Search

We hypothesized that administration of androgen receptors antagonist flutamide following trauma-hemorrhage (T-H) in metestrus females will maintain immune function and reduce remote organ damage under those conditions. Female B57BL/J6 mice (metestrus state, 8–12 weeks old) underwent laparotomy and hemorrhagic shock (35.0±5.0 mmHg for 90 min) and then received 17β-estradiol (E2; 50 μg/25 g), flutamide (625 μg/25 g), or E2 + flutamide. Four hours after resuscitation, plasma cytokine and chemokine (TNF-α, IL-6, IL-10, IFN-γ, and MCP-1) concentrations and their release in vitro by hepatic and pulmonary tissue macrophages (MΦ) were determined by flow cytometry. Organ damage was assessed by edema formation (wet-to-dry weight ratio) and neutrophil infiltration [myeloperoxidase (MPO) activity]. Administration of E2, flutamide, or E2 + flutamide following T-H resulted in a significant decrease in systemic TNF-α, IL-6, and MCP-1 concentrations under those conditions. This was accompanied by significantly decreased in vitro TNF-α release by Kupffer cells after administration of E2, flutamide, or E2 + flutamide. The in vitro release of proinflammatory cytokines by alveolar MΦ, however, was reduced significantly only by the addition of E2 or E2 + flutamide but not by the addition of flutamide. A significant decrease in pulmonary and hepatic edema formation as well as neutrophil infiltration in the lung was observed after E2, flutamide and E2 + flutamide administration. In contrast, hepatic neutrophil infiltration was only significantly reduced following E2 and E2 + flutamide administration. Thus, although flutamide does not produce synergistic, salutary effects with E2, its administration in females following T-H also produces salutary effects on the immune and organ function, similar to E2 administration under those conditions.

Recent findings from our laboratory have shown that acute alcohol (EtOH) intoxication before burn injury impairs intestinal immunity and barrier functions. To further delineate the mechanism of impaired intestinal barrier function, the present study examined the role of corticosterone (CORT) and interleukin (IL)-18, as CORT and IL-18 are elevated following a combined insult of EtOH intoxication and burn injury. Male rats (∼250 g) were gavaged with EtOH to achieve a blood EtOH level of ∼100 mg/dL prior to burn or sham injury (25% total body surface area). Immediately after injury, a group of rats was treated with CORT synthesis inhibitor metyrapone (25 mg/kg), with or without recombinant (r)IL-18 (50 μg/kg). Another group of rats was treated with caspase-1 inhibitor Ac-YVAD-CHO to block IL-18 production. On Day 1 after injury, there was a significant increase in blood CORT levels, intestinal levels of IL-18, neutrophil chemokines [cytokine-induced neutrophil chemoattractant 1 (CINC-1) and CINC-3], intercellular adhesion molecule-1, myeloperoxidase activity, and intestinal permeability in rats receiving a combined insult of EtOH and burn injury. Treatment of rats with CORT inhibitor or with caspase-1 inhibitor prevented the increase in all of the above parameters following a combined insult of EtOH and burn injury. Moreover, coadministration of rIL-18 in metyrapone-treated rats restored the above parameters, similar to those observed in rats receiving EtOH and burn injury. These findings suggest that a combined insult of EtOH and burn injury results in increased CORT levels, which in turn up-regulates intestinal IL-18 levels and thereby causes altered intestinal barrier function following a combined insult of EtOH intoxication and burn injury.

Thermal injury induces an inflammatory response that contributes to the development of secondary tissue damage. Neutrophil recruitment and activation are in part responsible for this tissue damage. Although gammadelta T cells have been shown to regulate the inflammatory responses in tissues that are prone to neutrophil-mediated injury post-burn, their role in the induction of secondary tissue injury post-burn remains unknown. To study this, gammadelta T cell-deficient (gammadelta TCR-/-) and wild-type (WT) mice were subjected to thermal injury or sham procedure, and tissue samples were isolated 1-24 h thereafter. Burn injury induced neutrophil accumulation in the lung and small intestines of WT mice at 1-3 h post-injury. No such increase in neutrophil tissue content was observed in gammadelta TCR-/- mice. An increase in tissue wet/dry weight ratios was also observed in these organs at 3 h post-burn in WT but not in gammadelta TCR-/- mice. A parallel increase in plasma and small intestine levels of the chemokines macrophage-inflammatory protein-1beta (chemokine ligand 4) and keratinocyte-derived chemokine (CXC chemokine ligand 1) were observed in injured WT mice but not in injured gammadelta TCR-/- mice. Increased activation (CD120b expression) of the circulating gammadelta T cell population was also observed at 3 h post-burn in WT mice. These results indicate the gammadelta T cells, through the production of chemokines, play a central role in the initiation of neutrophil-mediated tissue damage post-burn.

Studies have shown that cell-mediated immunity is markedly suppressed after thermal injury. T lymphocyte dysfunction and macrophage hyperactivity have been implicated as causative factors. Previous studies have primarily examined the effects of thermal injury on αβ T lymphocytes; however, the role of γδ T lymphocytes in the immune response after thermal injury is unclear. Therefore, wild-type mice and mice lacking the TCR δ gene (TCR δ−/−) were subjected to a third-degree scald burn and cell-mediated immune responses assessed at 7 days post-injury. TCR δ−/−mice had 75% mortality after burn injury compared with 25% mortality in the wild-type group. Plasma interluekin-6 (IL-6) levels were significantly elevated at 2, 4, and 18 h post-injury, whereas no difference was observed in tumor necrosis factor α (TNF-α) and prostaglandin E2 (PGE2) plasma levels. Plasma levels of these inflammatory mediators were similar in wild-type and TCR δ−/-_ mice post-injury. Splenic macrophage PGE2, IL-6, TNF-α, and IL-10 production was significantly increased in wild-type mice at 7 days post-injury, whereas macrophages from injured TCR δ−/− mice had a significantly attenuated capacity to produce IL-6 and TNF-α. In contrast, the increased release of PGE2 and IL-10 by macrophages post-injury was not reduced in TCR δ−/− mice. These results implicate a dual role for γδ T lymphocytes in the immunopathogenic response to burn injury: (1) they contribute to survival from the insult; and (2) they mediate the induction of a pro-inflammatory macrophage phenotype at 7 days post-injury. Thus, γδ T lymphocytes, in part through the modulation of macrophage activity, appear to contribute to the immune dysfunction after thermal injury. J. Leukoc. Biol. 67: 644–650; 2000.

17β-Estradiol (i.e., estrogen or E2) is a female sex steroid, which plays an essential role in female reproductive physiology. However, several lines of evidence indicate that in addition to its role in reproductive physiology, E2 is critical for maintaining many other organ functions in stress conditions. These include immune, cardiovascular, and neuronal functions, as well as regulation of skin, bone, and lipid metabolism. Studies have examined the role of E2 as an adjunct in post-trauma responses, and this article will review whether E2 as an adjunct to fluid resuscitation following trauma-hemorrhage plays any role in improving/restoring immune and cardiovascular functions.

Protein kinase B (Akt) is known to be involved in proinflammatory and chemotactic events in response to injury. Akt activation also leads to the induction of heme oxygenase (HO)-1. Up-regulation of HO-1 mediates potent, anti-inflammatory effects and attenuates organ injury. Although studies have shown that 17β-estradiol (E2) prevents organ damage following trauma-hemorrhage, it remains unknown whether Akt/HO-1 plays any role in E2-mediated attenuation of hepatic injury following trauma-hemorrhage. 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, E2 (1 mg/kg body weight), E2 plus the PI-3K inhibitor (Wortmannin), or the estrogen receptor (ER) antagonist (ICI 182,780). At 2 h after sham operation or trauma-hemorrhage, plasma α-GST and hepatic tissue myeloperoxidase (MPO) activity, IL-6, TNF-α, ICAM-1, cytokine-induced neutrophil chemoattractant-1, and MIP-2 levels were measured. Hepatic Akt and HO-1 protein levels were also determined. Trauma-hemorrhage increased hepatic injury markers (α-GST and MPO activity), cytokines, ICAM-1, and chemokine levels. These parameters were markedly improved in the E2-treated rats following trauma-hemorrhage. E2 treatment also increased hepatic Akt activation and HO-1 expression compared with vehicle-treated, trauma-hemorrhage rats, which were abolished by coadministration of Wortmannin or ICI 182,780. These results suggest that the salutary effects of E2 on hepatic injury following trauma-hemorrhage are in part mediated via an ER-related, Akt-dependent up-regulation of HO-1.

As studies indicate that genomic and nongenomic pathways are involved in mediating the salutary effects of 17β-estradiol (E2) following trauma-hemorrhage, we examined if the nongenomic effects of E2 on attenuation of intestinal injury after trauma-hemorrhage involve the PI-3K/Akt pathway. Male Sprague-Dawley rats (∼300 g body weight) underwent trauma-hemorrhage (mean blood pressure 40 mmHg for 90 min), followed by resuscitation. E2 conjugated to BSA (E2-BSA; 1 mg/Kg E2), with or without an estrogen receptor antagonist (ICI 182,780), a PI-3K inhibitor (Wortmannin), or vehicle, was injected i.v. during resuscitation. At 2 h after trauma-hemorrhage or sham operation, intestinal myeloperoxidase (MPO) activity, ICAM-1, cytokine-induced neutrophil chemoattractant (CINC)-1, CINC-3, and IL-6 levels were measured (n=6 rats/group). Intestinal PI-3K, phosphorylation of Akt (p-Akt), and Akt protein expressions were also determined. One-way ANOVA and Tukey’s test were used for statistical analysis. The results indicated that trauma-hemorrhage increased intestinal MPO activity and ICAM-1, CINC-1, CINC-3, and IL-6 levels. These parameters were improved significantly in the E2- or E2-BSA-treated rats subjected to trauma-hemorrhage. Although trauma-hemorrhage decreased intestinal PI-3K and p-Akt protein expressions, E2 or E2-BSA treatment following trauma-hemorrhage prevented such decreases in intestinal PI-3K and p-Akt protein expressions. Coadministration of ICI 182,780 or Wortmannin abolished the beneficial effects of E2-BSA on attenuation of intestinal injury following trauma-hemorrhage. Thus, the PI-3K/Akt pathway plays a critical role in mediating the nongenomic, salutary effects of E2 on attenuation of shock-induced intestinal tissue damage.

The depression in cell-mediated immune function following trauma-hemorrhage is shown to be restored by 17β-estradiol (E2) administration. However, it remains unknown which of the two estrogen-receptors, (ER)-α or ER-β, plays the predominant role in mediating the beneficial effects of E2. Female B57BL/J6 ER-β−/− transgenic mice [knockout (KO)] and corresponding ovariectomized wild-type (WT) mice were subjected to laparotomy and hemorrhagic shock (35.0±5.0 mmHg for 90 min) and treated with E2 (50 μg/25 g) or ER-α agonist propyl pyrazole triol (PPT; 50 μg/25 g) following trauma-hemorrhage. Four hours after resuscitation, systemic cytokine concentrations and cytokine release by splenocytes and splenic macrophages were determined by cytometric bead array. Trauma-hemorrhage resulted in a significant increase in plasma tumor necrosis factor α (TNF-α), interleukin (IL)-6, and IL-10. In contrast, the release of these cytokines by splenic macrophages was decreased significantly in WT and KO animals. Administration of E2 or PPT following trauma-hemorrhage produced a significant reduction in systemic TNF-α and IL-6 concentrations in WT and KO mice. Although the suppression in the productive capacity of these cytokines following trauma-hemorrhage by macrophages and splenocyte was also prevented in E2- and PPT-treated WT mice, the release of cytokines by macrophages and splenocytes in E2- and PPT-treated KO mice was not restored to the levels observed in sham animals. These findings collectively suggest that both receptors appear to play a significant role in mediating the immunoprotective effects of E2 in different tissue compartments following trauma-hemorrhage.

Although 17β-estradiol (E2) administration after trauma-hemorrhage (T-H) reduces tissue neutrophil sequestration in male rodents, it remains unknown which of the estrogen receptor (ER) subtypes mediates this effect and whether the same ER subtype is involved in all the tissues. We hypothesized that the salutary effects of E2 on attenuation of neutrophil accumulation following T-H are tissue and receptor subtype-specific. Male Sprague-Dawley rats underwent sham operation or T-H (mean blood pressure, 40 mmHg for 90 min and then resuscitation). E2 (50 μg/kg), ER-α agonist propyl pyrazole triol (PPT; 5 μg/kg), ER-β agonist diarylpropiolnitrile (DPN; 5 μg/kg), or vehicle (10% dimethyl sulfoxide) was administered subcutaneously during resuscitation. Twenty-four hours thereafter, tissue myeloperoxidase (MPO) activity (a marker of neutrophil sequestration), cytokine-induced neutrophil chemoattractant (CINC)-1, CINC-3, and intercellular adhesion molecule (ICAM)-1 levels in the liver, intestine, and lung were measured (n=6 rats/group). ER-α and ER-β mRNA levels in sham-operated rats were also determined. T-H increased MPO activity, CINC-1, CINC-3, and ICAM-1 levels in the liver, intestine, and lung. These parameters were improved significantly in rats receiving E2 after T-H. Administration of the ER-α agonist PPT but not the ER-β agonist DPN improved the measured parameters in the liver. In contrast, DPN but not PPT significantly improved these parameters in the lung. In the intestine, ER subtype specificity was not observed. ER-α mRNA expression was highest in the liver, whereas ER-β mRNA expression was greatest in the lung. Thus, the salutary effects of E2 administration on tissue neutrophil sequestration following T-H are receptor subtype and tissue-specific.

This study examined the role of protein phosphatase type-1 (PP1), type-2A (PP2A), and mitogen-activated protein kinase phosphatase-1 (MKP-1) in altered mesenteric lymph node (MLN) T cell function in a two-hit model of alcohol (EtOH) intoxication and burn injury. Male rats (250 g) were gavaged with EtOH to achieve a blood EtOH level of ∼100 mg/dL prior to burn or sham injury (25% total body surface area). MLN T cells harvested 24 h after injury show a significant decrease in p38 and extracellular signal-regulated kinase (ERK)-1/2 phosphorylation in T cells from rats receiving a combined insult of EtOH intoxication and burn injury compared with rats receiving EtOH intoxication or burn injury alone. Treatment of cells with inhibitors of PP1/PP2A [calyculin A (CA) and okadaic acid (OA)] prevented the suppression in T cells p38 and ERK-1/2 activation. In addition, the suppression in interleukin-2 and interferon-γ production was attenuated in T cells cultured in the presence of CA and OA. MKP-1 inhibitor triptolide did not prevent the suppression in T cells p38/ERK-1/2 and cytokine production. Furthermore, there was a significant decrease in PP1α phosphorylation (Thr320) and an increase in PP2A (Tyr307) phosphorylation in T cells following a combined insult of EtOH intoxication and burn injury. As phosphorylation of PP1 at Thr320 and PP2A at Tyr307 led to an inhibition of their enzymatic activities, the decrease in the PP1α phosphorylation correlates with an increase in its enzyme activity. Thus, these results suggest that activation of PP1 is likely to play a predominant role in T cell suppression following a combined insult of EtOH intoxication and burn injury.

Hemeoxygenase (HO)-1 induction following adverse circulatory conditions is known to be protective, and precastrated males have less intestinal damage than sham-operated males following trauma-hemorrhage (T-H). Previous studies have also shown that administration of flutamide up-regulated estrogen receptor (ER) expression in males following T-H. We hypothesized that flutamide administration in males following T-H up-regulates HO-1 via an ER-dependent pathway and protects against intestinal injury. Male Sprague-Dawley rats underwent T-H [mean blood pressure (MBP) 40 mmHg for 90 min and then resuscitation]. A single dose of flutamide (25 mg/kg body weight), with or without an ER antagonist (ICI 182,780), a HO enzyme inhibitor [chromium-mesoporphyrin (CrMP)], or vehicle, was administered subcutaneously during resuscitation. At 2 h after T-H or sham operation, intestinal myeloperoxidase (MPO) activity, intercellular adhesion molecule (ICAM)-1, cytokine-induced neutrophil chemoattractant (CINC)-1, and CINC-3 levels were measured. Intestinal ER-α, ER-β, androgen receptor, and HO-1 mRNA/protein levels were also determined. Results showed that T-H increased intestinal MPO activity, ICAM-1, CINC-1, and CINC-3 levels. These parameters were improved significantly in the flutamide-treated rats subjected to T-H. Flutamide treatment increased intestinal HO-1 and ER-β mRNA/protein levels as compared with vehicle-treated T-H rats. Administration of the ER antagonist ICI 182,780 or the HO inhibitor CrMP prevented the flutamide-induced attenuation of shock-induced intestinal damage. Thus, the salutary effects of flutamide administration on attenuation of intestinal injury following T-H are mediated via up-regulation of ER-β-dependent HO-1 expression.

Previous studies have shown that alcohol (EtOH) ingestion before burn injury impaired intestinal barrier and immune function. This study determined whether EtOH and burn injury up-regulate interleukin (IL)-18 and whether IL-18 up-regulation following EtOH and burn injury is a cause for neutrophilrecruitment and increased intestinal edema. Rats (250 g) were gavaged with EtOH to achieve a blood EtOH level in the range of 100 mg/dL prior to burn or sham injury (25% total body surface area). A group of rats was treated with Ac-YVAD-CHO (5 mg/kg), an inhibitor of caspase-1 (an enzyme that converts pro-IL-18, an inactive form of IL-18, to mature IL-18), at the time of injury. One day after injury, rats were killed. IL-18 production was determined in circulation and in the supernatants harvested from spleen, mesenteric lymph nodes, and Peyer's patch cell cultures as well as in intestinal tissue homogenates. Neutrophil accumulation in intestine was determined by measuring myeloperoxidase (MPO) activity. We found a significant increase in IL-18 levels in the lymphoid cell supernatants and intestinal tissue homogenates obtained from EtOH and burn-injured rats compared with the rats receiving burn or sham injury. This was accompanied by an increase in intestinal MPO and edema. No demonstrable change in intestinal morphology was observed in any group. Treatment of rats with caspase-1 inhibitor significantly attenuated the increase in IL-18 levels and intestinal MPO activity in EtOH and burn-injured rats. Inhibition of IL-18 also prevented an increase in intestinal tissue water content. As MPO is considered an index of neutrophil infiltration, results presented in this manuscript collectively suggest that IL-18 up-regulation is likely to contribute to the increased neutrophil infiltration and edema in intestinal tissue observed following EtOH and burn injury.

Signaling through the Fas/Fas ligand (FasL) pathway plays a central role in immune-cell response and function; however, under certain pathological conditions such as sepsis, it may contribute to the animal’s or patient’s morbidity and mortality. To determine the contribution of FasL to mortality, we conducted survival studies by blocking Fas/FasL with Fas receptor fusion protein (FasFP) in vivo. C3H/HeN mice received FasFP or the saline vehicle (veh) immediately (0 h) or delayed (12 h), after sepsis induced by cecal ligation and puncture (CLP). Subsequently, we examined the effect of FasFP treatment (12 h post-CLP) on macrophage apoptosis and functional capacities. Peritoneal and splenic macrophages and Kupffer cells from sham-veh-, CLP-veh-, sham-FasFP-, or CLP-FasFP-treated mice were harvested 24 h after CLP and stimulated with lipopolysaccharide (LPS) for 24 h. The results indicate that only delayed (12 h) but not 0 h administration of FasFP demonstrated a significant increase in survival. The ability of all macrophage populations to release interleukin (IL)-6 was significantly depressed, and IL-10 release was augmented after CLP. FasFP treatment attenuated the increased IL-10 release in Kupffer cells. However, althogh enhanced susceptibility to LPS-induced apoptosis could be suppressed in CLP mouse Kupffer cells by FasFP, FasFP did not change the peritoneal or splenic macrophage response. Furthermore, FasFP attenuated the elevated plasma levels of liver enzymes after sepsis. These data indicate that in vivo inhibition of Fas/FasL signaling has tissue-specific effects on the induction of macrophage apoptosis, functional changes, and liver damage, which may contribute to the host’s ability to ward off a septic challenge.

Prostaglandin E2 (PGE2) and macrophage (Mϕ) -derived reactive nitrogen intermediates (RNI) have been implicated in T cell dysfunction after thermal injury. Normally, Mϕ inducible nitric oxide synthase (iNOS) activity can be regulated by PGE2, however, it is unknown whether PGE2 modulates Mϕ iNOS activity after thermal injury. Splenic Mϕ isolated from mice 7 days after thermal injury produced higher levels of RNI than Mϕ from sham mice when stimulated with lipopolysaccharide (LPS) or interferon-γ (IFN-γ) and tumor necrosis factor α (TNF-α) in combination. PGE2, when added concurrently with LPS, suppressed RNI production by Mϕ from sham mice, whereas Mϕ from injured mice were unaffected. When Mϕ were pretreated with PGE2 before LPS, RNI production was suppressed in both populations. RNI production in response to IFN-γ or IFN-γ and TNF-α in combination was enhanced by PGE2 in both populations, however, the effect was markedly greater in Mϕ from injured mice. The PGE2-mediated changes in RNI production were paralleled by similar changes in iNOS protein expression, suggesting that the effect of PGE2 was at the level of enzyme expression rather than activity. Dibutryl cAMP induced similar effects as PGE2, suggesting the response to PGE2 after thermal injury is independent of potential changes in PGE2-induced adenylate cyclase activity and is cAMP-mediated. The results indicate that Mϕ from burned mice display an altered sensitivity to PGE2, resulting in enhanced iNOS activity. Thus, PGE2, which is elevated after thermal injury and can directly suppress T cell function, may also contribute to immune dysfunction through the enhancement of Mϕ iNOS activity. J. Leukoc. Biol. 64: 740–746; 1998.