Evidence for a regulatory role of alpha 4-integrins in the maturation of eosinophils generated from the bone marrow in the presence of dexamethasone.

BACKGROUND: Although eosinophils co-express multiple integrin receptors, the contributions of integrins to eosinophil development have not been explored. We previously described extensive aggregation and cytological immaturity in eosinophils developing in bone-marrow (BM) cultures exposed to dexamethasone. Here we examined the relationship of alpha 4 integrins with these effects of dexamethasone. OBJECTIVES: We evaluated: (a) the effects of exposure to dexamethasone in BM culture on eosinophil expression of alpha 4 integrin receptors and ligands; (b) the contribution of alpha 4 integrins to eosinophil aggregation and maturation. METHODS: Cultures were established with IL-5 (alone or with dexamethasone) for up to 7 days, and eosinophil production, alpha 4 integrin receptor/ligand expression, aggregation and morphology were evaluated before and after targeting alpha 4 integrin-dependent adhesions. Because prostaglandin E2 (PGE2) modifies the effects of dexamethasone on eosinophilopoiesis, PGE2 effects on alpha 4 integrin expression and function were also evaluated. RESULTS: Dexamethasone increased the yield of eosinophils up to day 7. The frequency of eosinophils expressing alpha 4, beta1 and beta 7 integrin receptors at day 7 was also increased by dexamethasone. Eosinophils also expressed the alpha 4 beta 1 ligand, VCAM-1. Dexamethasone increased the expression of alpha 4 integrin and VCAM-1 in aggregates containing eosinophils as early as day 3. PGE2, added up to day 3, modified the effects of dexamethasone to suppress the expression of alpha 4 integrin, decrease aggregation and promote cytological maturation of eosinophils recovered at day 7. Dissociation of immature eosinophils from clusters present at day 3 by reagents targeting alpha 4 or beta1 integrins or VCAM-1 also induced cytological maturation. The concordant effects of targeting alpha 4 integrins with drugs and antibodies support a relationship between alpha 4-mediated aggregation and maturational arrest. CONCLUSIONS: These observations support a novel role for alpha 4 integrin receptors and ligands in eosinophilopoiesis. In addition, increased alpha 4 expression following glucocorticoid exposure may contribute to the retention and accumulation of eosinophils in haemopoietic tissue.

Mandibular appliance modulates condylar growth through integrins.

Functional orthopedic therapy corrects growth discrepancies between the maxilla and mandible, possibly through postural changes in the musculature and modulation of the mandibular condylar cartilage growth. Using Wistar rats, we tested the hypothesis that chondrocytes respond to forces generated by a mandibular propulsor appliance by changes in gene expression, and that integrins are important mediators in this response. Immunohistochemical analyses demonstrated that the use of the appliance for different periods of time modulated the expression of fibronectin, alpha5 and alphav integrin subunits, as well as cell proliferation in the cartilage. In vitro, cyclic distension of condylar cartilage-derived cells increased fibronectin mRNA, as well as Insulin-like Growth Factor-I and II mRNA and cell proliferation. A peptide containing the Arginine-Glycine-Asparagine sequence (RGD), the main cell-binding sequence in fibronectin, blocked almost all these effects, confirming that force itself modulates the growth of the rat condylar cartilage, and that RGD-binding integrins participate in mechanotransduction.

Targeting Leishmania (L.) chagasi amastigotes through macrophage scavenger receptors: the use of drugs entrapped in liposomes containing phosphatidylserine.

OBJECTIVES: We devised liposome-entrapped antimony with the negatively charged lipid phosphatidylserine-liposome-entrapped antimony (Sb-LP)-in order to improve their targeting to infected macrophages through the interaction with scavenger receptors (SRs). METHODS: SR production was indirectly evaluated by its mRNA synthesis in infected and uninfected peritoneal macrophages using RT-PCR. The interaction and cytotoxicity of Sb-LP with SRs and their metabolism were determined by incubation with macrophages in the presence of cytochalasin B, chloroquine or different competitive ligands, with determination of the 50% inhibitory concentration (IC50) in vitro in infected macrophages. The intracellular trafficking of Sb-LP was evaluated by confocal microscopy using trapped fluorescent dyes. RESULTS: Our results showed an up-regulation of macrophage SR mRNA during the initial steps of Leishmania (L.) chagasi infection. By competitive ligand assays, we demonstrated the preferential uptake of Sb-LP by macrophage SRs. Sb-LP was 16-fold more effective (IC50=14.11 microM) than the free drug (IC50=225.9 microM) against L. (L.) chagasi-infected macrophages. The binding and uptake of Sb-LP in macrophages were shown to be energy-dependent and were reduced in the presence of cytochalasin B, showing the dependency of the cell microfilament system. Confocal analysis using trapped fluorescent dyes showed fluorescence of parasites or in their close proximity, compatible with the localized delivery of the liposomes. CONCLUSIONS: The uptake of Sb-LP was reduced in infected macrophages, despite their effectiveness and targeting ability, suggesting a low metabolic rate in infected macrophages that could be overcome by the higher efficiency of the liposomal formulation. These in vitro results suggest that liposomes could improve the therapeutic index of old drugs, such as pentavalent antimony, via targeted delivery to Leishmania-infected cells.

Monoclonal antibodies against noxiustoxin.

Noxiustoxin, a 39-amino acid residue peptide isolated from the venom of the Mexican scorpion Centruroides noxius, has previously been shown to affect voltage-dependent K+ channels. Here we describe the isolation and characterization of monoclonal antibodies (MAbs) against this toxin and their use in structure-function relationship studies. Six hybridoma clones (BNTX4, -12, -14, -16, -18, and -21) producing MAbs against noxiustoxin were isolated. The epitopes defined by the MAbs are overlapping or in close proximity because no MAb pair could bind simultaneously to the toxin. All the MAbs inhibited to various degrees the binding of the toxin to its receptor sites on rat brain synaptosomal membranes. The venom from other Centruroides species was shown to contain components cross-reacting with the MAbs, suggesting the existence of other NTX-like toxins.

On the mechanism of rat uterus desensitization to kallikrein.

An inactive form of kallikrein prepared by iodination with cold iodine, did not show any enzymatic or oxytocic action. However, a competitive pattern between this inactive and active kallikrein was observed in rat uterus preparation: When the inactive form was applied several times in the muscle, a single dose of active kallikrein was unable to cause contraction, but a double dose elicited a response. The rhythmic movement caused by a singular dose of active kallikrein, had its time curtailed by adding the inactive kallikrein to the bath. The inactive kallikrein did not interfere with bradykinin activity.

Chloramine T impairs the receptor-mediated endocytosis of plasma kallikrein by the liver.

Treatment of rat plasma-kallikrein with chloramine T (which does not affect neither its amidolytic activity nor its Mr) impairs the hepatic clearance of the enzyme in a dose-related manner. Preperfusion of the liver with chloramine T before the addition of plasma-kallikrein also diminishes the hepatic clearance of the enzyme.

Characterization of a class of nonformylated Enterococcus faecalis-derived neutrophil chemotactic peptides: the sex pheromones.

Bacteria produce a heterogeneous mixture of neutrophil chemotactic agents in culture filtrates. Formylmethionyl peptides have been shown to comprise a significant portion of the chemotactic activity in bacterial culture filtrates; however, not all of the chemotactic agents in bacterial culture filtrates are formylated peptides. To examine whether nonformylated peptides derived from bacteria could act as chemotactic agents, we studied several nonformylated hepta- and octapeptide Enterococcus faecalis-derived sex pheromones, their modified derivatives, and their competitive inhibitors for activation of rat peritoneal neutrophils. Several of these peptides, in particular cAM373 and cPD1, proved to be potent chemotactic agents in submicromolar concentrations as well as inducers of lysosomal granule enzyme secretion. Moreover, the more biologically active peptides were able to compete with fMet-Leu-[3H]Phe for binding to the formyl peptide receptor. These studies demonstrate that the formylmethionyl moiety may be an absolute requirement only for the binding of di- and tripeptides to the formyl peptide receptor. Larger peptides that may have or that may allow for additional contact points between the peptide and receptor may require N-formylation only relatively. Indeed, by removing this structural restraint, the formyl peptide receptor may interact with an unlimited number of peptide fragments of both infectious and host origins to then modulate neutrophil responses to infection and inflammation.