Macrophage-Derived Neutrophil Chemotactic Factor (MNCF) Induces Neutrophil Migration Through Lectin-Like Activity

Abstract

We have previously reported that rat peritoneal macrophages stimulated with LPS release a factor (MNCF) which induces neutrophil migration which is not blocked by glucocorticoids. The initial characterization of MNCF showed that it did not correspond to any of the current known chemotactic cytokines or to known chemotactic agents. Recent studies have shown that neutrophil migration to the inflammatory focus may involve protein molecules with carbohydrate-binding sites, the LEC-CAMs. This fact prompted us to investigate the possible lectin-like nature of MNCF. Thus, the supernatants of macrophage monolayers stimulated with LPS were submitted to affinity chromatography on immobilized sugar columns. We observed that the D-galactose (D-gal) binding fraction retained the MNCF activity. This fraction, consisting of four protein components, was submitted to chromatography on Sephadex G-75, yielding a homogeneous preparation of the active component. MNCF has a MW of 54 KDa (gel filtration and SDS-PAGE) and a pI of 2-3 (isoelectrofocusing and chromatofocusing). A direct mechanism of action of MNCF on neutrophil migration initially suggested by the absence of blockade of the phenomenon in vivo by glucocorticoids, was confirmed by the observation that a protein-carbohydrate interactión is involved in this activity. Homogeneous preparation of MNCF induced neutrophil migration in vivo and in vitro. The in vitro effect was blocked by D-gal which did not interfere with the chemotactic effects of known interleukins (IL-1β, TNF-α, IL-6 and IL-8). The present results strengthen our previous suggestion that MNCF is yetundescribed monokine which induces neutrophil migration through a direct mechanism involving the D-gal binding site of the molecule.