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

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Presentation on theme: "Immune dysfunction in trauma and sepsis: Novel aspects of ubiquitin Matthias Majetschak, MD, PhD DeWitt Daughtry Family Department of Surgery Div. of Trauma."— Presentation transcript:

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

3 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 Immune dysfunction in critical illness

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

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

6 Mediator release - trauma Days after trauma Systemic level Pro – inflammatory Anti-inflammatory IL-1: not detectable IL-2: not detectable IL-6 IL-8 TNF sIL2-R sTNF-R IL-1-RA IL-10 TGF  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). normal range

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

9 Intracellular: UBIQUITIN-PROTEASOME PATHWAY PubMed: 14,932 citations as of 04/2006 Ub P E P T I D E S Target Protein E1 E2 E3 ATP AMP + PP i Ub (n) Target Protein Proteasome ATP AMP + PP i Ub Target Protein DUB Intracellular:UBIQUITIN-PROTEASOMEPATHWAY 2 nd 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) ● present in normal plasma/serum, urine, CSF Ubiquitin ● elevated plasma/serum levels: parasitic infections Asseman et al. J Immunol Methods 1994 alcoholic liver cirrhosis Takagi et al. Alcohol Clin Exp Res 1999 type 2 diabetes Akarsu et al. Diabetes Care 2001 hairy cell leukemia Daino et al. Blood 2000 renal failure/hemodialysis Okada et al. Clin Chim Acta 1993 Akarsu et al. Nephron 2001 ● elevated CSF levels: M. Alzheimer Wang et al. Neuropathol 1991 Creutzfeldt-Jakob Manaka et al. Neurosci Lett 1992

12 ? EXTRACELLULARACTIONS ● 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 Ubiquitin

13 97.0 66.0 45.0 30.0 20.1 14.4 kDa 1510152025Ub front start Ub V Trauma Day 0 µg / lane Ubiquitin in serum / urine - trauma / sepsis - Majetschak et al. Blood 2003 min max 25 th perc 75 th perc median

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

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

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

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)

19 Ubiquitin release - hemolysis Patel et al. J. Surg Res 2006 Majetschak et al. Crit Care Med 2005  : 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 /10 6 lysed human erythrocytes (81±34  g/mL blood) 18±5 ng /10 6 lysed porcine erythrocytes (94±25  g/mL blood)

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

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

22 Anti-ubiquitin antibodies: ● stimulate LPS evoked TNF  production in trauma/sepsis patients ● have no effects in healthy volunteers

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

24 Exogenous ubiquitin inhibits ex-vivo LPS induced TNF  production TNF  secretionTNF  mRNA expression Majetschak et al. Blood 2003

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

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

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

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

29 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 Exogenous ubiquitin in-vivo

30 prepubertal swine, anaesthetized (ketamin/fentanyl) mechanically ventilated (FiO2: 0.5) fluid resuscitation (Lactated Ringer`s): MAP < 69 mmHg prepost ● 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) LPS 1.5 µg/kg Porcine model of endotoxic shock 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 ● 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 Porcine models of severe trauma and traumatic brain injury (TBI) Earle et al. Surgery 2005 Majetschak et al. J Trauma 2004 HEM RESUSCITATION i.v. ubiquitin or i.v. BSA Fx or TBI randomized, blinded

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

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

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

40 1 10 20 30 40 50 60 70 76 MQIFVKTLTG KTITLEVEPS DTIENVKAKI QDKEGIPPDQ QRLIFAGKQL EDGRTLSDYN IQKESTLHLV LRLRGG 50 59 L EDGRTLSDY Szewczuk et al. Biopolymers 2004 Immunosuppressive potency similar to cyclosporin Ubiquitin

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

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

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

44 bright field ex  488 nm / em 530 nm Ubiquitin – uptake into monocytes Majetschak et al. Immunol Cell Biol 2006 - ubiquitin+ ubiquitin

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

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

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

48 250 160 105 75 50 35 30 25 15 10 kDa free Ub — Ub-protein conjugates  Ctrl. — sepsis healthy Ubiquitin – uptake and conjugation to intracellular proteins Does it make sense? 2. Altered ubiquitin equilibrium during sepsis: Reduced conjugates Ub Ub-P Ub Ub-P SEPSIS 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 Ub Ub-P Ub Ub-P SEPSIS Law of mass action Could shift equilibrium towards conjugate formation Counteract further decrease in ubiquitin protein conjugates

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

51 Ubiquitin - systemic and local release after trauma/sepsis - continuous increase in CSF after TBI lethal outcome - endogenous immune modulator - broad therapeutic potential - 76 amino acids (8.5 kDa) - heat stable - highly conserved - present in all eukaryotic cells - ubiquitin-proteasome pathway

52 Acknowledgement University of Miami Lissette T. Busby Mayur B. Patel Steven A. Earle David R. King Kenneth G. Proctor Steven M. Cohn University of Heidelberg Norbert Ponelies Thomas Hirsch Ulrich Krehmeier Udo Obertacke University of Berlin Arwed Hosstmann Andreas Oberholzer Wolfgang Ertel University of Munich Siegfried Zedler Eugen Faist Supported by: DFG (German Science Foundation) MA 2474/1-1, MA 2474/2-1, MA 2472/2-2

53 “Finally, I believe ubiquitin is a ‘lucky’ molecule. Almost everyone who has studied it has made fascinating observations.” Martin Rechsteiner, 1988


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