Role of CCL-2, CCR-2 and CCR-4 in cerulein-induced acute pancreatitis and pancreatitis-associated lung injury Jean Louis Frossard, 1,2 Se ´bastien Lenglet, 3 Fabrizio Montecucco, 3 Sabine Steffens, 3 Katia Galan, 3 Graziano Pelli, 3 Laurent Spahr, 1,2 Francois Mach, 3 Antoine Hadengue 1 ABSTRACT Background and aims Acute pancreatitis is an inflammatory process of variable severity. Leucocytes are thought to play a key role in the development of pancreatitis and pancreatitis-associated lung injury. The interactions between inflammatory cells and their mediators are crucial for determining tissue damage. Monocyte chemoattractant protein-1 (or CCL-2), CCR-2 and CCR-4 are chemokines and chemokine receptors involved in leucocyte trafficking. The aim of the study was to evaluate the role of the CCL-2, CCR-2 and CCR-4 chemokine receptors in the pathogenesis of cerulein- induced pancreatitis and pancreatitis-associated lung injury. To address the role of CCL-2, CCR-2 and CCR-4 that attracts leucocytes cells in inflamed tissues, pancreatitis was induced by administering supramaximal doses of cerulein in mice that do not express CCL-2, CCR-2 or CCR-4. Methods The severity of pancreatitis was measured by serum amylase, pancreatic oedema and acinar cell necrosis. Lung injury was quantitated by evaluating lung microvascular permeability and lung myeloperoxidase activity. Chemokine and chemokine-receptor expression were quantitated by real-time PCR. The nature of inflammatory cells invading the pancreas and lungs was studied by immunostaining. Results The authors have found that pancreas CCL-2 and CCR-2 levels rise during pancreatitis. Both pancreatitis and the associated lung injury are blunted, but not completely prevented, in mice deficient in CCL-2, whereas the deficiency in either CCR-2 or CCR-4 does not reduce the severity of both the pancreatitis and the lung injury. The amounts of neutrophils and monocyte/ macrophages (MOMA)-2 cells were significantly lower in mice deficient in CCL-2 compared with their sufficient littermates. Conclusions These results suggest that CCL-2 plays a key role in pancreatitis by modulating the infiltration by neutrophils and MOMA-2 cells, and that its deficiency may improve the outcome of the disease. INTRODUCTION Acute pancreatitis is an inflammatory process in which the injury is mild in 70 to 80% of cases, but 20% of the patients have a severe injury. 1e3 The pathophysiology of the disease includes the acti- vation and release of pancreatic enzymes in the interstitium, pancreatic autodigestion and a multiple organ dysfunction. Inflammatory medi- ators produced within the gland increase the pancreatic injury, and spread out in distant organs, transforming a local inflammation into a systemic disease. The effects of these inflammatory media- tors on the pancreas and remote organs have been evaluated using genetically modified mice or blocking experiments. For example, intercellular adhesion molecule-1 (ICAM-1), 4 chemoattractant mediators such as macrophage inflammatory peptide 1a (MIP-1a) 5 and interleukin-8 (IL-8) 6 have been involved in neutrophil activation during pancreatitis. Chemokines are a family of cytokines with potent chemotactic and activating effects on different types of leucocyte. Chemokines mediate the trafficking of leucocyte including the lympho- cyte and macrophage. IL-8 (CXCL8) is the most potent neutrophil chemokine, acting through the C-X-C chemokine receptor-2 (CXCR2). Macro- phage inflammatory protein-2 (CXCL-2) is a second potent rodent neutrophil chemokine, a CXCR2 ligand homologous to human GRO-b. 78 The role of C-X-C chemokines in acute inflammatory condi- tions including acute pancreatitis has recently been studied. 9 10 CCL-2 is a member of the CC chemo- kine subfamily that controls recruitment of monocytes through its receptor. 11 It binds with high affinity to the chemokine receptor CCR-2. 12 Using mice deficient in either CCL-2 or CCR-2, these molecules were shown as major regulators of macrophage migration. 13 CCR-4 in addition to CCR-2 is of interest because CCL-2 binds to both CCR-2 and CCR-4. These observations have led us to postulate that chemokines such as MIP-2 and CCL-2 may play a key role in the pathogenesis of acute pancreatitis. To address this question, an acute oedematous form of pancreatitis was induced by administering supramaximal doses of the secretagogue cerulein in mice lacking CCL-2, CCR-2 or CCR-4. MATERIALS AND METHODS Reagents Cerulein was purchased from Research Plus (Bayonne, New Jersey). All other chemicals and reagents were purchased from Sigma (Basel, Switzerland). Animals All experiments were performed according to protocols approved by the Institutional Animal Care. Ccr2-null mice were generated as described previously. 14 The knockout strains were back- crossed to the inbred DBA/1j and BALB/c mice 1 Service of Gastroenterology and Hepatology, Geneva University Hospital, Geneva, Switzerland 2 Universite ´ de Gene `ve, Geneva University Hospital, Geneva, Switzerland 3 Service of Cardiology, Geneva University Hospital, Geneva, Switzerland Correspondence to Professor Jean Louis Frossard, Service of Gastroenterology, Geneva University Hospital, 1211 Geneva 14, Switzerland; jean-louis.frossard@hcuge.ch Accepted 12 January 2011 Published Online First 23 February 2011 J Clin Pathol 2011;64:387e393. doi:10.1136/jcp.2010.088500 387 Original article group.bmj.com on October 15, 2013 - Published by jcp.bmj.com Downloaded from group.bmj.com on October 15, 2013 - Published by jcp.bmj.com Downloaded from group.bmj.com on October 15, 2013 - Published by jcp.bmj.com Downloaded from group.bmj.com on October 15, 2013 - Published by jcp.bmj.com Downloaded from group.bmj.com on October 15, 2013 - Published by jcp.bmj.com Downloaded from group.bmj.com on October 15, 2013 - Published by jcp.bmj.com Downloaded from group.bmj.com on October 15, 2013 - Published by jcp.bmj.com Downloaded from