Immunoprotective effect of a calcium channel blocker on macrophage antigen presentation function, major histocompatability class II antigen expression, and interleukin-1 synthesis after hemorrhage

Hemorrhage induces a severe suppression of the immune system resulting in increased susceptibility to sepsis. Although studies indicate beneficial effects of calcium channel blockers on cell and organ functions after low-flow conditions, it remains unknown whether such agents have any effects on different immune responses after hemorrhage. To study this, C3H/HeN mice were bled to a mean blood pressure of 35 mm Hg and were maintained for 60 minutes, followed by resuscitation with their own shed blood and adequate fluid. The mice received either the water-soluble calcium channel blocker diltiazem (400 or 2400 micrograms/kg body weight) or saline solution (vehicle). Peritoneal macrophages were obtained by lavage 24 hours later. Antigen presentation was measured by coculturing peritoneal macrophages with the D10.G4.1 helper T-lymphocyte clone. Immune associated antigen (Ia) expression was determined by direct immunofluorescence. Interleukin (IL)-1, 6, and tumor necrosis factor-alpha (TNF) levels in peritoneal macrophage supernatants were measured by use of cytokine-specific cellular assays. Hemorrhage caused a significant decrease in peritoneal macrophage antigen presentation function, Ia expression, and IL-1 and IL-6 synthesis in the vehicle-treated group, whereas TNF levels were increased. However, both doses of diltiazem significantly improved peritoneal macrophage antigen presentation, Ia expression, and IL-1 synthesis. IL-6 synthesis was only increased with high doses of diltiazem, whereas both diltiazem doses decreased TNF production. These results indicate that the calcium channel blocker diltiazem can markedly improve macrophage functions after hemorrhage. The use of diltiazem might offer a new therapeutic modality in the treatment of immunosuppression and in decreasing the susceptibility to sepsis after hemorrhagic shock.     

L-arginine: a unique amino acid for improving depressed wound immune function following hemorrhage

OBJECTIVE: To determine whether L-arginine has any salutary effects on wound immune cell function following trauma-hemorrhage. BACKGROUND: Depressed wound immune function contributes to an increased incidence of wound infections following hemorrhage. Although administration of L-arginine has been shown to restore depressed cell-mediated immune responses following hemorrhage potentially by maintaining organ blood flow, it remains unknown whether L-arginine has any salutary effects on the depressed local immune response at the wound site. METHODS: Male mice were subjected to a midline laparotomy and polyvinyl sponges were implanted subcutaneously in the abdominal wound prior to hemorrhage (35 +/- 5 mm Hg for 90 min and resuscitation) or sham operation. During resuscitation mice received 300 mg/kg body weight L-arginine or saline (vehicle). Sponges were harvested 24 h thereafter, wound fluid collected and wound immune cells cultured for 24 h in the presence of LPS. Pro- (IL-1 beta, IL-6) and anti-inflammatory (IL-10) cytokines were determined in the supernatants and the wound fluid. In addition, wounds were stained for IL-6 immunohistochemically. In a separate set of animals, skin and muscle blood flow was determined by microspheres. RESULTS: The capacity of wound immune cells to release IL-1 beta and IL-6 in vitro was significantly depressed in hemorrhaged mice receiving vehicle. Administration of L-arginine, however, improved wound immune cell function. In contrast, in vivo the increased IL-6 release at the wound site was decreased in L-arginine-treated mice following hemorrhage. Moreover, IL-10 levels were significantly increased in the wound fluid in hemorrhaged animals receiving L-arginine compared to vehicle-treated mice. In addition, the depressed skin and muscle blood flow after hemorrhage was restored by L-arginine. CONCLUSIONS: Thus, L-arginine might improve local wound cell function by decreasing the inflammatory response at the wound site. Since L-arginine protected wound immune cell function this amino acid might represent a novel and useful adjunct to fluid resuscitation for decreasing wound complications following hemorrhage.     

Do female sex steroids adversely or beneficially affect the depressed immune responses in males after trauma-hemorrhage?

HYPOTHESIS: Administration of female sex steroids in males after trauma-hemorrhage has salutary effects on the depressed immune responses. DESIGN: Randomized laboratory experiment. INTERVENTIONS: Male C3H/HeN mice were subjected to midline laparotomy and hemorrhagic shock (35+/-5 mm Hg for 90 minutes, then resuscitation) or sham operation and received subcutaneous 17beta-estradiol (40 microg/kg body weight) or corn oil vehicle at the beginning of resuscitation. MAIN OUTCOME MEASURES: At 24 hours after hemorrhage, the animals were killed and plasma 17beta-estradiol and IL-6, splenocyte interleukin (IL) 2, IL-3, and IL-10 production as well as splenic and peritoneal macrophage IL-1beta, IL-10, and IL-6 release were measured. RESULTS: Splenocyte IL-2 and IL-3 release were significantly depressed after hemorrhage in vehicle-treated mice (P<.05, analysis of variance). Treatment with 17beta-estradiol after hemorrhage led to the restoration of splenocyte IL-2 and IL-3 release. The depressed proinflammatory cytokine (IL-1 and IL-6) release seen in splenic and peritoneal macrophages was restored in the 17beta-estradiol-treated hemorrhage group. In contrast, the sustained release of the anti-inflammatory cytokine IL-10 by splenocytes and splenic and peritoneal macrophages in vehicle-treated mice after hemorrhage was decreased in 17beta-estradiol-treated mice. The increase in circulating IL-6 levels after hemorrhage was significantly attenuated in 17beta-estradiol-treated mice. Although administration of 17beta-estradiol increased plasma 17beta-estradiol levels by approximately 100% in sham as well as hemorrhage groups, improved immune responses were seen only in posthemorrhage 17beta-estradiol-treated mice. There was no adverse effect of 17beta-estradiol treatment in the sham or posthemorrhage groups. CONCLUSION: Since administration of a single dose of 17beta-estradiol in males after trauma-hemorrhage restores the immune functions and decreases circulating levels of IL-6, hormones with estrogenic properties should be considered as safe and novel therapeutic agents for restoring the immune responsiveness in male trauma victims.     

Diltiazem administration after crystalloid resuscitation restores active hepatocellular function and hepatic blood flow after severe hemorrhagic shock.

Studies have shown that active hepatocellular function is depressed after hemorrhagic shock, despite crystalloid resuscitation. It is also known that calcium antagonists produce various beneficial effects on cell and organ function after ischemia and shock. However, it remains unknown whether such agents have any salutary effects on the depressed active hepatocellular function and hepatic blood flow in a nonheparinized model of trauma and hemorrhage. To study this, rats underwent a midline laparotomy (trauma-induced) and were bled to and maintained at a mean arterial pressure of 40 mm Hg until 40% of the maximum bleedout was returned in the form of Ringer's lactate. They were then resuscitated with four times the volume of shed blood with Ringer's lactate over 60 minutes, during and after which diltiazem (400 micrograms/kg body weight) was infused intravenously over 95 minutes. Active hepatocellular function (Vmax and Km) was determined with an in vivo indocyanine green clearance technique. Effective hepatic blood flow (EHBF) was determined by Fick principle and corrected by the indocyanine green extraction ratio. Hepatic microvascular blood flow (HMBF) was measured by laser Doppler flowmetry. Results indicate that Vmax, Km, EHBF, and HMBF decreased significantly at 1.5 and 4 hours after resuscitation. Diltiazem infusion restored the depressed Vmax, Km, EHBF, and HMBF and prevented the occurrence of hepatic edema. Thus, diltiazem may be a useful adjunct in the treatment of trauma and severe hemorrhage even in the absence of blood resuscitation.     

Estradiol administration after trauma-hemorrhage improves cardiovascular and hepatocellular functions in male animals

OBJECTIVE: To determine whether female sex steroids have any salutary effects on the depressed cardiovascular and hepatocellular functions following trauma and hemorrhage in male animals. SUMMARY BACKGROUND DATA: Studies indicate that gender difference exists in the immune and cardiovascular responses to trauma-hemorrhage, and that male sex steroids appear to be responsible for producing immune and organ dysfunction, but it remains unknown if female sex steroids produce any salutary effects on the depressed cellular and organ functions in males following trauma and hemorrhage. METHOD: Adult male Sprague-Dawley rats underwent a midline laparotomy (i.e., trauma induction), and were bled to and maintained at a mean arterial pressure of 40 mmHg until 40% of the maximum bleed-out volume was returned in the form of Ringer's lactate (RL). Animals were then resuscitated with RL at 4 times the shed blood over 60 minutes. 17beta-estradiol (50 microg/kg) or an equal volume of vehicle was injected subcutaneously 15 minutes before the end of resuscitation. The maximal rate of ventricular pressure increase or decrease (+/-dP/dtmax), cardiac output, and hepatocellular function (i.e., maximal velocity and overall efficiency of in vivo indocyanine green clearance) were assessed at 24 hours after hemorrhage and resuscitation. Plasma levels of interleukin (IL)-6 were also measured. RESULTS: Left ventricular performance, cardiac output, and hepatocellular function decreased significantly at 24 hours after trauma-hemorrhage and resuscitation. Plasma levels of IL-6 were elevated. Administration of 17beta-estradiol significantly improved cardiac performance, cardiac output, and hepatocellular function, and attenuated the increase in plasma IL-6 levels. CONCLUSION: Administration of estrogen appears to be a useful adjunct for restoring cardiovascular and hepatocellular functions after trauma-hemorrhage in male rats.     

Physiological levels of 5 alpha-dihydrotestosterone depress wound immune function and impair wound healing following trauma-hemorrhage

HYPOTHESIS: Studies indicate that a depressed wound immune function contributes to an increased rate of wound complications and impaired wound healing following trauma-hemorrhage (T-H). Androgen, ie, 5 alpha-dihydrotestosterone, is responsible for producing the depressed systemic cell-mediated immune responses following T-H in males. The aim of the present study was to determine whether depletion of 5 alpha-dihydrotestosterone in males before T-H has any salutary effects on wound immune cell function and wound healing in male mice following T-H. DESIGN: Mice were castrated or sham castrated 14 days before midline laparotomy (ie, tissue trauma) and subcutaneous polyvinyl sponge implantation, followed by hemorrhage (mean +/- SEM blood pressure, 35 +/- 5 mm Hg for 90 minutes and resuscitation) or sham operation. At 24 hours thereafter, wound immune cells from the sponges were harvested and cultured with lipopolysaccharide A. Release of interleukin 1 beta (IL-1 beta) and IL-6 (in picograms per milliliter) was determined in the supernatants by enzyme-linked immunosorbent assay. In addition, IL-6 was assessed at the wound site by immunohistochemistry. Ten days after T-H, wound-breaking strength was measured. RESULTS: Precastration prevented the significantly suppressed capacity of wound immune cells to release IL-1 beta and IL-6. In addition, precastration normalized the elevated IL-6 expression at the wound site in the T-H mice. Moreover, wound-breaking strength was improved in castrated mice 10 days after T-H. CONCLUSIONS: Male sex steroids appear to be responsible for wound immune cell dysfunction following trauma and severe blood loss. Because decreasing androgen levels resulted in improved wound healing, our results suggest that the use of androgen receptor-blocking agents, eg, flutamide, following T-H might represent a novel adjunct for decreasing the rate of wound complications under those conditions.     

Restoration of body temperature to normothermia during resuscitation following trauma-hemorrhage improves the depressed cardiovascular and hepatocellular functions

HYPOTHESIS: Rewarming the body to 37 degrees C during resuscitation following trauma-hemorrhage has salutary effects on cardiovascular and hepatocellular functions. DESIGN, INTERVENTIONS, AND MAIN OUTCOME MEASURES: Male rats underwent laparotomy (trauma induced) and were then bled to and maintained at a mean arterial pressure of 40 mm Hg until 40% of the maximum shed blood volume was returned in the form of Ringer lactate solution. Rats were exposed to ambient temperature and allowed to become hypothermic during hemorrhage. The animals were then resuscitated with 4 times the volume of shed blood with Ringer lactate solution for 60 minutes. In 1 group, the body temperature was rewarmed to 37 degrees C during resuscitation. In another group, the body temperature was maintained at hypothermia (32 degrees C) for 4 hours after resuscitation. In an additional group, the body temperature was kept at 37 degrees C during hemorrhage and resuscitation. At 4 hours after resuscitation, the rats were returned to a room with ambient temperature. Various in vivo heart performance variables (maximal rate of pressure increase and decrease), cardiac output, hepatocellular function, and plasma IL-6 level were determined at 24 hours after resuscitation. RESULTS: Either maintenance of normothermia during hemorrhage or prolonged hypothermia following resuscitation had deleterious effects on cardiovascular variables and hepatocellular function and up-regulated plasma IL-6 levels. In contrast, rewarming the body to 37 degrees C during resuscitation improved cardiac contractility, cardiac output, and hepatocellular function and reduced plasma IL-6 level. CONCLUSION: Since rewarming the body temperature to normothermia during resuscitation improved depressed cardiovascular and hepatocellular functions, this should be considered as a useful adjunct to fluid resuscitation after trauma-hemorrhage.     

Energetics of defective macrophage antigen presentation after hemorrhage as determined by ultraresolution 31P nuclear magnetic resonance spectrometry: restoration with adenosine triphosphate-MgCl2.

BACKGROUND. The purpose of this study was to determine whether a decrease in macrophage energetics contributes to the profound immune dysfunction that occurs after hemorrhage and, if so, whether adenosine triphosphate (ATP)-MgCl2 treatment has any beneficial effects on the above parameters. METHODS. C3H/HeN mice were bled to a mean blood pressure of 35 mm Hg, maintained at that pressure for 60 minutes, resuscitated with their own shed blood and Ringer's lactate, and treated with ATP-MgCl2 (80 mumol/kg body weight) or saline solution (vehicle). Peritoneal macrophages were harvested 1 hour after resuscitation and ATP levels were determined by 31P nuclear magnetic resonance spectrometry. In addition, macrophage functions were determined by measuring antigen presentation capacity (AP), as well as interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor (TNF) synthesis. RESULTS. Hemorrhage caused a significant decrease in peritoneal macrophage AP function, as well as IL-1, IL-6, and TNF synthesis, by 52% +/- 14%, 91% +/- 12%, 78% +/- 8%, and 89% +/- 8%, respectively, which was correlated with a 78% +/- 6% decrease in macrophage ATP levels (p less than 0.05). Treatment with ATP-MgCl2 after hemorrhage restored macrophage ATP levels (p less than 0.05) and significantly increased (p less than 0.05) macrophage AP, IL-1, IL-6, and TNF release by 110% +/- 21%, 130% +/- 38%, 124% +/- 17%, and 66% +/- 24%, respectively. CONCLUSIONS. The decreased macrophage ATP levels may be the cause of defective macrophage AP and cytokine release after hemorrhage, and both macrophage ATP levels and macrophage immune functions can be restored with adjuvant ATP-MgCl2 treatment after hemorrhage.     

ATP-MgCl2 restores the depressed hepatocellular function and hepatic blood flow following hemorrhage and resuscitation

Although ATP-MgCl2 produces a myriad of beneficial effects following organ ischemia and simple hemorrhagic shock in animal models which involved heparinization and/or blood resuscitation, it is not known whether ATP-MgCl2 has any salutary effect on the depressed active hepatocellular function (AHF) and hepatic microvascular blood flow (HMBF) in a nonheparinized model of trauma and severe hemorrhage in the absence of blood resuscitation. To determine this, rats underwent a midline laparotomy (i.e., trauma induced) and were bled to and maintained at a mean arterial pressure of 40 mm Hg until 40% of the maximum shed blood volume was returned in the form of Ringer's lactate (RL). The animals were then resuscitated with four times the volume of shed blood with RL. ATP-MgCl2, 50 mumoles/kg body weight (BW) each or an equivalent volume of normal saline, was infused intravenously for 95 min during and following crystalloid resuscitation. At 1.5 and 4 hr after resuscitation, AHF (Vmax, maximal velocity of indocyanine green clearance; Km, efficiency of the active transport process) was determined without blood sampling by using an in vivo indocyanine green clearance technique. HMBF was measured with laser Doppler flowmetry. Results indicate that Vmax, Km, and HMBF decreased significantly at 1.5-4 hr after hemorrhage and resuscitation. ATP-MgCl2 infusion restored the depressed Vmax, Km, and HMBF and prevented the occurrence of hepatic edema. The restoration of AHF with ATP-MgCl2 treatment may be due to its direct salutary effect on the active indocyanine green transport process and/or due to improvement in hepatic microcirculation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of cyclooxygenase-2 by NS-398 following hemorrhage and subsequent sepsis: no beneficial effects in either gender

BACKGROUND: Inhibition of cyclooxygenase-2 with a reduction of prostaglandin E(2)production by the specific antagonist NS-398 has been shown to have beneficial effects on immune function and survival in a trauma model. Immune function after experimental hemorrhagic shock and subsequent sepsis may be gender-related, with enhanced immunity and better survival in females. However, it remains unclear if the observed effect of NS-398 treatment is gender-related following hemorrhagic shock and subsequent sepsis. METHODS: Male and female CBA/J mice (age: 2-3 months) were subjected to hemorrhagic shock (35 +/- 5 mm Hg for 90 min and fluid resuscitation) or sham operation. At resuscitation and after 20 and 40 h each received either NS-398 10 mg/kg or placebo i.p. At 48 h after resuscitation, either splenocytes and peritoneal macrophages (pM phi) were harvested (n = 8 per group), or polymicrobial sepsis was induced by cecal ligation and puncture (CLP). Following CLP, either 10-day survival (n = 15 per group) was determined or pM phi and splenocytes were harvested 4 h after CLP (n = 8 per group). Cytokine release of pM phi, and splenocyte proliferation and responsiveness in vitro were assessed. RESULTS: Treatment with NS-398 led to lower PGE(2) levels as compared to placebo-treated animals, reaching significance (p < 0.05) in males. Placebo-treated males had significantly depressed proinflammatory immune response (IL-1, IL-6, IL-2, IFN-gamma) after hemorrhagic shock and experienced further suppression by CLP (all, p < 0.05). In contrast, young females displayed unchanged cytokine release after hemorrhagic shock, but a comparable suppression following CLP. Treatment with NS-398 did not influence cytokine release nor survival. CONCLUSIONS: Despite a significant reduction of PGE(2) concentration, NS-398 treatment has no beneficial effects on cytokine release and survival in this model of hemorrhage and subsequent sepsis.     

Inhibition of macrophage-activating cytokines is beneficial in the acute septic response.

Interferon-gamma and other cytokines enhance macrophage (M phi) antimicrobial function and have been considered for therapeutic use in sepsis. Systemic sequelae of macrophage activation, however, are unclear. This study examined the effects of M phi activating cytokines (interferon-gamma [IFN-gamma] and interleukin-4 [IL-4]) and monoclonal antibodies directed against these cytokines in modulating the acute septic response. CFW/Swiss Webster mice (n = 345) received endotoxin (lipopolysaccharide [LPS]: 60 mg/kg body weight intraperitoneally) and were randomized to five treatment groups: IFN-gamma (10(4) units), IL-4 (10(4) units), IgG1 isotype antibody (TRFK5: 200 micrograms), anti-IFN-gamma (200 micrograms), or anti-IL-4 (200 micrograms) monoclonal antibodies (MAbs) given simultaneously or 2 hours after LPS. Animals were divided into two groups and studied for mortality or measurement of peritoneal M phi superoxide anion release (O2-), tumor necrosis factor (TNF), and IL-6 production 6 hours after administration of LPS +/- experimental regimens. Serum TNF and IL-6 also were assessed at 2 and 4 hours after LPS, respectively. Administration of LPS resulted in a 27% survival compared with 10% in the IFN-gamma and 13% in the IL-4 groups. Treatment with anti-IFN-gamma offered protection against LPS lethality (93%-100% survival, p less than 0.001 vs. other groups) when given either simultaneously or 2 hours after LPS. Anti-IFN-gamma also significantly decreased PM phi O-2 and TNF release. Thus anti-IFN-gamma may have an important role in the modulation of the acute septic response.     

Depressed gut absorptive capacity early after trauma-hemorrhagic shock. Restoration with diltiazem treatment.

Although bacterial translocation occurs after trauma-hemorrhage, it is unknown whether gut absorptive capacity (GAC) is altered under those conditions. The aim of this study, therefore, was to determine this and also whether diltiazem (DZ) has any effect on GAC after hemorrhage. Rats (n = 12; 270 to 300 g) were lightly anesthetized with ether and a 6-cm midline laparotomy performed (i.e., trauma induced). A nasogastric tube was inserted, cannulation of a carotid and femoral artery and a jugular and the portal veins performed, and the animals allowed to recover from anesthesia. They were then bled to a mean blood pressure of 40 mm Hg within 10 minutes and maintained at this pressure by further bleeding or return of fluid (lactated Ringer's solution [LRS] ) until 40% of the shed blood volume (SBV) had been returned in the form of LRS (end of hemorrhage, EH; time from onset of hemorrhage to EH = 93.4 +/- 4.4 minutes). The rats then were resuscitated with LRS, 3 times the SBV over 45 minutes, followed by 2x over 60 minutes. One group received 400 micrograms/kg DZ, and another group received saline, with the 2x LRS. Sham animals were not hemorrhaged. Gut absorptive capacity was determined by the 1-hour D-xylose absorption test at 2 and 4 hours after EH. Results demonstrate that GAC is significantly depressed after hemorrhage and resuscitation. Administration of diltiazem restored GAC to normal levels. Thus DZ is a useful adjuvant to treatment after trauma-hemorrhagic shock, because it restores GAC to normal and allows for early enteral nutrition.     

Sirtinol attenuates hepatic injury and pro-inflammatory cytokine production following trauma-hemorrhage in male Sprague-Dawley rats

Background: Although studies have demonstrated that sirtinol administration following adverse circulatory conditions is known to be protective, the mechanism by which sirtinol produces the salutary effects remains unknown. We hypothesized that sirtinol administration in male rats following trauma-hemorrhage decreases cytokine production and protects against hepatic injury.
Methods: Male Sprague–Dawley rats underwent trauma-hemorrhage (mean blood pressure 40 mmHg for 90 min, then resuscitation). A single dose of sirtinol (1 mg/kg of body weight) or vehicle was administered intravenously during resuscitation. Twenty-four hours thereafter, tissue myeloperoxidase (MPO) activity (a marker of neutrophil sequestration), cytokine-induced neutrophil chemoattractant (CINC)-1, CINC-3, intercellular adhesion molecule (ICAM)-1, and interleukin (IL)-6 levels in the liver and plasma alanine aminotransferase (ALT) concentrations were measured ( n =6 Sprague–Dawley rats/group).
Results: Trauma-hemorrhage increased hepatic MPO activity, CINC-1, CINC-3, ICAM-1, and IL-6 levels and plasma ALT concentrations. These parameters were significantly improved in the sirtinol-treated rats subjected to trauma-hemorrhage.
Conclusion: The salutary effects of sirtinol administration on attenuation of hepatic injury following trauma-hemorrhage are, at least in part, related to reduction of pro-inflammatory mediators.     

Impact of alcohol intoxication on hemodynamic, metabolic, and cytokine responses to hemorrhagic shock

BACKGROUND: Alcohol intoxication is associated with a high incidence of traumatic injury, particularly in the young healthy population. The impact of alcohol intoxication on the immediate pathophysiologic response to injury has not been closely examined. We hypothesized that acute alcohol intoxication would aggravate the immediate outcome from hemorrhagic shock by impairing homeostatic counterregulation to blood loss. METHODS: Chronically catheterized male Sprague-Dawley rats were randomized to receive an intragastric infusion of ethyl alcohol (1.75 g/kg followed by 250-300 mg/kg/h) or isocaloric dextrose (3-mL bolus + 0.375 mL/h) for 15 hours. Before initiating fixed-pressure hemorrhage followed by fluid resuscitation, an additional intragastric bolus of ethyl alcohol (1.75 g/kg) was administered. Hemodynamic, metabolic, cytokine, and acid-base parameters were assessed during the hemorrhage period and at completion of resuscitation. Lungs were obtained for cytokine determinations. RESULTS: Basal mean arterial pressure was significantly lower in alcohol-intoxicated (blood-alcohol concentration, 135 +/- 12 mg/dL) animals than in controls during baseline (20%) and after the initial fluid resuscitation period (30%). Hemorrhage decreased arterial HCO3 and Pco2, and increased Po2 without significant alteration in arterial blood pH. Alcohol intoxication blunted the decrease in Pco2 and increase in Po2 and decreased blood pH during baseline and throughout the course of the hemorrhage period. Hemorrhage produced marked and progressive elevations in plasma glucose and lactate levels in controls, and this was inhibited by alcohol intoxication. Hemorrhage elevated plasma tumor necrosis factor-alpha (TNF-alpha) (686 +/- 252 pg/mL) and interleukin (IL)-10 (178 +/- 25 pg/mL), and did not alter IL-6 and IL-1 levels. Alcohol blunted the hemorrhage-induced rise in plasma TNF-alpha (142 +/- 48 pg/mL) and enhanced the hemorrhage-induced increase in IL-10 (678 +/- 187 pg/mL). Hemorrhage produced a two- to threefold increase in lung content of TNF-alpha, IL-1alpha, and IL-6 without significantly altering lung IL-10. Alcohol exacerbated the hemorrhage-induced increase in lung TNF-alpha, and did not alter the IL-1alpha, IL-6, and IL-10 lung responses. CONCLUSION: These results indicate marked alterations in the hemodynamic and metabolic responses to hemorrhagic shock by alcohol intoxication. Furthermore, our findings suggest that alcohol modulates the early proinflammatory responses to hemorrhagic shock. Taken together, these alterations in metabolic and inflammatory responses to hemorrhage are likely to impair immediate outcome and predispose to tissue injury.     

Oral and portal venous tolerance in the interferon-gamma knockout (GKO) mouse

BACKGROUND: The role of the proinflammatory cytokine interferon-gamma (IFN-gamma) in the development of oral or portal venous tolerance is poorly defined. By using knockout mice for IFN-gamma (GKO mice), we investigated the effect of both oral and portal venous administration of alloantigen on the systemic delayed type hypersensitivity (DTH) response to alloantigen rechallenge. MATERIALS AND METHODS: C57BL/6 (B6) control and GKO mice (also on a B6 background) were given either saline or BALB/c spleen cells (25 million) by oral gavage (PO) or by injection into the portal vein (PV) on day 0. The injection of 10 million BALB/c spleen cells subcutaneously into the dorsal flanks was performed after 7 days followed by footpad injection of 10 million BALB/c spleen cells on day 14. Specific footpad swelling was measured 24 h later using a micrometer. Splenocyte responsiveness was measured by in vitro mixed lymphocyte culture and cytotoxic T lymphocyte assay. Cytokine production of interleukin (IL)-2, IL-4, IL-10, and IFN-gamma was measured by enzyme-linked immunosorbent assay. RESULTS: Although B6 mice given PO or PV saline demonstrated a DTH response of 0.47 +/- 0.04 mm and 0.49 +/- 0.05 mm, respectively, in GKO mice, a greater DTH response of 0.72 +/- 0.08 mm and 0.75 +/- 0.05 mm was measured after either PO or PV saline (P < 0.05, P < 0.05, respectively). In vitro MLC and CTL confirmed the heightened DTH response to BALB/c observed in GKO versus B6. Despite this heightened response in control GKO mice, the DTH response was completely suppressed in both GKO and B6 mice, after both PO and PV BALB/c feedings (P < 0.001, P < 0.001, respectively, in both GKO and B6 mice). The unchanged brisk response to third-party C3H/HeJ demonstrated antigenic specificity. CONCLUSIONS: Although DTH responsiveness to alloantigen is increased in the absence of IFN-gamma, both oral and portal venous alloantigen-specific tolerance can still be established in GKO mice similar to that in control B6 mice. The achievement of a tolerogenic immune response by both oral and portal venous routes indicates that lack of IFN-gamma does not preclude the induction of tolerance in this murine model.     

The immunosuppressive mechanism of total lymphoid irradiation. I. The effect on IL-2 production and IL-2 receptor expression

Total lymphoid irradiation is an effective immunosuppressive therapy used to prepare patients for organ transplantation and to treat several autoimmune diseases. We have used the mouse model to investigate the mechanism of TLI-induced immunosuppression. Mice were irradiated with 250-rad daily fractions to a total dose of 3500 rads. Splenocytes from control and TLI-treated mice were analyzed for IL-2 production, IL-2 receptor expression after mitogen stimulation, and percent L3T4 positive cells. At two weeks following the completion of TLI, IL-2 production from whole spleen populations had decreased 90% compared with controls (TLI mean IL-2 units = 25 +/- 9.0, control mean = 277 +/- 104). IL-2 receptor expression on splenocytes following Con A stimulation was decreased 80% compared with controls (TLI mean percentage of IL-2 receptor expressing cells = 16.2 +/- 4.1, control mean = 82.0 +/- 7.5). L3T4+ cells that expressed IL-2 receptor were also decreased following TLI (TLI mean = 4.4 +/- 1.4, control mean = 22.0 +/- 3.8), as were proliferative responses to Con A and PHA. Addition of recombinant mouse IL-2 did not restore IL-2 receptor expression or proliferative responses. Whole TLI-treated splenocytes did not suppress IL-2 production or proliferative responses of normal splenocytes. These immunologic abnormalities recovered over time, and by 8 weeks post TLI IL-2 production, IL-2 receptor expression, L3T4+ cell numbers and proliferative responses had returned toward normal. These results suggest that TLI therapy transiently depletes IL-2 producing, IL-2 receptor expressing, and mitogen responsive lymphocytes. The immunosuppression is not mediated through a suppressor cell and is independent of IL-2 production.     

What is the role of interleukin 10 in polymicrobial sepsis: anti-inflammatory agent or immunosuppressant?

BACKGROUND: Controversy exists concerning the role of interleukin 10 (IL-10) in sepsis. When IL-10 is used in models of endotoxemia, it appears to protect (by anti-inflammatory effects), whereas in models of polymicrobial sepsis it seems to be deleterious (by immunosuppression?). However, little direct evidence for such an immunosuppressive role is available for polymicrobial sepsis. Thus the aim of this study was to determine whether IL-10 contributes to lymphocyte immunosuppression in a model of cecal ligation and puncture (CLP) and whether neutralization of IL-10 has any salutary effects on survival after sepsis. METHODS: To assess the former, polymicrobial sepsis was induced in male C57BL/6J wild-type (+/+) and C57BL/6J-IL-10 knockout(-/-) mice by CLP. Splenocytes were harvested 24 hours later and stimulated with concanavalin A to assess their proliferative capacity and their ability to release the Th1 lymphokines interleukin 2 and interferon gamma (by enzyme-linked immunosorbent assay, nanograms/millilter). To further verify the immunosuppressive role of IL-10, splenocytes were obtained from male C3H/HeN mice 24 hours after CLP and then stimulated in the presence or absence of anti-IL-10 monoclonal antibody (Mab, 4 micrograms/mL). To assess the in vivo effects of IL-10 neutralization on survival after CLP, C3H/HeN mice (16 per group) were given 250 micrograms of anti-IL-10 Mab (intraperitoneally) either immediately after CLP (before the initiation of the hyperdynamic phase) or 12 hours after CLP (the beginning of the hypodynamic state). Control mice were given nonspecific rat immunoglobulin G. RESULTS: These data indicate that IL-10 deficiency (-/-) prevents the depression of the proliferative capacity and Th1 lymphokine production after sepsis. Analysis of the interleukin 2-interferon gamma production patterns and proliferative capacity in lymphocytes treated with anti-IL-10 Mab confirmed the role of IL-10 in suppressing lymphocyte responsiveness in CLP. Interestingly, however, only delayed administration (12 hours after CLP) of anti-IL-10 markedly increased survival of mice (Fisher's exact test, P < .05). CONCLUSION: The results not only illustrate IL-10's role in septic immune dysfunction but document that anti-IL-10 administration beyond the initial proinflammatory hyperdynamic state of polymicrobial sepsis improves survival of animals subjected to sepsis.     

Suppression of interleukin 2 production in an animal model of thermal injury is related to prostaglandin synthesis

We performed studies using an animal model of thermal injury to confirm the observed decrease in interleukin 2 (IL-2) production in burned patients and to explore the underlying mechanisms. Ten mice subjected to a 25% scald were compared with ten anesthetized littermates (controls) and six untreated mice (normal mice) 1, 3, 5, 7, 10, 14, and 21 days after burn. Production of IL-2 by splenocytes was stimulated by concanavalin A alone, or in the presence of the cyclooxygenase inhibitor indomethacin or flurbiprofen. The IL-2 content of the resulting supernatant was determined by the response of the IL-2-dependent cell line CTLL-2. The IL-2 production was significantly suppressed in the burned mice at three days (mean +/- SEM, 30.9% +/- 5.2%), five days (19% +/- 5.5%), seven days (41.6% +/- 6.4%), and 21 days (20% +/- 4.5%). Significant enhancement of IL-2 production by indomethacin was seen in the burned group (mean, 95%), but not in controls (mean, 23.8%) or normal mice (mean, 17.2%), and similar effects were seen with flurbiprofen. In separate experiments the effects of exogenous prostaglandin E2 on lymphocyte blastogenesis and IL-2 production were studied, and an increased susceptibility to the inhibitory effects of prostaglandin E2 was observed following thermal injury.     

IL-12p40 and IL-18 in crescentic glomerulonephritis: IL-12p40 is the key Th1-defining cytokine chain, whereas IL-18 promotes local inflammation and leukocyte recruitment

Experimental crescentic glomerulonephritis (GN) is characterized by T helper 1 (Th1) directed nephritogenic immune responses and cell-mediated glomerular injury. IL-12p40, the common cytokine chain for both IL-12 and IL-23, is important in the generation and potentially the maintenance of Th1 responses, whereas IL-18 is a co-factor for Th1 responses that may have systemic and local proinflammatory effects. For testing the hypothesis that both endogenous IL-12p40 and endogenous IL-18 play pathogenetic roles in crescentic GN, accelerated anti-glomerular basement membrane GN was induced in mice genetically deficient in IL-12p40 (IL-12p40-/-), IL-18 (IL-18-/-), or both IL-12p40 and IL-18 (IL-12p40-/-IL-18-/-). Compared with wild-type C57BL/6 mice, IL-12p40-/- mice failed to make a nephritogenic Th1 response and developed markedly reduced crescent formation and renal leukocytic infiltration, despite renal production of chemoattractants and adhesion molecules. IL-18-/- mice developed an intact antigen-specific systemic Th1 response, a similar degree of crescent formation, but fewer glomeruli affected by other severe histologic changes and fewer leukocytes in glomeruli and interstitium. IL-18 was expressed within diseased kidneys. Local production of TNF, IL-1beta, IFN-gamma, CCL3 (MIP-1alpha), and CCL4 (MIP-1beta) was reduced in IL-18-/- mice, demonstrating a local proinflammatory role for IL-18. Combined deletion of IL-12p40 and IL-18 did not result in synergistic effects. Consistent with the hypothesis that inflammation leads to fibrosis, all three groups of deficient mice expressed lower levels of intrarenal TGF-beta1 and/or alpha1(I) procollagen mRNA. These studies demonstrate that in severe experimental crescentic GN, IL-12p40 is the key Th1-defining cytokine chain, whereas IL-18 has local proinflammatory roles.     

Ibuprofen restores cellular immunity and decreases susceptibility to sepsis following hemorrhage.

Although hemorrhage depresses splenocyte (SPL) functions and increases susceptibility to sepsis, it is not known whether increased tumor necrosis factor (TNF) or prostaglandin (PG) production are responsible for it. To study this, mice (C3H/HeN) were bled to a mean blood pressure of 35 mm Hg, maintained at that pressure for 60 min, resuscitated, and treated with ibuprofen (1.0 mg/kg body weight) or vehicle (saline). Hemorrhage reduced (P less than 0.05) SPL proliferation by 60%, SPL release of interleukin-2 (IL-2) by 47%, interferon-gamma (IFN-gamma) by 67%, TNF by 54%, and interleukin-6 (IL-6) by 46% compared to sham. In addition, splenic macrophage (sM phi) release of interleukin-1 (IL-1) and TNF was decreased by 58 and 67% (P less than 0.05), respectively. However, ibuprofen treatment increased (P less than 0.05) SPL proliferation, lymphokine (IL-2, IFN-gamma, and IL-6) synthesis, and IL-1 release by sM phi compared to hemorrhage alone. Furthermore, ibuprofen enhanced the release of TNF by SPL (+175%, P less than 0.05) and sM phi (+68%) compared to the vehicle group. Ibuprofen also decreased (P = 0.011) the susceptibility to sepsis following hemorrhage. These results indicate that PGs are involved in hemorrhage-induced suppression of cellular immunity and in the increased mortality of such animals following a septic challenge.