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We examined the role of interleukin (IL)-18 and cytokine-induced neutrophil chemokines (CINC)-I and CINC-3 in the neutrophil release of superoxide anion ([O.sub.2.sup.-]) and elastase following alcohol/ethanol (EtOH) and bum injury. Male rats (~250 g) were gavaged with EtOH to achieve a blood EtOH level of ~100 mg/dl before ~12.5% total body surface area burn or sham injury. Immediately after injury, rats were administered with anti-rat IL-18 antibody (80 [micro]g/kg) or isotype control. After 20 min, anti-IL-18 antibody-treated rats were given either recombinant (r) rat CINC-1 or CINC-3. On day 1 after injury, the combined insult of EtOH and bum injury caused a significant increase in neutrophil elastase and [O.sub.2.sup.-] production as well as an increase in neutrophil accumulation, myeloperoxidase activity, and edema in the intestine. Treatment of rats with anti-IL-18 antibody normalized the above parameters. However, administration of rCINC- 1 in anti-IL- 18 antibody-treated rats increased the above parameters to levels similar to those observed following EtOH and bum injury. In contrast, administration of rCINC-3 did not influence the above parameters except neutrophil elastase. These findings indicate that IL-18 and CINC-1 may independently modulate neutrophil tissue-damaging actions following EtOH and burn injury. However, the finding that the treatment of rats with anti-IL-18 antibodies inhibits CINC-I and CINC-3 supports the notion that IL-18 plays a critical role in increased neutrophil tissue-damaging action following a combined insult of EtOH intoxication and burn injury. thermal injury; ethanol; reactive oxygen species; proteases; intestine permeability; cytokines

Although trauma-hemorrhage (T-H) induces suppressed splenic dendritic cell (DC) maturation and antigen presentation capacity, it remains unclear whether IL-15 modulates splenic DC functions. The aim of this study therefore was to investigate the effect of IL-15 on splenic DC functions after T-H. Male C3H/HeN mice (6-8 wk old) were randomly assigned to T-H or sham operation. T-H was induced by midline laparotomy and ~90 min of hemorrhagic shock (blood pressure 35 mmHg), followed by fluid resuscitation (4 X the shed blood volume in the form of Ringer lactate). Two hours later, mice were killed, splenic DCs were isolated, and the effects of exogenous IL-15 on their costimulatory factors, major histocompatibility class II expression, ability to produce cytokines, and antigen presentation were measured. The results indicate that IL-15 production capacity of splenic DCs was reduced following T-H. Ex vivo exposure to IL-15 attenuated the suppressed production of TNF-[alpha], IL-6, and IFN-[gamma] from splenic DCs following T-H. In addition, expression of surface antigen studies demonstrate that exogenous IL-15 attenuated T-H-induced downregulation of the activation of DC. The suppressed splenic DC antigen presentation function following T-H was also attenuated by IL-15 treatment. Moreover, IL-15 enhanced IL-12-induced IFN-[gamma] production and antigen presentation by splenic DCs. These data suggest that ex vivo treatment with IL-15 following T-H provides beneficial effects on splenic DCs. The depression in IL-15 production by splenic DCs could contribute to the host's enhanced susceptibility to infections following T-H. antigen presentation; tumor necrosis factor-[alpha]; major histocompatibility class II

Although angiotensin II (Ang II) plays a key role in development of organ ischemia-reperfusion injury, it remains unclear whether it is involved in development of intestinal injury following trauma-hemorrhage (T-H). Studies have shown that 17[beta]-estradiol (E2) administration following T-H improves small intestinal blood flow; however, it is unclear whether Ang II plays a role in this E2-mediated salutary effect. Male Sprague-Dawley rats underwent laparotomy and hemorrhagic shock (removal of 60% total blood volume, fluid resuscitation after 90 min). At onset of resuscitation, rats were treated with vehicle, E2, or E2 and estrogen receptor antagonist ICI 182,780 (ICI). A separate group of rats was treated with Ang II subtype I receptor (AT1R) antagonist losartan. At 24 h after T-H, plasma Ang II, IL-6, TNF-[alpha], intercellular adhesion molecule (ICAM)-I, cytokine-induced neutrophil chemoattractant (CINC)-1 and CINC-3 levels, myeloperoxidase (MPO) activity, and AT1R expression were determined. T-H significantly increased plasma and intestinal Ang II, IL-6, TNF-[alpha] levels, intestinal ICAM-1, CINC-1, CINC-3 levels, MPO activity, and AT1R protein compared with shams. E2 treatment following T-H attenuated increased intestinal MPO activity, Ang II level, and AT1R protein expression. ICI administration abolished the salutary effects of E2. In contrast, losartan administration attenuated increased MPO activity without affecting Ang II and AT1R levels. Thus Ang II plays a role in producing small intestine inflammation following T-H, and the salutary effects of E2 on intestinal inflammation are mediated in part by Ang II and AT1R downregulation. shock; estrogen receptor antagonist; myeloperoxidase; chemokines; cytokines

p38 MAPK has been reported to regulate the inflammatory response in various cell types via extracellular stimuli, p38 MAPK activation also results in the induction of heine oxygenase (HO)-1, which exerts potent anti-inflammatory effects. Although studies have shown that 17[beta]-estradiol ([E.sub.2]) prevented organ dysfunction following trauma-hemorrhage, it remains unknown whether p38 MAPK/HO-1 plays any role in [E.sub.2]-mediated attenuation of intestinal injury under those conditions. To study this, male rats underwent trauma-hemorrhage (mean blood pressure ~40 mmHg for 90 min) followed by fluid resuscitation. At the onset of resuscitation, rats were treated with vehicle, [E.sub.2] (1 mg/kg body wt), the p38 MAPK inhibitor SB-203580 (2 mg/kg body wt) or [E.sub.2] plus SB-203580. Two hours thereafter, intestinal myeloperoxidase (MPO) activity and lactate, TNF-[alpha], IL-6, ICAM-1, cytokine-induced neutrophil chemo-attractant (CINC)-1, and macrophage inflammatory protein (MIP)-2 levels were measured. Intestinal p38 MAPK and HO-1 protein levels were also determined. Trauma-hemorrhage led to an increase in intestinal MPO activity and lactate, TNF-[alpha], IL-6, ICAM-1, CINC-1, and MIP-2 levels. This was accompanied with a decrease in intestinal p38 MAPK activity and increase in HO-1 expression. Administration of [E.sub.2] normalized all the above parameters except HO-1, which was further increased following trauma-hemorrhage. Administration of SB-203580 with [E.sub.2] abolished the [E.sub.2]-mediated restoration of the above parameters as well as the increase in intestinal HO-1 expression following trauma-hemorrhage. These results suggest that the p38 MAPK/HO-1 pathway plays a critical role in mediating the salutary effects of [E.sub.2] on shock-induced intestinal injury. p38 MAPK inhibitor; CINC-1; MIP-2; MPO

Studies have shown that p38 MAPK and nitric oxide (NO), generated by endothelial NO synthase (eNOS), play key roles under physiological and pathophysiological conditions. Although administration of 17[beta]-estradiol ([E.sub.2]) protects cardiovascular injury from trauma-hemorrhage, the mechanism by which E2 produces those effects remains unknown. Our objective was to determine whether the [E.sub.2]-mediated activation of myocardial p38 MAPK and subsequent eNOS expression/phosphorylation would protect the heart following trauma-hemorrhage. To study this, male Sprague-Dawley rats underwent soft-tissue trauma (midline laparatomy) and hemorrhagic shock (mean blood pressure 35-40 mmHg for 90 min), followed by fluid resuscitation. Animals were pretreated with specific p38 MAPK inhibitor SB-203580 (SB; 2 mg/kg), and nonselective NO synthase inhibitor [N.sup.G]-nitro-L-arginine methyl ester (L-NAME; 30 mg/kg) 30 min before vehicle (cyclodextrin) or E2 (100 [micro]g/kg) treatment, followed by resuscitation, and were killed 2 h thereafter. Cardiovascular performance and other parameters were measured. [E.sub.2] administration following trauma-hemorrhage increased cardiac p38 MAPK activity, eNOS expression and phosphorylation at [Ser.sup.1177], and nitrate/nitrite levels in plasma and heart tissues; these were associated with normalized cardiac performance, which was reversed by SB administration. In addition, [E.sub.2] also prevented trauma-hemorrhage-induced increase in cytokines (IL-6 and TNF-[alpha]), chemokines (macrophage inflammatory protein-2 and cytokine-induced neutrophil chemoattractant-1), and ICAM-I, which was reversed by L-NAME administration. Administration of [E.sub.2] following trauma-hemorrhage attenuated cardiac tissue injury markers, myeloperoxidase activity, and nitrotyrosine level, which were reversed by treatment with SB and u-NAME. The salutary effects of E2 on cardiac functions and tissue protection following trauma-hemorrhage are mediated, in part, through activation of p38 MAPK and subsequent eNOS expression and phosphorylation. nitric oxide synthase; cytokine; chemokine; myeloperoxidase

Although splenic dendritic cell (DC) functions are markedly altered following trauma-hemorrhage, the mechanism(s) responsible for the altered DC functions remains unknown. We hypothesized that trauma-hemorrhage inhibits DC function via suppressing toll-like receptor 4 (TLR4) expression and mitogen-activated protein kinases (MAPKs). To examine this, male C3H/HeN (6-8 wk) mice were randomly assigned to sham operation or trauma-hemorrhage. Trauma-hemorrhage was induced by midline laparotomy and ~90 min of hypotension [blood pressure (BP) 35 mmHg], followed by fluid resuscitation (4x the shed blood volume in the form of Ringer lactate). Two hours later, mice were euthanized, splenic DCs were isolated, and the changes in their MAPK activation, TLR4-MD-2 expression, and ability to produce cytokines were measured. The results indicate that trauma-hemorrhage downregulated the lipopolysaccharide (LPS)-induced MAPK activation in splenic DCs. In addition to the decrease in MAPK activation, surface expression of TLR4-MD-2 was suppressed following trauma-hemorrhage. Furthermore, LPS-induced cytokine production from splenic DCs was also suppressed following trauma-hemorrhage. These findings thus suggest that the decrease in TLR4-MD-2 and MAPK activation may contribute to the LPS hyporesponsiveness of splenic DCs following trauma-hemorrhage. Hyporesponsiveness of splenic DCs was also found alter stimulation with the TLR2 agonist zymosan. Our results may thus explain the profound immunosuppression that is known to occur under those conditions. toll-like receptor 4 expression; splenic dendritic cell; toll-like receptors; lipopolysaccharide

Although 1713-estradiol administration following trauma-hemorrhage prevents the suppression in splenic macrophage cytokine production, it remains unknown whether the salutary effects are mediated via estrogen receptor (ER)-[alpha] or ER-[beta] and which signaling pathways are involved in such 17[beta]-estradiol effects. Utilizing ER-[alpha]- or ER-[beta]-specific agonists, this study examined the role of ER-[alpha] and ER-[beta] in 17[beta]-estradiolmediated restoration of macrophage cytokine production following trauma-hemorrhage. In addition, since MAPK and NF-[kappa]B are known to regulate macrophage cytokine production, we also examined the activation of those signaling molecules. Male rats underwent traumahemorrhage (mean arterial pressure of 40 mmHg for 90 min) and fluid resuscitation. The ER-[alpha] agonist propyl pyrazole triol (PPT; 5 [micro]g/kg), the ER-[beta] agonist diarylpropionitrile (DPN; 5 [micro]g/kg), 17[beta]-estradiol (50 [micro]g/kg), or vehicle (10% DMSO) was injected subcutaneously during resuscitation. Twenty-four hours thereafter, splenic macrophages were isolated, and their IL-6 and TNF-[alpha] production and activation of MAPK and NF-[kappa]B were measured. Macrophage IL-6 and TNF-[alpha] production and MAPK activation were decreased, whereas NF-[kappa]B activity was increased, following trauma-hemorrhage. PPT or 17[beta]-estradiol administration after trauma-hemorrhage normalized those parameters. DPN administration, on the other hand, did not normalize the above parameters. Since PPT but not DPN administration following trauma-hemorrhage was as effective as 17[beta]estradiol in preventing the suppression in macrophage cytokine production, it appears that ER-[alpha] plays the predominant role in mediating the salutary effects of 17[beta]-estradiol on macrophage cytokine production following trauma-hemorrhage and that such effects are likely mediated via normalization of MAPK but not NF-[kappa]B signaling pathways. shock; mitogen-activated protein kinase; nuclear factor-[kappa]B; propyl pyrazole triol; diarylpropionitrile

Although 17[beta]-estradiol (E2) administration following trauma-hemorrhage prevents the suppression in splenocyte cytokine production, it remains unknown whether the salutary effects of 17[beta]-estradiol are mediated via estrogen receptor (ER)-[alpha] or ER-[beta]. Moreover, it is unknown which signaling pathways are involved in 17[beta]-estradiol's salutary effects. Utilizing an ER-[alpha]- or ER-[beta]-specific agonist, we examined the role of ER-[alpha]- and ER-[beta] in E2-mediated restoration of T-cell cytokine production following trauma-hemorrhage. Moreover, since MAPK, NF-[kappa]B, and activator protein (AP)-1 are known to regulate T-cell cytokine production, we also examined the activation of MAPK, NF-[kappa]B, and AP-1. Male rats underwent trauma-hemorrhage (mean arterial pressure 40 mmHg for 90 min) and fluid resuscitation. ER-[alpha] agonist propyl pyrazole triol (PPT; 5 [micro]g/kg), ER-[beta] agonist diarylpropionitrile (DPN; 5 [micro]g/kg), 17[beta]-estradiol (50 [micro]g/kg), or vehicle (10% DMSO) was injected subcutaneously during resuscitation. Twenty-four hours thereafter, splenic T cells were isolated, and their IL-2 and IFN-[gamma], production and MAPK, NF-[kappa]B, and AP-1 activation were measured. T-cell IL-2 and IFN-[gamma] production was decreased following trauma-hemorrhage, and this was accompanied with a decrease in T-cell MAPK, NF-[kappa]B, and AP-1 activation. PPT or 17[beta]-estradiol administration following trauma-hemorrhage normalized those parameters, while DPN administration had no effect. Since PPT, but not DPN, administration following trauma-hemorrhage was as effective as 17[beta]-estradiol in preventing the T-cell suppression, it appears that ER-[alpha] plays a predominant role in mediating the salutary effects of 17[beta]-estradiol on T cells following trauma-hemorrhage, and that such effects are likely mediated via normalization of MAPK, NF-[kappa]B, and AP-1 signaling pathways. shock; MAPK; NF-[kappa]B; activator protein-l; propyl pyrazole triol; diarylpropionitrile doi:10.1152/ajpcell.00488.2006

p38 mitogen-activated protein kinase (MAPK) activates a number of heat shock proteins (HSPs), including HSP27 and [[alpha].sub.B]-crystallin, in response to stress. Activation of HSP27 or [[alpha].sub.B]-crystallin is known to protect organs/cells by increasing the stability of actin microfilaments. Although our previous studies showed that 17[beta]-estradiol (E2) improves cardiovascular function after trauma-hemorrhage, whether the salutary effects of E2 under those conditions are mediated via p38 MAPK remains unknown. Male rats (275-325 g body wt) were subjected to soft tissue trauma and hemorrhage (35-40 mmHg mean blood pressure for ~90 min) followed by fluid resuscitation. At the onset of resuscitation, rats were injected intravenously with vehicle, [E.sub.2] (1 mg/kg body wt), [E.sub.2] + the p38 MAPK inhibitor SB-203580 (2 mg/kg body wt), or SB-203580 alone, and various parameters were measured 2 h thereafter. Cardiac functions that were depressed after trauma-hemorrhage were returned to normal levels by [E.sub.2] administration, and phosphorylation of cardiac p38 MAPK, HSP27, and [[alpha].sub.B]-crystallin was increased. The E2-mediated improvement of cardiac function and increase in p38 MAPK, HSP27, and [[alpha].sub.B]-crystallin phosphorylation were abolished with coadministration of SB-203580. These results suggest that the salutary effect of [E.sub.2] on cardiac function after trauma-hemorrhage is in part mediated via upregulation of p38 MAPK and subsequent phosphorylation of HSP27 and [[alpha].sub.B]-crystallin. heat shock proteins; [[alpha].sub.B]-crystallin; p38 mitogen-activated protein kinase inhibitor doi: 10.1152/ajpheart.01303.2006.

Although studies have shown that 17[beta]-estradiol ([E.sub.2]) prevents neutrophil infiltration and organ damage following trauma-hemorrhage, the mechanism by which [E.sub.2] inhibits neutrophil transmigration remains unknown. Macrophage migration inhibitory factor (MIF) is thought to play a central role in exacerbation of inflammation and is associated with lung injury. MIF regulates the inflammatory response through modulation of Toll-like receptor 4 (TLR4). Activation of TLR4 results in the release of proinflammatory cytokines and chemokines, which induce neutrophil infiltration and subsequent tissue damage. We hypothesized that E2 mediates its salutary effects in the lung following trauma-hemorrhage via negative regulation of MIF and modulation of TLR4 and cytokine-induced chemotaxis. C3H/HeOuJ mice were subjected to trauma-hemorrhage (mean blood pressure 35 [+ or -] 5 mmHg for ~90 min, then resuscitation) or sham operation. Mice received vehicle, [E.sub.2], or [E.sub.2] in combination with recombinant mouse MIF protein (rMIF). Trauma-hemorrhage increased lung MIF and TLR4 protein levels as well as lung and systemic levels of cytokines/chemokines. Treatment of animals with [E.sub.2] following trauma-hemorrhage prevented these changes. However, administration of rMIF protein with [E.sub.2] abolished the [E.sub.2]-mediated decrease in lung TLR4 levels, lung and plasma levels of IL-6, TNF-[alpha], monocyte chemoattractant protein-1, and keratinocyte-derived chemokine (KC). Administration of rMIF protein also prevented [E.sub.2]-mediated reduction in neutrophil influx and tissue damage in the lungs following trauma-hemorrhage. These results suggest that the protective effects of [E.sub.2] on lung injury following trauma-hemorrhage are mediated via downregulation of lung MIF and TLR4-induced cytokine/chemokine production. hemorrhagic shock; 17[beta]-estradiol; Toll-like receptor 4; myeloperoxidase; cytokines; chemokines doi:10.1152/ajplung.00479.2006

In this study, we examined whether IL-18 plays a role in lung inflammation following alcohol (EtOH) and burn injury. Male rats (~250 g) were gavaged with EtOH to achieve a blood EtOH level of ~100 mg/dl before burn or sham injury (~12.5% total body surface area). Immediately after injury, rats were treated with vehicle, caspase-1 inhibitor AC-YVAD-CHO to block IL-18 production or with IL-18 neutralizing anti-IL-18 antibodies. In another group, rats were treated with anti-neutrophil antiserum ~16 h before injury to deplete neutrophils. On day 1 after injury, lung tissue IL-18, neutrophil chemokines (CINC-1/CINC-3), ICAM-1, neutrophil infiltration, MPO activity, and water content (i.e., edema) were significantly increased in rats receiving a combined insult of EtOH and burn injury compared with rats receiving either EtOH intoxication or burn injury alone. Treatment of rats with caspase-1 inhibitor prevented the increase in lung tissue IL-18, CINC-1, CINC-3, ICAM-1, MPO activity, and edema following EtOH and burn injury. The increase in lung IL-18, MPO, and edema was also prevented in rats treated with anti-IL-18 antibodies. Furthermore, administration of anti-neutrophil antiserum also attenuated the increase in lung MPO activity and edema, but did not prevent the increase in IL-18 levels following EtOH and burn injury. These findings suggest that acute EtOH intoxication before burn injury upregulates IL-18, which in turn contributes to increased neutrophil infiltration. Furthermore, the presence of neutrophils appears to be critical for IL-18-meditaed increased lung tissue edema following a combined insult of EtOH and burn injury. inflammatory mediators; adhesion molecule; chemokines doi:10.1152/ajplung.00408.2006

Leukocyte infiltration, mediated by chemokines, is a key step in the development of organ dysfunction. Lung and liver neutrophil infiltration following traumahemorrhage is associated with upregulation of monocyte chemoattractant protein-1 (MCP-1). Because MCP-1 is not a major attractant for neutrophils, we hypothesized that MCP-I influences neutrophil infiltration via regulation of keratinocyte-derived chemokines (KC). To study this, male C3H/HeN mice were pretreated with MCP-1 antiserum or control serum and subjected to trauma-hemorrhage or sham operation. Animals were killed 4 h after resuscitation. One group of trauma-hemorrhage mice receiving MCP-1 antiserum was also treated with mufine KC during resuscitation. Plasma levels and tissue content of MCP-1 and KC were determined by cytometric bead arrays. Immunohistochemistry was performed to determine neutrophil infiltration; organ damage was assessed by edema formation. Treatment with MCP-1 antiserum significantly decreased systemic, lung, and liver levels of MCP-1 and KC following trauma-hemorrhage. This decrease in MCP-1 levels was associated with decreased neutrophil infiltration and edema formation in lung and liver following traumahemorrhage. Restitution of KC in mice treated with MCP-1 antiserum restored tissue neutrophil infiltration and edema. These results lead us to conclude that increased levels of MCP-1 cause neutrophil accumulation and distant organ damage by regulating KC production during the postinjury inflammatory response. neutrophils; inflammation; cell trafficking

Neutrophil infiltration is a key step in the development of organ dysfunction following trauma-hemorrhage (T-H). Although we have previously shown that 17[beta]-estradiol (E2) prevents neutrophil infiltration and organ damage following T-H, the mechanism by which E2 inhibits neutrophil transmigration remains unknown. We hypothesized that E2 prevents neutrophil infiltration via modulation of keratinocyte-derived chemokine (KC), a major attractant for neutrophils. To examine this, male C3H/HeN mice were subjected to T-H or sham operation and thereafter resuscitated with Ringer lactate and E2 (1 mg/kg body wt) or vehicle. Animals were killed 2 h after resuscitation, and Kupffer cells were isolated. Plasma levels and Kupffer cell production capacities of KC, TNF-[alpha], and IL-6 were determined by BD Cytometric Bead Arrays; lung mRNA expression of KC was measured with real-time PCR; myeloperoxidase activity assays were performed to determine neutrophil infiltration, and organ damage was assessed by edema formation. Treatment with E2 decreased systemic levels and restored Kupffer cell production of KC, TNF-[alpha], and IL-6, as well as KC gene expression and protein in the lung. This was accompanied with a decrease in neutrophil infiltration and edema formation in the lung. These results suggest that E2 prevents lung neutrophil infiltration and organ damage in part by decreasing KC during posttraumatic immune response. cytokines; estrogen; neutrophil infiltration; organ damage

Studies have shown salutary effects of 17[beta]-estradiol following traumahemorrhage on different cell types. 17[beta]-Estradiol also induces improved circulation via relaxation of the aorta and has an anti-apoptotic effect on endothelial cells. Because mitochondria play a pivotal role in apoptosis, we hypothesized that 17[beta]-estradiol will maintain mitochondrial function and will have protective effects against [H.sub.2][O.sub.2]-induced apoptosis in endothelial cells. Endothelial cells were isolated from rats' aorta and cultured in the presence or absence of [H.sub.2][O.sub.2], a potent inducer of apoptosis. In additional studies, endothelial cells were pretreated with 17[beta]-estradiol. Flow cytometry analysis revealed [H.sub.2][O.sub.2]-induced apoptosis in 80.9% of endothelial cells; however, prior treatment of endothelial cells with 17[beta]-estradiol resulted in an ~40% reduction in apoptosis. This protective effect of 17[beta]-estradiol was abrogated when endothelial cells were cultured in the presence ICI-182780, indicating the involvement of estrogen receptor (ER). Fluorescence microscopy revealed a 17[beta]-estradiol-mediated attenuation of [H.sub.2][O.sub.2]-induced mitochondrial condensation. Western blot analysis demonstrated that [H.sub.2][O.sub.2]-induced cytochrome c release from mitochondrion to cytosol and the activation of caspase-9 and -3 were decreased by 17[beta]-estradiol. These findings suggest that 17[beta]-estradiol attenuated [H.sub.2][O.sub.2]-induced apoptosis via ER-dependent activation of caspase-9 and -3 in rat endothelial cells through mitochondria. cytochrome c; estrogen receptors; mitochondria

Although studies have indicated that hemorrhagic shock and resuscitation produces hepatic damage by mechanisms involving adhesion molecules in endothelial cells and hepatocytes, it is not known if there is any difference in the extent of hepatic damage following bone fracture, soft tissue trauma, and hemorrhage (Fx-TH) between young and middle-aged animals. To study this, young (6-8 wk) and middle-aged (~12 mo) C3H/HeN male mice were subjected to a right lower leg fracture, soft tissue trauma, (i.e., midline laparotomy), and hemorrhage (blood withdrawal to decrease the blood pressure to 35 [+ or -] 5 mmHg for 90 min) followed by resuscitation with four times the shed blood volume in the form of lactated Ringer solution. Mice were euthanized 24 h later, and liver tissues were harvested. Total bilirubin levels in the hepatocyte extract increased markedly following Fx-TH in both groups of mice; however, the increase in middle-aged mice was significantly higher compared with young mice. TNF-[alpha] and IL-6 levels in the hepatocyte extract following Fx-TH increased significantly in middle-aged mice but remained unchanged in young mice. IL-10 levels significantly decreased in middle-aged mice following Fx-TH but remained unchanged in young mice. Kupffer cells from middle-aged mice produced significantly higher IL-6 and IL-10 levels compared with young mice. Protein levels and mRNA expression of ICAM-1 in hepatocytes were also significantly higher in middle-aged mice compared with young mice following Fx-TH. These results collectively suggest that the extent of hepatic damage following Fx-TH is dependent on the age of the subject. tumor necrosis factor-a; interleukin-10; interleukin-6; hepatocellular damage; intracellular adhesion molecule-1

Since cardiac function is depressed in males but not in proestrus (PE) females following trauma-hemorrhage (T-H), we examined whether different estrus cycles influence cardiac function in female rats under those conditions. We hypothesized that females in the PE cycle only will have normal cardiac function following T-H and resuscitation. Sham operation or T-H was performed in five groups of rats (250-275 g) including PE, estrus (E), metestrus (ME), diestrus (DE), and ovariectomized (OVX) females (n = 6-7 per group). Cardiac function was determined 2 h after T-H, following which cardiomyocytes were isolated and nuclei extracted. Cardiomyocyte IL-6 and NF-[kappa]B expressions were measured using Western blotting. Moreover, plasma IL-6, estradiol, and progesterone levels were measured using ELISA or EIA kits. Results (1-way ANOVA) indicated that following T-H, 1) cardiac function was depressed in DE, E, ME, and OVX groups but maintained in the PE group; 2) the PE group had the highest plasma estrogen level; 3) plasma IL-6 levels increased significantly in DE, E, ME, and OVX groups, but the increase was attenuated in the PE group; 4) cardiomyocyte IL-6 protein level increased significantly in DE, E, ME and OVX groups after TH, but the increase was attenuated in the PE group; and 5) cardiomyocyte NF-[kappa]B expression increased significantly but was attenuated in the PE group. These data collectively suggest that the estrus cycle plays an important role in cardiac function following TH. The salutary effect seen in PE following TH is likely due to a decrease in NF-[kappa]B-dependent cardiac IL-6 pathway. female reproductive cycle; ovariectomy; estrogen; trauma-hemorrhagic shock; interleukin-6; nuclear factor-[kappa]B

Traumatic and/or surgical injury as well as hemorrhage induces profound suppression of cellular immunity. Although local anesthetics have been shown to impair immune responses, it remains unclear whether lidocaine affects lymphocyte functions following trauma-hemorrhage (T-H). We hypothesized that lidocaine will potentiate the suppression of lymphocyte functions after T-H. To test this, we randomly assigned male C3H/HeN (6-8 wk) mice to sham operation or T-H. T-H was induced by midline laparotomy and ~90 rain of hemorrhagic shock (blood pressure 35 mmHg), followed by fluid resuscitation (4x shed blood volume in the form of Ringer lactate). Two hours later, the mice were killed and splenocytes and bone marrow cells were isolated. The effects of lidocaine on concanavalin A-stimulated splenocyte proliferation and cytokine production in both sham-operated and T-H mice were assessed. The effects of lidocaine on LPS-stimulated bone marrow cell proliferation and cytokine production were also assessed. The results indicate that T-H suppresses cell proliferation, Th1 cytokine production, and MAPK activation in splenocytes. In contrast, cell proliferation, cytokine production, and MAPK activation in bone marrow cells were significantly higher 2 h after T-H compared with shams. Lidocaine depressed immune responses in splenocytes; however, it had no effect in bone marrow cells in either sham or T-H mice. The enhanced immunosuppressive effects of lidocaine could contribute to the host's enhanced susceptibility to infection following T-H. shock; bone marrow cells

Previous studies showed that females in the proestrus stage of the reproductive cycle maintain organ functions after trauma-hemorrhage. However, it remains unknown whether the female reproductive cycle is an important variable in the regulation of lung injury after trauma-hemorrhage and, if so, whether the effect is mediated via upregulation of heme oxygenase (HO)-1. To examine this, female Sprague-Dawley rats during diestrus, proestrus, estrus, and metestrus phases of the reproductive cycle or 14 days after ovariectomy underwent soft tissue trauma and then hemorrhage (mean blood pressure 40 mmHg for 90 min followed by fluid resuscitation). At 2 h after trauma-hemorrhage or sham operation, lung myeloperoxidase (MPO) activity and intercellular adhesion molecule (ICAM)-1, cytokine-induced neutrophil chemoattractant (CINC)-1, CINC-3, and HO-1 protein levels were measured. Plasma 17[beta]-estradiol concentration was also determined. The results indicated that trauma-hemorrhage increased lung MPO activity and ICAM-1, CINC-1, and CINC-3 levels in ovariectomized females. These parameters were found to be similar to sham-operated animals in proestrus female rats subjected to trauma-hemorrhage. Lung HO-1 protein level in proestrus females was increased significantly compared with female rats subjected to trauma-hemorrhage during diestrus, estrus, and metestrus phases of the reproductive cycle and ovariectomized rats. Furthermore, plasma 17[beta]-estradiol level was highest in proestrus females. Administration of the HO inhibitor chromium mesoporphyrin prevented the attenuation of shock-induced lung damage in proestrus females. Thus these findings suggest that the female reproductive cycle is an important variable in the regulation of lung injury following trauma-hemorrhage and that the protective effect in proestrus females is likely mediated via upregulation of HO-1. hemorrhagic shock; cytokine-induced neutrophil chemoattractant-1; cytokine-induced neutrophil chemoattractant-3; intercellular adhesion molecule-1 doi:10.1152/ajplung.00537.2004

Tissue hypoxia is a common sequel of trauma-hemorrhage but can occur even without blood loss under hypoxic conditions. Although hypoxia is known to upregulate Kupffer cells (KC) to release cytokines, the precise mechanism of release remains unknown. We hypothesized that Src family kinases play a role in mediating KC mitogen-activated protein kinase (MAPK) signaling and their cytokine production after hypoxia. Male C3H/HeN mice received either Src inhibitor PP1 (1.5 mg/kg body wt) or vehicle 1 h before hypoxia. KCs were isolated 1 h after hypoxia, lysed, and immunoblotted with antibodies to Src, p38, ERK1/2, or JNK proteins. In addition, KCs were cultured to measure interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1) production. Hypoxia produced a significant increase in KC Src and MAPK (p38, ERK, JNK) activity compared with normoxic controls. This was associated with an increase in IL-6 and MCP-1 production. Treatment with PP1 abolished the increase in KC Src activation as well as p38 activity. However, PP1 did not prevent the increase in KC ERK1/2 or JNK phosphorylation. Furthermore, administration of PP1 prevented the hypoxia-induced increase in IL-6 but not MCP-1 release by KC. Additional in vitro results suggest that p38 but not ERK1/2 or JNK are critical for KC IL-6 production. In contrast, the production of MCP- 1 by KC was found to be independent of MAPK. Thus hypoxia increases KC IL-6 production by p38 MAPK activation via Src-dependent pathway. Src kinases may therefore be a novel therapeutic target for preventing immune dysfunction following low-flow conditions in trauma patients. innate immunity; macrophages; cell signaling doi:10.1152/ajpcell.00076.2006.

Although androstenediol (adiol or 5-androstene-3[beta],17[beta]-diol), a metabolite of dehydroepiandrosterone (DHEA), has protective effects following trauma-hemorrhage (T-H), it remains unknown whether administration of adiol has any salutary effects on the inflammatory response and outcome following a combined insult of T-H and sepsis. Male rats underwent T-H shock [mean arterial pressure (MAP) 40 mmHg for 90 min] followed by resuscitation. Adiol (1 mg/kg body wt) or vehicle was administered at the end of resuscitation. Sepsis was induced by cecal ligation and puncture (CLP) at 20 h after T-H or sham operation. Five hours after CLP, plasma and tissue samples were analyzed for cytokines (IL-6 and IL-10), MPO, neutrophil chemotactic factor (CINC-3), and liver injury (alanine aminotransferase and lactate dehydrogenase). In another group of rats, the gangrenous cecum was removed at 10 h after CLP, the cavity was irrigated with warm saline and closed in layers, and mortality was recorded over 10 days. T-H followed by CLP produced a significant elevation in plasma IL-6 and IL-10 levels, enhanced neutrophil cell activation, and resulted in liver injury. Adiol administration prevented the increase in cytokine production, neutrophil cell activation, and attenuated liver injury. Moreover, rats subjected to the combined insult, receiving vehicle or adiol, had a 50% and 6% mortality, respectively. Since adiol administration suppresses proinflammatory cytokines, reduces liver damage, and decreases mortality after the combined insult of T-H and sepsis, this agent appears to be a novel adjunct to fluid resuscitation for decreasing T-H-induced septic complications and mortality. liver enzymes; proinflammatory cytokines; neutrophils activation; myeloperoxidase doi:10.1152/ajpgi.00390.2005

Although 17[beta]-estradiol (E2) administration after trauma-hemorrhage attenuates lung injury in male rodents, it is not known whether the salutary effects are mediated via estrogen receptor (ER)-[alpha], or ER-[beta]. We hypothesized that the salutary effects of E2 lung are mediated via ER-[beta]. Male Sprague-Dawley rats underwent trauma-hemorrhage (mean blood pressure 40 mmHg for 90 min. then resuscitation). E2 (50 [micro]g/kg), ER-[alpha], agonist propyl pyrazole triol (PPT; 5 [micro]g/kg), ER-[beta] agonist diarylpropiolnitrile (DPN; 5 [micro]g/kg), or vehicle (10% DMSO) was injected subcutaneously during resuscitation. At 24 h after trauma-hemorrhage or sham operation, bronchoalveolar fluid (BALF) was collected for protein concentration. LDH activity, and nitrate/nitrite and IL-6 levels. Moreover, lung tissue was used for inducible nitric oxide synthase (iNOS) mRNA/protein expression, nitrate/nitrite and IL-6 levels, and wet/dry weight ratio (n = 6 rats/group). One-way ANOVA and Tukey's test were used for statistical analysis. The results indicated that E2 downregulated lung iNOS expression after trauma-hemorrhage. Protein concentration, LDH activity, and nitrate/nitrite and IL-6 levels in BALF and nitrate/nitrite and IL-6 levels in the lung increased significantly after trauma-hemorrhage: however, administration of DPN but not PPT significantly improved all parameters. Moreover, DPN treatment attenuated trauma-hemorrhage-mediated increase in iNOS mRNA/protein expression in the lung. In contrast, no significant change in the above parameters was observed with PPT. Thus the salutary effects of E2 on attenuation of lung injury are mediated via ER-[beta], and ER-[beta]-induced downregulation of iNOS likely plays a significant role in the DPN-mediated lung protection after trauma-hemorrhage. shock; propyl pyrazole triol; diarylpropiolnitrile

Although studies indicate that 17[beta]-estradiol administration after trauma-hemorrhage (T-H) improves cardiac and hepatic functions, the underlying mechanisms remain unclear. Because the induction of heat shock proteins (HSPs) can protect cardiac and hepatic functions, we hypothesized that these proteins contribute to the salutary effects of estradiol after T-H. To test this hypothesis, male Sprague-Dawley rats (~300 g) underwent laparotomy and hemorrhagic shock (35-40 mmHg for ~90 min) followed by resuscitation with four times the shed blood volume in the form of Ringer lactate. 17[beta]-estradiol (1 mg/kg body wt) was administered at the end of the resuscitation. Five hours after T-H and resuscitation there was a significant decrease in cardiac output, positive and negative maximal rate of left ventricular pressure. Liver function as determined by bile production and indocyanine green clearance was also compromised after T-H and resuscitation. This was accompanied by an increase in plasma alanine aminotransferase (ALT) levels and liver perfusate lactic dehydrogenase levels. Furthermore, circulating levels of TNF-[alpha], IL-6, and IL-10 were also increased. In addition to decreased cardiac and hepatic function, there was an increase in cardiac HSP32 expression and a reduction in HSP60 expression after T-H. In the liver, HSP32 and HSP70 were increased after T-H. There was no change in heart HSP70 and liver HSP60 after T-H and resuscitation. Estradiol administration at the end of T-H and resuscitation increased heart/liver HSPs expression, ameliorated the impairment of heart/liver functions, and significantly prevented the increase in plasma levels of ALT, TNF-[alpha], and IL-6. The ability of estradiol to induce HSPs expression in the heart and the liver suggests that HSPs, in part, mediate the salutary effects of 17[beta]-estradiol on organ functions after T-H. heme oxygenase-l; organ dysfunction; tumor necrosis factor-[alpha]; estrogen; injury

Although previous studies have shown that flutamide improves cardiovascular function after trauma-hemorrhage, the mechanisms responsible for the salutary effect remain unknown. We hypothesized that flutamide mediates its beneficial effects via an estrogen-dependent pathway through upregulation of peroxisome proliferator-activated receptor-[gamma] coactivator 1 (PGC-1). PGC-1, a key regulator of cardiac mitochondrial ATP production, induces mitochondrial DNA (mtDNA)-encoded genes such as cytochrome-c oxidase (COX) subunit I, II, and III (COX I, COX II, and COX III), which regulates mitochondrial oxidative phosphorylation. To test this hypothesis, male rats underwent trauma-hemorrhage (mean arterial pressure of 35-40 mmHg for ~90 min) followed by resuscitation. At the onset of resuscitation, rats received vehicle, flutamide (25 mg/kg body wt), flutamide in combination with estrogen receptor (ER) antagonist ICI-182,780 (3 mg/kg body wt), or ICI-182,780 alone. Flutamide administration after trauma-hemorrhage restored the depressed cardiac function and increased cardiac testosterone, estrogen levels, and arornatase activity. These increases were accompanied by normalized cardiac ER-[alpha] and ER-[beta] protein levels, PGC-1, and COX I mRNA expression, mitochondrial COX activity, and ATP contents. However, cardiac dihydrotestosterone, 5[alpha]-reductase II, androgen receptor protein levels, and mtDNA-encoded genes COX II and COX III were unaffected by flutamide treatment. The flutamide-mediated restoration of cardiac function, the increases in aromatase activity and estrogen levels, ER-[alpha], ER-[beta], PGC-1, COX I, COX activity, and ATP contents were, however, abolished when ER antagonist ICI-182,780 was administrated along with flutamide. These findings suggest that the salutary effect of flutamide on cardiac function after trauma-hemorrhage is mediated via an estrogen-dependent pathway through upregulation of PGC-1. hemorrhagic shock; heart; sex hormones; estrogen receptor-[alpha]; estrogen receptor-[beta]; peroxisome proliferator-activated receptor-[gamma] coactivator 1

Flutamide, an androgen receptor antagonist, is thought to improve cardiovascular function by blocking the androgen receptor after trauma-hemorrhage (T-H). Although 17[beta]-estradiol (E2) and flutamide improve cardiac function after T-H, whether E2 and flutamide produce their salutary effect via the same or a different mechanism is unknown. We hypothesized that E2 and flutamide mediate their effects via estrogen receptor (ER)-mediated upregulation of peroxisome proliferator-activated receptor coactivator 1 (PGC-1). PGC-1, a key regulator of cardiac mitochondrial function, induces mitochondrial genes by activating transcription factors such as nuclear respiratory factor 2 (NRF-2), which regulates mitochondrial proteins [i.e., mitochondrial transcription factor A (Tfam), cytochrome-c oxidase subunit IV, and [beta]-ATP synthase]. Adult male rats underwent T-H [5-cm midline incision and hemorrhage (blood pressure = 40 mmHg for ~90 min)] and resuscitation. At the onset of resuscitation, rats received vehicle, flutamide (25 mg/kg), or E2 (50 [micro]g/kg). Another group received the ER antagonist ICI-182780 (3 mg/kg) with or without flutamide. Flutamide or E2 administration after T-H restored depressed cardiac function. Moreover, E2 and flutamide normalized expression of cardiac PGC-1, NRF-2, Tfam, cytochrome-c oxidase subunit IV, and the mitochondrial DNA-encoded gene cytochrome-c oxidase subunit I and [beta]-ATP synthase, mitochondrial ATP, and cytochrome-c oxidase activity. However, if the ER antagonist ICI-182780 was administered with flutamide, flutamide-mediated PGC-I upregulation was totally abolished. These results indicate that E2 and flutamide upregulate PGC-I via the ER. Thus PGC-I upregulation appears to be the common mechanism by which E2 and flutamide mediate their salutary effects on cardiac function after T-H. hemorrhagic shock; adenosine triphosphate; sex hormones; androgen receptors; adenosinetriphosphatase

Previous studies showed that alcohol (EtOH) intoxication before bum injury suppresses mesenteric lymph node (MLN) T cell functions and increases gut bacterial translocation. In this study, we examined whether corticosterone (Cort) plays any role in suppressing MLN T cell function and bacterial accumulation after EtOH intoxication and burn injury. Rats were gavaged with EtOH to achieve a blood EtOH level of ~100 mg/dl before receiving 25% total body surface area burn or sham injury. A group of rats was treated with the Cog synthesis inhibitor metyrapone (25 mg/kg) at the time of injury and on day 1 after injury. Two days after injury, a significant increase in blood Cort levels and suppression of MLN T cell proliferation and IL-2 production was observed in rats receiving combined insult of EtOH intoxication and burn injury compared with rats receiving EtOH intoxication or burn injury alone. There was no change in T cell apoptosis after combined insult of EtOH and burn injury. Furthermore, T cell suppression was accompanied by a significant decrease in p38 and ERK1/2 activation (phosphorylation). There was no difference in JNK activation after EtOH and burn injury. Treatment of rats with metyrapone prevented the suppression of MLN T cell proliferation, IL-2 production, and p38 and ERK1/2 phosphorylation. Restoration of T cell function in metyrapone-treated animals was also associated with the decrease in bacterial accumulation in MLN. These findings suggest that EtOH intoxication before burn injury augments Cog release, which suppresses MLN T cell function by inhibiting p38 and ERK1/2 activation and promotes bacterial accumulation in MLN after EtOH and burn injury. T lymphocyte; mitogen-activated protein kinases; cell signaling; thermal injury

A growing body of evidence indicates that heme degradation products may counteract the deleterious consequences of hypoxia and/or ischemia-reperfusion injury. Because heine oxygenase (HO)-1 induction after adverse circulatory conditions is known to be protective, and because females in the proestrus cycle (with high estrogen) have better hepatic function and less hepatic damage than males after trauma-hemorrhage, we hypothesized that estrogen administration in males after trauma-hemorrhage will upregulate HO activity and protect the organs against dysfunction and injury. To test this hypothesis, male Sprague-Dawley rats under-went 5-cm laparotomy and hemorrhagic shock (35-40 mmHg for 93 [+ or -] 2 min), followed by resuscitation with four times the shed blood volume in the form of Ringer lactate. 17[beta]-Estradiol and/or the specific HO enzyme inhibitor chromium mesoporphyrin (CrMP) were administered at the end of resuscitation, and the animals were killed 24 h thereafter. Trauma-hemorrhage reduced cardiac output, myocardial contractility, and serum albumin levels. Portal pressure and serum alanine aminotransferase levels were markedly increased under those conditions. These parameters were significantly improved in the 17[beta]-estradiol-treated rats. Estradiol treatment also induced increased HO-1 mRNA expression, HO-1 protein levels, and HO enzymatic activity in cardiac and hepatic tissue compared with vehicle-treated trauma-hemorrhage rats. Administration of the HO inhibitor CrMP prevented the estradiol-induced attenuation of shock-induced organ dysfunction and damage. Thus the salutary effects of estradiol administration on organ function after trauma-hemorrhage are mediated in part via upregulation of HO-1 expression and activity. hemorrhagic shock; heme oxygenase-1; chromium mesoporphyrin; liver; heart

Recent studies from our laboratory have shown that alcohol and burn injury impair intestinal barrier and immune functions. Although multiple factors can contribute to impaired intestinal barrier function, such an alteration could result from a decrease in intestinal blood flow (BF) and oxygen delivery (D[O.sub.2]). Therefore, in this study, we tested the hypothesis that alcohol ingestion before burn injury reduces splanchnic blood flow and oxygen delivery. Rats (250 g) were gavaged with alcohol to achieve a blood ethanol level in the range of 100 mg/dl before burn or sham injury (25% total body surface area). Day 1 after injury, animals were anesthetized with methoxyflurane. Blood pressure, cardiac output (CO), [+ or -] dP/dt, organ BF (in ml x [min.sup.-1] x 100 [g.sup.-1]), and D[O.sub.2] (in mg x [ml.sup.-1] x 100 [g.sup.-1]) were determined. CO and organ BF were determined using a radioactive microsphere technique. Our results indicate that blood pressure, CO, and +dP/dt were decreased in rats receiving a combined insult of alcohol and burn injury compared with rats receiving either burn injury or alcohol alone. This is accompanied by a decrease in BF and D[O.sub.2] to the liver and intestine. No significant change in BF to the coronary arteries (heart), brain, lung, skin, and muscles was observed after alcohol and burn injury. In conclusion, the results presented here suggest that alcohol ingestion before burn injury reduces splanchnic BF and D[O.sub.2]. Such decreases in BF and D[O.sub.2] may cause hypoxic insult to the intestine and liver. Although a hypoxic insult to the liver would result in a release of proinflammatory mediators, a similar insult to the intestine will likely perturb both intestinal immune cell and barrier functions, as observed in our previous study. hemodynamic; liver; intestine; shock; trauma; ethanol; cardiovascular response; thermal injury

Recent studies have shown that administration of dehydroepiandrosterone (DHEA) after trauma-hemorrhage (T-H) improves cardiovascular and hepatic function in male animals. Although androstenediol, one of the DHEA metabolites, has been recently reported to produce salutary effects on cardiac function and splanchnic perfusion after T-H, it remains unknown whether androstenediol per se has any salutary effects on hepatic function under those conditions. To study this, male Sprague-Dawley rats underwent laparotomy and ~90 min of hemorrhagic shock (35-40 mmHg), followed by resuscitation with four times the shed blood volume in the form of Ringer lactate. Androstenediol (1 mg/kg body wt iv) was administered at the end of resuscitation, and the animals were killed 24 h later. T-H significantly reduced portal blood flow, bile production, and serum albumin levels. Portal pressure, serum alanine aminotransferase, hepatic nitrate/nitrite, inducible nitric oxide synthase (iNOS), and endothelin-1 markedly increased after T-H. The alterations in these parameters induced by T-H were significantly attenuated in rats treated with androstenediol. Endothelial NOS (eNOS) expression, which was not different between T-H and sham, was found to be significantly elevated in T-H androstenediol-treated rats. These data suggest that improvement in hepatic perfusion by androstenediol after T-H is likely due to a decrease in endothelin-1 and induction of eNOS. Moreover, the decrease in hepatic damage after androstenediol administration is likely related to liver iNOS downregulation. Thus androstenediol appears to be a novel and useful adjunct for restoring hepatic function in male animals after adverse circulatory conditions. hemorrhagic shock; nitric oxide; nitric oxide synthase-2; adiol; 5-androstene-3[beta] 17[beta]-diol.

Mechanism of cardiac depression after trauma-hemorrhage: 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, 2004; doi:10.1152/ajpheart. 00624.2003.--A prolonged depression of cardiovascular function occurs in males after trauma-hemorrhagic shock (T-H). Although a correlation between increased circulatory IL-6 levels and poor outcome has been reported after T-H, it remains unknown whether T-H increases IL-6 levels locally in cardiomyocytes and whether there is a correlation between altered cardiac function and local IL-6 production after T-H. T-H was induced in normal, castrated (2 wk before T-H), and 17[beta]-estradiol ([E.sub.2])-treated (0.5 mg so, 1 wk before T-H) adult male rats. At 2 h after T-H or sham operation, cardiac output, heart rate, mean arterial pressure, positive and negative first derivative of pressure ([+ or -] dP/dt), stroke volume, and total peripheral resistance were determined. Cardiomyocytes were isolated and divided into two parts: one was used for measurements of intracellular IL-6 levels using fluorescein-activated cell sorting, and the other was used to isolate RNA to determine IL-6 gene expression by quantitative real-time PCR. In addition, cardiac IL-6 protein levels were measured in freshly isolated hearts by Western blotting. Cardiac output, stroke volume, +dP/dt, -dP/dt, and total peripheral resistance were markedly altered after T-H. These parameters, except -dP/dt, improved significantly in the castrated group; however, all these parameters were restored in [E.sub.2]-treated males. Cardiomyocyte IL-6 mRNA expression and intracellular IL-6 production increased after T-H. Cardiac IL-6 protein levels increased after T-H in freshly isolated heart. Castration and [E.sub.2] treatment attenuated cardiomyocyte intracellular IL-6 levels and cardiac IL-6 protein levels after T-H; however, only [E.sub.2] treatment attenuated cardiomyocyte IL-6 gene expression. Thus there is an inverse correlation between cardiomyocyte IL-6 levels and cardiac function after T-H. The salutary effects of [E.sub.2] on cardiac function after T-H may be due in part to decreased IL-6 synthesis in cardiomyocytes. estrogen; ribonucleic acid; quantitative real-time polymerase chain reaction; Western blot; cytokines; fluorescein-activated cell sorting; interleukin-6

Recent studies have shown that dehydroepiandrosterone (DHEA) administration after trauma-hemorrhage (T-H) improves cardiovascular function and decreases cytokine production in male animals. Although androstenediol, one of the metabolites of DHEA, is reported to have estrogen-like activity, it remains unknown whether androstenediol per se has any salutary effects on cytokines and cardiovascular function after T-H. To examine this effect, male Sprague-Dawley rats underwent laparotomy and were bled to and maintained at a mean arterial blood pressure of 35-40 mmHg for ~90 min. The animals were resuscitated with four times the volume of maximal bleedout volume in the form of Ringer lactate. Androstenediol (1 mg/kg body wt iv) or vehicle was administered at the end of resuscitation. Twenty-tour hours after resuscitation, cardiac function and organ blood flow were measured by using [sup.85]Sr-microspheres. Circulating levels of nitrate/nitrite and IL-6 were also determined. Cardiovascular function and organ blood flow were significantly depressed after T-H. However, these parameters were restored by androstenediol treatment. The elevated plasma IL-6 levels after T-H were also lowered by androstenediol treatment. In contrast, plasma levels of nitrate/nitrite were the highest in the androstenedioltreated T-H animals. Because androstenediol administration after T-H decreases cytokine production and improves cardiovascular function, this agent appears to be a novel and useful adjunct for restoring the depressed cardiovascular function and for cytokine production in males after adverse circulatory conditions. hemorrhagic shock; nitric oxide; nitric oxide synthase; adiol; 5-androstene-3[beta], 17[beta]-diol

We evaluated the dependency of neutrophil [O.sup.-.sub.2] production on PTK-Lyn and MAPK-ERK1/2 in rats after thermal injury. Activation of PTK-Lyn was assessed by immunoprecipitation. Phosphorylation of ERK1/2 was assessed by Western blot analysis. [O.sup.-.sub.2] production was measured by isoluminol-enhanced luminometry. Imaging technique was employed to measure neutrophil [[[Ca.sup.2+]].sub.i] in individual cells. Thermal injury caused marked upregulation of Lyn and ERK1/2 accompanying enhanced neutrophil [O.sup.-.sub.2] production. Treatment of rats with PTK blocker (AG556) or MAPK blocker (AG1478) before burn injury caused complete inhibition of the respective kinase activation. Both AG556 and AG1478 produced an ~66% inhibition in [O.sup.-.sub.2] production. Treatment with diltiazem (DZ) produced an ~37% inhibition of [O.sup.-.sub.2] production without affecting Lyn or ERK1/2 activation with burn injury. [Ca.sup.2+] mobilization was upregulated with burn injury but not affected by treatment of burn rats with AG556. Unlike the partial inhibition of burn-induced [O.sup.-.sub.2] production by AG556, AG1478, or DZ, platelet-activating factor antagonist (PAFa) treatment of burn rats produced near complete inhibition of [O.sup.-.sub.2] production. PAFa treatment also blocked activation of Lyn. The findings suggest that the near complete inhibition of [O.sup.-.sub.2] production by PAFa was a result of blockade of PTK as well as [Ca.sup.2+] signaling. Overall, our studies show that enhanced neutrophil [O.sup.-sub.2] production after thermal injury is a result of potentiation of [Ca.sup.2+]-linked and -independent signaling triggered by inflammatory agents such as PAF. burn; rat; polymorphonuclear neutrophil; protein kinase C signaling; platelet-activating factor blockade; Lyn blockade; extracellular signal-regulated kinase 1/2 blockade

The mechanism of alcohol-mediated increased infection in burn patients remains unknown. With the use of a rat model of acute alcohol and burn injury, the present study ascertained whether acute alcohol exposure before thermal injury enhances gut bacterial translocation. On day 2 postinjury, we found a severalfold increase in gut bacterial translocation in rats receiving both alcohol and burn injury compared with the animals receiving either injury alone. Whereas there were no demonstrable changes in intestinal morphology in any group of animals, a significant increase in intestinal permeability was observed in ethanol- and burn-injured rats compared with the rats receiving either injury alone. We further examined the role of intestinal immune defense by determining the gut-associated lymphoid (Peyer's patches and mesenteric lymph nodes) T cell effector responses 2 days after alcohol and burn injury. Although there was a decrease in the proliferation and interferon-[gamma] by gut lymphoid T cells after burn injury alone; the suppression was maximum in the group of rats receiving both alcohol and burn injuries. Furthermore, the depletion of CD[3.sup.+] cells in healthy rats resulted in bacterial accumulation in mesenteric lymph nodes; such CD[3.sup.+] cell depletion in alcohol- and burn-injured rats furthered the spread of bacteria to spleen and circulation. In conclusion, our data suggest that the increased intestinal permeability and a suppression of intestinal immune defense in rats receiving alcohol and burn injury may cause an increase in bacterial translocation and their spread to extraintestinal sites. T lymphocyte; infection immunity bacteria; inflammation; shock

Previous research has shown that prostaglandin E2 (PGE2)-mediated suppression of T cell functions could result from an attenuation of p59fyn protein tyrosine kinase activity. Research was conducted to examine the effects of an adenylate cyclase agonist (forskolin) and antagonist (SQ-22536), including those of cAMP analogues, on T cell p59fyn kinase activity. Findings showed that PGE2-mediated suppression of p59fyn autophosphorylation and kinase activity in T cells is dependent on the activation of adenylate cyclase and independent of the elevation in cAMP levels.

The role of platelet-activating factor (PAF) in [Ca.sup.2+] signaling and [Ca.sup.2+]-related enhancement of reactive oxygen intermediate (ROI) generation in neutrophils of burn-injured rats was ascertained by evaluating the effect of treatment of the rats with a PAF receptor antagonist. The treatment of rats with the antagonist also allowed us to evaluate the role of PAF in the priming of neutrophil ROI response with burn in vivo. A full skin thickness burn injury was produced in anesthetized rats by exposing 30% of total body surface area to 98 [degrees] C water for 10 s. Sham and burn rats were killed 1 day later, and their blood was collected to obtain neutrophils. Fluorescence-activated cell sorter analysis was used to quantify ROI production by the neutrophils. Cytosolic-free [Ca.sup.2+] concentration ([[Ca.sup.2+].sub.i]) imaging technique was employed to measure neutrophil [[Ca.sup.2+].sub.i] in individual cells and microfluorometry for the assessment of [[Ca.sup.2+].sub.i] responses in suspensions of neutrophils. There was an overt enhancement of ROI generation by burn rat neutrophils. ROI release was accompanied by a marked elevation of [[Ca.sup.2+].sub.i] signaling. The treatment of rats with PAF receptor antagonist before burn prevented the upregulation of both [[Ca.sup.2+].sub.i] and ROI generation in neutrophils. These studies indicate that enhanced ROI production in neutrophils in the early stages after burn injury results from a PAF-mediated priming of the [[Ca.sup.2+].sub.i] signaling pathways in vivo. burn; rat; polymorphonuclear neutrophils; platelet-activating factor; reactive oxygen intermediates; calcium-protein kinase C signaling; platelet-activating factor blockade

Neutrophil depletion prevents intestinal mucosal permeability alterations in burn-injured rats. Am J Physiol Regulatory Integrative Comp Physiol 278: R1224-R1231, 2000.--Cutaneous thermal injury increases intestinal mucosal permeability. The mechanisms of this functional disturbance are not fully understood. We investigated whether accumulation of neutrophils in the intestine contributes to the increase in mucosal permeability. Labeled and unlabeled lactulose and mannitol were infused into a segment of rat ileum or jejunum. Blood concentrations of [[.sup.3]H]lactulose and [[.sup.14]C]mannitol were measured after 30, 60, and 90 min. On day 1 postburn, lactulose permeability increased fourfold in the ileum and twofold in the jejunum compared with sham-burned rats; mannitol permeability increased twofold in the ileum and 1.5-fold in the jejunum. A greater increase in permeability occurred on day 3 postburn in the ileum, but not in the jejunum. The depletion of neutrophils in burned rats prevented the increase in permeability in both segments on day 1 postburn. Histological studies of intestines from burned, with or without neutrophil depletion, and sham-burned rats showed similar morphology. However, numerous neutrophils were found in the extravascular compartment in day 1 postburn, but not in neutrophil-depleted and sham-burned rats. These findings support the concept that the burn-induced increase in mucosal permeability is produced during the accumulation of neutrophils in the intestine and can be abrogated by the depletion of neutrophils. jejunum; ileum; epithelial paracellular transport; bacterial translocation; lactulose; mannitol; antineutrophil antibody

Choudhry, Mashkoor A., Philip E. Hockberger, and Mohammed M. Sayeed. [PGE.sub.2] suppresses mitogen-induced [Ca.sup.2+] mobilization in T cells. Am. J. Physiol. 277 (Regulatory Integrative Comp. Physiol. 46): R1741-R1748, 1999.--[PGE.sub.2]-mediated suppression of T cell proliferation during sepsis could result from altered [Ca.sup.2+] signaling. The present study evaluated the effects of [PGE.sub.2] on [Ca.sup.2+] release from intracellular stores and its influx through the plasma membrane in splenic T cells from Sprague-Dawley rats. Intracellular [Ca.sup.2+] concentration ([[[Ca.sup.2+]].sub.i]) responses in individual T cells were assessed using the [Ca.sup.2+] imaging technique, and the release of [Ca.sup.2+] from intracellular stores and [Ca.sup.2+] influx were spectro-fluorometrically quantified in T cell suspensions. Under unstimulated conditions, nearly 85% of T cells exhibited [[[Ca.sup.2+]].sub.i] [is less than or equal to] 50 nM. After stimulation with concanavalin A (Con A), an increase in [[[Ca.sup.2+]].sub.i] was recorded in ~60% of the cells. The pretreatment oft cells with [PGE.sub.2] had no apparent effect on [[[Ca.sup.2+]].sub.i] in resting cells; it significantly suppressed the Con A-induced increase in [[[Ca.sup.2+]].sub.i] in all of the Con A-responsive cells. [Ca.sup.2+] release from the intracellular stores contributed to the early spike in [[[Ca.sup.2+]].sub.i], and the late phase of elevation in [[[Ca.sup.2+]].sub.i] was dependent on [Ca.sup.2+] influx through the plasma membrane. Our data suggest that [PGE.sub.2] causes an overall suppression of the Con A-induced [[[Ca.sup.2+]].sub.i] elevation in T cells via inhibiting both [Ca.sup.2+] influx and its release from the intracellular stores. concanavalin A; T lymphocytes; calcium ion signaling; intracellular calcium ion release; adenosine 3,5-cyclic monophosphate; prostaglandin [E.sub.2]