Extracellular ubiquitin inhibits the TNF-α response to endotoxin in peripheral blood mononuclear cells and regulates endotoxin hyporesponsiveness in critical illness by Matthias Majetschak, Ulrich Krehmeier, Mark Bardenheuer, Christof Denz, Michael Quintel, Gregor Voggenreiter, and Udo Obertacke Blood Volume 101(5):1882-1890 March 1, 2003 ©2003 by American Society of Hematology

Exogenous ubiquitin inhibits TNF-α secretion of blood and PBMNCs Exogenous ubiquitin inhibits TNF-α secretion of blood and PBMNCs.(A) Dose-dependent inhibition of TNF-α secretion of human whole blood by exogenous ubiquitin. Exogenous ubiquitin inhibits TNF-α secretion of blood and PBMNCs.(A) Dose-dependent inhibition of TNF-α secretion of human whole blood by exogenous ubiquitin. Whole-blood cultures (in duplicates) from healthy donors (n = 13-18) were incubated for 4 hours with 0 to 1 μg/mL exogenous ubiquitin in the presence of 100 ng/mL LPS. Data represent means ± SEM. *P < .05 versus cultures without ubiquitin. (B) Dose-dependent inhibition of TNF-α secretion of human PBMNCs by exogenous ubiquitin. PBMNC cultures (in duplicates) from healthy donors (n = 10-15) were incubated for 4 hours with 0 to 1 μg/mL exogenous ubiquitin in the presence of 100 ng/mL LPS. Data represent means ± SEM. *P < .05 versus cultures without ubiquitin. (C) Kinetics of the LPS-stimulated TNF-α secretion of human whole blood in the presence of 0 (■), 500 ng/mL (▪), and 1000 ng/mL (●) exogenous ubiquitin. Cultures (in duplicates) were incubated for 2, 4, 8, and 16 hours. (D) Kinetics of the LPS-stimulated TNF-α secretion of 105 human PBMNCs in the presence of 0 (■), 500 ng/mL (▪), and 1000 ng/mL (●) exogenous ubiquitin. Cultures (in duplicates) were incubated for 2, 4, 8, and 16 hours. (E) TNF-α mRNA levels in human PBMNCs stimulated with 100 ng/mL LPS in the presence of 0, 500, and 1000 ng/mL ubiquitin for 2 hours. *P < .05 versus stimulation without ubiquitin. (F) Dose-dependent inhibition of TNF-α secretion of porcine (▪) and murine ( ) whole blood by exogenous ubiquitin. Whole-blood cultures (in duplicates; n = 3) were incubated for 4 hours with 0 to 1 μg/mL exogenous ubiquitin in the presence of 100 ng/mL (porcine) and 1 μg/mL (murine) LPS. Data represent means ± SEM. *P < .05 versus cultures without ubiquitin. Matthias Majetschak et al. Blood 2003;101:1882-1890 ©2003 by American Society of Hematology

Effect of exogenous ubiquitin on LPS-induced TNF-α, IL-6, and IL-8 secretion of human whole blood and PBMNCs.Whole blood (▪) and PBMNC ( ) cultures (in duplicates) from healthy donors (n = 3) were incubated for 4 hours and 24 hours with 0 to 1 μg/mL exogeno... Effect of exogenous ubiquitin on LPS-induced TNF-α, IL-6, and IL-8 secretion of human whole blood and PBMNCs.Whole blood (▪) and PBMNC ( ) cultures (in duplicates) from healthy donors (n = 3) were incubated for 4 hours and 24 hours with 0 to 1 μg/mL exogenous ubiquitin in the presence of 100 ng/mL LPS. Data represent means ± SEM. *P < .05 versus cultures without ubiquitin. TNF-α/IL-6/IL-8 production (% control) is percent of TNF-α/IL-6/IL-8 secretion in cultures without exogenous ubiquitin. (A) TNF-α secretion, 4 hours of incubation. (B) TNF-α secretion, 24 hours of incubation. (C) IL-6 secretion, 4 hours of incubation. (D) IL-6 secretion, 24 hours of incubation. (E) IL-8 secretion, 4 hours of incubation. (F) IL-8 secretion, 24 hours of incubation. Matthias Majetschak et al. Blood 2003;101:1882-1890 ©2003 by American Society of Hematology

Detection of ubiquitin in serum and urine Detection of ubiquitin in serum and urine.(A) Ubiquitin serum and urine concentrations in healthy volunteers and critically ill patients. Detection of ubiquitin in serum and urine.(A) Ubiquitin serum and urine concentrations in healthy volunteers and critically ill patients. The boxes extend from the 25th to 75th percentile; the horizontal line shows the median. Error bars show the range of data. Data are measurements of ubiquitin concentrations in serum samples from 35 healthy uninjured donors, 23 severely injured blunt trauma patients on the day of admission, and 24 patients with sepsis. Ubiquitin urine concentrations were determined in specimens from 19 sepsis patients and 10 healthy individuals. *P < .05 versus concentrations in specimens from healthy volunteers. (B) Detection of free ubiquitin in serum specimen by immunoblotting. Serum proteins were separated by SDS-PAGE, transferred to PVDF membranes, and probed for ubiquitin with antiubiquitin AS (1:200). Lane 1, healthy donors' serum (15 μg); lanes 2-5, patients' serum (lane 2, 10 μg; lane 3, 15 μg; lane 4, 20 μg; lane 5, 25 μg); lane 6, ubiquitin (Ub; 10 ng). (C) Detection of free ubiquitin in urine specimen (10 μL) by immunoblotting. Lane 1, ubiquitin (Ub; 5 ng); lane 2, healthy donors' specimen; lanes 3 and 4, patients' specimens. Matthias Majetschak et al. Blood 2003;101:1882-1890 ©2003 by American Society of Hematology

Comparison of ubiquitin serum levels with LPS-stimulated whole blood TNF-α production and neutralization of the inhibitory activity for TNF-α production in patients' serum with antiubiquitin antibody.(A) Ubiquitin serum concentrations in volunteers (n = 12)... Comparison of ubiquitin serum levels with LPS-stimulated whole blood TNF-α production and neutralization of the inhibitory activity for TNF-α production in patients' serum with antiubiquitin antibody.(A) Ubiquitin serum concentrations in volunteers (n = 12) and trauma patients (n = 10) during 14 days after trauma. Data represent means ± SEM. (B) LPS-induced whole blood TNF-α secretion. Volunteers and trauma patients are the same as described in panel A. Whole blood cultures were incubated for 4 hours in the presence of 100 ng/mL LPS. Data represent means ± SEM. (C-D) Antiubiquitin antibodies neutralize the inhibitory activity of ubiquitin on LPS-induced whole blood (C) and PBMNCs (D) TNF-α production. TNF-α production (%) indicates percent of the TNF-α secretion in cultures without exogenous ubiquitin and without antibodies. Data represent means ± SEM from 3 different whole blood and PBMNC cultures obtained from healthy volunteers. Cultures without (■) or with 500 ng/mL exogenous ubiquitin (▪) in the presence of LPS (100 ng/mL for 4 hours). Control indicates cultures without addition of antibody. AS: antiubiquitin AS diluted 1:103, 1:102, and 1:10 in the cell cultures. UbP4D1 and UbN-19: diluted 1:103 in the cell cultures. (E-F) Effect of antiubiquitin antibody on the inhibitory activity of trauma (E) and sepsis (F) patients' serum on LPS-induced TNF-α production of whole blood and PBMNCs. Whole blood (■) and PBMNCs (▪) were cultured with 100 ng/mL LPS for 4 hours. TNF production (%) indicates percent of the TNF-α secretion in cultures containing additional healthy volunteers' serum (30%, vol/vol, in the cell culture mixture) without antibodies. Data represent means ± SEM from 4 different cultures obtained from healthy volunteers. C indicates control, healthy volunteers' serum; TS, trauma patients' serum (n = 4, 30%, vol/vol, in the cell culture mixture); SS, sepsis patients' serum (n = 4, 30%, vol/vol, in the cell culture mixture). Antiubiquitin AS diluted 1:102 and 1:10 in the cell cultures. UbP4D1 and UbN19 were diluted 1:103 in the cell cultures. Matthias Majetschak et al. Blood 2003;101:1882-1890 ©2003 by American Society of Hematology

Endogenous ubiquitin regulates the inhibitory activity for TNF-α production in trauma patients' serum.(A) Trauma patients' serum was applied to an antiubiquitin antibody column and the adsorbed protein was eluted by acidification. Endogenous ubiquitin regulates the inhibitory activity for TNF-α production in trauma patients' serum.(A) Trauma patients' serum was applied to an antiubiquitin antibody column and the adsorbed protein was eluted by acidification. Run-through and elutions were collected and tested for inhibitory activity of LPS-induced TNF-α production in healthy donors' whole blood. Whole blood cultures were incubated with the fractions (50%, vol/vol, in the cell culture mixtures) obtained by affinity chromatography in the presence of LPS for 4 hours in a constant volume of 200 μL. Data (percent control) are means ± SD of the TNF-α secretion in the cell culture supernatants from 2 experiments (in duplicates). C indicates control cell culture in the presence of 25% additional volunteers' serum in a constant volume of 200 μL; TS, cell culture in the presence of 25% trauma patients' serum in a constant volume of 200 μL; RT, cell cultures containing the run-through fraction. pH 7 to pH 3, cell cultures containing the eluted fractions. (B) Immunoblot analysis of the fractions obtained by antiubiquitin affinity chromatography. Fractions were separated by SDS-PAGE, transferred to PVDF membranes, and probed for ubiquitin with antiubiquitin AS (1:200; lanes 1-4) and monoclonal UbP4D1 (1:500; lanes 5-8). Lane 1, patients' serum, 10 μg; lane 2, run-through, 20 μg; lane 3, pH 3/4 eluate, 20 μL; lane 4, ubiquitin, 10 ng; lane 5, patients' serum, 50 μg; lane 6, run-through, 50 μg; lane 7, pH3/4 eluate, 200 μL, pH3/4 eluate 10-fold concentrated by boiling; lane 8, ubiquitin 80 ng. Matthias Majetschak et al. Blood 2003;101:1882-1890 ©2003 by American Society of Hematology

Antiubiquitin antibodies restore reduced TNF-α–producing capacities in blood from trauma and sepsis patients.(A-B) Effect of antiubiquitin antibodies on LPS-induced TNF-α secretion in blood of multiply-injured (A) and sepsis (B) patients. Antiubiquitin antibodies restore reduced TNF-α–producing capacities in blood from trauma and sepsis patients.(A-B) Effect of antiubiquitin antibodies on LPS-induced TNF-α secretion in blood of multiply-injured (A) and sepsis (B) patients. Whole blood was incubated with LPS (100 ng/mL) for 4 hours. AS indicates antiubiquitin AS diluted 1:102 and 1:10 in the cell cultures; UbP4D1 and Ub N19, diluted 1:103 in the cell cultures; volunteers, whole blood cultures from healthy donors; TNF production (%), percent of the TNF-α secretion in trauma (A) and sepsis (B) patients' whole blood incubated without antibodies. Values are mean ± SEM from 5 healthy donors, 5 trauma patients, and 5 sepsis patients. (C) Effect of antiubiquitin AS (1:10) on LPS-induced TNF-α secretion in blood of uninjured donor and trauma and sepsis patients. Individual values from panels A and B. Data are pg/mL TNF-α. Matthias Majetschak et al. Blood 2003;101:1882-1890 ©2003 by American Society of Hematology