ANALYSIS OF THE MECHANISMS INVOLVED IN THE DEVELOPMENT OF CUTANEUS LOXOSCELISM IN VITRO

PAIXÃO-CAVALCANTE, D.(1), TAMBOURGI, D.V.(1), GONÇALVES DE ANDRADE, R.M.(1), MAGNOLI, F.C.(1), VAN DEN BERG, C.W.(1,2)

(1)Laboratory of Immunochemistry, Butantan Institute, São Paulo, SP, Brazil and (2)Department of Pharmacology, Therapeutics and Toxicology, University of Wales, College of Medicine, Cardiff, UK.

Envenomation by spiders belonging to the genus Loxosceles commonly results in impressive local necrotic skin lesions with scar formation and ulcers that heal slowly. To examine whether Loxosceles intermedia venom and its sphingomyelinase (SMAse) toxin P2 affect the morphology and viability as well as the expression of surface proteins in cultures of dermal and epidermal cells,  human primary keratinocytes, the epidermal carcinoma cell line A431 and human and rabbit primary fibroblasts were treated with different concentrations of venom, purified SMAse P2, phorbolmyristic acid (PMA), Staphylococus aureus Smase, TNF-alpha or C2-ceramide. Alterations in cell morphology were assessed by phase contrast light microscopy. Cell viability was determined by incubating the cells with 10% Alamar Blue for 1 h at 37ºC. The absorbance of the supernatants was read at 560 nm and 595 nm and the cell viability was expressed as (A₅₆₀-A₅₉₅)_treatment/(A₅₆₀-A₅₉₅)_control x 100. To assess the expression of cell surface antigens, cells were treated with different concentrations of L. intermedia venom for 1 h at 37ºC. Thereafter, the cells were incubated with specific antibodies against membrane cofactor protein (MCP), CD59, decay accelerating factor (DAF), MHC class I, alpha₂-microglobulin and epidermal growth factor receptor followed by rabbit anti-mouse Ig/FITC antibody and analysis by flow cytometry. Treatment with L. intermedia venom and purified SMAseP2 decreased cell viability and altered the morphology of all cells studied. S. aureus Smase, TNF-alpha, PMA and C2-ceramide did not produce such effects. Venom treatment also decreased the expression of MCP, MHC I and alpha₂-microglobulin in all cells. L intermedia venom and P2 protein have toxic effects on the main cell types present in skin.

Supported by: FAPESP and The Wellcome Trust.

CORRESPONDENCE TO:

Danielle Paixão Cavalcante, Avenida Carceal Motta ,1310, São Paulo, SP, CEP: 05101-210, Brasil, Email: danipaixao@yahoo.com.br