RESUMO

BACKGROUND: Alveolar macrophages (AM) may participate in brochopulmonary hyper-reactivity by secreting cytokines that recruit mature eosinophils, or induce eosinophil production from recruited circulating progenitors. OBJECTIVE: To define whether AM products can contribute to lung eosinophil production in immunized guinea pigs (GP), by analysing the effect of AM culture supernatants (AM-SN) on in vitro eosinophilopoiesis. METHODS: Liquid and semi-solid bone marrow (BM) cultures were seeded with SN from 95% pure AM exposed to LPS. RESULTS: AM-SN increased very significantly the long-term viability, cell proliferation and eosinophil production in liquid culture and supported formation of eosinophil-bearing mixed colonies, by acting on progenitors depleted of mature eosinophils. The effect on eosinophil production was not duplicated by natural or recombinant sources of GM-CSF (which nevertheless supported GM colony formation by GP BM), not by rhIL-8 (which was active on GP cells) and was not due to residual LPS. FPLC separation of active AM SN yielded a peak of apparent m.w. 43 kDa, active on both liquid and semi-solid cultures. The active moiety was heat- and trypsin-resistant. Neutralizing monoclonal antibodies to hGM-CSF, mGM-CSF, hIL-3 and mIL-3 failed to deplete the activity in AM-SN. Ovalbumin immunization induced its production by AM even without LPS challenge. CONCLUSIONS: The lack of T lymphocytes among factor-producing AM, the properties of the active material, the inability of GM-CSF to reproduce these effects, and the failure of MoAbs to GM-CSF and to IL-3 to neutralize the activity indicate it is not due to the major eosinopoietic factors GM-CSF, IL-3 or IL-5.

Assuntos

RESUMO

An antivenom protein has been identified in the blood of the snake Crotalus durissus terrificus and proved to act by specifically neutralizing crotoxin, the main lethal component of rattlesnake venoms. The aim of this study was to purify the crotoxin inhibitor from Crotalus serum (CICS), and to analyze its mechanism of action. CICS has been purified from blood serum of the Crotalus snake by gel filtration on Sephadex G-200, ion-exchange chromatography on DEAE-Sephacel, and FPLC gel filtration on a Superose 12 column. It is an oligomeric glycoprotein of 130 kDa, made by the non-covalent association of 23-25-kDa subunits. Two different subunit peptides were identified by SDS/PAGE, however, their N-terminal sequences are identical. They are characterized by the absence of methionine residues and a high content of acidic, hydrophobic and cysteine residues. The neutralizing effect of purified CICS towards the neurotoxic effects of crotoxin has been demonstrated in vivo by lethality assays. CICS binds to the phospholipase subunit CB of crotoxin, but not to the acidic chaperon subunit CA; it efficiently inhibits the phospholipase activity of crotoxin and its isolated CB subunit and evokes the dissociation of the crotoxin complex. The molecular mechanism of the interaction between CICS and crotoxin seems to be very similar to that of crotoxin with its acceptor. It is, therefore, tempting to suggest that CICS acts physiologically as a false crotoxin acceptor that would retain the toxin in the vascular system, thus preventing its action on the neuromuscular system.

Assuntos

RESUMO

Crotoxin, the major toxic component of the South American rattlesnake, Crotalus durissus terrificus, is a neurotoxic phospholipase A2 which exerts its pathophysiological action by blocking the neuromuscular transmission. Crotoxin acts primarily by altering the acetylcholine release from the nerves terminals through a mechanism which has not yet been elucidated. It also acts on postsynaptic membranes by stabilizing the acetylcholine receptor in an inactive conformation very similar to the desensitized state. Crotoxin is made of two dissimilar subunits: a basic and weakly toxic phospholipase A2 component-B, and an acidic and non toxic component-A which does not possess any enzymatic activity. Binding experiments showed that crotoxin subunits dissociate when crotoxin interacts with biological membranes: Component-B binds, whereas component-A appears free in solution. The phospholipase A2 subunit binds in a non saturable, non specific manner, on any kind of biological membranes, whereas in the presence of component-A it interacts only with a limited number of high affinity binding sites present on synaptic membranes but not on erythrocyte membranes. Although the target site (acceptor) of crotoxin has not yet been formally identified, binding experiments carried out with small unilamellar phospholipid vesicles of different compositions indicate that some negatively charged phospholipids like mono and diphosphoinositide phosphates might be an important component of crotoxin acceptor site. Crotoxin is in fact a mixture of several isoforms which have very similar but not identical polypeptide sequences. An individual Crotalus durissus terrificus snake is able to synthesize several crotoxin isoforms which may result of the expression of several isogenes and/or of post-translational events. When compared in quantitative manner, the crotoxin isoforms slightly but significantly differ in their enzymatic and pharmacological properties. Finally, immunochemical investigations carried out with polyclonal antibodies prepared against both crotoxin subunits, showed that non precipitating anti-component-B- antibodies (Fab) inhibit the phospholipase A2 activity of crotoxin and neutralize its lethal potency, suggesting that the catalytic and toxic sites of crotoxin are closely related.

Assuntos

RESUMO

Crotoxin, the major toxic component of the South American rattlesnake, Crotalus durissus terrificus, is a neurotoxic phospholipase A2 which exerts its pathophysiological action by blocking the neuromuscular transmission. Crotoxin acts primarily by altering the acetylcholine release from the nerves terminals through a mechanism which has not yet been elucidated. It also acts on postsynaptic membranes by stabilizing the acetylcholine receptor in an inactive conformation very similar to the desensitized state. Crotoxin is made of two dissimilar subunits: a basic and weakly toxic phospholipase A2 component-B, and an acidic and non toxic component-A which does not possess any enzymatic activity. Binding experiments showed that crotoxin subunits dissociate when crotoxin interacts with biological membranes: Component-B binds, whereas component-A appears free in solution. The phospholipase A2 subunit binds in a non saturable, non specific manner, on any kind of biological membranes, whereas in the presence of component-A it interacts only with a limited number of high affinity binding sites present on synaptic membranes but not on erythrocyte membranes. Although the target site (acceptor) of crotoxin has not yet been formally identified, binding experiments carried out with small unilamellar phospholipid vesicles of different compositions indicate that some negatively charged phospholipids like mono and diphosphoinositide phosphates might be an important component of crotoxin acceptor site. Crotoxin is in fact a mixture of several isoforms which have very similar but not identical polypeptide sequences. An individual Crotalus durissus terrificus snake is able to synthesize several crotoxin isoforms which may result of the expression of several isogenes and/or of post-translational events. When compared in quantitative manner, the crotoxin isoforms slightly but significantly differ in their enzymatic and pharmacological properties. Finally, immunochemical investigations carried out with polyclonal antibodies prepared against both crotoxin subunits, showed that non precipitating anti-component-B- antibodies (Fab) inhibit the phospholipase A2 activity of crotoxin and neutralize its lethal potency, suggesting that the catalytic and toxic sites of crotoxin are closely related.