Inflammatory effects of CcH1, an P-I metalloproteinase from Cerastes cerastes snake venom: functional role of proteolytic domain in leukocyte recruitment

H Boukhalfa-Abib1,2, F Laraba-Djebari1,2. 1USTHB, Faculty of Biological Sciences, Laboratory of Cellular and Molecular Biology, Department of Cellular and Molecular Biology, Algeria, 2Pasteur Institute of Algeria, Laboratory of Research and Development on Venom, Algeria

Snakebite envenomations constitute a health problem in many regions of the world. Those caused by viperidae are characterized by prominent local tissue damage, including, necrosis of skeletal muscle, edema, hemorrhage and pain, as well as by systemic alterations such as hemorrhage, cardiovascular shock, acute renal failure and coagulopathies. Local and systemic effects caused by snake venoms have been associated with diverse components of the venom, such as phospholipases A2 and metalloproteinases. Snake Venom Metalloproteinases (SVMPs) play a relevant role in the multifactorial inflammatory response induced by snakebite. The P-I class SVMP CcH1 isolated from Cerastes cerastes snake venom is a weak hemorrhagic protein which induce a variety of local tissue-damage. This molecule is able to hydrolyze laminin and type IV collagen in a dose-dependent manner. Moreover, upon intramuscular injection, CcH1 induces formation of blisters and infiltration of leukocytes into dermis, indicating an important pro-inflammatory action of CcH1. However, the mechanisms involved in leukocyte migration are still unknown. Leukocyte migration is a key event in inflammation, a defense mechanism of host defense against injurious agents. During the acute phase of inflammation, neutrophils are the first cells to accumulate in tissues. In the present study, we analyze the inflammatory reaction induced by CcH1 in the peritoneal cavity of mice. The statistical significance of differences between groups was analyzed by a Student t-test. Obtained results showed that the injection of CcH1 (20 µg/20 g of Body Weight (BW) of male NMRI mice (20 ± 2 g BW; n = 4), induced a marked inflammatory cell infiltrate into peritoneal cavity of animals with predominance of neutrophils in the early phase followed by mononuclear cells in the late period. Our results further demonstrated that inactivation of CcH1 catalytic activity with EDTA abrogated the toxin-induced leukocyte influx. This indicates that the proteolytic activity of CcH1 is required for its ability to stimulate leukocyte migration. To further, we evaluated the number of blood circulating leukocytes in animals (20±2 g BW ; n = 4) injected with CcH1 (20 µg/20 g BW) in order to understand the ability of CcH1 to induce leukocyte infiltration. Obtained results showed a rapid and significant increase in blood neutrophil count in the early stage the induced inflammatory reaction, whereas the other circulating leukocyte count is no affected. This selective increase in the number of circulating neutrophils apparently resulted in the migration of some cells, since this effect is in correlation with the increase of leukocyte accumulation into peritoneum after injection of CcH1. In conclusion, as a P-I class metalloproteinase, CcH1 may contain only metalloproteinase domain, suggesting that the catalytic domain of SVMPs can induce by itself the crucial inflammatory events. This study will contribute to the understanding of the inflammatory mechanisms induced by SVMPs, which will be helpful for the pharmacological modulation of events which are associated with Cerastes cerastes venom-induced local injury.