Irshad H. Chaudry

Cecal ligation and puncture.

The model of cecal ligation and puncture (CLP) in rodents has been used extensively to investigate the clinical settings of sepsis and septic shock. This model produces a hyperdynamic, hypermetabolic state that can lead to a hypodynamic, hypometabolic stage, and eventual death. Blood cultures are positive for enteric organisms very early after CLP. The model has been widely used over the past 26 years and is highly versatile in adapting to a range of severity and testing objectives. It is inexpensive to prepare and technically straightforward. Aspects of sepsis research investigated using CLP include energetics, metabolism, resuscitation, antibiotic therapy, microbial factors, cardiovascular responses, immune function, mediator release, and cytokine expression patterns. The challenge of the small circulating blood volume in rodents can be overcome by using micromethods that enable analysis of small volumes, or alternatively, by using a large number of animals to obtain serial samples.

Female Sex Hormones Regulate Macrophage Function After Trauma-Hemorrhage and Prevent Increased Death Rate From Subsequent Sepsis

ObjectiveTo determine whether reduction of circulating female sex hormones by ovariectomy causes suppression of macrophage (M&phgr;) function after trauma-hemorrhage and increases susceptibility to subsequent sepsis. Summary Background DataStudies indicate that immune functions are markedly depressed in males but not in proestrus females after trauma-hemorrhage. Although male sex steroids are immunosuppressive, it remains unknown whether female sex hormones are immunoprotective after trauma-hemorrhage. MethodsCirculating female sex hormones were reduced by ovariectomy of 8-week-old female CBA/J mice. Two weeks afterward, ovariectomy and proestrus sham-ovariectomy mice were subjected to laparotomy (i.e., soft tissue trauma) and hemorrhagic shock (35 ± 5 mm Hg for 90 minutes, then resuscitated) or sham operation. Two hours afterward, splenic and peritoneal M&phgr; and Kupffer cells were isolated and cytokine production was assessed. In a second series of experiments, animals were subjected to sepsis by cecal ligation and puncture at 24 hours after trauma-hemorrhage or sham operation, and survival was assessed. ResultsRelease of interleukin-1 and interleukin-6 by splenic and peritoneal M&phgr; from proestrus mice was maintained after trauma-hemorrhage, whereas release of interleukin-1 and interleukin-6 by M&phgr; from ovariectomized mice was depressed by approximately 50%. In contrast, trauma-hemorrhage resulted in a fourfold increase of Kupffer cell release of tumor necrosis factor-alpha in ovariectomized females and a fivefold increase in plasma concentrations of tumor necrosis factor-alpha. Release of tumor necrosis factor-alpha and plasma concentrations were unchanged in proestrus mice under such conditions. When proestrus and ovariectomized animals were subjected to sepsis by cecal ligation and puncture at 24 hours after trauma-hemorrhage or sham operation, ovariectomized mice had a significantly higher death rate than proestrus mice. ConclusionsThese findings suggest that female sex hormones play a critical role in maintaining immune responses after trauma-hemorrhage by suppressing the elaboration of tumor necrosis factor-alpha and prevent the increased lethality from subsequent sepsis. Thus, female sex hormones may be a useful adjunct in preventing trauma-induced immunodepression and increased susceptibility to subsequent sepsis.

Influence of sex and age on mods and cytokines after multiple injuries.

Although salutary effects of female sex steroids have been demonstrated not only in proestrus females but also in male animals treated with estradiol in different models of trauma, it remains unknown whether sex hormones influence post-traumatic immune response in humans. We therefore investigated the effect of sex and age on organ dysfunction and clinical course in patients with multiple injuries. Polytraumatized patients (injury severity score > 16) between 16 and 65 years old admitted to the Hannover Medical School Level 1 trauma center between January 1997 and December 2001 were included. Marshall Score for multiple organ dysfunction syndrome (MODS) was calculated for at least 14 days. The length of stay in intensive care unit and the ventilator days were recorded in addition to the number of transfusions the patient received. A total of 106 males and 37 females were included in the study. Patients with MODS had increased plasma levels of IL-6, IL-8, and IL-10. Furthermore, patients with organ dysfunction had more frequent sepsis and higher mortality rates. In addition, MODS was associated with extended length of stay in the intensive care unit and increased ventilator days. Females not older than 50 years with an injury severity score greater than 25 suffered significantly less MODS and sepsis compared with age-matched males. Moreover, they had significantly lower plasma cytokines. Thus, in this prospective study, sex difference was confirmed in MODS and sepsis, with a benefit observed in females. Although the levels of sex steroids were not measured, it seems that the protective effects may be due to estrogen effects on plasma cytokines. This suggestion is based on the fact that such salutary effects were apparent predominantly in premenopausal females.

Gender differences in acute response to trauma-hemorrhage.

To understand the pathogenesis of a disease, experimental models are needed. A good experimental model is the one that simulates responses observed in the clinical setting. In recent years, clinical studies have indicated that gender might be a factor that plays a significant role in the outcome of patients with shock, trauma, and sepsis. These observations are now being evaluated in experimental setting. Studies performed in a rodent model of trauma-hemorrhage have concluded that alterations in immune and cardiac functions after trauma-hemorrhage are more markedly depressed in adult males, and ovariectomized and aged females. However, both are maintained in castrated males and in proestrus females. Moreover, the survival rate of proestrus females subjected to sepsis after trauma-hemorrhage is significantly higher than age-matched males or ovariectomized females. Although these observations suggest gender-specific response after trauma-hemorrhage, the mechanisms responsible for gender specificity remain largely unknown. Furthermore, in other injuries such as burn, experimental studies dealing with sexual dimorphism are limited. Therefore, more studies in clinical and experimental settings are required to determine whether gender-specific responses are global across the injuries or are observed in specific injury situations. Studies are also needed to delineate underlying mechanisms responsible for differences between males and females after trauma-hemorrhage. The information gained from the experimental studies will help in designing innovative therapeutic approaches for the treatment of trauma patients.

Surgical trauma and immunosuppression: pathophysiology and potential immunomodulatory approaches

BackgroundSeveral studies indicate that organ failure is the leading cause of death in the postoperative phase after major surgery. An excessive inflammatory response followed by a dramatic depression of cell-mediated immunity after major surgery appears to be responsible for the increased susceptibility to subsequent sepsis. In view of this, most of the scientific and medical research has been directed towards measuring the progression and interrelationship of mediators after major surgery. Furthermore, the effect of those mediators on cell-mediated immune responses has been studied.ObjectiveThis article focuses on the effect of surgical injury and blood loss on cell-mediated immune responses in experimental studies utilizing models of trauma and hemorrhagic shock. The findings from those experimental studies will also be correlated with data from surgical patients.ResultsRecently, a gender-dimorphic immune and organ responsiveness in the susceptibility to and morbidity from shock, trauma, and sepsis has been found. Androgens have been shown to be responsible for the immunosuppression after trauma–hemorrhage in males. In contrast, female sex steroids exhibit immunoprotective properties after trauma and severe blood loss.ConclusionIn view of these findings, clinically relevant therapeutic strategies have been developed using the testosterone receptor blocker flutamide and/or estrogen or agents with estrogenic effects, i.e., dehydroepiandrosterone, which might yield safe and useful therapeutic approaches for the treatment of immune depression in surgical patients.

The role of MAPK in Kupffer cell toll-like receptor (TLR) 2-, TLR4-, and TLR9-mediated signaling following trauma-hemorrhage

Severe injury deranges immune function and increases the risk of sepsis and multiple organ failure. Kupffer cells play a major role in mediating posttraumatic immune responses, in part via different Toll‐like receptors (TLR). Although mitogen‐activated protein kinases (MAPK) are key elements in the TLR signaling pathway, it remains unclear whether the activation of different MAPK are TLR specific. Male C3H/HeN mice underwent midline laparotomy (i.e., soft tissue injury), hemorrhagic shock (MAP ∼35 mm Hg for 90 min), and resuscitation. Kupffer cells were isolated 2 h thereafter, lysed and immunoblotted with antibodies to p38, ERK1/2, or JNK proteins. In addition, cells were preincubated with specific inhibitors of p38, ERK1/2, or JNK MAPK followed by stimulation with the TLR2 agonist, zymosan; the TLR4 agonist, LPS; or the TLR9 agonist, CpG DNA. Cytokine (TNF‐α, interleukin‐6 (IL‐6), monocyte chemoattractant protein‐1 (MCP‐1), and KC) production was determined by cytometric bead array after 24 h in culture. MAPK activity as well as TNF‐α, MCP‐1, and KC production by Kupffer cells were significantly increased following trauma‐hemorrhage. TLR4 activation by LPS stimulation increased the levels of all measured cytokines. CpG‐stimulated TLR9 signaling increased TNF‐α and IL‐6 levels; however, it had no effect on chemokine production. Selective MAPK inhibition demonstrated that chemokine production was mediated via p38 and JNK MAPK activation in TLR2, ‐4, and ‐9 signaling. In contrast, TNF‐α and IL‐6 production was differentially regulated by MAPK depending on the TLR pathway stimulated. Thus, Kupffer cell TLR signaling employs different MAPK pathways in eliciting cytokine and chemokine responses following trauma‐hemorrhage. J. Cell. Physiol. 210: 667–675, 2007.

Divergent Immune Responses in Male and Female Mice after Trauma-Hemorrhage: Dimorphic Alterations in T Lymphocyte Steroidogenic Enzyme Activities

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

Trauma-hemorrhage induces depressed splenic dendritic cell functions in mice

Although Kupffer cell, splenic, and peritoneal macrophage functions are markedly altered following trauma-hemorrhage (T-H), it remains unclear whether T-H also affects splenic dendritic cell (sDC) functions. We hypothesized that sDC functions will also be compromised following T-H. Male C3H/HeN (6- to 8-wk) mice were randomly assigned 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 (four times the shed blood volume in the form of Ringer’s lactate). Two hours later, the mice were sacrificed; sDC were isolated; and the changes in their apoptosis, MHC class II expression, and ability to produce costimulatory cytokines and Ag presentation were measured. The results indicate that sDC Ag presentation capacity was significantly decreased and MHC class II expression was also significantly decreased following T-H. Moreover, LPS-induced IL-12 production and LPS- or IL-12-induced IFN-γ production following T-H were significantly decreased. Thus, the markedly decreased MHC class II expression and cytokine (IL-12, IFN-γ) production following T-H may be the cause for the depressed sDC Ag presentation under those conditions. This depression in Ag presentation could contribute to the host’s enhanced susceptibility to sepsis following T-H.

Inhibition of IL-18 reduces myeloperoxidase activity and prevents edema in intestine following alcohol and burn injury

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

Alcohol intoxication and post-burn complications.

Results from the studies discussed in this article suggest that alcohol (EtOH) intoxication is a major public health problem. While the effects of injury and EtOH intoxication independent of each other have been studied in detail, only few studies have evaluated the effect of a combined insult of EtOH intoxication and burn injury on host defense. An analysis of the studies conducted in the clinical setting suggests that intoxicated patients require frequent intubations, experience delayed wound healing and longer hospital stay. Furthermore, there is a greater risk of mortality in these patients compared to those who sustained injuries in the absence of EtOH intoxication. On the other hand, there are a few studies that do not support this notion. The results obtained in experimental models clearly suggest that acute EtOH intoxication before burn injury impairs host defense and increases susceptibility to infection. Additionally, experimental data from our laboratory also indicate that EtOH intoxication before burn injury suppresses intestinal immune defense, impairs gut barrier functions and increases bacterial growth. This results in increased bacterial translocation in EtOH and burn injury. In addition, a decrease in cardiac function is also reported following a combined insult of EtOH intoxication and burn injury. Altogether, these findings suggest that EtOH intoxication before burn injury diminishes host resistance resulting in increased susceptibility to infection. Moreover, the findings of a higher incidence of infectious complications in burn and trauma patients who sustained injury in the presence of EtOH compared to those in its absence suggest that EtOH intoxication at the time of injury is a risk factor. Therefore blood EtOH should be monitored in burn/trauma patients at the time of admission in the emergency room.