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• MDSCs are found in the burn wound and uninjured skin, but not the skin of naïve mice. • TLR expression is suppressed on burn wound MDSCs. • Burn wound MDSCs constitutively produced both pro- and anti-inflammatory cytokines. • Burn wound M-MDSCs expressed both iNOS and ARG1. Myeloid-derived suppressor cells (MDSCs) have been identified in the burn wound, however their characterization is incomplete. To study this, mice were subjected to a major burn and skin cells were isolated 3 days thereafter for analysis. Significant infiltration of the burn wound with MDSCs was observed as compared with uninjured skin. The skin of naïve mice did not contain MDSCs. Characterization of the cells showed that 33% of MDSCs in the wound were monocytic (M)-MDSCs, which was significantly less than that found in uninjured skin (52%). In contrast, polymorphonuclear (PMN)-MDSCs were greater in the burn wound as compared with uninjured skin. Burn wound TLR expression by both MDSCs subsets was decreased as compared with uninjured skin. Wound MDSCs produced pro- and anti-inflammatory cytokines and iNOS was present in both MDSC subsets, whereas ARG1 was only present in M-MDSCs. In conclusion, both M- and PMN-MDSCs infiltrate burn wound with after injury, however, they displayed decreased TLR expression, suggesting receptor down-regulation. [ABSTRACT FROM AUTHOR]

Background: Burn-induced inflammation leads to impaired immune responses resulting in increased morbidity and mortality. T-cells are central in the immune response and circulating CD4 and CD8 T-cells have been used to evaluate immune status; however, the role of these T-cell subsets in the burn wound is unknown. Methods: Male C57BL/6 mice were subjected to a major 3rd degree scald burn or sham treatment. Twenty-four hours later, full thickness skin samples from sham mice and the burn wounds were collected and single cells were isolated and analyzed for αβ TCR, γδ TCR, CD3, CD4, CD8 and CD69 expressions by flow cytometry. Results: The burn wound contained significantly greater numbers of T-cells than skin from sham mice, due to a profound infiltration of αβ T-cells. These infiltrating αβ T-cells were primarily suppressor T-cells with a CD8+ or CD8-CD4- phenotype. The 15-fold increase in CD8+ αβ T-cells caused a decrease in the CD4:CD8 ratio from 0.7 in sham skin to 0.3 in the burn wound. In contrast, the majority of the γδ T-cells in sham skin were CD4-CD8-, which decreased 9-fold in the burn wound. CD69 expression was suppressed on burn wound αβ T-cells, but increased on γδ T-cells in the burn wound. Conclusions: The infiltrating burn wound αβ T-cells likely act to quell inflammation. In contrast wound γδ T-cells were activated with elevated CD4 and CD69 expression. Thus, these two distinct T-cell subsets likely differentially regulate the burn wound inflammatory response. [ABSTRACT FROM AUTHOR]

Background: Gamma delta T-cells have been shown to be important to the early immunoinflammatory response to injury, independent of infection. This unique T-cell population acts to regulate cell trafficking and the release of cytokines and growth factors. We propose this sterile inflammatory response is in part associated with damage associated molecular patterns (DAMPs) generated by major injury, such as burn, and mediated via toll-like receptors (TLRs). It is unknown whether DAMPs can activate resident γδ T-cells that reside in skin. Methods: Gamma delta T-cells were isolated from the skin of male C57BL/6 mice by enzymatic digestion. Mitochondrial DAMPs (MTDs) were generated from mitochondria isolated from mouse livers by sonication and centrifugation. Dermal γδ T-cells were incubated with MTDs (0–500 μg/ml) for 24 hr and cells and supernatants were collected for analysis. Results: MTDs activated dermal γδ T-cells, as evidenced by increased TLR2 and TLR4 expression following in vitro exposure. MTDs also induced the production of inflammatory cytokines (IL-1β, IL-6), and growth factors (PDGF and VEGF) by γδ T-cells. Conclusions: These findings herein support the concept that MTDs released after tissue/cellular injury are capable of activating dermal γδ T-cells. We propose that the activation of this unique T-cell population is central in the initiation of sterile inflammation and also contributes to the subsequent healing processes. [ABSTRACT FROM AUTHOR]

Gamma delta T-cells have been shown to be important in the early immunoinflammatory response to injury, which can be independent of infection. This sterile inflammatory response is believed to be, in part, associated with danger-associated molecular patterns (DAMPs). Mitochondrial DAMPs (MTDs) have been shown to be important in trauma-induced neutrophil activation, but it is unknown whether MTDs activate other innate immune cells, such as γδ T-cells. To study this, splenic CD3+ γδ T-cells were isolated from αβ T-cell-deficient C57BL/6 mice and mitochondria isolated from wild type mouse livers. MTDs were isolated from mitochondria by sonication and centrifugation. Gamma delta T-cells were incubated with various concentrations of MTDs (0–500 µg/ml) for 24 h. T-cells were phenotyped for TLR expression by flow cytometry and the supernatants assayed for cytokine and growth factor content. MTDs caused a dose-dependent increase in TLR2 and TLR4 expression by γδ T-cells. Both the percentage of cells positive for TLRs and the degree of expression increased. MTDs also induced the production of IL-1β, IL-6, IL-10, RANTES, fibroblast growth factor-basic and vascular endothelial growth factor by γδ T-cells. These findings support the concept that the MTDs released after tissue/cellular injury are capable of activating γδ T-cells, thus initiating sterile inflammation, as well as subsequent healing processes. [ABSTRACT FROM AUTHOR]

Aging is an important and critical factor that contributes to the clinical outcome of burn patients. The very young and the elderly are more likely to succumb after major burn as compared to their adult counterparts. With the aging population, improved understanding of the mechanisms underlying age-associated complications after burns becomes even more demanding. It is widely accepted that elderly burn patients have significantly increased morbidity and mortality. Irrespective of the type of burn injury, the aged population shows slower recoveries and suffers more complications. Age-associated immune dysfunction, immunosenescence, may predispose the elderly burn patients to more infections, slower healing and/or to other complications. Furthermore, pre-existing, age-related medical conditions such as, pulmonary/cardiovascular dysfunctions and diabetes in the elderly are other important factors that contribute to their poorer outcomes after major burn. The present review describes the impact of aging on burn patients outcomes. [ABSTRACT FROM AUTHOR]

Healing of the burn wound is a critical component of the burn patient''s successful recovery. While inflammation is a critical component of the healing process, it is unknown whether the inflammatory response differs between non-burn and burn wounds. To study this, mice were subjected to major burn injury or sham procedure. Wound cells were collected by implantation of polyvinyl alcohol sponges beneath the burn site in injured mice or beneath uninjured skin in sham mice (i.e., non-burn wound). Three days thereafter, skin, wound fluid, and infiltrating cells were collected for analysis. Significant levels of tumor necrosis factor (TNF)-α, interleukin (IL-6), monocyte chemoattractant protein (MCP)-1, and keratinocyte-derived chemokine (KC) were observed in burn wound tissue and the wound fluid from both non-burn and burn wounds. Burn injury induced 3-fold higher levels of KC and 50-fold higher levels of IL-6 in the wound fluid compared with non-burn injury. Significant numbers of the cells from both burn and non-burn wounds were CD11b+, GR1+, and F4/80+, suggestive of a myeloid suppressor cell phenotype, whereas CD3+ T-cells were negligible under both conditions. LPS induced TNF-α, IL-6, IL-10, MCP-1, KC, and nitric oxide production in both cell populations, however, IL-6, IL-10, MCP-1, and KC levels were suppressed in burn wound cell cultures. These findings indicate that significant differences in the wound inflammatory response exist between burn and non-burn cutaneous wounds and that the unique characteristics of the inflammatory response at the burn site may be an important contributing factor to post-burn wound healing complications. [Copyright &y& Elsevier]

Severe burn induces an immunopathological response that contributes to the development of a systemic inflammatory response (SIRS) and subsequent multiple organ failure. While, multiple immune cells type (T-cells, macrophages, neutrophils) are involved in this response, recent evidence suggests that a unique T-cell subset, gammadelta T-cells are central in the response to injury. While gammadelta T-cells represent only a small percentage of the total T-cell population, they display specific functional characteristics that uniquely position them in the immune/inflammatory axis to influence a number of important aspects of the body's response to burn. This review will focus on the potential regulator role of gammadelta T-cells in immunopathological response following burn and thereby their potential as therapeutic targets for affecting inflammation and healing. [ABSTRACT FROM AUTHOR]

Abstract: Severe burn induces an immunopathological response that contributes to the development of a systemic inflammatory response (SIRS) and subsequent multiple organ failure. While, multiple immune cells type (T-cells, macrophages, neutrophils) are involved in this response, recent evidence suggests that a unique T-cell subset, γδ T-cells are central in the response to injury. While γδ T-cells represent only a small percentage of the total T-cell population, they display specific functional characteristics that uniquely position them in the immune/inflammatory axis to influence a number of important aspects of the body’s response to burn. This review will focus on the potential regulator role of γδ T-cells in immunopathological response following burn and thereby their potential as therapeutic targets for affecting inflammation and healing. [Copyright &y& Elsevier]

Abstract: Burn injury is associated with profound inflammation and activation of the innate immune system involving γδ T-cells. Similarly, Toll-like receptors (TLR) are associated with activation of the innate immune response; however, it is unclear whether TLR expression is altered in γδ T-cells after major burn injury. To study this, male C57BL/6 mice were subjected to burn injury (25% TBSA) and 1 or 7 days thereafter, blood and spleen cells were isolated and subjected to FACs analysis for TLRs and other phenotypic markers (γδ TCR, αβ TCR, CD69, CD120b). A marked increase in the number of circulating γδ T-cells was observed at 24h post-burn (14% vs. 4%) and a higher percentage of these cells expressed TLR-2. TLR-4 expression was also increased post-burn, but to a lesser degree. These changes in TLR expression were not associated with altered CD69 or CD120b expression in γδ T-cells. The mobilization of, and increased TLR expression in, γδ T-cells was transient, as phenotypic changes were not evident at 7 days post-burn or in γδ T-cells from the circulation or spleen. The increases in TLR expression were not observed in αβ T-cells after burn injury. In conclusion, 24h after burn injury mobilization of γδ T-cells with increased TLR expression was observed. This finding suggests that this unique T-cell population is critical in the innate immune response to injury, possibly through the recognition of danger signals by TLRs. [Copyright &y& Elsevier]

For>100 years, researchers and medical professionals have been aware of a gender difference in immune responsiveness following insults like trauma, shock, burn or sepsis. Strong experimental evidence indicates that female animals are protected under such conditions compared to their male counterparts. 17β-estradiol (estrogen) is reported to be responsible for those protective effects as studies have shown that estrogen administration plays a major role in the restoration and maintenance of cellular and organ function following injury. Numerous experimental studies have also shown a variety of salutary effects of estrogen under low-flow conditions; however, clinical studies are rare and observational studies are inconclusive, even though a trend towards an advantage for females has been shown. Nonetheless, further clinical studies are needed as estrogen administration is simple and a single-dose administration of this hormone is not expected to produce any adverse effects. Thus, treatment of traumatized patients with estrogen could prove to be a simple, yet powerful, therapeutic option which may find its way into programs for the management and care of the traumatized host. [ABSTRACT FROM AUTHOR]

A major complication associated with burn injury is delayed wound healing. While healing of the burn injury site is essential, healing of distal injury sites caused by surgical interventions and other processes also is important. The impact of burn injury on healing of these distal wound sites is not understood clearly. To study this, mice were subjected to major burn injury or a sham procedure. Immediately following, excisional wounds were made on the dorsal surface caudal to the burn site and wound closure was monitored over a 7-d period by planimetry. In a second series of experiments, plasma and excisional wounds were collected for in vitro analysis of cyto- and chemokine levels, L-arginine metabolism,and hypoxia-inducible factor (HIF)-1α expression.At 1-7 d post-injury, a significant inflammatory response was evident in both groups, but the healing process was delayed in the burninjured mice. At 3 d post-injury, wound levels of tumor necrosis factor-á (TNF-α), interleukin-6 (IL-6), and keratinocyte-derived chemokine were suppressed in the burn group.This difference in the wound inflammatory response was independent of changes in L-arginine metabolism (nitrate levels, inducible nitric oxide synthase expression, arginase activity), but correlated with a marked reduction in HIF-1α protein levels. In conclusion, these findings suggest that HIF-1α and the inflammatory response play a significant role in wound healing, and reduced levels of HIF-1α contribute to the impaired healing response post-burn. [ABSTRACT FROM AUTHOR]

Abstract: Background: Immune and infectious complications are associated with burn injury. Opiate analgesics also can induce similar complications, however, their impact on postburn infectious complications is unknown. Methods: A retrospective survey of records from 1997 to 2002 from an academic burn center was conducted. Information on all opiate analgesic use was obtained and expressed as opiate equivalents (OEs). Total OEs were summed for each patient and then compared between cases and controls. Results: Patients who developed infections were more likely to be in the high OE group. This association was modified by burn severity. Patients with small burns and infection were more likely to be in the high OE group, whereas patients with moderate to large burns and infections were not associated significantly with opiate use. Conclusions: The results of this preliminary study suggest that opiate analgesics can contribute to the development of postburn infectious complications when the burn injury is of a less severe nature. [Copyright &y& Elsevier]

Pro-inflammatory cytokine release after shock is central in the development of subsequent multiple organ dysfunction syndrome. Some studies suggest that interleukin-10 (IL-10) is an immunosuppressive mediator after injury or sepsis, while others suggest that IL-10 is an important regulator of the pro-inflammatory response. We hypothesized that in a model of trauma and hemorrhagic shock (TH), IL-10 regulates pro-inflammatory cytokine activity via an autocrine effect on cytokine mRNA transcription in Kupffer cells early after TH. To study this, male C3H/HeN mice were sham-operated or subjected to TH. Plasma levels of TNF-α, IL-6 and PGE2 were elevated following TH. A sharp peak in IL-10 levels was observed at 2 h after the insult. Kupffer cell (KC) depletion prior to TH reduced plasma IL-6, IL-10 and TNF-α levels, whereas treatment with anti-IL-10 after TH increased IL-6 and TNF-α levels. Kupffer cell mRNA expression for IL-6, IL-10 and TNF-α was elevated in the TH group and further increased by anti-IL-10 treatment. These findings indicate that KC-dependent IL-10 regulates the early systemic inflammatory response after TH. Thus, while IL-10 is an important mediator of immunosuppression following traumatic injury, it also is beneficial with regard to its ability to counter-regulate the early inflammatory response under such conditions. [Copyright &y& Elsevier]

Studies have shown that 17β-estradiol has salutary effects on immune functions after trauma–hemorrhage (TH). It remains unknown, however, whether 17β-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±5 mmHg for 90 min followed by resuscitation) and 24 h later sepsis was induced by cecal ligation and puncture (CLP). Splenic macrophages (SMφ) and peripheral blood mononuclear cells (PBMC) were isolated and cultured with LPS. 5α-Dihydrotestosterone-treated mice showed a depressed pro-inflammatory cytokine production after TH-sepsis in both SMφ and PBMC. In contrast, the 17β-estradiol treated groups showed suppressed pro-inflammatory cytokine production in the PBMC population under those conditions. In summary, 17β-estradiol was able to prevent immune dysfunction after TH and subsequent sepsis. However, the beneficial effects of 17β-estradiol were limited to tissue-fixed Mφ, suggesting compartmentalization of the response. Thus, events occurring in the tissue-fixed cells are not necessarily reflected in the circulating PBMC population. [Copyright &y& Elsevier]

Trauma-hemorrhage initiates inflammation in males that leads to a systemic inflammatory response syndrome (SIRS) and immunosuppression, which in turn may develop into multiple organ dysfunction syndrome (MODS). In contrast, females, in the proestrus state (which is associated with elevated estrogen levels), do not show immune depression following trauma-hemorrhage. Studies in experimental models have demonstrated that the underlying basis for this gender-dimorphic immune response following trauma-hemorrhage is due to the marked effects of sex hormones. Sex hormones influence the onset and severity of immune-mediated pathophysiological conditions primarily by modulating immune cell function. Nonetheless, despite extensive studies, the mechanisms of sex hormone action are yet to be precisely defined. Earlier studies had suggested that the sex hormone-mediated modulation of immune activity was primarily via the thymus. In recent years, however, it has become apparent that sex hormones can also influence the immune system by acting on several nonthymic targets such as monocytes, macrophages, lymphoid cells, endothelial cells and the central nervous system. However, more research is needed to define the mechanism responsible for gender-specific immune response to trauma-hemorrhage. These studies will likely contribute to the therapeutic modalities that include manipulation of sex hormones, and/or their receptor agonist/antagonists in the treatment of traumatized patients. [Copyright &y& Elsevier]

The activation of a pro-inflammatory cascade after burn injury appears to be important in the development of subsequent immune dysfunction, susceptibility to sepsis and multiple organ failure. Macrophages are major producers of pro-inflammatory mediators and their productive capacity for these mediators is markedly enhanced following thermal injury. Thus, macrophage hyperactivity (as defined by increased productive capacity for pro-inflammatory mediators) appears to be of critical importance in the development of post-burn immune dysfunction. This review will focus on the current state of knowledge with regards to the role of macrophages in the development of post-burn immune dysfunction. Particular areas of discussion include: nitric oxide synthase (NOS) and cyclooxygenase (COX) enzyme systems, macrophages and the T-helper (Th)-1/Th-2 cytokine responses, alterations in macrophages signal transduction and a potential role for γ/δ T-cells in the development of macrophage hyperactivity following thermal injury. A more comprehensive understanding of the relationship between macrophage activity and post-burn immune dysfunction will hopefully provide the basis for improved therapeutic regimes in the treatment of burn patients. [Copyright &y& Elsevier]

Examines the effectivity of estrogen pretreatment in the management hypoxia-induced immune depression in males. Discussion on the physiological effects of hypoxemia; Impact of elevated systemic estradiol levels on immune depression; Usefulness of the sex hormone in the prevention of hypoxemia during surgery.

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±5 mmHg for 90 min, 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. [Copyright &y& Elsevier]

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-γ production. Splenic macrophage nitric oxide, PGE2, TNF-α, IL-6 and IL-10 production was enhanced post-burn and IL-12 production was suppressed. PBMC from such animals displayed enhanced PGE2 production and suppressed IL-6 and IL-12 production. These results indicate that while an immunosuppressive Th2 phenotype (increased IL-10 and/or suppressed IL-2, IFN-γ) 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 &y& Elsevier]

Resistance of macrophages to the suppressive effect of interleukin-10 following thermal injury. Am J Physiol Cell Physiol 281: Cl180-Cl187, 2001.—The activation of a macrophage (Mφ)-dependent proinflammatory cascade following thermal injury plays an imporrant role in the development of immunosuppression and increased susceptibility to subsequent sepsis in burn patients. In contrast, although interleukin (IL)-10, an antiinflammatory cytokine that can downregulate Mφ activity, has also been implicated in postburn immune dysfunction, its role in the regulation of M[sub φ] 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 Mφs were isolated 7 days later. Lipopolysaccharide (LPS)-stimulated IL-10, IL-6, tumor necrosis factor (TNF)-α, 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-α release, but not NO release, in both groups. The addition of exogenous IL-10 to the Mφ 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, Mφs from injured mice were significantly better able to maintain inflammatory mediator-productive capacity. The resistance of Mφs from injured mice to IL-10-mediated suppression correlated with decreased IL-10 receptor (IL10R) expression and increased CDllb expression. These findings suggest that Mφs, following thermal injury, display resistance to suppression by IL-10 due in part to downregulation of IL-10R expression. [ABSTRACT FROM AUTHOR]

Focuses on the resistance of macrophages to the suppressive effect of interleukin-10. Association of inflammatory cytokine with postburn immune dysfunction; Increase of tumor necrosis factor; Suppression of immune responses following thermal injury.

We previously reported an early surge in high mobility group box protein 1 (HMGB1) levels in a polytrauma (PT) rat model. This study investigates the association of HMGB1 levels in mediating PT associated dysregulated immune responses and its influence on the cellular levels of receptor for advanced glycation end products (RAGE) and toll-like receptor 4 (TLR4). Using the same PT rat model treated with anti-HMGB1 polyclonal antibody, we evaluated changes in circulating inflammatory cytokines, monocytes/macrophages and T cells dynamics and cell surface expression of RAGE and TLR4 at 1, 3, and 7 days post-trauma (dpt) in blood and spleen. Notably, PT rats demonstrating T helper (Th)1 and Th2 cells type early hyper-inflammatory responses also exhibited increased monocyte/macrophage counts and diminished T cell counts in blood and spleen. In blood, expression of RAGE and TLR4 receptors was elevated on CD68+ monocyte/macrophages and severely diminished on CD4+ and CD8+ T cells. Neutralization of HMGB1 significantly decreased CD68+ monocyte/macrophage counts and increased CD4+ and CD8+ T cells, but not γδ+TCR T cells in circulation. Most importantly, RAGE and TLR4 expressions were restored on CD4+ and CD8+ T cells in treated PT rats. Overall, findings suggest that in PT, the HMGB1 surge is responsible for the onset of T cell exhaustion and dysfunction, leading to diminished RAGE and TLR4 surface expression, thereby possibly hindering the proper functioning of T cells. [ABSTRACT FROM AUTHOR]

Focuses on a study which determined whether hypoxemia increases nitric oxide (NO) production by macrophages in vivo and explores whether the increase was due to elevated macrophage-inducible NO synthase. How hypoxemia induces NO production by macrophages; Effects of hypoxia on NO production by splenic macrophages; How macrophage iNOS messenger RNA is expressed following hypoxemia.

Focuses on a study which investigated whether hypoxemia increases nitric oxide (NO) production by macrophages in vivo and whether the increase is due to elevated macrophage-inducible nitric oxide synthase (iNOS). Effect of hypoxia on NO production by splenic macrophages; Expression of macrophage iNOS messenger RNA following hypoxemia; How hypoxia induces increased levels of circulating interferon-gamma.

Reports on cyclooxygenase 2-mediated suppression of macrophage interleukin-12 production after thermal injury. Suppression of postagladin production by cyclooxygenase-2 inhibitor, NS-398; Prevention of the suppression in plasma interleukin-12 level due to NS-398 level.

Introduction: The high incidence of morbidity and mortality following major burn can be in part attributed to immune dysfunction and wound healing complications. Inflammation plays a major role in the complex process of wound repair. Recently, a novel class of T-helper cells, termed Th-17 cells, has been found to secrete the pro-inflammatory cytokines IL-17 and IL-22. The Th-17 response also involves other cytokines, such as IL-6 and TGF-β, which have been shown to be associated with burn-induced inflammation. Nonetheless, the relationships between the Th-17 response and post-burn inflammation are unknown. Methods: C57BL/6 male mice (n =5–6/group) were subjected to a major burn (25% TBSA) or sham procedure. Three hours thereafter, skin samples were collected (uninjured skin and burn skin) and processed for the determination of Th-17 cytokine (IL-6, IL-17, IL-22, IL-23, IL-27, and TGF-β) levels by ELISA. Results: At 3h after burn a significant (∼3-fold) increase in tissue levels of IL-17 and IL-22 was observed at the burn site as compared to sham skin. The burn-induced Th-17 response was independent of statistically significant changes in other Th-17 cytokines (i.e., IL-6, IL-23, IL-27 and TGF-β). Conclusions: These findings indicate the development of a robust Th-17 response at the burn site that may play an important role in subsequent immune and wound healing derangements. [Copyright &y& Elsevier]

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

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

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

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

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

Immune responses in proestrus females are not altered after trauma-hemorrhage, whereas they are markedly depressed in males. Elevated levels of female sex steroids appear to be responsible for maintaining immune responses but it remains unknown, whether estrogen per se is responsible. To study this, proestrus female C3H/HeN mice were subjected to laparotomy (i.e., soft tissue trauma) and hemorrhagic shock (35±5 mmHg for 90 min, then resuscitated) or sham operation and received the estrogen receptor antagonist EM-800 or vehicle during resuscitation. Two hours following trauma-hemorrhage, splenocyte proliferation, IL-2, IL-3, IFN-γ release, and splenic macrophage IL-6 release was maintained in vehicle-treated females. In EM-800-treated females, however, these immune parameters were significantly depressed. Following trauma-hemorrhage, Kupffer cell TNF-α release and circulating TNF-α were increased only in EM-800-treated females. These findings indicate that the ability of proestrus females to maintain immune function following trauma-hemorrhage is estrogen-dependent and mediated via estrogen receptors. [Copyright &y& Elsevier]

Burn injury induces immune dysfunction and alters numerous physiological parameters. While clinical studies indicate that burn injury size profoundly impacts patient immune status, only limited experimental studies have systematically addressed its impact on immune functional parameters. In the present study, mice were subjected to burn injuries of varying sizes and splenic immune cells (splenocytes and macrophages) were isolated 7 days thereafter. Burn injury suppressed splenic T-cell proliferation in an injury size-dependent manner that correlated with the release of the immunosuppressive mediators PGE2 and nitric oxide. In addition, a shift towards an immunosuppressive Th-2 cytokine profile and a hyperactive macrophage phenotype (increased release of inflammatory mediators) was observed post-injury, however, this effect was in part independent of burn size. Thus, unlike patient survival data, burn injury-induced changes in immune function do not necessarily correlate with the size of the injury. [Copyright &y& Elsevier]

Burns are associated with activation of the innate immunity that can contribute to complications. Damage-associated molecular patterns (DAMPs) released after tissue injury play a critical role in the activation of the innate immunity, which appears to be mediated via toll-like receptors (TLRs). Previous findings have shown that TLRs and TLR-mediated responses are up-regulated after burn. Nonetheless, it is unclear what impact burn has on circulating levels of DAMPs. To study this, male C57BL/6 mice were subjected to a major burn or sham procedure. Three hours to 7days thereafter, plasma was collected and assayed for the representative DAMPs (i.e., HMGB1, cytochrome C, DNA and S100A) and extracellular cleavage products (fibronectin and hyaluronan). HMGB1, cytochrome C, fibronectin and hyaluronan levels were elevated in a time-dependent manner after burn as compared to sham levels. A significant elevation in TNF-α, IL-6 and IL-10 cytokine plasma levels was also found after burn. All cytokine levels were increased as early as 3h and remained elevated up to 24h. Circulating CD11b+ monocytes were increased at 24h after burn and showed increased expression of TLR-2. In conclusion, these findings support the concept that burn-induced elevations in circulating DAMPs are in part responsible for monocyte activation and the development of inflammatory complications under such conditions and warrants further investigation. [ABSTRACT FROM AUTHOR]

Background Inflammation and activation of the innate immune system are often associated with traumatic injury and may involve alterations in toll-like receptor (TLR)-mediated responses. Methods A prospective observational study was designed and conducted. Twenty-one severely injured (ISS = 16-41) trauma intensive care unit (ICU) patients and six healthy volunteers that served as controls were enrolled. Anticoagulated whole blood was collected at 2-12 d after ICU admission and incubated in the presence of media alone (baseline), zymosan (TLR2 agonist) or lipopolysaccharide (LPS; TLR4 agonist) for 3 h. Supernatant levels of inflammatory cytokines (IL-1β, IL-6, IL-10, and TNFα) were determined. Results TLR2-mediated and TLR4-mediated activation of whole blood cell cultures from both healthy volunteers and subjects-induced elevated cytokine levels over that observed in unstimulated cultures. Baseline values of IL-6 were significantly elevated in subject cultures as compared to healthy volunteers. Healthy volunteer cultures had 2-3-fold greater levels of IL-6 and TNFα than subject cultures when stimulated with zymosan (TLR2 agonist) or LPS (TLR4 agonist). IL-1β and IL-10 levels did not differ significantly between healthy volunteers and subjects. Conclusions The ability of circulating leukocytes from trauma ICU patients to be activated by TLR agonists is markedly suppressed and may play a role in the development of subsequent infectious complications. [ABSTRACT FROM AUTHOR]

Background The overall immunopathology of the T-helper cell (Th)-17 immune response has been implicated in various inflammatory diseases including pulmonary inflammation; however its potential role in acute respiratory distress syndrome (ARDS) is not defined. This study aimed to evaluate the Th-17 response in bronchoalveolar lavage fluid (BALF) and blood and from trauma patients with pulmonary complications. Methods A total of 21 severely injured intensive care unit (ICU) subjects, who were mechanically ventilated and undergoing bronchoscopy, were enrolled. BALF and blood were collected and analyzed for Th-1 (interferon [IFN]γ), Th-2 (interleukin [IL]-4, -10), Th-17 (IL-17A, -17F, -22, 23) and pro-inflammatory (IL-1β, IL-6, tumor necrosis factor [TNF]α) cytokine levels. Results Significant levels of the Th-17 cytokines IL-17A, -17F and -21 and IL-6 (which can be classified as a Th-17 cytokine) were observed in the BALF of all subjects. There were no significant differences in Th-17 cytokines between those subjects with ARDS and those without, with the exception of plasma and BALF IL-6, which was markedly greater in ARDS subjects, as compared with controls and non-ARDS subjects. Conclusions Trauma patients with pulmonary complications exhibited a significant Th-17 response in the lung and blood, suggesting that this pro-inflammatory milieu may be a contributing factor to such complications. [ABSTRACT FROM AUTHOR]

Burn induces an immunopathological response involving multiple immune cell types that includes γδ T-cells. Nonetheless, the role of γδ T-cells at the wound site after burn is not clearly defined. Wild type and γδ T-cell receptor deficient (δ TCR-/-) mice were subjected to a major burn or sham procedure. At 1–7 d thereafter, skin samples were collected and T-cell populations analyzed. The majority of T-cells in the skin of sham mice were γδ T-cells. After burn, however, an increase in the total T-cells was observed at the wound site and these cells were predominantly αβ T-cells. Their influx was γδ T-cell dependent, as it was markedly reduced in injured δ TCR-/- mice. Burn wound γδ T-cells were activated with increased expression of TLRs and CD69. In contrast, the infiltrating αβ T-cells TLR and CD69 expressions were attenuated after burn. Thus, burn is associated with of γδ T-cell activation at the injury site, which initiates a massive infiltration of the wound with αβ T-cells that likely facilitate the transition from the inflammatory to the proliferative phase of healing. [ABSTRACT FROM AUTHOR]

BACKGROUND: Traumatic hemothorax (HTX) has been demonstrated to predictably contain low fibrinogen, low hematocrit, and low platelet counts. When analyzed on its own, shed HTX demonstrates coagulopathy. However, when mixed with normal pooled plasma (NPP) at physiologically relevant dilutions, HTX demonstrates accelerated coagulation. We hypothesize that when HTX is mixed with a patient's own plasma, the mixture will demonstrate hypercoagulability. The accelerated coagulation of this mixture would have important implications for the autotransfusion of HTX as a method of resuscitating a trauma patient. METHODS: Adult trauma patients from whom greater than 140 mL of HTX was evacuated within 1 hour of tube thoracostomy were included. HTX was sampled at 1 hour after evacuation, and a portion of the sample was centrifuged and stored as frozen plasma for later analysis. The remainder of the sample was analyzed (coagulation, hematology, electrolytes), and values were compared with concurrent venous values extracted via chart review. A citrate tube containing the patient's venous blood was additionally spun down and frozen for subsequent mixing study analysis. Coagulation was further evaluated by mixing serial dilutions of the previously frozen HTX with NPP. Additionally, the previously frozen HTX was mixed in serial dilutions with the previously frozen sample of patient plasma (PTP). RESULTS: Subjects (10) were enrolled based on inclusion criteria and collection of a discarded venous sample. In HTX samples analyzed alone, no thrombus was formed in any coagulation test (activated partial thromboplastin time [aPTT] > 180). The median aPTT value of PTP alone was 25.5. In 1-hour specimens mixed at a clinically relevant dilution of 1:4, HTX mixed with NPP had a mediana PTT value of 26.0, whereas HTX mixed with PTP had a median aPTT value of 21.7. Thus, the mixture of HTX 1 PTP demonstrated a statistically significantly lower aPTT than the mixture of HTX 1 NPP (P = 0.01). Additionally, the mixture of HTX and PTP shows a statistically significantly lower aPTT value than PTP alone (P = 0.03), indicating a hypercoagulable state. CONCLUSIONS: HTX demonstrates coagulopathy when analyzed independently, but is hypercoag-ulable when mixed with NPP or PTP. Furthermore, mixing studies show a statistically significantly lower aPTT when HTX is mixed with PTP versus HTX mixed with NPP. Thus, autotransfusion of HTX would likely produce a hypercoagulable state in vivo, and should not be used in place of other blood products to resuscitate a trauma patient. The autotransfusion of HTX may, however, be of use in a resource-limited environment where other blood products are not available. [ABSTRACT FROM AUTHOR]

BACKGROUND: The evacuated hemothorax has been poorly described because it varies with time, it has been found to be incoagulable, and its potential effect on the coagulation cascade during autotrans- fusion is largely unknown. METHODS: This is a prospective descriptive study of adult patients with traumatic chest injury ne-cessitating tube thoracostomy. Pleural and venous samples were analyzed for coagulation, hematology, and electrolytes at 1 to 4 hours after drainage. Pleural samples were also analyzed for their effect on the coagulation cascade via mixing studies. RESULTS: Thirty-four subjects were enrolled with a traumatic hemothorax. The following measured coagulation factors were significantly depleted compared with venous blood: international normalized ra-tio (>9 vs 1.1) (P < .001) and activated partial thromboplastin time (aPTT) (>180 vs 24.5 seconds) (P < .001). Mixing studies showed a dose-dependent increase in coagulation dilutions through 1:8 (P < .05). CONCLUSIONS: An evacuated hemothorax does not vary in composition significantly with time and is incoagulable alone. Mixing studies with hemothorax plasma increased coagulation, raising safety concerns. [ABSTRACT FROM AUTHOR]

Abstract: Background: The high incidence of morbidity and mortality following major burn can in part be attributed to immune derangements and wound healing complications. Inflammation plays an important role in wound healing, of which inducible nitric oxide synthase (iNOS) derived nitric oxide is a central mediator. T-cells of the γδ TCR lineage have also been shown to be important in healing of the burn wound site. Nonetheless, the role of γδ T-cells in the regulation of the burn wound iNOS expression is unknown. Methods: Wildtype (WT) and δ TCR−/− male C57BL/6 mice were subjected to burn (3rd degree, 12.5% TBSA) or sham treatment. Three days after injury, skin samples from non-injured and the burn wound were collected and analyzed for the expression of iNOS and cytokines and chemokine levels. In a second series of experiments, WT mice were subjected to burn and left untreated or treated with the iNOS inhibitor, L-Nil. Skin cytokine and chemokine levels were assessed 3days thereafter. Results: Burn induced an 18-fold increase in iNOS expression at the wound site as compared to the uninjured skin of WT sham mice. In δ TCR−/− mice iNOS expression at the wound site was significantly lower than that of the WT group. Burn also induced increased levels of IL-1β, IL-6, G-CSF, TNF-α, KC, MCP-1, MIP-1α and MIP-1β at the wound site in WT and δ TCR−/− mice, but G-CSF, TNF-α, and MIP-1β levels were greater in δ TCR−/− mice. Inhibition of iNOS activity in WT mice with L-Nil suppressed burn wound levels of IL-1β, G-CSF, and MIP-1α, whereas IL-6, TNF-α, KC, MCP-1 and MIP-1β were unaffected. Conclusions: T-cells of the γδ TCR lineage significantly contribute to the up-regulation of iNOS expression which contributes to wound inflammation. [Copyright &y& Elsevier]

Abstract: Rationale: Cytokines are central mediators of the immune-inflammatory response to injury and subsequent multiple organ dysfunction syndrome (MODS). Although previous studies evaluated cytokine levels after trauma, differences between patients with burn and non-burn trauma have not been assessed systematically. Methods: A prospective database of trauma patients admitted between May 2004 and September 2007 to the burn or surgical intensive care units within 24h of injury with an anticipated stay of at least 72h was analyzed. Sequential clinical and laboratory parameters were collected in the first week, including multiplex analysis data for plasma levels of inflammatory cytokines (IL-6, and IL-8). Patients with known pre-injury coagulopathy were excluded. A Marshall score of 10 or greater was defined as MODS. Results: A total of 179 patients were enrolled (67 burn and 112 non-burn). Plasma IL-6 and IL-8 levels were markedly elevated in both burn and non-burn patients compared to healthy volunteers. Burn subjects had higher levels of IL-6 and IL-8 than the non-burn on days 1 through 7 after injury. Subjects with burns and at least 30% total body surface area were older and had a lower injury severity score, a higher prevalence of MODS, and correspondingly higher mortality. Multivariate analysis of injury type, MODS, and time did not demonstrate an influence of MODS. Conclusions: Burns were associated with a greater and more sustained immune-inflammatory response than non-burn trauma as evidenced by elevated plasma IL-6 and IL-8 levels during the first week. There was no association between MODS and plasma cytokine levels. [Copyright &y& Elsevier]

Abstract: Burn is associated with profound inflammation and activation of the innate immune system in multiple organ beds, including the lung. Similarly, toll-like receptors (TLR) are associated with innate immune activation. Nonetheless, it is unclear what impact burn has on TLR-induced inflammatory responses in the lung. Methods: Male C57BL/6 mice were subjected to burn (3rd degree, 25% TBSA) or sham procedure and 1, 3 or 7days thereafter, bronchoalveolar lavage (BAL) fluid was collected and cells were isolated and cultured in vitro with specific TLR agonists as follows: Zymosan (TLR-2), LPS (TLR-4) and CpG-ODN (TLR-9). Supernatants were collected 48h later and assayed for inflammatory cytokine levels (IL-1β, IL-6, IL-10, IL-17, TNF-α, KC, MCP-1, MIP-1α, MIP-1β and RANTES) by Bioplex. Results: BAL fluid from sham and burn mice did not contain detectable cytokine levels. BAL cells, irrespective of injury, were responsive to TLR-2 and TLR-4 activation. Seven days after burn, TLR-2 and TLR-4 mediated responses by BAL cells were enhanced as evidenced by increased production of IL-6, IL-17, TNF-α, MCP-1, MIP-1β and RANTES. Conclusions: Burn-induced changes in TLR-2 and TLR-4 reactivity may contribute to the development of post-burn complications, such as acute lung injury (ALI) and adult respiratory distress syndrome (ARDS). [Copyright &y& Elsevier]

The article compares the high-frequency percussive ventilation with contemporary low tidal volume ventilation in select burn intensive care units. The clinical outcomes of high-frequency percussive ventilation and low tidal volume ventilation are found similar in burn patients with respiratory failure. The low tidal volume ventilation failed to achieve ventilation and oxygenation goals as compared with high-frequency percussive ventilation.

p38 MAPK has been reported to regulate the inflammatory response in various cell types via extracellular stimuli. p38 MAPK activation also results in the induction of heme oxygenase (HO)-1, which exerts potent anti-inflammatory effects. Although studies have shown that 17β-estradiol (B2) prevented organ dysfunction following trauma-hemorrhage, it remains unknown whether p38 MAPK/HO-1 plays any role in E2-mediated attenuation of intestinal injury under those conditions. To study this, male rats underwent trauma-hemorrhage (mean blood pressure ~40 mmHg for 90 min) followed by fluid resuscitation. At the onset of resuscitation, rats were treated with vehicle, E2 (1 mg/kg body wt), the p38 MAPK inhibitor SB-203580 (2 mg/kg body wt) or E2 plus SB-203580. Two hours thereafter, intestinal myeloperoxidase (MPO) activity and lactate, TNF-α, IL-6, ICAM-1, cytokine-induced neutrophil chemoattractant (CINC)-1, and macrophage inflammatory protein (MIP)-2 levels were measured. Intestinal p38 MAPK and HO-1 protein levels were also determined. Trauma-hemorrhage led to an increase in intestinal MPO activity and lactate, TNF-α, IL-6, ICAM-1, CINC-1, and MIP-2 levels. This was accompanied with a decrease in intestinal p38 MAPK activity and increase in HO-1 expression. Administration of E2 normalized all the above parameters except HO-1, which was further increased following trauma-hemorrhage. Administration of SB-203580 with E2 abolished the E2-mediated restoration of the above parameters as well as the increase in intestinal HO-1 expression following trauma-hemorrhage. These results suggest that the p38 MAPK/HO-1 pathway plays a critical role in mediating the salutary effects of E2 on shock-induced intestinal injury. [ABSTRACT FROM AUTHOR]

Studies have shown that p38 MAPK and nitric oxide (NO), generated by endothelial NO synthase (eNOS), play key roles under physiological and pathophysiological conditions. Although administration of 17β-estradiol (E2) protects cardiovascular injury from trauma-hemorrhage, the mechanism by which E2 produces those effects remains unknown. Our objective was to determine whether the E2-mediated activation of myocardial p38 MAPK and subsequent eNOS expression/phosphorylation would protect the heart following trauma-hemorrhage. To study this, male Sprague-Dawley rats underwent soft-tissue trauma (midline laparatomy) and hemorrhagic shock (mean blood pressure 35-40 mmHg for 90 min), followed by fluid resuscitation. Animals were pretreated with specific p38 MAPK inhibitor SB-203580 (SB; 2 mg/kg), and nonselective NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME; 30 mg/kg) 30 min before vehicle (cyclodextrin) or E2 (100 µg/kg) treatment, followed by resuscitation, and were killed 2 h thereafter. Cardiovascular performance and other parameters were measured. E2 administration following trauma-hemorrhage increased cardiac p38 MAPK activity, eNOS expression and phosphorylation at Ser1117, and nitrate/nitrite levels in plasma and heart tissues; these were associated with normalized cardiac performance, which was reversed by SB administration. In addition, E2 also prevented trauma-hemorrhage-induced increase in cytokines (IL-6 and TNF-α), chemokines (macrophage inflammatory protein-2 and cytokine-induced neutrophil chemoattractant-1), and ICAM-1, which was reversed by L-NAME administration. Administration of E2 following trauma-hemorrhage attenuated cardiac tissue injury markers, myeloperoxidase activity, and nitrotyrosine level, which were reversed by treatment with SB and L-NAME. The salutary effects of E2 on cardiac functions and tissue protection following trauma-hemorrhage are mediated, in part, through activation of p38 MAPK and subsequent eNOS expression and phosphorylation. [ABSTRACT FROM AUTHOR]

Abstract: Although 17β-estradiol administration following trauma–hemorrhage attenuates Kupffer cell, splenic and peritoneal macrophage functions, it remains unknown whether 17β-estradiol has any salutary effects on splenic dendritic cell (DC) functions and if so, whether such effects are mediated via the estrogen receptors (ER). We hypothesized that 17β-estradiol administration following trauma–hemorrhage has salutary effects on splenic DC functions. Male C3H/HeN (6–8 weeks) mice were randomly assigned to sham operation or trauma–hemorrhage. Trauma–hemorrhage was induced by midline laparotomy and ∼90min of hemorrhagic shock (blood pressure [BP] 35mmHg), followed by fluid resuscitation (4× the shed blood volume in the form of Ringer''s lactate). Estrogen receptor (ER)-α agonist propyl pyrazole triol (PPT; 5μg/kg), ER-β agonist diarylpropionitrile (DPN; 5μg/kg), 17β-estradiol (50μg/kg), or vehicle (10% DMSO) was injected subcutaneously during resuscitation. Two hours later, the mice were sacrificed, splenic DCs were isolated and the changes in their apoptosis, co-stimulating factors and MHC class II expression, ability to produce cytokines, and antigen presentation capacity were measured. Apoptosis of splenic DC increased following trauma–hemorrhage; however, 17β-estradiol administration after trauma–hemorrhage normalized the rate of apoptosis. Moreover, splenic DC cytokines production, co-stimulating factors and MHC class II expression, and antigen presentation capacity were significantly decreased following trauma–hemorrhage; however, 17β-estradiol as well as PPT also prevented these depressions. In contrast, DPN did not attenuate splenic DC functions following trauma–hemorrhage. Since PPT administration following trauma–hemorrhage was more effective in normalizing splenic DC functions than DPN, the salutary effects of 17β-estradiol on splenic DC functions are mediated predominantly via ER-α. [Copyright &y& Elsevier]

Abstract: Trauma-hemorrhage produces immunodepression in males but not in proestrus females and this difference is due to the presence of high estrogen in proestrus females. Although skin is the largest immunological organ of the body and is considered the first line of defense, no study to-date has examined whether trauma-hemorrhage has any effects on keratinocytes which are the major epidermal cell type (>90%) of skin. We therefore examined whether epidermal keratinocytes inflammatory response and the signal transduction pathways involved in the inflammatory response are altered following trauma-hemorrhage. C3H/HeN mice were subjected to trauma-hemorrhage and 2h thereafter; keratinocytes were harvested and stimulated with LPS for 24h (5μg/ml). Inflammatory mediators, Toll-like receptor (TLR) and myeloid differentiation adaptor protein (MyD88) expression, and the activation of mitogen-activated protein kinase (MAPK) were determined. Trauma-hemorrhage increased the production of IL-6, IL-10, IL-12 and TNF-α enhanced the expression of TLR4, MyD88 as well as the activation of MAPK proteins (p38, ERK and JNK) in epidermal keratinocytes. However, administration of a single dose of 17β-estradiol following trauma-hemorrhage prevented the increase in these inflammatory parameters under those conditions. These findings suggest that 17β-estradiol normalizes epidermal keratinocytes inflammatory responses following trauma-hemorrhage by preventing the upregulation of TLR4-mediated MAPK activation. [Copyright &y& Elsevier]

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

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

Toll-like receptor 4 (TLR4) mediates mitochondrial DNA (mtDNA) damage and biogenic responses. Mitochondrial transcription factor A (Tfam) is an essential regulator for mtDNA transcription and ATP production. Increased ATP levels were associated with normalization of immune function following trauma-hemorrhage. Moreover, administration of 17β-estradiol following trauma-hemorrhage upregulates cardiac Tfam and ATP levels. We therefore hypothesized that the salutary effect of 17β-estradiol on Kupffer cell function following trauma-hemorrhage is mediated via negative regulation of TLR4, which downregulates iNOS, upregulates Tfam and mtDNA-encoded gene cytochrome c oxidase I (mtCOI), and consequently increases cellular ATP levels. Male C3H/HeN, C3H/HeOuJ (intact TLR4), and C3H/HeJ (TLR4 mutant) mice were subjected to trauma-hemorrhage (mean BP 35 ± 5 mmHg ∼90 min, then resuscitation) or sham operation. At the beginning of resuscitation, mice received 17β-estradiol (25 µg/25 g) or vehicle intravenously and were sacrificed 2 h thereafter. Kupffer cell TLR4, iNOS, IL-6 and TNF-α production capacities were increased, and ATP, Tfam, and mtCOI levels were decreased following trauma-hemorrhage. Administration of 17β-estradiol following trauma-hemorrhage prevented the increase in Kupffer cell TLR4, iNOS, and cytokine production. This was accompanied by normalized ATP, Tfam, and mtCOI levels. Furthermore, the decreased Kupffer cell ATP and mtCOI levels were not observed in TLR4 mutant mice following trauma-hemorrhage. Taken together, these findings suggest that downregulation of TLR4-dependent ATP production is critical to 17β-estradiol-mediated immunoprotection in Kupffer cells following trauma-hemorrhage. J. Cell. Physiol. 211: 364–370, 2007. © 2007 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]