Lopap: A non-inflammatory and cytoprotective molecule in neutrophils and endothelial cells

Abstract

Lopap (Lonomia obliqua prothrombin activator protease) is a member of the lipocalin family isolated from the extract of L obliqua bristles. Lopap displays serine protease-like activities, including coagulation disturbance, cytokine secretion and antiapoptotic activity in human cultured endothelial cells. Here, we have investigated the effects of the recombinant protein (rLopap) on the inflammatory and apoptotic processes of neutrophils and endothelial cells from male Wistar rats. We found that rLopap did not induce in vivo leukocyte-endothelial interactions in the microvasculature, initial steps of leukocyte recruitment during inflammation. Incubation of rLopap with neutrophils or endothelial cells prevented apoptosis evoked by serum deprivation and induced nitric oxide (NO) production in both cell types, and increased the expression of ICAM-1 by endothelial cells. Simultaneous incubation of endothelial cells or neutrophils with rLopap and N omega-nitro-L-arginine methyl ester (L-NAME), a non-specific inhibitor of NO synthases, inhibited NO production and impaired the protection on apoptosis. Differently, incubation of endothelial cells with monoclonal antibody anti ICAM-1 did not change the protection on apoptosis evoked by rLopap. Together, these results indicate that rLopap does not display inflammatory properties in vivo but inhibits apoptosis of neutrophils and endothelial cells depending, at least in part, on NO production.

Toxins from Lonomia obliqua — Recombinant Production and Molecular Approach

“…These effects resemble the consumption coagulopathy triggered by the whole venom, indicating the involvement of this prothrombin activator as a key toxin in envenoming [26]. In addition, Lopap is able to modulate endothelial cell responses promoting cell survival, IL-8, nitric oxide (NO) and PGI 2 release, expression of the cell adhesion molecules ICAM-1 and E-selectin, but not VCAM-1 and PCAM-1 [45,77,78]. Lopap also displays cytoprotective activity in neuthrophils and do not modify expressions of L-selectin and β2 integrin.…”

“…Lopap also displays cytoprotective activity in neuthrophils and do not modify expressions of L-selectin and β2 integrin. Secretion of the proinflammatory cytokines IL-6 and TNF-α is not changed by Lopap treatment in both cell cultures [78]. Lopap seems to have no effect on modulation of coagulation and fibrinolysis through endothelial cell response, since it does not modify von Willebrand factor (vWF) and tissue plasminogen activator (t-PA) release or tissue factor procoagulant activity on endothelial cell surface [45,77].…”

New Insight into the Mechanism of <i>Lonomia obliqua</i> Envenoming: Toxin Involvement and Molecular Approach

“…Several studies have shown that, besides its prothrombin activation activity, Lopap demonstrates to exert action on endothelial cell responses [46,48,49] . Endothelial cells respond to external stimuli by altering the secretion or surface expression of biomolecules that regulate vascular tone and permeability, leukocyte traffic, platelet function, blood coagulation and fibrinolysis [50,51] .…”

“…On the other hand, NO and PGI 2 produced by endothelial cells have also been implicated as modulators of the leukocyte migration into inflammatory sites, by acting on leukocyte-endothelial interaction steps [53,54] and on expression and activity of adhesion molecules [55,56] . In a recent study [49] it was demonstrated that Lopap does not induce in vivo and in vitro inflammatory properties in neutrophils and endothelial cells (rolling, adherence into vessel wall endothelium, and pro-inflammatory cytokine secretion), thus the biological significance of expression of cytokines and cell adhesion molecules induced by Lopap remains to be clarified. Lopap is also unable to induce in HUVECs the release of pro-thrombotic mediators such as tissue factor and von Willebrand factor, and pro-fibrinolytic mediators such as tissue plasminogen activator (t-PA) [48] .…”

“…Fritzen et al [48] suggest that this effect is probably related to the Lopap-induced NO, since NO was also described as an endothelial survival factor, inhibiting apoptosis [75,76] . Recently, Waisman et al [49] showed that Lopap induces anti-apoptotic effects on endothelial cells and neutrophils, depending on NO production. Studies carried out with different cell lineages have demonstrated that NO modulates apoptosis [77,78] .…”