- Email: [email protected]
Inflammatory mediators generated at the site of inoculation of Loxosceles gaucho spider venom
Toxicon 56 (2010) 972–979
Contents lists available at ScienceDirect
Toxicon journal homepage: www.elsevier.com/locate/toxicon
Inflammatory mediators generated at the site of inoculation of Loxosceles gaucho spider venom Katia C. Barbaro a, *, Marcela S. Lira a, Claudia A. Araújo a, Alessandra Pareja-Santos b, Bianca C.L.F. Távora a, José Pedro Prezotto-Neto a, Louise F. Kimura a, Carla Lima b, Mônica Lopes-Ferreira b, Marcelo L. Santoro c a b c
Laboratory of Immunopathology, Institute Butantan, Av. Vital Brasil 1500, 05503-900 São Paulo, SP, Brazil Special Laboratory of Applied Toxinology (CEPID), Institute Butantan, Av. Vital Brasil 1500, 05503-900 São Paulo, SP, Brazil Laboratory of Pathophysiology, Institute Butantan, Av. Vital Brasil 1500, 05503-900 São Paulo, SP, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history: Received 24 February 2010 Received in revised form 24 June 2010 Accepted 28 June 2010 Available online 6 July 2010
Patients bitten by Loxosceles spiders generally manifest marked local inflammatory reaction and dermonecrosis. This report evaluated edema formation, leukocyte infiltration and release of inflammatory mediators at the injection site of Loxosceles gaucho venom. BALB/c mice were i.d. injected with venom and thereafter paws were disrupted and homogenized to obtain differential counts of migrated cells, as well to assay the levels of cytokines, chemokines and lipid mediators. Increased footpad thickness was detected as soon as 30 min after venom injection, and 24 h later was similar to that of the control group. Loxosceles venom mildly augmented the recruitment of leukocytes to the footpad in comparison with PBS-injected mice. Moreover, it stimulated the release of IL-6, MCP-1 and KC at 2 and 24 h after venom injection. In addition, higher levels of PGE2 were detected 30 min after venom injection in comparison with control group. However, the venom failed to increase levels of IL-1b, TNF-a, TXB2 and LTB4. Our results demonstrate that L. gaucho venom evokes an early complex inflammatory reaction, stimulating the secretion of pro-inflammatory cytokines and lipid mediators (PGE2), and recruiting leukocytes to the footpad which contribute to the local reaction induced by L. gaucho venom. Ó 2010 Elsevier Ltd. All rights reserved.
Keywords: Spider venom Loxosceles Loxoscelism Brown spider Local reaction Inflammatory mediators
1. Introduction Loxoscelism is a syndrome caused in humans by bites from the brown spider Loxosceles spp. In general, the clinical manifestations are characterized by inflammation and
Abbreviations: IL-1b, interleukin 1 beta; IL-6, interleukin 6; IL-8, interleukin 8; TNF-a, tumor necrosis factor alpha; MCP-1, monocyte chemoattractant protein-1; KC, keratinocyte chemoattractant; TXB2, thromboxane B2; LTB4, leukotriene B4; PGE2, prostaglandin E2; RANTES, regulated upon activation, normal T-cell expressed, and secreted; GMCSF, granulocyte macrophage colony-stimulating factor; GRO-a, growth regulated oncogene-alpha. * Corresponding author. Tel.: þ55 11 37267222x2278/2134; fax: þ55 11 37261505. E-mail addresses: [email protected], [email protected] (K.C. Barbaro). 0041-0101/$ – see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.toxicon.2010.06.022
dermonecrosis at the site of the bite, although in some cases systemic hemolysis and coagulopathy are present, leading to acute renal failure (Hogan et al., 2004; Abdulkader et al., 2008). In Brazil, eight Loxosceles species are found, however, Loxosceles gaucho, Loxosceles laeta and Loxosceles intermedia are commonly implicated in accidents reported in humans (Barbaro and Cardoso, 2003), which occur mainly in the southern and southeastern regions of the country. In 2006, Loxosceles spiders were responsible for approximately 40% of 19,105 notified cases of spider envenomation reported to the Brazilian Ministry of Health (SINAN, 2006). Loxosceles venoms are rich source of proteases, hydrolases, lipases, peptidases, collagenases, alkaline phosphatases, 5-ribonucleotidases, phosphohydrolases and other components. A family of phospholipases D has been mainly
K.C. Barbaro et al. / Toxicon 56 (2010) 972–979
implicated in the genesis of dermonecrosis that occurs after the bite (Hogan et al., 2004; Silva et al., 2004; Barbaro et al., 2005; Kalapothakis et al., 2007). Venom components interact with the cellular membrane, degrade components of extracellular matrix, take part in complement system activation and recruit polymorphonuclear leukocytes and platelets, among others, thus contributing to the establishment of local injury (Futrell, 1992; Hogan et al., 2004; Silva et al., 2004). Such venom components may be important to induce an early inflammatory reaction, eliciting the release of many endogenous pro-inflammatory mediators that contribute to the development of the lesion (Hogan et al., 2004). Loxosceles venoms have been demonstrated to stimulate cytokine production. Patel et al. (1994) showed that Loxosceles reclusa venom stimulated the production of IL-8 and the secretion of GM-CSF by endothelial cells, the migration of neutrophils to the injection site, and the release of granule content after neutrophil adhesion. Moreover, Málaque et al. (1999) observed that L. gaucho venom causes alterations in primary cultures of keratinocytes and stimulates TNF-a production. In addition, Loxosceles deserta has been shown to stimulate the expression of vascular endothelial growth factor (VEGF) in human keratinocyte culture (Desai et al., 2000). Induction of release of chemokines (IL8, GRO-a, MCP-1, RANTES) by endothelial and epithelial cells has also been implicated in the pathogenesis of loxoscelism (Gomez et al., 1999). Recently, Souza et al. (2008) reported high levels of IL-6 and TNF-a in a patient bitten by Loxosceles spp. spider. Thus, based on these findings, a number of cytokines and chemokines seem to contribute to the pathogenesis of dermonecrotic arachnidism. The poor efficiency of current strategies (serotherapy and corticoid administration) to treat loxoscelism in Brazil is attributed to at least two causes. One of them is the rapid tissue injury that occurs after Loxosceles envenomation (Barbaro and Cardoso, 2003; Hogan et al., 2004; Silva et al., 2004). The other is the delayed medical assistance, since patients seek medical care usually 12–24 h after the spider bite (Barbaro et al., 1992b; Málaque et al., 2002). Thus, the effectiveness of loxoscelism treatment should take into consideration both the intrinsic venom toxic activity and the local inflammatory reaction that develops after envenomation. Loxosceles venom induces dermonecrosis in rabbits, guinea pigs and humans but not in mice (Futrell, 1992; Barbaro et al., 1996a,b; Domingos et al., 2003; Hogan et al., 2004). However, Loxosceles venom causes an important inflammatory reaction at the injection site and, depending on the dose, it can be lethal to animals (Barbaro et al., 1994). Domingos et al. (2003) demonstrated that the size and availability of local sphingomyelin may be important in determining the outcome of Loxosceles envenomation in different mammalian species. Since we are interested to verify the inflammatory reaction in the absence of dermonecrosis, in the present work we investigated the effect of L. gaucho venom in evoking an acute inflammatory response by means of the evaluation of edema formation and the release of inflammatory mediators (cytokines, chemokines and lipid mediators) after injection of L. gaucho venom in mouse paws.
973
2. Materials and methods 2.1. Animals and venom BALB/c mice (male, 18–20 g) were provided by the Butantan Institute animal house. The procedures involving animals were conducted according to national laws and policies controlled by Butantan Institute Animal Investigation Ethical Committee (protocol n 316/06). Specimens of adult L. gaucho spiders were collected in São Paulo State, Brazil. The spiders were kept in quarantine for 1 week without food before venom extraction. Venoms were obtained as previously described (Barbaro et al., 1992a). Protein concentrations were determined by the bicinchoninic acid (BCA) assay (Smith et al., 1985). Standard curves were constructed using bovine serum albumin (Sigma Chemicals, St Louis, MO, USA) diluted in duplicate. 2.2. Experimental model Mice (n ¼ 4–8) were injected (30 mL) in the right hind paw with L. gaucho venom (3, 5, or 10 mg), PBS (negative control), or lipopolysaccharide (LPS) from Escherichia coli (1 mg, Sigma Chemicals, St Louis, MO, USA, positive control). Edema-forming activity was studied at three time intervals (0.5, 2 and 24 h), and thereafter the right paws were removed at the level of the tibiotarsal joint, disrupted and homogenized. Following centrifugation (400 g) at 4 C for 10 min, supernatants were recovered to assay the release of cytokines, chemokines and lipid mediators by enzymatic immunoassay. Cell pellets were recovered to perform cell counts. 2.3. Edema measurement after footpad injection Mice were injected (30 mL) in the footpad with different doses of L. gaucho venom or PBS. Edema formation was measured using a plethysmographer (Ugo Basile, IT). Results expressed the difference in paw volume (mL) prior to (control) and after (experimental) injection. Edema was measured at different time points (0.5, 2 and 24 h) after injection. 2.4. Leukocyte recruitment to footpads Total leukocyte counts from footpad homogenates were performed by Trypan blue exclusion (Sigma–Aldrich, USA) using a hemocytometer; differential counts were accomplished using cytocentrifuge slides stained with HEMA-3 (Fischer Scientific Company, MI, USA). For differential cell counts, 100 leukocytes were enumerated and identified as macrophages, lymphocytes or neutrophils, based on staining and morphologic features. 2.5. Assays for cytokines, chemokines and lipid mediators Levels of cytokines (IL-1b, IL-6 and TNF-a), chemokines (KC and MCP-1) and lipid mediators (LTB4, PGE2 and TXB2) were measured in supernatants of paw tissue extracts (as described in section 2.2) or in supernatants of murine
974
K.C. Barbaro et al. / Toxicon 56 (2010) 972–979 35 *
30
* *
25
*
*
Edema (μL)
PBS 3 µg 5 µg 10 µg
20
*
15 10
5 β
0 2
0.5
24
Time (hours)
Fig. 1. Edematogenic activity (mean S.D.) of L. gaucho venom in mice (n ¼ 4–8). Edema (in mL) was calculated by the difference between the volume of hind paws after injection of L. gaucho venom (at 0.5, 2 and 24 h) and the volume before injection (control). *p < 0.05, in relation to the control group (PBS). b: Animals died before 24 h.
dendritic cells stimulated with LPS (1 mg), using commercial specific two-site sandwich enzyme linked immunosorbent assays (ELISA), according to manufacturer’s instructions (IL-1b, IL-6, TNF-a, MCP-1: B&D Biosciences, CA, USA; KC: R&D Systems, MN, USA; LTB4, PGE2 and TXB2: Cayman Chemicals, MI, USA). Samples were quantified using standard curves of eicosanoids, and recombinant murine cytokines and chemokines. Results were expressed as mean S.D. for duplicate samples. 2.6. Histopathological analyses L. gaucho venom (3, 5, or 10 mg) or PBS was injected in the right paw of mice. At 0.5 and 2 h the right paws of mice were removed and fixed in 4% paraformaldehyde in PBS 0.1 M, pH 7.2 for 24 h. The paw was serially sectioned and the samples were dehydrated in ethanol and embedded in paraffin. Sections of 6–7 mm were obtained in a Microm HM340 microtome and stained with hematoxylin–eosin.
soon as 30 min after venom injection (5 and 10 mg), reaching an approximately 3-fold increase with 10 mg venom at 0.5 and 2 h. At 24 h, the total cell count (10 mg group) was still approximately 1.8-fold higher than that of the PBS group. The differential count of migrated cells into the paw tissue was also analyzed, and showed an increase in macrophages, lymphocytes and neutrophils as soon as 0.5 h in animals injected with 5 and 10 mg of L. gaucho venom (Fig. 2B–D). The recruitment of inflammatory cells is still augmented, more than 2-fold, when compared with the control group at 2 h after venom injection. At 24 h, only lymphocyte counts returned to basal levels. By histological analysis, the presence of edema was observed as soon as 0.5 h after venom injection in all doses tested (Fig. 3), and inflammatory infiltrates were scant. After 2 h, besides edema, an increase in inflammatory infiltrate was observed mainly in 5 and 10 mg doses. L. gaucho venom stimulated the secretion of proinflammatory cytokines. The levels of IL-6 increased at 2 h and 24 h after venom injection (Fig. 4). No difference was observed in IL-1b and TNF-a levels between experimental and PBS groups for all venom doses and time intervals tested (data not shown). However, LPS, an agent known to increase the production of IL-1b and TNF-a, did stimulate the secretion of these mediators in the foot paws and in dendritic cells culture (data not shown). The levels of both chemokines, MCP-1 and KC, were significantly increased 2 h after venom injection (Figs. 5 and 6); after 24 h, the levels of these chemokines were remarkably higher (about 10-fold) when compared with PBS group. In order to determine the participation of lipid mediators in the inflammatory reaction, the levels of PGE2, LTB4 and TXB2 were also assayed. Fig. 7 shows that L. gaucho venom induced a high PGE2 release from as soon as 30 min to 2 h after venom injection; the levels of PGE2 remained increased in animals injected with 5 mg of venom at 24 h after venom injection. No increase in TXB2 or LTB4 levels were observed when compared with the PBS group in all samples tested (data not shown). 4. Discussion
2.7. Statistical analyses Results were expressed as means SD. One-way or two-way ANOVA, followed by Bonferroni test, was used to analyze data, employing SigmaStat 3.5 software. Values with p < 0.05 were considered statistically significant. 3. Results In order to determine the ability of L. gaucho venom to evoke edema at the site of venom administration, mice were injected i.d. in the footpad with either venom alone or vehicle (PBS). The results showed that at 0.5 and 2 h after injection, edema was statistically significant in venomtreated groups in comparison with PBS group (Fig. 1). No statistical difference in paw volume was observed 24 h after injection between venom (all doses) and PBS groups. Our results demonstrated that L. gaucho injection evokes a complex inflammatory reaction with leukocyte recruitment to the footpad (Fig. 2A), which is observed as
Although some symptoms of envenomation caused by spiders from Loxosceles genus are well characterized, e.g. formation of dermonecrotic lesions, several caveats are still unanswered about by which mechanisms L. gaucho venom could trigger an acute immune response after the bite. Herein we studied soluble pro-inflammatory mediators likely involved in the development of the inflammatory response induced by L. gaucho venom, and we show that this venom differentially triggers the release of some pivotal inflammatory mediators, such as IL-6, MCP-1, KC, PGE2, but not of other ones (IL-1b, TNF-a, LTB4, TXB2). This report is the first to demonstrate that the inflammatory response induced by L. gaucho venom in mouse [an animal species that does not manifest the classical dermonecrotic effect induced by Loxosceles venom (Barbaro et al., 1996a; Domingos et al., 2003)] is not triggered by classical proinflammatory mediators such as IL-1b, TNF-a, LTB4, TXB2. Preliminary studies showed that edema decreased rapidly and was similar to that of the animals injected with
K.C. Barbaro et al. / Toxicon 56 (2010) 972–979
B 100
cells/mL (x 105)
80
*
* *
*
60
PBS 3 µg 5 µg 10 µg
*
40 20
50
0 0.5
2 Time (hours)
PBS 3 µg 5 µg 10 µg
* *
8 *
D Neutrophils (x 105)
Lymphocytes (x 105)
10
40
* *
*
6 4
*
30
*
20
*
10 0
24
12
PBS 3 µg 5 µg 10 µg
*
Macrophages (x 105)
A
C
975
0.5
2 Time (hours)
24
50 *
40 30
*
* *
*
PBS 3 µg 5 µg 10 µg
*
20 10
2
β
0
0 0.5
2 Time (hours)
24
0.5
2 Time (hours)
24
Fig. 2. Effect of L. gaucho venom in leukocyte recruitment to mouse footpads (n ¼ 4–8). After 0.5, 2 and 24 h of venom injection (3, 5 or 10 mg), animals were euthanized, and the footpad tissue was processed for counting of total leukocytes (A), macrophages (B), lymphocytes (C) and neutrophils (D). Mice only injected with PBS were considered as the control group. The results represent the mean S.D. *p < 0.05 compared with control-group. b: Animals died before 24 h.
PBS at 8 h after venom injection (data not shown), suggesting that L. gaucho venom induces a rapid inflammatory reaction in mouse footpads. Based on these findings, we investigated the acute inflammation at early time point (0.5 and 2 h), as well as at 24 h, when edema was no more evident. The experimental model established herein used venom injection into footpads rather than into the peritoneal cavity – the most used model to test inflammatory effects of venoms – in order to mimic human envenomation caused by Loxosceles spp. In mice injected with L. gaucho venom, the peak of edema occurred at 0.5 h, decreasing steadily thereafter. The velocity of edema induction in mice differs from that observed in human envenomation, where the inflammatory reaction begins more slowly (6–24 h) after the bite and takes many days to decrease (Barbaro et al., 1992b; Málaque et al., 1999; Barbaro and Cardoso, 2003). Mast cells have been reported to play an important role in edema formation. Rattmann et al. (2008) showed that L. intermedia venom degranulates mast cells, releasing histamine and serotonin. Furthermore, Paludo et al. (2009) showed that L. intermedia venom also contains histamine that, in association with venom phospholipase-D, can induce mast cell degranulation (Rivera and Gilfillan, 2006), contributing to local inflammatory events. Histopathologically, an inflammatory cell infiltrate was observed in footpads injected with L. gaucho venom. At the
site of the injury, leukocyte recruitment is a crucial event to initiate the immune response against the insulting agent, such as toxins and pathogens. Macrophages were increased at all time intervals, but lymphocytes were found only at the initial intervals. Neutrophils play a crucial role on Loxosceles dermonecrotic lesions (Smith and Micks, 1970; Rees et al., 1984; Ospedal et al., 2002; Silva et al., 2004; Hogan et al., 2004). In our experiments, the number of migrated neutrophils increased as soon as 30 min of L. gaucho venom injection, demonstrating the importance of these cells in eliciting the local reaction. In agreement with our results, neutrophils and mononuclear cells were the predominant cells in peripheral blood at 2 and 4 h after Androctonus australis hector venom injection (Adi-Bessalem et al., 2008). Neutrophil recruitment was also found in experimental models using Bothrops spp. venoms (Lomonte et al., 1993; Farsky et al., 1997). Edema induced by L. gaucho venom was noticed to decrease rapidly in murine hind paws. This profile is typical of L. gaucho venom, since edema induced by centipede (Malta et al., 2008) or stingray (Barbaro et al., 2007) venoms persists up to 48 h. In addition, we could detect cellular infiltrate, mainly macrophages and neutrophils, into the foot paws after 24 h of L. gaucho venom injection, indicating that a local inflammatory response is still present, despite the absence of swelling. This might explain why inflammatory mediators such as IL-6, KC, MCP-1, and PGE2 are still found into the foot paws.
976
K.C. Barbaro et al. / Toxicon 56 (2010) 972–979
Fig. 3. Micrographs of mouse paw tissues after L. gaucho venom injection. Mice were injected with PBS or L. gaucho venom (3, 5 or 10 mg) and the histological analysis was performed at 0.5 and 2 h. Hematoxylin–eosin stain. Bar size ¼ 50 mm.
K.C. Barbaro et al. / Toxicon 56 (2010) 972–979 1000
IL-6 (pg/mL)
PBS 3 µg 5 µg 10 µg
* 100
*
*
* *
β 10 0.5
24
2
Time (hours) Fig. 4. Analysis of IL-6 levels in footpad of mice (n ¼ 4–8) injected with L. gaucho venom. After 0.5, 2 and 24 h of venom injection (3, 5 or 10 mg), footpad homogenates were collected for IL-6 assay. The results represent the mean S.D. *p < 0.05 compared with the control group (PBS). b: Animals died before 24 h.
In addition to the recruitment of inflammatory cells, our results suggested that pro-inflammatory mediators (IL-6, MCP-1, KC, PGE2) could participate in the development of the tissue injury in L. gaucho envenomation. However, no increase was noticed in IL-1b, TNF-a, TXB2 and LTB4 production. Pro-inflammatory mediators are primarily responsible for initiating an effective immune response against toxins and pathogens, but, the overproduction of these mediators contributes to shock, multiple organ failure and death (Van der Meide and Schellekens, 1996). Several cytokines have been involved in severe envenomation, e.g. TNF-a, IL-1b and IL-6. TNF-a has been reported to be a crucial mediator in the pathogenesis of various envenomations (Petricevich, 2004). The role of IL-6 is controversial, since it has either pro- or anti-inflammatory properties (Barton, 1997). As a down-regulator of inflammatory responses, IL-6 can inhibit the production of IL-1b and TNF-a by increasing, respectively, the synthesis of IL10000 PBS 3 µg 5 µg 10 µg
MCP-1 (pg/mL)
*
*
1000
*
*
*
100
β
10 0.5
2
24
Time (hours) Fig. 5. MCP-1 levels in footpad homogenates of mice (n ¼ 4–8) injected with L. gaucho venom. After 0.5, 2 and 24 h of venom injection (3, 5 or 10 mg), footpad homogenates were collected for MCP-1 assay by ELISA. The results represent the mean S.D. *p < 0.05 compared with the control group (PBS). b: Animals died before 24 h.
977
1Ra and soluble TNF receptor p55 (Jones, 2005). The origin of IL-6 secretion induced by L. gaucho venom at the inflammatory site has not been elucidated yet. In addition, high levels of PGE2 produce potent vasodilation and suppress TNF-a and IL-1b production. Thus, the absence of detectable levels of IL-1b and TNF-a at the site of the injury might inhibit the effects of IL-6 and PGE2, retarding perhaps an exaggerated inflammatory response and delaying thereby tissue necrosis in the host. In order to ensure that the absence of IL-1b and TNF-a in L. gaucho-injected mice is a differential feature of this venom, the supernatants of LPS-injected mice or of LPSstimulated mouse dendritic cells were tested as a positive control in the cytokine assays. We found that LPS stimulated the production of IL-6, IL-1b and TNF-a (data not shown), suggesting that L. gaucho stimulates the secretion of IL-6, but not of IL-1b and TNF-a. Furthermore, in agreement with our data, L. gaucho venom alone was also shown to be unable to induce TNF-a release by J774A.1 cells, a murine macrophage lineage (Domingos et al., 2003). Besides, the intramuscular injection of Bothrops asper snake venom was unable to change TNF-a production (Chaves et al., 2005) showing that each animal venom might stimulate a different cytokine profile. Chemokines have a pivotal role in the selective induction and amplification of the inflammatory response, by favoring leukocyte migration by means of activation of Gprotein-coupled receptors, and expression of adhesion molecules and glycosaminoglycans (Rossi and Zlotnik, 2000). In addition, they increase the affinity of leukocyte integrins to their ligands on the vascular wall during diapedesis, and regulate polymerization and depolymerization of actin in leukocytes for their movement and migration toward the inflammatory site (Luo et al., 2007). MCP-1, also known as CCL2, is a potent chemoattractant to monocytes and T cells, and binds to CCR2 expressed by these cells (Melgarijo et al., 2009), and might be involved in monocyte and lymphocyte migration to the site of injection of L. gaucho venom. This might be also due to the high production of PGE2, which has been demonstrated to recruit monocytes to the site of inflammation and promote their differentiation to macrophages (Kurth et al., 2001). KC (homologue to IL-8 or CXCL8 in humans) promotes neutrophil chemotaxis and degranulation by binding to the receptors CXCR1 or, preferentially, CXCR2. The blockage of CXCR1 or CXCR2 has been shown to inhibit the neutrophil influx in inflammation (Souza et al., 2004; Cugini et al., 2005), suggesting a critical role of KC in recruiting neutrophils. As we demonstrated, the kinetics of KC is associated with neutrophil migration to the footpads. However, neutrophils have also been found in footpads as soon as 30 min after L. gaucho venom, which might be attributed to PGE2 release. In fact, the association between PGE2 secretion and neutrophil influx has been demonstrated following injection of BaP1, a metalloproteinase from B. asper venom, which stimulates PGE2 and cell influx, mostly neutrophils, in mouse joints (Fernandes et al., 2007). Moreover, the absence of LTB4 in our model might be due to the production of PGE2, since this eicosanoid was found to induce IL-6 production in macrophages (Bagga et al., 2003) and to inhibit 5-lipooxygenase (5-LOX),
978
K.C. Barbaro et al. / Toxicon 56 (2010) 972–979
Acknowledgements This paper was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP grant 06/564954). The authors thank Mr Santos-Neto M.O. for technical assistance. We also thank CNPq for the grant to K.C.B. (304800/2007-4). IBAMA provided animal collection permission no. 15383-2 and CGEN provided the license for genetic patrimony access (02001.005110/2008). Conflict of interest statement The authors declare that there are no conflicts of interest. Fig. 6. KC levels in footpad homogenates of mice (n ¼ 4–8) injected with L. gaucho venom. After 0.5, 2 and 24 h of venom injection (3, 5 or 10 mg), footpad homogenates were collected for KC assay by ELISA. The results represent the mean S.D. *p < 0.05 compared with the control group (PBS). b: Animals died before 24 h.
decreasing consequently LTB4 production (Levy et al., 2001). Furthermore, the absence of TXB2 release might be associated with the absence of IL-1b in our model. IL-1b has been shown to stimulate COX2 expression in several types of cells, including macrophages, up-regulating the release of PGE2 and TXB2 (Dinarello, 2009). Taken together, our results show that L. gaucho venom elicits marked PGE2 release, and in association with IL-6, MCP-1 and KC secretion, induce vascular leakage (edema) and leukocyte migration to the site of injection. These findings suggest that these mediators contribute to the inflammatory reaction induced by L. gaucho venom in mouse footpad, and help to understand the pathophysiology of loxoscelism. Thus, novel approaches for alternative treatments, such as anti-inflammatory drugs, might be useful to diminish the local lesion caused by Loxosceles venom.
Fig. 7. Prostaglandin E2 (PGE2) release in the footpad of mice after L. gaucho venom injection. After 0.5, 2 and 24 h of venom injection (3, 5 or 10 mg), animals were sacrificed and the footpads were processed in order to obtain footpad homogenate fluid for PGE2 quantification by ELISA (n ¼ 4–8). The results represent the mean S.D. *p < 0.05 compared with the control group (PBS). b: Animals died before 24 h.
References Abdulkader, R.C.R.M., Barbaro, K.C., Barros, E.J.G., Burdmann, E.A., 2008. Nephrotoxicity of insect and spider in Latin America. Sem. Nephrol. 28, 373–382. Adi-Bessalem, S., Hammoudi-Triki, D., Laraba-Djebari, F., 2008. Pathophysiological effects of Androctonus australis hector scorpion venom: tissue damages and inflammatory response. Exp. Toxicol. Pathol. 60, 373–380. Bagga, D., Wang, L., Farias-Eisner, R., Glaspy, J.A., Reddy, S.T., 2003. Differential effects of prostaglandin derived from omega-6 and omega-3 polyunsaturated fatty acids on COX-2 expression and IL-6 secretion. Proc. Natl. Acad. Sci. U.S.A. 100, 1751–1756. Barbaro, K.C., Cardoso, J.L.C., Eickstedt, V.R.D., Mota, I., 1992a. Dermonecrotic and lethal components of Loxosceles gaucho spider venom. Toxicon 30, 331–338. Barbaro, K.C., Cardoso, J.L.C., Eickstedt, V.R.D., Mota, I., 1992b. IgG antibodies to Loxosceles gaucho venom in human envenoming. Toxicon 30, 1117–1121. Barbaro, K.C., Eickstedt, V.R.D., Mota, I., 1994. Antigenic cross-reactivity of venoms from medically important Loxosceles (Araneae) species in Brazil. Toxicon 32, 113–120. Barbaro, K.C., Ferreira, M.L., Cardoso, D.F., Eickstedt, V.R.D., Mota, I., 1996a. Identification and neutralization of biological activities in the venoms of Loxosceles spiders. Braz. J. Med. Biol. Res. 29, 1491–1497. Barbaro, K.C., Sousa, M.V., Morhy, L., Eickstedt, V.R.D., Mota, I., 1996b. Compared chemical properties of dermonecrotic and lethal toxins from spiders of the genus Loxosceles (Araneae). J. Prot. Chem. 15, 337– 343. Barbaro, K.C., Cardoso, J.L.C., 2003. Mecanismo de ação do veneno de Loxosceles e aspectos clínicos do loxoscelismo. In: Cardoso, J.L.C., França, F.O.S., Wen, F.H., Málaque, C.M.S., Haddad Jr., V. (Eds.), Animais Peçonhentos no Brasil: Biologia, Clínica e Terapêutica dos acidentes. Savier, São Paulo, pp. 160–174. Barbaro, K.C., Knysak, I., Martins, R., Hogan, C., Winkel, K., 2005. Enzymatic characterization, antigenic cross-reactivity and neutralization of dermonecrotic activity of five Loxosceles spider venoms of medical importance in the Americas. Toxicon 45, 489–499. Barbaro, K.C., Lira, M.S., Malta, M.B., Soares, S.L., Garrone Neto, D., Cardoso, J.L.C., Santoro, M.L., Haddad Junior, V., 2007. Comparative study on extracts from the tissue covering the stingers of freshwater (Potamotrygon falkneri) and marine (Dasyatis guttata) stingrays. Toxicon 50, 676–687. Barton, B.E., 1997. IL-6: insights into novel biological activities. Clin. Immunol. Immunopathol. 85, 16–20. Chaves, F., Teixeira, C.F., Gutierrez, J.M., 2005. Role of TNF-alpha, IL-1beta, IL-6 in local tissue damage induced by Bothrops asper venom: an experimental assessment in mice. Toxicon 45, 171–178. Cugini, D., Azzolini, N., Gagliardini, E., Cassis, P., Bertini, R., Colotta, F., Noris, M., Remuzzi, G., Benigni, A., 2005. Inhibition of the chemokine receptor CXCR2 prevents kidney graft function deterioration due to ischemia/ reperfusion. Kidney Int. 67, 1753–1761. Desai, A., Lankford, H.A., Warren, J.S., 2000. Loxosceles deserta spider venom induces the expression of vascular endothelial growth factor (VEGF) in keratinocytes. Inflammation 24, 1–9. Dinarello, C.A., 2009. Immunological and inflammatory functions of the interleukin-1 family. Ann. Rev. Immunol. 27, 519–550. Domingos, M.O., Barbaro, K.C., Tynan, W., Penny, J., Lewis, D.J., New, R.R., 2003. Influence of sphingomyelin and TNF-a release on lethality and
K.C. Barbaro et al. / Toxicon 56 (2010) 972–979 local inflammatory reaction induced by Loxosceles gaucho spider venom in mice. Toxicon 42, 471–479. Farsky, S.H., Walber, J., Costa-Cruz, M., Cury, Y., Teixeira, C.F., 1997. Leukocyte response induced by Bothrops jararaca crude venom: in vivo and in vitro studies. Toxicon 35, 185–193. Fernandes, C.M., Teixeira, C.F.P., Gutierrez, J.M., Rocha, F.A., 2007. The snake venom metalloproteinase BaP1 induces joint hypernociception through TNF-a and PGE2 dependent mechanisms. Br. J. Pharmacol. 151, 1254–1261. Futrell, J., 1992. Loxoscelism. Am. J. Med. Sci. 304, 261–267. Gomez, H.F., Miller, M.J., Desai, A., Warren, J.S., 1999. Loxosceles spider venom induces the production of alpha and beta chemokines: implications for the pathogenesis of dermonecrotic arachnidism. Inflammation 23, 207–215. Hogan, C.J., Barbaro, K.C., Winkel, K., 2004. Loxoscelism: old obstacles, new directions. Ann. Emerg. Med. 44, 608–624. Jones, S.A., 2005. Directing transition from innate to acquired immunity: defining a role for IL-6. J. Immunol. 175, 3463–3468. Kalapothakis, E., Chatzaki, M., Gonçalves-Dornelas, H., de Castro, C.S., Silvestre, F.G., Laborne, F.V., de Moura, J.F., Veiga, S.S., ChávezOlórtegui, C., Granier, C., Barbaro, K.C., 2007. The Loxtox protein family in Loxosceles intermedia (Mello-Leitão) venom. Toxicon 50, 938–946. Kurth, I., Willimann, K., Schaerli, P., Hunziker, T., Clark-Lewis, I., Moser, B., 2001. Monocyte selectivity and tissue localization suggests a role for breast and kidney-expressed chemokine (BRAK) in macrophage development. J. Exp. Med. 194, 855–861. Levy, B.D., Clish, C.B., Schmidt, B., Gronert, K., Serhan, C.N., 2001. Lipid mediator class switching during acute inflammation: signals in resolution. Nat. Immunol. 7, 612–619. Lomonte, B., Tarkowski, A., Hanson, L.A., 1993. Host response to Bothrops asper snake venom. Analysis of edema formation, inflammatory cells, and cytokine release in a mouse model. Inflammation 17, 93–105. Luo, B.H., Carman, C.V., Springer, T.A., 2007. Structural basis of integrin regulation and signaling. Ann. Rev. Immunol. 25, 619–647. Málaque, C.M.S., Ori, M., Santos, S.A., Andrade, D.R., 1999. Production of TNF-a by primary cultures of human keratinocytes challenged with Loxosceles gaucho venom. Rev. Inst. Med. Trop. S. Paulo 41, 179–182. Málaque, C.M., Castro-Valência, J.E., Cardoso, J.L.C., França, F.O., Barbaro, K. C., Fan, H.W., 2002. Clinical and epidemiological features of definitive and presumed loxoscelism in São Paulo, Brazil. Rev. Inst. Med. Trop. S. Paulo 44, 139–143. Malta, M.B., Lira, M.S., Soares, S.L., Rocha, G.C., Knysak, I., Martins, R., Guizze, S.P.G., Santoro, M.L., Barbaro, K.C., 2008. Toxic activities of Brazilian centipede venoms. Toxicon 52, 255–263. Melgarijo, E., Medina, M.A., Sanchez-Jimenez, F., Urdiales, J.L., 2009. Monocyte chemoattractant protein 1: a key mediator in inflammatory processes. Int. J. Biochem. Cell Biol. 41, 998–1001.
979
Ospedal, K.Z., Appel, M.H., Fillus Neto, J., Mangili, O.C., Veiga, S.S., Gremski, W., 2002. Histopathological findings in rabbits after experimental acute exposure to the Loxosceles intermedia (brown spider) venom. Int. J. Exp. Pathol. 83, 287–294. Paludo, K.S., Biscaia, S.M., Chaim, O.M., Otuki, M.F., Naliwaiko, K., Dombrowski, P.A., Franco, C.R., Veiga, S.S., 2009. Inflammatory events induced by brown spider venom and its recombinant dermonecrotic toxin: a pharmacological investigation. Comp. Biochem. Physiol. C Toxicol. Pharmacol. 149, 323–333. Patel, K.D., Modur, V., Zimmerman, G.A., Prescott, S.M., McIntyre, T.M., 1994. The necrotic venom of the brown recluse spider induces dysregulated endothelial cell-dependent neutrophil activation. J. Clin. Invest. 94, 631–642. Petricevich, V.L., 2004. Cytokine and nitric oxide production following severe envenomation. Curr. Drug. Targets Inflamm. Allergy 3, 325– 332. Rattmann, Y.D., Pereira, C.R., Cury, Y., Gremski, W., Marques, M.C., da Silva-Santos, J.E., 2008. Vascular permeability and vasodilation induced by the Loxosceles intermedia venom in rats: involvement of mast cell degranulation, histamine and 5-HT receptors. Toxicon 51, 363–372. Rees, R.S., Nanney, L.B., Yates, R.A., King, L.E., 1984. Interaction of brown recluse spider venom on cell membranes: the inciting mechanism? J. Invest. Dermatol. 83, 270–275. Rivera, J., Gilfillan, A.M., 2006. Molecular regulation of mast cell activation. J. Allergy Clin. Immunol. 117, 1214–1225. Rossi, D., Zlotnik, A., 2000. The biology of chemokines and their receptors. Ann. Rev. Immunol. 18, 217–242. Silva, P.H., da Silveira, R.B., Appel, M.H., Mangili, O.C., Gremski, W., Veiga, S.S. , 2004. Brown spiders and loxoscelism. Toxicon 44, 693–709. SINAN-Animais Peçonhentos/SVS/MS. http://dtr2004.saude.gov.br/sinanweb/ tabnet/dh?sinan/animaisp/bases/animaisbr.def. Smith, C.W., Micks, D.W., 1970. The role of polymorphonuclear leukocytes in the lesion caused by the venom of the brown spider Loxosceles reclusa. Lab. Invest. 22, 141–144. Smith, P.K., Krohn, R.I., Hermanson, G.T., Mallia, A.K., Gartner, F.H., Provenzano, M.D., Fujimoto, E.K., Goeke, N.M., Olson, B.J., Klenk, D.C., 1985. Measurement of protein using bicinchoninic acid. Anal. Biochem. 150, 76–85. Souza, D.G., Bertini, R., Vieira, A.T., Cunha, F.Q., Poole, S., Allegretti, M., Colotta, F., Teixeira, M.M., 2004. Repertaxin, a novel inhibitor of rat CXCR2 function, inhibits inflammatory responses that follow intestinal ischemia and reperfusion. Br. J. Pharmacol. 143, 132–142. Souza, A.L., Málaque, C.M., Sztajnbok, J., Romano, C.C., Duarte, A.J., Seguro, A.C., 2008. Loxosceles venom-induced cytokine activation, hemolysis, and acute kidney injury. Toxicon 51, 151–156. Van der Meide, P.H., Schellekens, H., 1996. Cytokines and the immune response. Biotherapy 8, 243–249.
Contents lists available at ScienceDirect
Toxicon journal homepage: www.elsevier.com/locate/toxicon
Inflammatory mediators generated at the site of inoculation of Loxosceles gaucho spider venom Katia C. Barbaro a, *, Marcela S. Lira a, Claudia A. Araújo a, Alessandra Pareja-Santos b, Bianca C.L.F. Távora a, José Pedro Prezotto-Neto a, Louise F. Kimura a, Carla Lima b, Mônica Lopes-Ferreira b, Marcelo L. Santoro c a b c
Laboratory of Immunopathology, Institute Butantan, Av. Vital Brasil 1500, 05503-900 São Paulo, SP, Brazil Special Laboratory of Applied Toxinology (CEPID), Institute Butantan, Av. Vital Brasil 1500, 05503-900 São Paulo, SP, Brazil Laboratory of Pathophysiology, Institute Butantan, Av. Vital Brasil 1500, 05503-900 São Paulo, SP, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history: Received 24 February 2010 Received in revised form 24 June 2010 Accepted 28 June 2010 Available online 6 July 2010
Patients bitten by Loxosceles spiders generally manifest marked local inflammatory reaction and dermonecrosis. This report evaluated edema formation, leukocyte infiltration and release of inflammatory mediators at the injection site of Loxosceles gaucho venom. BALB/c mice were i.d. injected with venom and thereafter paws were disrupted and homogenized to obtain differential counts of migrated cells, as well to assay the levels of cytokines, chemokines and lipid mediators. Increased footpad thickness was detected as soon as 30 min after venom injection, and 24 h later was similar to that of the control group. Loxosceles venom mildly augmented the recruitment of leukocytes to the footpad in comparison with PBS-injected mice. Moreover, it stimulated the release of IL-6, MCP-1 and KC at 2 and 24 h after venom injection. In addition, higher levels of PGE2 were detected 30 min after venom injection in comparison with control group. However, the venom failed to increase levels of IL-1b, TNF-a, TXB2 and LTB4. Our results demonstrate that L. gaucho venom evokes an early complex inflammatory reaction, stimulating the secretion of pro-inflammatory cytokines and lipid mediators (PGE2), and recruiting leukocytes to the footpad which contribute to the local reaction induced by L. gaucho venom. Ó 2010 Elsevier Ltd. All rights reserved.
Keywords: Spider venom Loxosceles Loxoscelism Brown spider Local reaction Inflammatory mediators
1. Introduction Loxoscelism is a syndrome caused in humans by bites from the brown spider Loxosceles spp. In general, the clinical manifestations are characterized by inflammation and
Abbreviations: IL-1b, interleukin 1 beta; IL-6, interleukin 6; IL-8, interleukin 8; TNF-a, tumor necrosis factor alpha; MCP-1, monocyte chemoattractant protein-1; KC, keratinocyte chemoattractant; TXB2, thromboxane B2; LTB4, leukotriene B4; PGE2, prostaglandin E2; RANTES, regulated upon activation, normal T-cell expressed, and secreted; GMCSF, granulocyte macrophage colony-stimulating factor; GRO-a, growth regulated oncogene-alpha. * Corresponding author. Tel.: þ55 11 37267222x2278/2134; fax: þ55 11 37261505. E-mail addresses: [email protected], [email protected] (K.C. Barbaro). 0041-0101/$ – see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.toxicon.2010.06.022
dermonecrosis at the site of the bite, although in some cases systemic hemolysis and coagulopathy are present, leading to acute renal failure (Hogan et al., 2004; Abdulkader et al., 2008). In Brazil, eight Loxosceles species are found, however, Loxosceles gaucho, Loxosceles laeta and Loxosceles intermedia are commonly implicated in accidents reported in humans (Barbaro and Cardoso, 2003), which occur mainly in the southern and southeastern regions of the country. In 2006, Loxosceles spiders were responsible for approximately 40% of 19,105 notified cases of spider envenomation reported to the Brazilian Ministry of Health (SINAN, 2006). Loxosceles venoms are rich source of proteases, hydrolases, lipases, peptidases, collagenases, alkaline phosphatases, 5-ribonucleotidases, phosphohydrolases and other components. A family of phospholipases D has been mainly
K.C. Barbaro et al. / Toxicon 56 (2010) 972–979
implicated in the genesis of dermonecrosis that occurs after the bite (Hogan et al., 2004; Silva et al., 2004; Barbaro et al., 2005; Kalapothakis et al., 2007). Venom components interact with the cellular membrane, degrade components of extracellular matrix, take part in complement system activation and recruit polymorphonuclear leukocytes and platelets, among others, thus contributing to the establishment of local injury (Futrell, 1992; Hogan et al., 2004; Silva et al., 2004). Such venom components may be important to induce an early inflammatory reaction, eliciting the release of many endogenous pro-inflammatory mediators that contribute to the development of the lesion (Hogan et al., 2004). Loxosceles venoms have been demonstrated to stimulate cytokine production. Patel et al. (1994) showed that Loxosceles reclusa venom stimulated the production of IL-8 and the secretion of GM-CSF by endothelial cells, the migration of neutrophils to the injection site, and the release of granule content after neutrophil adhesion. Moreover, Málaque et al. (1999) observed that L. gaucho venom causes alterations in primary cultures of keratinocytes and stimulates TNF-a production. In addition, Loxosceles deserta has been shown to stimulate the expression of vascular endothelial growth factor (VEGF) in human keratinocyte culture (Desai et al., 2000). Induction of release of chemokines (IL8, GRO-a, MCP-1, RANTES) by endothelial and epithelial cells has also been implicated in the pathogenesis of loxoscelism (Gomez et al., 1999). Recently, Souza et al. (2008) reported high levels of IL-6 and TNF-a in a patient bitten by Loxosceles spp. spider. Thus, based on these findings, a number of cytokines and chemokines seem to contribute to the pathogenesis of dermonecrotic arachnidism. The poor efficiency of current strategies (serotherapy and corticoid administration) to treat loxoscelism in Brazil is attributed to at least two causes. One of them is the rapid tissue injury that occurs after Loxosceles envenomation (Barbaro and Cardoso, 2003; Hogan et al., 2004; Silva et al., 2004). The other is the delayed medical assistance, since patients seek medical care usually 12–24 h after the spider bite (Barbaro et al., 1992b; Málaque et al., 2002). Thus, the effectiveness of loxoscelism treatment should take into consideration both the intrinsic venom toxic activity and the local inflammatory reaction that develops after envenomation. Loxosceles venom induces dermonecrosis in rabbits, guinea pigs and humans but not in mice (Futrell, 1992; Barbaro et al., 1996a,b; Domingos et al., 2003; Hogan et al., 2004). However, Loxosceles venom causes an important inflammatory reaction at the injection site and, depending on the dose, it can be lethal to animals (Barbaro et al., 1994). Domingos et al. (2003) demonstrated that the size and availability of local sphingomyelin may be important in determining the outcome of Loxosceles envenomation in different mammalian species. Since we are interested to verify the inflammatory reaction in the absence of dermonecrosis, in the present work we investigated the effect of L. gaucho venom in evoking an acute inflammatory response by means of the evaluation of edema formation and the release of inflammatory mediators (cytokines, chemokines and lipid mediators) after injection of L. gaucho venom in mouse paws.
973
2. Materials and methods 2.1. Animals and venom BALB/c mice (male, 18–20 g) were provided by the Butantan Institute animal house. The procedures involving animals were conducted according to national laws and policies controlled by Butantan Institute Animal Investigation Ethical Committee (protocol n 316/06). Specimens of adult L. gaucho spiders were collected in São Paulo State, Brazil. The spiders were kept in quarantine for 1 week without food before venom extraction. Venoms were obtained as previously described (Barbaro et al., 1992a). Protein concentrations were determined by the bicinchoninic acid (BCA) assay (Smith et al., 1985). Standard curves were constructed using bovine serum albumin (Sigma Chemicals, St Louis, MO, USA) diluted in duplicate. 2.2. Experimental model Mice (n ¼ 4–8) were injected (30 mL) in the right hind paw with L. gaucho venom (3, 5, or 10 mg), PBS (negative control), or lipopolysaccharide (LPS) from Escherichia coli (1 mg, Sigma Chemicals, St Louis, MO, USA, positive control). Edema-forming activity was studied at three time intervals (0.5, 2 and 24 h), and thereafter the right paws were removed at the level of the tibiotarsal joint, disrupted and homogenized. Following centrifugation (400 g) at 4 C for 10 min, supernatants were recovered to assay the release of cytokines, chemokines and lipid mediators by enzymatic immunoassay. Cell pellets were recovered to perform cell counts. 2.3. Edema measurement after footpad injection Mice were injected (30 mL) in the footpad with different doses of L. gaucho venom or PBS. Edema formation was measured using a plethysmographer (Ugo Basile, IT). Results expressed the difference in paw volume (mL) prior to (control) and after (experimental) injection. Edema was measured at different time points (0.5, 2 and 24 h) after injection. 2.4. Leukocyte recruitment to footpads Total leukocyte counts from footpad homogenates were performed by Trypan blue exclusion (Sigma–Aldrich, USA) using a hemocytometer; differential counts were accomplished using cytocentrifuge slides stained with HEMA-3 (Fischer Scientific Company, MI, USA). For differential cell counts, 100 leukocytes were enumerated and identified as macrophages, lymphocytes or neutrophils, based on staining and morphologic features. 2.5. Assays for cytokines, chemokines and lipid mediators Levels of cytokines (IL-1b, IL-6 and TNF-a), chemokines (KC and MCP-1) and lipid mediators (LTB4, PGE2 and TXB2) were measured in supernatants of paw tissue extracts (as described in section 2.2) or in supernatants of murine
974
K.C. Barbaro et al. / Toxicon 56 (2010) 972–979 35 *
30
* *
25
*
*
Edema (μL)
PBS 3 µg 5 µg 10 µg
20
*
15 10
5 β
0 2
0.5
24
Time (hours)
Fig. 1. Edematogenic activity (mean S.D.) of L. gaucho venom in mice (n ¼ 4–8). Edema (in mL) was calculated by the difference between the volume of hind paws after injection of L. gaucho venom (at 0.5, 2 and 24 h) and the volume before injection (control). *p < 0.05, in relation to the control group (PBS). b: Animals died before 24 h.
dendritic cells stimulated with LPS (1 mg), using commercial specific two-site sandwich enzyme linked immunosorbent assays (ELISA), according to manufacturer’s instructions (IL-1b, IL-6, TNF-a, MCP-1: B&D Biosciences, CA, USA; KC: R&D Systems, MN, USA; LTB4, PGE2 and TXB2: Cayman Chemicals, MI, USA). Samples were quantified using standard curves of eicosanoids, and recombinant murine cytokines and chemokines. Results were expressed as mean S.D. for duplicate samples. 2.6. Histopathological analyses L. gaucho venom (3, 5, or 10 mg) or PBS was injected in the right paw of mice. At 0.5 and 2 h the right paws of mice were removed and fixed in 4% paraformaldehyde in PBS 0.1 M, pH 7.2 for 24 h. The paw was serially sectioned and the samples were dehydrated in ethanol and embedded in paraffin. Sections of 6–7 mm were obtained in a Microm HM340 microtome and stained with hematoxylin–eosin.
soon as 30 min after venom injection (5 and 10 mg), reaching an approximately 3-fold increase with 10 mg venom at 0.5 and 2 h. At 24 h, the total cell count (10 mg group) was still approximately 1.8-fold higher than that of the PBS group. The differential count of migrated cells into the paw tissue was also analyzed, and showed an increase in macrophages, lymphocytes and neutrophils as soon as 0.5 h in animals injected with 5 and 10 mg of L. gaucho venom (Fig. 2B–D). The recruitment of inflammatory cells is still augmented, more than 2-fold, when compared with the control group at 2 h after venom injection. At 24 h, only lymphocyte counts returned to basal levels. By histological analysis, the presence of edema was observed as soon as 0.5 h after venom injection in all doses tested (Fig. 3), and inflammatory infiltrates were scant. After 2 h, besides edema, an increase in inflammatory infiltrate was observed mainly in 5 and 10 mg doses. L. gaucho venom stimulated the secretion of proinflammatory cytokines. The levels of IL-6 increased at 2 h and 24 h after venom injection (Fig. 4). No difference was observed in IL-1b and TNF-a levels between experimental and PBS groups for all venom doses and time intervals tested (data not shown). However, LPS, an agent known to increase the production of IL-1b and TNF-a, did stimulate the secretion of these mediators in the foot paws and in dendritic cells culture (data not shown). The levels of both chemokines, MCP-1 and KC, were significantly increased 2 h after venom injection (Figs. 5 and 6); after 24 h, the levels of these chemokines were remarkably higher (about 10-fold) when compared with PBS group. In order to determine the participation of lipid mediators in the inflammatory reaction, the levels of PGE2, LTB4 and TXB2 were also assayed. Fig. 7 shows that L. gaucho venom induced a high PGE2 release from as soon as 30 min to 2 h after venom injection; the levels of PGE2 remained increased in animals injected with 5 mg of venom at 24 h after venom injection. No increase in TXB2 or LTB4 levels were observed when compared with the PBS group in all samples tested (data not shown). 4. Discussion
2.7. Statistical analyses Results were expressed as means SD. One-way or two-way ANOVA, followed by Bonferroni test, was used to analyze data, employing SigmaStat 3.5 software. Values with p < 0.05 were considered statistically significant. 3. Results In order to determine the ability of L. gaucho venom to evoke edema at the site of venom administration, mice were injected i.d. in the footpad with either venom alone or vehicle (PBS). The results showed that at 0.5 and 2 h after injection, edema was statistically significant in venomtreated groups in comparison with PBS group (Fig. 1). No statistical difference in paw volume was observed 24 h after injection between venom (all doses) and PBS groups. Our results demonstrated that L. gaucho injection evokes a complex inflammatory reaction with leukocyte recruitment to the footpad (Fig. 2A), which is observed as
Although some symptoms of envenomation caused by spiders from Loxosceles genus are well characterized, e.g. formation of dermonecrotic lesions, several caveats are still unanswered about by which mechanisms L. gaucho venom could trigger an acute immune response after the bite. Herein we studied soluble pro-inflammatory mediators likely involved in the development of the inflammatory response induced by L. gaucho venom, and we show that this venom differentially triggers the release of some pivotal inflammatory mediators, such as IL-6, MCP-1, KC, PGE2, but not of other ones (IL-1b, TNF-a, LTB4, TXB2). This report is the first to demonstrate that the inflammatory response induced by L. gaucho venom in mouse [an animal species that does not manifest the classical dermonecrotic effect induced by Loxosceles venom (Barbaro et al., 1996a; Domingos et al., 2003)] is not triggered by classical proinflammatory mediators such as IL-1b, TNF-a, LTB4, TXB2. Preliminary studies showed that edema decreased rapidly and was similar to that of the animals injected with
K.C. Barbaro et al. / Toxicon 56 (2010) 972–979
B 100
cells/mL (x 105)
80
*
* *
*
60
PBS 3 µg 5 µg 10 µg
*
40 20
50
0 0.5
2 Time (hours)
PBS 3 µg 5 µg 10 µg
* *
8 *
D Neutrophils (x 105)
Lymphocytes (x 105)
10
40
* *
*
6 4
*
30
*
20
*
10 0
24
12
PBS 3 µg 5 µg 10 µg
*
Macrophages (x 105)
A
C
975
0.5
2 Time (hours)
24
50 *
40 30
*
* *
*
PBS 3 µg 5 µg 10 µg
*
20 10
2
β
0
0 0.5
2 Time (hours)
24
0.5
2 Time (hours)
24
Fig. 2. Effect of L. gaucho venom in leukocyte recruitment to mouse footpads (n ¼ 4–8). After 0.5, 2 and 24 h of venom injection (3, 5 or 10 mg), animals were euthanized, and the footpad tissue was processed for counting of total leukocytes (A), macrophages (B), lymphocytes (C) and neutrophils (D). Mice only injected with PBS were considered as the control group. The results represent the mean S.D. *p < 0.05 compared with control-group. b: Animals died before 24 h.
PBS at 8 h after venom injection (data not shown), suggesting that L. gaucho venom induces a rapid inflammatory reaction in mouse footpads. Based on these findings, we investigated the acute inflammation at early time point (0.5 and 2 h), as well as at 24 h, when edema was no more evident. The experimental model established herein used venom injection into footpads rather than into the peritoneal cavity – the most used model to test inflammatory effects of venoms – in order to mimic human envenomation caused by Loxosceles spp. In mice injected with L. gaucho venom, the peak of edema occurred at 0.5 h, decreasing steadily thereafter. The velocity of edema induction in mice differs from that observed in human envenomation, where the inflammatory reaction begins more slowly (6–24 h) after the bite and takes many days to decrease (Barbaro et al., 1992b; Málaque et al., 1999; Barbaro and Cardoso, 2003). Mast cells have been reported to play an important role in edema formation. Rattmann et al. (2008) showed that L. intermedia venom degranulates mast cells, releasing histamine and serotonin. Furthermore, Paludo et al. (2009) showed that L. intermedia venom also contains histamine that, in association with venom phospholipase-D, can induce mast cell degranulation (Rivera and Gilfillan, 2006), contributing to local inflammatory events. Histopathologically, an inflammatory cell infiltrate was observed in footpads injected with L. gaucho venom. At the
site of the injury, leukocyte recruitment is a crucial event to initiate the immune response against the insulting agent, such as toxins and pathogens. Macrophages were increased at all time intervals, but lymphocytes were found only at the initial intervals. Neutrophils play a crucial role on Loxosceles dermonecrotic lesions (Smith and Micks, 1970; Rees et al., 1984; Ospedal et al., 2002; Silva et al., 2004; Hogan et al., 2004). In our experiments, the number of migrated neutrophils increased as soon as 30 min of L. gaucho venom injection, demonstrating the importance of these cells in eliciting the local reaction. In agreement with our results, neutrophils and mononuclear cells were the predominant cells in peripheral blood at 2 and 4 h after Androctonus australis hector venom injection (Adi-Bessalem et al., 2008). Neutrophil recruitment was also found in experimental models using Bothrops spp. venoms (Lomonte et al., 1993; Farsky et al., 1997). Edema induced by L. gaucho venom was noticed to decrease rapidly in murine hind paws. This profile is typical of L. gaucho venom, since edema induced by centipede (Malta et al., 2008) or stingray (Barbaro et al., 2007) venoms persists up to 48 h. In addition, we could detect cellular infiltrate, mainly macrophages and neutrophils, into the foot paws after 24 h of L. gaucho venom injection, indicating that a local inflammatory response is still present, despite the absence of swelling. This might explain why inflammatory mediators such as IL-6, KC, MCP-1, and PGE2 are still found into the foot paws.
976
K.C. Barbaro et al. / Toxicon 56 (2010) 972–979
Fig. 3. Micrographs of mouse paw tissues after L. gaucho venom injection. Mice were injected with PBS or L. gaucho venom (3, 5 or 10 mg) and the histological analysis was performed at 0.5 and 2 h. Hematoxylin–eosin stain. Bar size ¼ 50 mm.
K.C. Barbaro et al. / Toxicon 56 (2010) 972–979 1000
IL-6 (pg/mL)
PBS 3 µg 5 µg 10 µg
* 100
*
*
* *
β 10 0.5
24
2
Time (hours) Fig. 4. Analysis of IL-6 levels in footpad of mice (n ¼ 4–8) injected with L. gaucho venom. After 0.5, 2 and 24 h of venom injection (3, 5 or 10 mg), footpad homogenates were collected for IL-6 assay. The results represent the mean S.D. *p < 0.05 compared with the control group (PBS). b: Animals died before 24 h.
In addition to the recruitment of inflammatory cells, our results suggested that pro-inflammatory mediators (IL-6, MCP-1, KC, PGE2) could participate in the development of the tissue injury in L. gaucho envenomation. However, no increase was noticed in IL-1b, TNF-a, TXB2 and LTB4 production. Pro-inflammatory mediators are primarily responsible for initiating an effective immune response against toxins and pathogens, but, the overproduction of these mediators contributes to shock, multiple organ failure and death (Van der Meide and Schellekens, 1996). Several cytokines have been involved in severe envenomation, e.g. TNF-a, IL-1b and IL-6. TNF-a has been reported to be a crucial mediator in the pathogenesis of various envenomations (Petricevich, 2004). The role of IL-6 is controversial, since it has either pro- or anti-inflammatory properties (Barton, 1997). As a down-regulator of inflammatory responses, IL-6 can inhibit the production of IL-1b and TNF-a by increasing, respectively, the synthesis of IL10000 PBS 3 µg 5 µg 10 µg
MCP-1 (pg/mL)
*
*
1000
*
*
*
100
β
10 0.5
2
24
Time (hours) Fig. 5. MCP-1 levels in footpad homogenates of mice (n ¼ 4–8) injected with L. gaucho venom. After 0.5, 2 and 24 h of venom injection (3, 5 or 10 mg), footpad homogenates were collected for MCP-1 assay by ELISA. The results represent the mean S.D. *p < 0.05 compared with the control group (PBS). b: Animals died before 24 h.
977
1Ra and soluble TNF receptor p55 (Jones, 2005). The origin of IL-6 secretion induced by L. gaucho venom at the inflammatory site has not been elucidated yet. In addition, high levels of PGE2 produce potent vasodilation and suppress TNF-a and IL-1b production. Thus, the absence of detectable levels of IL-1b and TNF-a at the site of the injury might inhibit the effects of IL-6 and PGE2, retarding perhaps an exaggerated inflammatory response and delaying thereby tissue necrosis in the host. In order to ensure that the absence of IL-1b and TNF-a in L. gaucho-injected mice is a differential feature of this venom, the supernatants of LPS-injected mice or of LPSstimulated mouse dendritic cells were tested as a positive control in the cytokine assays. We found that LPS stimulated the production of IL-6, IL-1b and TNF-a (data not shown), suggesting that L. gaucho stimulates the secretion of IL-6, but not of IL-1b and TNF-a. Furthermore, in agreement with our data, L. gaucho venom alone was also shown to be unable to induce TNF-a release by J774A.1 cells, a murine macrophage lineage (Domingos et al., 2003). Besides, the intramuscular injection of Bothrops asper snake venom was unable to change TNF-a production (Chaves et al., 2005) showing that each animal venom might stimulate a different cytokine profile. Chemokines have a pivotal role in the selective induction and amplification of the inflammatory response, by favoring leukocyte migration by means of activation of Gprotein-coupled receptors, and expression of adhesion molecules and glycosaminoglycans (Rossi and Zlotnik, 2000). In addition, they increase the affinity of leukocyte integrins to their ligands on the vascular wall during diapedesis, and regulate polymerization and depolymerization of actin in leukocytes for their movement and migration toward the inflammatory site (Luo et al., 2007). MCP-1, also known as CCL2, is a potent chemoattractant to monocytes and T cells, and binds to CCR2 expressed by these cells (Melgarijo et al., 2009), and might be involved in monocyte and lymphocyte migration to the site of injection of L. gaucho venom. This might be also due to the high production of PGE2, which has been demonstrated to recruit monocytes to the site of inflammation and promote their differentiation to macrophages (Kurth et al., 2001). KC (homologue to IL-8 or CXCL8 in humans) promotes neutrophil chemotaxis and degranulation by binding to the receptors CXCR1 or, preferentially, CXCR2. The blockage of CXCR1 or CXCR2 has been shown to inhibit the neutrophil influx in inflammation (Souza et al., 2004; Cugini et al., 2005), suggesting a critical role of KC in recruiting neutrophils. As we demonstrated, the kinetics of KC is associated with neutrophil migration to the footpads. However, neutrophils have also been found in footpads as soon as 30 min after L. gaucho venom, which might be attributed to PGE2 release. In fact, the association between PGE2 secretion and neutrophil influx has been demonstrated following injection of BaP1, a metalloproteinase from B. asper venom, which stimulates PGE2 and cell influx, mostly neutrophils, in mouse joints (Fernandes et al., 2007). Moreover, the absence of LTB4 in our model might be due to the production of PGE2, since this eicosanoid was found to induce IL-6 production in macrophages (Bagga et al., 2003) and to inhibit 5-lipooxygenase (5-LOX),
978
K.C. Barbaro et al. / Toxicon 56 (2010) 972–979
Acknowledgements This paper was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP grant 06/564954). The authors thank Mr Santos-Neto M.O. for technical assistance. We also thank CNPq for the grant to K.C.B. (304800/2007-4). IBAMA provided animal collection permission no. 15383-2 and CGEN provided the license for genetic patrimony access (02001.005110/2008). Conflict of interest statement The authors declare that there are no conflicts of interest. Fig. 6. KC levels in footpad homogenates of mice (n ¼ 4–8) injected with L. gaucho venom. After 0.5, 2 and 24 h of venom injection (3, 5 or 10 mg), footpad homogenates were collected for KC assay by ELISA. The results represent the mean S.D. *p < 0.05 compared with the control group (PBS). b: Animals died before 24 h.
decreasing consequently LTB4 production (Levy et al., 2001). Furthermore, the absence of TXB2 release might be associated with the absence of IL-1b in our model. IL-1b has been shown to stimulate COX2 expression in several types of cells, including macrophages, up-regulating the release of PGE2 and TXB2 (Dinarello, 2009). Taken together, our results show that L. gaucho venom elicits marked PGE2 release, and in association with IL-6, MCP-1 and KC secretion, induce vascular leakage (edema) and leukocyte migration to the site of injection. These findings suggest that these mediators contribute to the inflammatory reaction induced by L. gaucho venom in mouse footpad, and help to understand the pathophysiology of loxoscelism. Thus, novel approaches for alternative treatments, such as anti-inflammatory drugs, might be useful to diminish the local lesion caused by Loxosceles venom.
Fig. 7. Prostaglandin E2 (PGE2) release in the footpad of mice after L. gaucho venom injection. After 0.5, 2 and 24 h of venom injection (3, 5 or 10 mg), animals were sacrificed and the footpads were processed in order to obtain footpad homogenate fluid for PGE2 quantification by ELISA (n ¼ 4–8). The results represent the mean S.D. *p < 0.05 compared with the control group (PBS). b: Animals died before 24 h.
References Abdulkader, R.C.R.M., Barbaro, K.C., Barros, E.J.G., Burdmann, E.A., 2008. Nephrotoxicity of insect and spider in Latin America. Sem. Nephrol. 28, 373–382. Adi-Bessalem, S., Hammoudi-Triki, D., Laraba-Djebari, F., 2008. Pathophysiological effects of Androctonus australis hector scorpion venom: tissue damages and inflammatory response. Exp. Toxicol. Pathol. 60, 373–380. Bagga, D., Wang, L., Farias-Eisner, R., Glaspy, J.A., Reddy, S.T., 2003. Differential effects of prostaglandin derived from omega-6 and omega-3 polyunsaturated fatty acids on COX-2 expression and IL-6 secretion. Proc. Natl. Acad. Sci. U.S.A. 100, 1751–1756. Barbaro, K.C., Cardoso, J.L.C., Eickstedt, V.R.D., Mota, I., 1992a. Dermonecrotic and lethal components of Loxosceles gaucho spider venom. Toxicon 30, 331–338. Barbaro, K.C., Cardoso, J.L.C., Eickstedt, V.R.D., Mota, I., 1992b. IgG antibodies to Loxosceles gaucho venom in human envenoming. Toxicon 30, 1117–1121. Barbaro, K.C., Eickstedt, V.R.D., Mota, I., 1994. Antigenic cross-reactivity of venoms from medically important Loxosceles (Araneae) species in Brazil. Toxicon 32, 113–120. Barbaro, K.C., Ferreira, M.L., Cardoso, D.F., Eickstedt, V.R.D., Mota, I., 1996a. Identification and neutralization of biological activities in the venoms of Loxosceles spiders. Braz. J. Med. Biol. Res. 29, 1491–1497. Barbaro, K.C., Sousa, M.V., Morhy, L., Eickstedt, V.R.D., Mota, I., 1996b. Compared chemical properties of dermonecrotic and lethal toxins from spiders of the genus Loxosceles (Araneae). J. Prot. Chem. 15, 337– 343. Barbaro, K.C., Cardoso, J.L.C., 2003. Mecanismo de ação do veneno de Loxosceles e aspectos clínicos do loxoscelismo. In: Cardoso, J.L.C., França, F.O.S., Wen, F.H., Málaque, C.M.S., Haddad Jr., V. (Eds.), Animais Peçonhentos no Brasil: Biologia, Clínica e Terapêutica dos acidentes. Savier, São Paulo, pp. 160–174. Barbaro, K.C., Knysak, I., Martins, R., Hogan, C., Winkel, K., 2005. Enzymatic characterization, antigenic cross-reactivity and neutralization of dermonecrotic activity of five Loxosceles spider venoms of medical importance in the Americas. Toxicon 45, 489–499. Barbaro, K.C., Lira, M.S., Malta, M.B., Soares, S.L., Garrone Neto, D., Cardoso, J.L.C., Santoro, M.L., Haddad Junior, V., 2007. Comparative study on extracts from the tissue covering the stingers of freshwater (Potamotrygon falkneri) and marine (Dasyatis guttata) stingrays. Toxicon 50, 676–687. Barton, B.E., 1997. IL-6: insights into novel biological activities. Clin. Immunol. Immunopathol. 85, 16–20. Chaves, F., Teixeira, C.F., Gutierrez, J.M., 2005. Role of TNF-alpha, IL-1beta, IL-6 in local tissue damage induced by Bothrops asper venom: an experimental assessment in mice. Toxicon 45, 171–178. Cugini, D., Azzolini, N., Gagliardini, E., Cassis, P., Bertini, R., Colotta, F., Noris, M., Remuzzi, G., Benigni, A., 2005. Inhibition of the chemokine receptor CXCR2 prevents kidney graft function deterioration due to ischemia/ reperfusion. Kidney Int. 67, 1753–1761. Desai, A., Lankford, H.A., Warren, J.S., 2000. Loxosceles deserta spider venom induces the expression of vascular endothelial growth factor (VEGF) in keratinocytes. Inflammation 24, 1–9. Dinarello, C.A., 2009. Immunological and inflammatory functions of the interleukin-1 family. Ann. Rev. Immunol. 27, 519–550. Domingos, M.O., Barbaro, K.C., Tynan, W., Penny, J., Lewis, D.J., New, R.R., 2003. Influence of sphingomyelin and TNF-a release on lethality and
K.C. Barbaro et al. / Toxicon 56 (2010) 972–979 local inflammatory reaction induced by Loxosceles gaucho spider venom in mice. Toxicon 42, 471–479. Farsky, S.H., Walber, J., Costa-Cruz, M., Cury, Y., Teixeira, C.F., 1997. Leukocyte response induced by Bothrops jararaca crude venom: in vivo and in vitro studies. Toxicon 35, 185–193. Fernandes, C.M., Teixeira, C.F.P., Gutierrez, J.M., Rocha, F.A., 2007. The snake venom metalloproteinase BaP1 induces joint hypernociception through TNF-a and PGE2 dependent mechanisms. Br. J. Pharmacol. 151, 1254–1261. Futrell, J., 1992. Loxoscelism. Am. J. Med. Sci. 304, 261–267. Gomez, H.F., Miller, M.J., Desai, A., Warren, J.S., 1999. Loxosceles spider venom induces the production of alpha and beta chemokines: implications for the pathogenesis of dermonecrotic arachnidism. Inflammation 23, 207–215. Hogan, C.J., Barbaro, K.C., Winkel, K., 2004. Loxoscelism: old obstacles, new directions. Ann. Emerg. Med. 44, 608–624. Jones, S.A., 2005. Directing transition from innate to acquired immunity: defining a role for IL-6. J. Immunol. 175, 3463–3468. Kalapothakis, E., Chatzaki, M., Gonçalves-Dornelas, H., de Castro, C.S., Silvestre, F.G., Laborne, F.V., de Moura, J.F., Veiga, S.S., ChávezOlórtegui, C., Granier, C., Barbaro, K.C., 2007. The Loxtox protein family in Loxosceles intermedia (Mello-Leitão) venom. Toxicon 50, 938–946. Kurth, I., Willimann, K., Schaerli, P., Hunziker, T., Clark-Lewis, I., Moser, B., 2001. Monocyte selectivity and tissue localization suggests a role for breast and kidney-expressed chemokine (BRAK) in macrophage development. J. Exp. Med. 194, 855–861. Levy, B.D., Clish, C.B., Schmidt, B., Gronert, K., Serhan, C.N., 2001. Lipid mediator class switching during acute inflammation: signals in resolution. Nat. Immunol. 7, 612–619. Lomonte, B., Tarkowski, A., Hanson, L.A., 1993. Host response to Bothrops asper snake venom. Analysis of edema formation, inflammatory cells, and cytokine release in a mouse model. Inflammation 17, 93–105. Luo, B.H., Carman, C.V., Springer, T.A., 2007. Structural basis of integrin regulation and signaling. Ann. Rev. Immunol. 25, 619–647. Málaque, C.M.S., Ori, M., Santos, S.A., Andrade, D.R., 1999. Production of TNF-a by primary cultures of human keratinocytes challenged with Loxosceles gaucho venom. Rev. Inst. Med. Trop. S. Paulo 41, 179–182. Málaque, C.M., Castro-Valência, J.E., Cardoso, J.L.C., França, F.O., Barbaro, K. C., Fan, H.W., 2002. Clinical and epidemiological features of definitive and presumed loxoscelism in São Paulo, Brazil. Rev. Inst. Med. Trop. S. Paulo 44, 139–143. Malta, M.B., Lira, M.S., Soares, S.L., Rocha, G.C., Knysak, I., Martins, R., Guizze, S.P.G., Santoro, M.L., Barbaro, K.C., 2008. Toxic activities of Brazilian centipede venoms. Toxicon 52, 255–263. Melgarijo, E., Medina, M.A., Sanchez-Jimenez, F., Urdiales, J.L., 2009. Monocyte chemoattractant protein 1: a key mediator in inflammatory processes. Int. J. Biochem. Cell Biol. 41, 998–1001.
979
Ospedal, K.Z., Appel, M.H., Fillus Neto, J., Mangili, O.C., Veiga, S.S., Gremski, W., 2002. Histopathological findings in rabbits after experimental acute exposure to the Loxosceles intermedia (brown spider) venom. Int. J. Exp. Pathol. 83, 287–294. Paludo, K.S., Biscaia, S.M., Chaim, O.M., Otuki, M.F., Naliwaiko, K., Dombrowski, P.A., Franco, C.R., Veiga, S.S., 2009. Inflammatory events induced by brown spider venom and its recombinant dermonecrotic toxin: a pharmacological investigation. Comp. Biochem. Physiol. C Toxicol. Pharmacol. 149, 323–333. Patel, K.D., Modur, V., Zimmerman, G.A., Prescott, S.M., McIntyre, T.M., 1994. The necrotic venom of the brown recluse spider induces dysregulated endothelial cell-dependent neutrophil activation. J. Clin. Invest. 94, 631–642. Petricevich, V.L., 2004. Cytokine and nitric oxide production following severe envenomation. Curr. Drug. Targets Inflamm. Allergy 3, 325– 332. Rattmann, Y.D., Pereira, C.R., Cury, Y., Gremski, W., Marques, M.C., da Silva-Santos, J.E., 2008. Vascular permeability and vasodilation induced by the Loxosceles intermedia venom in rats: involvement of mast cell degranulation, histamine and 5-HT receptors. Toxicon 51, 363–372. Rees, R.S., Nanney, L.B., Yates, R.A., King, L.E., 1984. Interaction of brown recluse spider venom on cell membranes: the inciting mechanism? J. Invest. Dermatol. 83, 270–275. Rivera, J., Gilfillan, A.M., 2006. Molecular regulation of mast cell activation. J. Allergy Clin. Immunol. 117, 1214–1225. Rossi, D., Zlotnik, A., 2000. The biology of chemokines and their receptors. Ann. Rev. Immunol. 18, 217–242. Silva, P.H., da Silveira, R.B., Appel, M.H., Mangili, O.C., Gremski, W., Veiga, S.S. , 2004. Brown spiders and loxoscelism. Toxicon 44, 693–709. SINAN-Animais Peçonhentos/SVS/MS. http://dtr2004.saude.gov.br/sinanweb/ tabnet/dh?sinan/animaisp/bases/animaisbr.def. Smith, C.W., Micks, D.W., 1970. The role of polymorphonuclear leukocytes in the lesion caused by the venom of the brown spider Loxosceles reclusa. Lab. Invest. 22, 141–144. Smith, P.K., Krohn, R.I., Hermanson, G.T., Mallia, A.K., Gartner, F.H., Provenzano, M.D., Fujimoto, E.K., Goeke, N.M., Olson, B.J., Klenk, D.C., 1985. Measurement of protein using bicinchoninic acid. Anal. Biochem. 150, 76–85. Souza, D.G., Bertini, R., Vieira, A.T., Cunha, F.Q., Poole, S., Allegretti, M., Colotta, F., Teixeira, M.M., 2004. Repertaxin, a novel inhibitor of rat CXCR2 function, inhibits inflammatory responses that follow intestinal ischemia and reperfusion. Br. J. Pharmacol. 143, 132–142. Souza, A.L., Málaque, C.M., Sztajnbok, J., Romano, C.C., Duarte, A.J., Seguro, A.C., 2008. Loxosceles venom-induced cytokine activation, hemolysis, and acute kidney injury. Toxicon 51, 151–156. Van der Meide, P.H., Schellekens, H., 1996. Cytokines and the immune response. Biotherapy 8, 243–249.