Role of Matrix Metalloproteinases in HaCaT Keratinocytes ... - Core

ORIGINAL ARTICLE

Role of Matrix Metalloproteinases in HaCaT Keratinocytes Apoptosis Induced by Loxosceles Venom Sphingomyelinase D Danielle Paixaõ-Cavalcante1, Carmen W. van den Berg2, Matheus de Freitas Fernandes-Pedrosa1, Rute M. Gonçalves de Andrade1 and Denise V. Tambourgi1 Envenomation by the spider Loxosceles (brown spider) can result in dermonecrosis and severe ulceration. We have previously shown that Loxosceles sphingomyelinase D (SMaseD), the enzyme responsible for these pathological effects, induced expression of matrix metalloproteinase-9 (MMP-9), which is possibly one of the main factors involved in the pathogenesis of the cutaneous loxoscelism. The aim of this study was to further investigate the molecular mechanisms triggered by Loxosceles SMaseD involved in the initiation of the dermonecrotic lesion, using HaCaT cultures, a human keratinocyte cell line, as an in vitro model for cutaneous loxoscelism. We show here that SMaseD from Loxosceles spider venom induces apoptosis in human keratinocytes, which is associated with an increased expression of metalloproteinase-2 and -9, and that the use of metalloproteinase inhibitors, such as tetracycline, may prevent cell death and potentially may prevent tissue destruction after envenomation. Journal of Investigative Dermatology (2006) 126, 61–68. doi:10.1038/sj.jid.5700049

INTRODUCTION Envenomation by spiders of the Loxosceles species (brown spiders) can produce severe clinical symptoms, including dermonecrosis, thrombosis, vascular leakage, hemolysis, and persistent Ramos-cerrille inflammation (Futrell, 1992). Loxosceles is the most poisonous spider in Brazil and children who develop the more severe systemic effects after envenomation nearly always die. At least three different Loxosceles species of medical importance are known in Brazil (L. intermedia, L. gaucho, and L. laeta) and more than 3,000 cases of envenomation by L. intermedia alone are reported each year. In North America, several Loxosceles species, including L. reclusa (brown recluse), L. apachea, L. arizonica, L. unicolor, L. deserta, and L. boneti, are known to be the principal cause of numerous incidents of envenomation (Ginsburg and Weinberg, 1988; Gendron, 1990; Bey et al., 1997; Ramos-Cerrillo et al., 2004). In South Africa, L. parrami and L. spinulosa are responsible for cutaneous loxoscelism (Newlands et al., 1982), and in 1

Laborato´rio de Imunoquı´mica, Instituto Butantan, Saõ Paulo, Brazil and Department of Pharmacology, Therapeutics and Toxicology, Cardiff University, Wales College of Medicine, Cardiff, UK 2

Correspondence: Dr Denise V. Tambourgi, Laborato´rio de Imunoquı´mica, Instituto Butantan, Av. Professor Vital Brazil, 1500, Saõ Paulo CEP 05508-900, Brazil. E-mail: [email protected] Abbreviations: C, complement; EGFR, EGF receptor; FBS, fetal bovine serum; MCP, membrane cofactor protein; MoAb, monoclonal antibodies; MMP, matrix metalloproteinase; PI, propidum iodide; rP2, recombinant SMaseD P2; SMaseD, sphingomyelinase D Received 26 April 2005; revised 25 August 2005; accepted 31 August 2005

& 2006 The Society for Investigative Dermatology

Australia, a cosmopolitan species, L. rufescens, is capable of causing ulceration in humans. The Loxosceles spider bite, which initially causes only minor discomfort, can result in chronic ulcer formation and extensive tissue destruction that may take many months to heal (Atkins et al., 1958; Wasserman and Anderson, 1983–1984). Local histopathologic alterations in experimentally envenomed animals and in humans bitten by Loxosceles spiders include increased endothelium thickness and edema, vasodilatation, vessel wall degeneration, hemorrhage into the dermis, dermis–epidermis dissociation, and vacuolization of basal layer keratinocytes (Mackinnon and Witkind, 1953; Pizzi et al., 1957). The accumulation of polymorphonuclear leukocytes is associated with the activation of the complement system (C) (Patel et al., 1994; Tambourgi et al., 2005). We have purified and cloned several isoforms of sphingomyelinase D (SMaseD) from L. laeta and L. intermedia venoms and have shown that they are responsible for all the local and systemic effects induced by whole venom (Tambourgi et al., 1998, 2004; Fernandes-Pedrosa et al., 2002). The venoms of various Loxosceles species contain several functionally active isoforms of SMaseD, with identity varying from 40 to 90% (Fernandes Pedrosa et al., 2002; Ramos-Cerrillo et al., 2004; Tambourgi et al., 2004). SMaseD from Loxosceles venoms hydrolyze sphingomyelin, resulting in the formation of ceramide-1-phosphate (Cer-1-P or N-acyl-sphingosine-1-phosphate) (Forrester et al., 1978; Kurpiewski et al., 1981; Tambourgi et al., 1998) and also catalyze the release of choline from lysophosphatidylcholine in the presence of Mg2 þ (van Meeteren www.jidonline.org

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D Paixaõ-Cavalcante et al. SMaseD Induces Apoptosis in HaCaT by Activation of MMPs

et al., 2004). Lysophosphatidylcholine is an abundant phospholipid in plasma, where it is tightly bound to albumin; removal of its choline headgroup yields lysophosphatidic acid, a potent lipid mediator with numerous biological activities in many different cells (Moolenaar, 1999; Chunn et al., 2002). We have focused our recent investigations on the effects of Loxosceles SMaseD on erythrocytes and nucleated cells and have found that these enzymes induce activation of membrane-bound metalloproteinases (Tambourgi et al., 2000; van den Berg et al., 2002). In the case of erythrocytes, this led to an increased susceptibility to activation of C via the alternative pathway because of metalloproteinase-induced cleavage of glycophorins, which are important regulators of C activation (Tambourgi et al., 2000). However, on nucleated cells, this led to a decrease in C susceptibility, despite the cleavage of the C-regulator membrane cofactor protein (MCP) (van den Berg et al., 2002). On erythrocytes, activation of the classical pathway of C, possibly by SMaseD-induced loss of membrane asymmetry (Tambourgi et al., 2002), resulting in exposure of phosphatidyl serine on the external lipid monolayer, also contributed to the enzyme-induced hemolysis. SMaseD-induced exposure of phosphatidyl serine on the surface of other cells may also explain other pathological effects observed after envenomation, for example, intravascular coagulation; exposure of phosphatidyl serine on the cell surface of platelets and endothelial cells is known to initiate the coagulation cascade and initiate platelet aggregation. Loss of E membrane asymmetry may be due to the change of lipid composition induced by the binding and hydrolytic activity of the spider SMaseD (Rees et al., 1984; Tambourgi et al., 1995, 1998, 2000, 2002, 2004; Fernandes Pedrosa et al., 2002). The ability of SMaseD to bind to different species of erythrocytes (such as humans, sheep, rats, rabbits, and guinea-pigs) as well as to several cell types (such as epidermoid cells, hepatocytes, monocytes, B and T cells, endothelial cells, platelets, and neutrophils) has already been described (Rees et al., 1984; Tambourgi et al., 1995, 2000; van den Berg et al., 2002; D.V. Tambourgi, personal communication). Although no specific receptor has been described yet for this interaction, the SMaseD binding ability is certainly an important step for the mechanism of action of the Loxosceles spider venom. We have also shown that the C system plays an important role in the development of cutaneous loxoscelism through the C5a- and MAC-dependent recruitment of neutrophils (Tambourgi et al., 2005). Furthermore, we have shown that in rabbits, Loxosceles SMaseD induced the expression of matrix metalloproteinase-9 (MMP-9), which is possibly one of the main factors involved in the pathogenesis of the cutaneous loxoscelism (Tambourgi et al., 2005). The discovery of the involvement of metalloproteinases in the Loxosceles venominduced dermonecrosis is novel and has potential therapeutic significance given the availability of metalloproteinase inhibitor drugs. It has been shown that venom from L. gaucho induced tumor necrosis factor-alpha and venom from L. deserta induced vascular endothelial growth factor 62

Journal of Investigative Dermatology (2006), Volume 126

production by human keratinocytes (Malaque et al., 1999; Desai et al., 2000), both of which may contribute to the pathology induced in vivo. Uncontrolled proteolytic tissue destruction appears to be a pathogenic factor in nonhealing wounds in which MMPs are involved. Overexpression of MMPs has been implicated in the pathogenesis of several diseases, such as tumor progression, atherosclerotic plaque rupture, aortic aneurisms, acute respiratory distress syndrome, and asthma (Ohnishi et al., 1998). Tetracycline and chemically modified tetracyclines have been shown to exert a number of anti-inflammatory and immunomodulatory activities, independent of their antibiotic properties (Kuzin et al., 2001). These include the ability to inhibit metalloproteinases, a class of enzymes involved in crucial cellular functions such as remodeling of the extracellular matrix. Tetracycline can specifically inhibit the expression, the activation from proenzyme precursor, as well as the enzymatic activity of metalloproteinases (Hanemaaijer et al., 1998). Tetracyclines may have considerable potential for human therapy of MMP-related human diseases (Acharya et al., 2004). The aim of this study was to investigate the role of endogenous MMP in an in vitro model for cutaneous loxoscelism, using cultured immortalized human keratinocytes. Furthermore, we also investigated the ability of tetracycline and its derivatives, doxycycline and minocycline, to inhibit the clinical symptoms of Loxosceles envenomation. We used the venom and recombinant SMaseD of L. intermedia as a model for SMaseD of various Loxosceles species in this study. Understanding the molecular mechanisms of action of Loxosceles venom will aid in the development of therapies to alleviate the clinical symptoms of spider envenomation. RESULTS L. intermedia venom and recombinant SMaseD P2 reduce cell viability

In order to analyze the toxic effects of Loxosceles venom or its main component, SMaseD, HaCaT cells were incubated with increasing concentrations of Loxosceles venom or recombinant SMaseD P2 (rP2) during 3 days and the cell viability was analyzed by the Alamar Blue method. Figure 1 shows that both venom and the recombinant SMaseD P2 (rP2) were able to induce loss of cell viability; the maximum percentage of death (60%) was reached at a concentration of 20 mg/ml. The recombinant SMaseD was more efficient in provoking the loss of cell viability than whole venom. The data show that SMaseD is a major and possibly unique enzyme responsible for HaCaT cell death induced by Loxosceles venom in vitro. Presence of MMPs in HaCaT cell supernatants

We have recently shown that in rabbits, Loxosceles SMaseD induces the expression of MMP-9, which is possibly a main factor involved in the pathogenesis of the cutaneous loxoscelism (Tambourgi et al., 2005). In order to investigate the possible association of cell death and MMP expression, supernatants recovered from HaCaT cells after 3 days of


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In order to further investigate the possible association of loss of cell viability and MMPs expression, HaCaT cells were treated with Loxosceles venom or rP2 (15 mg/ml) in the presence of increasing concentrations of tetracycline, doxycycline, or minocycline. Figure 3 shows that tetracycline at a concentration of 40 mg/ml could fully protect HaCaT cells from cell death induced by Loxosceles venom (Figure 3a). Doxycycline and minocycline were less effective in protecting cells from death induced by venom. The maximum protection, against the toxic effects of rP2, achieved by using tetracycline, doxycycline, or minocycline was 90% (Figure 3b). The tetracyclines, in the doses used, did not affect HaCaT cell viability (data not shown).

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treatment with venom or rP2 (15 mg/ml) were analyzed by gelatin zymography and Western blot. Figure 2a shows an increased expression/secretion, when compared to nontreated cells, of a band with a Mr around 68 kDa, corresponding to the active form of MMP-2, and a band with Mr around 90 kDa, corresponding to the active form of MMP-9 in the supernatants of cells treated with venom or rP2. A band around 72 kDa, corresponding to the proform of MMP-2, with the same intensity, was observed in treated and nontreated samples. The increased expression of MMP-2 and -9 in the supernatants of human HaCaT cells, treated with venom or rP2, was confirmed by Western blot using specific monoclonal antibodies (MoAbs) (Figure 2b and c) and ELISA (data not shown). In order to evaluate if inhibitors of metalloproteinases could modulate the expression of these gelatinases, cells were incubated with medium, venom, or rP2 in the presence or absence of tetracycline (50 mg/ml) and analyzed by Western blot. Figure 2b and c shows that tetracycline largely prevented secretion of MMP-2 and -9 by HaCaT cells.

Figure 2. Loxosceles venom SMaseD induces the expression of gelatinases in HaCaT cell line. Zymography analysis of HaCaT cell culture supernatants after 3 days of treatment with medium (C), 15 mg/ml of venom (V), or rP2 (a). The supernatants of HaCaT cell cultures treated with 15 mg/ml of venom (V) or rP2 or medium (C), in the presence or absence of tetracycline (50 mg/ml) for 3 days, were run on 12.5% SDS-PAGE gel under nonreducing conditions and Western blotted using MoAbs against human MMP-2 (b) or anti-MMP-9 (c).

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Figure 1. Effect of L. intermedia venom and rP2 treatment on human keratinocytes cell viability. HaCaT cell cultures (1 105 cells/ml) were incubated with increasing concentrations of Loxosceles venom (K) or rP2 (J), and after 3 days of treatment, cell viability was analyzed by the Alamar Blue assay. Results are representative of two independent experiments and are expressed as the mean of triplicates 7standard deviation. The asterisks indicate values statistically different (Po0.05) from the control.

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Figure 3. Effect of tetracycline, doxycycline, and minocycline on loss of cell viability of human keratinocytes induced by L. intermedia venom or rP2. HaCaT cells (1 105 cells/ml) were cultivated in 24-well plates in DMEM without FBS. At day 0, cells were treated with 15 mg/ml of venom (a) or rP2 (b) and simultaneously incubated with different concentrations of tetracycline (m), doxycycline (n), or minocycline (J). Three days of treatment, the cell viability was analyzed by the Alamar Blue assay. Results are representative of two independent experiments and are expressed as the mean of triplicates 7standard deviation. The asterisks indicate values statistically different (Po0.05) from the control.

inhibitor with the enzyme, thus reducing their ability to bind and act on the cell surface. In order to evaluate this possibility, HaCaT cells were treated with 15 mg/ml of venom or rP2 (2 hours at 371C) in the presence of tetracycline (50 mg/ ml). Cultures incubated with EDTA (10 mM) were also used as control, since we have previously shown that this agent could prevent SMaseD action and binding to cells, as the toxin is dependent on Mg2 þ for membrane association and lipase activity (Tambourgi et al., 1998, 2000; van Meeteren et al., 2004; Murakami et al., 2005). Binding of SMaseD to the cell surface was analyzed by flow cytometry using a specific antibody (anti-F35). Figure 4a shows that the association of SMases to the cell surface was impaired by the use of EDTA, but not by tetracycline. These results indicate that tetracycline, in the concentration used, did not interfere with the association of the toxins with the cell membrane.

Binding of SMaseD to HaCaT

The protection against the toxic effects of SMaseD from Loxosceles venom obtained with the use of tetracycline in keratinocyte cultures could be due to the interaction of this

Venom and rP2 effect on cell surface markers

We have previously shown that endogenous membranebound metalloproteinases activated by Loxosceles venom are www.jidonline.org

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Figure 4. Effect of L. intermedia venom and SMaseD on the expression of keratinocyte cell surface antigens. HaCaT cells (107 cells/ml) were treated with buffer (C), Loxosceles venom (V), or rP2 (15 mg/ml) for 2 hours at 371C in the presence or absence of tetracycline (50 mg/ml) or EDTA (10 mM). Binding of the SMaseD (a), using anti-F35 polyclonal antibody, and expression of MCP (b), b2-microglobulin (c), and EGFR (d), using specific MoAbs, were analyzed by flow cytometry. Results are representative of three different experiments expressed as the mean of duplicates7standard deviation.

responsible for the release of some cell surface proteins such as MCP, major histocompatibility complex I, and b2-microglobulin on human endothelial cells (van den Berg et al., 2002). We have also tested if venom and the recombinant SMaseD from L. intermedia spider could interfere with the expression of these membrane-bound cell surface molecules on HaCaT cells. By flow cytometry cell surface analysis, a reduction in the expression of MCP and b2microglobulin was detected after a 2-hour incubation of HaCaT with 15 mg/ml of Loxosceles venom or rP2 (Figure 4b and c). The EGF receptor (EGFR) plays an important role in wound healing. We show here that EGFR is also removed from the cell surface after incubation of the HaCaT cells with Loxosceles venom or rP2 (Figure 4d). Cleavage of all these cell surface molecules could be prevented by EDTA (10 mM), but not by tetracycline (50 mg/ml) (Figure 4b–d). Binding of Annexin V and propidum iodide

Detection of a reduction in the expression of EGFR caused by Loxosceles venom/SMaseD stimulus is novel and has important implications since it has been reported that reduction of the expression of this receptor is associated with the induction of apoptosis (Shao et al., 2003). In order to investigate if the removal of EGFR was associated with HaCaT cell death via apoptosis, cells incubated with venom or rP2 (15 mg/ml) were analyzed by flow cytometry, after staining with Annexin-FITC and propidium iodide (PI), or by DNA fragmentation. A significant increase in Annexin V-FITC and PI binding on the HaCaT cell surface (typical of late apoptosis) was observed after 24 hours of venom or rP2 treatment, as compared to control cells, incubated with 64


Dalbecco’s modified Eagle’s medium (DMEM) (Figure 5). The exposure of phosphatidylserine on the outside of the cell membrane was more evident after rP2 treatment. Induction of apoptosis was confirmed by DNA gel electrophoresis. DNA fragmentation was observed in samples obtained from cells treated with venom or rP2 (Figure 6). The same pattern of degradation was observed after incubation with hydrogen peroxide, a well-known inducer of apoptosis (Cardoso et al., 2004). Tetracycline prevented apoptosis in cells treated with venom rP2, but not with hydrogen peroxide (Figure 6). DISCUSSION Envenomation by Loxosceles spiders is a well-documented cause of necrotic skin lesions in humans. SMaseD is the primary enzyme in Loxosceles venom responsible for their major clinical effects in humans. We have previously shown that SMaseD induces in rabbit skin the expression of MMP-9, which was associated with the pathogenesis of the cutaneous loxoscelism (Tambourgi et al., 2005). The mammalian MMPs (also known as matrixins) belong to a family of zinc-dependent neutral secreted or surface-bound endopeptidases, which are involved in the degradation of the extracellular matrix, an important feature in many normal and abnormal biological conditions. MMPs have been identified to have a role in various physiological conditions such as fetal tissue development and postnatal tissue repair, and certain pathological conditions like periodontitis, autoimmune disorders of skin and dermal photoaging, rheumatoid arthritis, osteoarthritis, chronic ulcerations, uterine involution, wound healing, bone resorption, and


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Figure 5. Binding of Annexin V and PI to HaCaT treated with L. intermedia venom and rP2. HaCaT cell cultures, in DMEM without FBS, were treated with 15 mg/ml of venom or rP2 for 24 hours at 371C. As control, the cells were incubated with buffer. After incubation, cells were detached, washed in cold PBS, and incubated with Annexin V-FITC and PI as recommended by the manufacturer (R&D Systems Inc.) and analyzed by flow cytometry.

tumor progression and metastasis. Their activity can be regulated at four levels: induction of MMP genes, vesicle trafficking and secretion, activation of latent proforms, and complexing with specific tissue inhibitors of metalloproteinases (Acharya et al., 2004). Data we present here show that venom and the recombinant SMaseD P2 from L. intermedia spider induce an increased expression/secretion of MMP-2 and -9 in the keratinocytic cell line HaCaT. The augmentation of MMPs expression/secretion coincided with the reduction of cell viability, which occurred by apoptosis, since loss of membrane asymmetry and DNA degradation were detected in these cells. Several agents have been developed to block the synthesis of MMPs, prevent them from interacting with the molecules that direct their activities to the cell surface, or inhibit their enzymatic activity (Blavier et al., 1999; Egeblad and Werb, 2002). Inhibitors of MMPs can be broadly classified as being nonsynthetic (eg endogenous tissue inhibitors of metalloproteinases) or synthetic (eg collagen peptidomimetics, nonpeptidomimetics, bisphosphonates, tetracycline derivatives). The tetracycline derivatives inhibit not only the activity but also the production of MMPs and are thus being investigated for the treatment of disorders in which the MMP system is amplified (Golub et al., 1998; Greenwald et al., 1998). This family of agents comprises both the classic tetracycline antibiotics, such as tetracycline, doxycycline, and minocycline, and the newer tetracycline analogs that have been chemically modified to eliminate their antimicrobial activity. These agents inhibit the collagenases, MMP-1, -3, and -13, and the gelatinases, MMP-2 and -9, via multiple mechanisms (Ryan et al., 1996; Golub et al., 1998), including blocking the activity of mature MMPs by chelation of the zinc atom at the enzyme binding site, interfering with the proteolytic activation of pro-MMP into their active form, and reducing the expression of MMPs and protecting MMPs from proteolytic and oxidative degradation. Some tetracycline derivatives have been evaluated in preclinical cancer models and have entered early clinical trials in patients with malignant diseases. In our experimental model, the classic tetracycline antibiotics (tetracycline, doxycycline, and minocycline) modulated the toxic action of L. intermedia venom SMaseD

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Figure 6. DNA gel eletrophoresis. HaCaT cells, cultured in DMEM without FBS, were treated with 15 mg/ml of venom (V) or rP2 for 24 hours at 371C in the presence ( þ ) or absence of tetracycline (50 mg/ml). Cells incubated with PBS (C) and hydrogen peroxide (H2O2) (100 mM) were used as negative and positive controls, respectively. After incubation, cells were detached, washed in cold PBS, and the DNA was isolated using Trizol reagent and analyzed by agarose gel electrophoresis.

on HaCaT cells, inhibiting not only the increased expression/ secretion of MMP-2 and -9 but also the DNA degradation and binding of Annexin V, thus protecting cells from loss of cell viability. Loxosceles venom and SMaseD also induced a significant reduction in the expression of MCP, b2-microglobulin, and EGFR; however, tetracycline was unable to prevent the release of these proteins from the keratinocyte cell surface. We have previously shown that MCP was released into the supernatant as a truncated form from endothelial cell cultures, by activation of a metalloproteinase. This event could be inhibited by 1,10 phenanthroline, but not by phenylmethanesulfonyl fluoride or by the MMP-specific www.jidonline.org

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metalloproteinase inhibitor, tissue inhibitors of metalloproteinase-2, suggesting that MMPs were not involved and that the metalloproteinase activated on cell surface was a member of adamalysins (van den Berg et al., 2002). Taken together, these data indicate that two groups of metalloproteinases are induced/activated by Loxosceles, i.e., secreted MMPs which expression/secretion can be controlled by tetracyclines, and a membrane-associated metalloproteinase, possibly from the adamalysin family. Our observation that Loxosceles venom SMaseD also causes reduction in the expression of EGFR on the HaCaT cell surface is remarkable since EGFR signaling can be modulated during inflammatory processes, thereby affecting cell proliferation and thus having implications in wound repair. EGFR is a transmembrane protein with intrinsic tyrosine kinase activity, which transduces important signals from the surface of epithelial cells to the intracellular domain. In scar formation, EGFR expression is greater that in normal skin. The overexpression of EGFR contributes to the migration of cells from the ulcer margin and formation of granulation tissue and microvessels (angiogenesis) during the ulcer healing process (Cheng et al., 2002; Gibson, 2004; McGaffin et al., 2004; Satish et al., 2004). The difficult healing observed in the lesions induced by Loxosceles venom may be attributed to the decrease in EGFR expression. In conclusion, we have demonstrated that SMaseD from L. intermedia venom provokes apoptosis in the HaCaT immortalized keratinocyte cell line by an indirect process that involves an augmented expression and secretion of MMP-2 and -9. The discovery of the ability of metalloproteinase inhibitor drugs, such as tetracyclines, to control the noxious effects of Loxosceles venom associated with the dermonecrotic process is novel and of potential therapeutic significance. The use of this inhibitor in experimental in vivo models of cutaneous loxoscelism is the subject of further study. MATERIALS AND METHODS Chemicals, reagents, and buffers Tween 20, BSA, paraformaldehyde, gelatin, Triton X-100, tetracycline, doxycycline, minocycline, and EDTA were purchased from Sigma (St Louis, MO). 5-Bromo-4-chloro-3-indolyl-phosphate and nitroblue tetrazolium were from Promega Corp. (Madison, WI). Annexin V-FITC and PI were from R&D Systems Inc. (Minneapolis, MN). Trizol reagent was from Invitrogen Inc. (Burlington, Canada). Buffers were veronal-buffered saline, pH 7.4 (10 mM Na barbitone, 0.15 mM CaCl2, and 0.5 mM MgCl2), PBS, pH 7.2, 10 mM Na phosphate, 150 mM NaCl, and FACS buffer (PBS, 1% BSA, and 0.01% sodium azide).

Antibodies MoAbs against EGFR (Ab-10/111:16) were from Novacastra Labs (New Castle, UK) and those against human MMP-2 and -9 were from R&D Systems (Minneapolis, MN). MoAb against MCP (GB24) was kindly donated by Dr John Atkinson (St Louis, MO) and that against b2-microglobulin (b2-m/01) by Dr Vaclav Horejsi (Prague, Czech Republic). Rabbit polyclonal antibody against F35 (fraction containing SMaseD isoforms from L. intermedia venom) was made in-house (Tambourgi et al., 1995). Rabbit anti-mouse IgG-FITC (RAM-FITC) 66


and goat anti-rabbit IgG-FITC (GAR-FITC) were from Amersham Pharmacia Biotech (Buckinghamshire, UK). Goat anti-mouse IgGalkaline phosphatase (GAM-IgG-AP) was from Promega Corp.

Venom L. intermedia spiders were provided by ‘‘Laborato´rio de Imunoquı´mica, Instituto Butantan, SP, Brazil’’. The venom was obtained by electrostimulation by the method of Bucherl (1969), with slight modifications. Briefly, electric stimuli of 15–20 V were repeatedly applied to the spider sternum and the venom drops were collected with a micropipette, vacuum–dried, and stored at 20oC. Stock solutions were prepared in PBS at 1.0 mg/ml.

SMase expression Recombinant L. intermedia SMaseD named as P2 (accession number AY304472) was expressed in E. coli strain BL21 (DE3) as a fusion protein composed of the mature SMaseD with an N-terminal extension containing a 6 histidine tag and was purified as described (Tambourgi et al., 2004). rP2 was purified from the soluble fraction of cell lysates on an Ni (II) chelating sepharose fast flow column (Pharmacia). Recombinant proteins were eluted with the elution buffer (100 mM Tris-HCl, pH 8.0; 300 mM NaCl; 0.8 M imidazole) and dialyzed against PBS. The protein content of the samples was evaluated by the Lowry method (Lowry et al., 1951).

Cell culture and maintenance The human keratinocyte cell line, HaCaT, kindly donated by Dr Fusening (Heidelberg, Germany), was maintained in DMEM (GibcoBRL, Gaithersburg, MD), supplemented with 10% (vol/vol) heatinactivated (561C, 30 minutes) fetal bovine serum (FBS; Gibco-BRL), 100 IU/ml penicillin, and 100 IU/ml streptomycin at 371C in humidified air with 5% CO2. Institutional approval was not required since experiments were performed in vitro using immortalized cells.

Viability assay The cell viability was analyzed by Alamar Blue assay (Nakayama et al., 1997). Briefly, HaCaT cells were subcultured in 24-well plates in DMEM plus FBS at 1 105 cells/ml (0.5 ml/well). One day before starting the treatment, the cells were maintained overnight in DMEM without FBS, followed by incubation with different concentrations of venom or rP2 in the presence or absence of increasing amounts of the different metalloproteinases inhibitors, for example, tetracycline, minocycline, doxycycline, or EDTA diluted in DMEM without FBS. DMEM without FBS was used as the control. After 3 days of incubation, the culture supernatants were collected and the cells were incubated with 250 ml/well of DMEM plus 10% of Alamar Blue reagent (Biosource International, Camarillo, CA) for 1 hour at 371C. Alamar Blue added to wells not containing cells was used as the background control. Supernatants of each sample (100 ml) were collected and mixed with 100 ml of water and the absorbance was measured in a spectrophotometer (Multiskan-EX, Labsystems, Finland) at 540 and 620 nm. The relative cell viability was calculated as ½ðsample ODð5402620 nmÞ background control ODð5402620 nmÞ Þ/ ðcontrol ODð5402620 nmÞ background ODð540620 nmÞ Þ100: The Student’s t-test was used to determine the significance of the differences between the mean values of cell viability from control


and experimental samples. The minimal level of significance was considered as Po0.05.

Flow cytometry Analyses of the expression of cell surface antigens and binding of venom SMaseD to the keratinocytes were assessed by flow cytometry. HaCaT cells were harvested and incubated at 1 107 cells/ml in veronal-buffered saline with L. intermedia venom or rP2 (15 mg/ml, for 2 hours at 371C) in the presence or absence of tetracycline (50 mg/ml) or EDTA (10 mM). Cells were washed and resuspended in FACS buffer at 3 106 cells/ml. Fifty microliters of cells were incubated with 50 ml of MoAbs against MCP, b2m, or EGFR or with polyclonal serum against F35 (between 1 and 10 mg/ ml) and incubated for 30 minutes at 41C. The cells were washed three times and incubated with FITC-labeled secondary antibodies for 30 minutes at 41C. The cells were washed and fixed in FACS buffer containing 1% paraformaldehyde and analyzed by flow cytometry (FACScalibur, Becton Dickinson, CA). Analysis of Annexin V-FITC and PI cell staining was carried out after 24 hours of incubation of HaCaT cell cultures, in DMEM without FBS at 371C in humidified air with 5% CO2, with venom or rP2 (15 mg/ml). After incubation, cells were detached, washed in cold PBS, and incubated with Annexin V-FITC and PI as recommended by the manufacturer (R&D Systems Inc.). Cells incubated with DMEM were used as negative control.

Analysis of DNA fragmentation HaCaT cells were treated with venom or rP2 (15 mg/ml) for 24 hours and DNA was isolated using Trizol reagent (Invitrogen Inc.) according to the manufacturer’s instructions. After 5 minutes of incubation, chloroform was added and the samples were centrifuged at 12,000 r.p.m. for 15 minutes at 41C. The interface, containing DNA, was collected and the DNA precipitated, washed, and solubilized in water and analyzed by agarose gel electrophoresis. As positive and negative controls, DNA samples were obtained from cells treated with 100 mM of hydrogen peroxide or medium, respectively. Samples of DNA (10 mg/well) were loaded on a 1% agarose gel containing 1 mg/ml of ethidium bromide. The electrophoresis was performed for 1 hour at 90 V and the DNA was visualized by UV fluorescence.

Gelatin zymography Gelatinase activity was analyzed by zymography as described (Kleiner and Stetler-Stwenson, 1994). Supernatants from HaCaT cells, collected after 3 days of incubation with venom or rP2 (15 mg/ ml), were analyzed by gelatin zymography. Supernatants of cells incubated with DMEM were used as negative control. The supernatants were mixed with SDS-PAGE sample buffer and run on a 10% polyacrylamide gel containing 0.1% gelatin. The gels were washed twice for 30 minutes at room temperature in 2.5% Triton X-100, and incubated overnight at 371C in zymography buffer (50 mM Tris-HCl, 200 mM NaCl, 10 mM CaCl2, 0.05% Brij-35; pH 8.3). Gels were stained in Coomassie brilliant blue solution (40% methanol, 10% acetic acid, and 0.1% Coomassie brilliant blue).

Electrophoresis and Western blotting Supernatants of HaCaT cells incubated with DMEM, venom, or rP2 (15 mg/ml), in the presence or absence of tetracycline (50 mg/ml),

were run on 12.5% SDS-PAGE under nonreducing conditions. Gels were blotted onto nitrocellulose and the membranes were blocked with PBS/5% BSA and incubated with mAb against human MMP-9 and anti-MMP-2 (10 mg/ml) for 1 hour at 371C. Membranes were washed three times with PBS/0.05% Tween 20 for 5 minutes and then incubated with GAM-IgG-AP (1/5,000) in PBS/5% BSA for 1 hour at 371C. After washing three times with PBS/0.05% Tween 20 for 10 minutes, blots were developed using nitroblue tetrazolium/ 5-bromo-4-chloro-3-indolyl-phosphate according to the manufacturer’s instructions (Promega). CONFLICT OF INTEREST The author states no conflict of interest.

ACKNOWLEDGMENTS This research was supported by grants from FAPESP, CNPq, CAT/CEPID/ Butantan, and The Wellcome Trust.

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