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Although 17beta-estradiol (E2) administration following trauma-hemorrhage (T-H) improves immune functions in male rodents, it remains unclear whether E2 has salutary effects on Peyer's patch (PP) T cell functions. We hypothesized that T-H induces PP T cell dysfunction and E2 administration following T-H will improve PP T cell function. T-H was induced in male C3H/HeN mice (6-8weeks) by midline laparotomy and approximately 90min of hemorrhagic shock (blood pressure 35mmHg), followed by fluid resuscitation (4x the shed blood volume in the form of Ringer's lactate). Estrogen receptor (ER)-alpha agonist propyl pyrazole triol (PPT; 5microg/kg), ER-beta agonist diarylpropionitrile (DPN; 5microg/kg), E2 (50microg/kg), or vehicle was injected subcutaneously at resuscitation onset. Two hours later, mice were sacrificed and PP T cells isolated. PP T cell capacity to produce cytokines in response to in vitro stimulation, PP T cell proliferation and MAPK (p38, ERK-1/2, JNK) activation were measured. Results indicate PP T cell proliferation, cytokine production and MAPK activation decreased significantly following T-H. E2, PPT or DPN administration normalized these parameters. Since PPT or DPN administration following T-H was effective in normalizing PP T cell functions, the salutary effects of E2 are mediated via ER-alpha and ER-beta., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Apoptotic cell death is considered to be an underlying mechanism in immunosuppression and multiple organ dysfunction after trauma-hemorrhage and sepsis. Although studied intensively over the last decade, the role of other cell death mechanisms under similar pathophysiological conditions has remained elusive. Recently, autophagy has emerged as an important mediator of programmed cell death pathways. Here, we review recent advances in our understanding of apoptosis and autophagy and the crosstalk between these processes. We explore the coexistence of these two processes and the effects of autophagy on apoptosis after trauma-hemorrhage and sepsis. The inter-relationship between autophagy and apoptosis might unveil novel therapeutic approaches for the detection and treatment of trauma-hemorrhage and sepsis.

Although 17beta-estradiol (E2) administration following trauma-hemorrhage (T-H) reduces liver injury by decreasing neutrophil accumulation via estrogen receptor (ER)-alpha, it remains unclear whether cytokine-induced neutrophil chemoattractant (CINC)-1 production by Kupffer cells (KC) is directly modulated by ER-alpha under such condition. Male rats underwent laparotomy and hemorrhagic shock (40 mmHg for 90 min), followed by resuscitation with four times the shed blood volume in the form of Ringer's lactate. ER-alpha agonist propyl pyrazole triol (PPT; 5 microg/kg), ER-beta agonist diarylpropionitrile (DPN; 5 microg/kg), E2 (50 microg/kg), or vehicle (10% DMSO) was administered subcutaneously during resuscitation; rats were sacrificed 24h thereafter. KC were isolated and cultured with ER agonists to examine if they directly affect CINC-1 production. T-H increased plasma alanine aminotransferase (ALT; hepatic injury) and hepatic myeloperoxidase (MPO) activity. E2, PPT and DPN administration reduced increased ALT; however, PPT was more effective than DPN. PPT and E2, but not DPN significantly attenuated increased hepatic MPO activity and CINC-1 levels. PPT addition in vitro (10(-7) and 10(-6)M) significantly reduced KC CINC-1 production. In summary, the salutary effects of E2 against hepatic injury are mediated predominantly via ER-alpha which directly modulates KC CINC-1 production and hepatic neutrophil accumulation following T-H.

Although studies have shown 17beta-estradiol (E2) administration following trauma-hemorrhage (T-H) attenuates alterations in T cell cytokine production, it remains unknown whether such effects of E2 are mediated via genomic or non-genomic pathways. In this study, we determined the non-genomic effects of E2 on splenic T cell cytokine production and the role of MAPK following T-H. Male Sprague-Dawley rats underwent T-H (mean BP 40 mmHg for 90 min, then resuscitation). E2, E2 conjugated with BSA (E2-BSA, 1 mg/kg E2) with or without an estrogen receptor antagonist (ICI 182 780), or vehicle was administered during resuscitation. Two hours thereafter, T cell production of IL-2 and IFN-gamma and activation of MAPK (p38, ERK-1/2 and JNK) were determined. The effect of selective MAPK inhibitors on cytokine production was also examined in vitro. IL-2 and IFN-gamma production capacity and MAPK activation decreased in T cells following T-H. However, E2 administration normalized these parameters. Although E2-BSA administration also attenuated suppression in cytokine production, the values were lower compared to sham. In contrast, E2-BSA prevented T-H-induced suppression in MAPK activation to the same extent as E2. Co-administration of ICI 182 780 abolished E2-BSA effects. These findings suggest E2 effects on T cell cytokine production following T-H are mediated at least in part via non-genomic pathway and these non-genomic effects are likely mediated via MAPK pathways.

Since splenic immune functions are depressed in metestrus females following trauma-hemorrhage, we hypothesized that administration of the androgen receptor antagonist flutamide at the onset of resuscitation will maintain the immune function of the spleen following trauma-hemorrhage. Female C57BL6/J mice (metestrus state, 8-12 weeks old), underwent laparotomy and hemorrhagic shock (35.0+/-5.0 mm Hg for 90 min) and received 17beta-estradiol (50 microg/25 g), flutamide (625 microg/25 g) or 17beta-estradiol+flutamide. Four hours after resuscitation, the in vitro productive capacity of different cytokines (TNF-alpha, IL-6, IL-10, and IFN-gamma) by splenic MPhi and splenocytes were determined by flow cytometry. A significantly decreased cytokine production by both splenocytes and splenic MPhi was observed following trauma-hemorrhage compared to shams. Administration of 17beta-estradiol, flutamide and 17beta-estradiol+flutamide following trauma-hemorrhage resulted in a significant increase in the in vitro IL-6 release by splenic MPhi. The TNF-alpha productive capacity, however, was only restored by 17beta-estradiol and 17beta-estradiol+flutamide administration following trauma-hemorrhage. No significant effect of either treatment was observed with regard to the suppressed splenic MPhi IL-10 release. Anti-CD3 stimulation, administration of 17beta-estradiol and 17beta-estradiol+flutamide, but not the administration of flutamide alone resulted in a significant increased release of TNF-alpha, IL-6 and IFN-gamma compared to vehicle-treated animals. No significant effect of either treatment was found on IL-10 productive capacity. These results collectively suggest that flutamide administration following trauma-hemorrhage in females has beneficial effects on splenic immune function. However, flutamide administration in combination with estrogen does not provide any significant, additional effects over 17beta-estradiol administration alone.

Although administration of androstenediol (a metabolite of dehydroepiandrosterone) following trauma-hemorrhage (T-H) produces beneficial effects on inflammatory cytokines and organ function, it remains unknown whether this metabolite has any salutary effects in preventing alterations in immune cell cytokine production following a combined insult of T-H and sepsis. To examine this, male rats underwent laparotomy, hemorrhagic shock (mean BP 40 mmHg for 90 min) and resuscitation or sham operation. Androstenediol (1 mg/kg BW i.v.) or vehicle was administered at the end of resuscitation. Twenty hrs after T-H or sham operation, sepsis was induced by cecal ligation and puncture (CLP). Five hours thereafter, plasma cytokine levels and cytokine production of various immune cells were determined. In a separate set of experiments, survival was monitored for 10 days after the induction of sepsis. Administration of androstenediol markedly decreased plasma IL-6 and TNF-alpha levels following T-H and CLP. Furthermore, it prevented the increased production of IL-6 and TNF-alpha by Kupffer cells and alveolar macrophages and attenuated the decrease in IL-6 and TNF-alpha production by splenic macrophages; however, it had no significant effects on the depressed IL-6 and TNF-alpha production by PBMC following T-H and CLP. The depressed IL-2 and IFN-gamma production by splenocytes under those conditions was attenuated by the administration of androstenediol. Furthermore, survival rate following T-H and subsequent sepsis was improved by androstenediol treatment. Since androstenediol administration following T-H attenuated cytokine production and reduced mortality in a double-hit model of T-H and sepsis, this agent appears to be a novel and useful adjunct for maintaining the immune cell functions following T-H and for decreasing the mortality rate from subsequent susceptibility to sepsis.

Studies have shown that following bacterial infection or endotoxin administration, immune functions are regulated differently in mice of different genetic background. Since the susceptibility to sepsis following trauma-hemorrhage is dependant on the severity of injury, it is important to determine whether genetic background of the animal influence immune functions after trauma-hemorrhage. The aim of our studies, therefore, was to assess differences in the immune functions in genetically different strains of age-matched C3H/HeN and C57BL/6 male mice following trauma-hemorrhage. The analysis for immune functions included: proliferation of splenocyte and bone-marrow cells, IL-2 and IFN-gamma release by splenocytes, and TNF-alpha and IL-10 release by splenic, peritoneal, liver (Kupffer cell), and bone-marrow macrophages. The results show significant differences in splenocyte and bone-marrow functions, and in the release of the mediators of immune function by immune competent cells: (a) between the two genetic strains, and (b) in each mouse strain following trauma-hemorrhage. Thus, genetic background appears to significantly influence the severity of immune responses in males following trauma-hemorrhage.

Patients without spleens have an increased risk of infection. Previous studies have shown that splenectomy (Spx) influences Kupffer cells (KC), peritoneal macrophages (pMphi) and alveolar macrophages (aMphi) phagocytosis and bactericidal activity. This study examined the effect of Spx on the cytokine production by peripheral blood monocular cells (PBMC), KC, aMphi, pMphi and cells from mesenteric lymph nodes (MLN). We also determined if Spx influences survival following sepsis induced by cecal ligation and puncture (CLP). C57BL/6J male mice (8-10 weeks old) underwent sham operation or Spx. Two weeks after sham or Spx, KC, pMphi, aMphi, PBMC and cells from MLN were isolated and stimulated with LPS. Cell-free supernatants were analyzed for TNF-alpha, IL-6, and IL-10. A significant increase in KC, pMphi and aMphi TNF-alpha and IL-6 release was observed following Spx. The production of IL-10 was also significantly higher in KC under those conditions. In contrast, the release of TNF-alpha, IL-6 and IL-10 was significantly decreased in PBMC after Spx. Similarly, TNF-alpha and IL-6 was also decreased in MLN after Spx. Overall survival after CLP was not different in mice subjected to either sham or Spx. However, the mean time to death was significantly decreased in mice subjected to Spx compared to sham injured mice. These findings suggest that Spx modulates immune cell functions in various tissue compartments of the body and results in early deaths following sepsis.

Although immune responses following soft-tissue trauma-hemorrhage are markedly different in young (6-8 weeks) and aged (18-20 months) mice, it remains unknown if there are any differences in immune responses in middle-aged and young mice following bone fracture, soft-tissue trauma-hemorrhage (Fx-TH). To study this, young (6-8 weeks) and middle-aged (approximately 12 months) C3H/HeN male mice were subjected to sham operation or Fx-TH followed by resuscitation with Ringer's lactate. The mice were sacrificed 2 h thereafter and splenocytes, bone marrow cells (BM) and Kupffer cells (KC) were harvested, purified and stimulated with ConA (for splenocytes) or LPS (for BM and KC) in vitro. Splenocyte release of Th1 (IL-2 and IFN-gamma) cytokines was decreased and Th2 (IL-4 and IL-10) cytokine release was increased following Fx-TH in both young and middle-aged mice. However, the decrease in IL-2 and increase in IL-10 were significantly more in middle-aged mice compared to young mice (p < 0.05). Furthermore, splenocyte proliferation was decreased more in middle-aged mice compared to young mice following Fx-TH (p < 0.05). Additionally, TNF-alpha production was more in BM from middle-aged compared to BM from young mice after Fx-TH (p < 0.05). The production of IL-6 and IL-10 was also significantly higher in KC from middle-aged mice compared to young ones following Fx-TH. These results suggest that at middle age, the immune responses to Fx-TH are significantly different from those observed in young mice in different compartments of the body. Although the mechanism of the difference in various compartments in middle-aged vs. young mice following Fx-TH remains unknown, the decreased IL-2 production along with other altered T cell and macrophage functions may contribute to an increased susceptibility to sepsis in middle-aged vs. young individuals., (Copyright 2004 Elsevier Ltd.)

Studies have shown that 17beta-estradiol has salutary effects on immune functions after trauma-hemorrhage (TH). It remains unknown, however, whether 17beta-estradiol has a similar effect in a double-hit model of TH and subsequent sepsis. It is also unknown if under those conditions the circulating immune cells accurately represent immunological responses occurring in fixed tissues, such as the spleen. To study this, pre-castrated mice were hormonally treated and then subjected to soft-tissue trauma (i.e. midline laporatomy), hemorrhagic shock (MAP 35+/-5mmHg for 90 min followed by resuscitation) and 24 h later sepsis was induced by cecal ligation and puncture (CLP). Splenic macrophages (SMphi) and peripheral blood mononuclear cells (PBMC) were isolated and cultured with LPS. 5alpha-Dihydrotestosterone-treated mice showed a depressed pro-inflammatory cytokine production after TH-sepsis in both SMphi and PBMC. In contrast, the 17beta-estradiol treated groups showed suppressed pro-inflammatory cytokine production in the PBMC population under those conditions. In summary, 17beta-estradiol was able to prevent immune dysfunction after TH and subsequent sepsis. However, the beneficial effects of 17beta-estradiol were limited to tissue-fixed Mphi, suggesting compartmentalization of the response. Thus, events occurring in the tissue-fixed cells are not necessarily reflected in the circulating PBMC population.

Kupffer cells have been reported as a major source of proinflammatory cytokines (i.e. IL-6, TNF-alpha), which have been implicated in the pathogenesis of trauma-hemorrhage. Previous studies have shown a protective effect of 17beta-estradiol on immune function and physiological responses following trauma-hemorrhage. In this study, we investigated whether 17beta-estradiol has a direct effect on Kupffer cell cytokine production following trauma-hemorrhage. Male Sprague-Dawley rats were subjected to trauma (midline laparotomy) and hemorrhage (35-40 mmHg for 90 min followed by fluid resuscitation) or sham operation. Two hours later, Kupffer cells were isolated and cultured with 17beta-estradiol in the presence and absence of lipopolysaccharide stimulation. Kupffer cell IL-6 and TNF-alpha production increased following trauma-hemorrhage. Incubation with 17beta-estradiol attenuated the production of IL-6 by cells from both sham and trauma-hemorrhage animals in a dose-dependent manner. The suppression of IL-6 production by 17beta-estradiol was paralleled by a decrease in mRNA levels. In contrast to IL-6, the effects of 17beta-estradiol on TNF-alpha production were minimal. In conclusion, these results indicate the direct downregulation of Kupffer cell IL-6 production by 17beta-estradiol at a molecular level, which might explain in part the previously observed salutary effects of estradiol treatment following trauma-hemorrhage.

Male gender and age appear to be causative factors in development of immunodepression and septic complications following trauma-haemorrhage. Studies have demonstrated that administration of the sex hormone prolactin following trauma-haemorrhage in male mice prevents immunodepression. Since the thymus is the primary location of the T-cell-lymphopoiesis, we investigated the effect of trauma-haemorrhage to thymic prolactin-receptor (PRLr)-expression in male and proestrus female mice in three different age groups (young, adult, aged) by flow cytometry and PCR. C3H/HeN mice were subjected to laparotomy (i.e., soft-tissue trauma) and hemorrhagic shock (35+/-5mmHg for 90min, then resuscitated) or sham operation. Twenty-four hours later thymocytes were isolated. Trauma-haemorrhage upregulated PRLr expression in young and mature mice of both genders, however, the increase was attenuated in females. In contrast, in aged mice PRLr expression was elevated in both genders, independent of trauma-haemorrhage and was not further increased under such conditions. These findings suggest that the gender dimorphism in the immune response to trauma-haemorrhage may in part be related to differences in thymic PRLr expression under such conditions.

Studies have shown that both animal tissue-fixed immune cells and human peripheral blood mononuclear cell (PBMC) functions are altered after burn injury. Additional studies suggest that the burn injury-induced alterations in these divergent cell populations from different species are similar. It remains unknown, however, whether the observed changes in animal tissue-fixed immune cell function following thermal injury also occurs to a similar extent in the PBMC population. The aim of our study was to compare PBMC and tissue-fixed immune cell functions from the same animal using a murine burn model. At 7 days post-burn, mice were more susceptible to sepsis and delayed type hypersensitivity responses were suppressed. Splenocytes isolated from injured mice displayed suppressed proliferation and increased IL-10 production. In contrast, PBMC from injured mice displayed suppressed proliferation, IL-2 and IFN-gamma production. Splenic macrophage nitric oxide, PGE(2), TNF-alpha, IL-6 and IL-10 production was enhanced post-burn and IL-12 production was suppressed. PBMC from such animals displayed enhanced PGE(2) production and suppressed IL-6 and IL-12 production. These results indicate that while an immunosuppressive Th(2) phenotype (increased IL-10 and/or suppressed IL-2, IFN-gamma) was induced in both the splenic and PBMC compartments post-injury, differential expression and dimorphism in the response also exists. Thus, the assessment of only PBMC function in burn patients may not accurately reflect the patient's actual immune status at the tissue level., (Copyright 2002 Elsevier Science Ltd. All rights reserved.)

The object of the study was to determine whether male and female sex steroids produce divergent effects on Th1 and Th2 cytokine release following trauma-haemorrhage. Recent studies indicate that androgens are responsible for the depressed splenocyte Th1 cytokine release in males following trauma-haemorrhage. In contrast, female mice maintain their Th1 cytokine release capacity following trauma-haemorrhage. Nonetheless, the effect of male and female sex steroids on Th1 and Th2 cytokine release following trauma-haemorrhage remains unknown. Male C3H/HeN mice were castrated and treated with pellets containing either vehicle, 5alpha-dihydrotestosterone (DHT), 17beta-estradiol (estradiol), or a combination of both steroid hormones, for 14 days prior to soft-tissue trauma (i.e. laparotomy) and haemorrhagic shock (35+/-5 mmHg for 90 min followed by adequate fluid resuscitation) or sham operation. Untreated male and female mice, as well as DHT treated female mice, served as control groups. Twenty-four hours later the animals were sacrificed, plasma obtained and splenocytes harvested. Plasma DHT and estradiol levels in treated animals were comparable with intact male and female mice, respectively. A significant depression of splenocyte Th1 cytokines, i.e. IL-2, IFN-gamma, was observed in DHT treated castrated animals, DHT treated females, and untreated males following trauma-haemorrhage, as opposed to maintained Th1 cytokine release in estradiol treated and estradiol/DHT treated castrated animals and females. The release of the anti-inflammatory cytokine IL-10 was markedly increased in DHT treated mice and males subjected to trauma-haemorrhage compared to shams, but decreased in estrogen treated mice and females under such conditions. These results suggest that male and female sex steroids differentially affect the release of Th1 and Th2 cytokines following trauma-haemorrhage and should be further studied for their potential to modulate splenocyte function in trauma victims., (Copyright 2001 Academic Press.)

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., (Copyright 2001 Academic Press.)

Recent studies suggest that increased activation-induced lymphocyte apoptosis (AICD) is detected in mouse splenocytes during polymicrobial sepsis which may contribute to lymphocyte immune dysfunction [i.e., decreased interleukin (IL-)2 and interferon-gamma (IFN-gamma) production] leading to the associated morbidity seen in those animals. Thus, we wanted to examine the hypothesis that immune suppressive agents, such as IL-4, IL-10 or prostaglandin E2(PGE2), known to be elevated in septic animals, also contribute to this increase in AICD. Here we demonstrate that the inclusion of monoclonal antibody (mAb) to IL-10, but not anti-IL-4 or ibuprofen (IBU), blunted this sepsis induced increase in splenocyte AICD. Additionally, septic mice deficient in the IL-10 gene product (-/-) showed neither an increase in AICD nor a loss of IL-2/IFN-gamma release capacity. Interestingly, mAb to IL-10 did not altered the extent of AICD in a Th2-cell line, but exogenous IL-10 did potentiate Th1-like cell line AICD. This was consistent with the finding that the increased AICD seen in septic mouse splenocytes was restricted largely to the CD4+ cells producing IL-2 (Th1-cells) and that mAb to IL-10 treatment suppressed this change. Furthermore, IL-10 appears to mediate its AICD effect by upregulation of the Fas receptor and Fas receptor signaling protein components, but not by altered expression of Bcl/Bax/Bad family members, in septic mouse splenocytes. To the extent that these processes contribute in a pathological fashion to the animal's capacity to survive sepsis we have previously observed that in vivo post-treatment of mice with mAb IL-10 markedly attenuated septic mortality. Collectively, these data indicate that in the septic mouse the Th2 cytokine IL-10 not only serves to actively induce Th1 lymphocyte immune dysfunction but also plays a role in their apoptotic depletion. These processes in turn appear to contribute to the animal's inability to ward off lethal septic challenge., (Copyright 2001 Academic Press.)

Despite significant advances in the management of trauma victims, traumatic injury with the ensuing sepsis and multiple organ failure remains the leading cause of death between the ages of 18 and 44 in the USA. Recently, interest in the clinically and experimentally observed gender dimorphic response to traumatic injury has led to the possibility of modulating cell and organ functions following trauma and hemorrhagic shock by the administration of sex steroids. Here, we review the effects of the adrenal steroid dehydroepiandrosterone (DHEA), a precursor of sex steroid synthesis, on organ and immune functions following trauma-hemorrhage, and its potential as a novel therapy for improving the depressed cell and organ functions in trauma patients.

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., (Copyright 2000 Academic Press.)

Although a profound depression in immune function occurs following injury, the mechanism responsible for this is not fully understood. Furthermore, steroid hormones are known to be important mediators in the regulation of immune function. Although dehydroepiandrosterone (DHEA), the most plentiful steroid hormone, has been shown to stimulate immune function in normal animals, it is unknown whether DHEA has any salutary or detrimental effects on immune responses after trauma and haemorrhage. To study this, male mice were subjected to trauma, haemorrhage and resuscitation, following which they received either DHEA or vehicle subcutaneously. DHEA administration restored the normally depressed splenocyte proliferation as well as interleukin 2, interleukin 3, and interferon gamma elaboration following trauma and haemorrhage. In an attempt to determine the mechanisms mediating this effect, T cells were stimulated in vitro in the presence of DHEA and a variety of hormone antagonists. The stimulatory effect of DHEA on splenocyte proliferation was unaltered by the testosterone receptor antagonist flutamide, while the oestrogen antagonist tamoxifen completely abrogated its effect. In addition, DHEA administration normalized the elevated serum corticosterone level typically seen following injury. These results indicate, therefore, that DHEA improves splenocyte function after trauma and haemorrhage by directly stimulating T cells and also by preventing a rise in serum corticosterone., (Copyright 1999 Academic Press.)

Previous studies indicate a profound suppression of tumour necrosis factor alpha (TNF-gamma), IL-1 beta and IL-6 release capacity by peritoneal macrophage (PM phi), splenic macrophage (SM phi) and Kupffer cells (KC) during late sepsis. Such a loss of functional capacity may reduce the animal's ability to ward off infection. Prolactin is known to enhance monocyte, T- and B-lymphocyte immune responses under normal conditions and has beneficial effects on cell-mediated immunity after haemorrhage. In the respect, the dopamine antagonist, metoclopramide, has been reported to increase circulating prolactin levels. Nonetheless, it remains unknown whether prolactin or metoclopramide have any salutary effect on macrophage (M phi) cytokine gene expression following sepsis. To study this, male C3H/HeN mice were subjected to sepsis and immediately thereafter were treated with prolactin (100 micrograms/25 g body weight, s.c.), metoclopramide (100 micrograms/100 g BW, s.c.) or given saline. PM phi, SM phi and KC (only SM phi and KC in metoclopramide-treated animals) were isolated at 24 h after sepsis. The monolayers were stimulated with or without LPS 10 micrograms/ml for 1 h in vitro. Total RNA was extracted and mRNA was detected by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). A significant depression of constitutive and inducible mRNA levels of IL-1 beta, IL-6 and TNF-alpha in all three M phi populations were observed, when compared with shams (with exception of KC IL-6 mRNA in unstimulated cells). Prolactin as well as metoclopramide treatment after the onset of sepsis caused significant elevation of constitutive and inducible cytokine gene expression in all macrophages examined. Thus, prolactin and metoclopramide enhance the depressed M phi gene expression and may be useful in improving cell-mediated immunity during sepsis.

Although haemorrhagic shock produces immunodepression in both humans and experimental animals, no information is available concerning gender differences in the immune and endocrine response to shock. To study this, male and female (proestrus and diestrus) C3H/HeN mice (25 g body weight) were bled and maintained at a mean arterial blood pressure of 35 +/- 5 mmHg for 1 h and then adequately resuscitated. The animals were killed at 2 h after resuscitation to obtain splenocytes, macrophages (M phi, peritoneal and splenic), as well as whole blood. IL-1 release by M phi, splenocyte proliferative capacity and splenocyte IL-3 release in female mice was significantly increased. Male mice, however, showed decreased release of all interleukins (IL-1, 2, 3, 6) as well as splenocyte proliferative capacity after shock. Plasma corticosterone levels decreased in proestrus female mice, as opposed to increased levels in males following shock. Corticosterone may therefore, be in part responsible for the observed gender differences. To the authors' knowledge, this is the first study which shows that immune responsiveness in female mice is enhanced after haemorrhagic shock, as opposed to decreased responsiveness in males. Thus, unlike males which exhibit increased susceptibility to sepsis/infections, females should be able to better tolerate the deleterious effects of shock.

Kupffer cells are an important source of proinflammatory cytokines and contribute to the systemic inflammatory response observed following haemorrhagic shock. The systemic release of cytokines, such as TNF-alpha, IL-1 beta, IL-6, etc., has been associated with the decreased host immune and organ dysfunction following hypotension. Studies indicate that anterior pituitary hormone prolactin (PRL) plays an important role in the regulation of lymphocyte proliferation and macrophage function in vivo, as well as in vitro. However, it is not known what effects PRL administration has on Kupffer cells proinflammatory mediator release following haemorrhage. Therefore, it was the aim of this study to determine the effect of in vivo PRL administration on cytokine gene expression in Kupffer cells after haemorrhage. To study this, C3H/HeN male mice were bled to and maintained at a mean arterial pressure of 35 mmHg for 60 minutes, then resuscitated with shed blood, and segregated into two groups: one group was treated with PRL (100 micrograms/25 g body weight subcutaneously) while the other group received saline-vehicles. This was followed with lactated Ringer's solution (2 x the volume of shed blood). Two hours thereafter, the animals were sacrificed, Kupffer cells were isolated and stimulated with or without 10 micrograms/ml LPS for 1 hour. Total RNA was extracted and cytokine mRNA was detected by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). The results demonstrated that haemorrhage markedly increased the level of mRNA for IL-1 beta, IL-6, TGF-beta and TNF-beta in Kupffer cells. However, in vivo PRL treatment significantly decreased the cytokine gene expression in Kupffer cells following haemorrhage. This indicates that PRL may be useful in blunting the systemic inflammatory response associated with cell and organ depression following shock.

TNF-alpha, IL-1, IL-6 and TGF-beta are important macrophage-derived mediators which play the pleiotropic role in inflammatory, metabolic, hematopoietic and immunologic processes. Studies have shown that haemorrhagic shock without significant tissue trauma induces profound immunosuppression which is associated with elevated plasma levels of TNF-alpha IL-1, IL-6 as well as TGF-beta. Furthermore, Kupffer cells but not the splenic M phi isolated from post-haemorrhaged animals showed an increased capacity to release inflammatory cytokines in response to LPS stimulation in vitro. However, it remains unknown whether the innate (i.e. in the absence of LPS stimulation) cytokine genes expression in Kupffer cells and splenic M phi is affected by haemorrhage. To determine this, C3H/HeN male mice were bled to and maintained at a mean arterial blood pressure of 35 mmHg for 60 min, and then adequately resuscitated. Splenic macrophages and Kupffer cells were isolated at 1 h after haemorrhage. Total RNA was extracted and cytokine mRNA was detected by semi-quantitative reverse transcription and polymerase chain reaction (RT-PCR). The results demonstrate that haemorrhage significantly elevated the mRNA accumulation of TNF-alpha, IL-1 beta, TGF-beta while IL-6 gene expression in Kupffer cells and splenic M phi was only slightly increased. Since Kupffer cells but not the splenic M phi showed increased cytokine release, it could be concluded that the differential regulation of cytokine release by these two macrophage populations following haemorrhage may be due to the divergence of the cytokine at the translational level.(ABSTRACT TRUNCATED AT 250 WORDS)

Haemorrhagic shock results in a severe depression of the cellular and humoral immunity, thus rendering the host increasingly susceptible to sepsis. To study the effect of elevated TNF release following haemorrhagic shock on depressed macrophage and splenocyte functions, C3H/HeN mice were pretreated intraperitoneally with either anti-murine TNF-Ab or saline. Twenty hrs later, mice were bled to and maintained at a mean BP of 35 mmHg for 60 min followed by adequate fluid resuscitation. Pretreatment with anti-TNF-Ab completely neutralized elevated TNF plasma levels following haemorrhage. This was associated with an increased (P < 0.05) capacity of pMø isolated 24 h after haemorrhagic shock to release TNF, while the ability to secrete IL-6 and PGE2 was reduced. Haemorrhagic shock-induced suppression of pMø antigen presentation capacity and MHC class II antigen expression, as well as depression of splenocyte proliferation and lymphokine production was also attenuated (P < 0.05) by anti-TNF-Ab pretreatment. These data indicate that elevated circulating TNF levels play a pivotal role in the depression of essential macrophage and splenocyte functions following haemorrhagic shock.

Although studies indicate that simple hemorrhage induces profound depression of cell-mediated immunity and enhances the host's susceptibility to sepsis, the mechanism for this remains unknown. Since the Kupffer cells (KC) are positioned to have constant exposure to various immunomodulators and antigens released during hypotension, we have examined whether antigen presentation by KC, a critical component in eliciting an antigen specific immune response or those processes associated with it, are depressed following hemorrhage. C3H/HeN mice were bled to and maintained at a mean BP of 35 mmHg for 60 min, and then resuscitated with their own blood and adequate fluids. The mice were killed at varying periods of time after hemorrhage to obtain KC from the liver, and assessed for their capacity to present antigen to a sensitized clone Th/cell line (D10.G4.1). Hemorrhaged mice exhibited a marked decrease in antigen presenting capacity beginning as little as 2 h and lasting up to 3-5 days post-hemorrhage. The ability of KC to express mouse interleukin 1 (mIL-1) showed a significant decline at 2 h following hemorrhage, but this effect was not apparent at 24 h post-hemorrhage. In contrast, KC capacity to produce IL-1, IL-6 and tumour necrosis factor (TNF) (cytokines which can co-stimulate T cell antigen presentation) was markedly enhanced during the first 24 h following hemorrhage. A marked decrease was observed in both the mean of the average fluorescence per KC and the percent of Ia antigen-positive KC which persisted for at least 3 days after hemorrhage. The ability of ibuprofen (a cyclooxygenase blocker) to partially restore the antigen presenting capacity of KC from hemorrhaged mice in vitro indicates that prostaglandins are involved in this dysfunction. Thus, the depression of KC antigen presentation, as well as the enhanced capacity of these cells to release inflammatory mediators (TNF, IL-1, IL-6 and prostanoids) which may produce cell and organ dysfunction, could contribute to the host's enhanced susceptibility to sepsis following hemorrhage.

Although tumor necrosis factor (TNF) and interleukin 6 (IL 6) are purported to be important mediators of inflammatory responses following trauma, it is not known if the serum levels of these cytokines are altered by simple hemorrhage. The objective of this study therefore was to determine whether or not: 1) there is any elevation of TNF or IL 6, and 2) if endotoxin, an important upregulator of these cytokines, is also increased following hemorrhage. To study this, C3H/HeN mice were bled to, and maintained at a mean blood pressure of 35 mmHg for 60 min, and then resuscitated with their own shed blood and adequate fluid. Mice were sacrificed at 30 min into hemorrhage and at 2, 4 or 24 hr post-hemorrhage to obtain serum samples. IL 6 and TNF levels were measured using cytokine dependent cellular assays. Using a quantitative Limulus amebocyte lysate assay, endotoxin levels were determined. TNF levels were significantly elevated at 30 min into hemorrhage, remaining so at 2 hr after resuscitation, but absent by 4 hr. Although there was a trend toward elevated IL 6 levels at 2 hr following hemorrhage, which was sustained up to 24 hr, the values were not significantly different from sham controls. When compared to controls, no marked increase in endotoxin was seen at any time point during or following hemorrhage. These results indicate that hemorrhage, in the absence of significant tissue trauma, causes enhanced TNF release which is not the result of increased endotoxin.