Molecular Mechanisms of Apoptosis in Neutrophil

Granulocytes Compared to Septic Granulocytes

Dr. med. Ladislav Mica

A little dog looking for broken bubbles with the tip of his nose….

1

Table of Contents:

Abbreviation Index 5

Author’s contribution to this Work 8

1.0 Introduction 9

1.1 The Toll-like receptors 12

1.2 The MAP-Kinases 13

1.3 The IAP-proteins 15

1.4 The Caspases 16

1.5 The Proteasome 20

1.6 The Bcl-2 Proteins 21

2.0 Material and Methods 25

2.1 Patients 25

2.2 Isolation and Culture of Neutrophil Granulocytes 25

2.3 Quantification of Apoptosis 26

2.4 Flow Assisted Cytometry of Toll-like Receptors 2 and 4 in PMN 26

2.5 Analysis of MAP-Kinases in PMN 27

2.5.1 Experimental Protocol 27

2.5.2 Westernblot Analysis of Neutrophil MAP-Kinases 27

2.6 Analysis of cIAP2 Protein in PMN 28

2.6.1 Experimental Protocol 28

2.6.2 Caspase-3 Activity Measurement 29

2.6.3 Detection of cIAP2 mRNA by RT-PCR 29

2.6.4 Westernblot Analysis of cIAP2 Protein 30

2.7 Analysis of Bcl-2 Proteins in PMN 30

2.7.1 Experimental Protocol 30

2

2.7.2 Analysis of PMN Apoptosis 31

2.7.3 Western-Blot Analysis of Bcl-2 Proteins 32

2.7.4 Detection of mRNA of Bcl-2 Proteins by RT-PCT 33

2.8 Statistical Analysis 33

3.0 Results 35

3.0.1 Apoptosis is Reduced in PMN from Septic Patients 35

3.1.1 Expression of TLR-2 and TLR-4 on Freshly Isolated PMN 35

3.1.2 Ligand Binding and Receptor Expression 36

3.2 MAP-Kinases and PMN Apoptosis 38

3.2.1 Effect of Herbimycin on Neutrophil Apoptosis 38

3.2.2 Participation of Phosphatases in the Regulation of PMN Apoptosis 39

3.2.3 Effect of MAP-Kinase Inhibitors on LPS and IFN--Mediated PMN

Apoptosis 41

3.2.4 Detection of Phosphorylated ERK and p38 MAP-Kinase in PMN 43

3.3 cIAP2 and Activity of Caspase-3 in Neutrophil Granulocytes 46

3.3.1 LPS Induces cIAP2 mRNA and Protein in PMN 46

3.3.2 LPS Induces Ubiquitination of Caspase-3 in PMN 47

3.3.3 Reduction of Spontaneous and CD95-Induced Apoptosis by LPS 49

3.3.4 LPS Reduces Caspase-3-like Activity 50

3.4 The Correlation of Mcl-1 with Apoptosis of PMN 51

3.4.1 Expression of Bcl-2 mRNA and Protein in PMN 51

3.4.2 Expression of Mcl-1 mRNA and Protein in PMN 53

3.4.3 Expression of BAX mRNA and Protein in PMN 54

3.4.4 Expression of Bid mRNA and Protein in PMN 56

3

4.0 Discussion 58

4.1 Cell Membrane: The Needle Ear 59

4.2 The Phosphate Cascades: Quick Resuscitation Action 61

4.3 cIAP2: Blocking the Road to Death 63

4.4 Bcl-2 Proteins: The Balanced Suicide Machinery 65

4.5 The Strategy 67

4.6 Hypothetical Molecular Targets 68

4.7 Work to be done 70

5.0 References 73

6.0 Books 87

7.0 Databases 87

8.0 Curriculum Vitae 88

9.0 Authors Publications 91

9.1 Oral presentations 93

Certificate of Competency 100

4

Abbreviation Index

AIF: Apoptosis Inducing Factor, endonuclease

APACHE II: Acute Physiology and Chronic Health Evaluation

ATP: Adenosin Tri-Phosphate

BAX: BCL2-Associated X protein

Bcl-2: B-Cell Leukemia Type 2 Protein

BH-Domain: Bcl-Homology Domain

Bid: BH3 Interacting Domain Death Agonist

BIR: Baculoviral IAP Repeat

CARD: Caspase Recruitment Domain

Caspase: Cysteinyl-Aspartate Specific Protease

CD14: Cluster of Differentiation 14, co-receptor of TLR 4 for LPS

CD95: Fas, Cluster of Differentiation 95

c-myc: Mastergene, regulates Transcription, protooncogene

Cyto-c: Cytochrome-c, intercristary space protein, proapoptotic in cytosole

Diablo: Direct IAP-Binding Protein with Low pI

DIC: Disseminated Intravasal Coagulopathy

E1: Ubiquitin Activating Enzyme

E2: Ubiquitin Transfer Enzyme

E3: Ubiquitin Ligase

E-Box proteins: Ubiquitin Ligation Associated Proteins

ERK: Extracellular Signal Regulated Kinase

FADD: Fas-Associated Death Domain, CD95 –Associated Death Domain

GM-CSF : Granulocyte-Macrophage Colony Stimulating Factor

I-B: Inhibitory Factor-B

IAP: Inhibitor of Apoptosis Protein

5

ICE : Interleukin-1 Converting Enzyme, Caspase-1

IFN-: Interferon-

IGF-1: Insulin-like Growth Factor-1

IRAK: Interleukin-1 Receptor-Associated Kinase

LPS: Lipopolysaccharide

MALP2: Macrophage Activating Lipoprotein 2

MAP-Kinase: Mitogen Activated Protein Kinase

Mcl-1: Induced Myeloid Leukemia Cell Differentiation Protein

MHC: Major Histocompability Complex

MKK: Mitogen-Activated Protein Kinase Kinase

MODS: Multi Organ Dysfunktion Syndrome

MOF: Multi Organ Failure

mTOR: Mammalian Target of Rapamycin

MyD88: Myeloic Differentiation Factor 88

NF-B: Nuclear Factor-B

p38: Mitogen-Activated Protein Kinase

PAS: Phagophore Assembly Site

PBMC: Peripheral Blood Mononuclear Cell

PMN: Polymorph Nuclear Cells

RING: E2, Ubiquitin ligase sub-domain of a protein

SIRS: Systemic Inflammatory Response Syndrome

Smac: Second Mitochondria-Derived Activator of Caspase

STAT3: Signal Transducer and Activator of Transcription 3

TAB: ABC-Channel for oligopeptides, Endoplasmic reticulum

Toll: [German] = ”great”, Drosophila scientist’s irony

TRAF: TNF Receptor Associated Factor

6

Ubc: E2, Ubiquitin conjugating enzyme

Ubl : E3, Ubiquitin ligase

UVRAG: UV Radiation Resistance-Associated Gene Protein

7

Author’s contribution to this work

FACS analysis was partially performed by the author and L. Härter PhD.

RT-PCR analysis was totally performed by the author.

Western-Blot analysis was partially performed by the author and L. Härter PhD.

Caspase-3 Activity assay was completely performed by the author.

Cell culture and harvesting was partially performed by the author an L. Härter PhD and U.

Steckholzer BS.

Acquisition of samples from patients was partially performed by the author and L. Härter PhD

and U. Steckholzer BS.

Statistical analysis was performed by the author and L. Härter PhD with Sigma Stat.

8

1. Introduction

Despite of increased security in traffic and civil life polytrauma remains the most often cause

of death under the age of 40 years (1-4). The brain injury, sudden blood loss and penetrating

or blunt trauma are the leading injuries causing immediate death after trauma. Direct

mechanical forces on the organism cause primary tissue damage. The damaged tissue suffers

low supply with oxygen leading to damage increase and to unspecific stimulation of

neutrophil granulocytes (PMN) and monocytes (PBMC) by released mediators from the tissue

(1-4, 6-10). Once, over helming the organism with antigenic load the cells of the first line of

defense, PMN and PBMC, are systemically activated leading to systemic inflammation. This

systemic inflammation was defined in 1991, through the consensus conference of the

American College of Chest Physicians/Society of Critical Care Medicine (ACCP/SCCM), as

systemic inflammatory response syndrome (SIRS) (5) (Table 1.).

9

Temperature > 38,8°C or < 36,8°C3.

Number of leukocytes > 12,000/mm3

or < 4000/mm3 or 10% juvenile neutrophil granulocytes4.

Breathing rate > 20/min, respectively, Hyperventilation with decrease of the arterial CO2 partial pressure (PaCO2) under 32 mmHg.

2.

Heart rate > 90 bpm1.

For the definition of SIRS, two or more parameters must be fulfiled. Sepsis is definedas SIRS with detection of bacteremia or bacterial focus. (5)

Table 1: Clinical parameters of the systemic inflammatory response syndrome (SIRS)

Temperature > 38,8°C or < 36,8°C3.

Number of leukocytes > 12,000/mm3

or < 4000/mm3 or 10% juvenile neutrophil granulocytes4.

Breathing rate > 20/min, respectively, Hyperventilation with decrease of the arterial CO2 partial pressure (PaCO2) under 32 mmHg.

2.

Heart rate > 90 bpm1.

For the definition of SIRS, two or more parameters must be fulfiled. Sepsis is definedas SIRS with detection of bacteremia or bacterial focus. (5)

Table 1: Clinical parameters of the systemic inflammatory response syndrome (SIRS)

Neutrophil granulocytes are cells from the monopoetic cell line capable in phagocytosis and

unspecific immune response (Innate Immunity). Bacterial and viral decay products stimulate

specific receptors on cellular surface a lead to pro-inflammatory activation. During SIRS

PMN are accumulated in different organs leading to secondary damage of the organs by

endothelial damage and disseminated intravascular coagulopathy (DIC), causing necrosis and

apoptosis of parenchymal cells by degranulation of PMN. This hyper inflammation causes

MODS (multi organ dysfunction syndrome) and MOF (multi organ failure) (11-15). Both the

severity of trauma and the antigenic load as well as the over flooding by pro-inflammatory

cytokines are leading to this hyper stimulation of PMN.

Figure 1: Cellular signaling. A ligand binds to a receptor. The interaction between these two proteins leads to conformational changes and the activation of second messenger systems carrying the message (Order) into the nucleus. In the nucleus the signal (multi protein complex) results in the activation of transcription. The newly synthetised proteins result in an answer of the cell, A: intracellular changes, B: behavior.

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Receptor

Ligand

Nucleus

Order

The Cell

A

B

Receptor

Ligand

Nucleus

Order

The Cell

A

B

Under physiologic conditions PMN are distributed in a circulating pool and in a marginal pool

adhering on venous walls. Stress hormones cause the set free of the marginal pool of the

venous walls. Stress-conditions also stimulate the set free of immature neutrophil

granulocytes from the bone marrow (left shift). The circulating PMN have only a short half

lifespan of about 16 hours, about 60% die after this period of time (17). The death of

neutrophil granulocytes is a well controlled process called Apoptosis (Greek: απόπτωσις

falling leaf). This process was first described by Kerr and Wylie in the early 1972 (16) and

called shrinkage necrosis, describing the microsopical characteristics of the cell during

apoptosis. The main characteristics described were cellular shrinkage, forming of bubbles in

the cellular membrane (blebbing) and kariorrhexis. The growing field of molecular biology

led to a discovery of a plenty of factors at the beginning of 1990. The discovery of death as a

well defined molecular process in the cell changed the view on cellular life and centered the

process of dieing into the existence.

In this work we will in an endotoxin model analyze the signaling through the Toll-like

receptors in the outer cellular membrane. This name was given to a group of proteins by the

society of the scientists of the species Drosophila melanogaster. Once ligand binds to a

receptor, the results is an intracellular signal, activating a second messenger systems and

leading to a cellular response. In the case of neutrophil granulocytes the signaling pathway of

MAP-kinases (Mitogen Activated Phosphatases) was analyzed. Different factors are

deactivated by the proteasome. The proteasome is the “intracellular trash can” of the cell.

All intracellular proteins, once tagged with Ubiquitin, are destined to be destroyed by the

proteasome. The interaction between cIAP2 and Caspase-3 was analyzed at the level of the

proteasome. The role of the most important members of Bcl-2 family proteins was analyzed.

The Bcl-2 proteins consist of a pro- and anti-apoptotic group acting like the complementary

system against the outer mitochondrial membrane.

11

1.1 The Toll-like receptors

The first line of defense in immunology consists of neutrophil granulocytes (PMN) and

mononuclear cells (PBMC). The pathogene recognition is initiated by receptors of the innate

immunity. These receptors were primary recognized in Drosophila melanogaster and were

called TOLL-receptors. Similar receptors with the same function were discovered on human

phagocyting cells and called Toll-like receptors (18-20). These receptors are like antennas

recognizing pathogens of microorganisms and viruses. Once activated by a pathogen the

signal results in production of pro-inflammatory cytokines and the inhibition of apoptosis in

PMN. To date up to 10 different receptors have been characterized with different ligand

specifities (21, 22). In this study we focused on the Toll-like receptor 2 and 4, both on the

surface of PMN. The TLR2 has been found to signal for the toxins of gram positive bacteria

like MALP2, macrophage activating lipoprotein 2 from Mycoplasma fermentans (23, 24). The

TLR4 together with CD14 is activated by LPS, lipopolysyccharide from gram negative

bacteria like Escherichia coli (23, 24).

A reduced cytokine response on repetitive stimulation with LPS has already been shown. This

effect called endotoxin tolerance (25) is known for many years but the molecular mechanism

has not been elucidated yet. The hypothesis of down regulation of TLR during endotoxin

tolerance was supported by a murine model. In contrast Medvedew reported incrased TLR

receptors on LPS tolerant monocytes (26). Seemingly a paradoxon, the mechanisms toward

endotoxin tolerance seems not to originate from cellular surface. Therefore we investigated in

this study the expression dynamics of TLR2 and TLR4 upon stimulation with specific ligands.

We will show an upregulation of TLR2 and TLR4 on PMN from healthy individuals upon

stimulation with LPS, MALP2 and patients with sepsis.

12

Figure 2: The different Toll-like receptors are depicted on the top. This innate immunity system recognizes different bacterial and viral products. Once bound to the receptor the intracellular signal results in a phosphorylation of MyD88, a signal transducer. MyD88 caries the signal to IRAK1 and IRAK4 resulting in the activation of the MAP kinases pathway (see 1.2). In this study we focused only on TLR2 and TLR4 with the ligands MALP2 and LPS.(http://www.genome.jp/dbget-bin/www_bget?pathway+hsa04620)

1.2 The MAP-kinases

The activation of Toll-like receptors by their ligands (in this study LPS for TLR2 and MALP2

for TLR4) leads to a signal transduction across the cellular membrane and the activation of

MyD88 as the first player in a highly diverse phosphorylation cascade. This protein called

MyD88 represents a MAP4K very upstream of an activation signal, in mammalian cells

usually pro-inflammatory, pro-mitogenic signals from cytokine receptors, signals from

integrins via Ras and Src and signals via Rho/Rac system. These signals normally do not

occur as simple lonely stimuli but the cell is normally exposed to an orchestra of different

stimuli activating these MAP-kinases. The way of the activation of MAP-kinase pathway is

very diverse and leads via different steps (see figurere 3) to the activation of ERK (p42/44),

p38 or JNK (c-Jun terminal kinase) (26). In this study we focused only on the ERK, p38 and

JNK kinases representing the final path of the MAP-kinase pathway. Although, the activation

of JNK upon stimulation with LPS in PMN, has not been shown yet (27).

13

Lipoproteins

Flagellin CpGs LPS dsDNA ssRNA

TLR

-1

TLR

-6

TLR

-2

TLR

-4

TLR

-3

TLR

-7

TLR

-5

TLR

-5

TLR

-8

TLR

-9

TLR

-2

TLR

-4

Bacteria Viruses

MyD88 MyD88MyD88 MyD88

MALP2

Cell membrane

Lipoproteins

Flagellin CpGs LPS dsDNA ssRNA

TLR

-1

TLR

-6

TLR

-2

TLR

-4

TLR

-3

TLR

-7

TLR

-5

TLR

-5

TLR

-8

TLR

-9

TLR

-2

TLR

-4

Bacteria Viruses

MyD88MyD88 MyD88MyD88MyD88MyD88 MyD88MyD88

MALP2

Cell membrane

Figure 3: Schematically depicted signalling cascade of a TLR receptor. Upon stimulation with its ligand the signal is being transduced via MyD88, IRAK, TRAF, TAB and TAK to MKK, the MKK factors transmit a phosphate group to the final kinases JNK, p38 and ERK. The phosphorylation of p38, ERK or JNK leads to the activation Transcription factors and to a cellular response. (http://www.genome.jp/dbget-bin/www_bget?pathway+hsa04620)

The use of highly specific inhibitors revealed the involvement of p38 MAP kinase in

spontaneous apoptosis in PMN (29) as well as the delay in apoptosis after incubation with

LPS (30) or other pro-inflammatory stimuli like GM-CSF (32). It was also shown that p38 is

14

TL

R

MyD88

IRAK4

IRAK1

TRAF6

TAB1

TAB2

TAK1

MKK 1/2MKK 3/6MKK 4/7

JNK p38 ERK

+p+p

+p+p+p

+p

MAP4K

MAP3K

MAP2K

MAPK

Cell membrane

TL

R

MyD88

IRAK4

IRAK1

TRAF6

TAB1

TAB2

TAK1

MKK 1/2MKK 3/6MKK 4/7

JNK p38 ERK

+p+p

+p+p+p

+p

TL

R

MyD88MyD88

IRAK4

IRAK1

TRAF6

TAB1

TAB2

TAK1

TAB1

TAB2

TAK1

MKK 1/2MKK 3/6MKK 4/7

JNKJNK p38p38 ERKERK

+p+p+p+p

+p+p+p+p+p+p

+p+p

MAP4K

MAP3K

MAP2K

MAPK

Cell membrane

activated under apoptotic conditions (28). The MEK1 activated ERK kinase has also been

shown to regulate cellular survival in PMN from healthy donors after stimulation with LPS.

However, only traces were found in the literature and the role of MAP-kinases in PMN

remained to be discussed within the disease of sepsis.

1.3 The IAP-proteins

Inhibitor of Apoptosis proteins (IAP) were first identified in baculoviruses. All IAP-proteins

contain several motifs called BIR-domains, baculoviral IAP repeats. An IAP protein normally

consists of 1-3 BIR domains. The BIR domains allow the IAP to bind specifically to proteases

and to inhibit them. The BIR domains of some IAPs allow them to bind to caspases (see

chapter 1.4), that are the main executioners in apoptotic cell death. The inhibition of these

proteases, caspases, provides the simplest explanation for the inhibition of cell death by IAPs.

However, another domain has been recently shown to participate in apoptotical death

regulation (31). The RING-domain is able to bind monoubiquitin molecules and to transfer

these ubiquitins to a substrate. Once covalently bound to lysine residues, the target is destined

to be degradated by the proteasome. Ubiquitination is an active process consuming ATP and

requiring the E-Box proteins. The E-Box proteins are subdivided into three groups. The E1

proteins provide the initial reaction by activating the ubiquitin molecule. The molecule is

linked on the C-terminal glycine carboxylate to a SH-group of the activating enzyme, this step

is ATP consuming. In a transacylation reaction, the Ubiquitin is transferred from E1-Ub to a

cysteine SH within the active site of the conjugating enzyme E2 and forms E2-Ub. The E3

enzymes are the direct ubiquitin ligases (Ubl) recognizing the substrate and bringing E2 and

the substrate together. This results in an attachment of a polyubiquitin chain and subsequent

degradation of the targeted protein in the proteasomal pathway (see chapter 1.5). In this study

the E2/E3 protein is represented by the cIAP2 protein, which once activated harbors an

ubiquitin in the RING domain, resulting in cIAP2-Ub.

15

Figure 4: Ubiquitination pathway, exponential activation of ubiquitin-ligases (Ubl) by less ubiquitin conjugating enzymes (Ubc). Many substrates (S) can be ubiquitinated by one ubiquitin ligase. On the right side the molecule of cIAP2: the RING domain together with the CARD domain are comparable to E2 and E3 proteins. (Kraus, Biochemistry of Signal Transduction)

A RING domain transfers the ubiquitin residues without additional recognition of E3, this role

is played by the BIR domains. cIAP2 recognizes with the BIR domain the activated caspase-

3, which is one of the main executioners of the apoptotic pathway, a final common path.

16

BIR BIR BIR RING

BIR BIR BIR CARD RING

BIR RING

BIR

BIR BIR BIR

BIR

BIR BIR BIR CARD RING

BIR RING XIAP

ILP2

cIAP1

cIAP2

ML-IAP

NAIP

Survivin

Apollon

BIRBIR BIRBIR BIRBIR RINGRING

BIRBIR BIRBIR BIRBIR CARDCARD RINGRING

BIRBIR RINGRING

BIRBIR

BIRBIR BIRBIR BIRBIR

BIRBIR

BIRBIR BIRBIR BIRBIR CARDCARD RINGRING

BIRBIR RINGRING XIAP

ILP2

cIAP1

cIAP2

ML-IAP

NAIP

Survivin

Apollon

E1

Ubc1 Ubc2 Ubc3 Ubc4 Ubc5 UbcX

Ubl

1U

bl2

Ubl

3

Ubl

XU

blY

Ubl

Z………

S1 S2 S3 SX SY SZSubstrates

E2

E3

BIR

BIR

BIR

CA

RD

RIN

G

cIAP2E1

Ubc1 Ubc2 Ubc3 Ubc4 Ubc5 UbcXUbc1 Ubc2 Ubc3 Ubc4 Ubc5 UbcX

Ubl

1U

bl2

Ubl

3

Ubl

1U

bl2

Ubl

3

Ubl

XU

blY

Ubl

Z

Ubl

XU

blY

Ubl

Z………

S1 S2 S3S1 S2 S3 SX SY SZSX SY SZSubstrates

E2

E3

BIR

BIR

BIR

BIR

BIR

BIR

BIR

BIR

CA

RD

CA

RD

RIN

GR

ING

cIAP2

Figure 5: Different members of human IAP protein family. RING: RING domain, ubiquitin ligase. CARD: Caspase recruitment domain, binding to Caspases. BIR: Baculoviral IAP repeats, enzyme inhibition. The different BIR domains are able to inhibit different enzymes, especially the caspases during apoptosis. In this study we examined the expression pattern of cIAP2.

The activated caspase-3 is competitively inhibited by BIR3 and the RING domain may attach

ubiquitin molecules to the activated caspase-3 modifying this enzyme to be degradated by the

proteasomal pathway. Therefore, we investigated the role of cIAP2 in neutrphil granulocytes

throwing a small light to apoptotic resistance of PMN upon pro-apoptotic stimuli during

sepsis.

1.4 The Caspases

17

The caspases (cysteinyl-aspartate specific protease) are directly responsible for the

biochemical changes during apoptosis in a dying cell. The caspases belong to a group of

intracellular enzymes expressed as zymogenes. These enzymes share the specifity for

aspartate from which the name is derived from. During apoptosis these zymogenes are

processed to active enzymes by an activation cascade and degrade intracellular structures and

proteins (34). The first discovered caspase, nowadays caspase-1, was ICE (interleukin-1

converting enzyme) in 1992 (33). This enzyme was responsible for activating limited

proteolysis of the pro-inflammatory cytokine interleukin-1. Several other proteins were

sequenced with ICE-sharing homologies responsible for the execution of cell death and

inflammation. About 15 members of the caspase family were identified until now; caspase-14

seems to play only a role in the differentiation of keratinocytes (35). Caspase-12 is expressed

in homo sapiens only as a catalytically inactive form and is not represented in the list of

human caspases. The implementation of caspase-12 is discussed in the inflammatory pathway

in a murine model (36).

All caspases consist of a bigger (p20), smaller (p10) subunit and a prodomain witch varies in

size and function. These zymogenes have to be activated by limited proteolysis between p20

and p10, normally the prodomain is also removed. This activation results in the formation of a

heterotetramere (2p20 + 2p10) with two catalytically active centers. The center specifically

recognizes the cleavage site by a sequence of four amino-acids. Aspartate is at the firs

position, the whole enzyme family can also be distinguished into groups according to the

optimal cleavage peptide sequence (34).

Two pathways of the caspase-activation are known, the extrinsic receptor mediated pathway

and the intrinsic mitochondrial pathway. The activation of a death receptor leads to an

intracellular activation of an initiator caspase (usually caspase 8 or 10) (37). The initiator

caspase activates downstream events leading to the activation of effector caspases and the

execution of death. The intrinsic pathway is characterized by the activation of the pro-

18

apoptotic Bcl-2 system leading to pore-forming in the outer mitochondrial membrane and the

leakage of cytochrome-c and other pro-apoptotic factors (see chapter 1.6). Cytochrome-c

together with Apaf-1 forms a heteropentamere called the apoptosome (40). The apoptosome

activates caspase-9 which leads downstream to the activation of caspase-3 as the main

effector caspase, and the execution of cell death.

In this study we focus only on caspase-3 representing the final common path of an possible

extrinsic or intrinsic activation.

Figure 6: Depicted are assorted human caspases involved in apoptotic process. All caspases have a big p20 and a small p10 subunit, additional oligomerisation units (DED: Death Effector Domain and CARD: Caspase Recruitment Domain) have only the initiator caspases. Arrow indicates the cleavage region of activation. The asterisk shows the approximate position of the active cystein. (www.expasy.ch)

19

Human caspases in apoptosis

p10 p20 DEDDED Caspase-8

p10 p20 DED DED Caspase-10

p10 p20 CARD Caspase-9

p10 p20 CARD Caspase-2

p10 p20 Caspase-3

p10 p20 Caspase-6

p10 p20 Caspase-7

Initi

ator

cas

pase

sE

ffec

tor

casp

ases

Human caspases in apoptosis

p10 p20p20 DEDDEDDEDDED Caspase-8

p10 p20p20 DEDDED DEDDED Caspase-10

p10 p20p20 CARDCARD Caspase-9

p10 p20p20 CARDCARD Caspase-2

p10 p20p20 Caspase-3

p10 p20p20 Caspase-6

p10 p20p20 Caspase-7

Initi

ator

cas

pase

sE

ffec

tor

casp

ases

1.5 The Proteasome

The proteasome is a multienzymic complex forming a tunnel to allow optimal access of the

enzymes to degrade a polyubiquitinated protein. After the activation or the damage of a

protein its tertiary structure changes and opens a specific ubiquitination signal, the degron

sequence (38). This sequence of aminoacids is responsible for the binding of an E2 protein

and the attachment of a polyubiquitin chain. The polyubiquitinated protein is recognized by

the 19S regulatory domain of the proteasome and bound to it to allow ATP-dependent

unfolding of this protein. The unfolded protein is drawn into the proteasome (20S protease

complex) and degraded in aminoacids and oligopeptides. The oligopeptides are used to be

presented on MHC I complexes and/or to be recycled. The proteasomal pathway is the

deagradation way for intracellular proteins (38, Lodish, Molecular Cell Biology).

The proteasome, seemingly the intracellular trash can, is also involved in transcription

regulation. Different inhibitors of transcription factors (e.g. IB) (39) are upon activation

degraded by the proteasome and the transcription factor is set free to transduce into the

nucleus. In the nucleus the transcription is terminated by degradation of the transcription

factor by the proteasomal pathway. The primarily attached polyubiquitin chain is

disconnected and recycled (Lodish, Molecular Cell Biology).

The participation of the proteasome has been recently shown, where the inhibition led to

inhibition of the anti-apoptotic LPS-effect, connecting this event with activation-inhibition of

NF-B. Usually I-B is being polyubiquitinated and degraded by the proteasome and NF-B

may translocate into the nucleus to activate transcription of target genes. If the degradation of

I-B is inhibited, the NF-B inhibition persists and the transcription does not take place

(www.genome.jp). Both, the initiation and the termination of transcription are regulated by

the proteasomal mechanism. In this study we schow an induction of cIAP2 protein and

caspase-3-activity depending on the proteasome. In PMN only poor data are published to this

double-edged theme.

20

Figure 7: A polyubiquitinated protein is recognized by the regulatory complex. The protein is unfolded in an ATP dependent manner and drawn into the proteasome. The multimeric complex cuts the protein into aminoacids and oligopetides leading to the deactivation of the protein. (http://www.genome.jp/dbget-bin/www_bget?pathway+hsa03050)

1.6 The Bcl-2 proteins

The Bcl-2 protein class is a widely spread class of proteins and can be subdivided in two

subgroups. The proaoptotic group consists of several members similar the anti-apoptotic

group, the clue of the pro-apoptotic activity is the absence of the anti-apoptotic BH4 domain

of these proteins (40). To quantitatively realize the intracellular role of these proteins we

could compare them to the complementary system of blood plasm. In the Bcl-2 system we

21

Protein

Prot

ein

Lid

Lid19S Regulatory Complex

20S Protease Complex

Recycling of ubiquitinmonomeres

Oligopeptides

Polyubiquitin chain

Protein

Prot

ein

Lid

Lid19S Regulatory Complex

20S Protease Complex

Recycling of ubiquitinmonomeres

Oligopeptides

Polyubiquitin chain

have two different activating ways. The extrinsic pathway is characterized by binding of

ligand to a receptor (e.g. CD95L and CD95) initiating limited proteolysis (e.g. BID to tBID)

to set a BH3 domain free to start the pro-apoptotic Bcl-2 process.

Figure 8: Bcl-2 proteins are subdivided into two groups. The Bcl-2 members are anti-apoptotic and have an obligatory BH4 domain (Bcl-2, Mcl-1). A membrane anchor is facultative. The BH3 proteins do not have a BH4 domain, the proteins are characterized by the pro-apoptotic BH3 domain (BID, BAX). A membrane anchor is also here facultative. Some members have only the BH3 domain and are called BH3-only proteins (Bim). (www.expasy.ch)

Table 2: Depicted are the Members of the Bcl-2 protein family. Examined proteins in this study are indicated in bold letters. On the left side is the anti-apoptotic group and on the right side is the pro-apoptotic group. (www.expasy.ch)

22

BH1 BH2BH3

Antiapototic

BH1 BH2BH3

Proapototic

Membrane anchor (facultative)

BH3

BH4 BH1BH1 BH2BH3BH3

Antiapototic

BH1BH1 BH2BH3BH3

Proapototic

Membrane anchor (facultative)

BH3BH3

BH4

Antiapoptotic:

Bcl-2Bcl-xlBcl-wDiva/BooBfl-1Bok/MtdMcl-1 Bak

Proapoptotic:

HrkBadBikNoxaBcl-xSBimBNIP3BidNix BaxPuma

Antiapoptotic:

Bcl-2Bcl-xlBcl-wDiva/BooBfl-1Bok/MtdMcl-1 Bak

Proapoptotic:

HrkBadBikNoxaBcl-xSBimBNIP3BidNix BaxPuma

This free BH3 domain leads to the activation of other down steam located pro-apoptotic Bcl-2

members and to an assembly of BAX in the outer mitochondrial membrane.

Figure 9: Shown is the activation cascade of the Bcl-2 system subdivided into the extrinsic and the intrinsic pathway. Extrinsic Pathway: The binding of a ligand to a receptor results in the activation of caspases (caspase 8 or 10), the activated caspases activate BID by limited proteolysis to tBID (truncated BID). tBID activates BAX which polymerises upon binding to cardiolipin and VDAG and forms channels into the outer mitochondrial membrane. Now, different pro-apoptotic factors are released to enhance the apoptotic process.Intrinsic pathway: Cellular stress leads to break down of microtubuli or other intracellular structures guarded by BH3-only proteins. These BH3 proteins are now released and inhibit the anti-apoptotic Bcl-2 or Mcl-1. This inhibition shifts the cell towards apoptosis.

23

Cell membrane FADD

CD95

Caspase-8, 10

BID tBID

BAX

BAX

Bcl-2

AIF

Smac/Diablo

Cyto-c

Bim

Bim

Mcl-1

Mitochondrion

Microtubuli

Extrinsic pathway Intrinsic pathway

Cell membrane FADD

CD95

Caspase-8, 10Caspase-8, 10

BID tBID

BAX

BAX

Bcl-2Bcl-2

AIFAIF

Smac/DiabloSmac/Diablo

Cyto-cCyto-c

Bim

BimBim

Mcl-1Mcl-1

Mitochondrion

Microtubuli

Extrinsic pathway Intrinsic pathway

The oligomerisation of BAX in the outer mitochondrial membrane leads to pore forming and

to set free of components from the intercristary space (cytochrome c, AIF, smac, Diablo).

These intercristary factors are responsible for activation of the apoptotic pathway by caspase

activation (cytochrome c, smac, Diablo) (41) and DNA degradation (AIF) (41). The intrinsic

pathway consists of many BH3-only members co-associated with cyto-skeletal structures (e.g.

microtubuli, endoplasmic reticula, golgi apparatus and nuclear membrane).

These BH3-only proteins (e.g. bim, nix, bik) are set free by disturbances of these internal

structures leading to the activation of BAX (41). In contrast the anti-apoptotic Bcl-2 and Mcl-

1 inhibit the assembly of BAX to form pores in the outer mitochondrial wall (see figure 9).

The specific interaction between the different Bcl-2 members remains still unclear. How this

highly complex intracellular “complement system” precisely works is subject of further

studies.

Therefore, we analysed the expression pattern of Bid, BAX, Bcl-2 and Mcl-1 in PMN. Bid

stands for receptor mediated (extrinsic) Bcl-2 dependent apoptosis and Mcl-1 inhibits BH3-

only proteins from the intrinsic pathway.

24

2. Material and Methods

2.1 Patients

The study population consisted patients admitted to the Surgical Intensive Care Unit of the

University Hospital Zürich. The Acute Physiology and Chronic Health Evaluation (APACHE

II) score at admission was 23.7 ± 5.6 (range 15–36) points (42). Severe sepsis was diagnosed

if all criteria of SIRS, evidence of two or more organ dysfunctions, and a proven septic focus

were present (43). Infection was due to pneumonia, peritonitis, meningitis, or abort. Isolated

microorganisms included mostly gram-negative bacteria, but also gram-positive bacteria in a

ratio 3:1. The overall mortality of these septic patients was 45%, these patients died due to

septic multiple organ failure. The group of healthy individuals was comparable with that of

patients with sepsis with regard to age and sex. All patients were enrolled into this study

under informed consent guidelines approved by the Human Ethical Committee of the

University of Zurich.

2.2 Isolation and Culture of Neutrophil Granulocytes

Fresh heparinized blood from healthy individuals (n=7) was diluted 1:1 (v/v) with HBSS

(Invitrogen Corp., Paisley, United Kingdom), layered over Ficoll-Histopaque® (Histopaque®-

1077, Sigma), and centrifuged at 4 °C, 800 x g for 20 min. The erythrocyte/granulocyte

containing pellet was diluted in 1:10 (v/v) ammonium chloride-EDTA (155 mM NH4Cl, 10

mM NaHCO3, 11 mM EDTA, pH 7.6) and stored for 30 min on ice for the lysis of

erythrocytes as described previously.(44-46) After centrifugation the neutrophils were

adjusted to a density of 1 x 106 cells/mL in RPMI 1640 (Invitrogen Corp., Paisley, United

Kingdom) containing 10 % FCS, Gentamycin (Invitrogen Corp.) and Glutamax (Invitrogen

Corp.) (0.1 mg/mL each) in polypropylene Falcon® tubes (Becton Dickinson Basel,

Switzerland) and cultured at 37 °C and 5 % CO2 for the times indicated.

25

2.3 Quantification of Apoptosis.

Determination of apoptosis and secondary necrosis utilizes the high affinity of Annexin-V for

phosphatidylserine which is exposed on the surface of apoptotic cells (47). After incubation

period of 16-22 hours PMN were washed with phosphate-buffered saline, resuspended in

binding buffer (10 mM HEPES/ NaOH, 140 mM NaCl, 2.5 mM CaCl2, pH 7.4) and incubated

with 0.25 mg/mL FITC-conjugated Annexin-V and 10 mg/mL PI. The mixture was kept on

ice for 5 min., and the cell fluorescence was measured by two-parameter flow cytometry

(FACS Calibur, Becton Dickinson, Basel, Switzerland) (48). When green fluorescence (FITC)

was plotted against red fluorescence (PI) three distinct cell populations could be detected in a

dotplot: viable cells (FITC-/PI-), apoptotic cells (FITC+/PI-) and secondary necrotic cells

(FITC+/PI+) (48). A minimum of 10,000 events was counted per sample and data reported as

the percentage of apoptotic cells (Annexin-V-FITC+/PI-).

2.4 Flow Assisted Cytometry of Toll-like Receptors 2 and 4

Isolated cells were maintained in RPMI 1640-medium with 10% fetal calf serum (FCS; Gibco

BRL) supplemented with 1.5mmol/L L-Glutamax (Gibco BRL) at a concentration of 1x106

neutrophils/mL in 24-well cell culture plates (Costar Co., Cambridge, MA) at 37°C in a

humidified atmosphere (5% CO2). Cells were incubated for 4 and 16 hours with or without

LPS (1g/mL) or MALP-2 (2nM).

Measurement of TLR-2 / -4 expression were carried out within the first 24 hours after

diagnosis of sepsis, defined by the following clinical parameters (defined septic focus and

fulfillment of all criteria of SIRS (fever, tachycardia, tachypnea or hypocapnia, leukocytosis)

(49). For measurement of TLR expression on human leukocytes neutrophils (1 x 106/mL

each) were washed in sample buffer (PBS containing glucose (1g/L)) at the end of

experiment. Phycoerythrine (PE) fluorescence of individual cells was measured using a

FACS-Calibur flow cytometer (Becton Dickinson AG, Basel, Switzerland), while gating on

26

physical parameters to exclude cell debris. A minimum of 10,000 events per gate was counted

per sample. Results are reported as the mean fluorescence corrected by subtracting the

fluorescence of cells stained with the respective PE-labeled isotype control antibody

(eBioscience, Wembley, UK).

2.5 Analysis of MAP-Kinases

2.5.1 Experimental Protocol

Isolated neutrophils were maintained in RPMI 1640-medium with 10 % fetal calf serum

(FCS; Gibco BRL) supplemented with 1.5 mmol/L L-Glutamax (Gibco BRL) at a

concentration of 1 x 106 cells/mL in 24-well cell culture plates (Costar Co., Cambridge, MA)

at 37 °C in a humidified atmosphere (5 % CO2). Cells were preincubated for one hour with or

without herbimycin (1 50 PD98059 (Alexis Läufelfingen, CH) (50 51 and

SB203580 (Alexis Läufelfingen, CH) (5 52 following stimulation with or without

LPS (1 g/mL) or IFN- (biological activity 3.0 x 107 U/mg, Boehringer-Ingelheim, Austria)

(10 ng/mL). The concentrations of IFN- were similar to those detected in the circulation of

patients with severe sepsis (53). Herbimycin and vanadate were used in concentrations which

have been found to completely inhibit protein tyrosine kinase and protein-phosphotyrosine

phosphatase, respectively (50, 54).

2.5.2 Westernblot Analysis of Neutrophil MAP-Kinases

For analysis of phosphorylated MAP kinases in neutrophils, cells (1 × 106) were centrifuged

and frozen immediately in liquid nitrogen at the end of experiment and were stored at −80°C

until further processing. Neutrophils (1 × 106) were resuspended in 100 L of Laemmli buffer

and were subsequently boiled for 10 min. Equal amounts of whole cell lysate (10 L/lane

corresponding to 1 × 105 cells) were separated by SDS-PAGE in 10% polyacrylamide gels in

a Mini Protean II chamber (Bio-Rad, Hercules, CA). Proteins were subsequently

27

electrotransferred onto a nylon membrane (Immobilon P; Millipore, Bedford, MA) in a Mini

Trans Blot transfer chamber (Bio-Rad), and membranes were blocked overnight at 4°C in

Tris-buffered saline supplemented with 0.1% Tween-20 and 2% milk diluent (KPL,

Gaithersburg, MD). The phosphorylated MAP kinases ERK and p38 were detected using

specific antibodies from New England Biolabs (Beverly, MA) and a peroxidase- coupled goat

anti-rabbit secondary antibody (Dako, Glostrup, Denmark). The total MAP kinases p42/44

ERK and p38 protein was detected using antibodies from Santa Cruz (Santa Cruz

Biotechnology, Santa Cruz, CA) and respective peroxidasecoupled secondary antibodies

(Dako). Specific binding was visualized by enhanced chemiluminescence (Amersham,

Buckinghamshire, UK) following the manufacturer’s recommendations. The molecular

weight of the protein bands was determined by use of prestained low-molecular-weight

markers (Sigma Chemical) on the same gel.

2.6 Analysis of cIAP2 Protein

2.6.1 Experimental Protocol

PMN were preincubated either with or without LPS (1 μg/mL) for 6 hours and then stimulated

with an agonistic aCD95 antibody (100 ng/mL) for another 16 hours or incubated in medium

alone for a total of 22 hours. The proteasome was inhibited with the proteasome-inhibitor

(PSI, 30 μM, [N-carbobenoxy-L-isoleucyl-L-γ-t-butyl-L-glutamyl-L-alanyl-L-leucinal]

Calbiochem). 1 hour prior to activation of CD95 (39). Following incubation cells were

harvested for westernblot and caspase-3-activity measurements, shock frozen in liquid N2 and

stored at –80°C until further use. In parallel, neutrophil apoptosis after 22 hours was measured

by flow cytometry. The timepoints for cIAP2 mRNA expression analysis were set at 0, 1, 2

and 4 hours, either with or without LPS incubation. The expression of cIAP2 protein was

analyzed after incubation with medium, LPS or aCD95 at 0, 2 and 4 hours.

2.6.2 Caspase-3-Activity Measurement.

28

Caspase-3 activity was measured in cellular extracts from neutrophil samples. After

incubation, neutrophils were lysed by freeze-thaw procedure in hypotonic extraction buffer

(25 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid), 5 mM MgCl2, 0.1 %

Triton-X 100, pH 7.5, with addition of 1 mM Pefablock® pepstatin, leupeptin, and aprotinin (1

M each)), subsequently centrifuged (15 min, 14,000 x g, 4 °C), and the supernatant stored at

–80 °C until further used (46). The fluorometric cleavage assay for caspase-3-like activity

(DEVD-afc, Calbiochem) was carried out in microtiter plates (Greiner, Nuertingen,

Germany) according to the method described by Thornberry (33) using the fluorometric plate

reader Victor-2 (Fluostar, Dr. Gurath GmbH, Germany) with the excitation wavelength set at

385 nm and an emission wavelength of 505 nm (33). The protein concentrations of the

respective samples were measured with a commercially available kit (Pierce Assay, Pierce,

United Kingdom), and caspase-3 activity was calculated as units (U) per mg protein with 1

U/mg being equal to the cleavage of 1 µmol 7-amino-4-trifluromethylcoumarin (afc) per mg

protein and minute (46).

2.6.3 Detection of cIAP2 mRNA by RT-PCR.

29

For detection of specific mRNA expression DNA was isolated from neutrophils by Trizol

method following standard procedure. Briefly, neutrophils (2.5 x 107) were centrifuged after

the experiments and frozen (-80°C) until further use. For isolation of mRNA cells were

thawed in Trizol (Invitrogen) and total mRNA isolated by TRIZOL-method following

manufacturers recommendation. After DNAse (DNase I, Roche) and RNase Inhibitor (RNase

Inhibitor, Invitrogen) treatment, mRNA was transcribed to cDNA by RT-reaction. The primer

for cIAP2 and -Actin were designed with the program Primer Express (Applied Biosystems,

Foster City, CA) using the cIAP2 sequence (NCBI accession number: XM_040715) as a

template. For detection of -Actin the primers derived from the sequence (NCBI accession

number: XM_063487) (see Table 3.) were used. All primers were purchased from Microsynth

(Microsynth, Balgach, Switzerland). A total of 5 g mRNA was transcribed with reverse

transcriptase (Superscript TM-II, Invitrogen) into cDNA. For the PCR reaction 0.1 g cDNA

was amplified with specific primers, nucleotides and polymerase (Taq Polymerase,

Invitrogen) for a total of 38 or 34 cycles, respectively. The amplified cDNA was separated by

electrophoresis on a 1.8 % agarosegel and visualized under UV after ethidiumbromide (0.5 %)

staining.

2.6.4 Westernblot Analysis of cIAP2 Protein

30

Whole cell lysates (corresponding to 1.25 x 105 cells) were loaded on 10 % polyacrylamide

gels (Mini Protean II, BioRad, Hercules, CA) in Laemmli buffer (Sample Buffer Laemmli,

Sigma). Proteins were separated at 40 mA for 50 min and then transferred onto PVDF

membranes (BioRad) for 60 min at 100V. Membranes were blocked in TBST with 0.2 %

Bovine Serum Albumin (Sigma) overnight, and then incubated either with polyclonal goat

anti-human caspase-3 (Santa Cruz Biotechnology, Santa Cruz, CA), polyclonal rabbit anti-

human Ubiquitin (Santa Cruz) or polyclonal goat anti-human cIAP2 (Santa Cruz) for 4 hours

at room temperature, washed with TBST and then exposed to a secondary HRP conjugated

antibody for 1 hour at room temperature. Chemiluminescence was detected by ECL

(Amersham Biosciences, Buckinghamshire, United Kingdom) on a scientific imaging film

(Kodak X-Omat AR Film, Kodak, Lausanne, Switzerland).

2.7 Analysis of Bcl-2Proteins

2.7.1. Experimental Protocol

Heparinized blood (20 U heparin/mL; heparin was tested for endotoxin: < 5 pg/mL heparin)

obtained from the patients at day of sepsis diagnosis, or from healthy controls was diluted 1:1

with RPMI 1640 medium (Gibco BRL, Life Technologies, Paisley, Scotland). Neutrophils

were isolated by density centrifugation in Histopaque-1077 (Sigma Chemical Co.) followed

by two washing steps in phosphate buffered saline (PBS) as previously described (56-58).

Lysis of residual erythrocytes was performed using nine volumes of an ice-cold isotonic

ammonium chloride solution (NH4Cl 155 mM, KHCO3 10 mM, EDTA 0.1 mM) to one

volume of cell pellet at 0 °C for 20 minutes. The final neutrophil preparation contained > 95%

neutrophils as was determined by flow cytometry analysis using fluorescein isothiocyanate-

labeled monoclonal antibody anti-CD15 (Coulter, Hialeah, FL). Cell viability was > 98% as

determined by trypan blue exclusion. The cell loss in neutrophil cultures was < 5%

31

irrespective of the experimental design or the added proteins using trypan blue exclusion and

microscopic cell counting.

Isolated neutrophils were maintained in RPMI 1640-medium with 10% fetal calf serum (FCS;

Gibco BRL) supplemented with 1.5 mmol/L L-Glutamax (Gibco BRL) at a concentration of 1

x 106 cells/mL in 24-well cell culture plates (Costar Co., Cambridge, MA) at 37 °C in a

humidified atmosphere (5% CO2). Cells were stimulated with or without LPS (1 g/mL) or

( aCD95) (100 ng/mL) for the times indicated.

2.7.2 Analysis of PMN Apoptosis

For measurement of DNA fragmentation neutrophils (1 x 106/mL) were washed in sample

buffer (PBS containing glucose (1 g/L)) at the end of experiment. Cells were fixed in 1 mL of

70% ethanol over 12 hours at 4°C. Fixed cells were incubated in 1 mL propidium iodide (PI)

staining solution (sample buffer with 50 µg/ml propidium iodide and 100 U/ml RNase A

(Boehringer)) at room temperature. Propidium iodide fluorescence of individual cells was

measured using an FACS-Calibur flow cytometer (Becton Dickinson), while gating on

physical parameters to exclude cell debris. A minimum of 10,000 events was counted per

sample. Results are reported as the percentage of hypodiploid (fragmented) nuclei reflecting

the relative proportion of apoptotic cells (55-58).

2.7.3 Western-Blot Analysis of Bcl-2 Proteins

For analysis of Bcl-2 proteins in neutrophils, cells (1 x 106) were resuspended in lysis buffer

(PBS supplemented with AEBSF (1 mM) and Leupeptin (1 mg/mL)), centrifuged and frozen

immediately in liquid nitrogen at the end of experiment and stored at –80 °C until further

processing. Neutrophils (1 x 106) were resuspended in Lämmli buffer (100 l) and

subsequently boiled for 10 min. Equal amounts of whole cell lysate (10 l/lane corresponding

to 1 x 105 cells) were separated by SDS-PAGE in 10 % polyacrylamide gels in a Mini Protean

II chamber (BioRad, Hercules, CA). Proteins were subsequently electrotransferred onto a

32

nylon membrane (Immobilon P, Millipore, Bedford, MA) in a Mini Trans Blot transfer

chamber (BioRad) and membranes were blocked overnight at 4 °C in TBS supplemented with

0.1 % Tween-20 and 0.2 % BSA (Sigma). The proteins Bcl-2 and BAX were detected using

specific antibodies from New England Biolabs (Beverly, MA) and a peroxidase coupled goat-

anti-rabbit secondary antibody (Dako, Glostrup, Denmark). The Bid protein was detected

using antibody from Santa Cruz (Santa Cruz, CA, USA) and Mcl-1 by an antibody from

Trevigen Inc. (Gaithersburg, MD, USA) and their respective peroxidase coupled secondary

antibodies (Dako, Denmark). Specific binding was visualized by enhanced

chemiluminescence (Amersham, Buckinghamshire, UK) on X-ray film (Kodak) following

manufacturers recommendations. The molecular weight of the protein bands were determined

by use of prestained low-molecular-weight markers (Sigma) on the same gel. The amount of

specific protein was quantified by densitometry. Staining of X-ray film was measured with an

Imaging system (AlphaInnotech, San Leandro, CA) and the relative density of bands is given

in mean ± SD (%) of the 0 hour value (set at 100 %) after subtraction of the specific

background.

2.7.4 Detection of mRNA of Bcl-2 Proteins by RT-PCR

For detection of specific mRNA expression DNA was isolated from neutrophils by Trizol

method following standard procedure. Briefly, neutrophils (2.5 x 107) were centrifuged after

the experiments and frozen (-80°C). Cells were thawed in Trizol (Invitrogen). Total mRNA

was isolated from whole cells by TRIZOL method and transcribed to cDNA after DNAse

treatment by RT-reaction. PCR was carried out with primers specific for Bcl-2, BAX, Bid and

Mcl-1 (see Table 3.), and visualized with ethidiumbromide after electrophoresis on a 1.8 %

agarose gel. The primer for Bcl-2, BAX, Bid and Mcl-1 were designed with the program

33

Primer Express (Applied Biosystems) and the published sequence (M14745, NM_138761,

NM_001196, and NM_021960 respectively). A total of 5 g mRNA was transcribed with

reverse Transcriptase (Superscript TM-II, Invitrogen) into cDNA. For the PCR reaction 0.1

g cDNA were incubated with specific primers and polymerase (Taq Polymerase, Invitrogen)

for 35 (34) cycles. The amplified cDNA, together with an amplified house-keeping gene -

Actin was separated by agarose electrophoresis and visualized under UV after

ethidiumbromide staining. Detection limit of PCR was defined as the lowest cyclenumber that

yielded a clear positive band for the positive control sample (e.g. THP-1 cells).

2.8 Statistical Analysis

Data are presented as mean ± SEM. Mean values were compared using Student two-tailed t-

test for independent means. Differences were regarded as significant, if p < 0.05.

34

5’-CTTCCGTAATTAGGAACCTG-3’5’-CTTGCATATAATGAAGTGAA-3’

NM_021960Mcl-1

5’-AAGTTCCTACCACTGTGCAATG-3’ 5’-CAAGTAGATGAGGGAACTGGC-3’

XM_040715 cIAP2

5‘-ATGGACTGTGAGGTCAAC-3‘5‘-AGTCCATCCCATTTCTGG-3‘

NM_001196BID

5’-AGATGTCCAGGCAGCTGCAC-3’5’-TGTTGACTTCACTTGTGGCC-3’

M14745Bcl-2

5’-GACCCGGTGCCTCAGGA-3’5‘-ATGGTCACGGTCTGCCA-3‘

NM_138761BAX

5’-AGCGGGAAAT GTGCATG-3’ 5’-CAGGGTACCTGGTGGTGCC-3’

XM_063487 -Actin

Forward primerReverse primer

NCBI accession numberProtein

5’-CTTCCGTAATTAGGAACCTG-3’5’-CTTGCATATAATGAAGTGAA-3’

NM_021960Mcl-1

5’-AAGTTCCTACCACTGTGCAATG-3’ 5’-CAAGTAGATGAGGGAACTGGC-3’

XM_040715 cIAP2

5‘-ATGGACTGTGAGGTCAAC-3‘5‘-AGTCCATCCCATTTCTGG-3‘

NM_001196BID

5’-AGATGTCCAGGCAGCTGCAC-3’5’-TGTTGACTTCACTTGTGGCC-3’

M14745Bcl-2

5’-GACCCGGTGCCTCAGGA-3’5‘-ATGGTCACGGTCTGCCA-3‘

NM_138761BAX

5’-AGCGGGAAAT GTGCATG-3’ 5’-CAGGGTACCTGGTGGTGCC-3’

XM_063487 -Actin

Forward primerReverse primer

NCBI accession numberProtein

Table 3: The List of used primers in our study. Depicted is the name of the protein, the NCBI accession number and the forward and reverse primer.

35

3.0 Results

3.0.1 Apoptosis is Reduced in PMN from Septic Patients

Spontaneous apoptosis in neutrophils from patients with sepsis (28.8% ± 4.9%) was

significantly lower after 16 hours of incubation compared with that in controls (64.0% ±

2.8%). Incubation with endotoxin (LPS, 1 g/mL) further reduced apoptosis in neutrophils

from patients (19.5% ± 4.4%) and controls (35.9% ± 3.2%). In contrast, incubation of

neutrophils from septic patients with the agonistic anti-CD95 antibody (100 ng/mL)

completely restored the life span of those cells to levels seen in unstimulated controls (64.3%

± 6.8%). Stimulation of neutrophils from healthy controls with the agonistic anti-CD95

antibody significantly (p < 0.05) enhanced apoptosis after 16 hours to 92.9% ± 0.8%.

3.1.1 Expression of TLR-2 and TLR-4 on Freshly Isolated Leukocytes

In comparison with cells from healthy controls, leukocytes from patients expressed

significantly (p < 0.05) increased amounts of TLR-2 and TLR-4 receptors. TLR-specific

fluorescence was calculated by subtraction of fluorescence from the respective isotype control

(IgG) from specific TLR-2 or TLR-4 fluorescence. No differences between patients and

controls were seen for mean fluorescence of isotype IgG control on PMN (10.3 ± 1.9 and 10.0

± 1.5) or monocytes (10.8 ± 3.2 and 8.6 ± 0.7). On freshly isolated PMN from patients with

sepsis, the mean fluorescence was 78.0 ± 18.6 for TLR-2 and 11.4 ± 2.3 for TLR-4, whereas

on control PMN the mean fluorescence was 12.8 ± 2.2 for TLR-2 and 2.3 ± 0.4 for TLR-4.

Subgroup analysis of survivors (n = 14) versus nonsurvivors (n = 7) within the patient cohort

revealed no significant differences in TLR-2 (79.4 ± 25.7 vs. 75.1 ± 22.6 in survivors versus

nonsurvivors) or TLR-4 (9.8 ± 2.5 vs. 14.9 ± 4.9, respectively) expression.

36

IgGTLR-4 TLR-2

IgG

TLR-4TLR-2

A

B

ControlsPatients

TLR-2 TLR-4

mea

nflu

ores

cenc

e

0

20

40

60

80

100 *

* C

IgGTLR-4 TLR-2

IgG

TLR-4TLR-2

A

B

ControlsPatients

TLR-2 TLR-4

mea

nflu

ores

cenc

e

0

20

40

60

80

100 *

*

ControlsPatients

TLR-2 TLR-4

mea

nflu

ores

cenc

e

0

20

40

60

80

100 *

** C

Figure 10: Expression of TLR-2 and TLR-4 receptors on PMN. Freshly isolated human neutrophil granulocytes (PMN) from a healthy control (n = 12) (A) or a patient with sepsis (n = 21) (B) were incubated with PE-labeled anti-IgG (IgG), anti-TLR-2, or anti-TLR-4 antibodies. Specific fluorescence was measured in FACS. Representative PE-fluorescence histograms for each group are shown. Expression of TLR-2 and TLR-4 receptors on freshly isolated PMN (C). Mean fluorescence was determined by subtraction of nonspecific PE-fluorescence from TLR specific fluorescence. *p < 0.05, patients versus controls.

3.1.2. Ligand Binding and Receptor Expression

The increased TLR-2 and TLR-4 expression on leukocytes from patients with sepsis might be

a result of previous contact with endotoxins. Therefore, the expression of TLR-2 and TLR-4

on PMN was investigated after incubation with their respective ligands, MALP-2 and LPS.

Isolated PMN (1 × 106/mL) were incubated with either medium, MALP-2 (2 nM) or LPS (1

g/mL) for 4 and 16 h, and TLR-2 and TLR-4 expression was measured in FACS.

37

Medium

0 2 4 6 8 10 12 14 16

mea

nflu

ores

cenc

e

0

100

200

300

LPS

0 2 4 6 8 10 12 14 16

mea

nflu

ores

cenc

e

0

100

200

300

MALP-2

hours

0 2 4 6 8 10 12 14 16

mea

nflu

ores

cenc

e

0

100

200

300

TLR-2

*

*

*

*

**

*

**

Medium

0 2 4 6 8 10 12 14 160

20

40

60

80

100

LPS

0 2 4 6 8 10 12 14 160

20

40

60

80

100

MALP-2

hours

0 2 4 6 8 10 12 14 160

20

40

60

80

100

TLR-4

**

*

*

*

*

*

*

*

Medium

0 2 4 6 8 10 12 14 16

mea

nflu

ores

cenc

e

0

100

200

300

LPS

0 2 4 6 8 10 12 14 16

mea

nflu

ores

cenc

e

0

100

200

300

MALP-2

hours

0 2 4 6 8 10 12 14 16

mea

nflu

ores

cenc

e

0

100

200

300

TLR-2

*

*

*

*

**

*

**

Medium

0 2 4 6 8 10 12 14 160

20

40

60

80

100

LPS

0 2 4 6 8 10 12 14 160

20

40

60

80

100

MALP-2

hours

0 2 4 6 8 10 12 14 160

20

40

60

80

100

TLR-4

**

*

*

*

*

*

*

*

Figure 11: TLR-2 and TLR-4 receptor expression on incubated PMN. Isolated neutrophil granulocytes (PMN) from healthy controls (●) or patients with sepsis (○) were incubated with medium, LPS (1 μg/mL), or MALP-2 (2 nM) for 4 and 16 h. Mean fluorescence was determined by subtraction of nonspecific PE-fluorescence from TLR-2- or TLR-4-specific fluorescence. *p < 0.05, patients versus controls.

In all cells the expression of TLRs increased after 4 h, independent of the stimulus and cell

population, with a stronger increase in cells from patients than in cells from controls.

However, in leukocytes from patients with sepsis, a non-significant decline of TLR

expression was seen between 4 h and 16 h of incubation on PMN. Compared with medium

alone, incubation with the TLR-2 ligand MALP-2 (2 nM) or TLR-4 ligand LPS (1 g/mL)

had no effect on expression of TLR-2 or TLR-4 on PMN. This was seen not only in cells from

controls but also in cells from patients with sepsis after 4 h as well as 16 h of incubation. This

38

indicates that ligand binding may not be responsible for up-regulation or downregulation of

respective TLRs on leukocytes in patients with sepsis.

3.2 MAP-Kinases and PMN Apoptosis

3.2.1 Effect of Herbimycin on PMN apoptosis

The influence of tyrosine kinase blockade in neutrophil apoptosis was investigated using the

kinase inhibitor herbimycin. Spontaneous neutrophil apoptosis in patients with sepsis (36.0%

± 3.1%) was significantly lower than in neutrophils from healthy individuals (72.1% ± 2.5%)

after 16 h of incubation. Ex vivo stimulation of neutrophils with LPS (1 g/mL) for 16h

significantly reduced neutrophil DNA fragmentation in controls from 72.1% ± 2.5% to 39.9%

± 3.9% and in patients from 36.0% ± 3.1% to 20.8% ± 2.8%. Neutrophils incubated with IFN-

(10 ng/mL) for 16 h showed a significant reduction (p < 0.05) of spontaneous neutrophil

DNA fragmentation to 26.2% ± 1.8% in healthy controls and to 15.4% ± 1.1% in patients

with sepsis. Preincubation of cells with herbimycin (1 M) abrogated (p < 0.05) the LPS

(70.9% ± 2.8%) or IFN- (60.3% ± 3.5%) effect on neutrophil DNA fragmentation in

neutrophils from healthy controls and reconstituted apoptosis in neutrophils from patients

with sepsis to the level of spontaneous apoptosis (40.7% ± 3.7% and 23.9% ± 2.2%,

respectively). However, herbimycin failed to fully reconstitute the rate of DNA fragmentation

to levels seen in healthy controls, indicating that other mechanisms might be involved in

patients with sepsis. Furthermore, the anti-apoptotic effect of IFN- could not be fully

abrogated by the use of herbimycin, indicating that the LPS- and IFN--mediated effect might

be regulated by different kinases. Herbimycin itself had no effect on spontaneous neutrophil

apoptosis.

39

MediumLPSIFN-

Apoptosis[% PI]

controlsn=9

SIRSn=9

sepsisn=9

0

20

40

60

80

100

†

*

*

*

*

*

†

MediumLPSIFN-

Apoptosis[% PI]

controlsn=9

SIRSn=9

sepsisn=9

0

20

40

60

80

100

†

*

*

*

*

*

†

Figure 12: Influence of LPS and IFN- on apoptosis of neutrophil granulocytes. Both, LPS and IFN-, Inhibit spontaneous apoptosis of PMN in healthy individuals (controls), patients with SIRS and patients with sepsis. Data are given as Mean ± SEM,*p < 0.05 spontaneous vs. stimulus, † p < 0.05 controls vs. patients

3.2.2 Participation of Phosphatases in the Regulation of PMN apoptosis

Phosphatases have been shown to regulate the activity of protein kinases (27). To investigate

whether phosphatases influence the activity of kinases in the signal transduction of

spontaneous and endotoxin-mediated apoptosis, neutrophils were preincubated with the

phosphatase inhibitor vanadate (5 M) for 1 h before stimulation with or without LPS (1

g/mL) for up to 16 h. However, vanadate had no effect on spontaneous or endotoxin-

mediated neutrophil apoptosis in healthy controls as well as in patients with sepsis.

40

PatientsControls

Apo

ptos

is [%

PI]

0

20

40

60

80

100

*

*

†

0

20

40

60

80

100Medium Herbimycin

IFN-IFN-- -

0

20

40

60

80

100

0

20

40

60

80

100

†

Apo

ptos

is [%

PI]

*

†

*

†

LPSLPS- -

‡

‡

‡

‡

A

B

PatientsControls

Apo

ptos

is [%

PI]

0

20

40

60

80

100

*

*

†

0

20

40

60

80

100Medium Herbimycin

IFN-IFN-- -

0

20

40

60

80

100

0

20

40

60

80

100

†

Apo

ptos

is [%

PI]

*

†

*

†

LPSLPS- -

‡

‡

‡

‡

A

B

Figure 13: Influence of herbimycin (1 μM/mL) on apoptosis of neutrophils from healthy individuals (controls, n = 10) and patients with sepsis (patients, n = 18) in the absence or presence of LPS (1 μg/mL, A) or IFN- (10 ng/mL, B). DNA fragmentation was analyzed by flow cytometry after staining with PI. Results are depicted as mean ± SEM. *p < 0.05, stimulus vs. medium control; †p < 0.05, herbimycin vs. stimulus; ‡p < 0.05, patient vs. respective control.

3.2.3 Effect of MAP-Kinase Inhibitors on LPS and IFN--Mediated PMN Apoptosis

41

To further analyze the involvement of MAP kinases in the regulation of neutrophil apoptosis,

cells from 10 patients were preincubated for 1 h with the ERK kinase inhibitor PD98059 (50

M) (19) or the p38 MAPK inhibitor SB203580 (5M) (60) before stimulation with LPS (1

g/mL) or IFN- (10ng/mL). In healthy controls, the LPS-reduced apoptosis (33.1% ± 3.3%)

was reconstituted (52.6% ± 3.7%) after incubation with PD98059 (p < 0.05), but not with the

p38 inhibitor SB203580 (35.3% ± 6.5%). In cells incubated with IFN- (24.7% ± 3.5%),

PD98059 only slightly increased apoptosis (34.1% ± 3.1%), but inhibition of p38 with

SB203580 significantly (p < 0.05) increased apoptosis (51.3% ± 7.7%). Whereas PD98059

alone (60.2% ± 2.4%) had no effect on spontaneous neutrophil apoptosis, incubation with

SB203580 increased spontaneous apoptosis in neutrophils to 82.4% ± 3.3%. In neutrophils

from patients with sepsis, incubation with LPS (16.9% ± 2.3%) reduced spontaneous

apoptosis (27.2% ± 3.1%) by 37.9% and incubation with IFN- (13.9% ± 2.0%) by 48.9%.

Inhibition of ERK with PD98059 fully restored the LPS-induced (26.4% ± 3.5%), but not the

IFN--induced (18.4% ± 3.1%) reduction of neutrophil apoptosis. The p38 MAPK inhibitor

SB203580 alone enhanced spontaneous apoptosis (42.0% ± 5.8%), and slightly decreased

LPS-induced apoptosis to 12.5% ± 2.9%. Similar to controls, the IFN-- mediated reduction

of apoptosis was abrogated (25.6% ± 5.8%) by incubation with SB203580. However, neither

PD98059 nor SB203580 restored reduced apoptosis in neutrophil from patients with sepsis to

levels seen in controls.

42

Apo

ptos

is [%

PI]

- LPS IFN-

0

20

40

60

80

100

*

*

**

Controls

***

*

*

0

20

40

60

80

100

- LPS IFN-

Apo

ptos

is [%

PI]

Patients

††

†

† †

Medium

PD98059

SB203580

Apo

ptos

is [%

PI]

- LPS IFN-

0

20

40

60

80

100

*

*

**

Controls

***

*

*

0

20

40

60

80

100

- LPS IFN-

Apo

ptos

is [%

PI]

Patients

††

†

† †

Medium

PD98059

SB203580

Figure 14: Influence of MAP kinase inhibitors PD98059 (50 μM) and SB203580 (5 μM) on apoptosis of neutrophils from healthy individuals (controls, n = 10,) and patients with sepsis (patients, n = 10,) incubated with medium (medium), LPS (1 μg/mL), or IFN- (10 ng/mL). DNA fragmentation was analyzed by flow cytometry after staining with PI. Data are depicted as mean ± SEM. *p < 0.05, stimulus vs. medium control; †p < 0.05, inhibitor vs. respective control.

3.2.4 Detection of Phosphorylated ERK and p38 MAP-Kinases in PMN

43

Phosphorylation of the MAP kinase p42/44 ERK and p38 was analyzed by Western blot in

neutrophil cell lysates separated by SDS-PAGE. As a loading control, the same samples run

in parallel were probed with antibodies specific for total p42/44 ERK or p38 protein. The

phosphorylated form of p42/44 ERK and p38 MAP kinases were both found in neutrophils

freshly isolated from patients with sepsis as well as from controls, with phosphorylation of

p38 and ERK being more intense in neutrophils from patients than from controls. In contrast

to medium alone, incubation with LPS led to phosphorylation of p42/44 ERK and p38 both in

patients and controls, whereas IFN- did not. The inhibition of p42/44 ERK by PD98059 was

clearly seen, as PD98059 inhibits MEK1 kinase and thus prevents phosphorylation of p42/44

ERK. No reduction of p38 phosphorylation was seen in cells incubated with SB203580,

which is to be expected, as SB203580 inhibits p38 kinase activity, but not p38

phosphorylation (61). Interestingly, in neutrophils from patients, but not in controls,

phosphorylation of p42/44 ERK and p38 was seen after pretreatment with SB203580 and

subsequent stimulation with IFN-.

44

42

total ERK

Phosphorylated-ERKMW(kDa)

42ERK1ERK2

A B0 C A B C A B C

LPS IFN-Medium

ERK1ERK2

Control

total ERK

Phosphorylated-ERKPatient

A B0 C A B C A B C

LPS IFN-Medium

ERK1ERK2

ERK1ERK2

42

42

A: MediumB: PD98059C: SB203580

42

total ERK

Phosphorylated-ERKMW(kDa)

42ERK1ERK2

A B0 C A B C A B C

LPS IFN-Medium

ERK1ERK2

Control

total ERK

Phosphorylated-ERKPatient

A B0 C A B C A B C

LPS IFN-Medium

ERK1ERK2

ERK1ERK2

42

42

A: MediumB: PD98059C: SB203580

A: MediumB: PD98059C: SB203580

Figure 15: Phosphorylation of p42/44 ERK in neutrophils from healthy volunteers (control) or patients with sepsis (patient). PMN (1 × 106) were preincubated for 1 h with DMSO (0.1%, A), PD98059 (10 μM, B), or SB203580 (5 μM, C). Subsequently, cells were stimulated for 15 min with medium alone (medium), LPS (1 μg/mL), or IFN- (100 ng/mL). Equal amounts of protein from whole-cell lysates (5 × 105 cells/lane) were separated by SDS-PAGE (12%), and the phosphorylated (upper panel) and total ERK kinase (lower panel) were detected by immunoblotting. The molecular weight (MW) is indicated on the right.

45

Phosphorylated-p38MW(kDa)

p38

p38

total p38

42

42

A B0 C A B C A B C

LPS IFN-Medium

Control

LPS IFN-Medium

Phosphorylated-p38

MW(kDa)

p38

p38

total p38

A B0 C A B C A B C

42

42

Patient

Phosphorylated-p38MW(kDa)

p38

p38

total p38

42

42

A B0 C A B C A B C

LPS IFN-Medium

ControlPhosphorylated-p38

MW(kDa)

p38

p38

total p38

42

42

A B0 C A B C A B C

LPS IFN-Medium

Control

LPS IFN-Medium

Phosphorylated-p38

MW(kDa)

p38

p38

total p38

A B0 C A B C A B C

42

42

Patient

LPS IFN-Medium

Phosphorylated-p38

MW(kDa)

p38

p38

total p38

A B0 C A B C A B C

42

42

Patient

Figure 16: Phosphorylation of p38 in neutrophils from healthy volunteers (control) or patients with sepsis (patients). PMN (1 × 106) were preincubated for 1 h with DMSO (0.1%, A), PD98059 (10 μM, B), or SB203580 (5 μM, C). Subsequently, cells were stimulated for 15 min with medium alone (medium), LPS (1 μg/mL), or IFN- (100 ng/mL). Equal amounts of protein from whole-cell lysates (5 × 105 cells/lane) were separated by SDS-PAGE (12%), and the phosphorylated (upper panel) and total p38 kinase (lower panel) were detected by immunoblotting. The molecular weight (MW) is indicated on the right.

3.3 cIAP2 and Activity of Caspase-3 in Neutrophil Ganulocytes

46

3.3.1 LPS Induces cIAP2 mRNA and Protein in PMN

Incubation of neutrophils (1 x 106 / mL) with LPS (1 g/mL) induced upregulation of cIAP2

mRNA within one hour and up to four hours. In contrast, no cIAP2 mRNA was detected in

freshly isolated neutrophils, or when cells were incubated with medium alone. After four

hours incubation with LPS the expression of cIAP2 mRNA seemed to decline.

Figure 17: Experimental protocol. Neutrophil granulocytes (1 x 106/mL) were stimulated with and without LPS (1 g/mL) for 6 hours, followed by simulation with or without an agonistic aCD95 antibody (, 100 ng/mL) for another 16 hours, resulting in a total of 22 hours incubation. One hour before stimulation with agonistic aCD95 antibody the specific proteasome inhibitor PSI (30 M) was added.

Figure 18: LPS induces cIAP2 mRNA expression. PMN (1 x 106/mL) were incubated either in medium or with LPS (1 g/mL) at 37 °C for 1, 2 or 4 hours and cells were harvested subsequently. Total mRNA was isolated and transcribed in cDNA and 5 g/ml cDNA used as a template for PCR. cIAP2 PCR was run with 38 cycles and -Actin PCR run with 35 cycles in parallel. Amplified cDNA was separated on 1.8 % agarose gel and visualized under UV light after ethidiumbromide staining. M: Marker, C: Positive control. Representative blot of six separate experiments.For detection of cIAP2 protein expression corresponding samples were analyzed by

immunoblotting. Specific westernblots showed cIAP2-protein in freshly isolated cells with an

47

LPS

0-h

PSI

5-h

aCD95

6-h 22-h

LPS

0-h

PSI

5-h

aCD95

6-h 22-h

cIAP-2

-Actin

M 0h LPSC

1h

+-

2h

+-

4h

+-

cIAP-2

-Actin

M 0h LPSC

1h

+-

2h

+-

4h

+-

increased expression after two to four hours incubation with LPS. However, a reduced level

of cIAP2-protein was detected when cells were incubated with the agonistic aCD95 antibody

as well as in PMN incubated with medium alone indicating a possible degradation or decay of

cIAP2 protein.

Figure 19: Alterations in cIAP2 protein expression after LPS or CD95 stimulation. PMN (1 x 106/mL) were incubated either in medium, LPS (1 g/mL) or with an agonistic aCD95 antibody (, 100 ng/mL) at 37°C for 2 or 4 hours. At the end of experiment cells were harvested and lysed immediately. Whole cell lysate corresponding to 5 x 105 cells were separated by SDS-PAGE (10 %), blotted onto PVDF membrane and incubated with specific antibodies. cIAP2 specific bands were visualized by ECL after incubation with secondary HRP-coupled antibodies. Unspecific bands were taken as loading-control. (27)

3.3.2 LPS Induces Ubiquitination of Caspase-3 in PMN

IAPs have been shown to possess ubiquitination activity, which would destine the target

protein for degradation by the proteasome. Therefore ubiquitination of caspase-3 was

analyzed in neutrophils by westernblot after incubation with LPS or aCD95. Comparison of

ubiquitin- and caspase-3-specific westernblots of whole cell lysates from PMN incubated with

LPS revealed a parallel accumulation of a caspase-3-antibody positive band with same size

like ubiquitin-antibody positive band indicating that caspase-3 might be ubiquitinated after

48

- LPS aCD95

cIAP-2

Loadingcontrol

2h 4h

- LPS aCD95

0h

- LPS aCD95

cIAP-2

Loadingcontrol

2h 4h

- LPS aCD95

0h

incubation with LPS. Preincubation with LPS and subsequent stimulation with aCD95 also

revealed an accumulation of ubiquitinated caspase-3.

Figure 20: Ubiquitination of caspase-3 after stimulation of CD95 in neutrophils. Isolated PMN (1 x 106/mL) were preincubated with LPS (1 g/mL) for 5 hours, followed by PSI (30 M) for another hour before stimulation of PMN with or without an agonistic aCD95 antibody (100 ng/mL) for 16 hours. Whole cell lysate was separated by 10 % polyacrylamide SDS-PAGE and subsequently blotted onto PVDF membrane. Westernblots were analyzed for ubiquitin (A) or caspase-3 (B). Procaspase-3 and activated caspase-3 were detected at app. 31.6 kDa and 20 kDa and ubiquitinated proteins at 85 kDa. Unspecific bands were taken as loading-control. (27) Depicted westernblot represents one from six independent analyses.

The caspase-3-specific westernblot allowed discrimination of the pro-caspase-3 and caspase-3

bands. Incubation with LPS led to an increase of procaspase-3 without an increase of the

caspase-3 band. In contrast, a prominent increase of the caspase-3 band was seen in cells

incubated with aCD95. Ubiquitination of proteins induces degradation by the proteasome,

therefore cells were incubated with the proteasome inhibitor PSI (30 mM) one hour prior to

stimulation with aCD95. Whereas PSI had no effect on LPS-treated samples, inhibition of the

49

A

B

Procaspase-3Caspase-3

Loadingcontrol

UbiquitinatedCaspase-3

Loadingcontrol

Ubiquitin

PSI-

+ -+ -

+ -+ LPS

-- +

+ -- +

+

aCD95- - - - + + + +

Lane1 2 3 4 5 6 7 8MW89

89

32

A

B

Procaspase-3Caspase-3

Loadingcontrol

UbiquitinatedCaspase-3

Loadingcontrol

Ubiquitin

PSI-

+ -+ -

+ -+ LPS

-- +

+ -- +

+

aCD95- - - - + + + +

Lane1 2 3 4 5 6 7 8MW89

89

32

proteasome reduced the procaspase-3 and ubiquitinated caspase-3 band in aCD95 treated

samples preincubated with LPS. Shown is one of six blots performed.

3.3.3 Reduction of Spontaneous and CD95-Induced Apoptosis by LPS

The effect of PSI indicates an involvement of the proteasome in regulation of neutrophil

apoptosis. Therefore, neutrophil apoptosis was measured in FACS. Incubation of PMN

(1x106/mL) with LPS (1 g/mL) alone significantly decreased (24.8 ± 4.8 % apoptotic cells,

p < 0.05) spontaneous apoptosis (66.1 ± 2.3 % apoptotic cells, p < 0.05) after 22 hours.

Figure 21: LPS reduces spontaneous and CD95-induced PMN apoptosis. Isolated PMN (1 x 106/mL) were preincubated with LPS (1 g/mL) for 5 hours, followed by PSI (30 M) for another hour before stimulation of PMN with or without agonistic aCD95 antibody (100 ng/mL) for 16 hours. PMN apoptosis was detected by flow cytometry (FACS), after staining with Annexin-V and PI. Data are given as mean ± SEM of Annexin-V positive and propidium iodide positive cells of six experiments. Statistical significance was evaluated with students-t-test. *p < 0.05 with vs. without LPS, †p < 0.05 with vs. without PSI, ‡p < 0.05 with vs. without aCD95.

Additionally, preincubation with LPS for 6 hours significantly inhibited the aCD95-induced

apoptosis (64.3 ± 4.2 % versus 90.8 ± 0.9 %, p < 0.05). Whereas preincubation with the

50

Apo

ptos

is [%

]

0

20

40

60

80

100

*

*†

‡‡ ‡

LPS - +- + - +- +PSI - -+ + - -+ +

aCD95 - - - - + + + +

Apo

ptos

is [%

]

0

20

40

60

80

100

*

*†

‡‡ ‡

LPS - +- + - +- +PSI - -+ + - -+ +

aCD95 - - - - + + + +

specific proteasome inhibitor (PSI) alone had no effect (54.4 ± 2.7 %) on spontaneous

neutrophil apoptosis (66.1 ± 2.3 %), PSI abolished the endotoxin induced inhibition of

spontaneous apoptosis (52.6 ± 2.4 %) and the endotoxin induced inhibition in aCD95-induced

apoptosis (88.7 ± 2.6 %, p < 0.05) further strengthening the involvement of the proteasome in

regulation of neutrophil apoptosis.

3.3.4 LPS Reduces Caspase-3-Like Activity

Parallel to apoptosis caspase-3-like activity was measured in neutrophils. The basal caspase-

3-like activity of 0.5 ± 0.1 U increased after 22 hours incubation with medium alone (11.2 ±

3.2 U). Incubation with LPS significantly reduced caspase-3-like activity in PMN (5.8 ± 1.1

U, p < 0.05). Inhibition of the proteasome by PSI had no effect on caspase-3-like activity in

PMN incubated with medium alone (10.8 ± 2.7 U). However, parallel to apoptosis, incubation

with PSI completely abolished the LPS-induced inhibition of caspase-3-like activity (12.5 ±

1.5 U). In addition, preincubation with LPS reduced the aCD95-induced increase of caspase-

3-like activity (9.8 ± 2.5 U versus 11.9 ± 2.6 U), albeit only moderately. Inhibition of the

proteasome with PSI completely abolished the LPS effect on the aCD95-induced caspase-3-

like activity (15.8 ± 2.9 U, p < 0.05).

51

0

5

10

15

20

U [

mol

/mg

x m

in]

*

†

†

LPS

- + - + - + - +PSI

- - + + - - + +

aCD95 - - - - + + + +

0

5

10

15

20

U [

mol

/mg

x m

in]

*

†

†

LPS

- + - + - + - +PSI

- - + + - - + +

aCD95 - - - - + + + +

Figure 22: LPS reduces caspase-3 activity in PMN. Isolated PMN (1 x 106/mL) were preincubated with LPS (1 g/mL) for 5 hours, followed by PSI (30 M) for another hour before stimulation of PMN with or without agonistic aCD95 antibody (100 ng/mL) for 16 hours. Caspase-3-like activity was measured in whole cell lysate by DEVD-afc-cleavage assay. One Unit [U] was defined as µmol cleaved DEVD-afc per mg protein per minute. Data are given as mean ± SEM of six separate experiments. Statistical significance was evaluated with students-t-test. *p < 0.05 with vs. without LPS, †p < 0.05 with vs. without PSI.

3.4 The Correlation of Mcl-1 with Apoptosis of PMN

3.4.1 Expression of Bcl-2 mRNA and Protein in PMN

The expression of the anti-apoptotic Bcl-2 protein and mRNA was determined in neutrophils

from patients and healthy controls. No Bcl-2 protein was detected in neutrophils from both

groups, either in freshly isolated cells or in cells incubated with medium, LPS, or agonistic

anti-CD95 antibodies after 16 hours as compared with a monocyte sample. Densitometric

analysis of three separate Western blots for each group yielded values for Bcl-2 protein

expression of 1.8% ± 1.2% compared with a monocyte sample (100%) for controls and 1.2%

± 1.0% for samples from patients with sepsis incubated for 16 hours. Incubation with LPS

(1.5% ± 1.4% in controls and 2.5% ± 1.6% in patients) or agonistic CD95 antibody (2.3% ±

2.2% in controls and 3.7% ± 2.2% in patients) had no significant effect on Bcl-2 protein

52

expression. Bcl-2 mRNA was monitored by RT-PCR with specific primers for 35 cycles. In

contrast to THP-1 cells (positive control) no mRNA for Bcl-2 was detected in neutrophils

from either group. All samples expressed equal amounts of -actin mRNA (data not shown).

Figure 23: Analysis of Bcl-2 mRNA by RT-PCR. PMN (1 x 106/mL) from controls or patients were stimulated with medium, LPS (1 g/mL, LPS) or with an agonistic CD95 antibody (100 ng/mL, aCD95) for 16 hours. Extracted mRNA was transcribed and tested in PCR with specific primers for 35 cycles. Bands were visualized under UV after electrophoresis on a 1.5 % agarose gel and ethidiumbromide staining.

Figure 24: Bcl-2 Westernblot of control and patient PMN. Cells (1 x 106/mL) were incubated for 0 or 16 hours with Medium (16-h), LPS (1 g/mL, LPS), or with an agonistic CD95 antibody (100 ng/mL, aCD95). Equal protein of whole cells lysate was separated by SDS-PAGE on a 15% gel and immunostained with anti-Bcl-2 antibodies and respective secondary antibodies. Bcl-2 protein, detected in monocytes, monocytes (1 x 106/mL) preparation was used as positive control (+). Depicted is one of three westernblots for each group.3.4.2 Expression of Mcl-1 mRNA and Protein in PMN

53

Control

Patient

+ 0 h 16 h LPS αCD95M

Control

Patient

+ 0 h 16 h LPS αCD95M

Control Patient

aCD95

LPS16

h0 h + aC

D95LPS

16 h

0 h +

Control Patient

aCD95

LPS16

h0 h + aC

D95LPS

16 h

0 h +

Both protein and mRNA of the anti-apoptotic protein Mcl-1 were found in neutrophils from

patients and controls. Freshly isolated PMN from patients with sepsis contained slightly

higher levels of Mcl-1 (118.5% ± 55.8%) compared with neutrophils from controls (set at

100%); but after 16 hours of incubation Mcl-1 protein decreased significantly in neutrophils

from patients (27.3% ± 13.8%, p < 0.05 versus initial value), with marginally higher levels

seen in cells stimulated with LPS (31.7% ± 12.8%, p < 0.05 versus initial value) than with

anti-CD95 (30.4% ± 14.1%, p < 0.05 versus initial value). In contrast, PMN from controls

expressed unchanged amounts of Mcl-1 protein after 16 hours incubation with medium

(117.5% ± 8.1%), LPS (106.0% ± 13.2%), or agonistic CD95 antibody (110.1% ± 17.2%).

Figure 25: Analysis of Mcl-1 mRNA by RT-PCR. PMN (1 x 106/mL) from controls or patients were stimulated with LPS (1 mg/mL, LPS), or with an agonistic CD95 antibody (100 ng/mL, aCD95) for 16 hours. Extracted mRNA was transcribed and tested in PCR with specific primers for 34 cycles. Bands were visualized under UV after electrophoresis on a 1.5% agarose gel and ethidiumbromide staining.

Expression level of Mcl-1 mRNA was monitored by RT-PCR with specific primers for 34

cycles. In contrast to protein levels mRNA was not found in samples from controls incubated

for 16 hours; but equal amounts of Mcl-1 mRNA were found in all samples from patients with

54

16h

LPS

0 h aCD95

M 16h

LPS

0 h aCD95

PatientControl

16h

LPS

0 h aCD95

M 16h

LPS

0 h aCD95

PatientControl

sepsis, indicating an increased expression of Mcl-1 mRNA in PMN from patients with sepsis.

All samples expressed equal amounts of -actin mRNA (data not shown).

Figure 26: Mcl-1 westernblot of control and patient PMN. Cells (1x106/mL) were incubated for 16 hours with Medium (0 h, 16 h), LPS (1 g/mL, LPS), or with an agonistic CD95 antibody (100 ng/mL, aCD95). Equal protein of whole cells lysate was separated by SDS-PAGE on a 10 % gel and immunostained with anti-Mcl-1 antibodies and respective secondary antibodies. Depicted is one of three westernblots for each group.

3.4.3 Expression of BAX mRNA and Protein in PMN

The expression of the pro-apoptotic BAX protein and mRNA was determined in neutrophils

from patients and healthy controls. In contrast to Bcl-2, BAX protein was detected in all

samples from both groups. Slight differences were detected between different treatments but

these were not significant. Densitometric analysis of Western blots yielded values for

incubation with medium (74.2% ± 16.1% and 76.7% ± 17.3%), with LPS (69.4% ± 6.6% and

53.1% ± 18.4%), or agonisticCD95 antibody (82.3% ± 9.2% and 51.6% ± 29.2%) for controls

and patients, respectively. Values were derived from three separate Western blots from each

group. Expression level of BAX mRNA was unchanged in neutrophils from those of controls;

in PMN from patients slight reduction in BAX mRNA levels was seen after stimulation with

LPS compared with medium alone.

55

aCD95

LPS16

h0 h

Control Patient

aCD95

LPS16

h0 haC

D95LPS

16 h

0 h

Control Patient

aCD95

LPS16

h0 h

Control

0 h 16 h

LPS

0 h 16 h

LPS

Patient

M

1 2

+

Control

0 h 16 h

LPS

0 h 16 h

LPS

Patient

M

1 2

+

Figure 27: Analysis of BAX mRNA by RT-PCR. PMN (1 x 106/mL) from controls or patients were stimulated with LPS (1 g/mL, LPS), or with an agonistic CD95 antibody (100 ng/mL) (aCD95) for 16 hours. Extracted mRNA was transcribed and tested in PCR with specific primers for 35 cycles. Bands were visualized under UV after electrophoresis on a 1.5 % agarose gel and ethidiumbromide staining.

Figure 28: BAX westernblot of control and patient PMN. Cells (1 x 106/mL) were incubated for 16 hours with Medium (0 h, 16 h), LPS (1 g/mL, LPS), or with an agonistic CD95 antibody (100 ng/mL, aCD95). Equal protein of whole cells lysate was separated by SDS-PAGE on a 15 % gel and immunostained with anti-BAX antibodies and respective secondary antibodies. Depicted is one of three westernblots for each group.

3.4.4 Expression of Bid mRNA and Protein in PMN

56

LPS16

h0 h aC

D95

Control

Patient

LPS16

h0 h aC

D95

Control

Patient

In contrast to BAX, Bid protein was not distributed equally in all samples. In freshly isolated

cells from patients slightly higher levels of Bid protein were found compared with those in

controls. Lower levels were seen in cells from controls, which corresponded to high rates of

apoptosis, after 16 hours incubation with medium (45.3% ± 9.4%) or anti-CD95 antibody

(16.4% ± 8.6%). In contrast incubation with LPS resulted in a slightly higher Bid protein

content (55.5% ± 10.3%). Preincubation of PMN for 1 hour with the pan-caspase inhibitor z-

VAD-fmk (20 mol/L) inhibited the decrease of Bid in PMN incubated with agonistic CD95

antibody (58.2% ± 14.2%), indicating that Bid is truncated by caspases. In parallel samples

from patients with sepsis levels of Bid protein were 40.0% ± 10.3% for medium, 64.8% ±

10.5% for LPS, and 26.8% ± 16.3% for CD95 stimulation, inversely reflecting the level of

apoptosis. No Bid mRNA was found in samples from patients, either in freshly isolated cells

or in cells stimulated for 16 hours.

Figure 29: Analysis of Bid mRNA by RT-PCR. PMN (1 x 106/mL) from controls or patients were stimulated with LPS (1 g/mL, LPS), or with an agonistic CD95 antibody (100 ng/mL, aCD95) for 16 hours. Extracted mRNA was transcribed and tested in PCR with specific primers for 35 cycles. Bands were visualized under UV after electrophoresis on a 1.5% agarose gel and ethidiumbromide staining.

57

0 h 16h

16h

LPS

0 h aCD95

Patient Control

M 0 h 16h

16h

LPS

0 h aCD95

Patient Control

M

In contrast Bid mRNA was detected in PMN from healthy controls, but only in freshly

isolated cells and not in cells stimulated thereafter. All mRNA isolates expressed equal

amounts of -actin mRNA (data not shown).

Figure 30: Bid western-blot of control and Patient PMN. Cells (1 x 106/mL) were pre-incubated with medium or z-VAD-fmk (zVAD, 20 M) for one hour and then incubated for 15 hours with Medium (0 h, 16 h), LPS (1 g/mL, LPS), or with an agonistic CD95 antibody (100 ng/mL, aCD95). Equal protein of whole cells lysate was separated by SDS-PAGE on a 15 % gel and immunostained with anti-Bid antibodies and respective secondary antibodies. Depicted is one of three western-blots for each group.

58

aCD95LPS

16 h0 h

Control Patient

aCD95LPS

16 h0 haCD95

LPS16 h

0 h

Control Patient

aCD95LPS

16 h0 h

4.0 Discussion

Trauma is most often cause of death in industrial nations in the population under 40 years (1-

4). This fact points to the socio-structural importance of trauma surgery due to an over-aging

of our nations. Forced initial investment in a polytraumatized patient sells a better outcome

and minimizes follow costs for integration. Modern medical procedures with modern drugs let

develop new unknown situations for the human organism. Optimized drug application and

surgical treatment could ameliorate the outcome of severely injured patients. This group of

patients is at a high risk to develop SIRS and sepsis accompanied with MODS (Multi Organ

Dysfunction Syndrome) and MOF (Multi Organ Failure). The antigenic flood of crashed

tissue and external foreign antigens stimulate directly and indirectly host’s first line of

defense. Over-activation of PMN and PBMC leads to a set free of pro-inflammatory cytokines

and hyperstimulation of progenitor cells in the bone marrow leading to an amplifying loop

and a flood of PMN and PBMC. These cells degranulate and infiltrate parenchymateus organs

at the local site of injury and in uninjured organs. This “metastatic” behavior is the cause for

MODS and MOF. Simultaneously compensatory mechanisms are started (CARS;

Compensatory Anti-inflammatory Response Syndrome) to suppress the over-activation of

PMN. Starting CARS occurs usually too late and leads to an immunosupression endangering

patient’s life (Timing of surgical procedures: Table 4). Two extreme poles in polytrauma

disease which, when regulated could improve patients’ outcome (Dynamics of disease: Table

5).

59

4.1. Cell membrane: The Needle Ear

Intracellular antigens may mimicry proper ligands of the toll like receptors and activate those

intracellular signaling cascades. The massive attack of low molecular weight peptides and

oligosaccharides from the crashed tissue and the external world occupies free receptors very

quickly. Even if there is a very high binding constante, the concentration makes this

difference disappear.

Table 4: Decision making and strategy in a polytraumatized patient. Aggressive initial treatment leads to antigenic control and a reduced hyperinflammation. CARS makes surgery life threatening and deletary infections probable after approximately the day 10. (Prof. Dr. med. Otmar Trentz).

The Toll-like receptors are the primary and very fast sensors of an organism to detect foreign

organism in a body and to set it in a defensive position. This system seems to be

60

Surgical intervention Timing

Life saving surgery

? "Damage control" Day 1

Early total care

"Second look", only! Day 2-3

Scheduled definitivesurgery Day 5-10

No surgery!

Secondary reconstructivesurgery

Week 3

Physiological status

Hyper- inflammation

Immunosuppression

Recovery

Response toresuscitation:

"Window of opportunity"

+

Surgical intervention Timing

Life saving surgery

? "Damage control" Day 1

Early total care

"Second look", only! Day 2-3

Scheduled definitivesurgery Day 5-10

No surgery!

Secondary reconstructivesurgery

Week 3

Physiological status

Hyper- inflammation

Immunosuppression

Recovery

Response toresuscitation:

"Window of opportunity"

+

phylogenetically very old resembling the first immunity system. The Toll-like receptor system

has been shown to be expressed on Neutrophil Granulocytes, TLR 2 as well as TLR4 (62,63).

Endotoxin tolerance was described several years before and was controversely discussed (25).

Table 5: The Dynamics of SIRS and CARS in time (89). The initial hyperinflammation is followed by an overinhibition (CARS) of the immune response. Only the two extremes are shown, both take place at the same time.

Instead of a loss of Toll-like receptors on cellular surface, there is an increase of these

receptors accompanied with a reduced cellular response upon stimulation with LPS or

MALP2. Interestingly the same cells incubated ex vivo with the specific ligands for TLR2

(MALP2) and for TLR4 (LPS) revealed neither a receptor up-regulation nor a down-

regulation. Cells analyzed directly from septic patients revealed a significant increase in

receptor concentration on cellular surface with a similar stimulation response of MALP2 and

LPS, but on a lower level. Internal control analysis of IL8 and TNF in the supernatant of the

61

Inflammatory

Anti-Inflammatory

SIRS

CARS

Infection

Late MODS

Early MODS

Injury

[time]

Sepsis

Inflammatory

Anti-Inflammatory

SIRS

CARS

Infection

Late MODS

Early MODS

Injury

[time]

Sepsis

incubation medium revealed a sufficient response to the specific stimuli (data not shown).

This findings exclude a procedural phenomenon of absent TLR induction in an ex vivo assay.

Therefore, the receptor concentration cannot be responsible for different stimulation answers

in PMN from healthy individuals and PMN from septic patients. If, the tolerance on

endotoxin does not root in the desenzitation of Toll-like receptors, then other intra or

extracellular mechanisms must be responsible for this effect. The myeloid differentiation

factor (MyD88) is recruited to the cytoplasmic domain of TLR2 and TLR4. MyD88 binds to

IRAK (IL-1 receptor associated kinase) after stimulation (64). IRAK coassociates with IKK

(inhibitory B-kinase) witch leads to phosphorylation of I-B and sequent polyubiquitination

and proteasomal degradation of I-B. The finding that MyD88 deficient mice cannot

transduce the MALP2 or LPS signal and are therefore endotoxin resistent. This could center

MyD88 in the scientific interest but we could not find any differences on the level of mRNA

in healthy individuals and septic patients (data not shown). The kinase pathway is not linear

but a widespread cascade-like signaling effect involving a lot of other factors sub-grouped as

MAP-Kinases.

4.2 The Phosphate Cascades: Quick Resuscitation Action.

Diversifying the signal of MyD88 the phosphate residues are translocated to a plenty of

different MAP(n)K upon binding of LPS or IFN to its receptor. The signal is shifted from

MAP(n)K to MAP(n-1)K very quickly, within minutes. The MyD88 dependent pathway

terminates in the activation of JNK, p38 and p42/44 ERK. These three kinases are the

activators of the transcription factor AP-1 (http://www.genome.jp; hsa04620). We

demonstrated in this study the anti-apoptotic effect of LPS and IFN in PMN. The LPS

induced anti-apototic effect seems to be predominantly regulated by the MAP-kinase p42/44

ERK, observed in both healthy volunteers and patients with sepsis. Specific inhibition of

p42/44 ERK abolished the anti-apoptotic effect of LPS and the specific inhibition of p38

62

inhibited the IFN induced anti-apoptotic effect in PMN. These results point onto a central

role of ERK and p38 in LPS and IFN- signaling. It has been shown that pro-inflammatory

cytokines play a pivotal role in anti-apoptotic signaling in PNM (65, 66). Recognition of these

ligands by their specific inhibitors initiates protein phosphorylation transmitting the signal on

intracellular effector proteins (67). We showed in our in vitro inflammation model that the

stimulation of PMN with LPS or IFN leads to activation of protein tyrosine kinases, and

their inhibition abrogates the anti-apoptotic effect of LPS and IFN. In the control groups

incubated only with the specific inhibitors, we could not find any significant differences in the

apoptotic rate compared to the control group, respectively. This data support the thesis that

the MAP-kinases behave passively and are activated only upon contact with proinflammatory

stimuli. This thesis is supported by two findings: no effect on spontaneous apoptosis by

inhibition of MAP kinases and no exposure of activity in unstimulated cells. The specificity of

MAP-kinase inhibition by PD98059 and SB203580 has been shown elsewhere (68, 69).

Although the anti-apoptotic effect of LPS and IFN has been completely abrogated by

Herbimycin, the LPS mediated anti-apoptotic effect seems to be mediated by p42/44 ERK and

the IFN effect by p38. This indicates indirectly the involvement of NF-B in the LPS

mediated survival (http://www.genome.jp; hsa04620) (70).

However, the full inhibition of MAP-kinase activation in PMN by Herbimycin could not fully

restore the apoptotic rate in septic PMN. Other mechanism besides MAP kinase signaling

must exist that promotes PMN survival under inflammatory conditions.

4.3 cIAP2: Blocking the Road to Death

In phylogenetically ancient times organisms integrated baculoviral DNA. The repetitive

infection let develop a symbiosis with integration in our genome. cIAP2 is a member of the

63

BIR (baculoviral IAP repeats) protein Family and regulates the activity of intracellular

proteases. The protein’s ring finger domain translocates ubiquitin residues by an isopetidyl

bondage and destines the protease to proteasomal degradation.

In this study we have chosen an ex vivo stimulation protocol with a preincubation time with

LPS of 5 hours, to allow the cIAP2 induction. cIAP2 has the ability to bind and inhibit

caspase–3 and the RING-domain can transduce ubiquitin residues to the inhibited caspase-3

to be degraded by the proteasomal pathway. The Blockade of the proteasome inhibits the

caspase-3 degradation upon stimulation with aCD95.

In the ex vivo experiment we shoved a hypothetical protection mechanism to randomly

activated caspases, in this case caspase-3. PMN taken from the septic patient have a

communication history with patient’s blood. The transcription factors are activated and

transcription takes place, this could explain the partial caspase resistance upon CD95

activation. Nolan (39) and coworkers showed that an early inhibition of the proteasome led to

a reduction of LPS-mediated survival, suggesting an involvement of transcription factors like

NF-κB due to lack of IB inactivation (39). Therefore, we have chosen in our experiments a

longer preincubation period up to 5 hours with LPS to allow gene transcription and protein

expression with a possible cIAP2 induction, mimicking PMN history in patient’s blood. As

we could show, cIAP2 mRNA and Protein was expressed during this time period. To test the

participation of the proteasome in this mechanism, we used the specific proteasome inhibitor

PSI one hour prior to stimulation with aCD95. This time point was chosen to differentiate

between a direct effect of proteasomal inhibition on NF-κB regulated gene transcription by

inhibition of I-κB degradation and the later effect of proteasomal degradation of ubiquitinated

proteins (71). Activation of the CD95 cascade activates caspase-3, which then can be

ubiquitinated by IAPs and destined for degradation by the proteasomal pathway. It has been

shown previously that IAP-protein class members are able to inhibit and ubiquitinate caspase-

3.(72,73) During this process the IAPs are being cleaved and degraded by activated caspase-3,

64

but also a ubiquitin molecule is being attached to caspase-3 which eventually leads the

ubiquitinated caspase-3 towards proteasomal degradation. Our data show that the

preincubation of PMN with LPS leads to an early induction of cIAP2 mRNA and protein, and

that cIAP2-mediated caspase-3 ubiquitination takes place upon caspase-3 activation (75) such

as after stimulation of CD95. In parallel, we observed in westernblot a reduction of cIAP2

protein after two to four hours stimulation of CD95, further supporting this hypothesis. The

binding of IAPs to caspase-3 is mediated by the different BIR-domains of the cIAP2 protein.

The BIR-domain fits exactly into the active center of caspase-3 and inhibits their activity (74),

in parallel the ring-finger domain of the IAP molecule, responsible for ubiquitination comes

in close contact with caspase-3 and attaches one ubiquitin molecule to the activated caspase-3.

This process takes place repetitively leading to a polyubiquitin chain on the activated caspase-

3. A polyubiquitinated protein, like caspase-3, is destined for proteasomal degradation.

However, as the cIAP2 protein itself was cleaved and degraded as early as four hours after

CD95 stimulation, the cIAP2-mediated inhibition of apoptosis could be inhibited by a

caspase-3-mediated feedback loop, which might also explain the residual apoptosis seen in

CD95-stimulated PMN preincubated with LPS.

Inhibition of the proteasome by PSI, in turn, seemingly increased the amount of ubiquitinated

caspase-3 after LPS-stimulation due to lack of proteasomal degradation. However, inhibition

of the proteasome may also elevate the level of cIAP2 protein which due its

autoubiquitination ability would be likewise degraded (73). These cIAP2 molecules could

shift the balance to a reduction of caspase-3 activity because of the direct cIAP2-dependent

caspase-3 inhibition (76). However, the remaining caspase-3 activity and apoptosis rate in

cells treated with PSI suggests that ubiquitin has no direct inhibitory effect on activated

caspase-3, besides targeting caspase-3 for proteasomal degradation. The LPS-effect on CD95-

induced apoptosis and caspase-3 activity was completely abolished after inhibition of the

proteasome indicating that the ubiquitinated caspase-3 retains its activity.

65

In this short analysis of the inhibition of caspase-3 upon stimulation with LPS we

demonstrated the central role of the proteasome in protein-protein interaction in the

cytoplasm. The proteasome is a multi-phased structure involved in the survival and death of

the cell. All seems to depend on the point of time the interaction takes place (38).

4.4 Bcl-2 Proteins: The Balanced Suicide Machinery

Other factors regulating the execution of apoptosis are the Bcl-2 proteins. A plenty of

members of these proteins has been identified until now (see Table 2.). This system of pro-

apoptotic and anti-apoptotic Bcl-2 proteins builds a protection and offensive line to the outer

mitochondrial membrane. A good model imagination is an intracellular complementary

system which is always running and apoptosis takes only place when the balance is shifted to

the advantage of the pro-apoptotic Bcl-2 members. Our group has shown that the spontaneous

apoptotic cell death of PMN is not caspase-dependent, in contrast to receptor mediated

apoptosis (78). Our results confirm the data of other scientific groups for Bcl-2 (78), BAX

(79), or both (80, 81). The very small amounts of Bcl-2 in PMN lysates might be explained by

tiny PBMC contamination during preparation (82). This finding is supported by densitometric

analysis of these Western-blots compared to 100% PBMC preparation.

At the level of BAX-protein we were not able to find any significant differences in PMN from

healthy individuals or septic patients. A small difference in the expression pattern of BAX-

mRNA was found. The lower mRNA expression in septic PMN could be explained by their

age. Septic PMN are much older due to their increased lifespan during sepsis and thus might

express lower gene-activity in general. The high expression of BAX-protein in both groups

may be attributed to its high stability despite the lack of mRNA (83).

In contrast to Bcl-2 and BAX we observed differences in Bid-protein levels in PMN from

healthy individuals and septic patients. A significant decrease of the Bid-protein was always

observed in experimental groups with a high apoptotic rate. This shows that Bid-protein might

66

inversely reflect the level of apoptosis. This thesis was enforced by the observation that Bid-

protein reduction occurred also in septic PMN subjected to receptor mediated apoptosis. This

reduction was abolished by the pancaspase inhibitor zVAF-fmk (83). Levels of mRNA

remained unchanged in both groups. These finding let Bid participate apoptotic procedures

but make it unlikely to regulate it.

In contrast to Bcl-2 we observed the most prominent differences for Mcl-1 protein and

mRNA. Previous observations could be confirmed by our findings (81, 84). In contrast to

other Groups (84) we found a reduction of 50% of Mcl-1 protein in PMN from healthy

individuals incubated with medium, LPS or aCD95. This was caused with a high probability

by the different incubation time (16h vs. 20h). The high apoptotic rate of PMN could not have

left many non non-apoptotic cells after 20 hours. The stimulation with aCD95 led not to a

decrease of Mcl-1 protein suggesting a caspase-independent pathway of Mcl-1 waste, or a

shorter incubation protocol. Here we were able to detect mRNA of Mcl-1 only in septic PMN

despite low Mcl-1 protein. However, how the Mcl-1 turnover takes place remains to be

elucidated. Interestingly, the incubation of PMN with LPS led not to an induction of Mcl-1

mRNA in our experiments. This point in the regulation of the Mcl-1 gene during sepsis shows

that PMN stimulation with LPS may not represent a general model of sepsis.

Different inductors of Mcl-1 have been found, IL-6 (85) and IGF-1 (86) controlled by STAT3

(87). A model for the survival of PMN during sepsis has already been proposed (81). The

reduction of apoptosis during sepsis is achieved by a multi-cytokine orchestra and the

induction of short-lived anti-apoptotic proteins such as Mcl-1 (88). This induction shifts the

cell towards survival and antagonizes the long-lived factors like Bid and BAX. These findings

are supported by our results, but further investigation and analysis has to be done to

understand the fine network of the Bcl-2 system.

4.5 The Strategy

67

During trauma healthy tissue is being crushed and intracellular as well as external pathogens

may gain access to blood circulation. The first line of defense (PMN), recognize several

motifs by their TLR receptors leading to activation of these cells with a significantly

prolonged survival. If the supply of pathogens persists and the cells are over-activated, they

flood the organism and accumulate in parenchymateous organs. The early phase we call

MODS in this phase the organ damage is pontetially reversible. Persisting pathogenic load

and therefore, sustained stimulation let the cells degranulate, and damages the site of

degranulation (MOF). The organs suffer irreversible damage and the patient dies in a

septically induced MOF.

In our study we analysed different possible mechanisms of spontaneous apoptosis and their

inhibition by LPS. The Toll-like receptors were up-regulated during sepsis and not inducible

by LPS or MALP2 indicating not an auto-regulation but a poly-factorial event. Binding of

LPS or MALP2 led to activation of MAP-kinases and to an increased survival rate of PMN.

Bacterial pathogens are most likely to signal by p42/44 ERK and IFN- by p38, the inhibition

of these kinases restored the effect of the stimuli. The intracellular “trash can”, the

proteasome, was already shown to regulate apoptosis by the inhitition of the actvation of NF-

B (39). However, the effect of the proteasome-inhibition may be a bi-cutting edge. On the

one hand the inhibition may induce apoptosis and on the other hand it may inhibit apoptosis.

We could show the induction of cIAP2 and the ubiquitination of caspase-3 upon activation.

The caspase remained active when the proteasome was blocked. This possibly opens a tiny

window on caspase resistance in PMN. The analysis of Bcl-2 system revealed not directly

conclusive data, but Mcl-1 seems to be a platform of the Bcl-2-system during sepsis. The

induction of Mcl-1 mRNA and the absence of Mcl-1 protein can be explained by its

degradation during the apoptotic process, thus inhibiting this process. The clinical strategy

will still consist of surgical intervention and hemodialysis to reduce the antigenic load.

Frequent second looks and daily dressing changes will reduce the antigenic load at the side of

68

injury. Dialysis reduces the antigenic load systemically. Both strategies lead often to success

in patients with severe sepsis. The clue approach remains: “Keep the receptors tidy”.

4.6 Hypothetical Molecular Targets

Insisting on new hypothetical molecular targets for immuno modulation may lead to blindness

for principally simple problems. Let’s switch to an other point of view, and consider a septic

neutrophil as a tumor cell and not anymore as an inflammatory cell. These cells only try to

survive and to escape their apoptotic destiny. The clue question is where to block and to

down-regulate to achieve a smart immuno modulation in the sense of survival. At the level of

cellular membrane we have observed an induction of TLR-receptors, but this does not explain

the endotoxin tolerance of septic PMN. “Keep the receptors tidy” is the actual therapy in

severe sepsis. The “yes or no” signaling way of receptors is not suitable for modulation, there

is the “maybe” missing. At the level of second messengers (MAPK) hypothetical

phosphorylation controlling could slow down the endotoxin induced survival cascade (see

chap. 4.2, 4.5). MAPKs are ubiquitous signaling components, therefore, an inhibition could

cause massive side effects. Caspases as executioners of apoptosis fail as targets due to an

overall apoptotic cell death upon systemical activation. Only two components of the apoptotic

network remain the IAP and Bcl-2 proteins. These proteins are involved in a fine network

inside the suicide machinery and could represent possible molecular targets for immuno

modulation. Recent publications (90) show a possible role for triphenylurea and derivates to

inhibit BIR-domains in IAP proteins. The immuno modulative effect was shown for XIAP

and pancreatic cell carcinoma (91). The positive effect could be linked to the targeting of a

“defensive” molecular structure. This target, in this case XIAP, comes to action only when

caspases are activated. BIR (IAP) inhibition leads to an effect only when BIR is needed. In

the case of a septic PMN could it restore the apoptotic rate and ameliorate the septic outcome

(see Chap 3.3). Systemic experiments still have to be done.

69

Lessons learned from CLL (chronic lymphatic leukemia) (92) point to the Bcl-2 system. For

the didactical reason the pro-apoptotic Bcl-2 system will be called BH3-system in this section,

due to its pro-apoptotic activity. BH3 proteins (see Chap. 3.4) belong to the pro-apoptotic

group of Bcl-2 proteins. Especially the BH3 only proteins (Bid and Bim) once activated co-

associate with Bak and BAX leading to pore formation in outer mitochondrial membrane and

consequently to apoptosis. In many hematological tumors, leukemias, are the anti-apoptotic

Bcl-2 proteins induced. That is also the reason where Bcl-2 has got its name from (B-Cell

Leukemia Type 2). The consequence of induction of survival by Bcl-2 is more or less a

resistant cell to apoptotic stimuli. Mimicing the BH3-effect by recently introduced substances,

ABT-737 and ABT-263 (93), revealed under experimental conditions inhibition of tumor

growth in vitro. The cell death was normalized ad detected as mitochondrial apoptosis.

Experimental procedures in SCID mice with transplanted FDC-P1 tumor cells and application

of ABT-737 led to complete tumor regression (93). C-myc driven B-cell lymphomas in mice

could be inhibited by the application of ABT-737 (94). The platform of the final common

path could be Mcl-1 indicated by siRNA screens in H196 SCLC cells (94). As in our work the

Mcl-1 was in septic PMN always induced (see Chap. 3.4.2). Application of BH3 mimics

could restore the spontaneous apoptosis of septic PMN without severe side effects, due to

targeting of a “passive” molecule. The cellular signaling and apoptotic mechanism would not

be disturbed.

70

ABT-737, chemical compound mimicing BH3-domain activity. Abbott GmbH & Co., Ludwigsahafen, Germany. Application of ABT-737 restored apoptotic activity in CLL, and led to tumor regression (92).

Figure 31:

Theoretically, these two molecular targets may provide a field for future sepsis therapies.

4.7 Work to be done

No one knows how life appeared on the Earth, the stepwise evolution from simple to complex

is very probable. If we assume prokaryotic life without mitochondria, mechanisms for

enzymatic digesting of phagocytosed material must have existed. These systems may still

persist to higher eucariotes only slightly changed in function. On the search for alternative

apoptotic pathways a lot of work has been done. Models of interaction of BAX with

lysosomal membrane and the resulting autophagocytic cell death by the released lysosomal

material have been developed (97). These models are not applicable for every cell type and

have to be revised. Alternative genes have been characterized and called ATG genes mainly

in yeast but also in mammals (95, 96). These factors can co-assembly to a multifactor

complex and construct autodigestive machinery. In other words the result would be the same

like in apoptotic cell death. Other hypothetical function could be the protection against

intracellular pathogens such as Shigella spp. and Mycobacteria spp. This could have a pro-

surviving effect rather than a pro-apoptotic effect. A lot of research has to be done to

characterize the different factors in mammalian cells and to type out their functions. The

alternative cell death programme in neutrophil granulocytes has not been found, yet. Hence

the additive molecular targets for immunological modulation beside BH3 and BIR are still to

be expected. The acquisition of multiple possible targets could lead to a possible therapy

model of balanced immuno modulation, individually fitted for each situation. The flow will

always go in the same direction it depends only how fast this happens. As physicians we wont

be able save patient’s live, we gain only time. Only experimental work in vivo and in vitro

will show if we precede the right direction. Always be critical to a nice battle plan and do not

71

loose your primary options of therapy: “No battle plan survives the first five minutes of

contact with the enemy“(Genenarfeldmarschall Helmuth Karl Bernhard Graf von Moltke,

*26.10.1800 - †24.04.1891)

Figure 32: Autophagic cell death: Assembly of a multimeric protein complex at PAS (pagophore assembly site). ATG related genes were identified in Yeast and Drosophila. Poor correlation to mamalian hypothetical autophagosome (95, 96). This complex has probably a prosurviving role rather than a pro-apoptototic role.

72

PAS

LC3-II

ATG5-ATG12,ATG16L

Beclin-1

VPS34

VPS15

BCL2BCL-XL

UVRAG

WIPImTORULK

ATG9

AUTOPHAGY

PAS

LC3-II

ATG5-ATG12,ATG16L

Beclin-1

VPS34

VPS15

BCL2BCL-XL

UVRAG

WIPImTORULK

ATG9

AUTOPHAGY

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inhibitor of apoptosis, cIAP2, functions as a ubiquitin protein ligase and promotes in vitro

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86

6.0 Books:

Janeway CA,Travers P, Walport M, Capra JD. Immunobiology, The Immune System in

Health and Disease. Fourth Edition. Garland. Churchill Livingstone. 1999 ISBN: 0-8153-

3217-3

Lodish H, Berk A, Matsudaira P, Kaiser CA, Krieger M, Scott MP, Zipursky SL, Darnell J.

Molecular Cell Biology. Fifth Edition. Freeman 2004 ISBN: 0-7167-4366-3

Krauss G. Biochemistry of Signal Transduction and Regulation. Third Edition. Wiley-VCH

2005 ISBN-13:978-3-527-30591-9

Gomperts BD, Kramer IM, Tatham PER. Signal Transduction. Academic Press 2003 ISBN:

0-12-289631-9

Stryer L. Biochemistry. Fourth Edition. Freeman 1995 ISBN: 0-7167-2009-4

Watson JD,Witkowski J, Gilman M, Zoller M. Recombinant DNA. Second Edition.

Scientific American Books 2001 ISBN: 0-7167-1994-0

7.0 Databases:

National Center for Biotechnology Information: http://www.ncbi.nlm.nih.gov/

Genome Network: http://www.genome.jp/

The ExPASy (Expert Protein Analysis System): http://www.expasy.ch/

87

8.0 Curriculum vitae:

Dr. med. Ladislav Mica

Feldstrasse 15

CH-6300 Zug

12.08.1974 Born in Brno (ČSSR)

Mother: Pavla Mica-Miluska, Dr.med. *10.12.1944

Father: Ladislav Mica, Dr.med *26.12.1946

August 1977 - August 1980 Kindergarden Herčíkova in Brno

01.09.1980 - 31.06.1983 Primary school Herčíkova 19 in Brno .

July 1983 Emigration to Switzerland.

August 1983-August 1987 Primary school in Switzerland

14.09.1987 - 06.06.1994 Gymnasium: Institut Dr. Pfister in Oberägeri. (ZG, CH)

06.06.1994 Maturity Typus B (Latin) at the Institut Dr. Pfister in

Oberägeri, ZG, Switzerland

21.10.1994 - 02.10.2000 Studies of human medicine in Zürich.

1997-2001 Concomitant experimental doctoral study

in the laboratory of Prof. Dr. med. W. Ertel: „In vivo

studies of hepatic microcirculation in a murine model.“

02.10.2000 Finishing exams at the University of Zürich

Curriculum laboris:

06.11.2000 - 31.03.2001 Clinical work at department of Trauma Surgery at the

University Hospital of Zürich (Chief: Prof. Dr. med. O.

Trentz).

01.04.2001 - 31.03.2002 Postgraduate Course of Experimental Medicine (Head:

Prof. Dr. med. J. Zapf, sponsoring: Swiss National

Foundation).

07.03.2002 Promotion to Doctor of Human Medicine (University

of Zürich)

88

01.04.2002 – 31.03.2003 Scientific work at the Department of Trauma Surgery

(Chief: Prof. Dr. med. O. Trentz).

01.04.2003 – 30.09.2004 Clinical work at the department of Trauma Surgery

University Hospital of Zürich (Chief: Prof. Dr. med. O.

Trentz).

01.10.2004 – 30.04.2005 Further Education in Intensive Care Medicine,

University Hospital of Zürich (Chief: Prof. Dr.med. R.

Stocker)

05.11.2005 First exams in general surgery (FMH, Foederatio

Medicorum Helveticorum)

01.05.2005 - 31.08.2006 Clinical work at the department of Trauma Surgery

University Hospital of Zürich (Chief: Prof. Dr. med. O.

Trentz).

01.09.2006 – 31.12.2008 Clinical work at the clinic for general surgery in

Kreisspital Männedorf (Zürich, CH) (Chiefs: Prof.

Dr.med. A. Hollinger & Dr.med. A. Vollenweider)

21.11.2008 Specialist in Surgery FMH

01.01.2009 – 31.12.2009 Clinical work at the department of Trauma Surgery

University Hospital of Zürich (Chief: Prof. Dr. med. H.P.

Simmen)

01.01.2010- Teamleader at the at the department of Trauma Surgery

University Hospital of Zürich (Chief: Prof. Dr. med. H.P.

Simmen)

Further Education:

Courses: Microsurgery (Zürich, CH) 21-25.09.1998

AO-Course Davos (CH) 14-19.12.2003

Vascular International (CH) 21-24.01.2006

Abdominal Surgery, Davos, (CH)

04-10.03.2006

ATLS (Zürich, CH) 23.01.2004

Wiener Handchrurgie (A) 20-25.05.2007

89

Experimental experience:

Molecular Biology:

DNA: Isolation, Southern Blot, Restriction mapping,

Transfection/Transformation, PCR.

RNA: Isolation, RT-PCR, siRNA.

Proteins: Concentration, Western Blot, 2D-Blot, Enzyme- Kinetics

Membranes: Transporter activities

Animal experiments:

Intravital microscopy

Murine microsurgery and anesthesia.

Cell cultures: Human cells: Fibroblasts, Granulocytes, THP-1, Jurkat

Additional qualifications:

Linguistic skills: Czech (mother language)

German (mother language)

English, fluently.

French, good knowledge

Latin, text only

Italian, passive comprehension.

International Cooperations:

Active participation (PD Dr.med. M. Keel, Dr.med. L.

Mica) in: A multi-center, randomized, double-blind,

parallel group, placebo controlled trial to evaluate the

efficacy and safety of activated recombinant factor

VII (rFVIIa/NovoSeven®/NiaStase®) in severely

injured trauma patients with bleeding refractory to

standard treatment. NovoNordisk, Start University

Hospital of Zürich 01.11.2005 – 11.06.2008.

90

9.0 Publications

Wanner GA, Mica L, Wanner-Schmid E, Hentze H, Kolb S, Trentz O, Ertel W. Inhibition of

caspase activity prevents CD95-mediated hepatic microvascular perfusion failure and restores

Kuppfer cell clearance capacity. FASEB J, 13:1239-1248, 1999

Härter L, Mica L, Stocker R, Trentz O, Keel M. Mcl-1 Correlates with Reduced Apoptosis in

Neutrophils from Patients with Sepsis. Journal of American College of Surgeons 964-973,

2003.

Mica L, Härter L, Trentz O, Keel M. Endotoxin reduces CD95-Induced neutrophil apoptosis

by cIAP2-mediated Caspase-3 Degradation. Journal of American College of Surgeons 199

(4) 595-602, 2004.

Härter L, Mica L, Stocker R, Trentz O, Keel M. Increased expression of toll-like receptor-2

and –4 on leukocytes from patients with sepsis. Schock 22 (5): 403-409, 2004 IMPACT: 2.808

Keel M, Mica L, Stover J, Stocker R, Trentz O, Härter L. Thiopental-induced apoptosis in

lymphocytes is independent of CD95 activation. Anesthesiology 103 (3): 576-584. 2005

Labler L, Wedler V, Mica L, Trentz O. Entrapment of the anterior tibial artery in a distal tibial

fracture after intramedullary nailing. Der Unfallchirurg 109(2):156-9. 2006 [German].

Von Känel R, Hepp U, Buddeberg C, Keel M, Mica L, Aschbacher K, Schnyder U. Altered

Blood Coagulation in Patients with Posttraumatic Stress Disorder. Psychosomatic Medicine

2006 in press. IMPACT: 3,857

Labler L, Mica L, Härter L, Trentz O, Keel M. Einfluss der V.A.C. Therapie auf zytokine und

Wachstumsfaktoren in traumatischen Wunden. Zentralblatt für Chirurgie. 131: 62-67. 2006

Von Känel R, Hepp U, Kraemer B, Traber R, Keel M, Mica L, Schnyder U. Evidence for

low-grade systemic proinflammatory activity in patients with posttraumatic stress disorder. J

Psychiat Res, 41(9):744-52.

91

Von Känel R, Hepp U; Traber R, Kraemer B, Mica L, Keel M, Mausbach BT, Schnyder U.

Measures of endothelial dysfunction in plasma of patients with posttraumatic stress Disorder.

J Psychiat Res, 15;158(3):363-373. 2008

Mica L, Gianom D, Bode B, Jaklin P, Hollinger A. Rare Cause of Dysphagy: Giant Polypoid

Esophageal Well-Differentiated Liposarcoma. Case Rep Gastroenterol 1: 7-14. 2007

Vacuum-assisted closure therapy increases local interleukin-8 and vascular endothelial growth

factor levels in traumatic wounds. Labler L, Rancan M, Mica L, Härter L, Mihic-Probst D,

Keel M. J Trauma. 2009 Mar;66(3):749-57.

Early serum procalcitonin, interleukin-6, and 24-hour lactate clearance: useful indicators of

septic infections in severely traumatized patients. Billeter A, Turina M, Seifert B, Mica L,

Stocker R, Keel M. World J Surg. 2009 Mar;33(3):558-66.

Avulsion of the Hamstring Muscle Group: A Follow-Up of 6 Adult Non-Athletes with Early

Operative Treatment: A Brief Report. Mica L, Schwaller A, Stoupis C, Penka I, Vomela J,

Vollenweider A. World J Surg. 2009; 33: 1605-1610.

The Severity of Injury and the Extent of Hemorrhagic Shock Predict the Incidence of

Infectious Complications in Trauma Patients. T. Lustenberger, M. Turina, B. Seifert, L.Mica

and M. Keel European Journal of Trauma and Emergency Surgery, Volume 35(6): 538-

546.

Susanne Habelt, Adrian Schwaller, Albert Hollinger, Ladislav Mica. Septic polyarthritis

caused by Streptococcus pneumoniae: primary pneumococcal pneumonia as a risk factor in

older patients? A case report BMJ Case Reports 2009 [doi:10.1136/bcr.02.2009.1604]

Ladislav Mica, Valentin Neuhaus, Enrico Pöschmann, Dilek Könü-Leblebicioglu, Urs

Schwarz, Guido A Wanner, Clément ML Werner, Hans-Peter Simmen. Hydrocephalus

communicans after traumatic upper cervical spine injury with a cerebrospinal fluid fistula: a

rare complication. BMJ Case Reports 2010: published online 15 July 2010,

doi:10.1136/bcr.02.2010.2731

9.1 Oral Presenations

92

Wanner GA, Mica L, Trentz O, Ertel W. Anti-Fas antibody induces hepatic microvascular

perfusion failure and decreases Kuppfer cell clearance capacity. Surg Forum 49:184-185,

1998

Wanner GA, Mica L, Hentze H, Trentz O, Ertel W. Hemmung der Caspase-Aktivität

verhindert das Fas-vermittelte Mikrozirkulationsversagen der Leber und die reduktion der

Clearance-Funktion der Kuppferzellen. Langenbecks Arch Chir Suppl I: 19-21,1999

Wanner GA, Mica L, Trentz O, Ertel W. Inhibition of caspase activity attenuates endotoxin-

mediated hepatic microvascular perfusion failure and leukocyte response. Surg Forum 50,: 3-

4, 1999

Mica L, Wanner GA, Trentz O, Ertel W. Activation of apoptosis by agonistic anti-CD95

antibodies induces hepatic microvascular perfusion failure and decrease Kuppfer cell

clearance capacity. Eur Surg Res 31 (suppl 1).2, 1999

Mica L, Wanner GA, Hentze H, Trentz O, Ertel W. Zonal heterogenity of CD95-mediated

hepatic microvascular perfusion failure-role of caspases. Shock 11 (suppl 1). 49, 1999

Wanner GA, Mica L, Hentze H, Künstle G, Kolb S, Trentz O, Ertel W. Rolle des CD95

Rezeptors und der Kaspasen-Aktivität für die Endotoxin-assoziierte Hepatotoxizität und

Letalität. Chirurgisches Forum 29, 509-511, 2000

Wanner GA, Mica L, Kolb S, Trentz O, Ertel W. Inhibition of caspase activity prevents

hepatic microvascular perfusion failure and leukocyte accumulation after ischemia and

reperfusion. Surg Forum 52, 48-50, 2001

Keel M, Mica L, Eid K, Trentz O, Ertel W. Die Bedeutung der

Crash-Laparotomie/Thorakotomie bei schwerverletzten Patienten im hämorrhagischen Shock.

Hefte zu der Unfallchirurg, Kirschner/Stürmer (Hrsg.), Springer-Verlag Berlin Heidelberg

340-341, 2001

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Mica L, Härter L, Trentz O, Keel M. Bid or Bcl-2 do not regulate neutrophil apoptosis in

patients with Sepsis. Surg Forum 195; 3S, S79-S80, 2002

Härter L, Mica L, Trentz O, Keel M. Toll-like receptor-2 and –4 on leukocytes from patients

with sepsis. Surg Forum 195; 3S, S83-S84 2002.

Mica L., Härter L., Trentz O., Keel M. Bid und Bcl-2 regulieren nicht die Apoptose

neutrophiler Granulozyten beim Patienten mit Sepsis. In: Hefte zu der Unfallchirurg,

Rehm/Stürmer/Prokop (Hrsg.), pp. 373-374, Springer-Verlag: Berlin Heidelberg 2002.

Keel M, Mica L, Trentz O, Härter L. Erhöhte expression der Toll-like Rezeptoren-2und –4

auf Leukozyten von Patienten mit Sepsis. In: Hefte zu der Unfallchirurg,

Rehm/Stürmer/Prokop (Hrsg.), pp. 376-377, Springer-Verlag: Berlin Heidelberg 2002.

Wanner GA, Mica L, Kolb S, Trentz O, Ertel W. Bedeutung der Kaspasenaktivität für das

hepatische Mikrozirkulationsversagen nach Ischämie und Reperfusion. Chirurgisches Forum

31: 333-335, 2002.

Mica L, Härter L, Trentz O, Keel M. Beschleunigte ubiquitinierung und degradation der

aktivierten caspase-3 in neutrophilen granulozyten von patienten mit sepsis. Chirurgisches

Forum 32, 13-15, 2003.

Mica L, Härter L, Trentz O, Keel M. Endotoxin-vermittelte Hemmung der Apoptose

neutrophiler Granulozyten ist Proteasom, jedoch nicht NF-κB abhängig. Swiss Surgery

Supplementum 1, 5, 2003.

Mica L, Härter L, Trentz O, Keel M. Das Proteasom reguliert die LPS-vermittelte Reduktion

der spontanen Apoptose neutrophiler Granulozyten unabhängig von NF-kB. In: 67.

Jahrestagung der deutschen Gesellschaft für Unfallchirurgie / 89. Tagung der deutschen

gesellschaft für Orthopädische Chirurgie / 44. Tagung des Berufsverbandes der Fachärzte

für Orthopädie. www.egms.de/en/meetings/dgu2003/03dgu0346.shtml, 2003

94

Mica L, Härter L, Trentz O, Keel M. Endotoxin Reduces CD95-Induced Neutrophil

Apoptosis by cIAP2-Mediated Caspase-3 Degradation. Surgical Forum 197 3S, S37-S38,

2003

Mica L, Härter L, Trentz O, Keel M. STAT-3 reguliert die verminderte Apoptose neutrophiler

Granulozyten beim Patienten mit Sepsis. Chirurgisches Forum 33, 249-251, 2004.

Mica L, Härter L, Trentz O, Keel M. STAT-3 regulates the reduced apoptosis in neutrophils

from patients with sepsis. 3rd Day of Clinical Research, University Hospital of Zürich, 73,

2004.

Härter L, Mica L, Trentz O, Keel M. Upregulation of Toll-like receptors in neutrophils is

regulated by STAT-3. 3rd Day of Clinical Research, University Hospital of Zürich, 23, 2004.

Mica L., Härter L., Trentz O., Keel M. STAT-3 regulates the reduced apoptosis in neutrophils

from patients with sepsis. European J Trauma, Supplement 1, 76;289, 2004

Härter L., Mica L., Trentz O., Keel M. IFN-g-induced upregulation of toll-like receptors is

regulated by STAT-3 in neutrophils. European J Trauma, Supplement 1, 76;288, 2004

Labler L., Mica L., Härter L., Keel M. VAC®-therapy induces a local release of interleukin-8

and vascular endothelial growth factor. European J Trauma, Supplement 1, 44;158, 2004

Mica L, Härter L, Trentz O, Keel M. Regulation of neutrophil apoptosis in patients with

sepsis by STAT 3. Swiss Knife, Special Edition 48, 2004

Labler L, Lustenberger T, Lüthi S, Mica L, Trentz O, Keel M. Hemorrhagic shock increases

infections of closed and open fractures. 6th European Congress of Trauma and Emergency

Surgery. Rotterdam 2004

Lustenberger T, Lüthi S, Labler L, Mica L, Trentz O, Keel M. The pre-hospital delay

influences the posttraumatic morbidity. 6th European Congress of Trauma and Emergency

Surgery. Rotterdam 2004

95

Lüthi S, Lustenberger T, Labler L, Mica L, Stocker R, Trentz O, Keel M. Craniotomy after

head injury influences incidence of systemic inflammation. 6th European Congress of

Trauma and Emergency Surgery. Rotterdam 2004

Mica L, Lustenberger T, Lüthi S, Labler L, Trentz O, Keel M. The Severity of Injury and

Hemorrhagic Shock Correlates with the Incidence of Posttraumatic Infectious Complications.

6th European Congress of Trauma and Emergency Surgery. Rotterdam 2004

Mica L, Härter L, Trentz O, Keel M. Endotoxin reduces CD95-induced Neutrophil apoptosis

by cIAP2- mediated caspase-3 degradation. 3rd Swiss Apoptosis Meeting 16-17. September

Bern 2004

Härter L, Mica L, Trentz O, Keel M. STAT-3 participates in endotoxin-induced survival in

neutrophils from patients with sepsis. 3rd Swiss Apoptosis Meeting 16-17. September Bern

2004.

Mica L, Härter L, Trentz O, Keel M. NF-B reguliert die LPS-induzierte Zytokinfreisetzung,

nicht aber die Reduktion der Apoptose in neutrophilen Granulozyten von Patienten mit

Sepsis. Chirurgisches Forum 34, 2005

Mica L, Härter L, Trentz O, Keel M. The IFN-g-induced upregulation of Toll-like receptors is

regulated by STAT-3. Swiss Knife, Special Edition 32, 14.04, 2005.

Keel M, Labler L, Lustenberger T, Mica L, Stocker R, Trentz O. Outcome after “Damage

Control” or “Early Total Care” Management in 622 Severely Injured Patients. Swiss Knife,

Special Edition 9, 1.02, 2005.

Keel M, Labler L, Lustenberger T, Mica L, Trentz O. Day-One-Surgery in 696 Severely

Injured Patients by General Trauma Surgeons. Swiss Knife, Special Edition 10, 1.06, 2005.

Mica L, Härter L, Trentz O, Keel M. STAT-3 reguliert die IFN--induzierte Expression der

Toll-like Rezeptoren -2 und -4 in Leukozyten. In: 1. Gemeinsamer Kongress Orthopädie und

Unfallchirurgie www.abstractserver.de/abstracts/ou2005/ab00838.htm, 2005.

96

Labler L, Mica L, Härter L, Trentz O, Keel M. Erhöhte Interleukin-8 und VEGF Spiegel in

VAC-behandelten Wunden von Patienten nach Trauma. In: 1. Gemeinsamer Kongress

Orthopädie und Unfallchirurgie www.abstractserver.de/abstracts/ou2005/ab00832.htm,

2005.

Keel M, Lustenberger T, Mica L, Lüthi S, Labler L, Trentz O. Der Schweregrad der

Verletzungen und des haemorrhagischen Schocks korrelieren mit der Inzidenz von

Infektionen und septischen KomplikationenIn: 1. Gemeinsamer Kongress Orthopädie und

Unfallchirurgie www.abstractserver.de/abstracts/ou2005/ab00847.htm, 2005.

Mica L, Labler L, Lustenberger T, Trentz O, Keel M. Outcome of polytraumatized elder

patients: a retrospective study of 780 patients. Eur J Trauma, Supplement 1: 37, 2006.

Labler L, Mica L, Trentz O, Imhof HG. Mild traumatic breain injury in eldery patients. Eur J

Trauma, Supplement 1: 31, 2006.

Härter L, Mica L, Trentz O, Keel M. Up-regulation of Toll-like receptors on monocytes

durino sepsis. Eur J Trauma, Supplement 1: 238, 2006.

L. Mica, L. Labler, O. Trentz, L. Härter, M. Keel. Increased survival of neutrophil

granulocytes in VAC-treated compared to Epigard-treated wounds. Swiss Knife Special

Edition 2006;35-36, 17.06

L. Mica, L. Härter, M. Keel, O. Trentz. LPS prevents lysosomal decay during spontaneous

apoptosis in neutrophil granulocytes. Swiss Knife Special Edition 2006; 36, 17.08

R. von Kaenel, U. Hepp, R. Traber, B. Kraemer, L. Mica, M. Keel, U Schnyder. Evidence for

endothelial dysfunction in posttraumatic stress disorder. American Psychosomatic Society A-

54, 2006

Mica L, Labler L, Härter L, Trentz O, Keel M. Increased survival of neutrophil granulocytes

in VAC-treated compared to Epigard-treated Wounds. 6th Day of Clinical Research,

University Hospital of Zürich, March, 2007.

97

Härter L, Labler L, Mica L, Trentz O, Keel M. VAC-therapy induces local activation of

neutrophil granulocytes in traumatic wounds. 6th Day of Clinical Research, University

Hospital of Zürich, March, 2007.

T. Lustenberger, L. Mica, M. Turina, O. Trentz, M. Keel. Severe Hemorhagic Shock

Increases Mortality in Patients with Traumatic Brain Injury. Swiss Knife Special Edition

2007;53, 21.16

M. Turina, A. Biletter, L. Mica, T. Lustenberger, O. Trentz, M. Keel. Serum Procalcitonin

and Interleukin-6 Correlate with Infectious Complikations in 1079 Severely Traumatized

Patients, with strongest Correlation Observed in Subsequently Septic Patients. Swiss Knife

Special Edition 2007;54, 24.3

L. Mica, L. Labler, O. Trentz, L. Härter and M. Keel Increased survival of neutrophil

granulocytes in VAC®-treated compared to Epigard®-treated wounds. EATES Graz 2007

T. Lustenberger, L. Mica, M. Turina, O. Trentz and M. Keel Traumatic brain injury increases

mortality and morbidity in patients with hemorrhagic shock. EATES Graz 2007

P.M. Lenzlinger, T. Lustenberger, L. Mica, M. Keel. Severe Chest Injury in Polytrauma.

European Journal of Trauma and emergency Surgery 2008;34 (Supp. I), 54

L. Mica, M. Rancan, L. Härter, T. Lustenberger, M. Keel. Increased G-CSF in Wound Fluid

from VAC-Treated Wounds is not Responsible for Increased Neutrophil Survival. European

Journal of Trauma and emergency Surgery 2008;34 (Supp. I), 63

L. Mica, D. Gianom, B. Bode, P. Jaklin, A. Hollinger. Rare cause of dysphagy: well

differentiated esophageal wall liposarcoma. Swiss Knife 2008; special edition, 73

L. Mica, A. Schwaller, C. Stoupis, I. Penka, J. Vomela, A. Vollenweider. Pelvis-near avulsion

of the hamsting muscle group. Swiss Knife Special Edition 2009; 36

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M033 – A. Frischknecht, T. Lustenberger, M. Bukur, M. Turina, A. Billeter, L. Mica, M.

Keel. Damage control in severely injured trauma patients. A ten-year experience. ESTES

2010 Bruxelles

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