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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

We have previously demonstrated that in a rat model of trauma-hemorrhage (T-H), glucosamine administration during resuscitation improved cardiac function, reduced circulating levels of inflammatory cytokines, and increased tissue levels of O-linked N-acetylglucosamine (O-GlcNAc) on proteins. The mechanism(s) by which glucosamine mediated its protective effect were not determined; therefore, the goal of this study was to test the hypothesis that glucosamine treatment attenuated the activation of the nuclear factor-[kappa]B (NF-[kappa]B) signaling pathway in the heart via an increase in protein O-GlcNAc levels. Fasted male rats were subjected to T-H by bleeding to a mean arterial blood pressure of 40 mmHg for 90 min followed by resuscitation. Glucosamine treatment during resuscitation significantly attenuated the T-H-induced increase in cardiac levels of TNF-[alpha] and IL-6 mRNA, I[kappa]B-[alpha] phosphorylation, NF-[kappa]B, NF-[kappa]B DNA binding activity, ICAM-1, and MPO activity. LPS (2 [micro]g/ml) increased the levels of I[kappa]B-[alpha] phosphorylation, TNF-[alpha], ICAM-1, and NF-[kappa]B in primary cultured cardiomyocytes, which was significantly attenuated by glucosamine treatment and overexpression of O-GlcNAc transferase; both interventions also significantly increased O-GlcNAc levels. In contrast, the transfection of neonatal rat ventricular myocytes with OGT small-interfering RNA decreased O-GlcNAc transferase and O-GlcNAc levels and enhanced the LPS-induced increase in I[kappa]B-[alpha] phosphorylation. Glucosamine treatment of macrophage cell line RAW 264.7 also increased O-GIcNAc levels and attenuated the LPS-induced activation of NF-[kappa]B. These results demonstrate that the modulation of O-GlcNAc levels alters the response of cardiomyocytes to the activation of the NF-[kappa]B pathway, which may contribute to the glucosamine-mediated improvement in cardiac function following hemorrhagic shock. nuclear factor-[kappa]B; cytokines; neonatal rat ventricular myocytes; O-linked N-acetylglucosamine

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 endothelin-1 (ET-1)-induced organ hypoperfusion after trauma-hemorrhage is improved by estrogen administration, it remains unclear whether estrogen receptor (ER) subtypes play any role in the attenuation of ET-l-induced vasoconstriction in any specific organ bed. To investigate this, isolated perfusion experiments in the heart, liver, small intestine, kidney, and lung were carried out in sham, at the time of maximum bleedout (MBO; i.e., 5-cm midline incision, with removal of 60% of circulating blood volume over 45 min to maintain a mean blood pressure of 40 mmHg), and 2 h after trauma-hemorrhage and resuscitation (T-H/R). Organ-specific ET-1-induced vasoconstriction was evaluated, and the effects of 17[beta]-estradiol ([E.sub.2]) and ER-specific agonists propylpyrazole triol (PPT; ER[alpha] agonist) and diarylpropionitrile (DPN; ER[beta] agonist) were determined. ET-1 induced the greatest vasoconstriction in sham animals, with the strongest response in the kidneys, followed by the small intestine and liver. ET-1-induced responses were weakest in the heart and lungs. ET-1-induced vasoconstriction was evident at the time of MBO but was significantly decreased at 2 h after T-H/R. ER[beta] plays an important role in cardiac performance, as evidenced by improved heart performance (+ dP/dt) in the presence of DPN. DPN also induced a greater effect than PPT in the reduction of ET-1-induced vasoconstriction in the kidneys and lungs. In contrast, PPT attenuated ET-1-induced vasoconstriction in the liver, whereas both DPN and PPT were equally effective in the small intestine. The increased +dP/dt values induced by [E.sub.2], DPN, or PPT were evident at the time of MBO but were significantly decreased at 2 h after T-H/R. These data indicate that the effects of ET-1 on vasoconstriction and the role of ER subtypes in estrogen-induced vasorelaxation are organ specific and temporally specific after trauma-hemorrhage. maximum bleedout; 17[beta]-estradiol; diarylpropionitrile; propylpyrazole triol

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

The aim of this study was to determine whether estrogen plays any role in the hepatic regeneration of nonligated lobe following portal branch ligation (PBL). Male rats were subjected to PBL on the left and middle lobes. Two and 7 days after PBL, the rats were killed and blood and liver samples were analyzed. Sham animals underwent only laparotomy. The serum estradiol levels were significantly elevated on day 2 following PBL and returned to normal levels on day 7. The expression of estrogen receptors (ER) in the liver evaluated by Western blotting did not show any change in the nonligated lobe compared with shams. Immunohistochemical study for ER showed a predominant ER expression in the hepatocyte nucleus in periportal area (zone 1), although there was no apparent difference in the amount and expression pattern between sham and PBL. However, chronic inhibition of ER by an ER antagonist (ICI 182,780) showed a significantly lower regeneration rate of the nonligated lobe compared with vehicle treatment. Liver regeneration-associated genes also were less activated in the ICI group. Moreover, portal venous flow, determined by fluorescent microspbere injection, was significantly lower in the ICI group compared with vehicle group. These changes correlated with the attenuated expression of endothelial nitric oxide synthase mRNA in both superior mesenteric arteries and veins. In conclusion, these results indicate that the estrogen's contribution on hepatic regeneration following PBL is at least partly mediated through maintaining mesenteric blood flow by mesenteric endothelial nitric oxide synthase upregulation rather than directly activating liver regeneration in the liver. portal vein embolization; nonligated lobe; estrogen receptor; endothelial nitric oxide synthase; mesenteric flow

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 endothelin-1 (ET-1) induces vasoconstriction, it remains unknown whether 17[beta]-estradiol ([E.sub.2]) treatment following trauma-hemorrhage alters these ET-1-induced vasoconstrictive effects. In addition, the role of the specific estrogen receptor (ER) subtypes (ER-[alpha] and ER-[beta]) and the endothelium-localized downstream mechanisms of actions of [E.sub.2] remain unclear. We hypothesized that [E.sub.2] attenuates increased ET-1-induced vasoconstriction following trauma-hemorrhage via an ER-[beta]-mediated pathway. To study this, aortic rings were isolated from male SpragueDawley rats following trauma-hemorrhage with or without [E.sub.2] treatment, and alterations in tension were determined in vitro. Doseresponse curves to ET-1 were determined, and the vasoactive properties of [E.sub.2], propylpyrazole triol (PPT, ER-[alpha] agonist), and diarylpropionitrile (DPN, ER-[beta] agonist) were determined. The results showed that trauma-hemorrhage significantly increased ET-1-induced vasoconstriction; however, administration of [E.sub.2] normalized ET-1-induced vasoconstriction in trauma-hemorrhage vessels to the shamoperated control level. The ER-[beta] agonist DPN counteracted ET-1-induced vasoconstriction, whereas the ER-[alpha] agonist PPT was ineffective. Moreover, the vasorelaxing effects of [E.sub.2] were not observed in endothelium-denuded aortic rings or by pretreatment of the tings with a nitric oxide (NO) synthase inhibitor. Cyclooxygenase inhibition with indomethacin had no effect on the action of [E.sub.2]. Thus, [E.sub.2] administration attenuates ET-1-induced vasoconstriction following trauma-hemorrhage via an ER-[beta]-mediated pathway that is dependent on endothelium-derived NO synthesis. estrogen receptor; nitric oxide; endothelium; aortic ring

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

Although gender differences in intestinal perfusion exist following trauma-hemorrhage (T-H), it remains unknown whether endothelin-1 (ET-1) plays any role in these dimorphic responses. To study this, male, proestrus female (female), and 17[beta]-estradiol (E2)-treated male rats underwent midline laparotomy, hemorrhagic shock (blood pressure 40 mmHg, 90 min), and resuscitation (Ringer lactate, 4x shed blood volume, 1 h). Two hours thereafter, intestinal perfusion flow (IPF) was measured using isolated intestinal perfusion. The IPF in sham-operated males was significantly lower than those in other groups and decreased markedly following T-H. In contrast, no significant decrease in IPF was observed in females and E2 males following T-H. The lower IPF in sham-operated males was significantly elevated by E[T.sub.A] receptor antagonist (BQ-123) administration and was similar to that seen in sham-operated females. The decreased IPF in males after T-H was also attenuated by BQ-123 administration. The intestinal ET-1 levels in sham-operated males were significantly higher than in other groups. Although plasma and intestinal ET-1 levels increased significantly after T-H in all groups, they were highest in males. Plasma E2 levels in females and E2 males were significantly higher than in males; however, they were not affected by T-H. There was a negative correlation between plasma ET-1 and E2 following T-H. Thus ET-1 appears to play an important role in intestinal perfusion failure following T-H in males. Because E2 can modulate this vasoconstrictor effect of ET-1, these findings may partially explain the previously observed salutary effect of estrogen in improving intestinal perfusion following T-H in males. hemorrhagic shock; BQ-123; tissue trauma: 17[beta]-estradiol

Prolactin (PRL) is involved in the regulation of immune functions under normal and pathological conditions. Trauma-hemorrhage (T-H) produces profound immunosuppression in male mice but not in proestrus female mice. Administration of PRL in males alter T-H, however, restores immune functions. In this study, PR[L.sup.+/+] and transgenic (PR[L.sup.-/-]) male and female mice were used to assess immune suppression after T-H and to determine the reasons for the hormone's beneficial effect. In vitro lymphoproliferation assay with Nb2 cells showed complete absence of PRL in the circulation of the transgenic PR[L.sup.-/-] mice of both sexes, whereas very high levels of the hormone were detected in the wild-type PR[L.sup.+/+] mice of both sexes. Moreover, T-H resulted in the appearance of significant levels of the hormone in circulation, but only in PR[L.sup.+/+] mice. Splenocyte proliferation in male PR[L.sup.-/-] mice was significantly lower than in PR[L.sup.+/+] mice after T-H. Marginal differences between PR[L.sup.+/+] and PR[L.sup.-/-] mice were observed in the release of IL-2 and IFN-[gamma] by splenocytes, while the release of IL-10 was significantly higher in PR[L.sup.-/-] than in PR[L.sup.+/+] mice. A significant observation of our study is the release of a ~25-kDa protein in the concanavalin A-stimulated splenocytes of male PR[L.sup.+/+] and PR[L.sup.-/-] mice that was active in the in vitro lymphoproliferation assay with Nb2 cells. It is unlikely that this protein is PRL because it is also present in the splenocyte extracts of PR[L.sup.-/-] transgenic mice. Nonetheless, because control of lymphoid cell proliferation is considered one of the characteristics of the immune system, the local release of this protein may be significant in the differences observed in splenocyte cytokine release after T-H in wild-type as well as transgenic mice. Nb2 cells; cytokines; immune functions

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

Role of IL-10 in regulating proinflammatory cytokine release by Kupffer cells following trauma-hemorrhage. Am J Physiol Gastrointest Liver Physiol 286: G942-G946, 2004. First published January 8, 2004; 10.1152/ajpgi.00502.2003.--IL-6 and TNF-[alpha] production by Kupffer cells is markedly stimulated following trauma-hemorrhage (T-H). Because IL-10 is an anti-inflammatory cytokine, the aim of this study was to determine whether IL-I0 regulates Kupffer cell proinflammatory cytokine release following T-H. To study this, we subjected adult male Sprague-Dawley rats to sham operation or T-H. The procedure involved a 5-cm midline laparotomy and ~90 min of hemorrhagic shock (35 mmHg), followed by resuscitation with four times the shed blood volume in the form of Ringer's lactate. At 2 h after the end of resuscitation, livers were perfused in vitro and perfusate was collected. In separate studies, Kupffer cells were isolated and incubated with different concentrations of anti-IL-10 MAb. IgG was used as control. After 16 h of incubation, IL-6 and TNF-[alpha] levels were measured by ELISA. Plasma IL-10 levels increased significantly following T-H. IL-10 levels in the perfusate and IL-10 production by cultured Kupffer cells were also significantly higher in the T-H group. When Kupffer cells were incubated with 10 [micro]g/ml of anti-IL-10 MAb, IL-6 and TNF-[alpha] production were significantly increased in both sham and T-H groups compared with those not treated with anti-IL-10 MAb. However, these changes were not observed when the cells were incubated with irrelevant (control) IgG. These results indicate that IL-10 production by Kupffer cells early after T-H may play a pivotal role in attenuating the proinflammatory cytokine environment, possibly in an autocrine/paracrine manner. isolated liver perfusion; anti-inflammatory cytokine; anti-interleukin-10 antibodies

Although gender differences exist in cardiovascular endothelial function, it remains unclear whether such differences are also seen in small intestinal endothelial function. To determine this, untreated male, age-matched proestrus female, castrated male, and 17[beta]-estradiol (E2)-treated noncastrated male rats were studied. Dose response curves to ACh and nitroglycerin (NTG) were determined by measuring changes in perfusion pressure by using an isolated small intestinal perfusion model. Endothelium-derived nitric oxide (NO) production/release was indirectly determined by the ability of intact endothelium to suppress serotonin ([10.sup.-5] M)-induced perfusion pressure changes. Intestinal tissue levels of NO were also measured. Moreover, plasma levels of androgen and E2 were determined and correlated with ACh ([10.sup.-8] M)-induced perfusion pressure reductions. ACh-induced intestinal perfusion pressure reductions in proestrus females, castrated males, and E2-treated noncastrated males were significantly higher than in untreated males. NTG-induced perfusion pressure reductions were not significantly different among groups. Perfusion pressures after administration of serotonin ([10.sup.-5] M) and intestinal tissue levels of NO in proestrus females, castrated males, and E2-treated noncastrated males were also significantly higher than in untreated males. Plasma androgen levels in proestrus females, castrated males, and in E2-treated noncastrated males were significantly lower compared with untreated males. There was a positive correlation between plasma androgen and ACh-reduced perfusion pressure; however, E2 levels did not show a similar relationship. Thus androgens appear to play an inhibitory role in small intestinal endothelial function. These properties in male vessels can be modulated by decreasing the level of circulating androgens or by E2 treatment. male; female; endothelium; acetylcholine; serotonin; nitroglycerin

Thromboxane [A.sub.2] (TX[A.sub.2]) and endothelin-1 (ET-1) have been proposed as the important vasoconstrictors that increase portal venous resistance in paracrine or autocrine fashion. We hypothesized that the hepatic damage following trauma-hemorrhage (T-H) is induced by the impaired hepatic circulation due to the increased production of vasoconstrictors such as ET-1 and TX[A.sub.2] by the liver. To test this, male Sprague-Dawley rats (n = 6/group) were subjected to trauma (i.e., midline laparotomy) and hemorrhage (35-40 mmHg for 90 min followed by fluid resuscitation) or sham operation. At 2 or 5 h after the end of resuscitation, the liver was isolated and perfused and portal inflow pressure, bile flow, and release of ET-1 and thromboxane [B.sub.2] (TX[B.sub.2]; a stable metabolite of TX[A.sub.]2) into the perfusate were measured. The level of portal pressure was higher at 5 h following T-H compared with 2 h after T-H and sham. The portal pressure was inversely correlated to the amount of bile production. Furthermore, the bile flow was significantly correlated to the hepatic damage as evidenced by release of lactate dehydrogenase into the perfusate. The level of ET-1 at 5 h following T-H in the perfusate after 30 min of recirculation did not show any difference from sham. However, the levels of TX[B.sub.2] in the T-H group were significantly higher than those in sham at that interval. These results indicate that the increased release of TX[A.sub.2] but not ET-1 following T-H might be responsible for producing the increased portal resistance, decreased bile production, and hepatic damage. endothelin; portal venous pressure; bile flow

Although immune functions are markedly depressed in males and not in proestrous females following trauma-hemorrhage (T-H), the mechanisms responsible for the divergent responses remain unknown. Because sex steroids modulate the activation of p38, our aim was to determine whether differences in the activation of p38 by phosphorylation (p38-P) might contribute to the sex-dimorphic immune response following T-H. The effects of testosterone and estradiol on the activation of p38 were also examined. Intact male mice (C3H/HeN), castrated males treated with vehicle, 5[alpha]-dihydrotestosterone (DHT), or 17[beta]-estradiol, and proestrous females were subjected to trauma (i.e., midline laparotomy) and hemorrhagic shock (35 [+ or -] 5 mmHg for 90 min and resuscitation) or sham operation. At 2 h thereafter, splenic (SM[phi]) and peritoneal macrophages (PM[phi]) were harvested and cultured (with 10 [micro]g/ml LPS), and Western blot analysis was carried out for quantification of p38 and p38-P. Sex, testosterone and estradiol plasma levels, and T-H did not alter the constitutive expression of p38 in SM[phi] and PM[phi]. In contrast, the activated form of p38 (p38-P) was markedly increased in SM[phi] and PM[phi] from female shams compared with male shams. Moreover, the phosphorylation of p38-P increased in males after T-H, whereas it decreased in females under those conditions. Castration before T-H prevented the increase in p38-P in males. Castrated animals treated with DHT displayed increased p38-P following T-H, whereas 17[beta]-estradiol had no effect on p38-P in castrated mice. Thus 1) sex influences the activation of p38 MAP kinase, 2) DHT is responsible for the increased activation of p38 in male mice, and 3) this sex-specific activation of p38 might be responsible for the sexually dimorphic immune response following T-H. cell signaling; sex steroids; hemorrhage; immune depression; macrophages

Trauma-hemorrhage and resuscitation (TH) produces profound immunodepression and enhances susceptibility to sepsis in males but not in proestrus females, suggesting gender dimorphism in the immune responses. However, the mechanism responsible for the maintenance of immune functions in proestrus females after TH is unclear. Splenic T lymphocytes express receptors for estrogen (ER), contain enzymes involved in estrogen metabolism, and are the major source of cytokine production; the metabolism of 17[beta]-estradiol was assessed in the splenic T lymphocytes of proestrus and ovariectomized mice by using appropriate substrates after TH. Analysis for aromatase and 17[beta]-hydroxysteroid dehydrogenases indicated increased 17[beta]-estradiol synthesis and low conversion into estrone in T lymphocytes of proestrus but not of ovariectomized mice. The effect of 17[beta]-estradiol on T lymphocyte cytokine release was reliant on ER expressions. This was apparent in the differences of ER expression, especially that of ER-[beta], and an association between increased 17[beta]-estradiol synthesis and sustained release of IL-2 and IL-6 in T lymphocytes of proestrus females after TH. Because 17[beta]-estradiol is able to regulate cytokine genes, and the splenic T lymphocyte cytokine releases is altered after TH, continued synthesis of 17[beta]-estradiol in proestrus females appears to be responsible for the maintenance of T lymphocyte cytokine release associated with the protection of immune functions after TH. inflammation; immune suppression; steroid synthesis; T lymphocytes; cytokines

Proestrous female rodents are protected from the deleterious effects of trauma-hemorrhage that are observed in males. We hypothesized that the gender dimorphic outcome after trauma-hemorrhage might be related to gender differences in endothelial function and organ perfusion under such conditions. Male and cycle-matched proestrous female Sprague-Dawley rats underwent a midline laparotomy, hemorrhagic shock (40 mmHg for ~90 min), and resuscitation (Ringer lactate, 4x shed blood volume over 60 min). Various parameters were measured 2 h after completion of resuscitation. In the first set of animals, the left ventricle was cannulated and heart performance (maximal rate of left ventricular pressure increase) as well as cardiac output and organ perfusion rates were determined with [sup.85]Sr microspheres. In the second set of animals, aortic vessel rings were harvested and relaxation in response to acetylcholine and nitroglycerin was measured. In the third set of animals, in situ isolated small intestine was perfused to measure the response of the splanchnic vessel bed to acetylcholine and nitroglycerin. After trauma-hemorrhage and resuscitation, females maintained cardiac output and demonstrated increased splanchnic and cardiac perfusion compared with males. Moreover, female intestines did not manifest the endothelial dysfunction that was observed in male intestines after hemorrhagic shock. We conclude that proestrous females show improved endothelial function and tissue perfusion patterns after hemorrhagic shock and that this gender-specific response might be a potential mechanism contributing to the beneficial effects of the proestrus stage under such conditions. cardiac output; endothelial function; coronary artery; gut; liver

Hyperglycemia is an early metabolic response to trauma and hemorrhage. The role of hepatic insulin resistance to the development of this hyperglycemia is not well understood. The aim of this study was to determine whether the liver becomes insulin resistant and to identify the particular hepatic insulin signaling pathways that may be compromised following trauma and hemorrhage. Male adult rats were bled to a mean arterial pressure of 40 mmHg and maintained at that pressure for 90 min followed by resuscitation with Ringer lactate. Data showed that trauma and hemorrhage rapidly induced profound hyperinsulinemia in combination with significant hyperglycemia, suggesting the development of insulin resistance. After trauma and hemorrhage, hepatic insulin signaling via the insulin-induced phosphatidylinositol 3 (PI3)-kinase-Akt pathway was abolished, whereas ERK1/2 signaling was relatively normal. The regulation (inhibition) of a hepatic-, insulin-, and the PI3-kinase-dependent gene, IGF binding protein-1, was also lost. The present study provides convincing evidence of a rapid onset hepatic insulin resistance following a combination of trauma and hemorrhage. liver; shock

The acute respiratory distress syndrome (ARDS) is a major cause of morbidity after injury. We hypothesized that alveolar macrophage (AM[PHI]) chemokine and cytokine release after hemorrhage and sepsis is regulated by NF-[kappa]B and MAPK. Adult male rats underwent soft tissue trauma and hemorrhagic shock (~90 min) followed by crystalloid resuscitation. Sepsis was induced by cecal ligation and puncture (CLP) 20 h after resuscitation. AM[PHI] were harvested, and TNF-[alpha], IL-6, and macrophage inflammatory protein (MIP)-2 release and serum IL-6 and TNF-[alpha] levels were measured at 5 h after HCLP. Lung tissues were analyzed for activation of NF-[kappa]B, myeloperoxidase activity, and wet/dry weight ratio. In control animals, AM[PHI] were stimulated with LPS with or without inhibitors of NF-[kappa]B and MAPK. Serum TNF-[alpha] and IL-6 levels and spontaneous AM[PHI] TNF-[alpha] and MIP-2 release were elevated (P < 0.05) after HCLP, concomitantly with the development of lung edema and leukocyte activation. Activation of NF-[kappa]B increased in lungs from the hemorrhage and CLP group compared with shams. Inhibition of NF-[kappa]B or the upstream MAPK significantly decreased LPS-stimulated AM[PHI] activation. Because enhanced release of inflammatory mediators by AM[PHI] may contribute to ARDS after severe trauma, inhibition of intracellular signaling pathways represents a target to attenuate organ injury under those conditions. mitogen-activated protein kinase; leukocytes; macrophage inflammatory protein; acute respiratory distress syndrome; extracellular signal-regulated kinase; nuclear factor-[kappa]B

Adrenomedullin (AM), a potent vasodilatory peptide, plays an important role in initiating the hyperdynamic response during the early stage of sepsis. Moreover, the reduced vascular responsiveness to AM appears to be responsible for the transition from the early, hyperdynamic to the late, hypodynamic phase of sepsis. Although the novel specific AM binding protein-1 (AMBP-1) enhances AM-mediated action in a cultured cell line, it remains to be determined whether AMBP-1 plays any role in modulating vascular responsiveness to AM during sepsis. To study this, adult male rats were subjected to sepsis by cecal ligation and puncture (CLP). The thoracic aorta was harvested for determination of AM-induced vascular relaxation. Aortic levels of AMBP-1 were determined by Western blot analysis, and AM receptor gene expression in the aortic tissue was assessed by RT-PCR. The results indicate that AMBP-1 significantly enhanced AM-induced vascular relaxation in aortic rings from sham-operated animals. Although vascular responsiveness to AM decreased at 20 h after CLP (i.e., the late, hypodynamic stage of sepsis), addition of AMBP-1 in vitro restored the vascular relaxation induced by AM. Moreover, the aortic level of AMBP-1 decreased significantly at 20 h after CLP. In contrast, AM receptor gene expression was not altered under such conditions. These results, taken together, suggest that AMBP-1 plays an important role in modulating vascular responsiveness to AM, and the reduced AMBP-1 appears to be responsible for the vascular AM hyporesponsiveness observed during the hypodynamic phase of sepsis. vasoactive peptide; complement factor H; adrenomedullin receptors; aorta; vascular relaxation; cecal ligation and puncture

Trauma-hemorrhage produces profound immuno-suppression in males but not in proestrus females. Prior castration or flutamide treatment of males following trauma-hemorrhage prevents immunosuppression, implicating 5[alpha]-dihydrotestosterone for the immunosuppressive effects. 5[alpha]-Dihydrotestosterone, a high-affinity androgen receptor-binding steroid, is synthesized in tissues as needed and seldom accumulates. The presence of steroidogenic enzymes in T lymphocytes suggests both synthesis and catabolism of 5[alpha]-dihydrotestosterone. We hypothesized, therefore, that the basis for high 5[alpha]-dihydrotestosterone activity in T lymphocytes of males following trauma-hemorrhage is due to decreased catabolism. Accordingly, catabolism of 5[alpha]-dihydrotestosterone was assessed in splenic T lymphocytes by examining the activity and expression of enzymes involved. Analysis showed increased synthesis and decreased catabolism of 5[alpha]-dihydrotestosterone in intact male T lymphocytes following trauma-hemorrhage. In contrast, reduced 5[alpha]-reductase activity and increased expression of 17[beta]-hydroxysteroid dehydrogenase oxidative isomers suggest inactivation of 5[alpha]-dihydrotestosterone in precastrated males. Thus our study suggests increased synthesis and decreased catabolism of 5[alpha]-dihydrotestosterone as a reason for loss of T lymphocyte functions in intact males following trauma-hemorrhage, as evidenced by decreased release of interleukin-2 and -6. gonadal steroids; androgen metabolism; cytokines

Hypoxemia depresses cell-mediated immune functions in males, whereas proestrous females do not show such a depression. We hypothesized that elevated systemic estradiol levels in proestrous females prevent hypoxemia-induced immune depression. To study this hypothesis, male C3H/HeN mice were pretreated with 17[beta]-estradiol ([E.sub.2], 40 [micro]g/kg body wt sc) or vehicle for 3 days before induction of hypoxemia and again immediately before induction of hypoxia. The mice were subjected to hypoxemia (95% [N.sub.2]-5% [O.sub.2]) or sham hypoxemia (room air) for 60 min, and plasma and spleen cells were collected 2 h later. In vehicle-treated mice, splenocyte proliferation and interleukin-2 and interleukin-3 production were depressed after hypoxemia. [E.sub.2]-pretreated animals, however, displayed no such depression in splenic T cell parameters after hypoxemia. Splenic macrophage cytokine production was also depressed in vehicle-treated mice subjected to hypoxia, whereas it was normal in [E.sub.2]-pretreated mice. In summary, these findings indicate that administration of [E.sub.2] before hypoxemia prevented the depression of cell-mediated immune functions. Thus administration of 17[beta]-estradiol in high-risk patients before major surgery might decrease hypoxemia-induced immune depression under those conditions. inflammation; cytokines; estradiol; gender

Cyclooxygenase 2-mediated suppression of macrophage interleukin-12 production after thermal injury. Am J Physiol Cell Physiol 282: C263-C270, 2002; 10.1152/ajpcell.00357. 2001.--Macrophage (M[??]) prostaglandin (PG)[E.sub.2] production has been implicated in immunosuppression and increased susceptibility to sepsis after thermal injury. Deficient interleukin (IL)-12 production has also been implicated in these postburn complications. The present study examined the relationship between M[??] cyclooxygenase (COX)-2 activity and IL-12 production after thermal injury. C57BL/6 female mice were subjected to a 25% total body surface area full-thickness burn. M[??] were isolated 7 days later, or the mice were subjected to sepsis by cecal ligation and puncture (CLP). IL-12 production by M[??] from injured mice was suppressed by >50%, whereas COX-2 expression and PG[E.sub.2] production were increased twofold. The COX-2 inhibitor NS-398 suppressed PG[E.sub.2] production and normalized IL-12 production in the injury group, whereas it had no effect on IL-10 production. Injured mice subjected to CLP had lower IL-12 plasma levels compared with sham-treated mice subjected to CLP. NS-398 treatment prevented the suppression in plasma IL-12 levels in the injury group. Thus elevated M[??] COX-2 activity, independent of IL-10, suppresses M[??] IL-12 production after thermal injury and may play an important role in the observed immunosuppression under such conditions. prostaglandin [E.sub.2]; interleukin-10; immunosuppression; sepsis; burns

Research was conducted to examine the effects of depletion of male sex steroids by castration of male mice before mycobacterium infection. Male C3H/HeN mice were castrated and treated with pellets having either a vehicle or a combination of both steroid hormones for 14 days prior to soft tissue trauma and hemorrhagic shock or a sham operation. Results showed that castration increased the host resistance to infection. Findings indicate that male and female sex steroids exhibit divergent immunomodulatory characteristics with respect to cell-mediated immune responses after trauma-hemorrhage.

A hypoxemia-induced increase in macrophage-inducible nitric oxide synthase (iNOS) activity following hemorrhage may be partly the reason for the immune cell dysfunction similar to that associated with hemorrhagic shock. Male Sprague-Dawley rats were used to investigate whether hypoxia per se in the absence of any blood loss is a sufficient stimulus to cause iNOS expression and NO production by macrophages. Results showed that elevated expression of iNOS mRNA at 1.5 h after hypoxia preceded the increase in NO production by macrophages.