Sandra H.P. Farsky

Melatonin and N-acetylserotonin inhibit leukocyte rolling and adhesion to rat microcirculation

The hormone melatonin produced by the pineal gland during the daily dark phase regulates a variety of biological processes in mammals. The aim of this study was to determine the effect of melatonin and its precursor N-acetylserotonin on the microcirculation during acute inflammation. Arteriolar diameter, blood flow rate, leukocyte rolling and adhesion were measured in the rat microcirculation in situ by intravital microscopy. Melatonin alone or together with noradrenaline did not affect the arteriolar diameter or blood flow rate. Melatonin inhibited both leukocyte rolling and leukotriene B(4) induced adhesion while its precursor N-acetylserotonin inhibits only leukocyte adhesion. The rank order of potency of agonists and antagonist receptor selective ligands suggested that the activation of MT(2) and MT(3) melatonin binding sites receptors modulate leukocyte rolling and adhesion, respectively. The effect of melatonin and N-acetylserotonin herein described were observed with concentrations in the range of the nocturnal surge, providing the first evidence for a possible physiological role of these hormones in acute inflammation.

Leukocyte response induced by Bothrops jararaca crude venom: in vivo and in vitro studies.

The effect of Bothrops jararaca crude venom (BjV) on the cellular component of inflammatory responses was investigated in vivo and in vitro. In vivo leukocyte accumulation and release of eicosanoids (thromboxane A2, TXA2, and leukotriene B4, LTB4) at the site of injection of the venom were assessed using the air pouch method in rats. Administration of BjV caused a significant cell accumulation, maximal values being obtained after 6-8 hr. Neutrophils were the predominant cell type in the inflammatory exudate. High concentrations of LTB4 were detected 1-4 hr after the injection of the venom. TXA2 concentrations were significantly increased only at the early stages of the response to the venom. In vitro chemotaxis assays were performed and showed that the venom per se was not able to induce oriented neutrophil migration because varying concentrations of the venom dissolved in Hanks balanced salt solution (HBSS) evoked a response equivalent to that of HBSS alone. Furthermore, the venom did not affect cellular intrinsic mechanisms involved with neutrophil locomotion because previous incubation of the cells with BjV produced no effect. However, high concentrations of the venom were able to generate serum chemotactic factor(s). Incubation of serum with the venom evoked a neutrophil migration similar to that observed with serum activated by lipopolysaccharide from Escherichia coli. Participation of chemotactic factors derived from the complement system is suggested by data showing loss of this activity when serum was heated (56 degrees C) before the addition of BjV. The present results suggest that leukocyte accumulation in the locality of a lesion induced by BjV is dependent on secretion or activation of endogenous components responsible for several steps in leukocyte recruitment instead of a direct effect of the venom on leukocytes.

Bothrops asper snake venom and its metalloproteinase BaP–1 activate the complement system. Role in leucocyte recruitment

The venom of the snake Bothrops asper, the most important poisonous snake in Central America, evokes an inflammatory response, the mechanisms of which are not well characterized. The objectives of this study were to investigate whether B. asper venom and its purified toxins--phospholipases and metalloproteinase--activate the complement system and the contribution of the effect on leucocyte recruitment. In vitro chemotaxis assays were performed using Boydens chamber model to investigate the ability of serum incubated with venom and its purified toxins to induce neutrophil migration. The complement consumption by the venom was evaluated using an in vitro haemolytic assay. The importance of complement activation by the venom on neutrophil migration was investigated in vivo by injecting the venom into the peritoneal cavity of C5-deficient mice. Data obtained demonstrated that serum incubated with crude venom and its purified metalloproteinase BaP-1 are able to induce rat neutrophil chemotaxis, probably mediated by agent(s) derived from the complement system. This hypothesis was corroborated by the capacity of the venom to activate this system in vitro. The involvement of C5a in neutrophil chemotaxis induced by venom-activated serum was demonstrated by abolishing migration when neutrophils were pre-incubated with antirat C5a receptor antibody. The relevance of the complement system in in vivo leucocyte mobilization was further demonstrated by the drastic decrease of this response in C5-deficient mice. Pre-incubation of serum with the soluble human recombinant complement receptor type 1 (sCR 1) did not prevent the response induced by the venom, but abolished the migration evoked by metalloproteinase-activated serum. These data show the role of the complement system in bothropic envenomation and the participation of metalloproteinase in the effect. Also, they suggest that the venom may contain other component(s) which can cause direct activation of C5a.

Skeletal muscle necrosis and regeneration after injection of Thalassophryne nattereri (niquim) fish venom in mice

Stings by Thalassophryne nattereri are responsible for envenomation of fishermen in north‐eastern Brazil. Its venom induces prominent local tissue damage, characterized by pain, oedema and necrosis. The pathogenesis of acute muscle damage induced by T. nattereri venom was studied in mice. Intramuscular injection induced myonecrosis within the first hours. Some muscle cells presented a hypercontracted morphology, but most necrotic fibres were not hypercontracted, being instead characterized by a disorganization of myofibrils, with Z line loss, mitochondrial swelling and sarcolemmal disruption. In addition, thrombosis was observed histologically in venules and veins, together with vascular congestion and stasis, evidenced by intravital microscopy. Venom induced a rapid increment in serum creatine kinase (CK) levels, concomitant with a reduction in gastrocnemius muscle CK activity, whereas no increments in muscle lactic acid were detected. A rapid cytolytic effect was induced by the venom on C2C12 murine myoblasts in culture. The inflammatory reaction in affected muscle was characterized by oedema and scarce cellular infiltrate of polymorphonuclear leucocytes and macrophages, with a consequent delay in the removal of necrotic material. Skeletal muscle regeneration was partially impaired, as evidenced by the presence of regenerating fibres of variable size and by the increase of fibrotic tissue in endomysium and perimysium. It is suggested that T. nattereri venom affects muscle fibres by a direct cytotoxic effect, and that the vascular alterations described preclude a successful regenerative process.

Hemostatic effects induced by Thalassophryne nattereri fish venom: a model of endothelium-mediated blood flow impairment

Accidents by Thalassophryne nattereri fish venom are characterised by severe local symptoms and signs including pain of fast onset, oedema and necrosis with impaired muscle regeneration. These effects have been related to alterations in hemostatic mechanisms and cytolytic effects rather than to conventional inflammatory pathways. In this work we evaluated the effects induced by the venom on microcirculatory vessels, platelets and blood coagulation. Effects evoked by topical application of venom on cremaster muscle were visualised through intravital microscopy. Stasis was observed, concomitantly with the presence of thrombi in venules and focal transient constrictions in arterioles, all of which impaired the blood flow. Significant alterations on vessel walls took place few minutes after venom application, characterised by increment in thickness, probably by deposition of fibrin. Increase in vascular permeability was also observed in venules. Additionally, the action of the venom was locally restricted since no alteration on systemic blood coagulation was observed. Venom lacked a direct pro-coagulant activity, but exerted a strong cytolytic effect on platelets and endothelial cells in vitro. These data suggest that venom action on endothelium may contribute to blood stasis and to the formation of platelet and fibrin thrombi, with the consequent ischemia, contributing to the local effects of the venom.

Role of crotoxin, a phospholipase A2 isolated from Crotalus durissus terrificus snake venom, on inflammatory and immune reactions

BACKGROUND: Crotoxin (CTX) is a potent neurotoxin from Crotalus durissus terrificus snake venom (CdtV) composed of two subunits: one without catalytic activity (crotapotin), and a basic phospolipase A2. Recent data have demonstrated that CdtV or CTX inhibit some immune and inflammatory reactions. AIM: The aim of this paper was to investigate the mechanisms involved in these impaired responses. MATERIALS AND METHODS: Male Swiss mice were bled before and at different intervals of time after subcutaneous injection of CTX or bovine serum albumin (BSA) (control animals). The effect of treatments on circulating leukocyte mobilisation and on serum levels of interleukin (IL)-6, IL-10, interferon (IFN)-gamma and corticosterone were investigated. Spleen cells from treated animals were also stimulated in vitro with concanavalin A to evaluate the profile of IL-4, IL-6, IL-10 or IFN-gamma secretion. Cytokine levels were determined by immunoenzymatic assay and corticosterone levels by radioimmunoassay. To investigate the participation of endogenous corticosteroid on the effects evoked by CTX, animals were treated with metyrapone, an inhibitor of glucocorticoid synthesis, previous to CTX treatment. RESULTS: Marked alterations on peripheral leukocyte distribution, characterised by a drop in the number of lymphocytes and monocytes and an increase in the number of neutrophils, were observed after CTX injection. No such alteration was observed in BSA-treated animals. Increased levels of IL-6, IL-10 and corticosterone were also detected in CTX-injected animals. IFN-gamma levels were not modified after treatments. In contrast, spleen cells obtained from CTX-treated animals and stimulated with concanavalin A secreted less IL-10 and IL-4 in comparison with cells obtained from control animals. Metyrapone pretreatment was effective only to reverse the neutrophilia observed after CTX administration. CONCLUSIONS: Our results suggest that CTX may contribute to the deficient inflammatory and immune responses induced by crude CdtV. CTX induces endogenous mechanisms that are responsible, at least in part, for these impaired responses.

Hormonal control of inflammatory responses.

Almost any stage of inflammatory and immunological responses is affected by hormone actions. This provides the basis for the suggestion that hormones act as modulators of the host reaction against trauma and infection. Specific hormone receptors are detected in the reactive structures in inflamed areas and binding of hormone molecules to such receptors results in the generation of signals that influence cell functions relevant for the development of inflammatory responses. Diversity of hormonal functions accounts for recognized pro- and anti-inflammatory effects exerted by these substances. Most hormone systems are capable of influencing inflammatory events. Insulin and glucocorticoids, however, exert direct regulatory effects at concentrations usually found in plasma. Insulin is endowed with facilitatory actions on vascular reactivity to inflammatory mediators and inflammatory cell functions. Increased concentrations of circulating glucocorticoids at the early stages of inflammation results in downregulation of inflammatory responses. Oestrogens markedly reduce the response to injury in a variety of experimental models. Glucagon and thyroid hormones exert indirect anti-inflammatory effects mediated by the activity of the adrenal cortex. Accordingly, inflammation is not only merely a local response, but a hormone-controlled process.

Pro and Antiinflammatory Properties of Toxins from Animal Venoms

Accidents evoked by venomous animals are common in tropical regions. In Brazil, envenomation evoked by snakes, spiders and scorpions are an important public health problem. Their venoms are composed of a great number of toxins, which are capable of acting on tissue and plasma components with consequent toxic and pharmacological effects. On the other hand, the diversity of venom composition makes them important source of toxins that can be employed as scientific tools. Here we describe the mechanisms of anti and pro-inflammatory properties of toxins of Bothrops and Crotalus genus snakes and Loxosceles and Phoneutria genus spider venoms. The emphasis was to summarise, both in vivo and in vitro, studies that focused on the action of phospholipases, metalloproteinases and sphingomyelinase D on vascular and cellular aspects of the process as well as the complex network of chemical mediators involved.

Characterization of local tissue damage evoked by Bothrops jararaca venom in the rat connective tissue microcirculation: an intravital microscopic study

The aim of this study was to investigate the dynamic of the effects after topical application of Bothrops jararaca venom (BjV) on the microcirculation of the internal spermatic fascia of Wistar rats. The administration of 1 microg of BjV induced marked disturbances in capillaries and postcapillary venules, characterized by vasodilatation, fibrin clot formation, hemorrhagic lesions and alterations in the leukocyte-endothelial interactions. The installation and development of these effects occurred simultaneously. The application of higher dose of venom (10 microg) induced more intense effects, observed by the time-course of the beginning, evolution and intensity of the effects. The time-course of events suggests that an interaction of different effects occurs during the development of local symptoms following the exposure of tissues to BjV.

Role of kinins and nitric oxide on the rabbit arthritis induced by Bothrops jararaca venom.

The effects of the nitric oxide synthase inhibitor N(omega)Nitro-L-arginine-methyl ester (L-NAME) and of the bradykinin B(2) receptor antagonist HOE 140 were evaluated in the inflammatory reaction induced by Bothrops jararaca venom (BjV) in New Zealand White rabbits. Arthritis was induced by injecting 0.5 ml of a sterile solution of BjV (1-64 microg/ml) into the knee intraarticular cavity. The contralateral joint was injected with bovine serum albumin (BSA) diluted in sterile saline. At selected times thereafter (4, 24 and 48 h), the vascular permeability and the leukocyte influx in both the synovial fluid and synovium were evaluated. BjV caused a dose-dependent increase in both leukocyte influx and protein extravasation which reached a maximal response at 16 microg. Bothrops jararaca venom also induced the increase in the leukocyte accumulation in the synovium and in the concentration of both NO(2)/NO(3) in the synovial fluid. Chronic administration of L-NAME (20 mg/kg/day in the drinking water for 2 weeks) markedly reduced the leukocyte accumulation (90%), protein leakage (44%), and NO(2)/NO(3) (50%) levels in the synovial fluid, measured at the 4th h. Hoe 140, given i.v. (0.3 mg/kg, 30 min before) also reduced leukocyte accumulation (75%), protein leakage (48%), and NO(2)/NO(3) (79%) levels in the synovial fluid, measured at the 4th h. Similar results were obtained with acute administration of L-NAME (30 mg/kg, i.v., 30 min before). These results indicate that arthritis induced by BjV is triggered by kinin formation and that the increase in both vascular permeability and leukocyte accumulation is modulated by NO release.