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.     

Interferon-gamma attenuates hemorrhage-induced suppression of macrophage and splenocyte functions and decreases susceptibility to sepsis.

Although it is known that interferon-gamma synthesis and macrophage functions are depressed after hemorrhage, it remains to be determined whether systemic administration of interferon-gamma has any effect on hemorrhage-induced depression of macrophage and splenocyte functions. To study this, C3H/HEN mice were bled to a mean blood pressure of 35 mm Hg, maintained for 60 minutes, and followed by adequate fluid resuscitation. The mice then received either 1000 units interferon-gamma or saline solution (vehicle). Peritoneal (pM phi) and splenic (sM phi) macrophages and splenocytes were isolated 24 hours later. PM phi antigen presentation was measured by coculturing pM phi with the D10.G4.1 cell clone. Major histocompatibility complex class II (Ia) antigen expression was determined by direct immunofluorescence. Cytokine release by pM phi, sM phi, and splenocytes was assessed with specific bioassays. For survival studies, mice were subjected to sepsis 3 days after hemorrhage. Treatment with interferon-gamma restored (p less than or equal to 0.05) hemorrhage-induced suppression of pM phi antigen presentation capacity and Ia antigen expression and increased (p less than or equal to 0.05) interleukin-1 and tumor necrosis factor release by pM phi and sM phi, as well as splenocyte proliferation (p less than or equal to 0.05). Interferon-gamma also decreased (p less than or equal to 0.007) the susceptibility to sepsis after hemorrhage. Thus interferon-gamma represents a potent agent for treating hemorrhagic shock-induced immunosuppression and for increasing the ability of the host defense system to combat bacterial infections after 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.     

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.     

Does endotoxin tolerance prevent the release of inflammatory monokines (interleukin 1, interleukin 6, or tumor necrosis factor) during sepsis?

Mice were subjected to sepsis by cecal ligation and puncture to determine whether macrophages from endotoxin-tolerant C3H/HeJ mice are also activated systemically to release inflammatory monokines associated with septic mortality. Blood levels of both tumor necrosis factor and interleukin 6 were significantly elevated during the first 1 to 4 hours of sepsis as compared with sham controls. Peritoneal macrophages from septic mice exhibited a marked spontaneous release of interleukin 1, interleukin 6, and tumor necrosis factor at 1 hour. However, the addition of endotoxin to macrophage cultures taken from septic mice had no further stimulatory effect. Sham controls alternatively showed no significant innate monokine release, but their macrophages did release increased monokine numbers in response to endotoxin. These results indicate that the spontaneous macrophage release of these monokines is comparable with that previously observed in endotoxin-sensitive mice, suggesting a common mechanism by which macrophages are primed by traumatic injury by an agent other than endotoxin to release monokines during sepsis. Thus, the administration of agents that decrease or prevent the deleterious effects of systemic inflammatory mediators during sepsis could be useful adjuvants in those clinical situations where the bacterial origin is unknown.     

Macrophage activation decreases macrophage prostaglandin E2 release in experimental trauma

Prostaglandin E2 (PGE2) derived from macrophages following trauma may contribute to trauma-induced immunosuppression. This study evaluated the effect of glucan, a macrophage-activating agent, on macrophage PGE2 release in a murine trauma model. ICR/HSD mice were administered D5W, glucan pre-trauma, or glucan post-trauma, and subjected to hindlimb crush and amputation injury. Splenic macrophages were isolated 24 hours following trauma, cultured (24 hrs), and macrophage PGE2 levels were determined. In-vitro marrow proliferation was assessed as a measure of immune function. Crush-amputation injury increased (184%) macrophage PGE2 release. In contrast, glucan administration (pre or post) reduced PGE2 levels in macrophage supernatants (71% and 85%, respectively). A 52% decrease in in-vitro bone marrow proliferation was observed following trauma. Glucan pre- or post-trauma eliminated the suppression of bone marrow proliferation. In conclusion, macrophage-activating immunomodulators may exert beneficial effects following trauma by: 1) reducing macrophage PGE2 synthesis and release; and 2) reducing traumatic suppression of bone marrow proliferation.     

Metoclopramide and cellular immune functions during polymicrobial sepsis

Metoclopramide (MCP) has been demonstrated to restore the depressed cellular immune function after hemorrhage by increasing the release of the immunomodulatory pituitary hormone prolactin. We investigated the effect of MCP on serum prolactin concentrations, on cellular immune functions (immune cell distribution, splenocyte proliferation, apoptosis and cytokine release) and on the survival 48 h after induction of a polymicrobial sepsis in mice. Administration of MCP increased circulating serum prolactin concentrations and splenocyte apoptosis rate and improved cellular cytokine release, but did not affect mortality of septic mice. We therefore conclude that administration of MCP modulated splenocyte apoptosis and cytokine release in a murine model of sepsis without an impact on the survival. Furthermore, this effect may be mediated by an increased endogenous prolactin release.     

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.     

Insights into the mechanisms of defective antigen presentation after hemorrhage

Although hemorrhage depresses macrophage antigen presentation (AP), a critical component in eliciting an antigen-specific immune response, it is not known which particular step in macrophage AP (i.e., uptake, ingestion, catabolism, or presentation of degraded antigens to T cells) is defective. To study this, C3H/HeN mice were bled to an arterial mean blood pressure of 35 mm Hg, maintained for 60 minutes, and then adequately resuscitated. Peritoneal and splenic macrophage cultures were prepared 2 and 24 hours after hemorrhage. Macrophage AP capacity was measured by coculturing macrophages with the T-helper cell clone D10.G4.1. To gain information about macrophage ability to digest the specific antigen conalbumin, lysosomal activity was bypassed by use of chemically denatured conalbumin peptides. To study macrophage ability to present conalbumin peptides, macrophages were fixed and D10.G4.1 proliferation in response to fragmented conalbumin was determined. AP of native conalbumin by peritoneal macrophages and splenic macrophages was depressed (p less than 0.05) by 50% (peritoneal macrophages) and 55% (splenic macrophages) 2 hours and 57% (peritoneal macrophages) and 35% (splenic macrophages) 24 hours after hemorrhage. In contrast, presentation of conalbumin peptides was only slightly decreased. In addition, the ability of fixed peritoneal macrophages and splenic macrophages to present conalbumin peptides was similar in hemorrhaged and sham mice. Because bypassing of macrophage lysosomal activity with degraded native antigens prevented the suppression of AP, the results suggest that hemorrhage-induced suppression of AP is not caused by a reduced macrophage capacity to present antigenic peptides but by decreased antigen catabolism by macrophages.     

Mechanism of immune dysfunction in sepsis: inducible nitric oxide-meditated alterations in p38 MAPK activation

BACKGROUND: After the onset of sepsis, there is a marked dysfunction in cell-mediated immunity that contributes to the morbidity and mortality seen in this condition. Although both nitric oxide (NO) from inducible NO synthase (iNOS) and the activation of p38 mitogen-activated protein kinase (p38 MAPK) appear to contribute to this immune dysfunction, the extent to which NO regulates p38 MAPK activity in sepsis remains unknown. METHODS: To examine this, we induced sepsis by cecal ligation and puncture (CLP) in iNOS knockout (iNOS -/-) or C57BL/6 control mice. Twenty-four hours after CLP or sham operation, splenic T cells and macrophages were isolated and then stimulated with monoclonal antibody against the T-cell marker CD3 (anti-CD3) or lipopolysaccharide. At 4 or 24 hours after stimulation, cytokine release was determined by enzyme-linked immunosorbent assay, and p38 MAPK phosphorylation (activation) was determined by immunoblotting with antibody specific to phosphorylated p38 MAPK. RESULTS: Splenic T-cell p38 MAPK activation and interleukin (IL)-10 release was increased by CLP, whereas Th1 cytokine (IL-2, interferon-gamma) release was depressed. iNOS gene deficiency inhibited p38 MAPK activation in splenic T cells taken from septic mice, and also suppressed IL-10 release in both sham and septic mice. Interestingly, although deficiency of iNOS restored IL-2 release after CLP, both sham and CLP T cells remained depressed in their ability to release interferon-gamma. Septic insult markedly suppressed C57BL/6 splenic macrophage release of proinflammatory agents tumor necrosis factor, IL-12, and IL-1, while augmenting the release of IL-10. However, although deficiency of iNOS concomitantly restored the ability to produce tumor necrosis factor while suppressing the rise in IL-10 release and p38 MAPK activation, it only partially restored IL-1 release and had no effect on IL-12 production seen after CLP. CONCLUSION: These data suggest that NO release from iNOS regulates aspects of sepsis-induced immune dysfunction by the activation of p38 MAPK.     

The contribution of soft-tissue trauma and/or bone fracture to depressed immunity following hemorrhagic shock in an experimental setting

Summary Bone fracture, soft-tissue trauma and hemorrhagic shock are frequent complications in trauma patients, and these patients are known to be immunocompromised. Nonetheless, it is difficult to differentiate the effect of soft-tissue trauma plus hemorrhage from that of bone fracture and hemorrhage on host immune function in the clinical setting. To determine this experimentally, closed bone fracture (right lower leg) and/or soft-tissue trauma (2.5 cm midline laparotomy) were induced prior to hemorrhagic shock (mean arterial BP of 35 ± 5 mm Hg for 90 min) in male C3H/HeN mice. All animals were killed at 72 h after initiation of the experiment and the spleens were collected aseptically. More significant depression of splenocyte IL-2 and IL-3 release occurred with the combined insult than after bony injury or tissue trauma alone with hemorrhage. The present study suggests that different traumatic insults, i. e. bone fracture as well as soft-tissue trauma in conjunction with hemorrhagic shock, produce comparable depression of host immune function. Moreover, combination of closed bone fracture and soft-tissue trauma prior to hemorrhagic shock leads to even more compromised immunity. This indicates that different mechanisms of immune depression may be involved following soft-tissue trauma or bony injury coupled with hemorrhage. The markedly depressed immune function following bony injury, soft-tissue trauma and hemorrhagic shock may contribute to the increased susceptibility of severely injured patients to sepsis and the ensuing multiple organ failure in the clinical situation.      

Mechanism of macrophage injury following traumatic hemorrhagic shock：through PTX—sensitive G—protein—mediated signal transduction pathway

OBJECTIVE: To study the mechanism of macrophage injury after trauma-hemorrhagic shock. METHODS: Wistar male rats underwent trauma (closed bone fracture) and hemorrhage (mean arterial blood pressure of 35 mm Hg+/-5 mm Hg for 60 minutes, following fluid resuscitation). Rats without trauma, hemorrhage or fluid resuscitation served as controls. Peritoneal macrophages were harvested at 6 hours and 1, 2, 3, 7 days after traumatic hemorrhagic shock to determine the effects of pertussis toxin (PTX, as a specific inhibitor to Gi(alpha) and cholera toxin (CTX, as a stimulant to Gs(alpha) on macrophage-Ia expression and TNF-alpha production and levels of Gi(alpha) and Gs(alpha). RESULTS: The macrophages from the injured rats revealed a significant decrease of Ia positive number and TNF-alpha release in response to LPS. Wi th pretreatment with PTX 10-100 ng/ml Ia positive cells and LPS-induced TNFalpha production in both control and impaired macrophages populations were dos e dependently increased. Both macrophages populations were not responding to CTX treatment (10-100 ng/ml). Western blot analyses showed that the levels of Gi(alpha) protein expression increased as much as 116.5%-148.8% of the control level fro m 6 hours through 7 days after traumatic hemorrhage. The levels of Gs protein expression were reduced at 6 hours and decreased to the lowest degree; 36% o f the control at day 1, began to return at day 2 and returned to the normal level at day 7, following traumatic hemorrhagic shock. CONCLUSIONS: PTX-sensitive G-protein may participate in th e modulation of macrophage-Ia expression and TNF-alpha release following traumatic hemorrhagic shock. Analyses of the alteration of Gi(alpha) and Gs protein express ions further supports the concept that G-protein is involved in trauma-induced macrophage signal transduction pathways.     

休克期一次性切痂对巨噬细胞分泌细胞因子的影响

目的 了解烧伤休克期一次性切痂大鼠血浆对巨噬细胞分泌TNFα、IL-6和IL-8的作用及对血浆中炎症介质TNFα、IL-6和IL-8影响,论证烧伤休克期切痂能否抑制巨噬细胞分泌TNFα、IL-6和IL-8。方法Ⅲ度30％TBSA大鼠随机分为伤后6h切痂组(E组)和烧伤未切痂组(C组),检测两组大鼠血浆中。TNFα、IL-6和IL-8含量,并将两组大鼠血浆分别与正常大鼠腹腔巨噬细胞共同孵育6h,检测培养液中。TNFα、IL-6和IL-8含量。结果 E组和c组伤后血浆中TNFα、IL-6和IL-8均明显升高,但E组于伤后12h上述3项指标下降并明显低于C组。两组大鼠血清均能刺激腹腔巨噬细胞分泌。TNFα、IL-6和IL-8,但E组的刺激能力明显低于C组。缩论烧伤休克期一次性切痂,能降低烧伤血浆诱导细胞分泌TNFα、IL-6和IL-8的能力,减少烧伤血浆中TNFα、IL-6和IL-8含量,有利于防止严重烧伤后炎症反应综合征的发生和发展。     

Pre-existing renal disease promotes sepsis-induced acute kidney injury and worsens outcome

While it is known that risk of death from sepsis is higher in patients with pre-existing chronic kidney disease its mechanism is unknown. To study this we established a two-stage mouse model where renal disease was first induced by folic acid injection followed by sub-lethal cecal ligation and puncture to induce sepsis. Septic mice with pre-existing renal disease had significantly higher mortality, serum creatinine, vascular permeability, plasma vascular endothelial growth factor (VEGF) levels, bacteremia, serum IL-10, splenocyte apoptosis and more severe septic shock when compared to septic mice without pre-existing disease. To evaluate the contribution of vascular and immunological dysfunction, we treated the folate-septic mice with soluble Flt-1 to bind VEGF and chloroquine to reduce splenocyte apoptosis. These treatments together resulted in a significant improvement in kidney injury, hemodynamics and survival. Our study shows that the sequential mouse model mimics human sepsis frequently complicated by pre-existing renal disease and might be useful in evaluating preventive and therapeutic strategies.     

Release of prostaglandin E2 and leukotriene B4 by alveolar macrophages from patients with sarcoidosis

BACKGROUND: Mediators released by alveolar macrophages, as well as by T cells, play an important part in modulating local immune processes in sarcoidosis. Among alveolar macrophage secretory products, arachidonic acid metabolites are known to regulate inflammatory and immune reactions. It has been suggested that cyclo-oxygenase and lipoxygenase pathway metabolites of arachidonic acid modulate the evolution of the granulomatous inflammatory response in the lung differently. METHODS: Alveolar macrophages recovered from the bronchoalveolar lavage (BAL) fluid of 32 patients with sarcoidosis in different states of disease activity and 10 normal subjects were evaluated for their ability to release prostaglandin E2 (PGE2) and leukotriene B4 (LTB4). Alveolar macrophages were cultured in the presence or absence of opsonised zymosan (500 micrograms/ml), and PGE2 and LTB4 levels in the culture supernatants were determined by enzyme immunoassay (EIA). RESULTS: Stimulated alveolar macrophages from patients with active sarcoidosis released higher LTB4 levels than those from normal subjects, but no differences in PGE2 release were observed between the two groups. The time course of LTB4 release by activated alveolar macrophages showed that normal cells produced similar levels of the hydroxyacid during the early and late times of culture while LTB4 release by activated cells from patients with sarcoidosis increased markedly after 60 minutes of culture, remaining elevated until 24 hours. Indomethacin (3 x 10(6) M) caused the expected inhibition of PGE2 formation without affecting LTB4 release. CONCLUSIONS: These results suggest that alveolar macrophages from the BAL fluid of patients with active sarcoidosis are primed to release LTB4, which may contribute to the locally heightened immune response.




     

Mechanisms of cellular recruitment in aseptic loosening of prosthetic joint implants

The association of macrophages engaged in polymethylmethacrylate (PMMA) particle phagocytosis with pockets of inflammatory cells is a pathognomonic feature of the aseptically loose interface not present at the well-fixed interface. The mechanism by which the presence of PMMA particles leads to cellular recruitment, bone resorption, and ultimate loosening is poorly understood. Granulocyte macrophage colony stimulating factor (GM-CSF) and interleukin 6 (IL-6), cytokines released by osteoblasts, stimulate the recruitment of macrophages into sites of inflammation. We show that exposure of macrophages to PMMA particles stimulated release of tumor necrosis factor (TNF), but no increase in prostaglandin E2 (PGE-2) or interleukin 1. Incubation of osteoblasts with conditioned medium from macrophages exposed to PMMA particles led to release of GMCSF, IL-6, and PGE-2. Incubation of the PMMA/macrophage medium with antibodies to TNF prior to osteoblast exposure inhibited release of GM-CSF, IL-6, and PGE-2 by the osteoblasts. Our data demonstrate that exposure of macrophages to PMMA particles leads to the release of TNF which then stimulates osteoblasts to produce GMCSF, IL-6, and PGE-2. Based upon the results of this study, we propose that the process of cellular recruitment in aseptic loosening is initiated when the mechanical failure of the cement mantle leads to the production of PMMA particles. These particles are phagocytized by macrophages leading to the production of TNF. TNF stimulates surrounding osteoblasts to produce GM-CSF, IL-6, and PGE-2 which leads to recruitment of macrophages and osteoclasts into the area of the bone-cement interface. The recruitment of these cells potentiates this process leading to bone resorption and ultimately, clinical loosening of prosthetic joint implants.     

Hypertonic resuscitation of hemorrhagic shock upregulates the anti-inflammatory response by alveolar macrophages

BACKGROUND: Resuscitated hemorrhagic shock predisposes patients to the development of acute respiratory distress syndrome (ARDS). Hypertonic saline (HTS) has been shown to inhibit immune cell activation in response to lipopolysaccharide (LPS) in vitro and to reduce lung damage when used for resuscitation of hemorrhagic shock in vivo. We hypothesize that HTS resuscitation of hemorrhagic shock may exert this anti-inflammatory effect by modulating alveolar macrophage function leading to an altered balance between the proinflammatory and the counter-inflammatory response. METHODS: A 2-hit rat model of shock resuscitation was used. Alveolar macrophages were harvested by bronchoalveolar lavage (BAL), and tumor necrosis factor (TNF)-alpha and interleukin (IL)-10 were quantified in the cell culture supernatants by enzyme-linked immunosorbent assay (ELISA). Alternatively, 1 hour after resuscitation, animals received endotracheal LPS followed by endotracheal anti-IL-10 neutralizing antibody. Lung injury was determined by measuring BAL neutrophil counts 4 hours after LPS in vivo administration. RESULTS: Systemic administration of HTS significantly modulates the responsiveness of alveolar macrophages. Specifically, HTS resuscitation inhibited LPS-induced TNF-alpha production while enhancing IL-10 release in response to LPS administered ex vivo and in vivo. Anti-IL-10 antibody in vivo partially reversed the lung protective effect of HTS resuscitation. CONCLUSIONS: HTS resuscitation exerts an immunomodulatory effect on alveolar macrophages by shifting the balance of pro- and counter-inflammatory cytokine production in favor of an anti-inflammatory response. The in vivo data suggest a causal role for HTS-induced augmented IL-10 as protective. These findings suggest a novel mechanism for the in vivo salutary effect of HTS resuscitation on lung injury after resuscitated hemorrhagic shock.     

Putative mechanism of hemorrhage-induced leukocyte hyporesponsiveness: induction of suppressor of cytokine signaling-3

BACKGROUND: After hemorrhagic shock, macrophages are less responsive to lipopolysaccharide (LPS) regarding cytokine production and receptor expression. However, mechanisms responsible for this are poorly understood. Suppressors of cytokine signaling (SOCS) proteins have been found to play a prominent role in LPS tolerance and cytokine desensitization in macrophages. Therefore, the purpose of this study was to determine whether hemorrhagic shock induced SOCS expression. METHODS: Male C3H/HeN mice were subjected to hemorrhage or sham hemorrhage. Twenty-four hours after each procedure, tissues were harvested, the cells were processed for protein, and SOCS expression was examined. RESULTS: Our data show that SOCS-1 expression does not change after hemorrhage, but SOCS-3 is up-regulated in a tissue and cell population (e.g., macrophage)-specific manner. CONCLUSION: These data suggest that cytokines or other inflammatory mediators present during the first 24 hours after the induction of shock have the ability to induce tolerance to LPS or cytokines and suppress the function of immune cells by up-regulating SOCS-3.     

Prostanoid production by lipopolysaccharide-stimulated Kupffer cells

Although some data suggest that macrophages in the reticuloendothelial system (RES) are important sources of thromboxane A2 (TxA2) and prostacyclin (PGI2) during endotoxic shock, we are unaware of data documenting the ability of hepatic macrophages (Kupffer cells) to release either TxA2 or PGI2 when exposed to lipopolysaccharide (endotoxin, LPS). In this study, Kupffer cells were examined for their ability to release prostaglandin E2 (PGE2), TxA2, and PGI2 following stimulation with 0, 1.0, 50.0, and 100.0 micrograms/ml of Escherichia coli LPS. Kupffer cells were obtained from rat livers by enzymatic digestion with 0.05% collagenase followed by enrichment of the macrophage population on the basis of differences in density and adherence among the various cell populations isolated. Based on several criteria (phagocytosis of opsonized sheep erythrocytes, positive staining for esterase and peroxidase, failure to replicate), 95% of adherent cells were Kupffer cells. After 4 days of incubation, cells were stimulated with various doses of LPS for 4 and 8 hr. Prostanoid concentrations in culture supernatants were determined by radioimmunoassay. Increasing doses of LPS significantly (P less than 0.001) increased the concentration of immunoreactive PGE2 (iPGE2) and iTxB2 (the stable metabolite of TxA2). The concentration of i6-keto-PFG1 alpha (stable metabolite of PGI2) increased following stimulation with 1.0 microgram/ml of LPS, but declined as the dose of LPS was increased. The results provide evidence that endotoxin-activated Kupffer cells, like other macrophage populations, release several metabolites of arachidonic acid. Kupffer cell-derived prostanoids, particularly TxA2, may be important mediators of some of the pathophysiologic manifestations of acute endotoxemia.     

Prolactin modulates survival and cellular immune functions in septic mice

BACKGROUND: The immunomodulatory properties of the pituitary hormone prolactin have been demonstrated. It was proposed that prolactin is important in maintaining normal immune response in several pathological states. We investigated the effect of prolactin administration on the survival and cellular immune functions during systemic inflammation. MATERIALS AND METHODS: Male NMRI mice were subjected to laparotomy (LAP) or sepsis induced by cecal ligation and puncture (CLP). Mice were treated with either saline (LAP/saline; CLP/saline) or prolactin (LAP/PRL, CLP/RPL; 4 mg/kg s.c.). Survival of septic mice was determined 24 and 48 h after CLP. Forty-eight hours after the septic challenge, the proliferative capacity, cytokine release (IL-2, IL-6, IFN-gamma) and apoptosis of splenocytes were determined. Additionally, monitoring of circulating leukocyte distribution was performed (WBC; CD3+, CD4+, CD8+, B220+, NK1.1+, F4/80+ cells by FASCan). RESULTS: CLP was accompanied by a mortality of 47% and induced a decrease in splenocyte proliferation and apoptosis rate. Administration of prolactin significantly increased the mortality of septic mice (81%). This was paralleled by a further decrease of splenocyte proliferation and an increased splenocyte apoptosis. In addition, administration of prolactin augmented the sepsis-induced inhibition of IL-2 release, attenuated the sepsis-induced inhibition of IFN-gamma release, and did not affect the release of IL-6. However, prolactin did not affect the sepsis-induced changes of circulating leukocyte subpopulations. CONCLUSIONS: We conclude that prolactin has profound immunomodulatory properties and that administration of prolactin in pharmacological doses is associated with a decreased survival and an inhibition of cellular immune functions in septic mice.