Loxosceles Sphingomyelinase Induces Complement-Dependent Dermonecrosis, Neutrophil Infiltration, and Endogenous Gelatinase Expression Denise V. Tambourgi,

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Loxosceles Sphingomyelinase Induces Complement-Dependent Dermonecrosis, Neutrophil Infiltration, and Endogenous Gelatinase Expression Denise V. Tambourgi, Danielle Paixão-Cavalcante, Rute M. Gonçalves de Andrade, Matheus de F. Fernandes-Pedrosa, Fábio C. Magnoli, B. Paul Morgan, Carmen W. van den Berg Journal of Investigative Dermatology Volume 124, Issue 4, Pages 725-731 (April 2005) DOI: 10.1111/j.0022-202X.2005.23654.x Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Loxoscelic skin lesion: analysis of the role of complement in the dermonecrosis. Histological analysis of skin of rabbits injected with 5 μg of Loxosceles intermedia venom and purified (P1) or recombinant (rec P1) sphingomyelinases. Control sites were injected with an equal volume of phosphate-buffered saline (PBS). The involvement of the C system in the development of the dermonecrotic lesion was evaluated in C6-deficient rabbits (C6 def) or in normal animals that were previously injected with cobra venom factor (CoVF) and 18 h later injected with 5 μg of Loxosceles venom. Panels correspond to the panoramic view of skin sections from rabbits injected with PBS (A), L. intermedia venom (B), purified P1 (C), rec P1 (D), and from C6-def rabbits (E) or CoVF-treated animals (F) injected with L. intermedia venom. Arrows indicate areas of leukocyte infiltration. Panels a–f show details of the collagenous area of the dermis of the same sections. White bars at the top of each panel indicate 100 μm. Journal of Investigative Dermatology 2005 124, 725-731DOI: (10.1111/j.0022-202X.2005.23654.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Determination of the number of neutrophils in the rabbit skin. (A) Number of neutrophils in the histological skin sections obtained from rabbits injected with 5 μg of Loxosceles intermedia venom and purified (P1) or recombinant (rec P1) sphingomyelinases or with buffer. (B) Neutrophil counts determined for sections obtained from normal or C6-deficient rabbits (C6 def) injected (+) or not (-) with L. intermedia venom and from normal animals that were previously injected with cobra venom factor (CoVF) (-) and 18 h later injected with 5 μg of Loxosceles venom (+). Results are representative of three different experiments and represented as mean±SD. Statistical analysis showed significant differences between the mean values of cell counts obtained for sections from control and experimental groups of rabbits (*p<0.001). Journal of Investigative Dermatology 2005 124, 725-731DOI: (10.1111/j.0022-202X.2005.23654.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Loxosceles intermedia venom induces the expression of matrix metalloproteinases (MMP)-9 in rabbit skin. Gelatinase activity of rabbit skin samples was analyzed by zymography. (A) Normal (control), venom (V), P1, or recombinant sphingomyelinase P1 (rec P1) (5 μg)-injected rabbits; (B) control, C6-deficient rabbits (C6-def), and cobra venom factor (CoVF)-treated rabbits injected with 5 μg of L. intermedia venom (+) or with phosphate-buffered saline (-). Gelatinase expression is indicated by the arrows; (C) skin extract of venom-injected rabbit was incubated with polyclonal (pAb) or monoclonal (mAb) anti-MMP-2 or -MMP-9 bound to protein Sepharose G beads or with beads without antibody (C). Supernatants were analyzed by zymography and compared with purified human MMP-2 (2) and human MMP-9 (9). Journal of Investigative Dermatology 2005 124, 725-731DOI: (10.1111/j.0022-202X.2005.23654.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Loxosceles venom induces the expression of gelatinases in rabbit fibroblasts and neutrophils. (A) Zymography analysis of neutrophils cell extracts after treatment with buffer (C) or with 10 μg per mL of Loxosceles intermedia venom (V). (B) Supernatants of cultured rabbit fibroblasts, 24 and 48 h after treatment with buffer (C) or 20 μg per mL of Loxosceles venom (V). Matrix metalloproteinase (MMP)-9 and MMP-2 expression is indicated by the arrows. Journal of Investigative Dermatology 2005 124, 725-731DOI: (10.1111/j.0022-202X.2005.23654.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions