Elsevier

Toxicon

Volume 45, Issue 3, 1 March 2005, Pages 335-346
Toxicon

Inflammatory events induced by Lys-49 and Asp-49 phospholipases A2 isolated from Bothrops asper snake venom: role of catalytic activity

https://doi.org/10.1016/j.toxicon.2004.11.004Get rights and content

Abstract

The inflammatory events induced in the peritoneal cavity of mice by two PLA2s isolated from Bothrops asper snake venom were investigated. MT-III, an Asp-49 catalytically active enzyme and MT-II, a catalytically inactive Lys-49 variant induced increase in vascular permeability. Inhibition of enzymatic activity of MT-III with p-bromophenacyl bromide reduced this effect. MT-III induced a larger neutrophil infiltrate than MT-II. This activity was significantly reduced after inhibition of catalytic activity. Reduction in the number of neutrophils was observed when antibodies against l-selectin, CD18 or LFA-1 were used, suggesting the involvement of these adhesion molecules in the effects of both PLA2s. There was no effect with antibodies against ICAM-1 and PECAM-1. Increase in the levels of LTB4 and TXA2, as well as of IL-1, IL-6 and TNF-α, were observed in the peritoneal exudates induced by MT-III. MT-II did not enhance levels of eicosanoids but increased those of cytokines. It is concluded that both PLA2s induce inflammatory events in this model. Since MT-III exerts a stronger proinflammatory effect, the enzymatic phospholipid hydrolysis may be relevant for these phenomena. However, the fact that MT-II induced inflammation suggests that molecular regions distinct from the catalytic site elicit inflammatory events perhaps by interacting with specific cell membrane acceptors.

Introduction

Venoms from snakes of the family Viperidae contain class II PLA2s which share structural features with secretory PLA2s of the class II-A present in inflammatory exudates in mammals (Kini, 1997, Kaiser et al., 1990). A number of venom PLA2s has been shown to induce edema (Vishwanath et al., 1987, Lomonte et al., 1993, Lloret and Moreno, 1993) and to promote inflammatory cell infiltration (Zhang and Gopalakrishnakone, 1999, de Castro et al., 2000), although comprehensive studies of the actions of venom PLA2s on the various events of inflammation are scarce. A particularly interesting subgroup of venom PLA2s includes homologues having a number of amino acid substitutions at the calcium-binding loop, especially lysine substituting aspartate at position 49, resulting in the inability of these enzymes to bind calcium and, consequently, in the abrogation of their catalytic activity (Ownby et al., 1999). Thus, these PLA2s homologues exert their activities independently of enzymatic phospholipid degradation (Lomonte et al., 1994, Landucci et al., 1998).

Bothrops asper constitutes the most important poisonous snake from a medical point of view in Central America (Gutierrez and Lomonte, 1995), and four myotoxic PLA2s have been isolated and characterized from this venom (Gutierrez et al., 1984, Lomonte and Gutierrez, 1989, Kaiser et al., 1990, Díaz et al., 1995). Two of them, myotoxins II and III have been sequenced, and the crystallographic structure of the former has been determined (Arni et al., 1995), paving the way for the performance of structure–function analyses. MT-III is a catalytically-active Asp-49 variant (Kaiser et al., 1990), whereas MT-II is a Lys-49, catalytically inactive homologue (Lomonte and Gutierrez, 1989). These proteins play a relevant role in the pathogenesis of local tissue damage induced by Bothrops asper venom, exerting myotoxic and edema-forming effects. Moreover, a conspicuous inflammatory cell infiltrate has been described histologically in muscle affected by these PLA2s (Lomonte et al., 1993). However, the mechanisms underlying the inflammatory cells tissue infiltration are still unknown.

Inflammation is a defense mechanism characterized by increase of vascular permeability, edema, and leukocyte migration from the vasculature into damaged tissues to destroy the injurious agent. During the acute phase of inflammation, neutrophils are the first cells to accumulate in tissues (Ryan and Majno, 1977). Migration of leukocytes into tissues is a multistep process, which is characterized by an initial weak interaction with the endothelium mediated by selectins adhesion molecules (l-selectin on neutrophils, E-selectin and P-selectin on endothelium) and their carbohydrated ligands, giving a rolling motion to leukocytes (Bevilacqua and Nelson, 1993). Rolling enables leukocyte activaction by chemotactic agents associated with the endothelial-cell membrane, inducing activation of β2 integrins (LFA-1 and MAC-1) on the white-cell surface. These molecules interact with endothelial members of the immunoglobulin superfamily (ICAM-1, -2 and -3) for firm leuko-endothelial adhesion. Leukocytes then begin to cross the endothelial layer through homologous interactions of platelet endothelial-cell adhesion molecule (PECAM-1) expressed in both leukocytes and the intercellular membranes of endothelial cells, and migrate following a chemoattractant gradient initiated in the injured tissue. The sequence of expression and function of the adhesion molecules is regulated by a range of inflammatory mediators (Hubbard and Rothlein, 2000, Kaplanski et al., 2003). In the present work, a comprehensive analysis of the inflammatory reaction elicited by the two PLA2s, MT-II and MT-III, in the mouse peritoneal cavity was performed, with special focus on leukocyte migration. Results indicate that, although both proteins are able to elicit an inflammatory reaction, the profile of inflammatory events and mechanisms elicited by these PLA2s differ. MT-III-induced cell influx is mediated by eicosanoids and cytokines whereas that event triggered by MT-II is mainly cytokine-dependent. However, both PLA2s upregulate similar leukocyte adhesion molecules. Finally, our findings suggest that, despite the fact that catalytic activity is not essential for the induction of inflammation in the case of a Lys-49 PLA2, it does play an important role in the development of inflammatory events after injection of an Asp-49 PLA2.

Section snippets

Chemicals and reagents

Heparin was obtained from Prod. Roche Quim. Farm. S.A. (Rio de Janeiro, Brazil) and Evans blue from Inlab (Brazil). Murine capture antibody anti-IL-6 (clone MP5-20F3), recombinant rIL-6 and detection antibody anti-IL-6 (clone MP5-32C11) were purchased from Pharmingen (CA, USA). 2,2′-azino-bis (3 ethylbenzthiazoline-6-sulfonic acid) ABTS was purchased from Southern Biotechnology Associates Inc. (AL, USA). Rat monoclonal antibodies anti-mouse l-selectin (anti-mouse CD62L; clone MEL-14),

Effects of PLA2s on vascular permeability

The increase of vascular permeability (VP) in the peritoneal cavities of animals was determined 15 min after the injection of two doses (0.5 and 1.0 mg/kg) of MT-II or MT-III into the cavities. Fig. 1A shows that a significant increase of VP was induced by 0.5 and 1.0 mg/kg of MT-II or MT-III. Doses of 1.0 mg/kg caused the most pronounced effect. Chemical inactivation of the catalytic site of MT-III abrogated the effect of this toxin on VP. Time course of the increase of VP induced by MT-II or

Discussion

PLA2s from snake venoms exert a large number of pharmacological activities (Kini, 1997) due to a process of accelerated evolution through which a high mutational rate in the coding regions of their genes has allowed the development of new functions, mainly associated with the exposed regions of the molecules (Kini and Chan, 1999). The integral analysis of the inflammation elicited by Asp-49 and Lys-49 PLA2s from B. asper venom in the mouse peritoneal cavity performed in the present study

Acknowledgements

The authors thank Dr Bruno Lomonte for his collaboration in the isolation of myotoxins and to Maria Zelma da Silva for technical assistance. This project was supported by grants from FAPESP-Brazil (Grants 98/00162-9; 98/15657-3), CNPq (301199/91-4) and Vicerrectoría de Investigación, Universidad de Costa Rica.

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