Methotrexate and cyclosporine treatments modify the activities of dipeptidyl peptidase IV and prolyl oligopeptidase in murine macrophages.

Analysis of the effects of cyclosporine A (25-28 mgkg(-1)) and/or methotrexate (0.1 mgkg(-1)) treatments on dipeptidyl peptidase IV (DPPIV) and prolyl oligopeptidase (POP) activities and on algesic response in two distinct status of murine macrophages (Mphis) was undertaken. In resident Mphis, DPPIV and POP were affected by neither individual nor combined treatments. In thioglycolate-elicited Mphis, methotrexate increased DPPIV (99-110%) and POP (60%), while cyclosporine inhibited POP (21%). Combined treatment with both drugs promoted a rise (51-84%) of both enzyme activities. Only cyclosporine decreased (42%) the tolerance to algesic stimulus. Methotrexate was revealed to exert prevalent action over that of cyclosporine on proinflammatory Mphi POP. The opposite effects of methotrexate and cyclosporine on POP activity might influence the availability of the nociceptive mediators bradykinin and substance P in proinflammatory Mphis. The exacerbated response to thermally induced algesia observed in cyclosporine-treated animals could be related to upregulation of those mediators.

A glycoprotein with anti-inflammatory properties secreted by an Aspergillus nidulans modified strain

Total RNA from lipopolysaccharide (LPS)-stimulated rat macrophages used to treat protoplasts from an Aspergillus nidulans strain originated the RT2 regenerated strain, whose culture supernatant showed anti-inflammatory activity in Wistar rats. The protein fraction presenting such anti-inflammatory activity was purified and biochemically identified. The screening of the fraction responsible for such anti-inflammatory property was performed by evaluating the inhibition of carrageenan-induced paw edema in male Swiss mice. Biochemical analyses of the anti-inflammatory protein used chromatography, carbohydrates quantification of the protein sample, amino acids content analysis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Total sugar quantification revealed 32 percent glycosylation of the protein fraction. Amino acid analysis of such fraction showed a peculiar pattern presenting 29 percent valine. SDS-PAGE revealed that the protein sample is pure and its molecular weight is about 40kDa. Intravenous injection of the isolated substance into mice significantly inhibited carrageenan-induced paw edema. The isolated glycoprotein decreased carrageenan-induced paw edema in a prostaglandin-dependent phase, suggesting an inhibitory effect of the isolated glycoprotein on prostaglandin synthesis.

Role of nitric oxide in the local and systemic pathophysiological effects induced by Bothrops asper snake venom in mice.

OBJECTIVE: To assess the role of nitric oxide in the most relevant local and systemic manifestations in mice injected with the venom of the snake Bothrops asper. Mice were pretreated with nitric oxide synthase inhibitors, and the modifications of the pathological effects induced by the venom were tested. RESULTS: Inhibition of NO synthesis did not affect acute local myonecrosis and hemorrhage in muscle tissue upon intramuscular injection of venom. Local footpad edema was reduced in mice pretreated with the NO synthase inhibitor L-NAME, and a reduction in the extent of inflammatory infiltrate in muscle tissue was observed after envenomation in mice pretreated with L-NAME and aminoguanidine. The most pronounced effect of NOS inhibition by L-NAME was an increment in the lethal activity of the venom, when injected by the intraperitoneal route. CONCLUSION: Nitric oxide does not seem to play a significant role in the local acute pathological alterations (hemorrhage and myonecrosis) induced by B. asper venom in mice, although it contributes to edema and inflammatory infiltrate. Nitric oxide exerts a protective role in the systemic pathophysiological manifestations leading to lethality.

Association of endothelin with lung hemorrhage induced by immune complexes in rats.

The participation of endothelins (ETs) in a model of neutrophil-dependent lung injury induced by intrabronchial instillation of rabbit antibodies to ovalbumin followed by i.v. injection of the antigens (Arthus reaction) was investigated. Hemorrhagic lesions were evaluated by measuring the extravasations of hemoglobin in lung parenchyma. From 5 min to 24 h after the Arthus reaction (AR), endothelin (ir-ET) levels in bronchoalveolar lavage fluid (BALF) and in plasma were measured by radioimmunoassay. BALF levels of ir-ET were not different between control and AR animals for the first 90 min after the antigen challenge but increased from 2 to 24 h after induction of AR. ET levels in the plasma did not change from the respective controls over the same 24 h period. Increased ir-ET in BALF was not affected by pretreatment with L-NAME (30 mg/kg, i.v.). A PAF antagonist (BN52021; 5 and 10 mg/kg, i.v.) increased ET content in BALF and decreased the intensity of the AR. Thiorphan (2 mg/kg, i.v.) inhibited the AR-induced hemorrhagic lesions in lungs. An ET(A) receptor antagonist, BQ-123 (1 mg/kg, i.v.) potentiated, whereas the ET(B) antagonist, BQ-788 (1 mg/kg, i.v.) inhibited the lung hemorrhage. It is concluded that ETs are released during and play a role in the lung AR.

Neutrophils do not contribute to local tissue damage, but play a key role in skeletal muscle regeneration, in mice injected with Bothrops asper snake venom.

Local tissue damage induced by crotaline snake venoms includes edema, myonecrosis, hemorrhage, and an inflammatory response associated with a prominent cellular infiltrate. The role of neutrophils in the local tissue damage induced by Bothrops asper snake venom and by myotoxin I, a phospholipase A2 isolated from this venom, was investigated. Male Swiss mice were pretreated with either an antimouse granulocyte rat monoclonal immunoglobulin G (IgG) antibody or with isotype-matched control antibody. No significant differences in these local effects were observed between mice pretreated with antigranulocyte antibodies and those receiving control IgG. Moreover, myotoxicity induced by B. asper myotoxin I was similar in neutrophil-depleted and control mice. The role of neutrophils in the process of skeletal muscle regeneration was also assessed. Muscle regeneration was assessed by quantifying the muscle levels of creatine kinase and by morphometric histological analysis of the area comprised by regenerating cells in damaged regions of skeletal muscle. Mice depleted of neutrophils and then injected with B. asper venom showed a more deficient regenerative response than mice pretreated with control IgG. Moreover, a drastic difference in the regenerative response was observed in mice injected with myotoxin I, because animals pretreated with control IgG showed a successful regeneration, whereas those depleted of neutrophils had abundant areas of necrotic tissue that had not been removed 7 days after injection, associated with reduced contents of creatine kinase. It is concluded that (1) neutrophils do not play a significant role in the acute local pathological alterations induced by the venom of B. asper, and (2) neutrophils play a prominent role in the process of skeletal muscle regeneration after injection of B. asper venom and myotoxin I, probably related to the phagocytosis of necrotic material and the recruitment of other inflammatory cells, two events directly associated with a successful muscle regenerative response.

Hyperalgesia induced by Asp49 and Lys49 phospholipases A2 from Bothrops asper snake venom: pharmacological mediation and molecular determinants.

The ability of Lys49 and Asp49 phospholipases A(2) (PLA(2)), from Bothrops asper snake venom, to cause hyperalgesia was investigated in rats, using the paw pressure test. Intraplantar injection of both toxins (5-20 micro g/paw) caused hyperalgesia, which peaked 1h after injections. Incubation of both proteins with heparin, prior to their injection, partially reduced this response. Chemical modification of Asp49 PLA(2) with p-bromophenacyl bromide (p-BPB), which abrogates its PLA(2) activity, also abolished hyperalgesia. Intraplantar injection of a synthetic peptide corresponding to the C-terminal sequence 115-129 of Lys49 PLA(2), caused hyperalgesia of similar time course, but varying magnitude, than that induced by the native protein. In contrast, a homologous peptide derived from the Asp49 PLA(2) did not show any nociceptive effect. Hyperalgesia induced by both PLA(2)s was blocked by the histamine and serotonin receptor antagonists promethazine and methysergide, respectively, by the bradykinin B(2) receptor antagonist HOE 140 and by antibodies to tumor necrosis factor alfa (TNFalpha) and interleukin 1 (IL-1). Pretreatment with guanethidine, atenolol, prazosin and yohimbine, inhibitors of sympathomimetic amines, or with indomethacin, inhibitor of the cyclo-oxygenase pathway, reduced Lys49 PLA(2)-induced hyperalgesia without interfering with the nociceptive activity of Asp49 PLA(2). The hyperalgesic response to both myotoxins was not modified by pretreatment with celecoxib, an inhibitor of the cyclo-oxygenase type II, by zileuton, an inhibitor of the lipoxygenase pathway or by N(g)-methyl-L-arginine (LNMMA), an inhibitor of nitric oxide synthase. These results suggest that Asp49 and Lys49 PLA(2)s are important hyperalgesic components of B. asper venom, and that Lys49 and Asp49 PLA(2)s exert their algogenic actions through different molecular mechanisms.

Inflammatory effects of snake venom myotoxic phospholipases A2.

Snake venom phospholipases A2 (PLA2) show a remarkable functional diversity. Among their toxic activities, some display the ability to cause rapid necrosis of skeletal muscle fibers, thus being myotoxic PLA2s. Besides myotoxicity, these enzymes evoke conspicuous inflammatory and nociceptive events in experimental models. Local inflammation and pain are important characteristics of snakebite envenomations inflicted by viperid and crotalid species, whose venoms are rich sources of myotoxic PLA2s. Since the discovery that mammalian PLA2 is a key enzyme in the release of arachidonic acid, the substrate for the synthesis of several lipid inflammatory mediators, much interest has been focused on this enzyme in the context of inflammation. The mechanisms involved in the proinflammatory action of secretory PLA2s are being actively investigated, and part of the knowledge on secretory PLA2 effects has been gained by using snake venom PLA2s as tools, due to their high structural homology with human secretory PLA2s. The inflammatory events evoked by PLA2s are primarily associated with enzymatic activity and to the release of arachidonic acid metabolites. However, catalytically inactive Lys49 PLA2s trigger inflammatory and nociceptive responses comparable to those of their catalytically active counterparts, thereby evidencing that these proteins promote inflammation and pain by mechanisms not related to phospholipid hydrolysis nor to mobilization of arachidonic acid. These studies have provided a boost to the research in this field and various approaches have been used to identify the amino acid residues and the specific sites of interaction of myotoxic PLA2s with cell membranes potentially involved in the PLA2-induced inflammatory and nociceptive effects. This work reviews the proinflammatory and nociceptive effects evoked by myotoxic PLA2s and their mechanisms of action.

Hyperalgesia induced by Asp49 and Lys49 phospholipases A2 from Bothrops asper snake venom Pharmacological mediation and molecular determinants

The ability of Lys49 and Asp49 phospholipases A2 (PLA2), from Bothrops asper snake venom, to cause hyperalgesia was investigated in rats, using the paw pressure test. Intraplantar injection of both toxins (5-20ìg/paw) caused hyperalgesia, which peaked 1h after injections. Incubation of both proteins with heparin, prior to their injection, partially reduced this response. Chemical modification of Asp49 PLA2 with p-bromophenacyl bromide (p-BPB), which abrogates its PLA2 activity, also abolished hyperalgesia. Intraplantar injection of a synthetic peptide corresponding to the C-terminal sequence 115-129 of Lys49 PLA2, caused hyperalgesia of similar time course, but varying magnitude, than that induced by the native protein. In contrast, a homologous peptide derived from the Asp49 PLA2 did not show any nociceptive effect. Hyperalgesia induced by both PLA2s was blocked by the histamine and serotonin receptor antagonists promethazine and methysergide, respectively, by the bradykinin B2 receptor antagonist HOE 140 and by antibodies to tumor necrosis factor alfa (TNFá) and interleukin 1 (IL-1). Pretreatment with guanethidine, atenolol, prazosin and yohimbine, inhibitors of sympathomimetic amines, or with indomethacin, inhibitor of the cyclo-oxygenase pathway, reduced Lys49 PLA2-induced hyperalgesia without interfering with the nociceptive activity of Asp49 PLA2. The hyperalgesic response to both myotoxins was not modified by pretreatment with celecoxib, an inhibitor of the cyclo-oxygenase type II, by zileuton, an inhibitor of the lipoxygenase pathway or by Ng-methyl-L-arginine (LNMMA), an inhibitor of nitric oxide synthase. These results suggest that Asp49 and Lys49 PLA2s are important hyperalgesic components of B. asper venom, and that Lys49 and Asp49 PLA2s exert their algogenic actions through different molecular mechanisms.

Inflammatory effects of snake venom myotoxic phospholipases A2

Snake venom phospholipases A2 (PLA2) show a remarkable functional diversity. Among their toxic activities, some display the ability to cause rapid necrosis of skeletal muscle fibers, thus being myotoxic PLA2s. Besides myotoxicity, these enzymes evoke conspicuous inflammatory and nociceptive events in experimental models. Local inflammation and pain are important characteristics of snakebite envenomations inflicted by viperid and crotalid species, whose venoms are rich sources of myotoxic PLA 2s. Since the discovery that mammalian PLA2is a key enzyme in the release of arachidonic acid, the substrate for the synthesis of several lipid inflammatory mediators, much interest has been focused on this enzyme in the context of inflammation. The mechanisms involved in the proinflammatory action of secretory PLA2s are being actively investigated, and part of the knowledge on secretory PLA2 effects has been gained by using snake venom PLA2s as tools, due to their high structural homology with human secretory PLA2s. The inflammatory events evoked by PLA2s are primarily associated with enzymatic activity and to the release of arachidonic acid metabolites. However, catalytically inactive Lys49 PLA2s trigger inflammatory and nociceptive responses comparable to those of their catalytically active counterparts, thereby evidencing that these proteins promote inflammation and pain by mechanisms not related to phospholipid hydrolysis nor to mobilization of arachidonic acid. These studies have provided a boost to the research in this field and various approaches have been used to identify the amino acid residues and the specific sites of interaction of myotoxic PLA2s with cell membranes potentially involved in the PLA2-induced inflammatory and nociceptive effects. This work reviews the proinflammatory and nociceptive effects evoked by myotoxic PLA2s and their mechanisms of action. © 2004 Published by Elsevier Ltd.

Evaluation of antivenoms in the neutralization of hyperalgesia and edema induced by Bothrops jararaca and Bothrops asper snake venoms.

Neutralization of hyperalgesia induced by Bothrops jararaca and B. asper venoms was studied in rats using bothropic antivenom produced at Instituto Butantan (AVIB, 1 ml neutralizes 5 mg B. jararaca venom) and polyvalent antivenom produced at Instituto Clodomiro Picado (AVCP, 1 ml neutralizes 2.5 mg B. aspar venom). The intraplantar injection of B. jararaca and B. asper venoms caused hyperalgesia, which peaked 1 and 2 h after injection, respectively. Both venoms also induced edema with a similar time course. When neutralization assays involving the independent injection of venom and antivenom were performed, the hyperalgesia induced by B. jararaca venom was neutralized only when bothropic antivenom was administered iv 15 min before venom injection, whereas edema was neutralized when antivenom was injected 15 min or immediately before venom injection. On the other hand, polyvalent antivenom did not interfere with hyperalgesia or edema induced by B. asper venom, even when administered prior to envenomation. The lack of neutralization of hyperalgesia and edema induced by B. asper venom is not attributable to the absence of neutralizing antibodies in the antivenom, since neutralization was achieved in assays involving preincubation of venom and antivenom. Cross-neutralization of AVCP or AVIB against B. jararaca and B. asper venoms, respectively, was also evaluated. Only bothropic antivenom partially neutralized hyperalgesia induced by B. asper venom in preincubation experiments. The present data suggest that hyperalgesia and edema induced by Bothrops venoms are poorly neutralized by commercial antivenoms even when antibodies are administered immediately after envenomation.

Evaluation of antivenoms in the neutralization of hyperalgesia and edema induced by Bothrops jararaca and Bothrops asper snake venoms

Neutralization of hyperalgesia induced by Bothrops jararaca and B. asper venoms was studied in rats using bothropic antivenom produced at Instituto Butantan (AVIB, 1 ml neutralizes 5 mg B. jararaca venom) and polyvalent antivenom produced at Instituto Clodomiro Picado (AVCP, 1 ml neutralizes 2.5 mg B. aspar venom). The intraplantar injection of B. jararaca and B. asper venoms caused hyperalgesia, which peaked 1 and 2 h after injection, respectively. Both venoms also induced edema with a similar time course. When neutralization assays involving the independent injection of venom and antivenom were performed, the hyperalgesia induced by B. jararaca venom was neutralized only when bothropic antivenom was administered iv 15 min before venom injection, whereas edema was neutralized when antivenom was injected 15 min or immediately before venom injection. On the other hand, polyvalent antivenom did not interfere with hyperalgesia or edema induced by B. asper venom, even when administered prior to envenomation. The lack of neutralization of hyperalgesia and edema induced by B. asper venom is not attributable to the absence of neutralizing antibodies in the antivenom, since neutralization was achieved in assays involving preincubation of venom and antivenom. Cross-neutralization of AVCP or AVIB against B. jararaca and B. asper venoms, respectively, was also evaluated. Only bothropic antivenom partially neutralized hyperalgesia induced by B. asper venom in preincubation experiments. The present data suggest that hyperalgesia and edema induced by Bothrops venoms are poorly neutralized by commercial antivenoms even when antibodies are administered immediately after envenomation

Bradykinin is involved in hyperalgesia induced by Bothrops jararaca venom.

Bradykinin is involved in hyperalgesia (pain hypersensitivity) induced by Bothrops jararaca venom-intraplantar injection of B. jararaca venom (5microg/paw) in rats caused hyperalgesia, which peaked 1h after venom injection. This phenomenon was not modified by promethazine (H(1) receptor antagonist), methysergide (5-HT receptor antagonist), guanethidine (sympathetic function inhibitor), anti-TNF-alpha or anti-interleukin-1 antibodies or by the chelating agent CaNa(2)EDTA. Venom-induced hyperalgesia was blocked by the bradykinin B(2) receptor antagonist HOE 140. On the other hand, des-Arg(9), [Leu(8)]-bradykinin, a bradykinin B(1) receptor antagonist, did not modify the hyperalgesic response. These results suggest that bradykinin, acting on B(2) receptor, is a mediator of hyperalgesia induced by B. jararaca venom.

Importance of metalloproteinases and macrophages in viper snake envenomation-induced local inflammation.

The inflammatory action of jararhagin, a hemorrhagic metalloproteinase from Bothrops jararaca venom, was studied in mice using dorsal air pouches. The injection of the toxin in 6-day-old air pouches resulted in a leukocyte accumulation comparable to that induced by LPS and whole venom. Polymorphonuclear and mononuclear cells were present in this infiltrate, with a predominance of neutrophils. Treatment of jararhagin with 1,10-phenantroline abolished its proteolytic activity and reduced the pro-inflammatory effect in approximately 50%. Cell influx was not observed when jararhagin was injected into 1-hr air pouches devoid of macrophages, except when it was injected together with 10(6) syngeneic peritoneal macrophages. Supernatants of macrophages stimulated in vitro with jararhagin did not induce leukocyte influx in 1-hr air pouches; the influx occurred after injection of the pellets of stimulated cultures. In summary, jararhagin is an important pro-inflammatory component of B. jararaca venom, and its activity is dependent upon the proteolytic activity and the presence of macrophages.

Hyperalgesia induced by Bothrops jararaca venom in rats: Role of eicosanoids and platelet activating factor (PAF)

In this study we investigated the ability of Bothrops jararaca venom (BjV) to induce hyperalgesia and the modulation of this effect by lipid mediators. It was found that intraplantar injection of BjV (1 to 25 ìg) caused a dose and time-related hyperalgesia. The peak of the hyperalgesic response was 1 hr after injection of the venom and persisted for 24 hr with the higher dose. The BjV-induced hyperalgesia was markedly attenuated by dexamethasone. Dexamethasone blocks the generation of biologically active metabolites from arachidonic acid by inhibiting PLA2 activation. Inhibition of the cyclo-oxygenase pathway by indomethacin, or inhibition of lipoxygenases by NDGA both significantly inhibited BjV-induced hyperalgesia. Two antagonists of PAF, WEB2170 and BN52021, also significantly inhibited the initial phase of the hyperalgesia. These results suggest that hyperalgesia induced by BjV is, at least partially, mediated by lipid mediators such as prostaglandins, leukotrienes and PAF.

Involvement of eicosanoids and PAF in immune-coplex alveolitis

An immune-complex-mediated hypersensitivity reaction induced in the rat lung was followed by release of the eicosanoids thromboxane, prostaglandin E2 and leukotriene B4 into bronchoalveolar space. Concomitantly, there was a decrease in the number of circulating platelets. The thrombocytopenia was inhibited by a cyclo-oxygenase inhibitor (indomethacin), a platelet activating factor (PAF) antagonist (BN-52021) and an inhibitor of thromboxane (econazozle), but was not affected by a lipoxygenase inhibitor (NDGA). These results suggest the involvement of eicosanoids and PAF in the immune complex hypersensitivity reaction in the rat lung and indicate the ocurrence of interactions between PAF and thromboxane