Patricia Bianca Clissa

Inflammatory pathogenesis of snake venom metalloproteinase-induced skin necrosis

Local tissue damage, characterized by edema, hemorrhage and necrosis, is a common consequence of envenoming by many vipers. We have investigated the contribution of inflammatory responses induced by the venom metalloproteinase jararhagin (isolated from Bothrops jararaca venom) in the development of these lesions. Local venom effects (edema, hemorrhage and necrosis) were inducedexperimentally in knockout mice deficient in the TNF receptors TNFR1 or TNFR2, IL‐1βR, IL‐6 and iNOS. Jararhagin‐induced dermal necrosis was abolished in mice deficient in the TNF receptors TNFR1 and TNFR2, and the same activity was significantly reduced in IL‐6–/– mice. There was no significant difference in edema and hemorrhage activities following jararhagin insult between knockout and WT strains, indicating that these local venom metalloproteinase‐induced effects are independent of these pro‐inflammatory mediators. The contribution of both TNF receptors and IL‐6 in local tissue necrosis raises important therapeutic issues regarding the treatment of local envenoming.

The effect of jararhagin, a metalloproteinase from Bothrops jararaca venom, on pro-inflammatory cytokines released by murine peritoneal adherent cells.

The release of pro-inflammatory cytokines (IL-1beta, IL-6 and TNF-alpha) from murine peritoneal adherent cells (MPAC) was studied after exposure to jararhagin, a metalloproteinase/disintegrin isolated from Bothrops jararaca venom. MPACs were treated with LPS (lipopolysaccharide), jararhagin, or EDTA-inactivated jararhagin for up to 24h. Following incubation, the culture supernatant was assayed by ELISA for the presence of cytokines, while the cells were analysed for viability and cytokine mRNA expression. The cells exposed to native jararhagin released TNF-alpha and IL-1beta after 4 and 24h respectively. When MPACs were exposed to Jararhagin treated with EDTA, TNF-alpha and IL-1beta production was sustained throughout the culture period and IL-6 production was observed. TNF-alpha, IL-6 and IL-1beta mRNA were detected 4h after stimulation with either native or EDTA-treated jararhagin. Addition of jararhagin to LPS stimulated cells resulted in a dramatic decrease in the release of IL-6 and TNF-alpha. RT-PCR showed that this inhibition does not occur at the transcriptional level and further experiments showed that jararhagin degraded soluble cytokines by proteolytic activity. This study suggests that jararhagin induces TNF-alpha, IL-1beta and IL-6 expression, which may be rapidly degraded by its proteolytic activity.

Characterisation of local inflammatory response induced by Thalassophryne nattereri fish venom in a mouse model of tissue injury.

The Thalassophryne nattereri fish venom induces a severe burning pain, oedema, and necrosis observed both clinically and experimentally. The present study was carried out in order to describe the pattern of local acute inflammatory response after T. nattereri venom injection. Our findings show that the edematogenic response induced by T. nattereri venom in footpad of mice was dose- and time dependent, and remained significantly elevated over 48 h after injection. Analysis of footpad homogenates were tested for the presence of TNF-alpha, IL-1beta and IL-6, and demonstrated augmented levels of these cytokines. Our results showed that the injection of venom developed an inadequate cellular inflammatory response evidenced by poor infiltration of mononuclear cells, preceded by decreased number of these cells in peripheral blood. In contrast, we observed an early intense recruitment of neutrophil to peritoneal cavity, accompanied by a significant decrease in the number of mononuclear cells. A drastic increase in the total amount of cells, mainly in neutrophils, followed by mononuclear cell recruitment was observed 24 h. In addition, we also demonstrated that T. nattereri venom affects the viability of mononuclear cells (J774A1) in culture. We conclude that the scarcity of inflammatory cellular influx into local lesions (intraplantar) induced by T. nattereri venom could be a consequence of an impaired blood flow in venules at injured tissue and cytotoxic effect of the venom on inflammatory cells can contribute to this impairment.

Different regions of the class P-III snake venom metalloproteinase jararhagin are involved in binding to α2β1 integrin and collagen.

SVMPs are multi-domain proteolytic enzymes in which disintegrin-like and cysteine-rich domains bind to cell receptors, plasma or ECM proteins. We have recently reported that jararhagin, a P-III class SVMP, binds to collagen with high affinity through an epitope located within the Da-disintegrin sub-domain. In this study, we evaluated the binding of jararhagin to alpha(2)beta(1) integrin (collagen receptor) using monoclonal antibodies and recombinant jararhagin fragments. In solid phase assays, binding of jararhagin to alpha(2)beta(1) integrin was detectable from concentrations of 20 nM. Using recombinant fragments of jararhagin, only fragment JC76 (residues 344-421), showed a significant binding to recombinant alpha(2)beta(1) integrin. The anti-jararhagin monoclonal antibody MAJar 3 efficiently neutralised binding of jararhagin to collagen, but not to recombinant alpha(2)beta(1) integrin nor to cell-surface-exposed alpha(2)beta(1) integrin (alpha(2)-K562 transfected cells and platelets). The same antibody neutralised collagen-induced platelet aggregation. Our data suggest that jararhagin binding to collagen and alpha(2)beta(1) integrin occurs by two independent motifs, which are located on disintegrin-like and cysteine-rich domains, respectively. Moreover, toxin binding to collagen appears to be sufficient to inhibit collagen-induced platelet aggregation.

Toxicity and immunogenicity of Crotalus durissus terrificus venom treated with different doses of gamma rays

Crotalus durissus terrificus venom (CDT venom) was irradiated with four different doses of gamma rays (2, 3, 5 and 10 kGy) from a 60Co source and their structural, toxic and immunogenic properties were analysed. Venom irradiated with 2 and 3 kGy were, respectively, 2.7 and 13.5 times less toxic than the native one, whereas the 5 or 10 kGy irradiated venom were at least 100 times less toxic than nonirradiated venom. Irradiated venom with all doses were immunogenic and the antibodies elicited by them were able to recognise the native venom in ELISA. However the toxoid produced with 2 kGy irradiation dose had its immunogenicity improved. Antisera raised against this toxoid had a higher neutralising capacity than those produced against the native venom. Irradiation of venom with 2 kGy dose was the most effective to inactivate the CDT venom toxicity and improve its immunogenicity.

Characterization of inflammatory reaction induced by neuwiedase, a P-I metalloproteinase isolated from Bothrops neuwiedi venom.

The Snake Venom Metalloproteinases (SVMPs) play a relevant role in the multifactorial inflammatory response induced by Bothrops envenomations. Neuwiedase, an SVMP isolated from Bothrops neuwiedi venom, is devoid of hemorrhagic activity on skin tests, but is able to induce myonecrosis and degrade fibrinogen, fibrin, type I collagen, fibronectin and laminin. In this study, we analyzed the inflammatory reaction induced by neuwiedase in gastrocnemius muscle, with special focus on cytokines release. Our results showed clear evidence of inflammatory infiltrate in the gastrocnemius muscle and an increase of MMP-9, and the cytokines KC, IL-1 beta and IL-6 in the early periods after toxin injection. The cytokine release was also evaluated in inflammatory and muscular cell culture. Both murine peritoneal adherent cells (MPACs) and muscle cells (C2C12) released pro-inflammatory cytokines after stimulus with neuwiedase. MPACs showed increased production of KC, IL-1 beta and IL-6 in the cell culture supernatant while in C2C12, the predominant chemokine expressed was KC. These data reinforce the importance of SVMPs in the inflammatory response caused by envenomation and point out the role of muscle cells in this event by releasing pro-inflammatory mediators able to attract leukocytes to the muscle, thus starting and amplifying the setting of the inflammatory reaction.

Insights of local tissue damage and regeneration induced by BnSP-7, a myotoxin isolated from Bothrops (neuwiedi) pauloensis snake venom.

Envenomations caused by Bothrops snake venoms are characterized by prominent local tissue damage due to myonecrosis, hemorrhage, edema and acute muscle damage which is widely correlated with phospholipases A2 (PLA2). In the present study, the progression of local tissue damage and inflammation induced by BnSP-7, a myotoxin isolated from Bothrops (neuwiedi) pauloensis snake venom, was evaluated. Local tissue damages characterized by edema, necrosis and inflammation were evaluated until 24 h after inoculation of BnSP-7. The regeneration of myofibers, analyzed by light microscopy, was observed from 72 h to 2 weeks post-inoculation of toxin. MMP-2 was expressed in gastrocnemius muscle at all time points tested, while the expression of MMP-9 increased expressively at the same time interval of regenerating muscle, suggesting the involvement of MMP-9 in the regeneration process. The production of pro-inflammatory cytokines was also increased, whereas IL-1 beta showed the highest level. Modification of BnSP-7 with BPB decreased the release of IL-8, IL-6 and IL-1 beta when compared to native BnSP-7. These data suggest that BnSP-7 acts as pro-inflammatory incentives (mediators), inducing MMP and cytokine production from the inflammatory and satellite cells, and thus it may play an important role in inflammatory process and, consequently, in the evolution of local tissue damage and regeneration.

Insularin, a disintegrin from Bothrops insularis venom: inhibition of platelet aggregation and endothelial cell adhesion by the native and recombinant GST-insularin proteins.

Insularin (INS) was obtained from Bothrops insularis venom by reversed-phase high-performance liquid chromatography using a C(18) column and characterized as a disintegrin by peptide mass fingerprint and inhibition of ADP-induced platelet aggregation. A cDNA coding for P-II a metalloproteinase/disintegrin was cloned from a cDNA library from B. insularis venom glands. The deduced protein sequence possesses 73 amino acid residues, including the N-terminal, internal peptides of native insularin, the ARGDNP-sequence and 12 cysteines in a conserved alignment. This cDNA fragment was subcloned in the pGEX-4T-1 vector and expressed in a prokaryotic expression system as a fusion protein with glutathione S-transferase (GST-INS). Both native and recombinant insularin inhibited ADP-induced platelet aggregation and endothelial cells (HUVEC) adhesion with similar activities indicating that GST-INS folded correctly and preserved the integrin-binding loop. Insularin may be a tool in studies that involve platelets and endothelial cell adhesion dependent on alphaIIbeta3 and alphavbeta3 integrins.

Jararhagin disruption of endothelial cell anchorage is enhanced in collagen enriched matrices.

Hemorrhage is one of the most striking effects of bites by viper snakes resulting in fast bleeding and ischemia in affected tissues. Snake venom metalloproteinases (SVMPs) are responsible for hemorrhagic activity, but the mechanisms involved in SVMP-induced hemorrhage are not entirely understood and the study of such mechanisms greatly depends on in vivo experiments. In vivo, hemorrhagic SVMPs accumulate on basement membrane (BM) of venules and capillary vessels allowing the hydrolysis of collagen IV with consequent weakness and rupture of capillary walls. These effects are not reproducible in vitro with conventional endothelial cell cultures. In this study we used two-dimension (2D) or three-dimension (3D) cultures of HUVECs on matrigel and observed the same characteristics as in ex vivo experiments: only the hemorrhagic toxin was able to localize on surfaces or internalize endothelial cells in 2D cultures or in the surface of tubules formed on 3D cultures. The contribution of matrigel, fibronectin and collagen matrices in jararhagin-induced endothelial cell damage was then analyzed. Collagen and matrigel substrates enhanced the endothelial cell damage induced by jararhagin allowing toxin binding to focal adhesions, disruption of stress fibers, detachment and apoptosis. The higher affinity of jararhagin to collagen than to fibronectin explains the localization of the toxin within BM. Moreover, once located in BM, interactions of jararhagin with α2β1 integrin would favor its localization on focal adhesions, as observed in our study. The accumulation of toxin in focal adhesions, observed only in cells grown in collagen matrices, would explain the enhancement of cell damage in these matrices and reflects the actual interaction among toxin, endothelial cells and BM components that occurs in vivo and results in the hemorrhagic lesions induced by viper venoms.

Jararhagin-induced mechanical hyperalgesia depends on TNF-α, IL-1β and NFκB in mice

Jararhagin is a hemorrhagic metalloprotease from Bothrops jararaca snake venom. The hyperalgesic mechanisms of jararhagin were investigated focusing on the role of proinflammatory cytokines (TNF-α and IL-1β) and the transcription factor NFκB. Intraplantar administration of jararhagin (1, 10, 100 and 1000 ng/paw) induced mechanical hyperalgesia, and increased TNF-α levels at 1, 3 and 5 h, and IL-1β levels at 0.5, 1 and 3 h after its injection in the paw tissue. Pre-treatment with morphine (2, 6, 12 μg/paw) inhibited jararhagin-induced mechanical hyperagesia. The systemic or local pre-treatment with etanercept (10 mg/kg and 100 μg/paw) and IL-1ra (30 mg/kg and 100 pg/paw) inhibited jararhagin-induced mechanical hyperalgesia. Co-administration of jararhagin (0.1 ng/paw) and TNF-α (0.1 pg/paw) or jararhagin (0.1 ng/paw) and IL-1β (1 pg/paw) enhanced the mechanical hyperalgesia. The systemic or local pre-treatment with PDTC (NFκB inhibitor; 100 mg/kg and 100 μg/paw) inhibited jararhagin-induced mechanical hyperalgesia as well as PDTC decreased the jararhagin-induced production of TNF-α and IL-1β. Thus, these data demonstrate the involvement of pro-inflammatory cytokines TNF-α and IL-1β and nuclear transcription factor NFκB in jararhagin-induced mechanical hyperalgesia indicating that targeting these mechanisms might contribute to reduce the pain induced by B. jararaca snake venom.