Bothrops asper and Bothrops jararaca snake venoms trigger microbicidal functions of peritoneal leukocytes in vivo

Venoms from snakes of the genus Bothrops cause pronounced local effects in the victims. These alterations result not only from the direct toxic action of venom components, but also from the prominent inflammatory reaction associated with these envenomations. In this study we investigated the ability of Bothrops asper (BaV) and Bothrops jararaca (BjV) venoms to induce cellular influx and microbicidal functions in leukocytes. BaV and BjV (5 μg/animal) caused a long lasting infiltration of leukocytes (3–48 h) when injected into mouse peritoneal cavity. Both venoms increased phagocytosis and production of hydrogen peroxide (H2O2) by polymorphonuclear (PMN) and mononuclear (MN) peritoneal leukocytes. In addition, nitric oxide (NO) production by macrophages was also enhanced after the venom injections. This effect was inhibited by treating animals with L-NAME and aminoguanidine, thus suggesting the induction of iNOS synthesis by the venoms. Western blot analysis confirmed the expression of iNOS in macrophages. BaV and BjV injection led to increased levels of IFN-γ at the site of inflammation. Since IFN-γ is an effective inducer of iNOS expression, an indirect action of the venoms on iNOS expression can be proposed. A marked formation of nitrotyrosine-containing proteins was also observed in macrophage homogenates. Based on these results, we suggest that reactive oxygen and nitrogen-derived species are involved in the pathogenesis of the local tissue damage characteristic of Bothrops sp envenomations.Fundação de Amparo à Pesquisa do Estado de São Paulo/[98/00162-9]/FAPESP/BrasilFundação de Amparo à Pesquisa do Estado de São Paulo/[97/13089-5]/FAPESP/BrasilFundação de Amparo à Pesquisa do Estado de São Paulo/[95/9699-7]/FAPESP/BrasilConselho Nacional de Desenvolvimento Científico e Tecnológico/[301199/91-4)]/CNPq/BrasilUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP

Inflammation induced by Bothrops asper venom: release of proinflammatory cytokines and eicosanoids, and role of adhesion molecules in leukocyte infiltration

Bothrops asper venom (BaV) causes systemic and local effects characterized by an acute inflammatory reaction with accumulation of leukocytes and release of endogenous mediators. In this study, the effects of BaV on the release of the cytokines IL-1, IL-6 and TNF-alpha and the eicosanoids LTB4 and TXA2 in the peritoneal cavity of mice were analyzed. We also investigated the participation of beta2 integrin chain, l-selectin, LFA-1, ICAM-1 and PECAM-1 adhesion molecules in the BaV-induced leukocyte accumulation. Levels of proinflammatory cytokines IL-6 and TNF-alpha, as well as eicosanoids LTB4 and TXA2 were significantly increased after BaV injection (250 microg/kg), whereas no increment in IL-1 was observed. Anti-mouse l-selectin, LFA-1, ICAM-1, PECAM-1 and beta2 integrin chain monoclonal antibodies resulted in a reduction of neutrophil accumulation induced by BaV injection compared with isotype-matched control injected animals. These data suggest that BaV is able to induce the activation of leukocytes and endothelium to express adhesion molecules involved in the recruitment of neutrophils into the inflammed site. Furthermore, these results showed that BaV induces the release of cytokines and eicosanoids in the local of the venom injection; these inflammatory mediators may be important for the initiation and amplification of the inflammatory reaction characteristic from Bothrops sp envenomation.Fundação de Amparo à Pesquisa do Estado de São Paulo/[98/0162-9]/FAPESP/BrasilFundação de Amparo à Pesquisa do Estado de São Paulo/[97/13089-5]/FAPESP/BrasilMinistério da Ciência, Tecnologia, Inovações e Comunicações/[301199/91-6]/CNPq/BrasilUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP

Secretory phospholipases A2 isolated from Bothrops asper and from Crotalus durissus terrificus snake venoms induce distinct mechanisms for biosynthesis of prostaglandins E2 and D2 and expression of cyclooxygenases

The effects of myotoxin III (MT-III), a phospholipase A2 (sPLA2) from Bothrops asper snake venom, and crotoxin B (CB), a neurotoxic and myotoxic sPLA2 from the venom of Crotalus durissus terrificus, on cyclooxygenases (COXs) expression and biosynthesis of prostaglandins (PGs) were evaluated, together with the mechanisms involved in these effects. Upon intraperitoneal injection in mice, both sPLA2s promoted the synthesis of PGD2 and PGE2, with a different time-course. MT-III, but not CB, induced COX-2 expression by peritoneal leukocytes without modification on COX-1 constitutive expression, whereas CB increased the constitutive activity of COX-1. MT-III increased the enzymatic activity of COX-1 and COX-2. Similar effects were observed when these sPLA2s were incubated with isolated macrophages, evidencing a direct effect on these inflammatory cells. Moreover, both toxins elicited the release of arachidonic acid from macrophages in vitro. Inhibition of cPLA2 by AACOCF3, but not of iPLA2 by PACOCF3 or BEL, significantly reduced PGD2, PGE2 and arachidonic acid (AA) release promoted by MT-III. These inhibitors did not affect MT-III-induced COX-2 expression. In contrast, cPLA2 inhibition did not modify the effects of CB, whereas iPLA2 inhibition reduced PGD2 and AA production induced by CB. These findings imply that distinct regulatory mechanisms leading to PGs' synthesis are triggered by these snake venom sPLA2s. Such differences are likely to explain the dissimilar patterns of inflammatory reaction elicited by these sPLA2s in vivo.UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP

Role of cyclooxygenases in oedema-forming activity of bothropic venoms

The venoms of Bothrops asper (BaV) and Bothrops jararaca (BjV), two of the most medically important poisonous snakes of Latin America, cause pronounced oedema in the victims through poorly understood mechanisms. In the present study, we examined the possible role of cyclooxygenases (COX) in the genesis of mouse paw oedema caused by BaV and BjV injections. BaV at 2.5 μg/paw and BjV at 0.75 μg/paw induced significant oedema that persisted for up to 6 h following subplantar injection. Treatment with indomethacin (2 mg/kg), rofecoxib, (10 mg/kg), or dexamethasone (2 mg/kg) significantly reduced the BaV- and BjV-induced oedema formation. Treatment with SC-560 (30 mg/kg) significantly reduced the oedema formation induced by BjV but had no effect on that induced by BaV. Both venoms induced significant increases in the levels of prostaglandin E2 (PGE2) and the expression of COX-1 and COX-2 in paw tissue. The peak of oedema formation and PGE2 release correlated with marked expression of COX-2 in the paw tissue. These results demonstrate that injection of BaV and BjV results in a rapid increase in oedema formation that is, at least partially, mediated by arachidonic acid metabolites formed by COX-2. In the case of BjV, COX-1-derived prostanoids also appear to contribute significantly to the inflammatory changes.Fundação de Amparo à Pesquisa do Estado de São Paulo/[02/13863-2]/FAPESP/BrasilCanadian Institutes of Health Research//CIHR/CanadáJanssen Pharmaceutica and the Canadian Association of Gastroenterology///CanadáFundação de Amparo à Pesquisa do Estado de São Paulo/[98/0162-9]/FAPESP/BrasilFundação de Amparo à Pesquisa do Estado de São Paulo/[02/10861-9]/FAPESP/BrasilUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP

Effects of Bothrops asper snake venom on the expression of cyclooxygenases and production of prostaglandins by peritoneal leukocytes in vivo, and by isolated neutrophils and macrophages in vitro

In this study, the ability of Bothrops asper snake venom (BaV) to increase the production of prostaglandins PGE2 and PGD2 was assessed in a mouse model in vivo and in inflammatory cells in vitro. In addition, the expressions of COX-1 and COX-2 were assessed. BaV induced an increment in the in vivo synthesis of PGE2 and PGD2, together with an enhanced expression of COX-2, but not of COX-1. However, enzymatic activities of COX-1 and COX-2 were increased. Incubation of isolated macrophages and neutrophils with a sub-cytotoxic concentration of BaV in vitro resulted in increased release of PGE2 and PGD2 by macrophages and PGE2 by neutrophils, concomitantly with an increment in the expression of COX-2, but not of COX-1 by both cell types. Our results demonstrate the ability of BaV to promote the expression of COX-2 and to induce the synthesis of proinflammatory prostaglandins. Macrophages and neutrophils may be important targets for this venom under in vivo situation.UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP

Inflammatory effects of BaP1 a metalloproteinase isolated from Bothrops asper snake venom: leukocyte recruitment and release of cytokines

The inflammatory events induced by BaP1, a 22.7 kDa metalloproteinase isolated from Bothrops asper snake venom, were studied. BaP1 i.p. injection in mice 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. Inhibition of enzymatic activity of BaP1 by chelation with EDTA resulted in a drastic reduction of this effect. In addition, BaP1 induced a significant increase of blood neutrophil numbers before its accumulation in peritoneal cavity, thus suggesting a stimulatory action of BaP1 on mechanisms of cell mobilization from bone marrow reserve compartments. A reduction in the number of neutrophils was observed in the exudate when antibodies against LECAM-1, CD18 and LFA-1 were used, suggesting the involvement of these adhesion molecules in the effects of BaP1. In contrast, there was no effect with antibodies against ICAM-1 and PECAM-1. Moreover, a conspicuous increment in the levels of IL-1 and TNF-alpha, but not of LTB4, was observed in peritoneal washes collected from mice injected with BaP1. It is concluded that BaP1 induces in vivo a marked leukocyte influx, which parallels an increased number of these cells in the blood, and is associated to the expression of specific leukocyte adhesion molecules and release of chemotactic inflammatory cytokines. Since BaP1 is a P-I class metalloproteinase, these results indicate that the proteolytic domain of metalloproteinases per se can trigger specific inflammatory events.Fundação de Amparo à Pesquisa do Estado de São Paulo/[98/00162-9]/FAPESP/BrazilFundação de Amparo à Pesquisa do Estado de São Paulo/[00/03637-0]/FAPESP/BrazilFundação de Amparo à Pesquisa do Estado de São Paulo/[03/08529-9]/FAPESP/BrazilUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP

Neutrophils do not contribute to local tissue damage, but play a key role in skeletal muscle regeneration, in mice injected with Bothrops asper snake venom

Local tissue damage induced by crotaline snake venoms includes edema, myonecrosis, hemorrhage, and an inflammatory response associated with a prominent cellular infiltrate. The role of neutrophils in the local tissue damage induced by Bothrops asper snake venom and by myotoxin I, a phospholipase A2 isolated from this venom, was investigated. Male Swiss mice were pretreated with either an antimouse granulocyte rat monoclonal immunoglobulin G (IgG) antibody or with isotype-matched control antibody. No significant differences in these local effects were observed between mice pretreated with antigranulocyte antibodies and those receiving control IgG. Moreover, myotoxicity induced by B. asper myotoxin I was similar in neutrophil-depleted and control mice. The role of neutrophils in the process of skeletal muscle regeneration was also assessed. Muscle regeneration was assessed by quantifying the muscle levels of creatine kinase and by morphometric histological analysis of the area comprised by regenerating cells in damaged regions of skeletal muscle. Mice depleted of neutrophils and then injected with B. asper venom showed a more deficient regenerative response than mice pretreated with control IgG. Moreover, a drastic difference in the regenerative response was observed in mice injected with myotoxin I, because animals pretreated with control IgG showed a successful regeneration, whereas those depleted of neutrophils had abundant areas of necrotic tissue that had not been removed 7 days after injection, associated with reduced contents of creatine kinase. It is concluded that (1) neutrophils do not play a significant role in the acute local pathological alterations induced by the venom of B. asper, and (2) neutrophils play a prominent role in the process of skeletal muscle regeneration after injection of B. asper venom and myotoxin I, probably related to the phagocytosis of necrotic material and the recruitment of other inflammatory cells, two events directly associated with a successful muscle regenerative responseFundação de Amparo à Pesquisa do Estado de São Paulo/[98/0162-9]/FAPESP/BrasilFundação de Amparo à Pesquisa do Estado de São Paulo/[00/08890-5]/FAPESP/BrasilFundação de Amparo à Pesquisa do Estado de São Paulo/[97/13089-5]/FAPESP/BrasilUniversidad de Costa Rica//UCR/Costa RicaUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP