immune dysfunction in trauma and sepsis: novel aspects of ubiquitin matthias majetschak, md, phd...

Immune dysfunction in trauma and sepsis:Novel aspects of ubiquitin

Matthias Majetschak, MD, PhD

DeWitt Daughtry Family Department of SurgeryDiv. of Trauma and Surgical Critical Care

Immune dysfunction

Ubiquitin - intracellular: the traditional view

Ubiquitin – extracellular: novel aspectssystemic and local release

immunological actions clinical relevant effects

possible mechanism

Ubiquitin

Immune dysfunction in trauma and sepsis:Novel aspects of ubiquitin

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

Ditschkowski et al., Ann Surg 1999

ISS < 16 (n = 11) ISS > 16, uncomplicated (n = 46)

ISS > 16, severe sepsis (n = 20)

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

0

500

1000

1500

2000

2500

3000

3500

TN

F (

U/m

L)

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

**

** *

Immune dysfunction – mediator dependent

normal serum trauma serum trauma serum0

10

20

30

40

50

60

70

80

90

100

110

120

LP

S e

voke

d T

NF

pro

du

ctio

n(%

co

ntr

ol)

normal serum trauma serum trauma serum0

10

20

30

40

50

60

70

80

90

100

110

120

LP

S e

voke

d T

NF

pro

du

ctio

n(%

co

ntr

ol)

day 0 day 14 day 0 day 14

Normal PBMNC Trauma PBMNC day 0

Majetschak et al. Crit Care Med 2000

(control) (control)

0 1 2 3 4 5 6 7 8 9 10 11 12 13 140 1 2 3 4 5 6 7 8 9 10 11 12 13 14

Mediator release - trauma

Days after trauma Days after trauma

Systemic level Systemic level

Pro – inflammatory Anti-inflammatory

IL-1: not detectableIL-2: not detectable

IL-6

IL-8

TNF

sIL2-R

sTNF-R

IL-1-RA

IL-10TGF

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 rangenormal range

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 mediatorsdid not reverse effects on cellular immune

responses

Highly redundant system, multiple factors involved

Unknown factors that regulate cell function

76 amino acids (8.5 kDa)

heat stable

highly conserved

present in all eukaryotic cells

Ubiquitin

Intracellular:Intracellular:UBIQUITIN-PROTEASOME PATHWAYUBIQUITIN-PROTEASOME PATHWAY

PubMed: 14,932 citations as of 04/2006PubMed: 14,932 citations as of 04/2006

Ub

PE P

T

ID

ES

Target Protein

E1E2 E3

ATP

AMP+ PPi

Ub(n)Target Protein

Pro

teas

om

e

ATP

AMP+ PPi

Ub

Target Protein

DUB

Intracellular:Intracellular:

UBIQUITIN-PROTEASOME UBIQUITIN-PROTEASOME

PATHWAYPATHWAY

2nd most common posttranslational protein modification following phosphorylation

The Nobel Prize in Chemistry 2004

"for the discovery of ubiquitin-mediated protein degradation"

Aaron CiechanoverIsrael

Avram HershkoIsrael

Irwin RoseUSA

EXTRACELLULAREXTRACELLULAR(PubMed: < 20 citations as of 04/2006)(PubMed: < 20 citations as of 04/2006)

● present in normal plasma/serum, urine, CSF

Ubiquitin

● elevated plasma/serum levels:

parasitic infectionsAsseman 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 leukemiaDaino et al. Blood 2000

renal failure/hemodialysisOkada et al. Clin Chim Acta 1993Akarsu et al. Nephron 2001

● elevated CSF levels:M. AlzheimerWang et al. Neuropathol 1991

Creutzfeldt-JakobManaka et al. Neurosci Lett 1992

??EXTRACELLULAREXTRACELLULARACTIONSACTIONS

● 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 activitiesKieffer et al. FASEB J 2004

● immunomodulation in critical illness Majetschak et al. Blood 2003

Ubiquitin

Ub

iqu

itin

[n

g/m

L]

serum volunteers serum trauma serum sepsis urine volunteers urine sepsis0

250

500

750

1000

**

*

97.0

66.0

45.0

30.0

20.1

14.4

kD

a

15 10 15 20 25 Ubfront

start

Ub

V Trauma Day 0

µg / lane

Ubiquitin in serum / urine

- trauma / sepsis -

Majetschak et al. Blood 2003

min

max

25th perc

75th percmedian

CS

F U

b [

ng

/mL

]

uninjured TBI

0

20

40

60

80

100

120

140

160

180

200 **

Ubiquitin in cerebrospinal fluid (CSF)after traumatic brain injury (TBI)

TBIDay 0

TBIDay 5

Ctrl. Ub


n = 10 n = 14

min

max

25th perc

75th percmedian

ctrl <9 9-16 17-25 26-35 >35

0

100

200

300

400

500

600

n=12

n=8

n=13

n=16n=18

n=12

* * *

*#

ISS

seru

m u

biq

uit

in (

ng

/mL

)

ctrl. > 8 5 - 8 < 5

0

100

200

300

400

500

600

700

n = 10 n = 5

n = 15

n = 21

GCSC

SF

ub

iqu

itin

[n

g/m

L]

* *

Ubiquitin levels on hospital admission

Trauma – Serum TBI – CSF

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14

0

100

200

300

400

500

38

37

1927 17

22

14

20

146

5

33

11

10

DAY

seru

m u

biq

uit

in (

ng

/mL

)

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14

0

200

400

600

800

1000 13

25

2823

2410

DAY

seru

m u

biq

uit

in (

ng

/mL

)

Trauma ISS > 16 Burns (> 2O, > 20% TBSA)

n =

n =

normal range normal range

Serum ubiquitin – time course

Time (h after TBI)

CS

F U

b [

ng

/mL

]

0 25 50 75 100 125 150 175

0

250

500

750

1000GCS 3

GCS 10

GCS 10

GCS 3

GCS 5GCS 5

CSF ubiquitin – time course in TBI patients

Died

Survived



Time (min)

CS

F U

b [

ng

/mL

]

0 30 60 90 120 150 180 210 240

050

100150200250300350400450500

TBI500

1000

1500

2000

2500

3000

3500

4000

Time (min)

OD

(4

14 n

m)

0 30 60 90 120 150 180 210 240

0.0

0.5

1.0

1.5

2.0

2.5

TBI

Ubiquitin release - hemolysis

experimental TBI, swine

: CSF with visible hemolysis (n = 4) : clear CSF (n = 3)

0 250 500 750 1000

0

1

2

3

4

5

Ub [ng/mL]O

D (

414

nm

)

Ubiquitin release - hemolysis

Patel et al. J. Surg Res 2006Majetschak et al. Crit Care Med 2005

: in-vitro erythrolysis (n = 3) : human CSF samples

0 10 20 30 40 50

0

500

1000

1500

2000

2500

3000

p<0.05 compared to day 0Ub

iqu

itin

in

pla

sma

fro

m p

RB

C u

nit

s (

n =

3,

ng

/mL

)

Days of pRBC storage

17±6 ng /106 lysed human erythrocytes (81±34 g/mL blood) 18±5 ng /106 lysed porcine erythrocytes (94±25 g/mL blood)

Day after Trauma

Ub

iqu

itin

[n

g/m

L]

LP

S e

vo

ke

d T

NF re

sp

on

se

(ng

/mL

)

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

0

100

200

300

400

500

0

1

2

3

4Ubiquitin

TNF

serum levels correlate with LPS evoked TNF responses

- Trauma patients -

Endogenous ubiquitin


● neutralize the inhibitory activity of trauma patients serum on LPS evoked TNFproduction

TN

F p

rod

ucti

on

(%

)

control Ub P4D1 Ub N-190

25

50

75

100

125

AS 1:103 AS 1:102 AS 1:10

normal serum trauma serum

Anti-ubiquitin antibodies



Anti-ubiquitin antibodies:

● stimulate LPS evoked TNF production in trauma/sepsis patients

● have no effects in healthyvolunteers T

NF

(p

g/m

L)

0

250

500

750

1000

1000

2000

3000

4000

5000

6000

7000

8000

- +AS 1:10

trauma

TN

F (

pg

/mL

)

0

250

500

750

1000

1000

2000

3000

4000

5000

6000

7000

8000

- +AS 1:10

sepsis

start

front

97.0

45.0

30.0

20.1

14.4

kD

a

97.0

66.0

45.0

30.0

20.1

14.4

kD

a

Ub

TS RT pH 4 Ub

TN

F (

% c

on

tro

l)

C TS RT pH 7 pH 6 pH 5 pH 4 pH 30

25

50

75

100

125

Anti-ubiquitinaffinity chromatography

- Trauma Serum -


Exogenous ubiquitininhibits ex-vivo LPS induced TNF production

TNF secretion TNF mRNA expression

Ubiquitin [ng/mL]

TN

F (

pg

/106 P

BM

NC

)

0 1 5 10 100 500 10000

1000

2000

3000

4000

5000

**

Ubiquitin [ng/mL]

TN

F m

RN

A (

am

ol/

mL

)

0 500 10000.0

2.5

5.0

7.5

10.0

12.5

15.0

17.5

*


0

2000

4000

6000

8000

10000

12000

14000

16000

18000

IL-6

(p

g/m

L)

0

1000

2000

3000

4000

5000 *

IL-8

(p

g/m

L)

Patel et al. J. Surg Res 2006

Exogenous ubiquitininhibits ex-vivo LPS induced IL-8 production

- + - +ubiquitin

(2 g/mL)ubiquitin

(2 g/mL)

Majetschak et al. Surgery 2004

Time (min)

TN

F (

pg

/106 l

eu

ko

cyte

s)

-15.0 0.0 30.0 120.0 180.0

0

50

100

150

200

250

300

Ub

*

100 ng/mL LPS ex-vivo1000 ng/mL LPS ex-vivo

EX-VIVO LPS EVOKED TNF RESPONSE

AFTER IN-VIVO UBIQUITIN

EFFECT OF SERUM ON LPS EVOKED WHOLE BLOOD TNF PRODUCTION

- in-vivo ubiquitin -

: p < 0.05 vs. t = 0 min (ANOVA)*

Time (min)

Eff

ec

t o

f s

eru

m o

nn

orm

al w

ho

le b

loo

d T

NF

p

rod

uc

tio

n(%

co

ntr

ol)

0 20 40 60 80 100 120 140 160

0

25

50

75

100

125 Ub

* * **

100 ng/mL LPS ex vivo

Ubiquitin

inhibits leukocyte functionin-vitro and in-vivo

clinical relevant effects ?

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

Time (min)

-40 -20 0 20 40 60 80 100 120 140 160 180

0

10

20

30

40

50

prepubertal swine, anaesthetized (ketamin/fentanyl)mechanically ventilated (FiO2: 0.5)fluid resuscitation (Lactated Ringer`s): MAP < 69 mmHg

pre post

● 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

Ubiquitin serum levels

Time (min)

Ub

seru

m l

evels

(n

g/m

L)

-40 -20 0 20 40 60 80 100 120 140 160 180

0

5000

10000

15000

20000Ub pre

Ub postUb/no LPS

ctrl (BSA)

Time (min)

-40 -20 0 20 40 60 80 100 120 140 160 180

0

20

40

60

80

100

120

Ub Ub

ctrl (BSA)

LPS

MAP (mmHg)

Fluid requirements

* : p < 0.05 vs. ctrl (ANOVA)

Time (min)

-40 -20 0 20 40 60 80 100 120 140 160 180

0

20

40

60

80

100

120

***

**

UbUb

LPS

ctrl (BSA)

cumulativeiv fluid (mL/kg)

Edema and erythema formation

Ubiquitinpre-treatment

control(BSA)

t = 180 min

Oxygenation

* : p < 0.05 vs. ctrl (ANOVA)

Time (min)

PaO

2 (

mm

Hg

)

-40 -20 0 20 40 60 80 100 120 140 160 180

0

50

100

150

200

250

300

350 **

*

Ub Ub

LPS

ctrl (BSA)

PaO2/FiO2 < 200

Mortality

Time (min)

Mo

rtali

ty (

%)

-40 -20 0 20 40 60 80 100 120 140 160 180

0

10

20

30

40

50

LPS

Ub Ub

9/18

5/18

4/18

2/18

1/18 0/60/6

p = 0.013

ctrl (BSA)

● 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 modelsof severe trauma and traumatic brain injury (TBI)

Earle et al. Surgery 2005Majetschak et al. J Trauma 2004

Time (min)

-30 0 30 60 90 120 150 180 210 240 270 300

051015202530354045

HEM RESUSCITATION

i.v. ubiquitin ori.v. BSA

Fx

or

TB

I randomized, blinded

- Severe Trauma / TBI - Fluid requirements

: p < 0.05 (ANOVA)*

Time (min)c

um

ula

tiv

e IV

flu

ids

[m

L/k

g]

-50 0 50 100 150 200 250 300

0

100

200

300

400

500

600

Ub/Alb

Ubiquitin (n=5)Albumin (n=5)

*

***

**

*

*

Time (min)

cu

mu

lati

ve

IV f

luid

s [

mL

/kg

]

-50 0 50 100 150 200 250

0

50

100

150

200

250

300

Ub/Alb

**

*

*Ubiquitin (n=5)Albumin (n=5)

HEM RESUSCITATION HEM RESUSCITATION

Fx

TB

I

Time (min)

PIP

[c

mH

20

]

-50 0 50 100 150 200 250 300

0

10

20

30

40

50

Ub/Alb

Albumin (n=5)Ubiquitin (n=5)

*

****

Time (min)

Pa

O2(m

mH

g)

-50 0 50 100 150 200 250 300

0

50

100

150

200

250

300 Ub/Alb

Albumin (n=5)Ubiquitin (n=5)

*

- Traumatic Brain Injury -

Pulmonary function & oxygenation

HEM RESUSCITATION

TB

I

HEM RESUSCITATION

TB

I

: p < 0.05 (ANOVA)*

Time (min)

ICP

[m

mH

g]

-50 0 50 100 150 200 250 300

0

5

10

15

20

25

30

35

40

45

Ub/Alb

* **

*Albumin (n=5)Ubiquitin (n=5)

*

**

HEM RESUSCITATION

TB

I

- Traumatic Brain Injury -

Intracranial pressure

: p < 0.05 (ANOVA)*

1 10 20 30 40 50 60 70 76MQIFVKTLTG KTITLEVEPS DTIENVKAKI QDKEGIPPDQ QRLIFAGKQL EDGRTLSDYN IQKESTLHLV LRLRGG

50 59

L EDGRTLSDY

Szewczuk et al. Biopolymers 2004

Immunosuppressive potency similar to cyclosporin

Ubiquitin

0 10 100 500 10000

2500

5000

7500

10000

12500

15000

Ubiquitin (ng/mL)

ML

R (

cpm

)

Earle et al. Transplantation, submitted

Effect of ubiquitin on mixed leukocyte reaction (MLR)

One-way MLRC3H/HEJ mouse splenocytes to -irradiated DBA2 splenocytes

0 2 4 6 8 10 12 14 16 18 20 22 24

0

10

20

30

40

50

60

70

80

90

100

i.p. ubiquitin/albumin

p < 0.001

Ubiquitin (n=14; 25 g/h)median survival: 17 days

Albumin (n=11; 25 g/h)median survival: 11 days

DAYS

all

og

en

eic

sk

in g

raft

su

rviv

al

(%)

Ub

Alb

Earle et al. Transplantation, submitted

Ubiquitin prolongs allogeneic skin graft survival

allogeneic syngeneicgraft (C3H/HEJ) graft (DBA2)

C3H/HEJ – DBA2 mice

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 ?

bright field

ex488 nm / em 530 nm

Ubiquitin – uptake into monocytes

Majetschak et al. Immunol Cell Biol 2006

- ubiquitin + ubiquitin

Fate of extracellular ubiquitin following uptake into monocytes (MonoMac 6)

97.4

58.1

39.8

29

20.1

14.3

Ub

ERK1/2

- - + + + + + +Ubb

LTA [ng/mL] 0 100 0 1 3 30 100 300

kDa


Ubb

Conjugation to intracellular target proteins

0 100 200 300 400 500 600 700 800 900 10000.0

0.5

1.0

1.5

2.0

2.5

3.0

cell culture with Ubb [ng/mL]

Ub

b u

pta

ke [

ng

/g

]

0 1 3 10 30 1000.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

LTA [g/mL]

Ub

b u

pta

ke

[n

g/

g]

0 1 10 100 10000.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

LPS [ng/mL]

Ub

b u

pta

ke [

ng

/g

]


Ubiquitin – uptake into monocytes

increased in inflammatory conditions(LPS / LTA)

shows saturation kinetics(Kd: 6 – 9 nM)

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

1.9 2.0 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 3.0 3.1 3.2 3.3 3.4 3.5

9.0

9.5

10.0

10.5

11.0

11.5

12.0

12.5

13.0

13.5

14.0

14.5

conjugated ubiquitin

free

ub

iqu

itin

0 100 200 300 400 500 600 700 800 900 1000

0

2

4

6

8

10

12

14

16

18

conjugated ubiquitin

free

ub

iqu

itin

PBMNC (n = 10) Muscle, heart, lung, liver, spleen, kidney (n = 5 each)

Ponelies et al. Shock 2005 Patel & Majetschak, Physiol Res, submitted

250

160

105

75

50

35

3025

15

10

kDa

free Ub —

Ub-protein conjugates

Ctrl. —

sepsis healthy

[ng

/g

pro

tein

]

ctrl. sepsis ctrl. sepsis ctrl. sepsis

0.0

2.5

5.0

7.5

10.0

12.5

15.0

17.5

20.0 p < 0.001

p < 0.001

total Ub free Ub conjugated Ub


Does it make sense?

2. Altered ubiquitin equilibrium during sepsis: Reduced conjugates

Ub

Ub-P

Ub

Ub-P

SEPSIS

Ponelies et al. Shock 2005


Does it make sense?

3. Monocytes contain 10 - 15 fg free ubiquitin / cell

Up to 10 fg free ubiquitin/cell can be taken up

UbUb

Ub-P

Ub

Ub-P

SEPSIS

Law of mass action

Could shift equilibrium towards conjugate formation

Counteract further decreasein ubiquitin protein conjugates

UbUbUb

Ub

InflammationInflammationTrauma - SepsisTrauma - Sepsis

P

E1,E2,E3

Ub(n) P

proteasomaldegradation

reversiblemodification

inhibition of pro-inflammatory responsesmodulation of cell function

maintenance of ubiquitin homeostasis

Ub

tissue

endothelium

lumenReceptor

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

Acknowledgement

University of MiamiLissette T. Busby

Mayur B. PatelSteven A. EarleDavid R. King

Kenneth G. ProctorSteven M. Cohn

University of HeidelbergNorbert PoneliesThomas Hirsch

Ulrich KrehmeierUdo Obertacke

University of BerlinArwed Hosstmann

Andreas OberholzerWolfgang Ertel

University of MunichSiegfried Zedler

Eugen Faist

Supported by:DFG (German Science Foundation)MA 2474/1-1, MA 2474/2-1, MA 2472/2-2

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

Martin Rechsteiner, 1988

immune dysfunction in trauma and sepsis: novel aspects of ubiquitin matthias majetschak, md, phd...

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