The presence of leucine aminopeptidase as an ecto-enzyme on the plasma membrane of neutrophils and its functions

Abstract

5'-Nucleotidase, alkaline phosphodiesterase, Mg2+-ATPase and leucine aminopeptidase are generally reported to be located on the plasma membrane of various mammalian cell types. Among these enzymes, the presence of leucine aminopeptidase (LAP) in neutrophils has not been clear. Therefore, we started with examining the existence of LAP in neutrophils and its subcellular localization with reference to 5'-nucleotidase and alkaline phosphodiesterase. 5'-Nucleotidase was hardly detected in human. Alkaline phosphodiesterase was found in guinea-pig, human and rabbit, but no evidence for the ecto-enzyme was obtained in guinea pig. On the other hand, LAP was observed with all the neutrophils examined and proved to be located as an ecto-enzyme by applying the criteria DePierre and Karnovsky have used to define ecto-enzymes: comparison of the enzyme activity of intact cells with the total cell activity using sonicates, localization of the products of intact cell enzymatic activity and inhibition of intact cell enzyme by a “nonpenetrating” reagent without the inhibition of a soluble cytoplasmic enzyme. Next, the possible functions of LAP were examined. Tuftsin, a phagocytosis-stimulating tetrapeptide, is liberated from leukokinin, neutrophilic γ-globulin with phagocytosis-stimulating activity, by leukokininase on the plasma membrane of neutrophils and these stimulatory effects are not maintained and fall off in time to control levels. On the other hand, the biological activity of tuftsin is reported to be susceptible to commercial leucine aminopeptidase. Therefore, we examined whether LAP in neutrophils acts as a tuftsin-inactivating enzyme or not. Experimental data showed that a tuftsin-inactivating activity was localized as an ecto-enzyme on the plasma membrane and its activity was parallel with the change in the LAP activity which was competitively inhibited by tuftsin, suggesting the possible responsibility of LAP for the tuftsin-inactivating activity of neutrophils.Phagocytosis is characterized by internalization of large portion of surface membrane of neutrophils and so ecto-enzymes might be interiorized along with plasma membrane and inactivated during phagocytosis. Then, we studied changes in LAP activity during phagocytosis of phagocytic particles. Inactivation of LAP was observed by exposure of neutrophils to complement-opsonized zymosan particles, but not to non-opsonized zymosan, IgG-coated zymosan or latex particles. Pretreatment of neutrophils with cytochalasin B, which prevents phagocytosis but not surface binding of particles, provoked inactivation to the same degree as when the cells were allowed to phagocytose the particles. However, the inactivation during phagocytosis was protected by serine protease inhibitors. These findings suggest that loss of LAP activity from phagocytosing cells may be mediated by certain serine protease inhibitor-sensitive factor (s) which are probably activated by the attachement of an opsonized zymosan particle to a specific membrane receptor, probably the C 3 b receptor. Abovementioned results suggested that LAP may modulate the regulation of phagocytic function of neutrophils, which led us to investigate the distribution of the LAP activity among leukocytes other than neutrophils. So-called “professional” phagocytes such as neutrophils, monocytes and macrophages showed higher enzyme activity than lymphocytes and eosinophils in all species examined although the difference was observed in the LAP activity between species. A chemical modification of intact guinea-pig leukocytes with diazotized sulfanilic acid, a poorly penetrating reagent, suggested that LAP was located as an ecto-enzyme in all cell types.