17 beta-Estradiol normalizes immune responses in ovariectomized females after trauma-hemorrhage.

Recent studies indicate that immune responses in proestrus females are maintained after trauma-hemorrhage but markedly depressed in ovariectomized females under such conditions. The current study tested the hypothesis that the decreased estrogen levels after ovariectomy are responsible for this immune depression. To study this hypothesis, ovariectomized female CBA/J mice were subjected to laparotomy (i.e., soft tissue trauma) and hemorrhagic shock (35 +/- 5 mmHg for 90 min, then resuscitated) or sham operation. The mice received either 17 beta-estradiol (E2; 100 microg/25 g body wt) or vehicle subcutaneously during resuscitation. Immune cells were isolated 24 h thereafter. Splenocyte proliferation and interferon-gamma, interleukin (IL)-2, and IL-3 release were significantly depressed after trauma-hemorrhage in vehicle-treated mice, whereas these functions were maintained in E2-treated mice. Peritoneal macrophage IL-1 beta and IL-6 release and splenic macrophage IL-6 and IL-12 release were also significantly depressed in vehicle-treated mice after trauma-hemorrhage, and release of these cytokines was restored by E2 treatment. In summary our findings indicate that the depressed splenic and peritoneal immune responses after trauma-hemorrhage can be normalized by a single dose of E2. Thus estrogen appears to be the causative factor in the maintenance of immunocompetence in females after trauma-hemorrhage, and its administration to ovariectomized or postmenopausal females should be helpful in preventing immune depression under such conditions.

Resistance of macrophages to the suppressive effect of interleukin-10 following thermal injury.

The activation of a macrophage (Mphi)-dependent proinflammatory cascade following thermal injury plays an important role in the development of immunosuppression and increased susceptibility to subsequent sepsis in burn patients. In contrast, although interleukin (IL)-10, an anti-inflammatory cytokine that can downregulate M phi activity, has also been implicated in postburn immune dysfunction, its role in the regulation of M phi function postburn remains unclear. To study this, C57BL/6 female mice were subjected to a 25% total body surface area third-degree scald burn, and splenic Mphis were isolated 7 days later. Lipopolysaccharide (LPS)-stimulated IL-10, IL-6, tumor necrosis factor (TNF)-alpha, and nitric oxide (NO) production were significantly increased in the burn group compared with shams. Blockade of endogenous IL-10 activity enhanced IL-6 and TNF-alpha release, but not NO release, in both groups. The addition of exogenous IL-10 to the M phi cultures dose dependently suppressed production of these inflammatory mediators in both groups. The timing of IL-10 addition to the cultures in relation to LPS stimulation, however, was critical. The suppressive effect of exogenous IL-10 was attenuated in both groups when the cells were exposed to IL-10 at 4-6 h after LPS stimulation; however, Mphis from injured mice were significantly better able to maintain inflammatory mediator-productive capacity. The resistance of Mphis from injured mice to IL-10-mediated suppression correlated with decreased IL-10 receptor (IL-10R) expression and increased CD11b expression. These findings suggest that Mphis, following thermal injury, display resistance to suppression by IL-10 due in part to downregulation of IL-10R expression.

Divergent immune responses in male and female mice after trauma-hemorrhage: dimorphic alterations in T lymphocyte steroidogenic enzyme activities.

Immune responses are suppressed in males, but not in proestrous females, after trauma-hemorrhage. Testosterone and 17beta-estradiol appear to be responsible for divergent immune effects. There is considerable evidence to suggest sex steroid hormone involvement in immune functions. As formation of active steroid depends on the activity of androgen- and estrogen-synthesizing enzymes, expression and activity of 5alpha-reductase, aromatase, and 3beta- and 17beta- hydroxysteroid dehydrogenases were determined in spleen and T lymphocytes of male and proestrous female mice after trauma-hemorrhage. All of the enzymes were present in spleen, specifically in T lymphocytes. 5alpha-Reductase expression and activity increased in male T lymphocytes, whereas aromatase activity, but not expression, increased in female T lymphocytes. Increased 5alpha-reductase activity in male T lymphocytes is immunosuppressive because of increased 5alpha-dihydrotestosterone synthesis, whereas in females increased aromatase activity triggering 17beta-estradiol synthesis is immunoprotective. This study also demonstrates the importance of 17beta-hydroxysteroid dehydrogenase oxidative and reductive functions. The immunoprotection of proestrous females is associated with enhanced reductase function of the enzyme. In males, decreased expression of oxidative isomer type IV, which impairs catabolism of 5alpha-dihydrotestosterone, probably augments immunosuppression. This study provides evidence for the involvement of intracrine sex steroid synthesis in gender dimorphic immune responses after trauma-hemorrhage.

Gender differences in the inflammatory response and survival following haemorrhage and subsequent sepsis.

Studies have shown gender dimorphism in cell-mediated immune responses following haemorrhage, with depressed responses in young males and maintained or enhanced responses in proestrus females. However, it remains unknown whether or not the sexually dimorphic immune response to haemorrhage provides any protection against a subsequent in vivo polymicrobial septic challenge. To study this, male and proestrus female C3H/HeN mice were subjected to haemorrhage (35+/-5 mmHg for 90 min followed by fluid resuscitation) or sham operation. Twenty-four hours thereafter, all mice were subjected to polymicrobial sepsis by cecal ligation and puncture (CLP) and survival was assessed over a 10 day period. Haemorrhage prior to CLP significantly increased mortality in males as compared to shams. In contrast, mortality in females following CLP was comparable between the sham and haemorrhage groups. Plasma levels of interleukin (IL-)6, tumour necrosis factor (TNF)-alpha and prostaglandin E(2)(PGE(2)) at 5 h after CLP were significantly increased in males subjected to prior haemorrhage. In contrast, plasma levels of IL-6 and TNF-alpha in females did not increase under such conditions. PGE(2)levels were comparable in males and females following CLP, however prior haemorrhage significantly reduced PGE(2)levels in females, whereas no change was observed in males. Liver and splenic expression of cyclooxygenase-2 protein paralleled the changes in plasma PGE(2). Female sex hormones, therefore, appear to play an important role not only in maintaining immune function following haemorrhage, but also provide a survival advantage against subsequent septic challenge.

Cyclooxygenase-2-mediated regulation of Kupffer cell interleukin-6 production following trauma-hemorrhage and subsequent sepsis.

Studies indicate that trauma-hemorrhage results in activation of Kupffer cells to release inflammatory mediators and it leads to immunosuppression and increased susceptibility to subsequent sepsis. The cyclooxygenase (COX) product prostaglandin (PG) E2 appears to be central to this process, however, non-selective inhibition of COX activity with non-steroidal anti-inflammatory agents that block both the constitutive (COX-1) and inducible (COX-2) isoforms of cyclooxygenase has not yielded promising results in trauma patients. Nonetheless, it remains unknown whether selective inhibition of COX-2 activity has any salutary effect following trauma-hemorrhage and subsequent induction of sepsis. To study this, male C3H/HeN mice were subjected to laparotomy (i.e., soft-tissue trauma) and hemorrhagic shock (35 +/- 5 mmHg for 90 min, then resuscitated) or to sham operation. Twenty-four hours later, the mice were subjected to sepsis by cecal ligation and puncture (CLP) or to sham CLP. The mice were treated with the COX-2 inhibitor NS-398 (10 mg/kg body weight, intraperitoneally) or vehicle immediately after trauma-hemorrhage or sham operation, 12 h thereafter, and following CLP or sham CLP. At 5 h after CLP, plasma PGE2, Interleukin-(IL) 6, and TNF-alpha levels were determined along with Kupffer cell IL-6 and TNF-alpha production in vitro. NS-398 treatment markedly suppressed the elevation in plasma PGE2 levels following CLP. The increase in plasma IL-6 levels after CLP were also significantly attenuated by NS-398 treatment. In vitro Kupffer cell IL-6 production after CLP was significantly reduced by in vivo NS-398 treatment. However, NS-398 had no effect on TNF-alpha levels, in vivo and in vitro. These findings indicate that activation of COX-2 following trauma-hemorrhage and subsequent sepsis up-regulates Kupffer cell IL-6 production. Thus, selective inhibition of COX-2 activity may reduce the deleterious consequences of sepsis under such conditions.

Does burn wound excision after thermal injury attenuate subsequent macrophage hyperactivity and immunosuppression?

Studies have shown that cell mediated immunity is suppressed markedly following thermal injury. Macrophages and the activation of an inflammatory cascade that includes interleukin (IL)-1, IL-6, tumor necrosis factor-alpha (TNFalpha) and PGE2 have been implicated as causative factors. Burn wound excision and grafting is a common clinical practice that decreases patient morbidity and mortality. It is not known, however, if the salutary effects of this procedure are related to modulation of macrophage activity post-burn. Therefore, C57BL/6 female mice were subjected to a third-degree scald burn covering 25% of their total body surface area followed by complete excision and allografting of the injury site at 8, 24, or 72 h post-burn. Splenic macrophage function was assessed 7 days post-burn. Thermal injury without burn excision and grafting significantly increased macrophage TNFalpha, IL-6, nitric oxide, and PGE2 production in response to lipopolysaccharide stimulation, whereas IL-1beta production was not increased. Burn wound excision and grafting normalized TNFalpha production to sham levels, independent of when post-burn the procedure was conducted. In contrast, the elevated production of other inflammatory mediators (IL-1beta, IL-6, nitric oxide, PGE2) post-burn was unaffected by burn wound excision and grafting. Moreover, splenic T-lymphocyte proliferation was also suppressed at 7 days post-burn and was not improved by burn wound excision and grafting. These results, therefore, suggest that the beneficial effects of burn wound excision and grafting are likely to be related to the normalization of macrophage TNFalpha production as well as the maintenance of skin barrier function.

Severe hypoxemia in the absence of blood loss causes a gender dimorphic immune response.

A gender dimorphic immune response has been observed after trauma and severe hemorrhage, a condition believed to be associated with tissue hypoxia. Although studies have shown that hypoxemia per se in males causes a systemic inflammatory response, it is unclear if the inflammatory response to hypoxemia exhibits gender dimorphic characteristics. To study this, male and female C3H/HeN mice in the proestrus state of the estrous cycle were subjected to hypoxemia (95% N(2)-5% O(2)) or sham hypoxemia (room air) for 60 min. Later (2 h), plasma interleukin (IL)-6 and tumor necrosis factor (TNF)-alpha levels were determined along with splenic immune responses. Plasma IL-6 and TNF-alpha concentrations after hypoxemia were significantly increased in males but not in females. Splenocyte proliferation was depressed in males after hypoxemia but not in females. A shift toward an immunosuppressive Th-2 cytokine profile was observed in males after hypoxemia [decreased interferon-gamma (Th-1) and increased IL-10 (Th-2)], whereas no such shift was observed in females. Splenic macrophage IL-6, IL-10, and IL-12 production were suppressed in males after hypoxemia; however, such suppression was not observed in females. These findings therefore indicate that a gender dimorphic immune response also exists after hypoxemia in the absence of blood loss and tissue trauma, similar to trauma-hemorrhage. Furthermore, because no systemic inflammatory response or alterations in T lymphocyte or macrophage functions are observed in proestrus females but such parameters are markedly altered after severe hypoxemia in males, these studies indicate that proestrus females can tolerate hypoxemia better than males.

Androgen and estrogen receptors in splenic T lymphocytes: effects of flutamide and trauma-hemorrhage.

The endogenous sex steroids, testosterone and beta-estradiol, play a major role in inflammatory processes. They regulate several cytokine genes by interaction with their intracellular receptors that are, essentially, transcription factors. Because T-lymphocyte functions are altered following trauma-hemorrhage in male mice, we investigated whether (i) receptors for androgen (AR) and estrogen (ER) are present in splenic T lymphocytes, (ii) receptor expressions are altered following trauma-hemorrhage, and (iii) pretreatment of male mice with the AR antagonist, flutamide, alters receptor expressions and IL-6 release. Analysis of nuclear extracts indicated the presence of AR and ER in splenic T lymphocytes. No difference in receptor expressions between males and females or following trauma-hemorrhage was observed. Pretreatment of males with flutamide, however, led to increased ER expression in T lymphocytes of sham and trauma-hemorrhaged animals. This suggested that flutamide is capable of inducing the expression of another receptor belonging to a different gonadal steroid. Because response elements for AR and ER are present in the promoter region of the IL-6 gene, release of IL-6 and expression of signal transducer and activator of transcription 3 (STAT3) were analyzed as functional parameters in splenic T lymphocytes. Trauma-hemorrhage decreased IL-6 release by T lymphocytes and the release was restored to sham levels with flutamide pre-treatment. Similarly, STAT3 expression was decreased in T lymphocytes following trauma-hemorrhage and the expression was restored by flutamide pre-treatment. These data collectively demonstrate the importance of gonadal steroids in the regulation of splenic T-lymphocyte functions.

The aromatase inhibitor, 4-hydroxyandrostenedione, restores immune responses following trauma-hemorrhage in males and decreases mortality from subsequent sepsis.

Studies have shown that immune responses are depressed in male mice, but not in proestrus females after trauma-hemorrhage (TH), resulting in increased mortality from subsequent sepsis in male mice compared with female mice. These gender-specific alterations in immune function are believed to be due to differences in sex steroid levels. Aromatase is a key enzyme in the sex steroid biosynthesis. Although earlier studies have shown that aromatase inhibitors prevent thymic atrophy in aged male rats, it remains unknown whether the use of 4-hydroxy-androstenedione (4-OHA) after TH in male mice has any salutary effects on the depressed immune responses. Male C3H/HeN mice were sham operated or subjected to trauma (i.e., midline laparotomy) and hemorrhagic shock (30+/-5 mmHg for 90 min) followed by adequate fluid resuscitation. 4-OHA (5 mg/kg) or vehicle was administrated s.c. just before resuscitation. At 2 h after resuscitation, the mice were killed, and spleens were harvested. Splenocyte proliferation, interleukin (IL-2), interferon (IFN-gamma), and IL-10 release and expression of androgen (AR) and estrogen receptors (ER)-alpha and -beta by immunoblot and reverse transcription-polymerase chain reaction (RT-PCR) were assessed. In another group, sepsis was induced by cecal ligation and puncture (CLP) 3 days after resuscitation, and survival was measured over a period of 10 days. A significant decrease in splenocyte proliferation, IL-2, and IFN-gamma release and increased release of IL-10 were observed in vehicle-treated mice. Animals treated with 4-OHA showed increased splenocyte proliferation, IL-2, and IFN-gamma release, and decreased IL-10 release. Immunoblot analysis showed decreased expression of the cytosolic AR, but no significant difference in the cytosolic and nuclear ER-alpha and -beta expression was observed in the vehicle-treated group after TH. In addition, AR and ER-beta mRNA expression was increased, whereas ER-alpha expression decreased in the vehicle-treated group after TH. ER-alpha expression decreased and ER-beta expression increased in the nucleus of 4-OHA treated mice as determined by immunoblot. There was no difference in the cytosolic AR expression in the 4-OHA-treated group after TH. AR and ER-beta mRNA expression was unaffected, whereas ER-alpha expression increased under such conditions. In additional groups, the increased mortality rate after TH and subsequent sepsis was significantly reduced by 4-OHA treatment. Thus, 4-OHA seems to be a novel and useful adjunct for restoring the depressed immune functions in males after TH and for decreasing mortality rates from subsequent sepsis.

L-arginine attenuates trauma-hemorrhage-induced liver injury.

OBJECTIVES: Liver injury is common after trauma-hemorrhage for which the underlying mechanism is not clear. Although administration of the essential amino acid L-arginine has been reported to restore the depressed cardiovascular functions and cell-mediated immune responses after trauma-hemorrhage, it remains unknown whether L-arginine protects against liver injury under those conditions. DESIGN: A prospective, controlled animal study. SETTING: A university research laboratory. SUBJECTS: Male Sprague-Dawley rats. INTERVENTIONS: Rats underwent sham operation or laparotomy and were bled to and maintained at a mean arterial blood pressure of 40 mm Hg until 40% of the maximum shed blood volume was returned in the form of lactated Ringer's solution. Hemorrhaged rats were then resuscitated with lactated Ringer's solution, four times the maximum shed blood volume over 1 hr. During resuscitation, animals received either 300 mg/kg of L-arginine or saline (vehicle) intravenously. At 3 and 5 hrs after resuscitation, rats were killed, blood was obtained, and the liver was fixed for histology (hematoxylin & eosin staining). Plasma glutathione S-transferase (a marker of liver damage), L-arginine, citrulline, and ornithine concentrations were assessed. MEASUREMENTS AND MAIN RESULTS: The increased concentrations of plasma glutathione S-transferase observed in vehicle-treated hemorrhage animals were normalized with L-arginine treatment at 5 hrs after resuscitation. Moreover, the histology indicated that L-arginine prevented liver edema and neutrophil infiltration after trauma-hemorrhage. Plasma L-arginine and citrulline were increased in L-arginine-treated rats. CONCLUSIONS: Because citrulline is a by-product of nitric oxide generation by nitric oxide synthase from L-arginine, this amino acid may be a useful adjunct for preventing hepatic injury after trauma-hemorrhage via endothelial derived nitric oxide production.

Effect of gender and sex hormones on immune responses following shock.

Several clinical and experimental studies show a gender dimorphism of the immune and organ responsiveness in the susceptibility to and morbidity from shock, trauma, and sepsis. In this respect, cell-mediated immune responses are depressed in males after trauma-hemorrhage, whereas they are unchanged or enhanced in females. Sex hormones contribute to this gender-specific immune response after adverse circulatory conditions. Specifically, studies indicate that androgens are responsible for the immunodepression after trauma-hemorrhage in males. In contrast, female sex steroids seem to exhibit immunoprotective properties after trauma and severe blood loss, because administration of estrogen prevents the androgen-induced immunodepression in castrated male mice. Nonetheless, the precise underlying mechanisms for these immunomodulatory effects of sex steroids after shock remain unknown. Although testosterone depletion, testosterone receptor antagonism, or estrogen treatment has been shown to prevent the depression of immune functions after trauma-hemorrhage, it remains to be established whether differences in the testosterone-estradiol ratio are responsible for the immune dysfunction. Furthermore, sex hormone receptors have been identified on various immune cells, suggesting direct effects. Thus, the immunomodulatory properties of sex hormones after trauma-hemorrhage might represent novel therapeutic strategies for the treatment of immunodepression in trauma patients.

Insights into the role of gammadelta T lymphocytes in the immunopathogenic response to thermal injury.

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 alphabeta T lymphocytes; however, the role of gammadelta T lymphocytes in the immune response after thermal injury is unclear. Therefore, wild-type mice and mice lacking the TCR delta gene (TCR delta-/-) were subjected to a third-degree scald burn and cell-mediated immune responses assessed at 7 days post-injury. TCR delta-/- mice had 75% mortality after burn injury compared with 25% mortality in the wild-type group. Plasma interleukin-6 (IL-6) levels were significantly elevated at 2, 4, and 18 h post-injury, whereas no difference was observed in tumor necrosis factor alpha (TNF-alpha) and prostaglandin E2 (PGE2) plasma levels. Plasma levels of these inflammatory mediators were similar in wild-type and TCR delta-/- mice post-injury. Splenic macrophage PGE2, IL-6, TNF-alpha, and IL-10 production was significantly increased in wild-type mice at 7 days post-injury, whereas macrophages from injured TCR delta-/- mice had a significantly attenuated capacity to produce IL-6 and TNF-alpha. In contrast, the increased release of PGE2 and IL-10 by macrophages post-injury was not reduced in TCR delta-/- mice. These results implicate a dual role for gammadelta 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, gammadelta T lymphocytes, in part through the modulation of macrophage activity, appear to contribute to the immune dysfunction after thermal injury.

Reversal of sexual dimorphism in splenic T lymphocyte responses after trauma-hemorrhage with aging.

Previous studies have demonstrated that hemorrhagic shock produces immunodepression in young male mice, whereas the immunoresponsiveness in young proestrus female mice is enhanced under such conditions. This sexually dimorphic immune response to hemorrhage appears to be related to high estrogen and testosterone levels in females and males, respectively. Nonetheless, it is unknown what impact the age-related decline in the sex steroid levels has on the immune response after hemorrhage. To study this, young (2-3 mo) and aged (18-19 mo) male and female CBA/J NIA mice were subjected to laparotomy (i.e., soft tissue trauma) and hemorrhage (35 +/- 5 mmHg for 90 min and fluid resuscitation) or sham operation. Twenty-four hours later, splenocyte responses were assessed in vitro. Splenic T lymphocyte responses [i.e., proliferation, interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) release] were depressed in young males and enhanced in young females after trauma-hemorrhage. In contrast, in the aged male and female groups these parameters of splenocyte function were reversed after trauma-hemorrhage (i.e., increased proliferation and IL-2 release in aged males compared with suppressed proliferation and IFN-gamma release in aged females). Furthermore, the release of the immunosuppressive cytokine IL-10 inversely correlated with the age- and gender-related changes in splenocyte responses after trauma-hemorrhage. Thus the sexually dimorphic immune response in young males and females to trauma-hemorrhage appears to reverse as sex hormone levels decline with age.

Immune dysfunction following trauma-haemorrhage: influence of gender and age.

Recent studies indicate that young female proestrus mice show an enhanced immune response following trauma-haemorrhage, as opposed to the immunodepression observed in males of comparable age. Testosterone is suggested as the cause of immunodepression in males, whereas oestradiol seems to be responsible for the enhanced immune response in females, however, sex hormone levels decrease with age. To determine if the sexual dimorphism in immune responses observed in young mice following trauma-haemorrhage changes with age, young (2-3 months) and aged (18-19 months) male and female CBA/J NIA mice were subjected to soft-tissue trauma (laparatomy) and haemorrhage (35+5 mmHg for 90 min and fluid resuscitation) or sham operation. Mice were killed 24 h later, and whole blood, as well as splenic and peritoneal macrophages (Mstraight phi) obtained. Plasma 17beta-oestradiol and free testosterone decreased in aged females and males, respectively. Mstraight phi from young females had enhanced IL-1beta and suppressed IL-10 production following trauma-haemorrhage, while aged females had unchanged production IL-1beta and IL-6 production and enhanced IL-10 release. In contrast, IL-1beta and IL-6 production by Mbeta from young males was suppressed and IL-10 production enhanced following trauma-haemorrhage, whereas Mstraight phi from aged males produced elevated levels of IL-1beta and IL-6 and suppressed levels of IL-10 following trauma-haemorrhage. Thus, the gender-related changes in the immune response to trauma-haemorrhage were reversed in aged mice.

Role of protein kinase C in cyclic AMP-mediated suppression of T-lymphocyte activation following burn injury.

Major burn injury induces T-lymphocyte dysfunction. Previous studies suggest that prostaglandin (PG) E2, which is elevated post-burn, is the causative factor via a cyclic AMP-dependent process. The present study was conducted to elucidate the mechanism by which cAMP induces T-lymphocyte dysfunction following burn injury. Splenocytes were isolated from mice 7 days after receiving a scald burn covering 25% of their total body surface or sham procedure. ConA-induced proliferation by splenocytes from burned mice was significantly suppressed. Macrophage depletion of the splenocyte cultures abrogated the suppression. Concanavalin A-stimulated proliferation by macrophage-depleted splenocytes was suppressed by PGE2 and dibutyryl cAMP in both groups. The IC50 of these cAMP-elevating agents, however, was approximately 100-fold lower for cells from burned mice, indicating an increased sensitivity to cAMP. PGE2 did not suppress PMA/Ca2+ ionophore-induced T-lymphocyte activation. Addition of PMA to ConA-stimulated cultures prevented the suppression of proliferative responses by PGE2, whereas Ca2+ ionophore had no effect. Thus, the suppression of T-lymphocyte activation following burn injury is macrophage-dependent, related to an increased sensitivity to cAMP and due to an uncoupling of cell surface receptors from protein kinase C activation.

Gender dimorphism in trauma-hemorrhage-induced thymocyte apoptosis.

Studies indicate that immune responses after trauma-hemorrhage are significantly depressed in males compared with enhanced immune responses in females under such conditions. Although androgen depletion in male mice by castration before soft tissue trauma and hemorrhagic shock prevents the depression of cell-mediated immunity, the underlying mechanism responsible for this remains unclear. Because the thymus is the primary location of T-cell lymphopoiesis and thymocytes express a large number of androgen receptors, we investigated whether differences in thymic apoptosis might contribute to the divergent immune response in males versus females after trauma-hemorrhage. To study this, male and female C3H/HeN mice were subjected to sham operation or soft tissue trauma (laparotomy) and hemorrhagic shock followed by fluid resuscitation. Animals were killed 72 h thereafter and thymocytes were isolated. Thymocyte interleukin 3 (IL-3) release was significantly suppressed in males, but not females, after trauma-hemorrhage. A parallel increase in thymic apoptosis that was primarily in the CD8+ thymocyte subset was observed in the males. Furthermore, in vitro treatment of thymocytes with 5a-dihydrotestosterone (DHT) increased the rate of apoptosis and decreased IL-3 release in a dose-dependent manner. Thus, the gender-dependent dimorphic immune response after trauma-hemorrhage may be in part due to an androgen-induced increase in thymic apoptosis in males under such conditions.

Sex steroids regulate pro- and anti-inflammatory cytokine release by macrophages after trauma-hemorrhage.

Studies indicate that macrophage immune responses in males are depressed after trauma-hemorrhage, whereas they are enhanced in females under such conditions. Nonetheless, the involvement of male and female sex steroids in this gender-dependent dimorphic immune response after trauma-hemorrhage remains unclear. To study this, male C3H/HeN mice were castrated and treated with pellets containing either vehicle, 5alpha-dihydrotestosterone (DHT), 17beta-estradiol, or a combination of both steroid hormones for 14 days before soft tissue trauma (i.e., laparotomy) and hemorrhagic shock (35 +/- 5 mmHg for 90 min followed by adequate fluid resuscitation) or a sham operation. Twenty-four hours later the animals were killed, plasma was obtained, and Kupffer cell and splenic and peritoneal macrophage cultures were established. For DHT-treated mice, we observed significantly decreased releases of the proinflammatory cytokines interleukin 1beta (IL-1beta) and IL-6 by splenic macrophage (-50 and -57%, respectively) and peritoneal macrophage (-51 and -52%, respectively) cultures after trauma-hemorrhage compared with releases by cultures of cells from mice subjected to a sham operation; in contrast, responses of splenic and peritoneal macrophage cultures from other groups subjected to trauma-hemorrhage did not change significantly. In addition, only DHT-treated animals exhibited increased Kupffer cell IL-6 release (+634%). The release of IL-10 in DHT-treated hemorrhaged animals was increased compared with that in sham-operated animals but was decreased in estrogen-treated mice under such conditions. These results suggest that male and female sex steroids exhibit divergent immunomodulatory properties with respect to cell-mediated immune responses after trauma-hemorrhage.

Hemorrhage decreases macrophage inflammatory protein 2 and interleukin-6 release: a possible mechanism for increased wound infection.

OBJECTIVE: To determine whether alteration in wound exudate cell immune function occurs after trauma-hemorrhage. BACKGROUND: Although clinical and experimental studies indicate that the rate of wound infection is increased after trauma and hemorrhagic shock, the underlying mechanism for this increased susceptibility remains unknown. METHODS: Male C3H/HeN mice were subjected to a midline laparotomy and polyvinyl alcohol sponges were implanted subcutaneously in the abdominal wound before hemorrhage (35+/-5 mm Hg for 90 minutes and resuscitation) or sham operation. The wound exudate cells from the sponges were harvested on the first, third, and fifth postoperative day and cultured for 24 hours in the presence of lipopolysaccharide (10 microg/ml) or heat-killed Staphylococcus aureus. Interleukin (IL)-1beta, IL-6, monocyte chemotactic protein 1, macrophage inflammatory protein 2, and nitrite levels were determined in the supernatants. The distribution of macrophages and polymorphonuclear leukocytes was assessed in the sponge with and without in vivo injection of S. aureus. The phagocytic activity of isolated wound exudate cells was determined using fluorescent S. aureus. RESULTS: The composition of exudate cells was unaltered by hemorrhagic shock; however, in vivo injection of S. aureus significantly decreased the percentage of macrophages under such conditions. Wound exudate cell phagocytic activity and the release of IL-1beta, IL-6, monocyte chemotactic protein 1, and macrophage inflammatory protein 2 was decreased on the first postoperative day. The release of IL-1beta and IL-6 was also decreased on the third postoperative day in hemorrhaged mice. On the fifth postoperative day, wound exudate cell cytokine production was comparable to that in shams. CONCLUSIONS: Because most wound infections occur early after severe trauma, these results suggest that the dysfunction of wound exudate cells after hemorrhage might contribute to the increased incidence of wound infections. Therefore, attempts to enhance or restore wound cell immune function might be helpful for decreasing the incidence of wound infections in trauma victims.

Splenic immune suppression in sepsis: A role for IL-10-induced changes in P38 MAPK signaling.

BACKGROUND: Studies have indicated that following the induction of sepsis, there is a late (24 h) generalized suppression of the immune response which is associated with increased anti-inflammatory mediator release (e.g., IL-10). However, the mechanisms by which this occurs are unknown. In this regard, recent studies indicate that p38 mitogen-activated protein kinase (p38 MAPK) may play a central role in transducing the signals from immunosuppressive agents which in turn may alter lymphoid cytokine release. The aim of this study, therefore, was to determine whether the anti-inflammatory mediator IL-10 alters splenocyte IL-2 and IFN-gamma release, as well as the expression and activation of p38 MAPK in septic animals. MATERIALS AND METHODS: Splenocytes (SPL) (or for some experiments purified T cells) were harvested from mice subjected 24 h earlier to either sepsis by cecal ligation and puncture (CLP) or Sham-CLP and stimulated with 2.5 microg concanavalin A (ConA)/ml in the presence or absence of either monoclonal antibody (Mab) to IL-10 (4 microg/ml) or IgG control. In subsequent studies, sepsis was induced in C57BL/6J and C57BL/6 IL-10 knockout mice, and SPL harvested and stimulated with ConA. SPL cytokine release was measured by ELISA, and the expression and phosphorylation of p38 MAPK were measured by Western analysis. RESULTS: The results indicate that Th1 cytokine (IL-2, IFN-gamma) release was depressed by sepsis, while p38 MAPK expression and activity were increased in SPL as well as in T-cells. Neutralization of IL-10 by in vitro use of anti-IL-10 Mab and in the IL-10 knockout animal restored the Th1 response and caused a downregulation of p38 MAPK expression and activity after CLP. Thus, IL-10 appears to contribute to the increase in p38 MAPK activity and expression and the corresponding suppression of Th1 response seen in late sepsis.

Does uninjured skin release proinflammatory cytokines following trauma and hemorrhage?

HYPOTHESIS: Uninjured skin contributes to the elevation in circulating levels of proinflammatory cytokines seen following severe injury. DESIGN: Male C3H/HeN mice underwent trauma, trauma-hemorrhage and resuscitation, or closed long-bone fracture. Serum, skin, and liver samples were harvested at designated times after experimental treatment. MAIN OUTCOME MEASURES: Levels of interleukin (IL) 1beta, IL-6, and tumor necrosis factor alpha (TNF-alpha) were determined in serum and skin cultures at 1, 8, and 24 hours after trauma-hemorrhage. The RNA was isolated from liver and skin samples at 1, 2, 4, 8, and 24 hours from all 3 experimental groups, and gene expression of the cytokines was determined. RESULTS: Remote (nontraumatized) skin from trauma-hemorrhage animals released significantly more IL-6 and TNF-alpha into culture supernatants at 1 and 24 hours and significantly more IL-1beta at 1, 8, and 24 hours than did skin from sham animals. Serum levels of all 3 cytokines were significantly elevated at 1 and 24 hours after trauma-hemorrhage relative to sham animals. Gene expression of all 3 cytokines was detected in skin and liver following trauma-hemorrhage. Furthermore, gene expression of all 3 cytokines was detected in uninjured skin after soft tissue trauma and closed long-bone fracture. CONCLUSIONS: Proinflammatory cytokine gene expression is up-regulated in uninjured skin following trauma, trauma-hemorrhage, and long-bone fracture. This increase in gene expression correlates with increased cytokine production by cultured skin as well as increased circulating cytokine levels. These results suggest that uninjured skin may also contribute to the rise in circulating cytokine levels seen after injury.