RESUMO
KM+ is a D-mannose binding lectin from Artocarpus integrifolia that induces neutrophil migration in vitro and in vivo. This attractant activity was shown to be caused by haptotaxis rather than chemotaxis. The inhibition by D-mannose of the neutrophil attraction exerted by KM+, both in vitro and in vivo, supports the idea that haptotaxis is triggered in vivo by the sugar binding sites interacting with glycoconjugates located on the neutrophil surface and in the extracellular matrix. In the present study an in vivo haptotaxis assay was performed by intradermally (i.d.) injecting 125I-KM+ (200 ng), which led to a selective staining of loose connective tissue and vascular endothelium. The radiolabelled area exhibited a maximum increase (five-fold) in neutrophil infiltration 3 h after injection, relative to i.d. 200 ng 125I-BSA. We characterized the ex vivo binding of KM+ to tissue elements by immunohistochemistry, using paraformaldehyde-fixed, paraffin-embedded, untreated rat skin. Bound KM+ was detected with an affinity-purified rabbit IgG anti-KM+ and visualized with an alkaline phosphatase based system. KM+ binding to connective tissue and vascular endothelium was inhibited by preincubating KM+ with 0.4 mM D-mannose and was potentiated by heparan sulfate (100 microg ml(-1)). An in vitro assay carried out in a Boyden microchamber showed that heparan sulfate potentiated the attractant effect of 10 microg KM+ by 34%. The present data suggest that KM+ induces neutrophil migration in vivo by haptotaxis and that the haptotactic gradient could be provided by the interaction of the KM+ carbohydrate recognition site(s) with mannose-containing glycoconjugate(s) in vascular endothelium and connective tissue. Heparan sulfate would act as an accessory molecule, enhancing the KM+ tissue binding and potentiating the induced neutrophil haptotaxis.