Chemokines as therapeutic targets in systematic inflammatory response syndrome

CHEMOKINES AS THERAPEUTIC TARGETS IN SYSTEMATIC INFLAMMATORY RESPONSE SYNDROME HE MIN (B.SC. WUHAN UNIVERSITY) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF PHARMACOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2008 ACKNOWLEDGEMENTS This work was carried out from 2004 to 2007 at Department of Pharmacology in National University of Singapore (NUS). I am very grateful for the privilege to join this exciting research program at NUS. I would like to express my great gratitude to my supervisor, Associate Professor Madhav Bhatia for his invaluable guidance and constant encouragement throughout the past four years. I am very grateful to him for sharing with me his deep knowledge, extensive research experience and positive attitude towards scientific research. I am also very grateful to everyone who has studied and worked at our lab in the past four years. It is a great pleasure for me to work as your colleague. Thank you all very much for your help in benchwork, in modules and in everyday life. I would like to give special thanks to our lab officer Mei Leng for taking good care of our lab and for helping me with some tricky animal experiments. I have enjoyed the friendly and encouraging atmosphere at Department of Pharmacology and Cardiovascular Biology Programme. I would like to take this opportunity to thank all the staffs and friends at Department of Pharmacology and Cardiovascular Biology Programme. I would also like to thank National University of Singapore for providing me generous research scholarship. Finally, I wish to thank my parents and my dear wife Cao Yang for their warm support and encouragement during the work with the thesis. He Min Jan 17th, 2008 I TABLE OF CONTENTS ACKNOWLEDGEMENTS  . I  TABLE OF CONTENTS   II  LIST OF ORIGINAL REPORTS  . VI  SUMMARY   VII  LIST OF TABLES   X  LIST OF FIGURES  . XI  LIST OF ABBREVIATIONS   XIV  I.  GENERAL INTRODUCTION   1  1.1 General overview   1  1.2 Literature review   4  1.2.1 Inflammation  . 4  1.2.2 Acute pancreatitis   6  1.2.2.1 Causes of acute pancreatitis  . 6  1.2.2.2 Pathophysiology of acute pancreatitis  . 7  1.2.2.2.1 First phase: the initial stage of pancreatic acinar cell damage   7  1.2.2.2.2 Second phase: local inflammation   9  1.2.2.2.3 Third phase: systemic inflammation  . 9  1.2.2.2.4 Inflammatory mediators in AP   10  1.2.2.3 Caerulein‐induced acute pancreatitis  . 12  II 1.2.2.3.1 Amylase   13  1.2.2.3.2 Pancreatic edema  . 14  1.2.2.3.3 Pancreatic acinar cells injury/necrosis   14  1.2.2.3.4 Lung injury   15  1.2.3 Sepsis  . 16  1.2.3.1 Pathophysiology of sepsis   16  1.2.3.1.1 Recognition of pathogens  . 16  1.2.3.1.2 Pro‐inflammatory cytokines  . 17  1.2.3.1.3 Substance P   18  1.2.3.1.4 Hydrogen sulfide   18  1.2.3.1.5 The coagulation cascade  . 19  1.2.3.1.6 Apoptosis and immune suppression  . 20  1.2.3.2 Animal models of sepsis  . 21  1.2.4 Chemokines  . 23  1.2.4.1 Chemokine classification   23  1.2.4.2 Chemokine receptors  . 28  1.2.4.3 Signal transduction   29  1.2.4.4 Chemokines in acute inflammation   30  1.2.4.4.1 Neutrophil and organ damage   30  1.2.4.4.2 Chemokine and leukocyte migration and activation  . 31  1.2.4.4.3 Other functions   32  1.2.4.5 Pharmacological agents targeting chemokines . 33  1.2.4.6 Blocking chemokines and their receptors in acute pancreatitis and sepsis  . 35  II.  MATERIALS AND METHODS  . 39  2.1 Induction of acute pancreatitis   39  2.2 Induction of sepsis   39  III 2.3 BX471  . 40  2.4 Mast cell depletion  . 41  2.5 Fractalkine   41  2.6 Water content   41  2.7 Amylase estimation   42  2.8 Myeloperoxidase estimation  . 42  2.9 Morphological examination   43  2.10 Cytokine and chemokine ELISA assay  . 43  2.11 Immunohistochemistry  . 43  2.12 Reverse transcriptase‐ polymerase chain reaction  . 45  2.13 Isolation of peritoneal mast cells  . 47  2.14 Intravital microscopy  . 47  2.15 Statistics  . 49  III.  TREATMENT WITH BX471, A SMALL MOLECULE ANTAGONIST OF  CCR1, PROTECTS AGAINST SYSTEMIC INFLAMMATION IN MOUSE MODELS  OF ACUTE PANCREATITIS AND SEPSIS   50  3.1 Introduction   50  3.2 Results   53  3.2.1 BX471 treatment in caerulein‐induced acute pancreatitis  . 53  3.2.2 BX471 Treatment in CLP‐induced sepsis   67  IV 3.3 Discussion  . 77  IV.  MAST CELL DEPLETION ATTENUATES CHEMOKINE PRODUCTION IN  CAERULEIN­INDUCED ACUTE PANCREATITIS AND CLP­INDUCED SEPSIS  . 83  4.1 Introduction   83  4.2 Results   85  4.2.1 Mast cell depletion by compound 48/80  . 85  4.2.2 Effect of mast cell depletion in caerulein‐induced acute pancreatitis   86  4.2.3 Effect of mast cell depletion in CLP‐induced sepsis   94  4.3 Discussion  . 99  V.  FRACTALKINE, A CX3C CHEMOKINE, IS CAPABLE OF MODULATING  INFLAMMATORY RESPONSE IN CAERULEIN­INDUCED ACUTE PANCREATITIS  AND CLP­INDUCED SEPSIS   103  5.1 Introduction  . 103  5.2 Results  . 105  5.2.1 FTK in caerule‐induced acute pancreatitis  . 105  5.2.2 FTK in CLP‐induced sepsis   112  5.3 Discussion   120  VI.  GENERAL DISCUSSION   124  6.1 Limitations  . 126  6.2 Future research   127  VII.  REFERENCES   130  V LIST OF ORIGINAL REPORTS He M., Moochhala S.M., Bhatia M. (2008) Administration of exogenous fractalkine, a CX3C chemokine, is capable of modulating inflammatory response in CLP-induced sepsis. Shock (Epub ahead of print). He M., Horuk R., Bhatia M. (2007) Treatment with BX471, a nonpeptide CCR1 antagonist, protects mice against acute pancreatitis-associated lung injury by modulating neutrophil recruitment. Pancreas 34, 233-41. He M., Horuk R., Moochhala S.M., Bhatia M. (2007) Treatment with BX471, a CC chemokine receptor antagonist, attenuates systemic inflammatory response during sepsis. Am J Physiol Gastrointest Liver Physiol 292, G1173-80. He M., Lau H., Ng S., Bhatia M. (2007) Chemokines in acute inflammation: regulation, function and therapeutic strategies. International Journal of Integrative Biology 1, 18-27. Bhatia M., Sun J., He M., Hedge A., Ramnath R.D. (2007) Chemokines in Acute Pancreatitis. Nova Science Publishers, Inc., New York. 103-116. Ramnath R.D., Ng S., He M., Sun J., Zhang H., Bawa M., Bhatia M. (2006) Inflammatory mediators in sepsis: Cytokines, chemokines, adhesion molecules and gases. Journal of Organ Dysfunction 2, 80-92. VI SUMMARY Exaggerated systemic inflammatory response syndrome may lead to multiple organ dysfunction, organ failure and eventually death. Acute pancreatitis is a mainly noninfective cause of systemic inflammatory response syndrome while sepsis is an infective cause of systemic inflammatory response syndrome. These two conditions share indistinguishable haemodynamic features as well as a very similar profile of inflammatory mediators, suggesting that the devastating consequences of the two diseases may result from similar pathogenic mechanisms. Among the numerous inflammatory mediators that have been characterized in the recent years, we may have the opportunity to discover promising drug targets for these diseases. Chemokines, a large family of small chemotactic cytokines, are critical inflammatory mediators in the development of both acute pancreatitis and sepsis. By binding to their seventransmembrane-domain G protein-coupled receptors, chemokines regulate the activation and migration of leucocytes during acute inflammation. Previous studies by gene targeting or inhibitors have shown that several members of chemokines and their receptors are very attractive drug targets for acute pancreatitis and sepsis. However, it is still not known whether small molecule antagonists targeting at specific chemokine receptors will have protective effect against damaging systemic inflammatory response during acute pancreatitis and sepsis. Moreover, other new approaches to manipulate chemokine system are yet to be discovered. In this study, chemokine system in the animal models of acute pancreatitis and sepsis was manipulated by three different strategies: 1. blockage of specific chemokine receptor by a small molecule antagonist; 2. depletion of mast cell by compound 48/80 3. administration of exogenous soluble form of CX3C chemokine fractalkine. The impacts of each VII strategy on the outcomes of the two diseases and the underlying mechanisms by which chemokines influence systemic inflammatory response have been investigated. In the first part of the study, treatment with BX471, a small molecule CCR1 antagonist, results in significant protection against acute pancreatitis associated lung injury and sepsis associated lung and liver injury by attenuating neutrophil infiltration. In both conditions, blocking CCR1 leads to a down-regulation of P-selectin, Eselectin and ICAM-1 in different organs, suggesting a complex interaction between chemokines and adhesion molecules on endothelial cells In the second part of the study, depletion of mast cells by compound 48/80 leads to down-regulation of various chemokines in lung in caerulein-induced acute pancreatitis and cecal ligation and puncture (CLP)-induced sepsis. Moreover, depletion of mast cells has a protective effect against acute pancreatitis-associated lung injury and sepsis-associated lung injury by attenuating neutrophil infiltration. In the third part of the study, administration of recombinant soluble fractalkine leads to an increase of neutrophil infiltration and an increase of several cytokines and chemokines in lung during acute pancreatitis. In contrast, treatment with soluble fractalkine attenuates leukocyte adhesion and infiltration in animal model of sepsis. 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Cystathionine-gamma-lyase ERK Extracellular signal-regulated kinases FAEEs Fatty acid ethanol esters FTK Fractalkine GAG Glycosaminoglycans GPCRs G protein-coupled receptor H2S Hydrogen sulfide i.p Intraperitoneal i.v Intravenous ICAM-1 Intercellular adhesion molecule-1 IKKs IkappaB kinase kinases IL Interleukin JNK c-Jun N-terminal kinases LPS Lipopolysaccharide MAPK Mitogen-activated protein kinases XIV MCP-1... of fibrin clots in the microvasculature, leading to impaired tissue perfusion and organ failure Activated protein C (APC) is converted from protein C when thrombin complexes with throbomudulin APC with its cofactor protein S acts as proteolytic inhibitor of the clotting factors Va and VIIa Moreover, APC increases the fibrinolytic response by inhibiting the activity of plasminogen-activator inhibitor-1... mediators including cytokines and chemokines TLRs activation can induce the expression adhesion molecules on the endothelial cells either directly or indirectly through pro -inflammatory cytokines TNF- and IL-1 (Parker et al., 2005) These consequences promote neutrophil migration to the site of inflammation during sepsis 1.2.3.1.2 Pro -inflammatory cytokines Pro -inflammatory cytokines including TNF-... molecules including selectins and ICAM-1 are all involved in this response Production of some cytokines and chemokine results in the infiltration of inflammatory cells such as neutrophils and macrophages The infiltration and activation of neutrophils leads to a further elevation of the inflammatory mediators and pancreatic acinar cell injury The severity of the disease seems to be determined by the... accurately examining the onset and development of experimental pancreatitis Cholecystokinin (CCK) is a peptide hormone of the gastrointestinal system responsible for stimulating the digestion of fat and protein Released from the 12 duodenum, the hormone acts on the pancreas to stimulate the secretion of a juice rich in digestive enzymes, including trypsinogen, chymotrypsinogen, amylase, lipase, and maltase CCK... better understood, in modern hospitals acute inflammation continues to be a main threat to patient’s health Local inflammation is tightly regulated by immune system and nervous system to combat invading pathogens or remove local injured cells If local inflammatory response fails to contain the insults, systemic inflammation may occur In general term, systemic inflammatory response syndrome (SIRS) is... acinar cell damage Under normal conditions, the digestive enzymes are synthesized and secreted by the acinar cells as inactive proenzymes called zymogens Zymogens are activated in the duodenum where enterokinase activates trypsinogen Subsequently, trypsin induces 7 the activation of itself as well as other digestive enzymes including chymotrypsiongen, procarboxypeptidase and proelastase In contrast,... phagocytes including macrophages and dendritic cells, and CD4 T cells to release antiinflammatory cytokines that contribute to immune suppression As inhibiting Fas-FasL signaling (e.g., Fas fusion protein (Chung et al., 2003), or Fas siRNA administration (Wesche-Soldato et al., 2005)), caspase inhibition (caspase gene deficiency and inhibitor (Hotchkiss et al., 2000) ), and the overexpression of downstream anti-apoptotic... and release of anti -inflammatory cytokines such as TGF- and IL-10 At the end of this resolution stage, inflamed tissue may restore its physiological function If the pathogens or noxious insults persist, the inflammatory cascade may progress into a chronic inflammation 5 1.2.2 Acute pancreatitis Acute pancreatitis (AP) is defined as an acute inflammatory process of the pancreas that frequently involved . kinase kinases IL Interleukin JNK c-Jun N-terminal kinases LPS Lipopolysaccharide MAPK Mitogen-activated protein kinases XV MCP-1 Monocyte chemotactic protein-1 MIP-1a Macrophage inflammatory. administration of recombinant soluble fractalkine leads to an increase of neutrophil infiltration and an increase of several cytokines and chemokines in lung during acute pancreatitis. In contrast,. a mainly non- infective cause of systemic inflammatory response syndrome while sepsis is an infective cause of systemic inflammatory response syndrome. These two conditions share indistinguishable