Sphingomyelinases Induce Direct Binding of C1q to the Erythrocyte Membrane and Cause Autologous Classical Complement Pathway Hemolysis 

^{1Tambourgi, D.V.; 1Fernandes-Pedrosa, M.F.; 1Gonçalves-de-Andrade, R.M.; 2Billington},S.J., ³Griffiths, M. and ⁴van den Berg, C.W.

1 Laboratório de Imunoquímica, Instituto Butantan, São Paulo, Brazil; 2 Department of Veterinary Science and Microbiology, University of Arizona, Tucson, USA, 3 Department of Medical Biochemistry and Immunology and 4 Department of Pharmacology, Therapeutics and Toxicology, University of Wales, College of Medicine, Cardiff, UK.

Bites by Loxosceles spiders can produce severe clinical symptoms, including dermonecrosis, thrombosis, vascular leakage, hemolysis and persistent inflammation. The causative factor is a sphingomyelinase D (SMase D) that cleaves sphingomyelin into choline and ceramide-1-phosphate. A similar enzyme, showing comparable bioactivity, is secreted by certain pathogenic corynebacteria and acts as a potent virulence factor. We have focused our recent investigations on the effects of SMases on erythrocytes and have found that the toxins led to an increased susceptibility to activation of Complement (C) via the classical pathway (CP) in the absence of antibodies. In the present study we have investigated the CP initiating components involved in the hemolysis induced by SMases from C. pseudotuberculosis (PLD) and from Loxosceles intermedia venom (P1). The possible involvement of C1q, SAP and CRP in the initiation of activation of the CP following incubation of human erytrocytes (E) with P1 and PLD was investigated. When P1 or PLD treated E were incubated with C8d-HS, an increase in C1q, SAP and CRP binding was observed compared to untreated E. Furthermore, purified C1q, SAP and CRP were found to bind to P1 or PLD treated E. Depletion of SAP or CRP from human serum did not inhibit C-mediated lysis, suggesting that pentraxins are not involved in the initiation of C-activation and that the activation of the CP is due to direct binding of C1q to the SMase treated cells. Annexin V-FITC was found to bind to the SMase treated E suggesting exposure of phosphatidyl-serine (PS), as a consequence of loss of membrane asymmetry. Exposure of PS has been shown to result in activation of the C-system and our results suggest that initiation of the CP after exposure of E to SMases is induced by binding of the C1q to PS.

Support: The Wellcome Trust, FAPESP and CNPq

Correspondence to: mpedrosa@butantan.gov.br