last, the effect of testosterone was tested on PMN from naive castrated male rats to determine whether testosterone would augment [Ca2+]i mobilization. Castrated male rats were used because our pilot studies indicated that female PMN appeared to be inherently more resistant to activation than male PMN.

T/HS model. Rats were anesthetized with intraperitoneal pentobarbital sodium (50 mg/kg) and subjected to T/HS as previously described (27). With the use of aseptic techniques, the right jugular vein and left femoral artery were isolated and cannulated with polyethylene (PE-50) tubing or 50-gauge silicone catheter containing heparinized saline (10 U), respectively. Next, a 3-cm midline laparotomy (trauma) was performed, followed by closure with a running 3-0 silk suture. The femoral artery catheter was then attached in line to a blood pressure monitor (BP-2 digital blood pressure monitor, Columbus Instruments, Columbus, OH) for continuous blood pressure monitoring. Venous blood was withdrawn into a heparinized 10 ml syringe at a rate of 1 ml/min and mean arterial pressure was maintained at 30 mmHg for 90 min. At the end of the shock period, animals were resuscitated with their shed blood at a rate of 1 ml/min. The mean arterial pressure returned to normal within a few minutes after resuscitation. Rectal temperature was monitored throughout the experiment and maintained at ~37°C by using an electric heating pad under the surgical platform. Our previous studies (16) indicated that the long-term mortality rate of this model ranges from 14 to 25%, with most deaths occurring during or just after the shock period. The T/SS animals were instrumented, underwent a laparotomy, but were not subjected to hemorrhagic shock. After the end of the T/HS or T/SS period, the rats were maintained under anesthesia for the duration of the experiment by the administration of pentobarbital sodium (25 mg/kg) every 2 h as needed.

Burn model. The rats underwent a 40% total body surface area burn by using the technique originally described by Walker and Mason (36). The rats were anesthetized with pentobarbital sodium (50 mg/kg) given intraperitoneally. The hair was shaved from the back and abdomen, and a depilatory agent was applied. The internal jugular vein was then cannulated with a 50-gauge silicon catheter by using aseptic technique, and a preburn blood sample was obtained. The rats were then placed in a template containing an opening exposing 20% of the body surface area. The back burn was produced by immersing the back of the animal through the template into boiling water (100°C) for 10 s, followed by an abdominal scald burn for 5 s. All the rats were resuscitated with an additional 3 ml of normal saline intraperitoneally before the abdominal burn and then 2 ml normal saline intravenously to prevent shock. Previous studies using this model have shown that it is not associated with abdominal visceral injury and that the burns are third degree (28). Rectal temperature was maintained at ~37°C with a heat lamp. The control (sham burn) groups were anesthetized, had their jugular vein catheterized, and were then placed in the plastic template and exposed to room-temperature water. After the burn or sham burn injury, the rats were administered the analgesic agent buprenorphine (0.2 mg/kg) for pain control and then allowed to awaken in their cages. At 3 h after the burn or sham burn, the rats were reanesthetized with pentobarbital sodium, and samples were harvested as described in Experimental design.

In vivo whole blood PMN adhesion molecule assay. Whole blood samples obtained preinjury, 1 h postinjury, and 3 h postinjury were divided into three 100-µl aliquots as previously described (8). The samples were incubated with 10 µl of anti-rat CD11b fluorescein isothiocyanate-labeled monoclonal antibody (BD Pharmingen, San Diego, CA). The samples were vortexed and then placed on ice for 45 min in the dark. They were then lysed by using PharmM Lyse (BD Pharmingen) for 15 min on ice and then centrifuged and washed three times with calcium and magnesium-free 1% phosphate-buffered saline at 400x g for 15 min at 4°C. Fluorescein isothiocyanate-conjugated mouse immunoglobulin G1 antibodies (BD Pharmingen) were used as isotype controls for nonspecific antibody binding.

Cells were analyzed for adhesion molecule expression using a FACSscan flow cytometer (Becton-Dickinson, San Jose, CA) and Cellquest software. Daily instrument calibration was performed using Calibrate beads (BD Pharmingen) and Rainbow calibration particles (BD Pharmingen) according to the manufacturer's recommendations. Neutrophils were identified by forward and side scatter analysis. Each sample was run in the setup mode until a granulocyte acquisition gate was established. Only gated events were acquired and viewed in the FL1 histogram without marker settings. Five thousand events were collected in granulocyte gate in all studies. Data were expressed as mean fluorescence intensities (MFI) of the FL1 channel.

In vitro neutrophil respiratory burst activity. Neutrophils were isolated as previously described (1). Briefly, heparinized whole blood samples (100 µl) from naive control male or proestrus female rats were placed into 5-ml polystyrene round-bottom tubes containing an equal volume of DMEM. The red blood cells were lysed with 1% PharmM lysing solution as previously described. The tubes were spun at 1,135 rpm for 5 min at 25°C. The supernatants were discarded, and the cells were washed twice with HBSS.

The white blood cell pellets were resuspended in 400 µl of HBSS. Plasma samples (25% vol/vol) from the burn, sham burn, T/HS, or T/SS groups were then added, and the tubes were placed into the incubator for 5 min, after which the dihydrorhodamine (15 ng/ml) was added to the tubes. Five minutes after dihydrorhodamine was added, the PMN were stimulated with PMA. After 15 min at 37°C, the PMN respiratory burst was measured by flow cytometry as described above.

In vitro PMN CD 11b adhesion molecule assay. Heparinized whole blood samples (100 µl) from control male and proestrus female rats were placed into 5-ml polystyrene round-bottom tubes containing an equal volume of DMEM. The red blood cells were lysed with 1% PharmM lysing solution as previously described (1). The tubes were spun at 1,135 rpm for 5 min at 25°C. The supernatants were discarded, and the cells were washed twice with HBSS. The white blood cell pellets were resuspended in 400 µl of HBSS to which the plasma samples at 25% vol/vol were added. The cells were then processed and analyzed for CD11b expression as described in In vivo whole blood PMN adhesion molecule assay.

Neutrophil isolation and cell calcium studies. Our methods are described in detail elsewhere (19). Briefly, anesthetized animals are exsanguinated by cardiac puncture into heparinized syringes (25 U/ml). Blood is centrifuged on Polymorphoprep media (Robbins, Sunnyvale, CA) adapted for use with rat PMN (13). The PMN layer is removed, and the osmolarity is restored. Cells were washed and resuspended in HEPES buffer without BSA. PMN are counted and assessed for purity by flow cytometry. These methods yield PMN of 96–99% purity and >98% viability by trypan blue exclusion.

Cells are incubated with 5 µg/ml fura-2 AM (Molecular Probes, Eugene, OR) for 30 min in the dark at 37°C. PMN were then divided into aliquots (2 x 106 PMN) and placed on ice in the dark. Immediately before study, the cells are centrifuged 5 s at 4,500 rpm and supernatants are removed. Cells are resuspended in HEPES buffer for study. [Ca2+]i is determined at 37°C with constant stirring by measuring fura-2 AM fluorescence at 505 nm using 340/380 nm dual wavelength excitation in a Fluoromax-2 spectrofluorometer (Jobin-SPEX, Edison, NJ). Sr2+ is used as a marker for divalent cation entry through nonspecific cation channels. The Kd for Sr2+ association with fura is not precisely known, so [Sr2+]i is reported as "apparent [Ca2+]i." The presence of Sr2+ in the media has no effect on the Rmax/Rmin measurements performed in the presence of 1 mM Ca2+ at the end of each experiment (20). The order of study of PMN isolates is alternated to avoid bias related to duration of dye loading or time of cell study. In these experiments, the isolated PMN were exposed to estradiol or diethylstilbesterol for 1 min before being assayed.

Statistical analysis. Data comparing differences across time or between multiple groups were analyzed by two-way analysis of variance with the Tukey-Kramer multiple comparisons or the Holm-Sidak tests. Comparisons of differences between the sexes were done using the unpaired t-test. Data are expressed as means (SD). Statistical significance was considered to be a P value ≤0.05.


   RESULTS
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 ABSTRACT
 METHODS AND MATERIALS
 RESULTS
DISCUSSION
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There was no difference in the blood pressure response to T/HS between the male or female rats or the rats subjected to castration or ovariectomy. Overall, the mean arterial blood pressure preshock was 116 mmHg (SD 14); it returned to 123 mmHg (SD 14) immediately after resuscitation and was 92 mmHg(SD 5) at the end of the postresuscitative period (data not shown for individual T/HS groups). The mean arterial blood pressure for the T/SS groups was 121 mmHg (SD 12) before sham shock injury and 113 mmHg (SD 7) at the time the animals were killed. None of the sham-shocked rats died during the experimental procedure, and the overall survival rate of the rats subjected to T/HS was 88% (36 of 41 rats survived).

Neutrophil activation after T/HS, as reflected in upregulation of CD11b expression, was greater in male rats than in proestrus female rats at both 1 h (311 ± 45 vs. 235 ± 50 MFI; P < 0.01) and 3 h (521 ± 120 vs. 261 ± 38 MFI; P < 0.001) after the end of the 90-min shock period. This increase in post-T/HS PMN activation appeared to be related to the presence of male sex hormones, because castration abrogated the increase in T/HS-induced CD11b upregulation as compared with the T/SS group (Fig. 1A), whereas ovariectomy did not reverse the protective effect of the proestrus state (Fig. 1B). In fact, CD11b expression was similar between the T/HS and the T/SS female groups at each time point (Fig. 1B). Nonetheless, the level of CD11b expression was increased post-T/HS or post-T/SS in both sexes, indicating that the stresses associated with the trauma and instrumentation portions of the model were capable of systemic neutrophil activation (P < 0.01 preshock or sham shock vs. postshock or sham shock). Furthermore, at the 3-h time point, but not at the preinjury or 1 h postinjury period, the level of CD11b expression was higher in each of the male groups (T/SS, T/HS, T/HS + castration) than in the comparable female group (Fig. 1; P < 0.05), further indicating that the male rats have an increased CD11b response after stress.


Figure 1
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  		Fig. 1. A and B: after trauma-hemorrhagic shock (T/HS), neutrophil CD11b expression is increased in male but not in proestrus female rats, and this increase in male rats is abrogated by castration. Data are expressed as means (SD); n = 6 rats per group. *P < 0.01 vs. all other groups. Because there was no statistical difference in the in vivo polymorphonuclear neutrophil (PMN) CD11b levels between sham-castrated or castrated male rats subjected to trauma-sham shock (T/SS), these two groups were combined for ease of data display. For the same reasons, sham-ovariectomized and ovariectomized T/SS groups were combined.

 
The mortality rate for the burned animals was 7% (3 of 39 rats died), whereas none of the sham-burned rats died. As was observed in the T/HS arm of the study, the process of anesthesia and handling of the rats was associated with an increase in CD11b expression in all of the groups of both sexes, as compared with the preburn CD11b values (Fig. 2). However, in contrast to T/HS, neutrophil CD11b expression was increased above the sham level in both the male and proestrus female rats at 3 h postburn (Fig. 2). However, PMN CD11b expression was still greater in the male than in the proestrus female rats at 3 h postburn (394 ± 42 vs. 294 ± 43 MFI; P < 0.01). Castration partially abrogated the magnitude of the postburn increase in CD11b expression, but ovariectomy had no effect (Fig. 2).


Figure 2
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  		Fig. 2. After thermal injury, neutrophil CD11b expression is increased in male rats to a greater extent than in female rats, and this increase in CD11b expression is reduced by castration. Data are expressed as means (SD); n = 6 rats per group. *P < 0.01 vs. all other groups; #P < 0.05 vs. sham burn groups.

 
Because the increase in PMN activation after T/HS or burn injury in the male rats as compared with the proestrus female rats could be related to an increased susceptibility of male PMN to activation and/or a greater increase in the PMN-activating properties of male plasma, we next tested the ability of plasma from the male and female T/HS and burn groups to activate normal naive male and female PMN. Plasma from both the male and female T/HS rats increased CD 11b expression (Fig. 3A) and the respiratory burst (Fig. 3B) of naive control male PMN; however, the increase in CD11b expression and the respiratory burst was greater in the naive male PMN incubated with male than female T/HS plasma (P < 0.01). Consistent with these in vivo observations, castration was associated with a decrease in the PMN-activating ability of male plasma, whether measured as CD11b upregulation or priming for an augmented respiratory burst (Fig. 3, A and B). In contrast to the in vivo observations in the female rats subjected to T/HS, plasma from the proestrus female rats increased PMN CD11b expression as well as the respiratory burst, and this increase in PMN activation was significantly augmented after ovariectomy (Fig. 3, A and B).


Figure 3
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  		Fig. 3. A: plasma from male rats subjected to T/HS increases naive male rat neutrophil CD11b expression, and this increase is partially abrogated by castration. Plasma from female rats subjected to T/HS increases naive male rat neutrophil CD11b expression, which is further increased after ovariectomy. B: plasma from male rats subjected to T/HS increases respiratory burst of naive male rat neutrophils, and this increase is abrogated by castration. Plasma from female rats subjected to T/HS increases respiratory burst of naive male rat neutrophils, which is further increased after ovariectomy. Data are expressed as means (SD); n = 6–10 rats per group. *P < 0.001 vs. T/SS; #P < 0.05 vs. T/SS and T/HS; **P < 0.05 vs. other groups.

 
Similar trends were observed after thermal injury, where the plasma of the burned male and female rats increased naive male PMN CD11b expression (Fig. 4A) and primed for an augmented respiratory burst (Fig. 4B). Just as was observed with T/HS plasma, plasma from the male burned rats increased CD11b expression and the respiratory burst of naive male PMN to a greater extent than did proestrus plasma (P < 0.01). Likewise, castration abrogated and ovariectomy increased the ability of burn plasma to activate naive PMN (Fig. 4, A and B).


Figure 4
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  		Fig. 4. A: plasma from male rats subjected to thermal injury increases naive male rat neutrophil CD11b expression, and this increase is abrogated by castration. Plasma from female rats subjected to T/HS increases naive male rat neutrophil CD11b expression, which is further increased after ovariectomy. B: plasma from male rats subjected to thermal injury increases respiratory burst of naive male rat neutrophils, and this increase is abrogated by castration. Plasma from female rats subjected to T/HS increases respiratory burst of naive male rat neutrophils, which is further increased after ovariectomy. Data are expressed as means (SD); n = 6–10 rats per group. *P < 0.01 vs. all other groups; #P < 0.01 vs. sham group.

 
Next, because the in vivo CD11b was not increased in the female rats after T/HS, we tested whether neutrophils from proestrus females may be more resistant to activation after T/HS than male neutrophils. Consistent with this notion, male, but not proestrus female neutrophils, were activated by both male and female T/HS plasma (Table 1). A similar response was observed when burn plasma was tested, with the control male, but not the proestrus female, neutrophils manifesting an increased CD11b response (417 ± 14 vs. 258 ± 8 MFI; P < 0.001).


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  		Table 1. Comparison of effects of male versus female plasma on male and proestrus female neutrophil CD11b expression

 
Having documented that both the neutrophil-activating ability of T/HS plasma from proestrus rats is less than that of male rats and that proestrus neutrophils are more resistant to activation than male neutrophils, we next examined whether the lack of neutrophil-activating activity of the proestrus female T/HS plasma was related to a lower level of neutrophil-activating factors into the mesenteric lymph of the female than the male rats. This appeared to be the case, because T/HS mesenteric lymph from male rats was able to upregulate CD11b expression and augment the respiratory burst (Table 2) of naive male neutrophils more than T/HS lymph collected from proestrus female rats. In contrast to male naive neutrophils, neutrophils from naive proestrus female rats were not activated by female T/HS lymph, and, although they were activated by male T/HS lymph, the degree of activation was less than that observed in male neutrophils (Table 2).


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  		Table 2. Comparison of effects of male versus female mesenteric lymph on male and proestrus female neutrophil CD11b expression and respiratory burst

 
Because sex hormone levels change over the estrus cycle, we next examined the ability of neutrophils obtained from female rats during metestrus, diestrus, and proestrus to be activated by male T/HS lymph. Male T/HS lymph was used, because it has greater neutrophil-activating ability than female T/HS lymph and our previous work indicates that the plasma neutrophil-activating activity derives from factors in the mesenteric lymph (4). The results of this study show that the ability of male T/HS lymph to activate female neutrophils varies during the various stages of the estrus cycle, with the greatest resistance being seen during the proestrus phase of the cycles, where estradiol and other female sex hormones are at their greatest levels (Fig. 5). However, at no stage of the estrus cycle are female neutrophils able to be activated by male T/HS lymph to the same degree as male neutrophils (Fig. 5).


Figure 5
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  		Fig. 5. Ability of T/HS lymph from male rats to activate naive female neutrophils varied over the estrus cycle, but response of naive female neutrophils was never as great as that of naive male neutrophils. Data are expressed as means (SD); n = 4–6 rats per group. *P < 0.01 vs. proestrus neutrophils; #P < 0.01 vs. all other neutrophil groups.

 
To further evaluate the role of estrogen as a mediator of the neutrophil response, we evaluated the effects of estrogens on neutrophil calcium signaling in vitro. These studies showed that the synthetic estrogen diethylstilbestrol caused a dose-dependent suppression of TG-mediated calcium entry (Fig. 6A) in male rat PMN. Most of this suppression reflected decreased cation entry through the calcium-specific rather than the nonspecific (i.e., Sr2+ permeable) pathways (Fig. 6B). We then evaluated the effects of the naturally occurring estrogen 17beta-E2 over a pharmacological dose range. We found that it, too, caused dose-dependent suppression of TG-mediated calcium influx in male rat PMN (Fig. 7A). We finally compared TG-stimulated SOCE in male and proestrus female rat PMN and compared the effects of increasing levels of 17beta-E2 on their SOCE responses. As seen in Fig. 7B, basal SOCE responses to TG of untreated male rat PMN are significantly greater than those of PMN from untreated proestrus females (P = 0.029). When male and female PMN were tested in the presence of external 17beta-E2, we saw that both groups showed significant suppression of SOCE by estrogens (P < 0.001) but that the male PMN responses started higher and were significantly more suppressed. Eventually, in the presence of increasing levels of environmental estrogen, the SOCE responses of male and proestrus female PMN became indistinguishable (Fig. 7B). However, in contrast, when testosterone was tested on PMN from naive castrated male rats, it had no effect on TG- or ionomycin-mediated calcium entry. This was true when testosterone was tested at physiological as well as pharmaco logical levels (Fig. 8).


Figure 6
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  		Fig. 6. A: diethylsilbestrol (DES) causes dose-dependent suppression of thapsigargin (TG)-mediated calcium influx in rat neutrophils. Fura-loaded rat PMN were suspended in cuvettes in Ca2+-free buffer in the presence of DES at concentrations shown for 1 min. Experiment was started and cells were treated at time (t) = 30 s with 1 µM TG. Calcium (1 mM) was added at t = 400 s to visualize influx. B: effect of DES on TG-mediated strontium (Sr2+) (1 mM) and Ca2+ movement is seen. There is some suppression of nonspecific (i.e., Sr2+ permeable) entry mechanism but more marked suppression of entry through calcium specific mechanisms. Thus DES predominantly suppressed divalent cationic entry through Ca2+-specific channels. n = 4 Representative experiments.

 

Figure 7
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  		Fig. 7. A: 17beta-Estradiol (17beta-E2) caused dose-dependent suppression of TG-mediated Ca2+ influx in male rat PMN at physiological concentrations (representative traces shown). B: analysis of effects of 17beta-E2 on store-operated calcium entry (SOCE) induced by TG in male and female rat PMN (n = 3–6 per group). AUC, area under the cytosolic calcium concentration curve for 100 s after recalcification of the medium. *P < 0.001 for all comparisons among male PMN except 50 vs. 100 nM 17beta-E2, which were not significant; **P < 0.01 for all comparisons except 0 nM (control) vs. 25 nM and 50 vs. 100 nM 17beta-E2, which were not significant. Thus both male and female PMN show suppression of SOCE by 17beta-E2. In the absence of 17beta-E2, control male PMN responses were significantly greater than control proestrus female PMN responses (P = 0.029), but no significant differences are seen between male and female PMN SOCE in the presence of high concentrations of external 17beta-E2.

 

Figure 8
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  		Fig. 8. Effects of testosterone on store-operated calcium influx into PMN from castrated male rats. A: cells were kept in calcium-free buffer (+BSA) in cuvettes and incubated for 3 min with testosterone at doses shown or its vehicle (veh) methanol. Experiments were started, and TG (1 µM final concentration) was added at t = 30 s. Calcium was readded to media at 400 s to visualize influx. B: we studied ionomycin-induced calcium influx. PMN were again treated with testrosterone at doses shown or vehicle for 3 min. PMN were then stimulated with ionomycin (Iono; 100 nM final concentration) at t = 30 s. Calcium was readded to media at 200 s. Testosterone had no noticeable effect on calcium influx generated by either store depletion agent at any dose. n = 3–4 Representative traces for each condition in the two sets of experiments.

 

   DISCUSSION
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 ABSTRACT
 METHODS AND MATERIALS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Emerging data have described a sexual dimorphism in the response to injury or illness, with differences in immune function manifested as both altered susceptibility to organ injury and to infection. These differences have been attributed to the effects of sex hormones, with the female sex hormone estradiol conferring protection and the male sex hormone testosterone increasing susceptibility to injury (3–5). Although much of the data supporting sex hormones as modulators of responses to shock, trauma, and sepsis are based on animal studies, more than 20 clinical studies investigating the role of sex in trauma or septic patients have appeared since 1998 (3). Although not all of these clinical studies found a clinical difference between male and female patients, many of the prospective studies documented that females had a better outcome than males (14, 15, 30, 33). This advantage of the female sex appeared to be observed primarily in premenopausal females and in patients with more severe injuries (i.e., injury severity scores of ≥15).

Because preclinical animal studies and some clinical studies indicated that females respond differently to shock, trauma, or sepsis than do males, the current study was designed to investigate the role of sex hormones as modulators of acute neutrophil activation. We used two distinct models of neutrophil activation, thermal injury and trauma-hemorrhagic shock, because neutrophil activation and neutrophil-mediated organ injury have been documented in both conditions (11). Additionally, the use of two different trauma models would potentially increase the robustness of the findings. Our observations that in vivo neutrophil activation is greater in male than proestrus female rats after T/HS or burn injury are consistent with studies investigating other aspects of the immunoinflammatory response (4, 24). Furthermore, our observations that castration abrogated T/HS and burn-induced in vivo neutrophil activation while ovariectomy had no effect indicate that testosterone has a greater in vivo effect on neutrophils than the female sex hormones. This observation is somewhat different from the macrophage, lymphocyte, and cytokine responses reported in the literature after T/HS, where both estradiol and testosterone exert modulatory effects on the immunoinflammatory response (4, 24).

Studies of calcium signaling in these cells suggest that estrogen-mediated alterations in PMN calcium entry through SOCE channels play a role in the differential capacity of male and female PMN to participate in inflammation. This key proinflammatory signaling pathway shows relative attenuation in proestrus female rat PMN as well as in male rat PMN subjected to estrogen treatment. These effects were seen both with estrogenic drugs and with authentic estrogen agonists. In the presence of high external estradiol levels, SOCE was suppressed to a very similar extent in both male and female PMN. Thus the effects of estrogens on calcium entry may underlie some of the observed phenotypic differences in male and female PMN responses observed after T/HS or burn injury. Furthermore, the fact that the estrogenic effects occurred so quickly (i.e., 1 min) suggests that these effects were being mediated through estrogen receptor signaling rather than through the more traditional genomic pathway. On the other hand, one limitation of these results is that, because of experimental constraints associated with the SOCE channel studies, only very short PMN incubation times could be tested, and supraphysiological doses of agents had to be employed to observe an estrogenic effect. Because of these in vitro constraints, the degree to which estrogen has a direct effect on neutrophil function in vivo cannot be answered definitively. Although the in vitro calcium studies were not definitive, because estrogens did modulate the ability of neutrophils to respond and a difference in the response of naive male and female neutrophils to different doses of estrogen was observed, these results are still highly supportive of the notion that estrogens can modulate in vivo neutrophil activation.

The reasons for the failure of ovariectomy to modulate the in vivo neutrophil response in the current study, when it has been reported to influence other arms of the immunoinflammatory response and our in vitro observations indicate that estrogen decreased SOCE, are unclear. However, our other in vitro studies testing the neutrophil-activating effects of plasma from male and proestrus females subjected to castration or ovariectomy prior to trauma-hemorrhage or burn injury on naive male neutrophils is consistent with castration having an anti-inflammatory and ovariectomy having a proinflammatory effect. In these studies, castration abrogated the neutrophil-activating capacity of male plasma, as would be expected from the in vivo results. However, in contrast to the in vivo response, ovariectomy augmented the in vitro neutrophil-activating capacity of T/HS and burn plasma when tested on naive male neutrophils. The endocrine results of ovariectomy and castration are not, of course, entirely parallel, because testosterone can be metabolized further to form estrogens in the presence of aromatase (31). Clearly though, clarification of the differential responses of PMN to hormonal ablation maneuvers will require much further study. This future work will require studies evaluating specific receptor-mediated activation of PMN, because our studies were performed with PMA, which is a nonspecific PMN activator. Thus it is possible that sex-based PMN responses to specific receptor agonists, such as endotoxin, different cytokines, and chemokines, may differ from what was observed with PMA. Furthermore, our in vitro calcium studies failing to find a direct effect of testosterone on neutrophil activation, whereas castration had such a protective effect in vivo, highlights the potential complexities of the in vivo state and suggests that at least some of the neutrophil-modulatory effects of testosterone may be indirect.

The in vivo increase in PMN activation after T/HS or burn injury in the male rats as compared with the proestrus female rats appears to be related to both an increased susceptibility of male PMN to activation as well as to male plasma having greater neutrophil-activating activity than proestrus female plasma. This notion is supported by several of our observations. First, both male and female T/HS and burn plasma samples activated naive control male neutrophils, and the neutrophil-activating activity of the plasma samples collected from the males was greater than that of the proestrus female rats. Second, in contrast to naive male neutrophils, naive proestrus female neutrophils were not activated by either male or female T/HS plasma or burn plasma. Thus, consistent with the in vivo response, the plasma from the male rats subjected to trauma-hemorrhage or thermal injury increased naive male rat PMN activation to a greater extent than plasma from proestrus females, whereas naive female proestrus neutrophils were resistant to activation.

One possible explanation for why the neutrophil-activating activity of the plasma from the proestrus females was less than that of the males subjected to trauma-hemorrhage could be related to our previous observation that proestrus female rats are more resistant to trauma-hemorrhage-induced gut injury, because gut injury is associated with biologically active mesenteric lymph (9). This appears to be the case, because the neutrophil-activating activity of T/HS lymph from male rats was much greater than that of proestrus female rats when tested in vitro on naive male neutrophils. Furthermore, when male and proestrus T/HS lymph was tested on naive female rats, only the male lymph activated the proestrus female neutrophils. This observation that lymph collected from proestrus females has less biological activity than lymph from male rats subjected to T/HS is consistent with other information showing that acute changes in organ function, neutrophil activation, and endothelial cell function after an episode of T/HS are related to factors liberated from the ischemic intestine into the mesenteric lymph, which reach the systemic circulation through the thoracic duct that empties into the subclavian vein (1, 2, 8, 9, 12, 27, 28, 37).

Taken together, the results of the current study indicate that male neutrophil activation in vivo after T/HS or burn injury is greater than in proestrus female neutrophils for at least two reasons. First, neutrophils from male rats appear to be activated to a greater extent by factors contained in post-T/HS and postburn plasma as well as T/HS lymph than are neutrophils from proestrus female rats. Second, male rat plasma and intestinal lymph appear to have greater neutrophil-activating capacity than proestrus female plasma after T/HS or thermal injury. Additionally, although sex hormones appear to modulate the acute neutrophil response to T/HS or burn injury, other sex-independent factors also appear to be important in the pathogenesis of neutrophil activation after T/HS or burn injury. This notion is based on several observations. First, castration did not completely abrogate neutrophil activation in vivo or the in vitro neutrophil-activating ability of post-T/HS or postburn plasma. Second, resistance to in vivo neutrophil activation was not affected by ovariectomy in proestrus female rats after trauma-hemorrhage or burn injury.

In considering the potential mechanisms by which sex hormones modulate neutrophil activation, it is important to point out that ovariectomy affects more than estrogen levels. Although the testes are the primary source of testosterone in the male and thus castration reduces testosterone, the hormonal response to ovariectomy is more complex. Although the ovaries are the primary source of estrogens in females, ovariectomy also decreases progesterone, prolactin, and dehydroepiandrosterone (DHEA) production (17, 29, 34). In fact, the plasma levels of estrogen, progesterone, and prolactin all peak during the proestrus stage of the reproductive cycle. Thus, although there are data to suggest that estrogen plays an important role in the sex-dimorphic response to T/HS (4, 21, 22), prolactin and DHEA have also been demonstrated to have immunomodulatory effects, and both have been shown to preserve organ function after injury (10, 23, 25, 38). Thus it is also possible that these hormones act in combination to provide the beneficial effects on neutrophil activation seen in the proestrus female. Additionally, the cellular mechanisms by which the sex hormones modulate PMN function after shock and/or trauma remain to be determined. However, there is increasing data suggesting that many of the immunoinflammatory effects of sex hormones, especially estrogens, act via activation of cell surface receptors and not via traditional genomic pathways (5). Consequently, future studies employing estrogen receptor knockout mice as well as specific estrogen receptor agonists and antagonists will be of importance in beginning to clarify the mechanisms by which sex hormones are signaling PMN to modulate their response.

In summary, the results of the current study indicate that a sexual dimorphism in the in vivo response of neutrophils to trauma exists, with male neutrophils being activated to a greater extent that proestrus female neutrophils. This sex-related difference appears to be related to both cellular and humoral factors. Evidence for a cellular difference between male and female neutrophil activation is based on the observation that naive female neutrophils are more resistant to activation after exposure to burn plasma or T/HS plasma or lymph than naive male neutrophils and that this resistance to T/HS lymph-induced activation varies across the estrus cycle. Additionally, the neutrophil-activating capacity of burn and trauma-hemorrhage plasma and trauma-hemorrhage lymph was much greater in male than in proestrus female rats, indicating that the humoral environment after trauma is more proinflammatory in male than in proestrus female rats. Last, it appears that estrogen is capable of abrogating calcium signaling via its effect on SOCE channels, whereas the fact that plasma and lymph from male, but not female, rats can active naive female neutrophils suggests that the observed in vivo resistance of proestrus neutrophils to trauma-induced activation is based on both cellular as well as humorally based differences between the sexes. The exact clinical significance of these observations will require patient-based studies, because increased PMN activation can be beneficial as well as deleterious, depending on the exact clinical situation. For example, although increased PMN activation during noninfectious inflammatory or ischemia-reperfusion states, such as trauma, hemorrhage shock, or burn injury, may lead to PMN-mediated bystander tissue injury, failure of PMN activation during an infectious episode could be equally deleterious.


   GRANTS
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 ABSTRACT
 METHODS AND MATERIALS
 RESULTS
 DISCUSSION
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REFERENCES
 
This study was supported in part by National Institutes of Health Grants GM-59841 (to EAD) and GM-059179 (to CJH).


   FOOTNOTES
 

Address for reprint requests and other correspondence: E. A. Deitch, Dept. of Surgery, MSB G506, UMDNJ-New Jersey Medical School, 185 South Orange Ave., Newark, NJ 07103 (e-mail: edeitch{at}umdnj.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.


   REFERENCES
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 ABSTRACT
 METHODS AND MATERIALS
 RESULTS
 DISCUSSION
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 REFERENCES
 

  1. Adams JM, Hauser CJ, Adams CA Jr, Xu DZ, Livingston DH, and Deitch EA. Entry of gut lymph into the circulation primes rat neutrophil respiratory burst in hemorrhagic shock. Crit Care Med 29: 2194–2198, 2001.[CrossRef][ISI][Medline]
  2. Adams CA, Magnotti LJ, Xu DZ, Lu Q, and Deitch EA. Acute lung injury after hemorrhagic shock is dependent on gut injury and sex. Am Surg 10: 905–913, 2000.
  3. Ananthakrishnan P and Deitch EA. Gut origin sepsis and MODS: the role of sex hormones in modulating intestinal and distant organ injury. XX vs XY 1: 108–117, 2003.
  4. Angele MK, Schwacha MG, Ayala A, and Chaudry IH. Effect of gender and sex hormones on immune responses following shock: a review article. Shock 14: 81–90, 2000.[ISI][Medline]
  5. Angele MK, Wichmann M, Eisenmenger S, Schildberg FW, and Chaudry IH. Immunologic effects of sex hormones following hemorrhagic shock: potential therapeutic options. XX vs XY 1: 39–45, 2003.
  6. Baker DEJ. The Laboratory Rat: Biology and Diseases. New York: Academic, Vol. 1, 1979, p. 54–165.
  7. Botha AJ, Moore FA, Moore EE, Kim FJ, Banerjee A, and Peterson VM. Post-injury neutrophil priming and activation: an early vulnerable window. Surgery 118: 358–364, 1995.[CrossRef][ISI][Medline]
  8. Caruso JM, Feketeova E, Dayal SD, Hauser CJ, and Deitch EA. Factors in intestinal lymph after shock increase neutrophil adhesion molecule expression and pulmonary leukosequestration. J Trauma 55: 727–733, 2003.[ISI][Medline]
  9. Caruso JM, Deitch EA, Xu DZ, Lu Q, and Dayal SD. Gut injury and gut-induced lung injury after trauma hemorrhagic shock is gender and estrus cycle specific. J Trauma 55: 531–539, 2003.[ISI][Medline]
  10. Catania RA, Angele MK, Ayala A, Cioffi WG, Bland KI, and Chaudry IH. Dehydroepiandrosterone restores immune function following trauma-hemorrhage by a direct effect on T lymphocytes. Cytokine 11: 443–450, 1999.[CrossRef][ISI][Medline]
  11. Deitch EA. Multiple organ failure: pathophysiology and potential future therapy. Ann Surg 216: 117–134, 1992.[ISI][Medline]
  12. Deitch EA, Shi HP, Lu Q, Feketeova E, Skurnick J, and Xu DZ. Mesenteric lymph from burned rats induces endothelial cell injury and activates neutrophils. Crit Care Med 32:533–538, 2004.[CrossRef][ISI][Medline]
  13. Fekete Z, Hauser CJ, Adams JM, Adams CAJ, Forsythe R, Hasko G, Xu DX, Livingston DH, and Deitch EA. Injury enhanced calcium mobilization in circulating rat neutrophils models human PMN responses. Shock 16: 15–20, 2001.[ISI][Medline]
  14. Gannon CJ, Napolitano LM, Pasquale M, Tracy JK, and McCarter RJ. Male gender increases risk of postinjury pneumonia. Surg Forum 52: 468–469, 2001.
  15. George RL, McGwin G, Windham ST Jr,, Melton SM, Metzger J, Chaudry IH, and Rue LW. Age-related gender differential in outcome after blunt or penetrating trauma. Shock 19:28–32, 2003.[CrossRef][ISI][Medline]
  16. Grotz MRW, Ding J, Guo W, Huang Q, and Deitch EA. Comparison of plasma cytokine levels in rats subjected to superior mesenteric artery occlusion or hemorrhagic shock. Shock 3: 362–368, 1995.[ISI][Medline]
  17. Guyton AC. Human Physiology and Mechanisms of Disease. Philadelphia, PA: Saunders, 1992, p. 613–623.
  18. Hauser CJ, Fekete Z, Adams JM, Garced M, Livingston DH, and Deitch EA. PAF mediated Ca2+ influx in human neutrophils occurs via store operated mechanisms. J Leukoc Biol 69: 63–68, 2001.[Abstract/Free Full Text]
  19. Hauser CJ, Fekete Z, Livingston DH, and Deitch EA. Chemokine stimulation of human neutrophil [Ca2+]i signaling in biologic environments. Shock 10: 324–328, 1998.[ISI][Medline]
  20. Itagaki K, Kannan KB, Livingston DH, Deitch EA, Fekete Z, and Hauser CJ. Store-operated calcium entry in human neutrophils reflects multiple contributions from independently regulated pathways. J Immunol 168: 4063–4069, 2002.[Abstract/Free Full Text]
  21. Jarrar D, Wang P, Cioffi WG, Bland KI, and Chaudry IH. The female reproductive system is an important variable in the response to trauma-hemorrhage. Am J Physiol Heart Circ Physiol 279: H1015–H1021, 2000.[Abstract/Free Full Text]
  22. Jarrar D, Wang P, Knoferl MW, Kuebler JF, Cioffi WG, Bland KI, and Chaudry IH. Insight into the mechanism by which estradiol improves organ functions after trauma-hemorrhage. Surgery 128: 246–251, 2000.[CrossRef][ISI][Medline]
  23. Jarrar D, Wang P, Song GY, Knoferl MW, Cioffi WG, Bland KI, and Chaudry IH. Metoclopromide: a novel adjunct for improving cardiac and hepatocellular functions after trauma-hemorrhage. Am J Physiol Endocrinol Metab 278: E90–E95, 2000.[Abstract/Free Full Text]
  24. Knoferl MW, Angele MK, Diodato MD, Schwacha MG, Ayala A, Cioffi WG, Bland KI, and Chaudry IH. Female sex hormones regulate macrophage function after trauma-hemorrhage and prevent increased death rate from subsequent sepsis. Ann Surg 235: 105–112, 2002.[CrossRef][ISI][Medline]
  25. Kuebler JF, Jarrar D, Wang P, Bland KI, and Chaudry IH. Dehydroepiandrosterone restores hepatocellular function and prevents liver damage in estrogen-deficient females following trauma and hemorrhage. J Surg Res 97: 196–201, 2001.[CrossRef][ISI][Medline]
  26. Lee CD, Xu DZ, Feketeova E, Kannan KB, Yun JK, Deitch EA, Fekete Z, Livingston DH, and Hauser CJ. Attenuation of shock-induced acute lung injury by sphingosine kinase inhibition. J Trauma 57: 955–960, 2004.[ISI][Medline]
  27. Magnotti LJ, Upperman JS, Xu DZ, Lu Q, and Deitch EA. Gut-derived mesenteric lymph but not portal blood increases endothelial cell permeability and promotes lung injury after hemorrhagic shock. Ann Surg 228: 518–527, 1998.[CrossRef][ISI][Medline]
  28. Magnotti LJ, Xu DZ, Lu Q, and Deitch EA. Gut-derived mesenteric lymph: a link between burn and lung injury. Arch Surg 134: 1333–1341, 1999.[Abstract/Free Full Text]
  29. Meijs-Roelofs HMA, DeGreef WJ, and Uilenbroek J. Plasma progesterone and its relationship to serum gonadotrophins in immature female rats. J Endocrinol 64: 329–336, 1975.[Abstract]
  30. Offner PJ, Moore EE, and Biffl WL. Male gender is a risk factor for major infections after surgery. Arch Surg 134: 935–940, 1999.[Abstract/Free Full Text]
  31. Schneider PC, Nickel EA, Samy TSA, Schwacha MG, Cioffi WG, Bland KI, and Chaudry IH. The aromatase inhibitor 4-hydroxyandrostenedione restores immune responses following trauma-hemorrhage in males and decreases mortality from subsequent sepsis. Shock 14: 647–353, 2000.
  32. Schorr W, Swandulla D, and Zeilhofer HU. Mechanisms of IL-8-induced Ca2+ signaling in human neutrophil granulocytes. Eur J Immunol 29: 897–904, 1999.[CrossRef][ISI][Medline]
  33. Schroder J, Kahlke V, Staubach K, Zabel P, and Stuber F. Gender differences in human sepsis. Arch Surg 133: 1200–1205, 1998.[Abstract/Free Full Text]
  34. Shaar CJ, Euker JS, Riegle GD, and Meites J. Effects of castration and gonadal steroids on serum leutinizing hormone and prolactin in old and young rats. J Endocrinol 66: 45–51, 1975.[Abstract]
  35. Tarlowe MH, Kannan KB, Itagaki K, Adams JM, Livingston DH, and Hauser CJ. Inflammatory chemoreceptor cross-talk suppresses leukotriene b(4) receptor 1-mediated neutrophil calcium mobilization and chemotaxis after trauma. J Immunol 171: 2066–2073, 2003.[Abstract/Free Full Text]
  36. Walker HI and Mason AD. A standard animal burn. J Trauma 8: 1049–1051, 1968.[ISI][Medline]
  37. Zallen GE, Moore EE, Johnson JL, Tamura DY, Ciesla DJ, and Silliman CC. Posthemorrhagic shock mesenteric lymph primes circulating neutrophils and provokes lung injury. J Surg Res 83: 83–89, 1999.[CrossRef][ISI][Medline]
  38. Zellweger R, Wichmann M, Ayala A, DeMaso C, and Chaudry IH. Prolactin: a novel and safe immunomodulating hormone for the treatment of immunodepression following severe hemorrhage. J Surg Res 63: 53–58, 1996.[CrossRef][ISI][Medline]




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