K-Effect of Bothrops alternatus snake venom on macrophage phagocytosis and superoxide production: participation of protein kinase C

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J. Venom. Anim. Toxins incl. Trop. Dis.

V.17, n.4, p.430-441, 2011.

Original paper - ISSN 1678-9199.

Effect of Bothrops alternatus snake venom on macrophage phagocytosis and superoxide production: participation of protein kinase C

Setubal SS (1), Pontes AS (1), Furtado JL (1), Kayano AM (1), Stábeli RG (1, 2), Zuliani JP (1, 2)

(1) Laboratory of Biochemistry and Biotechnology and Laboratory of Cell Culture and Monoclonal Antibodies, Tropical Pathology Research Institute (Ipepatro), Oswaldo Cruz Foundation (Fiocruz), Porto Velho, Rondônia State, Brazil; (2) Center of Biomolecules Applied to Medicine, Department of Medicine, Federal University of Rondônia (UNIR), Porto Velho, Rondônia State, Brazil.

Abstract: Envenomations caused by different species of Bothrops snakes result in severe local tissue damage, hemorrhage, pain, myonecrosis, and inflammation with a significant leukocyte accumulation at the bite site. However, the activation state of leukocytes is still unclear. According to clinical cases and experimental work, the local effects observed in envenenomation by Bothrops alternatus are mainly the appearance of edema, hemorrhage, and necrosis. In this study we investigated the ability of Bothrops alternatus crude venom to induce macrophage activation. At 6 to 100 μg/mL, BaV is not toxic to thioglycollate-elicited macrophages; at 3 and 6 μg/mL, it did not interfere in macrophage adhesion or detachment. Moreover, at concentrations of 1.5, 3, and 6 μg/mL the venom induced an increase in phagocytosis via complement receptor one hour after incubation. Pharmacological treatment of thioglycollate-elicited macrophages with staurosporine, a protein kinase (PKC) inhibitor, abolished phagocytosis, suggesting that PKC may be involved in the increase of serum-opsonized zymosan phagocytosis induced by BaV. Moreover, BaV also induced the production of anion superoxide (O2-) by thioglycollate-elicited macrophages. This BaV stimulated superoxide production was abolished after treating the cells with staurosporine, indicating that PKC is an important signaling pathway for the production of this radical. Based on these results, we suggest that phagocytosis and reactive oxygen species are involved in the pathogenesis of local tissue damage characteristic of Bothrops spp. envenomations.

Key words: snake venom, Bothrops, macrophages, phagocytosis, superoxide, protein kinase C, inflammation.

We are extremely grateful to The National Council for Scientific and Technological Development (CNPq), the State of Rondônia Planning Secretariat (SEPLAN-RO) and the Coordination for the Improvement of Higher Education Personnel (CAPES) for their financial support; and to Caroline Vargas Xavier and Fabianne Lacouth da Silva for technical assistance and critical reading of the manuscript.

The National Council for Scientific and Technological Development (CNPq) and the State of Rondônia Planning Secretariat (SEPLAN-RO) provided financial support. Moreover, Sulamita da Silva Setúbal was the beneficiary of a CNPq fellowship.

This study was approved by the Tropical Pathology Research Institute Ethics Committee on Animal Use under the protocol number 2008/8. Furthermore, animal tests were in accordance with the guidelines of the Brazilian College for Animal Experimentation (COBEA).

JULIANA PAVAN ZULIANI, Instituto de Pesquisas em Patologias Tropicais, Laboratório de Bioquímica e Biotecnologia e Laboratório de Cultivo Celular e Anticorpos Monoclonais, Rua da Beira, 7671, BR 364, km 3,5, Porto Velho, RO, 78912-000, Brasil. Phone: +55 69 3219 6010. Fax: +55 69 3219 6000. Email: jzuliani@pq.cnpq.br or jzuliani@ipepatro.org.br.