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Immune dysfunction in trauma and sepsis: Novel aspects of ubiquitin

Immune dysfunction in trauma and sepsis: Novel aspects of ubiquitin. Matthias Majetschak, MD, PhD DeWitt Daughtry Family Department of Surgery Div. of Trauma and Surgical Critical Care. Ubiquitin. Immune dysfunction in trauma and sepsis: Novel aspects of ubiquitin. Immune dysfunction

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Immune dysfunction in trauma and sepsis: Novel aspects of ubiquitin

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  1. Immune dysfunction in trauma and sepsis: Novel aspects of ubiquitin Matthias Majetschak, MD, PhD DeWitt Daughtry Family Department of Surgery Div. of Trauma and Surgical Critical Care

  2. Ubiquitin Immune dysfunction in trauma and sepsis: Novel aspects of ubiquitin Immune dysfunction Ubiquitin - intracellular: the traditional view Ubiquitin – extracellular: novel aspects systemic and local release immunological actions clinical relevant effects possible mechanism

  3. Immune dysfunction in critical illness Mediator dependent Promotes development of sepsis & multiple organ failure (MOF) Marshall Crit Care Med 2001, Adrie et al. Intensive Care Med 2000; Hack et al. Adv Immunol 1997 Sepsis remains one of the leading causes of mortality in critically ill patients Martin et al., N Engl J Med 2003; Vincent et al. Clin Infect Dis 2002; Bone Ann Intern Med 1996 25 – 35 % of severely injured blunt trauma patients develop sepsis/multiple organ failure (MOF) Regel et al. World J Surg 1996; Nast-Kolb et al. J.Trauma 2001; Sauaia et al. J Trauma 1998

  4. Immune dysfunction Majetschak et al., J Trauma 1997 Ditschkowski et al., Ann Surg 1999 LPS evoked whole blood TNF production * * * * * Days after trauma  ISS < 16 (n = 11) ISS > 16, uncomplicated (n = 46)  ISS > 16, severe sepsis (n = 20)  : uninjured, n = 18  : trauma, ISS > 16, n = 18-22

  5. Immune dysfunction – mediator dependent Normal PBMNC Trauma PBMNC day 0 (control) day 0 day 14 (control) day 0 day 14 Majetschak et al. Crit Care Med 2000

  6. Mediator release - trauma Pro – inflammatory Anti-inflammatory Systemic level Systemic level sIL2-R sTNF-R IL-1: not detectable IL-2: not detectable IL-1-RA IL-6 IL-10 IL-8 TGF TNF normal range normal range Days after trauma Days after trauma Hoch et al. (1993); Rabinovici et al. (1993); Svoboda et al. (1994); Cinnat et al. (1994/1995); Ertel et al. (1995); Neidhardt et al. (1997); Nast-Kolb et al. (1997); Gebhard et al. (2000); Majetschak et al. (2000a/b).

  7. Immune dysfunction – mediator dependent BUT: Most of the mediators did not correlate with - alterations of cellular immune functions - inhibitory serum activity Neutralization of any one of the mediators did not reverse effects on cellular immune responses Highly redundant system, multiple factors involved Unknown factors that regulate cell function

  8. Ubiquitin • 76 amino acids • (8.5 kDa) • heat stable • highly conserved • present in all • eukaryotic cells

  9. Intracellular:UBIQUITIN-PROTEASOME PATHWAY ATP E1 E2 E3 Target Protein Target Protein AMP + PPi Ub(n) Target Protein DUB ATP Proteasome AMP + PPi Ub P P E T I D E S Intracellular: UBIQUITIN-PROTEASOME PATHWAY Ub PubMed: 14,932 citations as of 04/2006 2nd most common posttranslational protein modification following phosphorylation

  10. The Nobel Prize in Chemistry 2004 "for the discovery of ubiquitin-mediated protein degradation" Aaron Ciechanover Israel Avram Hershko Israel Irwin Rose USA

  11. EXTRACELLULAR (PubMed: < 20 citations as of 04/2006) Ubiquitin ● present in normal plasma/serum, urine, CSF ●elevated plasma/serum levels: parasitic infectionsAsseman et al. J Immunol Methods 1994alcoholic liver cirrhosisTakagi et al. Alcohol Clin Exp Res 1999type 2 diabetesAkarsu et al. Diabetes Care 2001hairy cell leukemiaDaino et al. Blood 2000renal failure/hemodialysisOkada et al. Clin Chim Acta 1993 Akarsu et al. Nephron 2001 ● elevated CSF levels: M. AlzheimerWang et al. Neuropathol 1991Creutzfeldt-JakobManaka et al. Neurosci Lett 1992

  12. ? Ubiquitin EXTRACELLULAR ACTIONS ● lymphocyte differentiating properties Goldstein et al. Proc Natl Acad Sci USA 1975 ● inhibits platelet activities and IgG production Pancre et al. Eur J Immunol 1991; Nakamura et al. J Immunol 1996 ● inhibits growth and induces apoptosis Daino et al. Blood 2000 ● antimicrobial activities Kieffer et al. FASEB J 2004 ● immunomodulation in critical illness Majetschak et al. Blood 2003

  13. max 75th perc median 25th perc min V Trauma Day 0 start Ubiquitin in serum / urine - trauma / sepsis - 97.0 66.0 kDa 45.0 30.0 20.1 14.4 Ub 15 10 15 20 25 Ub front µg / lane Majetschak et al. Blood 2003

  14. max 75th perc median 25th perc min Ubiquitin in cerebrospinal fluid (CSF) after traumatic brain injury (TBI) TBI Day 0 TBI Day 5 Ctrl. Ub n = 14 n = 10 Majetschak et al. Crit Care Med 2005

  15. Ubiquitin levels on hospital admission Trauma – Serum TBI – CSF * *

  16. Serum ubiquitin – time course Trauma ISS > 16 Burns (> 2O, > 20% TBSA) n = n = normal range normal range

  17. CSF ubiquitin – time course in TBI patients Died Survived Majetschak et al. Crit Care Med 2005

  18. Ubiquitin release - hemolysis experimental TBI, swine : CSF with visible hemolysis (n = 4) : clear CSF (n = 3) Majetschak et al. Crit Care Med 2005

  19. Ubiquitin release - hemolysis : in-vitro erythrolysis (n = 3) : human CSF samples Ubiquitin in plasma from pRBC units (n = 3, ng/mL) Days of pRBC storage 17±6 ng /106 lysed human erythrocytes (81±34 mg/mL blood) 18±5 ng /106 lysed porcine erythrocytes (94±25 mg/mL blood) Patel et al. J. Surg Res 2006 Majetschak et al. Crit Care Med 2005

  20. Endogenous ubiquitin serum levels correlate with LPS evoked TNFa responses - Trauma patients - Majetschak et al. Blood 2003

  21. Anti-ubiquitin antibodies ● neutralize the inhibitory activity of trauma patients serum on LPS evoked TNFa production normal serum trauma serum Majetschak et al. Blood 2003

  22. Anti-ubiquitin antibodies: ● stimulate LPS evoked TNFa production in trauma/sepsis patients ● have no effects in healthy volunteers Majetschak et al. Blood 2003

  23. Anti-ubiquitin affinity chromatography - Trauma Serum - Majetschak et al. Blood 2003

  24. Exogenous ubiquitin inhibits ex-vivo LPS induced TNFa production TNFa secretion TNFa mRNA expression Majetschak et al. Blood 2003

  25. Exogenous ubiquitin inhibits ex-vivo LPS induced IL-8 production - + - + ubiquitin (2 mg/mL) ubiquitin (2 mg/mL) Patel et al. J. Surg Res 2006

  26. EX-VIVO LPS EVOKED TNFa RESPONSE AFTER IN-VIVO UBIQUITIN Majetschak et al. Surgery 2004

  27. EFFECT OF SERUM ON LPS EVOKED WHOLE BLOOD TNFa PRODUCTION - in-vivo ubiquitin - * : p < 0.05 vs. t = 0 min (ANOVA)

  28. clinical relevant effects ? Ubiquitin inhibits leukocyte function in-vitro and in-vivo

  29. Exogenous ubiquitin in-vivo Swine, n = 4; 1.3 mg/kg ubiquitin i.v. - no significant effects of ubiquitin on ● hemodynamics ● pulmonary function ● electrolytes, lactate or glucose levels ● leukocyte counts - no fluid requirement

  30. Porcine model of endotoxic shock prepubertal swine, anaesthetized (ketamin/fentanyl) mechanically ventilated (FiO2: 0.5) fluid resuscitation (Lactated Ringer`s): MAP < 69 mmHg ● Ubiquitin pre-treatment (n = 6; 1.3 mg/kg at t = -15 min) ● Ubiquitin post-treatment (n = 6; 1.3 mg/kg at t = 45 min) ● Control: BSA (n = 18; 1.3 mg/kg at t = -15 min/45 min) pre post LPS 1.5 µg/kg Majetschak et al. Surgery 2004

  31. Ubiquitin serum levels

  32. Fluid requirements * : p < 0.05 vs. ctrl (ANOVA)

  33. Edema and erythema formation Ubiquitin pre-treatment control (BSA) t = 180 min

  34. Oxygenation * : p < 0.05 vs. ctrl (ANOVA)

  35. Mortality

  36. Porcine models of severe trauma and traumatic brain injury (TBI) ● Ubiquitin – n = 5; 1.3 mg/kg, initial IV bolus prior to fluid resuscitation ● Control: BSA – n = 5; 1.3 mg/kg, initial IV bolus prior to fluid resuscitation i.v. ubiquitin or i.v. BSA randomized, blinded Fx or TBI RESUSCITATION HEM Earle et al. Surgery 2005 Majetschak et al. J Trauma 2004

  37. HEM RESUSCITATION - Severe Trauma / TBI - Fluid requirements Fx TBI HEM RESUSCITATION * : p < 0.05 (ANOVA)

  38. - Traumatic Brain Injury - Pulmonary function & oxygenation TBI TBI RESUSCITATION HEM RESUSCITATION HEM * : p < 0.05 (ANOVA)

  39. - Traumatic Brain Injury - Intracranial pressure TBI RESUSCITATION HEM * : p < 0.05 (ANOVA)

  40. Ubiquitin • 10 20 30 40 50 60 70 76 • MQIFVKTLTG KTITLEVEPS DTIENVKAKI QDKEGIPPDQ QRLIFAGKQL EDGRTLSDYN IQKESTLHLV LRLRGG 50 59 L EDGRTLSDY Immunosuppressive potency similar to cyclosporin Szewczuk et al. Biopolymers 2004

  41. Effect of ubiquitin on mixed leukocyte reaction (MLR) One-way MLR C3H/HEJ mouse splenocytes to g-irradiated DBA2 splenocytes Earle et al. Transplantation, submitted

  42. Ubiquitin prolongs allogeneic skin graft survival C3H/HEJ – DBA2 mice allogeneic syngeneic graft (C3H/HEJ) graft (DBA2) Ub Alb Earle et al. Transplantation, submitted

  43. possible mechanism ? Extracellular ubiquitin • inhibits leukocyte function in-vivo and in-vitro • reduces fluid shifts/capillary leak • - endotoxic shock • - trauma • prolongs allogeneic skin graft survival

  44. Ubiquitin – uptake into monocytes - ubiquitin + ubiquitin bright field lex488 nm / lem 530 nm Majetschak et al. Immunol Cell Biol 2006

  45. Fate of extracellular ubiquitin following uptake into monocytes (MonoMac 6) Conjugation to intracellular target proteins • - + + + + + + Ubb LTA [ng/mL] 0 100 0 1 3 30 100 300 Ubb kDa 97.4 58.1 39.8 29 20.1 14.3 Ub ERK1/2 Majetschak et al. Immunol Cell Biol 2006

  46. Ubiquitin – uptake into monocytes increased in inflammatory conditions (LPS / LTA) shows saturation kinetics (Kd: 6 – 9 nM) Majetschak et al. Immunol Cell Biol 2006

  47. Ubiquitin – uptake and conjugation to intracellular proteins Does it make sense? 1. Equilibrium in physiologic baseline conditions: essential for cell viability tightly regulated PBMNC (n = 10) Muscle, heart, lung, liver, spleen, kidney (n = 5 each) Ub-P Ub Ponelies et al. Shock 2005 Patel & Majetschak, Physiol Res, submitted

  48. Ubiquitin – uptake and conjugation to intracellular proteins Does it make sense? kDa Ub-P Ub-P 250 160 105 Ub Ub 75 50 Ub-protein conjugates  35 30 25 15 10 free Ub — Ctrl. — 2. Altered ubiquitin equilibrium during sepsis: Reduced conjugates SEPSIS sepsishealthy Ponelies et al. Shock 2005

  49. Ubiquitin – uptake and conjugation to intracellular proteins Does it make sense? 3. Monocytes contain 10 - 15 fg free ubiquitin / cell Up to 10 fg free ubiquitin/cell can be taken up Could shift equilibrium towards conjugate formation Ub-P Ub-P Counteract further decrease in ubiquitin protein conjugates SEPSIS Law of mass action Ub Ub Ub

  50. Inflammation Trauma - Sepsis Receptor E1,E2,E3 Ub(n) P P reversible modification proteasomal degradation inhibition of pro-inflammatory responses modulation of cell function maintenance of ubiquitin homeostasis endothelium tissue Ub Ub Ub Ub Ub lumen

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