Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 100(Suppl. I): 181-184, 2005 181 Inflammatory effects of snake venom metalloproteinases Catarina de Fátima Pereira Teixeira+, Cristina Maria Fernandes, Juliana Pavan Zuliani, Silvia Fernanda Zamuner

Laboratório de Farmacologia, Instituto Butantan, Av. Vital Brasil 1500, 05503-900 São Paulo, SP, Brasil Metalloproteinases are abundant enzymes in crotaline and viperine snake venoms. They are relevant in the pathophysiology of envenomation, being responsible for local and systemic hemorrhage frequently observed in the victims. Snake venom metalloproteinases (SVMP) are zinc-dependent enzymes of varying molecular weights having multidomain organization. Some SVMP comprise only the proteinase domain, whereas others also contain a disintegrin-like domain, cysteine-rich, and lectin domains. They have strong structural similarities with both mammalian matrix metalloproteinases (MMP) and members of ADAMs (a disintegrin and metalloproteinase) group. Besides hemorrhage, snake venom metalloproteinase induce local myonecrosis, skin damage, and inflammatory reaction in experimental models. Local inflammation is an important characteristic of snakebite envenomations inflicted by viperine and crotaline snake species. Thus, in the recent years there is a growing effort to understand the mechanisms responsible for SVMP-induced inflammatory reaction and the structural determinants of this effect. This short review focuses the inflammatory effects evoked by SVMP.

Key words: metalloproteinase - snake venom - inflammation

Envenomation by snakes of the family Viperidae is metalloproteinase (ADAM) (Fox & Long 1998). In turn, characterized by prominent local effects, including necro- SVMP are part of the “metzincin” family of zinc-depen- sis, hemorrhage, edema, and pain, which develop rapidly dent metalloproteinases, together with matrix metallo- after the accident and often result in permanent sequelae proteinases (MMP), astacins, and serralysins, all of them (Gutiérrez & Lomonte 1989, 1997, Dart et al. 1992, Warrell exhibiting an identical zinc-dependent motif, with the se- 1995). In addition, systemic alterations such as hemor- quence HEXXHXXGXXH and the presence of a methion- rhage, coagulopathy, shock, and acute renal failure may ine turn (Bode et al. 1993). SVMP can be divided into four occur. Both local and systemic effects of these snake ven- classes depending on their domain organization (Hite et oms have been associated with the action of a variety of al. 1994): P-I, comprising only the metalloproteinase do- venom components, which include metalloproteinases main; P-II, having a metalloproteinase domain followed (Ownby 1990, Gutiérrez & Lomonte 1997). by a disintegrin-like domain; P-III, comprising metal- Snake venom metalloproteinases (SVMP) comprise a loproteinase, disintegrin-like and cysteine-rich domains, subfamily of zinc-dependent enzymes of varying mo- and P-IV, containing additionally a lectin-like domain lecular mass, which are responsible for the hemorrhagic linked by disulfide bonds. The disintegrin-like domain effect characteristic of viperine and crotaline snake shows high sequence identity with venom disintegrins envenomations (Bjarnason & Fox 1994, Kamiguti et al. (Markland 1998), which are proteolytically released from 1998, Hati et al. 1999). In addition, more recent inv- P-II precursors. However, the disintegrin-like domain does estigations have evidenced that these enzymes are also not contain the typical RGD sequence found in involved in the pathogenesis of local myonecrosis disintegrins, showing different sequences in this region (Gutierrez et al. 1995, Rucavado et al. 1999), skin damage (Paine et al. 1992, Bjarnason & Fox 1994, Hite et al. 1994). (Rucavado et al. 1998), and inflammatory reaction All these metalloproteinases are synthesized as zy- (Gutiérrez et al. 1995, Moura da Silva et al. 1996). Although mogens, and are proteolytically processed to yield the anti-venom is the recognized therapy for systemic active enzyme (Bjarnason & Fox 1994). envenoming, it is unable to neutralize the toxins respon- Studies on the crystal structures of venom P-I class sible for the rapid onset of local tissue damage by snake metalloproteinases reveal similar structures, being ellip- venoms. Therefore, in the recent years there is a growing soidal molecules with a shallow active site clef that sepa- effort to understand the mechanisms responsible for rates a lower subdomain from the main domain composed SVMP-induced inflammatory reaction. of five stranded β-sheets and four α-helices (Gomis-Rüth The SVMP are members of the Reprolysin subfamily et al. 1993, Zhang et al. 1994, Kumasaka et al. 1996, Gong of zinc metalloproteinases, which also includes a group et al. 1998). The catalytic Zn2+ ion in the active-site clef is of mammalian homologous proteins, a disintegrin and surrounded by three His residues (142, 146, and 145) and a water molecule is anchored to Glu143 in a tetrahedral manner. Hemorrhagic activity has been associated with enzy- matic proteolytic activity, since chelation of the zinc atom Financial support: Fapesp (grant 02/13863-2), CNPq +Corresponding author. E-mail: [email protected] abolishes both proteolytic and hemorrhagic effects Received 8 November 2004 (Bjarnason & Tu 1978, Bjarnason & Fox 1994). The role of Accepted 30 DEcmber 2004 the others domains in the toxicity of high molecular weight 182 Inflammatory effects of snake venom • Catarina de Fátima Pereira Teixeira et al. enzymes is not clear, although it has been shown that lated from Bothrops jararaca venom has been shown to large hemorrhagic metalloproteinases having disintegrin- induce influx of leukocytes into mouse air pouch (Costa like and high-cysteine domains, are more active at induc- et al. 2002). This effect was dependent on the presence of ing hemorrhage than enzymes comprising only the metal- macrophages as well as on the proteolytic activity of this loproteinase domain (Bjarnason & Fox 1994, Hite et al. SVMP. Further investigations have also shown that 1994). Interestingly, there are metalloproteinases in snake jararhagin can directly stimulate the expression of mRNA venoms which are devoid of hemorrhagic activity (Willis encoding for TNF, IL-1 and IL-6 by elicited macrophages & Tu 1988, Markland 1998). The structural basis of this (Clissa et al. 2001). This suggests that macrophages are observation is not clear, although some comparative stud- important targets for SVMP, although the cell binding site ies have identified residues which may be required to ex- has not yet been characterized. Inactivation of the cata- ert this activity (Takeya et al. 1990, Hite et al. 1992, Ramos lytic activity of jararhagin had no effect on jararhagin- & Selistre-de-Araújo 2004). induced stimulatory effect on macrophages, pointing to a P-III class SVMP have strong similarities in sequence role for the disintegrin-like/cysteine domain in this effect and domain organization with ADAM (Fox & Long 1998). (Clissa et al. 2001). Investigations using knockout mice Members of the ADAM group have been implicated in deficient in TNF receptors and IL-6 have demonstrated the control of several physiological events including that both are relevant for development of jararhagin-in- cytokine shedding and cell migration. (Blobel 1997, duced necrosis but not edema nor hemorrhage (Laing et Peschon et al. 1998). In addition, increased expression of al. 2003). some mammalian MMP has been found in a variety of More recently, it was reported that HF3 a P-III class inflammatory conditions such as rheumatoid arthritis and SVMP isolated from B. jararaca venom induced pha- periodontal diseases (Cawston 1996, Cawston & gocytosis of opsonized zymosan particles by elicited Billington 1996). Therefore, mechanisms involved in the macrophages in vitro. This effect was inhibited by anti- bodies anti-α or anti-β or a combination of both anti- proinflammatory action of mammalian metalloproteinases m 2 α β and SVMP are under active investigation. This short re- bodies, thereby indicating that the integrin m 2 is in- view deals with the proinflammatory effects evoked by volved in the HF3-induced phagocytosis by macroph- SVMP and their mechanisms of action. ages (Silva et al. 2004). Not only native P-I or P-III class hemorrhagic Inflammatory effects metalloproteinases have been shown to induce in- Besides inducing hemorrhage and myonecrosis, flammatory reaction. The disintegrin and disintegrin-like venom metalloproteinases play a relevant role in the domains have also been shown to evoke such phenom- complex and multifactorial inflammatory response ena in some experimental models. Most disintegrins con- tain a RGD/KGD sequence within an amino acid hairpin characteristic of viperine envenomation. loop maintained by disulfide bridges. The RGD motif con- The first experimental evidence that SVMP cause in- fers disintegrins the ability to selectively bind to integrins flammation was provided by Gutierrez et al. (1995), who in different cell systems (Mariano-Oliveira et al. 2003). showed that a P-I class SVMP isolated from Bothrops Initially considered merely as antagonists of integrins, asper venom (BaP1) induced paw edema in mice. Upon the SVMP-derived disintegrins have been shown to in- intramuscular injection, this weakly hemorrhagic SVMP teract with and activate integrin-signalling pathways in induced formation of blisters and infiltration of leukocytes inflammatory cells. Integrin-signaling pathways mediate into dermis. This was associated with degranulation of important functions in leukocytes, including migration, mast cells and enlarged macrophages (Rucavado et al. spreading, activation of the respiratory burst, binding of 1998, 1999). Release of IL-1 and IL-6 induced by BaP1 in complement, cell adhesion, cytokine gene expression and the muscle tissue was further described, suggesting that apoptosis (Williams & Solonkin 1999, Lowell & Berton besides mast cell-stored mediators, cytokines could me- 1999, Larsson et al. 2000). Thus, ocellastusin, an RGD- diate the local inflammatory events induced by BaP1 containing short monomeric disintegrin, isolated from (Rucavado et al. 2002). In agreement with this hypoth- venom of Echis ocellatus, has been shown to induce esis, increased levels of IL-1 have also been detected in chemotaxis of human neutrophils in vitro (Smith et al. 2002). peritoneal exudates collected after BaP1 injection into Similarly, jarastatin, an RGD-disintegrin isolated from B. peritoneal cavity of mice, and this event was followed by jararaca venom showed a potent chemotactic activity on an increased expression of leukocyte adhesion molecules neutrophils and the ability to increase the levels of mRNA (our unpublished results). encoding for IL-8. Stimulation of leukocyte chemotaxis The ability of BaP1 to induce leukocyte migration has by jarastatin has been associated to its ability to trigger also been investigated in an in vitro model using Boyden’s intracellular signaling pathways mediated by integrin ac- chamber. In this study, the authors have demonstrated tivation (Coelho et al. 2004). Further studies have shown absence of a direct stimulatory effect of BaP1 on neutro- α β that this disintegrin interacts with m 2 integrin on neu- phil chemotaxis (Fasky et al. 2000). However, this SVMP trophil surface, thereby triggering integrin-mediated sig- was able to activate events of the classic and alternative naling, activating PI3K and MAPK pathways and inter- complement system, generating the chemotactic C5a fac- fering with neutrophil chemotaxis and production of tor. Accordingly, BaP1 treated serum was chemo-attrac- chemokines. In the same study, it was described that EC3, tant to neutrophils in vitro. a heterodimeric MLD-disintegrin, isolated from E. Hemorrhagic SVMPs are also able to induce in- carinatus suchoreki venom, is also able to induce chemot- flammatory events. Jararhagin, a P-III class SVMP iso- axis of human neutrophils in vitro, through its interaction Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 100(Suppl. I), 2005 183

α β with 9 1 integrin (Coelho et al. 2004). In addition, it was ACKNOWLEDGMENTS reported that alternagin-C, an ECD-disintegrin-like/cys- To Dr Solange MT Serrano for valuable suggestions and for teine-rich protein purified from B. alternatus venom, has critical reviewing the manunscript. a chemotactic activity on neutrophils and this effect in- volves actin cytoskeleton rearrangement, and FAK, PI3- REFERENCES kinase and Erk-2 activation,which are characteristic of Bjarnason JB, Fox JW 1994. Hemorrhagic metalloproteinases integrin-activated pathways (Mariano-Oliveira et al. 2003). from snake venoms. Pharmac Ther 62: 325-372. The interaction of disintegrins with macrophages has also Bjarnason JB, Tu AT 1978. Hemorrhagic toxins from Western been investigated. DC-HF3, a recombinant disintegrin-like diamondback rattlesnake (Crotalus atrox) venom: isolation cisteine rich protein derived from HF3 has been shown to and characterization of five toxins and the role of zinc in α β be able to stimulate m 2-mediated phagocytosis of op- hemorrhagic toxin. Biochemistry 17: 3395-404. sonized zymosan particles by macrophages (Silva et al. Blobel CP 1997. Metalloprotease-disintegrins: links to cell ad- 2004). However, in vivo inflammatory effects of SVMP- hesion and cleavage of TNF alpha and Notch. Review. Cell disintegrins have not yet been described, a study which 90: 589-592. must be addressed. Taken together, these findings indicate that the pro- Bode W, Gomis-Rüth FX, Stockler W 1993. Astacins, teolytic domain of SVMPs per se is able to trigger inflam- serralysins, snake venom and matrix metalloproteinases matory events in both in vivo and in vitro experimental exhibit identical zinc-binding environments (HEXX- HXXGXXH and Met-turn) and topologies and should be models. The SVMP disintegrin-like domains can stimu- grouped into a common family, the ‘metzincins’. Febs Let- late leukocyte functions in vitro through integrin-medi- ters 331: 134-140. ated pathways. Activation of distinct signaling pathways by these domains appears to be dependent on the struc- Cawston TE 1996. Metalloproteinase inhibitors and the pre- ture of each domain and on the type of cell surface recep- vention of connective tissue breakdown. Review. Pharmacol tors. Ther 70: 163-182. Cawston TE, Billington C 1996. Metalloproteinases in the rheu- Concluding remarks matic diseases. J Pathol 180: 115-117. The recent advance on venom metalloproteinases-in- Clissa PB, Laing GD, Theakston RD, Mota I, Taylor MJ, duced inflammatory reaction are briefly reviewed in this Moura-da-Silva AM 2001. The effect of jararhagin, a article. Exogenous administration of SVMP into experi- metalloproteinase from Bothrops jararaca venom, on pro- mental animals triggers a cascade of inflammatory events, inflammatory cytokines released by murine peritoneal ad- characterized by edema formation, leukocyte recruitment herent cells. Toxicon 39: 1567-1573. into tissues, and release of inflammatory mediators, which Coelho AL, De Freitas MS, Mariano-Oliveira A, Rapozo DC, mimic a number of systemic and local inflammatory disor- Pinto LF, Niewiarowski S, Zingali RB, Marcinkiewicz C, ders in humans. Recent studies have shown that both Barja-Fidalgo C 2004. RGD- and MLD-disintegrins, disintegrin and disintegrin-like/cysteine-rich domains of jarastatin and EC3, activate integrin-mediated signaling SVMP are able to trigger leukocyte functions and cell sig- modulating the human neutrophils chemotaxis, apoptosis naling inflammatory pathways in vitro, and that proteolytic and IL-8 gene expression. Exp Cell Res 292: 371-384. domain per se can induce inflammatory events in vivo. Costa EP, Clissa PB, Teixeira CF, Moura-da-Silva AM 2002. However, further studies are necessary to identify the Importance of metalloproteinases and macrophages in vi- domain structural features which are required for these per snake envenomation-induced local inflammation. Inflam- inflammatory effects. Moreover, studies on characteriza- mation 26: 13