Matthias Majetschak

Objective: To determine whether ubiquitin treatment modulates the lung cytokine response and attenuates lung ischemia-reperfusion injury. Design and Setting: Randomized and blinded treatment of unilateral lung ischemia-reperfusion injury in a research laboratory. Subjects: Twenty anesthetized and mechanically ventilated Brown Norway rats. Interventions: Unilateral clamping of the left lung for 90 mins followed by 60 mins of reperfusion. Intravenous administration of 1.5 mg/kg of ubiquitin (n = 10) or albumin (n = 10) 5 mins before reperfusion. Measurements and Main Results: Blood pressure was measured by the tail-cuff method. Oxygenation via the ischemic lung was assessed by Pao₂ measurements after right lung exclusion. Wet-to-dry weight ratios of the ischemic lungs were determined gravimetrically. Tissue homogenates (n = 5/group) were prepared from ischemic lungs at the end of reperfusion and assayed for malondialdehyde in combination with 4-hydroxyalkenals to assess lipid peroxidation, and for a panel of 22 cytokines/chemokines using a multiplex assay. Ubiquitin serum levels were determined by enzyme-linked immunosorbent assay. All animals were hemodynamically stable during the experimental procedure. Ubiquitin serum levels (mean ± sd) were 650 ± 40 ng/mL in controls and 1206 ± 181 ng/mL in the ubiquitin treatment group at the end of the experiment. Pao₂ after right lung exclusion was 45 (32–72) mm Hg with albumin and 61 (range, 36–132) mm Hg with ubiquitin (p = .0185). Wet-to-dry weight ratios of the injured lungs were 8.7 (range, 5.5–19.1) and 7.8 (range, 5.7 – 8.3) in the albumin and ubiquitin groups, respectively (p = .035). Malondialdehyde/4-hydroxyalkenals concentrations (mean ± sd, nmol/mg protein) were 2.5 ± 0.4 with ubiquitin and 3.0 ± 0.3 with albumin (p > .05). Concentrations of the interleukins 4, 10, and 13 were significantly increased in lung homogenates after ubiquitin treatment (p < .05). Conclusion: Ubiquitin treatment enhances the Th2 cytokine response in postischemic lungs during reperfusion, reduces lung edema formation, and improves pulmonary function during lung ischemia-reperfusion injury. This study further defines ubiquitin’s anti-inflammatory properties, and suggests that it could be used therapeutically to improve function of postischemic lungs.

Recent data suggest that ubiquitin (Ub) is systemically released after trauma, has pleiotropic effects on host defense mechanisms, and that Ub administration reduces fluid shifts into tissues during inflammation. Ub release after burns (B) has not been studied and its association with injury severity and outcome after blunt trauma (T) is unknown. Thus, we evaluated Ubs association with injury severity and outcomes after B and T. Injury severity was assessed with the Injury Severity Score (ISS) in T and burn size (% total body surface area, %TBSA) in B. A total of 129 T (ISS: 26 +/- 13) and 55 B (46% +/- 18% TBSA) were observed for sepsis/multiple organ failure (MOF) and survival. In B, sequential organ failure assessment scores were documented daily. Fifty volunteers served as controls (C) Ub serum levels were measured on day 0 (admission), 1, 3, 5, and 7 by enzyme-linked immunosorbent assay. Data were analyzed using bivariate or partial correlation analyses, t test, and analysis of variance with Tukey post-hoc test for multiple comparisons (two-tailed p < 0.05). Ub was significantly elevated in patients. Peak levels (ng/mL) were detectable on day 0 (C: 118 +/- 76; T: 359 +/- 205; B: 573 +/- 331) and increased with increased ISS, %TBSA, and presence of inhalation injury. In T, Ub normalized by day 3, but remained elevated in B. In B, Ub correlated significantly negative with sequential organ failure assessment scores (r: -0.143; p = 0.0147), sepsis/MOF development (r: -0.363; p = 0.001), and survival (r: -0.231; p = 0.009). Compared with B who recovered uneventfully, Ub levels were significantly lower on days 1 to 7 and on days 5/7 in B who developed sepsis/MOF or died, respectively. Ub concentrations reflect the extent of tissue damage. Along with Ubs previously described anti- inflammatory properties, this study suggests that its systemic release is protective, that burn patients who develop sepsis/MOF have a relative Ub deficiency and that Ub could play an important role during the physiologic response to burn injury.

Recently, it was shown that exogenous ubiquitin has anti-inflammatory actions in vivo and that the ubiquitin-decapeptide 50–59 has immunosuppressive effects similar to cyclosporine. Immunosuppressive effects of the native ubiquitin molecule and its therapeutic potential in transplantation are unknown. We tested the hypothesis that ubiquitin inhibits alloreactivity and increases allograft survival in a murine model of skin transplantation in fully mismatched strain combinations (C3H/HEJ-DBA2). Ubiquitin dose-dependently inhibited mixed leukocyte reaction in C3H/HEJ splenocytes in vitro. Intraperitoneal ubiquitin administration (25 μg/h for 14 days) was well-tolerated, dose-dependently increased ubiquitin serum concentrations and median allograft survival from 10 days (with albumin; control) to 17 days in DBA2 mice (survival ratio: 1.7, 95% confidence interval: 1.266–2.134; P=0.0005). The in vivo effects in this study combined with our previous work strongly indicate that ubiquitin is a potent immune modulator with broad therapeutic potential. Ubiquitin treatment could be a novel strategy to improve immunosuppressive regimens in transplantation.

Recent findings suggest that extracellular ubiquitin has pleiotropic effects on host defence mechanisms, but its cellular mechanism of action is not yet understood. Using fluorescence and in vivo confocal microscopy, we observed uptake of N‐terminal fluorescein‐labelled ubiquitin into human PBMC and MonoMac 6 cells. Immunoblotting experiments indicated that extracellular ubiquitin is then rapidly conjugated to a multitude of intracellular proteins. LPS and lipoteichoic acid significantly increased uptake and subsequent conjugation to intracellular proteins dose dependently. This mechanism showed saturation kinetics with a K _d value for ubiquitin in the low nanomolar range (B _max value of 0.14–0.27 μmol ubiquitin/mg protein. These results suggest that the monocytic ubiquitin system can be targeted with physiologically relevant concentrations of extracellular ubiquitin during inflammation. This concept could provide a simple explanation for a multitude of extracellular ubiquitin's actions and open up new strategies to influence ubiquitin‐dependent intracellular processes.

Recent studies suggest that extracellular ubiquitin could have a physiologic role in immunodepression in sepsis and trauma. The therapeutic potential of exogenous ubiquitin after trauma has not been examined. To fill this gap, we designed a series of experiments in a clinically relevant trauma model. Forty minutes after femur fractures and hemorrhage, swine received 1.3 mg of ubiquitin per kilogram or bovine serum albumin intravenously followed by fluid resuscitation to maintain systemic hemodynamics. Leukocyte function and the immunomodulatory capacity of serum were assessed measuring endotoxin-evoked tumor necrosis factor-α (TNF α) production ex vivo. TNF α and ubiquitin were quantified with enzyme-linked immunosorbent assay. Intravenous ubiquitin had no significant hemodynamic effect in normal animals. After injury, ubiquitin significantly reduced fluid requirements by at least 60% (p < 0.05). The injury was associated with transient immunodepression, as reflected by reduced endotoxin-evoked TNF α production by 40% to 50%. With ubiquitin, this response remained depressed for 100 to 160 minutes (p < 0.05), but fully recovered to baseline with albumin. Ubiquitin is apparently safe and effective for reducing fluid requirements as a measure of diffuse capillary leak. This immunomodulatory property suggests a new therapeutic approach after injury in particular, and for infectious and noninfectious inflammation in general.

Ubiquitin is suggested to play a key role in essential intracellular functions, such as heat shock response, protein breakdown, and regulation of immune responses. Ubiquitin has also been detected in the extracellular space, but the function and biologic significance is unclear. We describe a new function of extracellular ubiquitin and show that extracellular ubiquitin specifically inhibits ex vivo secretion of tumor necrosis factor-α (TNF-α) and TNF-α mRNA expression from peripheral blood mononuclear cells (PBMNCs) in response to endotoxin in a dose-dependent manner. In contrast, the TNF-α response to zymosan or Staphylococcus aureus as well as the interleukin-6 (IL-6) and IL-8 responses to endotoxin were unaffected by ubiquitin. Measurement of serum ubiquitin levels showed a significant 5- to 7-fold increase in sepsis and trauma patients, to the level required for inhibition of the PBMNC TNF-α response to endotoxin by ubiquitin. Elevated ubiquitin levels in serum were significantly correlated with a reduced TNF-α production. Antibodies to ubiquitin were able to (1) significantly increase (2- to 5-fold) the TNF-α response to endotoxin in whole blood from trauma and sepsis patients, (2) completely neutralize the inhibitory effect of trauma patients' serum on healthy donors' TNF-α production, and (3) partially neutralize the inhibitory effect of sepsis patients' serum on healthy donors' TNF-α production. Ubiquitin-depleted serum from trauma patients lost the inhibitory activity for TNF-α production, whereas extracted endogenous ubiquitin exerts the inhibitory activity. The results demonstrate that extracellular ubiquitin acts as a cytokinelike protein with anti-inflammatory properties and indicate that extracellular ubiquitin is involved in the regulation of immunodepression in critical illness.

The aim of this study was to evaluate whether the -174 G/C promoter polymorphism of the interleukin-6 (IL-6) gene is associated with the ex vivo, whole blood IL-6 response to endotoxin with the development of severe sepsis in severely injured, blunt trauma patients. Patients with a severe trauma and an injury severity score of 16 were included in the study. The IL-6 -174 G/C promoter polymorphism was determined by real-time polymerase chain reaction (PCR) assay using specific fluorescence-labelled hybridisation probes. Whole blood of the patients was stimulated with endotoxin and the IL-6 concentrations were measured by ELISA. There was no association between the IL-6 -174 genotypes and the ex vivo, stimulated IL-6 response: 25% of the patients developed severe sepsis later in the clinical course. These patients had higher IL-6 concentrations following whole blood stimulation on day 1 (p = 0.046) after the trauma than patients with uncomplicated post-traumatic recovery. The difference was even more significant on day 2 after the trauma (p = 0.02). High IL-6 responses in a whole blood stimulation assay with endotoxin on days 1 and 2 after a trauma are associated with severe post-traumatic sepsis. Genotyping for the IL-6 -174 G/C polymorphism does not allow early identification of trauma patients with a high, ex vivo IL-6 synthesis capacity.