Substance p regulates chemokine production in leukocytes implications in the pathogenesis of acute pancreatitis and associated lung injury

In a mouse model of caerulein-induced acute pancreatitis and associated lung injury, blockade of the biological actions of SP with a specific antagonist of its neurokinin-1 receptor NK-1

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ACKNOWLEDGEMENTS

First of all, I would like to express my heartfelt gratitude to my supervisor, Associate Professor Madhav Bhatia for providing me the great opportunity to work on this project and for his invaluable advice, guidance, continuous support and encouragement throughout the study

I would also like to thank Ms Shoon Mei Leng, the senior laboratory officer, for her assistance, support and her excellent work in maintaining our orderly conducive laboratory environment

My appreciation is also extended to every member of the Life Sciences Institute Cardiovascular Biology Program, Akhil Hegde, Cao Yang, He Min, Jenab Nooruddinbhai Sidhapuriw, Koh Yung Hua, Raina Devi Ramnath, Ramasamy Tamizhselvi, Selena Sio Weishan, and Zhi Liang for insightful discussions, technical advice and help in one way or another

Last but not least, I would like to give my special thanks to my parents Without their support and encouragement, this project could not have been completed

TABLE OF CONTENTS

ACKNOWLEDGEMENTS I SUMMARY VI LIST OF FIGURES IX ABBREVIATIONS XIII PUBLICATIONS XV CHAPTER I INTRODUCTION 1

1.1 G ENERAL OVERVIEW 1

1.2 A CUTE PANCREATITIS 2

1.2.1 Pathophysiology of acute pancreatitis and associated lung injury 3

1.2.1.1 Inflammatory mediators in acute pancreatitis and associated lung injury 4

1.2.1.2 Leukocytes in acute pancreatitis and associated lung injury 5

1.2.1.2.1 Leukocyte trafficking during inflammation 5

1.2.1.2.2 Neutrophils 6

1.2.1.2.3 Macrophages 8

1.2.2 Experimental models of acute pancreatitis 9

1.2.2.1 Principle of caerulein-induced acute pancreatitis model 9

1.2.2.2 Induction and characteristics of caerulein-induced acute pancreatitis model 10

1.3 SP 11

1.3.1 SP in acute pancreatitis and associated lung injury 11

1.3.2 SP in immunoregulation 12

1.3.2.1 SP in immunoregulation: neutrophils 12

1.3.2.2 SP in immunoregulation: macrophages 13

1.4 C HEMOKINES 14

1.4.1 Chemokines and their receptors in acute pancreatitis and associated lung injury 15

1.5 I NTRACELLULAR SIGNALING MOLECULES 16

1.5.1 NF-κB 17

1.5.2 MAPKs 18

1.5.3 PKC isoforms 19

1.5.4 PI3K-Akt 19

1.6 R ESEARCH RATIONALE AND OBJECTIVES 21

1.6.1 Research rationale 21

1.6.2 Objectives 22

CHAPTER II: SP REGULATES CHEMOKINE EXPRESSION IN LEUKOCYTES DURING ACUTE PANCREATITIS AND ASSOCIATED LUNG INJURY 24

2.1 I NTRODUCTION 24

2.2 M ATERIALS AND METHODS 25

2.2.1 Animals 25

2.2.2 Reagents 26

2.2.3 Caerulein-induced acute pancreatitis and associated lung injury model 26

2.2.4 Total RNA isolation and RT-PCR 27

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2.2.5 Immunohistochemistry 28

2.2.6 Statistical analysis 29

2.3 R ESULTS 30

2.3.1 Effects of caerulein hyperstimulation and CP96,345 treatment on pancreatic and lung MCP-1 expression 30

2.3.2 Effects of caerulein hyperstimulation and CP96,345 treatment on pancreatic and lung RANTES expression 30

2.3.3 Effects of caerulein hyperstimulation and CP96,345 treatment on pancreatic and lung MIP-1α expression 31

2.3.4 Effects of caerulein hyperstimulation and CP96,345 treatment on pancreatic and lung MIP-2 expression 31

2.3.5 Effects of caerulein hyperstimulation and CP96,345 treatment on immunohistochemical localization of MCP-1, MIP-1α and MIP-2 in the pancreas and lungs 32

2.4 D ISCUSSION 32

CHAPTER III IN VITRO EVIDENCE OF INDUCTION OF CHEMOKINE PRODUCTION AND CHEMOKINE RECEPTOR EXPRESSION IN MOUSE NEUTROPHILS BY SP AND THE CELLULAR MECHANISM 46

3.1 I NTRODUCTION 46

3.2 M ATERIALS AND METHODS 47

3.2.1 Animals 47

3.2.2 Reagents 47

3.2.3 Isolation of mouse primary neutrophils 48

3.2.4 Cell treatment 49

3.2.5 Cell migration assay 49

3.2.6 Total RNA isolation and RT-PCR 50

3.2.7 ELISA 51

3.2.8 Flow cytometry 51

3.2.9 Immunofluorescence staining 51

3.2.10 Nuclear extract preparation 52

3.2.11 NF-κB DNA-binding activity assay 52

3.2.12 Whole cell lysate preparation and Western blot analysis 53

3.2.13 Statistical analysis 54

3.3 R ESULTS 54

3.3.1 SP treatment activated mouse primary neutrophils 54

3.3.2 SP treatment activated the transcription factor NF-κB in mouse primary neutrophils 55

3.3.3 SP treatment enhanced chemokine expression in mouse primary neutrophils 56

3.3.4 SP treatment induced chemokine receptor expression in mouse primary neutrophils 57

3.3.5 SP treatment enhanced chemotactic responses of mouse primary neutrophils to rMIP-2 and rMIP-1α 58

3.4 D ISCUSSION 59

CHAPTER IV IN VITRO EVIDENCE OF INDUCTION OF CHEMOKINE PRODUCTION IN MOUSE MACROPHAGES BY SP AND THE CELLULAR MECHANISMS – ROLE OF NF-KB AND ERK1/2 72

4.1 I NTRODUCTION 72

4.2 M ATERIALS AND METHODS 73

4.2.1 Reagents 73

4.2.2 Cell culture and treatment 73

4.2.3 Isolation of peritoneal macrophages 74

4.2.4 ELISA 74

4.2.5 Whole cell lysate preparation and Western blot analysis 75

4.2.6 Nuclear extract preparation 75

4.2.7 NF-κB DNA-binding activity assay 75

4.2.8 EMSA 75

4.2.9 Statistical analysis 76

4.3 R ESULTS 76

4.3.1 SP induced chemokine production in mouse macrophages 76

4.3.2 NK-1R but not NK-2R antagonists abolished SP-induced chemokine production in mouse macrophages 78

4.3.3 SP enhanced NF-κB but not AP-1 activity via the NK-1R in mouse macrophages 78

4.3.4 SP activated NF-κB via the classical and atypical pathways in mouse macrophages 79

4.3.5 Inhibition of NF-κB activation abolished SP-induced chemokine production in mouse macrophages 80

4.3.6 SP activated ERK1/2 in mouse macrophages 80

4.3.7 Inhibition of ERK1/2 activation attenuated SP-induced chemokine production in mouse macrophages 81

4.3.8 Inhibition of ERK1/2 activation prevented SP-induced phosphorylation and nuclear translocation of NF-κB p65 in mouse macrophages 81

4.4 D ISCUSSION 82

CHAPTER V THE CELLULAR MECHANISMS OF SP-INDUCED CHEMOKINE PRODUCTION IN MOUSE MACROPHAGES – ROLE OF PKC ISOFORMS AND PI3K-AKT 101

5.1 I NTRODUCTION 101

5.2 M ATERIALS AND METHODS 101

5.2.1 Reagents 101

5.2.2 Cell culture and treatment 102

5.2.3 Isolation of peritoneal macrophages 102

5.2.4 ELISA 102

5.2.5 Whole cell lysate preparation and Western blot analysis 102

5.2.6 Subcellular fractionation 102

5.2.7 Nuclear extract preparation 103

5.2.8 NF-κB DNA-binding activity assay 103

5.2.9 Statistical analysis 103

5.3 R ESULTS 103

5.3.1 SP induced PKCα, δ and ε activation via NK-1R in mouse macrophages 103

5.3.2 PKCα, δ and ε were involved in SP-induced chemokine production in mouse macrophages 104

5.3.3 PKCα, δ and ε were involved in SP-induced ERK1/2 activation in mouse macrophages 105

5.3.4 PKCα, δ and ε were involved in SP-induced NF-κB activation in mouse macrophages 106

5.3.5 SP induced activation of PI3K-Akt pathway via NK-1R in mouse macrophages 106

5.3.6 PI3K-Akt pathway was involved in SP-induced ERK1/2 and NF-κB activation and chemokine production in mouse macrophages 107

5.3.7 PKCs and PI3K-Akt were two independent convergent pathways activated by SP in mouse macrophages 108

5.3.8 SP induced PKC and PI3K-Akt pathways in primary peritoneal macrophages 108

5.3.9 Signal transduction pathways induced by SP in mouse macrophages 109

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5.4 D ISCUSSION 109

CHAPTER VI NKA INDUCES CHEMOKINE PRODUCTION BY MOUSE MACROPHAGES VIA ERK1/2- AND PI3K-AKT-NFKB PATHWAYS 139

6.1 I NTRODUCTION 139

6.2 M ATERIALS AND METHODS 140

6.2.1 Reagents 140

6.2.2 Cell treatment 141

6.2.3 Isolation of primary peritoneal macrophages 141

6.2.4 Whole cell lysate preparation and Western blot analysis 141

6.2.5 Nuclear extract preparation 141

6.2.6 NF-κB DNA-binding activity assay 142

6.2.7 Total RNA isolation and RT-PCR 142

6.2.8 Immunofluorescence staining 142

6.2.9 ELISA 143

6.2.10 Statistical analysis 143

6.3 R ESULTS 143

6.3.1 NKA upregulated NK-1R expression in mouse macrophages lacking detectable NK-2R 143

6.3.2 NKA induced chemokine production via NK-1R in mouse macrophages 144

6.3.3 NKA induced NF-κB activation via NK-1R in mouse macrophages 145

6.3.4 NKA activated ERK1/2 and PI3K-Akt pathways in mouse macrophages 146

6.3.5 ERK1/2 and PI3K-Akt pathways were involved in NKA-induced NF-κB activation and chemokine production in mouse macrophages 146

6.4 D ISCUSSION 147

CHAPTER VII SUMMARY OF CONTRIBUTIONS AND FUTURE RESEARCH 171

7.1 S UMMARY OF CONTRIBUTIONS 171

7.2 F UTURE RESEARCH 174

REFERENCES 177

SUMMARY

The neuropeptide substance P (SP) is a well-recognized inflammatory mediator in acute pancreatitis However, the mechanism remains elusive Aiming to delineate its mechanistic actions, the present study attempts to trace the signal transduction cascade activated by SP in the regulation of release of inflammatory molecules that contribute to disease progression

In a mouse model of caerulein-induced acute pancreatitis and associated lung injury, blockade of the biological actions of SP with a specific antagonist of its neurokinin-1 receptor (NK-1R) significantly attenuated the chemokine (MCP-1, MIP-1α, and MIP-2) expression in pancreas and lungs, which correlated with less leukocyte infiltration and alleviated tissue damages Resident/infiltrating leukocytes (neutrophils and macrophages) were identified as major chemokine-expressing cells in both pancreas and lungs These preliminary observations suggested a potential mechanistic connection between SP, leukocytes and chemokines in acute pancreatitis – SP acting via resident/recruited leukocytes in tissues to induce the release of chemokines, which further aggravate the condition

In vitro studies on isolated cultured mouse neutrophils and macrophages supported that

SP, via NK-1R, exerts a direct stimulatory effect on chemokine (MCP-1, MIP-1α, and MIP-2) production by both cell types In neutrophils, SP-NK-1R also induced the expression of CD11b (a neutrophil activation marker) and chemokine receptors (CCR1 and CXCR2) As a functional consequence, SP-stimulated neutrophils exhibited

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enhanced migratory responses towards chemokines which implied an additional role for

SP in promoting leukocyte activation and migration in vivo

Mouse macrophages were also used as a model system to elucidate cellular mechanisms

of SP-induced chemokine production SP triggered a cascade of molecular events in the cells A key signaling molecule involved was the transcription factor NF-κB SP induced both classical and atypical pathways of NF-κB activation in mouse macrophages Inhibition of NF-κB abolished SP-induced chemokine production by macrophages Furthermore, extracellular signal-regulated kinase (ERK)1/2 lie upstream of NF-κB in SP-induced signal transduction cascade ERK1/2 were activated in the cells between 5 and 30 min following SP stimulation A specific ERK1/2 inhibitor prevented activation

of NF-κB and chemokine production induced by SP Two kinase pathways, PKCs and PI3K-Akt were further identified to be important for mediating SP-induced ERK1/2 activation PKC isoforms, including the conventional PKCα and novel PKCδ and ε, were selectively activated by SP-NK-1R at early time points (3 min) Inhibition of PKCα, δ, or

ε attenuated SP-NK-1R-indcued ERK1/2, NF-κB activation and chemokine production PI3K-Akt pathway was activated at a later time point (15 min) than PKCs and also preceded through ERK1/2 and NF-κB activation to induce chemokine production in macrophages PKCs and PI3K-Akt were two independent pathways and acted synergistically to trigger downstream events PKCs were likely to induce early ERK1/2 activation while PI3K-Akt contributed to the pathway at later time points Collectively, these data indicate that SP-NK-1R triggers two convergent signaling pathways, one led

by PKC isoforms and the other by PI3K-Akt, which induce ERK1/2 and NF-κB activation, leading to upregulated chemokine expression in leukocytes

Neurokinin A (NKA), another neuropeptide of the tachykinin family, was found to have similar inductive effects on chemokine production in macrophages, mediated via PI3K-Akt- and ERK1/2-dependent NF-κB activation

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LIST OF FIGURES

Figure 2.1 CP96,345 treatment attenuated caerulein-induced increase in MCP-1 mRNA

expression in the pancreas and lungs

Figure 2.2 Effects of caerulein hyperstimulation and CP96,345 treatment on

immunohistochemical localization of MCP-1 in the pancreas and lungs

Figure 2.3 CP96,345 treatment had no effect on caerulein-induced upregulation of

RANTES mRNA expression in the pancreas

Figure 2.4 CP96,345 treatment suppressed caerulein-induced upregulation of MIP-1α

mRNA expression in the pancreas and lungs

Figure 2.5 Effects of caerulein hyperstimulation and CP96,345 treatment on

immunohistochemical localization of MIP-1α in the pancreas and lungs

Figure 2.6 CP96,345 treatment attenuated caerulein-induced increase in the MIP-2

mRNA expression in the pancreas and lungs

Figure 2.7 Effects of caerulein hyperstimulation and CP96,345 treatment on

immunohistochemical localization of MIP-2 in the pancreas and lungs

Figure 3.1 SP upregulated CD11b integrin (neutrophils activation marker) expression

in primary mouse neutrophils

Figure 3.2 SP stimulated NF-κB binding activity, IκBα degradation and NF-κB

translocation in primary mouse neutrophils

Figure 3.3 Effect of SP stimulation on MIP-1α and MIP-2 expression by primary

mouse neutrophils

Figure 3.4 Effect of SP stimulation on CCR1, CCR2 and CXCR2 expression in

primary mouse neutrophils

Figure 3.5 SP stimulation enhanced chemotactic responses of primary mouse

neutrophils to rMIP-2 and rMIP-1α

Figure 4.1 SP enhanced chemokine production in RAW 264.7 macrophages

Figure 4.2 SP enhanced chemokine production in primary peritoneal macrophages

Figure 4.3 Effect of NK receptor antagonists on SP-induced MIP-2 and MCP-1

upregulation

Figure 4.4 SP enhanced NF-κB p65 but not AP-1 DNA-binding activity via the NK-1R

Figure 4.5 SP activated NF-κB via the classical and atypical pathways in RAW 264.7

Figure 4.8 Inhibition of ERK1/2 activation attenuated SP-induced chemokine

production in RAW 264.7 cells

Figure 4.9 SP-induced NF-κB DNA-binding activity was not dependent of ERK1/2

Figure 4.10 ERK1/2 was involved in Ser276-phosphorylation of NF-κB p65 subunit

Figure 5.1 SP induced membrane translocation of PKCα, δ, and ε in mouse

macrophages

Figure 5.2 SP induced phosphorylation of PKCα, δ, and ε in mouse macrophages

Figure 5.3 SP-induced PKC translocataion was NK-1R-dependent

Figure 5.4 PKCα, δ, and ε were involved in mediating SP-induced MIP-2 production

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Figure 5.9 Effects of a PI3K-Akt inhibitor LY294002 and an Akt inhibitor Triciribine

on SP-induced MIP-2 production in mouse macrophages

Figure 5.10 Effects of a PI3K-Akt inhibitor LY294002 and an Akt inhibitor Triciribine

on SP-induced MCP-1 production in mouse macrophages

Figure 5.11 PI3K-Akt was involved in mediating SP-induced ERK1/2 activation in

mouse macrophages

Figure 5.12 PI3K-Akt was involved in SP-induced NF-κB activation in mouse

macrophages

Figure 5.13 PKC and PI3K-Akt pathways activated by SP in mouse macrophages were

independent of each other

Figure 5.14 SP induced PKC and PI3K-Akt pathways to mediate chemokine expression

in primary peritoneal macrophages

Figure 6.1 NKA upregulated NK-1R mRNA and protein expression in mouse

macrophages

Figure 6.2 Lack of detectable NK-2R expression in mouse macrophages

Figure 6.3 NKA induced chemokine expression in RAW 264.7 macrophages via the

NK-1R

Figure 6.4 NKA induced MIP-2 and MCP-1 production in mouse primary macrophages

via the NK-1R

Figure 6.5 NKA activated the transcription factor NF-κB

Figure 6.6 NKA enhanced the DNA-binding activity of nuclear NF-κB via the NK-1R

Figure 6.7 NKA induced activation of ERK1/2 and PI3K-Akt signaling kinases in

RAW 264.7 macrophages

Figure 6.8 PI3K-Akt and ERK1/2 mediated NKA-induced signal transduction leading

to NF-κB activation

Figure 6.9 Blockade of ERK1/2 or PI3K-Akt pathway inhibits NKA-induced

chemokine synthesis in RAW 264.7 cells

Figure 6.10 ERK1/2 and PI3K-Akt pathways were important for NKA-induced

chemokine production in mouse primary macrophages

Figure 6.11 NKA-induced signal transduction cascade in mouse macrophages

Figure 7.1 Schematic summary of SP-initiated signal transduction cascade in mouse

macrophages

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ABBREVIATIONS

ARDS Acute respiratory distress syndrome

CINC Cytokine-induced neutrophil chemoattractant

DMEM Dulbecco’s modified Eagle’s medium

EDTA Ethylenediaminetetraacetic

ELISA Enzyme-linked immunosorbent assay

ERK Extracellular signal-regulated kinase

FITC Fluorescein isothiocyanate

fMLP Formyl-methionyl-leucyl-phenylalanine

GAPDH Glyceraldehyde 3-phosphate dehydrogenase

GRO Growth-regulated oncogene

HPRT Hypoxanthine-guanine phosphoribosyl transferase

MAPK Mitogen-activated protein kinase

MCP Monocyte chemoattractant protein

MIP Macrophage inflammatory protein

MODS Multiple organ dysfunction syndrome

PAF Platelet activating factor

PCR Polymerase chain reaction

PPT-A Preprotachykinin-A

RANTES Regulated on activation, T cell expressed and secreted

RIPA Radio-immunoprecipitation assay

RT-PCR Reverse transcriptase-polymerase chain reaction

SIRS Systemic inflammatory response syndromes

TIP Translocation inhibitor peptide

VCAM Vascular cell adhesion molecule

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PUBLICATIONS

Original reports, reviews, and book chapters

1 Sun J, Bhatia M Blockade of neurokinin-1 receptor attenuates CC and CXC

chemokine production in experimental acute pancreatitis and associated lung

injury Am J Physiol Gastrointest Liver Physiol 2007; 292: G143-G153

2 Sun J, Ramnath RD, Bhatia M Neuropeptide substance P upregulates chemokine

and chemokine receptor expression in primary mouse neutrophils Am J Physiol

Cell Physiol 2007; 293: C696-C704

3 Sun J, Ramnath RD, Zhi L, Tamizhselvi R, Bhatia M Substance P Enhances

NF-{kappa}B Transactivation and Chemokine Response in Murine Macrophages via

ERK1/2 and p38 MAPK Signaling Pathways Am J Physiol Cell Physiol 2008;

294: C1586-C1596

4 Sun J, Ramnath RD, Tamizhselvi R, Bhatia M Neurokinin A Engages

Neurokinin-1 Receptor to Induce NF-{kappa}B-dependent Gene Expression in

Murine Macrophages: Implications of ERK1/2 and PI3K-Akt Pathways Am J

Physiol Cell Physiol 2008; 295: C679-C691

5 Sun J, Ramnath RD, Tamizhselvi R, Bhatia M PKC and PI3K-Akt Pathways are

Involved in Substance P-Induced ERK1/2-NF-κB Activation and Chemokine

Expression in Mouse Macrophages FASEB J 2008 (In press)

6 Ramnath RD, Sun J, Adhikari S, Bhatia M Effect of mitogen-activated protein

kinases on chemokine synthesis induced by substance P in mouse pancreatic

acinar cells J Cell Mol Med 2007; 11: 1326-1341

7 Ramnath RD, Sun J, Adhikari S, Zhi L, Bhatia M Role of PKC-delta on

substance P-induced chemokine synthesis in pancreatic acinar cells Am J Physiol

Cell Physiol 2008; 294: C683-C692

8 Ramnath RD, Sun J, Bhatia M Role of calcium in substance P-induced

chemokine synthesis in mouse pancreatic acinar cells Br J Pharmacol 2008; 154:

1339-1348

9 Ramnath RD, Sun J, Bhatia M Involvement of Src family kinases in substance

P-induced chemokine production in mouse pancreatic acinar cells, its significance in

acute pancreatitis J Pharmacol Exp Ther 2008 (Accepted)

10 Tamizhselvi R, Sun J, Koh YH, Bhatia M Hydrogen sulfide acts as a mediator of

inflammation in acute pancreatitis by up- and down-regulating the production of cytokines via ERK 1/2 and NF-κB: in vitro studies using isolated mouse

pancreatic acinar cells 2008 (Submitted)

11 Ramnath RD, Ng SW, He M, Sun J, Zhang H, Bawa MS, Bhatia M

Inflammatory mediators in sepsis: cytokines, chemokines, adhesion molecules

and gases J Organ Dysfunction 2006; 2: 80-92 (Review)

12 Bhatia M, Sun J, He M, Hegde A, Ramnath RD Chemokines in acute

pancreatitis In: Progress in Chemokine Research, Chemokine Research Frontiers,

edited by Linkes WP ©2007 Nova Science Publishers, Inc

Abstracts published in journals

1 Sun J, Bhatia M Blockade of neurokinin 1 receptor attenuates CC and CXC

chemokine production in acute pancreatitis Pancreatoloty 2006; 6: 344

2 Sun J, Bhatia M Blockade of neurokinin 1 receptor attenuates CC and CXC

chemokine production in acute pancreatitis FEBS J 2006; 273: 284

3 Sun J, Bhatia M Blockade of neurokinin 1 receptor attenuates CC and CXC

chemokine production in acute pancreatitis and associated lung injury Acta

Pharmacol Sin 2006; 27: 267

4 Bhatia M, Sun J Effect of neurokinin 1 receptor antagonist treatment on

pancreatitis-induced increase in chemokine levels Inflammation Research 2005;

54: S177

5 Bhatia M, Sun J Effect of neurokinin 1 receptor antagonist treatment on acute

pancreatitis-induced increase in chemokine levels J Gastroenterol Hepatol 2005;

20: A231

6 Ramnath RD, Sun J, Bhatia M Role of MAP kinase in substance P-induced

chemokine synthesis in pancreatic acinar cells Pancreatology 2007; 7: 228

7 Ramnath RD, Sun J, Bhatia M Participation of phospholipase C in substance

P-induced chemokine production in pancreatic acinar cells Pancreatology 2008; 8

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International conference presentations and awards

1 Sun J, Bhatia M Blockade of neurokinin 1 receptor attenuates CC and CXC

chemokine production in acute pancreatitis 38th Meeting of the European

Pancreatic Club, 2006, Tampere, Finland, poster presentation on travel scholarship

2 Sun J, Bhatia M Blockade of neurokinin 1 receptor attenuates CC and CXC

chemokine production in acute pancreatitis 31st FEBS Congress Molecules in

Health & Disease, Istanbul, Turkey, poster presentation

CHAPTER I INTRODUCTION

1.1 General overview

Acute pancreatitis is a common clinical disorder with potentially devastating consequences The incidence of the condition varies geographically, with a yearly average of 35-80 cases per million population worldwide (Kong et al., 2004) The number has been reported to increase over the last few decades (Bhatia, 2002aa; Sinclair

et al., 1997) The condition is mild and self-limited in most cases However, about fourth of patients suffer a severe attack and the mortality rate among them is high up to 50% (Bhatia et al., 2000a) In recent years, the mortality rate has tended to decrease (Beger et al., 1997; Talamini et al., 1996) due to medical advances in intensive and critical care and management However, so far, few, if any, curative remedies are available for this condition Most acute pancreatitis cases are secondary to biliary disease

one-or excess alcohol consumption The exact mechanisms by which diverse etiological factors induce an attack remain to be fully understood, but it is generally believed that a local inflammatory reaction develops following acinar cell injury; if marked, it leads to a systemic inflammatory response syndrome (SIRS) and the development of multiple organ dynfunction syndrome (MODS), which is ultimately responsible for the majority of pancreatitis-associated morbidity and mortality (Bhatia et al., 2001) Impaired lung function is frequently the first indication of MODS and manifests clinically as acute respiratory distress syndrome (ARDS) (Bhatia and Moochhala, 2004) A large number of

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studies have shown that various inflammatory mediators, released locally or systemically, amplify the local as well as systemic inflammatory responses in acute pancreatitis The severity of the disease is therefore largely determined by the actions of these molecules Mediators involved in mediating local pancreatitis (Bhatia et al., 2000a) or in coupling pancreatitis to its systemic complications, particularly the lung injury (Bhatia and Moochhala, 2004), have been identified

SP, a neuropeptide belonging to the tachykinin family is a well-recognized proinflammatory mediator in acute pancreatitis and associated lung injury Both SP and its primary receptor neurokinin-1 receptor (NK-1R) are demonstrated as important determinants of disease severity (Bhatia et al., 1998b; Bhatia et al., 2003; Lau et al., 2005; Maa et al., 2000) Although the pathophysiological role of SP in acute pancreatitis has been known for long, the mechanism by which SP mediates the local and systemic inflammatory responses during disease progression remains unclear Earlier experimental evidence suggests interaction of this neuropeptide with other mediator molecules and cells may contribute to its proinflammatory actions (Lau et al., 2005; O’Connor et al., 2004; Poch et al., 1999; Saban et al., 1997; Sterner-Kock et al., 1999)

1.2 Acute pancreatitis

Acute pancreatitis refers to the acute form of inflammation of the pancreas, associated with autodigestion of the pancreas, pancreatic edema, necrosis, and hemorrhage Gallstones and alcoholism are the two most common causes of acute pancreatitis that account for more than 80% of all cases, although the ratio of these two causes has a wide geographical variation (Steinberg and Tenner, 1994) Other etiologies include certain

medications, trauma, metabolic disorders (e.g., hypertriglyceridemia and hypercalcemia), invasive procedures of the biliary and pancreatic duct (e.g., endoscopic retrograde cholangiopancreatography [ERCP]), and idiopathy.The severity of acute pancreatitis is characterized by wide severity variation ranging from a mild self-limited form with interstitial edema of the pancreas to a severe lethal form with extensive pancreatic necrosis, often with progress to SIRS, MODS, and eventually death Various inflammatory mediators have been identified as important determinants of the severity of the disease It is believed that the presence of pancreatic necrosis is a main prognostic factor in acute pancreatitis (Baron and Morgan, 1999; Isenmann et al., 1999) The development of intra- and extra-pancreatic necrosis strongly influences the risk of sustained and exaggerated systemic inflammatory response, and multiple organ failure, which is associated with a significant mortality rate (Rau et al., 2003)

1.2.1 Pathophysiology of acute pancreatitis and associated lung injury

Acute pancreatitis involves a complex cascade of events A critical initiating event is the intraacinar activation of pancreatic zymogens, which leads to acinar cell damage and autodigestion of the pancreas Trypsinogen, a serine protease, is believed to be the first enzyme to be activated Subsequently, other digestive proenzymes are cleaved and activated (Bhatia et al., 2000a; Gorelick et al., 1999) The pancreas has a variety of mechanisms to prevent intracellular zymogen activation and autodigestion However, in pancreatitis, these protective mechanisms are no longer effective (Grady et al., 1998) A local inflammatory response occurs as a result of acinar cell damage Inflammatory mediators spill over into the general circulation, leading to SIRS The severity of an

4   

attack of acute pancreatitis is esentially determined by the magnitude of the resultant systemic inflammatory response Although SIRS is principally considered as the normal host response to the insults, sustained or exaggerated SIRS leads to the development of distant organ damage and MODS, which is ultimately responsible for most pancreatitis-associated morbidity and mortality (Bhatia et al., 2001)

The first sign of MODS in acute pancreatitis is often impaired lung function which manifests clinically as ARDS The lungs become edematous and congested, leading to collapse of the smaller airways, with decreased lung compliance and respiratory failure (Ashbaugh et al., 1967) In severe acute pancreatitis, the very first changes can be seen a few hours after the onset of an attack Systemic leukocyte activation and migration play a predominant role in the pathogenesis of pancreatitis-associated lung injury (Gerard et al., 1997; Luster, 1998) Leukocytes are activated within the general circulation as a consequence of an overactive SIRS response Some then lodge within the pulmonary microcirculation As the condition develops, leukocytes migrate into the pulmonary interstitium causing increased endothelial permeability and tissue edema (Bhatia et al., 2001; Murakami et al., 1995) Other distant organs commonly affected in a severe acute pancreatitis include the kidneys and liver

1.2.1.1 Inflammatory mediators in acute pancreatitis and associated lung injury

Various inflammatory mediators are recognized to play a critical role in the pathogenesis

of acute pancreatitis and more so, of the subsequent inflammatory response and the resultant MODS (Bhatia et al., 2005) Inflammatory mediators important in local acute pancreatitis include cytokines (tumor necrosis factor-alpha [TNF-α], interleukin-1beta

[IL-1β], interleukin[IL]-6, and IL-10), platelet activating factor (PAF), CD40L, complement C5a, intercellular adhesion molecule-1 (ICAM-1), SP, hydrogen sulfide (H2S), neutral endopeptidase (NEP), and chemokines such as IL-8, cytokine-induced neutrophil chemoattractant (CINC)/growth-regulated oncogene-alpha (GRO-α), monocyte chemoattractant protein (MCP)-1 (Bhatia et al., 2001) Inflammatory mediators implicated in coupling of acute pancreatitis to distant organ damage particularly lung injury include TNF-α, IL-1, -4, -6, -8, -10, and -13, SP, PAF, adhesion molecules (e.g., ICAM-1, vascular cell adhesion molecule-1 [VCAM-1], E-selectin, P-selectin), granulocyte macrophage-colony stimulating factor (GM-CSF), C5a, chemokines, VEGF, IGF-I, KGF, reactive oxygen species (ROS), and reactive nitrogen species (RNS) (Bhatia and Moochhala, 2004) Among the mediators, SP and chemokines with their importance

in both local and distant inflammatory responses are the focuses of the current study These two mediators are reviewed in greater details in section 1.3 and 1.4, respectively

1.2.1.2 Leukocytes in acute pancreatitis and associated lung injury

Leukocyte trafficking to the site of injury is a central event in any inflammatory process During acute pancreatitis, excessive leukocyte activation and infiltration play a critical role in mediating local inflammatory reponse and more so, in the progression of local inflammation to the systemic inflammatory responses Notably, activated leukoyctes are key contributors to pancreatitis-associated distant organ damage such as lung injury and two leukocyte subpopulations, namely polymorphonuclear neutrophils and macrophages, have been particularly associated with the condition

1.2.1.2.1 Leukocyte trafficking during inflammation

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Leukocyte migration from blood vessel to areas of tissue injury during inflammation is a highly-regulated multistep process (Gerard and Rollins, 2001) Initial leukocyte rolling/tethering on endothelium is mediated by selectin family of adhesion molecules (E- and P-selectin) Selectins reduce the velocity of circulating leukocytes before they detect chemokines immobilized on the endothelial cells Chemokines subsequently bind to their receptors and chemokine signaling activates leukocyte integrins which can bind with endothelial cell surface adhesion molecules such as ICAM-1, leading to a firm adherence (Adams and Nash, 1996) Leukocytes are then believed to migrate along a chemokine gradient stabilized within the endothelial glycocalyx (Luster, 1998) Subsequent to infiltration to damaged tissues, leukocytes release various deleterious factors such as cytokines, proteolytic enzymes, and oxygen free radicals that enhance tissue destruction and propagate the disease to remote organs

The integrin CD11b is an adhesion molecule involved in the firm adhesion of leukocytes

to the endothelium and in transendothelial migration Surface CD11b upregulation has been widely accepted as a neutrophil activation marker and it was assessed to determine the activating effect of SP on neutrophils in the current study

1.2.1.2.2 Neutrophils

Polymorphonuclear neutrophils are the first leukocyte subpopulation that appear at the site of inflammation and have a central role in the clearance of infectious pathogens and the innate immunity Therefore, they have been implicated in mediating the acute-phase inflammation in different disease models including acute pancreatitis (Puneet et al., 2006; Sun and Bhatia, 2007) Neutrophils play a pathogenetic role in acute pancreatitis.Their

activation has been indicated as an important determinant of severity of acute pancreatitis (Bhatia et al., 2005)

Neutrophil infiltration into damaged tissues is associated with the development of pancreatic and pulmonary injury in acute pancreatitis In fact, the neutrophil granular enzyme myeloperoxidase (MPO) in tissues is a biomarker for assessing tissue damages associated with acute pancreatitis and associated lung injury (Bhatia et al., 1998a; Lau et al., 2005) The mechanism that initiates neutrophil infiltration is still poorly understood However, it is generally believed that recruitment of circulating neutrophils is induced by acinar cell injury and the release of pancreatic proteases Many other factors including adhesion molecules (ICAM-1 and P-selectin) (Genovese et al., 2006; Hartwig et al., 2004), cytokines like TGF-β1 (Schafer et al., 2005), chemokines like MCP-1 (Brady et al., 2002), and SP (Noble et al., 2006) may be involved in the recruitment The activated neutrophils, in turn, lead to more acinar cell injury and are responsible for the systemic inflammatory responses typically associated with severe acute pancreatitis

Strategies that interfere with neutrophil migration, such as depletion of circulating neutrophils or use of antibodies that prevent the adhesion or migration of neutrophils, have been demonstrated to reduce the severity of pancreatic and pulmonary damage in experimental models of acute pancreatitis (Bhatia et al., 1998a; Guo et al., 1995; Inoue et al., 1995; Inoue et al., 1996).Administration of anti-neutrophil sera to deplete circulating neutrophils in mice reduces the severity of pancreatitis and pancreatitis-associated lung injury (Pastor et al., 2006) Depletion of neutrophils is also associated with increased aciniar cell apoptosis which correlates with reduced severity of acute pancreatitis (Bhatia

et al., 1998c; Bhatia et al., 2005; Sandoval et al., 1996)

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1.2.1.2.3 Macrophages

During inflammation, activated resident macrophages secrete chemokines, which attract neutrophils, T cells, and additional macrophages and perpetuate the inflammatory responses (Smiley et al., 2001) Macrophages are implicated in many inflammatory conditions including polymicrobial sepsis, endotoxemia, acute pulmonary inflammation, acute intestinal inflammation, rheumatoid arthritis, and pancreatitis (Bhatia et al., 2006; Grimsholm et al., 2007; Hegde et al., 2007; Lau et al., 2005; Ng et al., 2008; Puneet et al, 2006; Sun and Bhatia, 2007; Zhang et al., 2007)

In acute pancreatitis and associated lung injury, macrophages are suggested to play a dual role in both the disease propagation and resolution Peritoneal macrophages have been shown to extend inflammation from the pancreas to the peritoneal cavity and subsequently induce lung injury in acute pancreatitis and thus play an important role in the progression of acute pancreatitis (Mikami et al., 2003) Depletion of macrophages in mice using anti-macrophage antisera reduced the number of infiltrating leukocytes in the pancreas and protected the animals from pancreatic damage during acute pancreatitis (Fink and Norman, 1996) On the other hand, studies also demonstrate a protective role

of macrophages in the condition Macrophages are found to be responsible for clearing apoptotic cells and stimulate an anti-inflammatory response during acute pancreatitis (Cao et al., 2007) Homing of peritoneal macrophages that overexpress heme oxygenase-

1 (HO-1) to the pancreas affords significant protection from pancreatic injury (Nakamichi

et al., 2005)

1.2.2 Experimental models of acute pancreatitis

Experimental models have been developed to induce acute pancreatitis in laboratory animals for investigation of the pathophysiology of the condition (Bhatia, 2002a; Bhatia

et al., 2005; Bilchik et al., 1990) Three most well-characterized models include 1) consecutive administration of a supramaximally stimulating dose of secretagogue cholecystokinin (CCK) analog caerulein to rodents which results in either mild (rats) or severe (mice) acute pancreatitis over hours (Lampel and Kern, 1977; Niederau et al., 1985); 2) feeding a choline-deficient diet supplemented with ethionine, the ethyl analog

of methionin (CDE diet) to young female which causes the development of severe necrotizing acute pancreatitis in these mice (Lombardi et al., 1975); 3) retrograde injection of bile salts into the pancreatic duct of rats, or the ligation of the common biliopancreatic duct in the opossum which leads to a severe acute pancreatitis (Senninger

et al., 1986)

Among these three models, caerulein hyperstimulation-induced acute pancreatitis is the most commonly employed, standard experimental model for the more frequent, edematous form of acute pancreatitis that closely mimics many characteristics of the human disease and it is the primary acute pancreatitis model used in our lab

1.2.2.1 Principle of caerulein-induced acute pancreatitis model

Caerulein is an amphibian decapeptide with the same biological activity as CCK CCK is

a peptide hormone of the gastrointestinal system responsible for stimulating the digestion

of fat and protein Released from the duodenum, CCK acts on the pancreas to stimulate the secretion of digestive enzymes CCK also acts as a neuropeptide, mediating satiety by

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acting on the CCK receptors that are distributed widely throughout the central nervous system CCK stimulates enzyme secretion in a dose dependent manner and maximal stimulation of protein secretion is associated with increased rate of protein synthesis However, if the maximal stimulation persists, the increase in protein synthesis will be outpaced by the rate of protein secretion and results in the reduction of enzyme stores of the exocrine pancreas (Scheele and Palade, 1975) However, supramaximal stimulation i.e increasing the dose of CCK by an order of magnitude over the levels that produce maximal secretion, generates paradoxical pancreatic responses, such as diminished secretion, accumulation of secretory proteins within the pancreas, and pancreatic injury (Lampel and Kern, 1977)

1.2.2.2 Induction and characteristics of caerulein-induced acute pancreatitis model

The model was most often induced by giving mice hourly intraperitoneal (i.p.) injections

of saline containing a supramaximally stimulating dose of caerulein (50 μg/kg) for 10 hours (Bhatia et al., 1998b) Maximal pancreatic injury occurs within the time course Caerulein hyperstimulation-induced acute pancreatitis is characterized by marked interstitial pancreatic edema, hyperamylaseamia, inflammation, and acinar cell injury Compared to other invasive acute pancreatitis models (such as duct ligation model) characterized with extensive and uncontrolled pancreatic destruction, it is non-invasive and highly reproducible (Bilchik et al., 1990; Lampel and Kern, 1977) and is now the most frequently employed model in studying experimental acute pancreatitis

1.3 SP

SP is an 11-amino acid peptide product of the preprotachykinin-A (PPT-A) gene It belongs to the tachykinin family of neuropeptides which also include neurokinin A (NKA), neurokinin B (NKB), and the newly discovered endokinins and hemokinins (Metwali et al., 2004; Page, 2004) SP is widely distributed throughout the central and peripheral nervous systems as a neurotransmitter and pain mediator (Haines et al., 1993; Maggi, 1997) and is released by unmyelinated, sensory nerve endings as well as by immune cells (Killingsworth et al., 1997; O'Connor et al., 2004;Susan and Helen, 2006) The biological actions of SP are mediated primarily by the NK-1R which belongs to the

G protein-coupled neurokinin receptor family

SP has proinflammatory effects on inflammatory and epithelial cells and participates in inflammatory conditions such as asthma, immune complex-mediated lung injury, arthritis, inflammatory bowel disease, polymicrobial sepsis, and acute pancreatitis (Bhatia et al., 2000a; Bowden et al., 1994; Castagliuolo et al., 1998; Puneet et al., 2006; Sun and Bhatia, 2007; Thurston et al., 1996)

1.3.1 SP in acute pancreatitis and associated lung injury

SP is a well-documented proinflammatory mediator in acute pancreatitis and associated lung injury Both SP and NK-1R are important determinants of the disease severity The pancreatic levels of SP and the expression of NK-1R on pancreatic acinar cells are increased in experimental acute pancreatitis in mice (Bhatia et al., 1998b) Gene deletion

of PPT-A and NK-1R reduce the severity of acute pancreatitis and associated lung injury (Bhatia et al., 1998b; Bhatia et al., 2003) NK-1R antagonist treatment protects mice

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against caerulein-induced acute pancreatitis and associated lung injury (Lau et al., 2005) Mice deficient in NEP, the enzyme that hydrolyses SP thereby terminating its action, are more susceptible to experimental acute pancreatitis and associated lung injury (Bhatia et al., 1997)

Interplay between SP and other inflammatory mediators has been demonstrated 1R is shown to mediate the proinflammatory effect of H2S during acute pancreatitis and associated lung injury (Bhatia et al., 2008) SP-NK-1R is suggested to regulate the expression of adhesion molecules including ICAM-1, E-selectin, and P-selectin in the pancreas and lungs during acute pancreatitis (Lau and Bhatia, 2007) SP-NK-1R is also shown to exert regulatory effects on tachykinins and neurokinin receptors during the condition: NK-1R antagonist treatment suppresses SP concentration, PPT-A gene and NK-1R expression in the pancreas; it also suppresses SP concentration, PPT-A and PPT-

SP-NK-C gene expression in the lungs, but further increases NK-1R and NK-2R expression

1.3.2 SP in immunoregulation

As a major mediator of immunoregulation, SP is known to affect multiple aspects of immune cell functions SP promotes lymphocyte proliferation, leukocyte migration and accumulation, secretion of proinflammatory cytokines including TNF-α, IL-6, IL-8, and other mediators from lymphocytes, macrophages, monocytes, and mast cells (Azzolina et al., 2003; Bost and Pascual, 1992; Fiebich et al., 2000; Maggie, 1997; Zhao et al., 2002)

1.3.2.1 SP in immunoregulation: neutrophils

SP affects the migratory responses and cytotoxic functions of neutrophils and induces degranulation, respiratory burst, and production of reactive inorganic oxidants in cells

(O’Connor et al., 2004; Serra et al., 1994; Serra et al., 1998; Wozniak et al., 1989) In addition, SP is a priming agent for neutrophils in a variety of cellular responses such as migration, IL-1β and TNF-α secretion, β2-integrin upregulation, leukotriene production, and calcium mobilization (Dianzani et al., 2001; Lloyds and Hallett, 1993; Saban et al., 1997; Sterner-Kock et al., 1999) Interaction of SP with neutrophils has also been demonstrated in vivo SP induces a rapid influx of neutrophils in human dermis SP mediates neutrophil adherence to alveolar epithelial cells and induces IL-1β and TNF-α release from the cells (DeRose et al., 1994; Serra et al., 1994) In NK-1R knockout mice, neutrophil accumulation to the lung is significantly inhibited during inflammation (Bozic

et al., 1996) Another knockout mouse study has reported that SP induces neutrophil accumulation in the inflamed not but normal skin (Pinter et al., 1999)

1.3.2.2 SP in immunoregulation: macrophages

SP has been shown to augment the production of cytokines (IL-6 and TNF-α) by macrophages via its C-terminal amino acid activity (O’Conner et al., 2004) It induces nitric oxide production, and oxidative burst of macrophages resulting in the production of reactive oxygen intermediates (Yaraee et al., 2007) It stimulates synthesis and release of arachidonic acid metabolites, prostaglandin E2, thromboxane B2, and toxic oxygen radicals in peritoneal macrophages (Bar-Shavit et al., 1980; O’Conner et al., 2004) SP also enhances antigen presentation and phagocytosis of murine macrophages during cellular immune responses

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1.4 Chemokines

Chemokines are a family of small (8-10 kDa), inducible, secreted cytokines (Baggiolini

et al., 1997; Murphy, 1994) They fall into four subfamilies (two major and two minor): CXC (α-subfamily), CC (β-subfamily), C (γ-subfamily), and CX3C (δ-subfamily) based

on their relative position of the first two cysteines (Lukacs et al., 1999; Zlotnik and Yoshie, 2000) Chemokines act as regulators of immune, inflammatory, and hematopoietic processes They play a major role in leukocyte trafficking, recruiting, and recirculation The two major subfamilies CXC and CC chemokines have been extensively investigated in various disease conditions The CXC chemokines such as IL-8, GRO-α, and the rodent CXC chemokines CINC and macrophage inflammatory protein (MIP)-2 are believed to act preferentially on neutrophils (Bhatia et al., 2000a; Lukacs et al., 1999; Rollins, 1999) and are primarily involved in neutrophil-mediated inflammation The CC chemokines MCP-1, MCP-2, MCP-3, regulated upon activation, normal T cell expressed and secreted (RANTES), MIP-1α, and MIP-1β are believed to act on monocytes, but not

on neutrophils (Bhatia et al., 2000a; Lukacs et al., 1999; Rollins, 1999) and tend to be involved in chronic inflammation (Baggiolini et al., 1997) They also influence basophil and eosinophil granulocytes, and T cells (Baggiolini et al., 1997) However, a recent work has shown that these narrow definitions are no longer valid (Bhatia et al., 2007) Chemokines bind to a family of seven-transmembrane-domain G-protein-coupled transmembrane receptors Chemokine receptors can be broadly subdivided into those that bind a single chemokine and those that bind a number of chemokines of either CXC or

CC type (Horuk, 1994; Wells et al., 1996) The expression pattern of the chemokine receptors is a major determinant of the selectivity of chemokines for target cells

1.4.1 Chemokines and their receptors in acute pancreatitis and associated lung injury

Chemokines, with a well-recognized role in inflammatory and immune responses, were first implicated in the condition acute pancreatitis in the early 1990s (Bhatia et al., 2007) Since then, a number of experimental and clinical studies have been conducted to identify different chemokine species that are important for the condition and to elucidate the mechanisms by which chemokines propagate inflammatory cascades (Bhatia et al., 2005; Brady et al., 2002; Marra, 2005; Pastor et al., 2003; Yang et al., 2000) These studies demonstrate that only chemokines from the CXC and CC subfamilies and a few chemokine receptors are of particular importance in this condition Among the CXC chemokines, IL-8, GRO-α, CINC, and MIP-2 are important in acute pancreatitis IL-8 is responsible for neutrophils chemoattraction, degranulation, and release of elastase (Makhija and Kingsnorth, 2002) MCP-1, MIP-1α, and RANTES are the key CC chemokine involved in the condition As for the chemokine receptors, CC chemokine receptor-1 (CCR1) and CCR5, the receptors for MIP-1α and RANTES, as well as CXCR2, the receptor for IL-8 and MIP-2, have been demonstrated to play an important role in modulating the severity of acute pancreatitis (Gerard et al., 1997)

The current study has investigated four chemokines, namely MCP-1, MIP-1α, RANTES and MIP-2 MCP-1 is a prototypic CC chemokine and known as the major chemokine in acute pancreatitis (Marra, 2005) Its mRNA expression in the pancreas was induced early

in a rat model of caerulein-induced acute pancreatitis while its plasma levels were significantly increased six hours after disease induction (Grady et al., 1997; Brady et al., 2002) Both MCP-1 and RANTES were secreted from rat pancreatic acinar cells when treated with ethanol and the secretagogue CCK (Yang et al., 2000) Caerulein

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hyperstimulation of rat pancreatic acini led to the MCP-1 synthesis in the cells (Bhatia et al., 2002b) Genetic deletion of CCR1, a receptor for MIP-1α and RANTES, protected mice from lung inflammation, a secondary effect to acute pancreatitis (Harrison and Geppetti, 2001) A clinical investigation of the role of MCP-1 and MIP-1α in human acute pancreatitis demonstrated that complicated acute pancreatitis was associated with significantly elevated local and systemic concentrations of MCP-1 MIP-1α levels remained unaffected by local conditions but showed a significant increase when MODS developed A close correlation between the severity of remote organ damage and MCP-1 levels suggests that MCP-1 might play a pivotal role in the pathological mechanism of complicated human acute pancreatitis (Rau et al., 2003) MIP-2 is a prototype murine CXC chemokine and a potent polymorphonuclear neutrophil chemoattractant and activator Neutrophils, as the early subpopulation of leukocytes infiltrating damaged tissues, produce CC chemokines that lead to subsequent monocyte/macrophage influx Neutrophils particularly play a predominant role in tissue destructive events through the release of proteolytic enzymes (such as polymorphonuclear elastase) and lethal oxygen metabolites MIP-2 levels were found to be increased in serum, pancreas and lung tissues

in acute pancreatitis in mice Administration of anti-MIP-2 antibodies partially protected the mice against pancreas and lung injury (Pastor et al., 2003)

1.5 Intracellular signaling molecules

The signaling molecules and pathways that are responsible for the initiation and progression of acute pancreatitis have been under intense scrutiny Important molecules implicated in the condition and potentially involved in SP-induced cellular inflammatory

responses are discussed in general below, including the transcription factor nuclear factor-kappaB (NF-κB), mitogen-activated protein kinases (MAPKs), protein kinase C (PKC) isoforms, phosphoinositide-3 kinase (PI3K) and Akt

1.5.1 NF-κB

The term NF-κB refers to a group of binary complexes of proteins with related binding and transactivation activities The prototypical NF-κB complex consists of a p65-p50 heterodimer P65/RelA, RelB, and c-Rel stimulate transcription, whereas p50 and p52 serve primarily to bind to DNA (Sizemore, 1999) NF-κB is the central regulator for expression of genes involved the inflammatory and immune responses (Ghosh and Karin, 2002)

promoter-In the classical pathway, activation of NF-κB, especially the p65-p50 heterodimer, depends on the phosphorylation of its endogenous inhibitor IκB, mainly by IκB kinases (IKKs) This leads to ubiquitination and proteasomal degradation of IκB The liberated NF-κB dimer then translocates to the nucleus, where it activates specific target genes (Ben-Neriah, 2002) Growing evidence indicates posttranslational modifications of NF-

κB, particularly phosphorylation and acetylation also play a significant role in the activation of the transcription factor (Chen et al., 2001; Kiernan et al., 2003) Activation

of NF-κB by specific stimuli is accompanied by increased phosphorylation of the p65/RelA subunit at specific serine residues and increased transactivation potential of NF-κB (Bird et al., 1997; Bohuslav et al., 2004; Naumann and Scheidereit, 1994; Vermeulen et al., 2003; Zhong et al., 1998) Phosphorylated p65 recruits coactivators, such as histone acetyltransferases CREB binding protein (CBP) and p300 to the NF-κB

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transcriptional complex thereby enhancing the transcriptional activity (Gerritsen et al., 1997; Merika et al., 1997; Sheppard et al., 1999; Wang and Baldwin 1998; Zhong et al., 1998;  Zhong et al., 2002) Acetylation of p65 is also proposed as an additional posttranlational mechanism of modulation of its activity (Chen and Greene 2003)

NF-κB activation has been demonstrated in acute pancreatitis It is also shown to regulate the transcription of many genes involved in the progression of the condition Thus NF-κB

is an important signaling molecule in the condition The role of NF-κB was investigated

in the current study for SP-induced cellular inflammatory mechanism

1.5.2 MAPKs

MAPKs are a family of proline-directed protein serine/threonine kinases activated by a cascade of intracellular phosphorylation events and transduce signals from the cell surface to the nucleus (Chang and Karin, 2001; Dong et al., 2002; Hazzalin and Mahadevan 2002) MAPKs consist of four subfamilies, the best characterized of which are the extracellular signal-regulated kinase (ERK)1/2, c-Jun N-terminal kinases (JNKs), and p38 MAPK Every MAPK subfamily is composed of a three sequentially acting kinase module, MEKK, MEK, and MAPK, each one activating the next via phosphorylation Their substrates, located in the cytoplasm as well as in the nucleus, include other kinases, transcription factors, phospholipases, and cytoskeletal proteins (Kyriakis and Avruch, 2001; Pearson et al., 2001; Roux and Blenis 2004) In general, ERK1/2 are mainly involved in anabolic processes, such as cell division, growth, and differentiation, whereas JNKs and p38 MAPK are mostly associated with cellular responses to stress conditions (Kefaloyianni et al., 2006; Kyriakis and Avruch, 2003;

Pearson et al., 2001; Roux and Blenis, 2004) For the importance of these kinases in regulation of inflammatory gene expression as demonstrated in various cellular systems, the role of MAPKs was investigated in the later part of the study for SP-induced cellular responses

1.5.3 PKC isoforms

The PKC family proteins are phospholipid-dependent serine/threonine kinases comprising 12 isoforms The PKC isoforms are subdivided into three groups based on their molecular structure and mode of activation, namely conventional PKCs (α, βI, βII, and γ), novel PKCs (δ, ε, η, and θ), and atypical PKCs (ζ, λ/ι, and μ) The conventional PKCs are activated by Ca2+ and by diacylglycerol (DAG) or phorbol esters The novel PKC isoforms are Ca2+ independent but can be activated by DAG and phorbol esters The atypical PKCs are unresponsive to Ca2+, DAG, or phorbol esters (Dempsey et al., 2000) PKC isoforms are involved in regulation of cellular processes including growth, migration, and inflammatory responses Earlier studies have suggested that PKC activation leads to downstream release of inflammatory mediators including lipid mediators, adhesion molecules, and chemokines in vitro (Parekh et al., 2000; Ramnath et al., 2008) In macrophages, PKCs are shown to be involved in nitric oxide production However, little is known about the involvement of PKC isoforms in SP-induced leukocyte inflammatory responses

1.5.4 PI3K-Akt

PI3K is a conserved family of signaling molecules involved in regulation of cellular proliferation and survival Akt, a serine/threonine kinase, is a direct downstream effector

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of PI3K PI3K activation leads to phosphorylation of phosphatidylinositides, which then activate Akt PI3K-Akt acts as a transducer for many pathways initiated by growth factors and cytokines and is modulated by multiple intracellular signaling pathways (Downward, 1998)

PI3K-Akt has an important role in regulating cellular growth, differentiation, adhesion, and inflammatory responses The role of PI3K in regulation of the inflammatory response has been controversial dependent on the cellular models and experimental conditions (Pengal et al., 2006) Some researchers have shown that PI3K-Akt pathway positively regulates proinflammatory signaling in monocytes/macrophages Using pharmacological inhibitors of PI3K-Akt and transfection of cells with a class I PI3K p110α, it is shown that PI3K-Akt is proinflammatory and required for LPS induction of gene expression in monocytes/macrophages Furthermore, it is shown that PI3K-Akt pathway is involved in the expression of cytokines, chemokines, reactive oxygen species, nitric oxide and other proinflamamtory mediators triggered by a range of stimuli in monocytes/macrophages (Kao et al., 2005; Mookerjee et al., 2006; Sebastian et al., 2008; Smith et al., 2007; Yoo

et al., 2005) In contrast, other studies have shown that the PI3K-Akt pathway negatively regulates LPS-activated MAPKs, various transcription factors and proinflammatory genes including cytokines, inducible NO synthase and nitrite in monocytes/macrophages (Diaz-Guerra et al., 1999; Fukao and Koyasu 2003; Guha and Mackman, 2002; Luyendyk et al., 2008; Martin et al., 2003; Tsukamoto et al., 2008) This pathway also induces expression of anti-inflammatory heme oxygenase-1 (Pischke et al., 2005) and IL-

10 (Pengal et al., 2006) PI3K-Akt pathway has been reported to act in the regulation of nuclear translocation and the transactivation potential of NF-κB (Madrid et al., 2000;

Madrid et al., 2001; Ozes et al., 1999; Sizemore et al., 1999) So far, the role of PI3K-Akt

in SP-induced cellular signaling in leukocytes has not yet been investigated

1.6 Research rationale and objectives

1.6.1 Research rationale

Acute pancreatitis is a common clinical condition with potentially fatal consequences The majority of death arises from the development of systemic inflammatory responses and MODS associated with the condition, in which various inflammatory mediators play

a key role For their importance, tremendous research efforts have been made on the identification of the inflammatory mediators involved and elucidation of their pathophysiological mechanisms

SP is a well-known proinflammatory neuropeptide in acute pancreatitis and associated lung injury Despite its established role in the pathophysiology of acute pancreatitis, the mechanism(s) by which SP acts to mediate the inflammatory responses

pancreatitis-in this disease remapancreatitis-ins largely elusive However, earlier obvervational studies have suggested possible mechanistic actions of the neuropeptide in the conditions Key implicative observations include:

1) Chemotactic property of SP on different leukocyte subpopulations;

2) SP-NK-1R blockade in mice results in reduced inflammatory leukocyte infiltration in pancreas and lungs during experimental acute pancreatitis (Lau et al., 2005); and

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3) SP-NK-1R blockade is also associated with decreased neutrophil granular enzyme myeloperoxidase activity in the tissues during experimental acute pancreatitis (Lau et al., 2005)

The regulatory effects of SP on leukocyte trafficking and inflammatory functions indicated lead us to posulate that other mediator molecules, particularly chemokines that have chemotactic and activating effects on leukocytes, are likely to be involved in SP-NK-1R-related pathogenesis of acute pancreatitis and associated lung injury

1.6.2 Objectives

The primary objective of the present study is to elucidate the mechanism of proinflammatory actions of SP in the pathogenesis of acute pancreatitis and associated lung injury, particularly pertaining to its interaction with other mediator molecules and cells that amplify the inflammatory responses and aggravate the condition

More specifically, the current study sought:

1) to identify the cellular and/or molecular targets of SP in propagating the inflammatory reponses during acute pancreatitis and associated lung injury;

2) to set up appropriate relevant cellular model(s) to look for direct in vitro evidence

of the proinflammatory effects of SP in the cells based on preliminary in vivo findings;

3) to elucidate the signaling mechanisms of SP-induced cellular inflammatory responses using the cellular models; and also

4) to examine effects of other neuropeptide(s) in cellular inflammatory responses using the cellular models