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Lysophosphatidic Acid Mediates the Release of Cytokines and Chemokines by Human Fibroblasts Treated with Loxosceles Spider Venom

2013; Elsevier BV; Volume: 133; Issue: 6 Linguagem: Inglês

10.1038/jid.2013.40

1523-1747

Carolina Campolina Rebello Horta, Bárbara Bruna Ribeiro Oliveira-Mendes, Anderson Oliveira do Carmo, Flávia Freire de Siqueira, Tatiana M. Barroca, Samyra Maria dos Santos Nassif Lacerda, Paulo Henrique Almeida Campos-Júnior, Luiz R. França, Rodrigo Lopes Ferreira, Evanguedes Kalapothakis,

Marine Invertebrate Physiology and Ecology

lysophosphatidic acid lysophosphatidylcholine L. similis venom phospholipase D recombinant L. intermedia dermonecrotic protein 1 TO THE EDITOR Loxosceles spiders are a genus of arachnids, whose bites cause necrotizing skin lesions. They are distributed worldwide in temperate and tropical regions. In Brazil, approximately 10,000 cases of Loxosceles spider bites are reported annually. L. intermedia, L. gaucho, and L. laeta are prevalent in most of the southern states of Brazil, whereas L. similis has been described mainly in the state of Minas Gerais. L. reclusa and L. deserta cause the majority of accidents in North America. The venoms of these species all have similar biochemical and pharmacological profiles (Barbaro et al., 2005Barbaro K.C. Knysak I. Martins R. et al.Enzymatic characterization, antigenic cross-reactivity and neutralization of dermonecrotic activity of five Loxosceles spider venoms of medical importance in the Americas.Toxicon. 2005; 45: 489-499Crossref PubMed Scopus (101) Google Scholar; Silvestre et al., 2005Silvestre F.G. de Castro C.S. de Moura J.F. et al.Characterization of the venom from the Brazilian Brown Spider Loxosceles similis Moenkhaus, 1898 (Araneae, Sicariidae).Toxicon. 2005; 46: 927-936Crossref PubMed Scopus (28) Google Scholar; Chatzaki et al., 2012Chatzaki M. Horta C.C. Almeida M.O. et al.Cutaneous loxoscelism caused by Loxosceles similis venom and neutralization capacity of its specific antivenom.Toxicon. 2012; 60: 21-30Crossref PubMed Scopus (15) Google Scholar. The envenomation, described as loxoscelism, is characterized by pain, local edema, and erythema, followed by dermonecrosis that require weeks to heal. The genesis of loxoscelism is attributed to a family of sphingomyelinase D enzymes, also known as Loxtox proteins (Kalapothakis et al., 2007Kalapothakis E. Chatzaki M. Gonçalves-Dornelas H. et al.The Loxtox protein family in Loxosceles intermedia (Mello-Leitão) venom.Toxicon. 2007; 50: 938-946Crossref PubMed Scopus (56) Google Scholar. Since the discovery that these enzymes have several phospholipid substrates besides sphingomyelin, they have been named phospholipase D (PLD; Van Meeteren et al., 2004Van Meeteren L.A. Frederiks F. Giepmans B.N. et al.Spider and bacterial sphingomyelinases D target cellular lysophosphatidic acid receptors by hydrolyzing lysophosphatidylcholine.J Biol Chem. 2004; 279: 10833-10836Crossref PubMed Scopus (109) Google Scholar; Lee and Lynch, 2005Lee S. Lynch K.R. Brown recluse spider (Loxosceles reclusa) venom phospholipase D (PLD) generates lysophosphatidic acid (LPA).Biochem J. 2005; 391: 317-323Crossref PubMed Scopus (104) Google Scholar; Chaim et al., 2011Chaim O.M. da Silveira R.B. Trevisan-Silva D. et al.Phospholipase-D activity and inflammatory response induced by brown spider dermonecrotic toxin: endothelial cell membrane phospholipids as targets for toxicity.Biochim Biophys Acta. 2011; 1811: 84-96Crossref PubMed Scopus (38) Google Scholar. Furthermore, the complex pathophysiological mechanisms underlying cutaneous loxoscelism have been discussed. Dragulev et al., 2007Dragulev B. Bao Y. Ramos-Cerrillo B. et al.Upregulation of IL-6, IL-8, CXCL1, and CXCL2 dominates gene expression in human fibroblast cells exposed to Loxosceles reclusa sphingomyelinase D: insights into spider venom dermonecrosis.J Invest Dermatol. 2007; 127: 1264-1266Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar showed that L. reclusa PLD upregulated the expression of proinflammatory cytokines/chemokines in human fibroblasts. The authors proposed that ceramide-1-phosphate formed by the hydrolysis of plasma membrane sphingomyelin is responsible for this effect. This speculation was challenged by Van Meeteren et al., 2007Van Meeteren L.A. Stortelers C. Moolenaar W.H. Upregulation of cytokine expression in fibroblasts exposed to Loxosceles sphingomyelinase D: what is the trigger?.J Invest Dermatol. 2007; 127: 1266-1267Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar, who hypothesized that lysophosphatidic acid (LPA), a product of lysophosphatidylcholine (LPC) hydrolysis, is the likely trigger for the observed response, rather than ceramide-1-phosphate. LPC is an abundant plasma component (approximately 150μM) and serves as a genuine physiological substrate for PLD (Van Meeteren et al., 2004Van Meeteren L.A. Frederiks F. Giepmans B.N. et al.Spider and bacterial sphingomyelinases D target cellular lysophosphatidic acid receptors by hydrolyzing lysophosphatidylcholine.J Biol Chem. 2004; 279: 10833-10836Crossref PubMed Scopus (109) Google Scholar. On the basis of this intriguing discussion, we investigated the role of LPA receptors in the release of cytokines/chemokines and in cell death caused by treatment of fibroblasts with L. similis venom (LsV). For comparison, we also used recombinant L. intermedia dermonecrotic protein 1 (recLiD1), a well-described toxin (Kalapothakis et al., 2002Kalapothakis E. Araujo S.C. de Castro C.S. et al.Molecular cloning, expression and immunological properties of LiD1, a protein from the dermonecrotic family of Loxosceles intermedia spider venom.Toxicon. 2002; 40: 1691-1699Crossref PubMed Scopus (63) Google Scholar; Felicori et al., 2006Felicori L. Araujo S.C. de Avila R.A. et al.Functional characterization and epitope analysis of a recombinant dermonecrotic protein from Loxosceles intermedia spider.Toxicon. 2006; 48: 509-519Crossref PubMed Scopus (42) Google Scholar. PLD activity was detected for LsV and recLiD1 (data not shown). HFF-1 human fibroblast cells (BCRJ/UFRJ, Rio de Janeiro, Brazil) were maintained in DMEM with 15% serum. Before treatment, cells were maintained in serum-free DMEM for 4hours. Subsequently, the medium was changed, and the cells were treated for 12hours with 10μgml−1 LsV, 10μgml−1 recLiD1 (both pre-incubated for 1hour at 37°C with 10μM LPC), 15μM LPA, 10μM LPC, or 10ngml−1 tumor necrosis factor-α in serum-free DMEM. Untreated cells served as negative controls. Treatments were performed in the absence or presence of 15μM Ki16425, an LPA receptor (LPA1/LPA3) antagonist, which was added 30minutes before the addition of each agent. After treatment, aliquots of conditioned medium from cell cultures were collected for analysis of cytokine/chemokine levels using ELISA kits for human IL-6, IL-8, IL-1β, RANTES (Life Technologies, Rockville, MD), CXCL1 (Abnova, Taipei, Taiwan), and CXCL2 (Immuno-Biological, Gunma, Japan). AlamarBlue assay (Life Technologies) was performed to confirm cell viability after stimulation, with 10μgml−1 LsV or recLiD1 for 12hours (data not shown). Exposure of HFF-1 to LsV or recLiD1 stimulated the release of IL-6, IL-8, CXCL1, and CXCL2. Cells produced lower levels of mediators after LPC stimulation (Figure 1). Loxosceles venoms have been shown to promote the release of inflammatory mediators in different experimental models (Barbaro et al., 2010Barbaro K.C. Lira M.S. Araújo C.A. et al.Inflammatory mediators generated at the site of inoculation of Loxosceles gaucho spider venom.Toxicon. 2010; 56: 972-979Crossref PubMed Scopus (27) Google Scholar. However, the participation of LPA receptors in this venom inflammatory response has not yet been investigated. In our assays, Ki16425 significantly inhibited the production of cytokines/chemokines by HFF-1 after treatment with LsV and recLiD1 (Figure 1). It is known that HFF-1 cells express LPA1, LPA2, and LPA3 receptors (Zhang et al., 1999Zhang Q. Peyruchaud O. French K.J. et al.Sphingosine 1-phosphate stimulates fibronectin matrix assembly through a Rho-dependent signal pathway.Blood. 1999; 93: 2984-2990Crossref PubMed Google Scholar. Control cells treated with LPA exhibited results similar to those of cells treated with LsV or recLiD1, and tumor necrosis factor-α induced secretion of the mediators tested (data not shown). These data indicate that LPA, formed by PLD activity of LsV and recLiD1, induces liberation of cytokines/chemokines via LPA receptor-mediated pathways. LPA is a bioactive phospholipid that is involved in many cellular functions, including cytokine/chemokine secretion (Fang et al., 2004Fang X. Yu S. Bast R.C. et al.Mechanisms for lysophosphatidic acid-induced cytokine production in ovarian cancer cells.J Biol Chem. 2004; 279: 9653-9661Crossref PubMed Scopus (166) Google Scholar. As Loxosceles PLD has several phospholipid targets, it is necessary to acknowledge that other mediators may also participate in these inflammatory effects. We next investigated whether LPA receptors were involved in cell death induced by longer incubation with LsV. Previous results from AlamarBlue assays revealed that cell survival decreased after 24–48hours of incubation with LsV (data not shown). Therefore, HFF-1 monolayers were stimulated with 10μgml−1 LsV, 10μgml−1 recLiD1 (both pre-incubated with 10μM LPC), 15μM LPA, or 10μM LPC in 15% serum DMEM for 48hours. Ki16425 was added as previously described. Untreated cells served as negative controls. After incubation, cells were trypsinized and washed with phosphate-buffered saline. The cells were then incubated in Apoptosis/Necrosis Detection Kit buffer containing 1% v/v 7-AAD and Annexin V-Cy3 (Enzo LifeSciences, Farmingdale, NY), and subjected to flow cytometry. Treatment with LsV or recLiD1 caused HFF-1 cell death after 48hours, primarily due to apoptosis, which was not inhibited by Ki16425 (Figure 2a). The lack of inhibition was expected, as treatment with LPA did not cause cell death. Control LPC did not injure cells either (data not shown). As LPA can act as a cell survival or apoptotic factor, dependent upon the cell type (Ye et al., 2002Ye X. Ishii I. Kingsbury M.A. et al.Lysophosphatidic acid as a novel cell survival/apoptotic factor.Biochim Biophys Acta. 2002; 1585: 108-113Crossref PubMed Scopus (116) Google Scholar, we performed the same procedures using human umbilical vein endothelial cells, which yielded similar results (Figure 2b). Thus, we did not find that LPA participates in apoptosis induced by LsV or recLiD1 in fibroblast and endothelial cells in vitro. These cells are known sites of Loxosceles venom interaction. Our results support the idea that PLD initiates the effects of Loxosceles venom through stimulation of acute inflammatory responses. In conclusion, LPA receptors are involved in the release of proinflammatory cytokines/chemokines provoked by LsV and recLiD1. The present work contributes to the open discussion regarding the participation of LPA in the pathophysiology of cutaneous loxoscelism and paves the way for investigation of new therapeutic strategies. This work was supported by CNPq, CAPES, and FAPEMIG. We thank IEF/MG for the permission for spider collection. This study was developed in Belo Horizonte, Minas Gerais, Brazil.