Effect of Loxosceles gaucho venom on cell morphology and behaviour in vitro in the presence and absence of sphingomyelin

Abstract

This study was performed to investigate whether the toxic effects of Loxosceles gaucho venom on cells might be exerted via stimulators of TNF-α release generated by sphingomyelinase D—a major component of the venom. It was demonstrated that L. gaucho venom alone is unable to induce TNF-α release by J774A.1 cells, while in the presence of exogenous sphingomyelin it induces a high level of TNF-α release which is significantly increased by incubation with non-inactivated serum. Ceramide phosphate also induces TNF-α release in J774A.1 cells, but (unlike sphingomyelin/sphingomyelinase) the level of release is not influenced by the presence or otherwise of non-inactivated serum. L. gaucho venom does not induce proliferation of J774A.1 cells and even at high concentrations it does not affect their viability. J774A.1 cells, which prior to venom treatment were elongated and clumped, round up after venom treatment, but, revert to their original morphology after incubation with fresh medium. TNF-α resistant MRC-5 cells and TNF-α sensitive MCF-7 cells are susceptible to the toxic effect of both L. gaucho venom and ceramide phosphate. The results obtained in this study demonstrate that exogenous sphingomyelin can modulate, in vitro, the release of TNF-α induced by L. gaucho venom in mouse macrophages. In addition, the results also indicate that ceramide phosphate and L. gaucho venom are toxic to several different cell types, via a variety of mechanisms, some, but not all, of which may involve TNF-α as an intermediary.

Introduction

Loxosceles ssp are responsible for the most severe clinical cases of spider envenomation which occur in Brazil, where three species of the genus are common: L. gaucho, L. laeta and L. intermedia (Eickstedt, 1994). Loxosceles venom causes mainly a local reaction around the bite that can develop into dermonecrosis. Systemic symptoms such as fever and exanthema can also appear, and, in severe cases, renal failure can occur leading to death, especially in children (Sezerino et al., 1998; Málaque et al., 2002). Sphingomyelinase D is the major component of Loxosceles venom, and has already been demonstrated to be the factor responsible for inducing dermonecrosis in humans, rabbits and guinea pigs (Futrell, 1992). Sphingomyelinase D cleaves the choline moiety of sphingomyelin, resulting in ceramide phosphate, in contrast to endogenous mammalian sphingomyelinases, which cleave the choline-phosphate moiety of sphingomyelin generating ceramide itself (Ballou et al., 1996).

It is well known that ceramides, the breakdown products resulting from the action of sphingomyelinases on sphingomyelin, are very important cellular regulators of various biological processes such as proliferation, migration, differentiation and apoptosis (Hannun, 1994). Sphingomyelin (N-acylsphingosine-1-phosphocholine or ceramide phosphocholine) the primary source of ceramide in signal transduction, is preferentially concentrated in the outer leaflet of the plasma membrane of mammalian cells (Barenholz and Thompson, 1980). However, the inner leaflet of the plasma membrane or lysosomal/endosomal compartment may be functionally a more important site for sphingomyelin hydrolysis and ceramide generation, given the necessity for interaction between ceramide and intracellular targets. Ceramides also regulate TNF-α production that causes vascular endothelial cells to express adhesion molecules for leukocytes and stimulates endothelial cells and macrophages to secrete chemokines such as IL-8, the most potent chemotactic factor for neutrophils.

It is well established in the scientific literature that dermonecrosis induced by Loxosceles venom is dependent on neutrophils and complement, although neutrophils are not directly activated by the venom (Futrell, 1992, Patel et al., 1994). This suggests that signal transduction molecules participate as intermediaries in the outcome of Loxosceles envenoming, and gives rise to a hypothesis that ceramide phosphate, generated by the action of Loxosceles sphingomyelinase D, stimulates production of TNF-α, which in turn recruits neutrophils to participate in an inflammatory reaction. Accordingly, in vitro experiments were conducted to examine the effect of exogenous sphingomyelin and ceramide phosphate on TNF-α release induced by L. gaucho venom, in order to test this hypothesis. A macrophage cell line was chosen as the experimental model since macrophages are a major source of endogenous TNF-α in vivo.

In addition, the toxic effects of L. gaucho venom and ceramide phosphate in TNF-α sensitive and resistant cell lines were also investigated.