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Effect of Bothrops Alternatus Snake Venom on Macrophage Phagocytosis Er

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Effect of Bothrops Alternatus Snake Venom on Macrophage Phagocytosis Er The Journal of Venomous Animals and Toxins including Tropical Diseases ISSN 1678-9199 | 2011 | volume 17 | issue 4 | pages 430-441 Effect of Bothrops alternatus snake venom on macrophage phagocytosis ER P and superoxide production: participation of protein kinase C A P Setubal SS (1), Pontes AS (1), Furtado JL (1), Kayano AM (1), Stábeli RG (1, 2), Zuliani JP (1, 2) RIGINAL O (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 ofBothrops 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. INTRODUCTION and human activity in rural areas where their concentration is higher (3-5). The majority of envenomations in Brazil are Bothrops alternatus snake (now in the caused by species of the genus Bothrops from genus Rhinocerophis) is found in northern and the Viperidae family (1). Bothrops snakes have northeastern Argentina, southern Paraguay and received a large amount of attention due to the the southern, southeastern and western part of high incidence of accidents, with over 90% of central Brazil (6, 7). Its preferred habitat is humid officially reported cases (2). The incidence of tropical, subtropical, and temperate forests. Its snakebites reported between 2000 and 2007, was average size is about one meter long, but it can 1920, of which 27% (703 cases) were recorded in reach two meters. the northern region, 28% in the southeast, 24% The severity of poisoning caused by this specie in the northeast and respectively 11 and 10% in can be partly explained by the large quantity the south and central-west regions (1). These of venom it produces. The maximum venom accidents are frequently related to climatic factors yield per specimen is 380 mg (dry weight) if Setubal SS, et al. Action of Bothrops alternatus PLA2 on macrophages compared with an average of 200 mg (dry weight) of phagocytes, macrophages, and neutrophils, of Bothrops atrox, Bothrops insularis, Bothrops which express a broad spectrum of receptors moojeni, and Bothrops jararacussu (8-10). In that participate in target cell recognition and common with other Bothrops species, Bothrops internalization of ligands (37). Complement alternatus venom contains a variety of proteins receptor type 3 (CR3, also called CD11b/ and enzymes including L-amino acid oxidase CD18, Mac-1, and integrin aMb2) recognizes (LAAO), phospholipase A2 (PLA2), thrombin- complement-coated particles (37-41). like enzyme (such as balterobin), thrombin CR3 requires an additional activation signal inhibitor (bothroalternin), metalloproteinases, for efficient particle binding and phagocytosis. disintegrins, and phosphodiesterases (11-23). These activation signals can be provided by Furthermore, although Bothrops alternatus a number of stimuli such as phorbol esters, is aggressive and dangerous, its venom is not as lipopolysaccharides (LPS), cytokines, growth well-known as other Bothrops venoms, especially factors, and chemokines, which activate Bothrops jararaca, which has been widely studied protein kinase C (PKC) and induce “inside- for its clinical importance in terms of snakebite out” signaling to integrin receptors (42-46). The incidence. inside-out signaling relies on the small GTP- In fact, the local effects observed in B. binding protein Rap1 and the actin-binding alternatus envenomation, according to clinical protein talin (47, 48). This signal is translated cases and experimental studies, mainly refer to changes in integrin tertiary structure and to the appearance of edema, hemorrhage, and clustering, which enhances receptor affinity and necrosis (24-27). The literature also shows thatB. valency (49, 50). alternatus venom causes systemic disturbances in Subsequent to phagocytosis, one of the most blood coagulation cascade in humans and other immediate macrophage responses is abrupt animals such as dogs, and 97% of accidents have production of the potent oxygen free radical, alterations in coagulation times (24). Its toxicity superoxide anion, referred to as oxidative burst. is similar to other Bothrops species (9, 28). The enzyme complex primarily responsible for Envenomation caused by different Bothrops producing this highly reactive oxygen species is species result in severe local tissue damage with NADPH oxidase complex. In resting cells, the hemorrhage, pain, myonecrosis, and inflammation oxidase is dormant and its components exist (8, 29-33). In addition, Bothrops venoms induce separately in the membrane and cytosol. When a significant leukocyte accumulation at the bite macrophages are stimulated, these components site (32-35). However, the activation state of these associate on the plasma or phagosomal cells is still unclear. membranes to form the active oxidase complex Macrophages are important cells of the (51, 52, 53). The resulting reactive oxygen innate immune system. They play a central species (ROS), such as superoxide anion and role in a wide variety of processes associated hydrogen peroxide, are used by macrophages with tissue maintenance, antigen presentation, to kill ingested microorganisms, and are thus inflammation, and tissue repair (36). Resident an essential component of the innate immune macrophages are one of the first lines of host system (51-53). This reaction parallels the release defense and are found in many tissues. Upon of a variety of inflammatory mediators that play stimulation, these quiescent cells become active crucial roles in host defense by microbial killing, and display diverse cellular functions such as the but may also cause injury to surrounding tissues production of numerous active substances. As (36, 51, 52, 54). macrophage activation can play beneficial and Despite the well-documented importance deleterious roles in tissues, these phenomena of macrophages in tissue maintenance, antigen may have an impact on the local pathological presentation, inflammation, and tissue repair, alterations induced by snake venoms. the activation state of these cells is still unclear One of the most important macrophage in snake envenomation. This study was therefore responses against infections is phagocytosis. designed to evaluate the effects of crude Bothrops This event consists of the uptake and destruction alternatus venom (BaV) on isolated thioglycollate of invading microorganisms which is initiated macrophages, particularly phagocytosis and by the engagement of receptors on the surface superoxide anion production. J Venom Anim Toxins incl Trop Dis | 2011 | volume 17 | issue 4 431 Setubal SS, et al. Action of Bothrops alternatus PLA2 on macrophages MATERIALS AND METHODS vials and incubated with 20 mL at different concentrations of venom from B. alternates Chemicals and Reagents (6, 12, 25, 50, and 100 mg/mL) diluted in assay Trypan blue, RPMI-1640, L-glutamine, medium or RPMI (control), for one hour at 37°C gentamicin, halothane, zymosan, staurosporine in a humidified atmosphere (5% CO2). Then, 20 (Sts), phorbol myristate acetate (PMA), DMSO, mL of 0.1% Trypan blue was added to 100 mL of Giemsa and nitroblue tetrazolium (NBT) were thioglycollate-elicited macrophage suspension. purchased from Sigma (USA). Fetal bovine serum Viable cell index was determined in a Neubauer’s (FBS) was obtained from Cultilab (Brazil). Low chamber by counting a total of 100 cells. Results endotoxin or endotoxin-free grades of salts and were expressed as percentage of viable cells. reagents were obtained from Merck (Germany). Adhesion Assay Animals and Venom Macrophage adhesion was assayed according Venom from Bothrops alternatus was acquired to the procedure described by Rosen and Gordon from the Bioactive Protein Serpentarium, (56). Thioglycollate-elicited
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