Immune response of severely injured patients--influence of surgical intervention and therapeutic impact.

BACKGROUND: [corrected] Severe injury leads to a severe deterioration of the patients' immune response. The changes of the immune response after severe injury include a broad range of immune functions and may result in a status of immunosuppression, which could favor infectious complications. Therefore, immunostimulating therapies have been introduced in the therapy for severely injured patients in clinical and experimental settings. OBJECTIVES: The article summarizes actual immunomodulating approaches in the treatment of trauma patients and therapeutic strategies avoiding additional immune deteriorations. RESULTS: Examples for an immunostimulating approach in trauma patients are interferon gamma and the granulocyte macrophage-colony-stimulating factor (GM-CSF), which are summarized in this review in detail. However, the effect of such an interference in the patients' immune response with all its different cellular targets is not yet clearly understood, and most studies focus on the reaction of circulating monocytes. In addition, further immunomodulating strategies, including nutritional support, are addressed. However, clinically established therapeutic immunomodulating strategies in trauma care so far do not exist. The impact of the accidental and also an additional surgical trauma on the immune response has been clearly demonstrated. Therefore, the idea of a "damage control orthopedic surgery" (DCOS) is not only necessary to prevent further deterioration of the homeostasis of, e.g., the coagulating system, but is also desirable in terms of minimizing the burden on the immune system. In addition, also the timing of secondary surgical treatment in trauma patient care should include an evaluation of the immune response, although the most reliable markers still need to be identified. CONCLUSION: Immunomodulating therapies in trauma patients exist on an experimental level with inconsistent results. The general management of trauma patients includes strategies that have been developed also on the basis of immunological considerations.

Regulation of cytokine synthesis in cardiac surgery: Role of extracorporeal circuit and humoral mediators in vivo and in vitro.

OBJECTIVE AND DESIGN: Cardiopulmonary bypass (CPB) impairs monocyte and neutrophil proliferation, cytokine synthesis, and antigen presentation. This study compares in vivo data with results from an extracorporeal circulation (ECC) model, distinguishing direct effects on cytokine synthesis from regulatory mechanisms. PATIENTS AND METHODS: Whole blood from 18 patients prior to, during and after CPB was stimulated with lipopolysaccharide (LPS). Tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, and IL-8 levels were measured. Additionally, blood from 4 volunteers was circulated in an ECC model. Cytokine levels were measured before and during mock ECC. RESULTS: LPS-induced cytokine synthesis was reduced after CPB (TNF-alpha: 11 %; IL-6: 29 %; IL-8: 48 % of preoperative values, all p < 0.001). In mock ECC, cytokine production (except IL-8) was suppressed: TNF-alpha production was lowest 60 min after starting ECC, IL-6 synthesis was lowest at 90 min (33 % and 15 % vs. pre-ECC levels; both p < 0.001). Patient sera contained cytokine-inhibitory activity after CPB, an activity not found in mock ECC. CONCLUSIONS: (1) In patients, CPB induces early transient LPS hyporesponsiveness; (2) blood contact with foreign surfaces induces LPS hyporesponsiveness; (3) serum cytokine-inhibitory activities are released after CPB, but not in mock ECC. Impaired leukocyte function may explain increased susceptibility to infections after CPB.

Differential immunostimulating effect of granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF) and interferon gamma (IFNgamma) after severe trauma.

OBJECTIVE: Severe trauma leads to an increased vulnerability to bacterial sepsis. In the present study, we compared the immunostimulating potential of granulocyte-colony stimulating-factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF) and interferon-gamma (IFN-gamma). DESIGN: Prospective clinical experimental study. SETTING: University hospital intensive care unit and research facility. PATIENTS: 6 patients with an Injury Severity Score (ISS) of more than 25 points. INTERVENTIONS: Heparinized blood samples of severely injured patients and 12 healthy volunteers were incubated in vitro with 10 ng/ml GM-CSF, 10 ng/ml G-CSF or 10 ng/ml IFN-gamma for 6 h. MEASUREMENTS: Flow cytometry: HLA-DR expression on monocytes, B- and T-lymphocytes. ELISA: LPS-induced TNFalpha and IL-10 production. RESULTS: In all patients reduced cytokine production and HLA-DR expression on monocytes was established. After administration of GM-CSF and IFN-gamma it in vitro, the level of HLA-DR expression on monocytes and the it ex vivo TNFalpha-synthesis increased while only GM-CSF increased significantly IL-10-liberation after LPS-stimulation. However, only IFN-gamma had the capacity to enhance HLA-DR on B- and T-lymphocytes. G-CSF it in vitro had no significant effect on the measured parameter. CONCLUSIONS: These data suggest that GM-CSF and IFN-gamma may serve to support immune functions in severely injured patients.

GM-CSF counteracts hemorrhage-induced suppression of cytokine-producing capacity.

OBJECTIVE AND DESIGN: To study whether a treatment with the hematopoietic growth factor GM-CSF restores the attenuated ex-vivo cytokine-producing capacity of macrophages after sublethal hemorrhagic shock. SUBJECTS: Male Sprague-Dawley rats. TREATMENT: 20 microg/animal of recombinant murine GM-CSF after shock via arterial line. METHODS: Hemorrhagic shock was established by pressure-controlled bleeding to a mean arterial pressure of 50 mm Hg for 35-40 min and consecutive resuscitation. 24 h after hemorrhage, lipopolysaccharide (LPS)-induced cytokine production of isolated macrophages derived from different compartments was measured. RESULTS: A significant reduction of LPS-induced TNFalpha production was found in whole blood cultures (1.0 +/- 0.7 ng/ml after sham vs. 0.23 +/- 0.08 ng/ml after shock operation), macrophages derived from spleen (0.88 +/- 0.23 ng/ml after sham vs. 0.03 +/- 0.1 ng/ml after shock operation), peritoneum (2.2 +/- 0.7 ng/ml after sham vs. 0.29 +/- 0.4 ng/ml after shock operation) and bronchoalveolar fluid (0.65 +/- 0.13 ng/ml after sham vs. 0.003 +/- 0.027 ng/ml after shock operation, mean +/- S.D.). In cells from animals treated with GM-CSF a significantly enhanced LPS-induced TNFalpha production in splenic, alveolar and peritoneal macrophages was found after shock compared to the cells derived from untreated animals (peritoneum: 289 +/- 366 ng/ml TNFalpha after shock vs. 2066 +/- 94 ng/ml TNFalpha after shock and GM-CSF; lung: 9 +/- 12 ng/ml TNFalpha after shock vs. 64 +/- 17 ng/ml TNFalpha after shock and GM-CSF; spleen: 58 +/- 96 ng/ml TNFalpha after shock vs. 548 +/- 47 ng/ml TNFalpha after shock and GM-CSF). Blood cultures collected from rats after hemorrhagic shock did not show a significant increase of TNFalpha-production after GM-CSF treatment. CONCLUSION: Hemorrhagic shock caused a depression of the TNFalphaa response to LPS which was partly counteracted by treatment with GM-CSF. Therefore, GM-CSF represents a promising approach to normalise trauma- and shock-induced immune dysfunction.

Endotoxin tolerance in rats: influence on LPS-induced changes in excretory liver function.

OBJECTIVE AND DESIGN: We investigated in a rat model of endotoxic shock whether endotoxin tolerance (ETT) prevents lipopolysaccharide (LPS) associated lethality and studied the initial function of liver response to LPS. ANIMALS: Male Sprague-Dawley rats. TREATMENT: ETT was induced by i.p. injection of LPS (Salmonella friedenau) intraperitoneally over 5 days. Rats (n = 6 each group) received 1 mg LPS/kg b. w. intravenously (Salmonella friedenau). The common bile duct was then canalized and bile was collected every 60 min for 6 h. 1 h after LPS-application liver biopsies were taken for determination of TNF-alpha by RT-PCR. Sham operated animals (n = 6 each group) were treated identically but without application of LPS. RESULTS: All ETT animals survived the duration of the experiment whereas non-tolerant animals (NETT) died before the end of the experiment (5/6). NETT animals showed a continuous decrease in bile flow after 240 min. The amount of bile acids was significantly lower (ANOVA) in NETT animals than in sham operated controls or ETT-animals. Analysis of TNF-alpha mRNA expression in the liver revealed an upregulation 1 h after LPS application, which was significantly lower in LPS-tolerant animals. CONCLUSIONS: Our results show that excretory liver failure and death subsequent to intravenous LPS application can be successfully counteracted by induction of ETT.

Tumor necrosis factor-alpha production in whole blood after cardiopulmonary bypass: downregulation caused by circulating cytokine-inhibitory activities.

OBJECTIVES: Cardiopulmonary bypass is associated with the release of proinflammatory cytokines (tumor necrosis factor alpha, interleukin 1beta, interleukin 6, and interleukin 8) and anti-inflammatory cytokines (interleukin 10 and transforming growth factor beta(1)). On the one hand this cytokine release is related to the postoperative systemic inflammatory response syndrome, and on the other hand it is related to deterioration of the immune system, for example in monocyte or polymorphonuclear neutrophil function, leading to an increased susceptibility to infections. To gain further insight into the alterations of immune cell reactivity and possible regulatory mechanisms, we studied lipopolysaccharide-induced tumor necrosis factor alpha synthesis in whole blood from cardiac surgical patients. METHODS: Fifteen patients undergoing elective heart surgery with cardiopulmonary bypass were included in the study. Ex vivo lipopolysaccharide-induced tumor necrosis factor alpha synthesis was measured in a whole blood assay before, during, and after bypass. Corresponding tumor necrosis factor alpha messenger RNA levels were determined by semiquantitative reverse transcriptase-polymerase chain reaction. In addition, the influence of patient serum on whole blood responsiveness and its relationship to anti-inflammatory cytokines were evaluated in vitro. RESULTS: Tumor necrosis factor alpha synthesis was significantly reduced after 30 minutes of cardiopulmonary bypass and showed the lowest values at the end of bypass (mean +/- SD 0.109 +/- 0.105 ng/10(6) white blood cells after 30 minutes of bypass and 0.050 +/- 0.065 ng/10(6) white blood cells at the end of bypass, vs 0.450 +/- 0.159 ng/10(6) white blood cells preoperatively, P <.001). As a further indication of reduced cytokine biosynthesis, diminished messenger RNA levels for tumor necrosis factor alpha were detected. Serum withdrawn from patients at the end of cardiopulmonary bypass reduced tumor necrosis factor alpha synthesis in heterologous blood from healthy volunteers highly significantly to 39.93% +/- 23.18% relative to control serum (P =.005) and preoperatively drawn serum (P =.024). This effect was dose dependent and was not specific for lipopolysaccharide-induced tumor necrosis factor alpha synthesis. Anesthesia and heparin administration did not influence tumor necrosis factor alpha production significantly. Ex vivo tumor necrosis factor alpha synthesis was negatively related to interleukin 10 serum levels, positively but weakly related to interleukin 4, and was not related to transforming growth factor beta(1) (Spearman correlation coefficients -0.565, P <.001, 0.362, P <.001, and -0.062, P =.460, respectively). However, interleukin 10 levels in patient serum after cardiopulmonary bypass were 300-fold below the quantities needed for half-maximal inhibition of tumor necrosis factor alpha synthesis in vitro. Moreover, the inhibitory activity could not be removed by immune absorption of interleukin 10. CONCLUSIONS: These results suggest that during cardiac operations cytokine-inhibitory serum activities are released or newly formed. These activities could not be explained by the actions of interleukins 4 and 10 or transforming growth factor beta(1). Although their exact nature remains undetermined, these substances may contribute to the diminished immune cell functions after cardiopulmonary bypass and thus need further characterization.

Effects of endotoxin tolerance on liver function after hepatic ischemia/reperfusion injury in the rat.

OBJECTIVE: It is known that endotoxin tolerance prevents lethality after ischemia/reperfusion injuries (e.g., myocardial infarction) in laboratory animals. We used a rat model of partial hepatic ischemia/reperfusion to investigate whether endotoxin tolerance prevents associated lethality and disorders of liver function. DESIGN: Prospective animal study. SETTING: University research facility. SUBJECTS: Male Sprague-Dawley rats. INTERVENTIONS: Hepatic ischemia was initiated by atraumatic clipping across the portal venous and hepatic arterial blood supply to the left lateral lobe for 90 mins. The common bile duct was canalized, and in a second set of experiments the bile duct of the left lateral lobe was canalized selectively. Bile flow, bile acids, and transaminases were determined during ischemia and 300 mins of reperfusion in endotoxin-tolerant and -nontolerant rats. MEASUREMENTS AND MAIN RESULTS: Endotoxin-nontolerant animals showed a 50% lethality after hepatic ischemia/reperfusion injuries. All endotoxin-tolerant rats survived and did not react with any change in bile flow, showing a constant flow. The amount of bile acids in the common bile duct was reduced during ischemia and regained the concentrations of sham-operated animals 60 mins after reperfusion. From 180 mins after reperfusion, the difference between endotoxin-tolerant and -nontolerant animals was statistically significant. When bile acid concentration was determined in the ischemic left lateral lobe, ischemia/reperfusion was found to significantly decrease in endotoxin-nontolerant rats 60 mins after reperfusion. In contrast, endotoxin-tolerant rats produced normal amounts of bile acids 60 mins after reperfusion. At 120 mins after reperfusion, the amount of bile acids in the formerly ischemic left lateral lobe was more than normal. CONCLUSIONS: In this model of partial hepatic ischemia/reperfusion, endotoxin tolerance prevents ischemia/reperfusion injury-associated lethality and local disorders of liver function. This phenomenon induced by endotoxin tolerance may be useful in liver surgery to prevent ischemia/reperfusion injury.

Cytokine synthesis in the liver of endotoxin-tolerant and normal rats during hemorrhagic shock.

In the present study the effects of endotoxin tolerance on hemorrhagic shock were investigated with particular focus on hepatic alterations. The following questions were addressed: (i) does hemorrhagic shock induce cytokine formation and heat shock response in the liver; and (ii) does endotoxin tolerance alter these reactions. Endotoxin tolerance was induced by repetitive daily injections of LPS for 5 days. Hemorrhagic shock was induced by hypovolemia (MAP 35 +/- 5 mmHg). After 3 h, the animals were resuscitated by re-infusion of homologous blood. m-RNA was isolated from liver biopsies and the mRNA levels of tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), interleukin-10 (IL-10) and heat shock protein 70 (HSP-70) were determined by RT-PCR. TNF-alpha was measured by ELISA in serum samples and in the supernatants of whole blood cultures. It was found that endotoxin tolerance reduced mortality caused by hemorrhagic shock from 80% to 20%. In parallel, TNF-alpha production in response to LPS in vivo and in vitro was significantly decreased. During hemorrhage and after resuscitation. increased mRNA levels were detected in hepatic biopsies for TNF-alpha, IL-6, IL-10 and HSP-70, with highest levels immediately after re-infusion. Endotoxin-tolerant rats produced significantly lower levels of TNF-alpha, while no differences were found for IL-10 and HSP-70. Within 30 min after reperfusion, significantly higher levels of IL-6 mRNA were found in hepatic biopsies from tolerant rats; these differences disappeared 2 h after reperfusion.

Sublethal hemorrhagic shock reduces tumor necrosis factor-alpha-producing capacity in different cell compartments.

Hemorrhagic shock results in a severe impairment of the immune response. Immunological alterations after hemorrhagic shock thus appear to be responsible for reduced resistance to infectious agents commonly observed after shock and severe injury. In the present study we examined the TNF-alpha-producing capacity of immune cells derived from different organs after sublethal shock in rats. Hemorrhagic shock was established by pressure controlled bleeding to a mean arterial pressure of 35 mm Hg for 35-40 min and consecutive resuscitation in male Sprague-Dawley rats. Twenty four hours after shock, TNF-a production in response to lipopolysaccharide (LPS, Salmonella friedenau) stimulation was measured in isolated spleen, bone marrow and blood cells. TNF-a production could be induced by stimulation with 1 ng/ml, in blood, spleen and bone marrow cells collected from sham-operated animals. A maximal stimulation was observed in all cell types after stimulation with 10 ng/ml LPS and could not be further increased with LPS doses of 100 ng/ml. Hemorrhagic shock of 35 mm Hg for 35-40 min, with consecutive resuscitation did not result in mortality, in contrast to a 4 hours lasting hemorrhagic shock resulting in 80% mortality. Blood, spleen or bone marrow cells, harvested from animals 24 hours after sublethal hemorrhagic shock, showed a significantly reduced TNF-alpha production in all cell populations after LPS stimulation. Serum collected from animals 2 hours after sublethal hemorrhagic shock contained an activity not present either before or 24 hours after shock, that downregulated LPS-induced TNF-alpha production in rat whole blood cultures and the murine macrophage cell line J774. The inhibitory activity present in serum, 2 hours after shock is not IL-10 since this mediator was not detectable in any serum sample. However, in the serum samples with TNF-alpha-inhibitory activity, elevated levels of PGE2 metabolites were found, which suggests the involvement of prostaglandins in trauma-induced immunosuppression. Altered TNF-a expression might be partially explained by an inhibitory activity in the serum already present 2 hours after shock. Since adequate, but not overwhelming TNF-alpha production is essential for host response, the altered cytokine formation might explain local and systemic susceptibility to infections after hemorrhagic shock.

Immunosuppression with FK506 increases bone induction in demineralized isogeneic and xenogeneic bone matrix in the rat.

The aim of the present study was to investigate a systemic induction of bone formation in rats by immunosuppression with FK506 (1 mg/kg body weight intraperitoneally [ip]) in a model of osteoinduction of isogeneic and xenogeneic demineralized bone matrix (DBM) for a period of 28 days. In particular, alterations of in vitro cytokine synthesis and changes of lymphocyte subsets were studied. DBM was implanted intramuscularly in the abdominal wall of Lewis rats (seven per group). Blood was sampled on days -7, 0, 7, and 28 for determination of in vitro tumor necrosis factor a (TNF-alpha) synthesis and lymphocyte subsets by flow cytometry (CD3+, CD4+, CD8+, CD45+, ED9+, and Ia+ antibodies). Ossicles of de novo formed bone and the tibias were removed on day 28 after double tetracycline labeling for histomorphometric analysis. Immunosuppression with FK506 significantly decreased lipopolysaccharide (LPS)-stimulated in vitro cytokine synthesis after 7 days and 28 days (p < 0.05). Compared with control animals FK506 treatment significantly increased the volume of induced bone in isogeneic (2.1 +/- 0.3 mm3 vs. 10.8 +/- 0.9 mm3) and xenogeneic (O mm3 vs. 4.7 +/- 0.8 mm3) DBM. Bone histomorphometry of the tibias revealed that immunosuppression increased both bone formation and bone resorption, accompanied by a significant reduction in the relative trabecular area (Tb.Ar). FK506 caused a decrease in the counts of CD8+ T cells probably because of destruction or dislocation of these cells. This suggests that the amount of CD8+ cells and the degree of T cell activation in terms of mean fluorescence intensity (MFI) may be associated with bone metabolism. In support of this, statistical analysis revealed a significant positive correlation between parameters of bone formation as well as bone resorption and the CD4+/CD8+ ratio. There was a significant negative correlation between parameters of remodeling of the metaphysis of the tibia and induced bone volume (BV), respectively, and MFI values of CD3+/Ia+ cells. These findings suggest an important role of T lymphocytes in bone formation and bone resorption in vivo. FK506 caused a marked increase of bone formation in DBM. However, the conclusion that immunosuppression increases fracture healing warrants further investigation.

Whole blood tumor necrosis factor-alpha production and its relation to systemic concentrations of interleukin 4, interleukin 10, and transforming growth factor-beta1 in multiply injured blunt trauma victims.

OBJECTIVE: To study the relation of whole blood endotoxin responsiveness to inhibitory mediators systemically released after severe blunt trauma. DESIGN: Prospective, observational study. SETTING: University trauma center. PATIENTS: Thirty-two patients with blunt trauma (mean injury severity score, 33 points). INTERVENTIONS: Standard emergency department, surgical care, and postoperative intensive care unit treatment. MEASUREMENTS AND MAIN RESULTS: Whole blood and serum were obtained immediately after admission to the emergency department (<8 hrs after trauma, denoted day 0) and on days 1, 2, 4, 6, 8, and 14 after trauma. Whole blood specimens were assayed for endotoxin-induced tumor necrosis factor (TNF)-alpha synthesis ex vivo and serum specimen were assayed for interleukin (IL)-4, IL-10, and transforming growth factor (TGF)-beta1 concentrations. Moreover, the TNF-alpha inhibitory capacity of recombinant human (rh) IL-4, rhIL-10, and TGF-beta1 as well as the inhibitory capacity of patients' serum from days 0, 1, 2, 4, 6, 8, and 14 were tested on uninjured donors' whole blood. Cytokines were determined by ELISA. Whole blood endotoxin responsiveness in multiply injured patients was significantly reduced during the observation period and was found to be significantly related to the total inhibitory activity detected in the corresponding sera. Exchange of patients' serum for uninjured donors' or recovered patients' serum restored TNF-alpha production of peripheral blood mononuclear cells from multiply injured patients. Serum levels of IL-4 and IL-10 were not related to trauma patients' whole blood TNF-alpha production upon endotoxin stimulation, whereas TGF-beta1 concentrations were positively related. Compared with the apparent half-maximal inhibition concentrations determined, serum levels of TGF-beta1, IL-10, and IL-4 were 20- to 20,000-fold below the quantities required to explain the inhibitory serum activity in multiply injured patients on day 0. CONCLUSIONS: Whole blood hyporesponsiveness to endotoxin in multiply injured patients is caused by soluble serum factors systemically released after trauma, whereas the intrinsic leukocyte function appears unaffected. Inhibitory mediators other than IL-4, IL-10, or TGF-beta1 are supposed to be of major biological relevance for the posttraumatic regulation of leukocyte function. Characterization of the causative suppressive mediators is supposed as a prerequisite for the development of immunologically based therapeutic approaches in critically ill patients.

Sex differences in posttraumatic cytokine release of endotoxin-stimulated whole blood: relationship to the development of severe sepsis.

BACKGROUND: In experimental trauma-hemorrhage and sepsis, a sexual dimorphism of cell-mediated immune functions has been described, which has been related to higher susceptibility to and mortality from sepsis in males. Therefore, in the present study, sex differences with regard to cytokine release of endotoxin stimulated whole blood and its relation to the development of severe posttraumatic sepsis were investigated in blunt trauma patients with multiple injuries. METHODS: Eighty-four patients (25 female; 59 male) sustaining blunt injuries with an Injury Severity Score > 16 were enrolled in the study. Whole blood and serum were obtained during a 14-day period of hospitalization. The capacity of peripheral blood mononuclear cells to produce cytokines (tumor necrosis factor-alpha, interleukin [IL]-6, IL-8) was tested by using a whole blood assay. Serum samples were assayed for anti-inflammatory cytokines (IL-4, IL-10, and transforming growth factor beta1) and sex hormones (testosterone, estradiol, progesterone). Patients were monitored daily for sepsis criteria according to the ACCP/ SCCM consensus conference 1992. RESULTS: Within the entire patient population, sex differences in posttraumatic cytokine release were not detectable. Male trauma patients developing severe sepsis (n = 16) presented with a significantly increased cytokine producing capacity in the early posttraumatic period (< or = 24 hours after admission to the emergency room) when compared with males with an uncomplicated recovery. In females, differences between the subgroups of patients with (n = 7) and without development of severe sepsis were not detectable. There were no differences in systemic levels of anti-inflammatory cytokines within the early posttraumatic period between the subgroups of male and female patients with and without development of severe sepsis. In females, differences in sex hormone levels were not detectable, whereas in males, development of severe sepsis later was found to coincide with significantly decreased testosterone and increased estradiol serum levels. CONCLUSION: The present study demonstrates a sex-specific regulation of leukocyte function in patients with multiple injuries within the early posttraumatic period. In male patients with multiple injuries, increased cytokine-producing capacities may correspond to enhanced inflammatory responses, which increase susceptibility to sepsis, whereas in female patients, other regulatory mechanisms may be involved.

Endotoxin tolerance protects against local hepatic ischemia/reperfusion injury in the rat.

Liver surgery and liver transplantation as well as circulatory shock are often associated with hepatic ischemia/reperfusion (I/R) injury. Recent evidence suggests that TNF-alpha plays a central role in I/R injury and, therefore, down-regulation of TNF-alpha seems to be a promising way to protect against the deleterious consequences of I/R. Endotoxin tolerance represents a state of unresponsiveness to endotoxin and is associated with diminished TNF-alpha production. Thus, the effect of endotoxin tolerance on hepatic I/R injury of the liver was investigated in a rat model. I/R injury was induced by temporary ischemia of the left lateral liver lobe for 90 min followed by a 3 h observation period of reperfusion. I/R injury resulted in functional hepatic disorder characterized by a decrease both in bile flow and bile acid concentration and 50% mortality. This was prevented by induction of endotoxin tolerance. Hepatic TNF-alpha mRNA expression after I/R of the liver was determined by RT-PCR. In untreated rats, TNF-alpha mRNA was induced in the liver 60 min after reperfusion and further increased until 3 h after reperfusion. In contrast, in endotoxin-tolerant rats, no increases in TNF-alpha mRNA expression were detected. This suggests that induction of endotoxin tolerance protects against hepatic I/R injury possibly via down-regulation of intra-organ TNF-alpha expression.

Interferon-gamma counteracts reduced endotoxin responsiveness of whole blood following trauma and cardiopulmonary bypass.

Accidental as well as surgical trauma has been reported to cause reduced endotoxin responsiveness of blood in terms of cytokine production. In this study, the effect of interferon-gamma (IFN-gamma) on tumour necrosis factor-alpha (TNF-alpha)-producing capacity of whole blood after severe trauma and cardiac surgery was investigated. Blood samples of severely injured patients were collected at the first day after trauma and of cardiac surgery patients before, 4 h and 2 days after cardiopulmonary bypass (CPB). The blood samples were incubated with INF-gamma (0-100 U/ml) for 20 h and subsequently lipopolysaccharide (LPS)-induced TNF-alpha production was determined. Compared to healthy donors, LPS-induced TNF-alpha production was significantly reduced in blood cultures of trauma patients on day 1 after trauma and 4 h after CPB. Pre-incubation with IFN-gamma in vitro increased endotoxin-induced TNF-alpha production in volunteers' and all patients' blood specimens in a dose-dependent manner. IFN-gamma prompted an elevation of cytokine synthesis in CPB patients' blood which equalled that of volunteers, whereas it caused a lower rise in TNF-alpha production in blood of multiply injured patients, reaching levels of untreated donors only after incubation with 100 U/ml IFN-gamma. These experiments show that hyporesponsiveness of whole blood induced by trauma or cardiac surgery with CPB is not irreversible, but can be counteracted by the immunostimulant IFN-gamma. IFN-gamma, therefore, could be applied clinically in trauma patients or after cardiac surgery to prevent or to resolve infection complications.

Cytosolic protein ubiquitylation in normal and endotoxin stimulated human peripheral blood mononuclear cells.

The ubiquitin-proteasome pathway is regarded as playing a crucial role in protein breakdown in inflammation and sepsis as well as in the regulation of inflammatory cell responses. In this pathway, ubiquitylation of target proteins is believed to act as a recognition signal for degradation by the 26S proteasome. As yet neither the ubiquitylation rate of cytosolic proteins, as a result of the total ubiquitin-protein ligase (tUbPL) activity, nor the specific ubiquitylation of calmodulin (ubiquitin-calmodulin ligase, uCaM-synthetase) has been determined in human mononuclear cells. Therefore, we studied cytosolic protein ubiquitylation in normal and in endotoxin (LPS)-stimulated human peripheral blood mononuclear cells (PBMNCs).PBMNCs from healthy volunteers were incubated with 0 or 100 ng/ml LPS for 18 h. Cytosolic extracts were obtained by hypotonic lysis and ultracentrifugation. TUbPL was measured as [(125)I]-[CT]-ubiquitin incorporation into the sum of cytosolic proteins. UCaM-synthetase activity was quantified with the fluphenazine (FP)-Sepharose affinity adsorption test. Endotoxin stimulation appears to inhibit tUbPL 3.7 +/- 2.7-fold to 48 +/- 43 fkat/mg (n = 6). UCaM-synthetase in cultures (n = 5) without endotoxin was determined to be 91 +/- 32 fkat/mg +Ca(2+) and 29 +/- 23 fkat/mg -Ca(2+). With endotoxin uCaM-synthetase was 138 +/- 73 fkat/mg +Ca(2+) and 14 +/- 22 fkat/mg -Ca(2+). Ca(2+)-specificity (ratio +/- Ca(2+)) of uCaM-synthetase increases from 3.1 without LPS to 10 after LPS stimulation, which was caused by a 2-fold decrease in minus Ca(2+) activity and a 1.5-fold increase in plus Ca(2+) activity. The data indicate specific regulatory effects of endotoxin on the cytosolic ubiquitylation systems in human PBMNCs.

Endotoxin and ischemic preconditioning: TNF-alpha concentration and myocardial infarct development in rabbits.

Ischemic preconditioning (IP) and prior exposure to lipopolysaccharides (LPS) reduce infarct size (IS) and serum tumor necrosis factor-alpha (TNF-alpha) concentration resulting from myocardial ischemia-reperfusion in rabbits. The decrease in TNF-alpha might relate to an induced TNF-alpha inhibitory serum activity (TNF-alpha-ISA). We analyzed TNF-alpha and TNF-alpha-ISA during 30 and 180 min ischemia and reperfusion, respectively, in anesthetized rabbits either untreated (group 1, n = 7), preconditioned (5 and 10 min ischemia and reperfusion, respectively, group 2, n = 9), or exposed to LPS 72 h before ischemia (group 3, n = 9). TNF-alpha-ISA was assessed by coincubating LPS-stimulated rabbit blood with serum of groups 1-3 and measuring TNF-alpha (WEHI assay). With a comparable area at risk, IS in group 1 was 36.9 +/- 11.1 (SD)%, and it was reduced to 13.1 +/- 11.6% and 17.3 +/- 11.3% (both P < 0.05) in groups 2 and 3, respectively. TNF-alpha was increased during ischemia-reperfusion in group 1 but remained unchanged in rabbits subjected to IP or LPS. TNF-alpha-ISA was detected during ischemia-reperfusion in group 2 (29% and 38% of maximum inhibition, respectively) and during baseline, ischemia and reperfusion in group 3 (51%, 46%, 48% of maximum inhibition, respectively) but was absent in group 1. Cardioprotection by IP and LPS is associated with a reduced TNF-alpha and an induced TNF-alpha-ISA during ischemia-reperfusion.

Endotoxin tolerance in rats: expression of TNF-alpha, IL-6, IL-10, VCAM-1 AND HSP 70 in lung and liver during endotoxin shock.

Endotoxin can induce a state of tolerance against its own pathological effects, commonly referred to as endotoxin tolerance. This phenomenon has been found to be associated with reduced serum levels of cytokines such as TNF-alpha, IL-1, IL-6 and IL-10. In the present study the expression of TNF-alpha, IL-6, IL-10, the adhesion molecule VCAM-1 and the heat shock protein 70 was determined in vivo in lung and liver of LPS-tolerant and naive rats by means of semiquantitative RT-PCR after i.v. LPS injection. TNFalpha, IL-6, IL-10, HSP 70 and VCAM-1 were induced in lung and liver after LPS injection. In liver and lung of endotoxin-tolerant rats TNF-alpha and IL-6 were induced to a lower degree after LPS treatment when compared to non-tolerant controls. The LPS-induced IL-10 expression was also slightly attenuated in the lung of tolerant rats, but in the liver no differences between tolerant and non-tolerant animals were observed. HSP 70 and VCAM-1 were expressed after systemic LPS treatment in liver and lung. The degree of induction, however, was the same in tolerant and untreated controls. The presented data show that endotoxin tolerance is reflected by a reduced cytokine expression in lung and liver in vivo. On the other hand, levels of expression of the adhesion molecule VCAM-1 and the stress protein HSP 70 do not appear to be changed by endotoxin tolerance.

Influence of granulocyte-macrophage colony-stimulating factor (GM-CSF) on whole blood endotoxin responsiveness following trauma, cardiopulmonary bypass, and severe sepsis.

Major surgery, multiple injury, and severe sepsis lead to an impaired immune response. The suppressed status of the immune system is reflected by a reduced TNFalpha production of whole blood after stimulation with endotoxin in vitro and by a decreased HLA-DR expression on monocytes. In the present study, the effect of the immunostimulating hematopoetic growth factor GM-CSF on whole blood cultures of multiple injury, cardiac surgery, and severe sepsis patients was investigated. Endotoxin-induced TNFalpha production and HLA-DR expression was reduced in blood cultures of these patients compared to healthy donors. Preincubation with GM-CSF in vitro increased cytokine production in volunteers' and all patients' blood specimens in a dose-dependent manner. The elevation of cytokine response in cardiopulmonary bypass patients' blood, caused by in vitro preincubation with GM-CSF, equaled that of normal patients, whereas GM-CSF caused a lower rise of TNFalpha-producing capacity in blood of multiple-injury and sepsis patients. Further, GM-CSF treatment in vitro increased the down-regulated HLA-DR expression on monocytes prepared after cardiac surgery to a degree comparable to preoperative levels. Finally, GM-CSF incubation in vitro elevated TNFalpha synthesis in normal monocytes and in cells treated with a combination of the anti-inflammatory mediators IL-10, TGFbeta, and PGE2. These experiments show that hyporesponsiveness of whole blood induced by trauma, sepsis, or cardiac surgery is not irreversible but can be, at least in vitro, overridden by the immunostimulating compound GM-CSF.

Relation of a TNF gene polymorphism to severe sepsis in trauma patients.

OBJECTIVE: To investigate the relation of the biallelic Nco1 restriction fragment length polymorphism in the first intron of the tumor necrosis factor (TNF) beta gene with the development of severe sepsis in multiply injured patients. SUMMARY BACKGROUND DATA: The biallelic Nco1 polymorphism of the TNFbeta gene has been described to be associated with autoimmune diseases and with the mortality rate in severe sepsis. Therefore, the Nco1 polymorphism may be associated with the clinical finding that despite comparable risk factors, posttraumatic sepsis develops in some patients but not others. METHODS: The study group consisted of 110 patients with severe blunt trauma (Injury Severity Score > or = 17). Typing of each patient for the biallelic Nco1 polymorphism was performed by analyzing restriction fragments of an Nco1-digested DNA fragment obtained using polymerase chain reaction. Genotypes were then related to the occurrence of severe posttraumatic sepsis and TNFalpha serum concentrations. RESULTS: Fifty-seven patients showed an uncomplicated posttraumatic recovery, and severe sepsis developed in 53 patients. The overall allele frequency (TNFB1 0.29, TNFB2 0.71) and genotype distribution (TNFB1 homozygous 7.3%, TNFB1/TNFB2 42.7%, TNFB2 homozygous 50%) were in agreement with the distribution in healthy volunteers. Genotype distribution in patients with an uncomplicated clinical course was significantly different from that in patients with severe posttraumatic sepsis. Development of severe posttraumatic sepsis was significantly increased in patients homozygous for the allele TNFB2. In patients with severe posttraumatic sepsis, TNFalpha serum concentrations were significantly higher in TNFB2-homozygous individuals compared with heterozygous and TNFB1 -homozygous individuals. The age- and injury-matched odds ratio for the homozygous TNFB2 genotype compared with the heterozygous genotype was 5.22 (p = 0.007, 95% confidence interval 1.6 to 17.9). CONCLUSIONS: In multiply injured patients, the Nco1 polymorphism within the TNFbeta gene is associated with the development of severe posttraumatic sepsis and with increased TNFalpha serum levels when severe sepsis has occurred. This suggests a genetic determination of the individual inflammatory response after infection or tissue damage, which significantly influences susceptibility to severe nosocomial infections.