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Lactoferrin, an iron-binding protein previously shown to occur in many external secretions, is identified as one of the major proteins present in human and guinea pig neutrophilic polymorphonuclear leukocytes. The identification of this protein in leukocyte extracts was based upon a comparison of its electrophoretic, antigenic, and iron-combining properties with the corresponding properties of the same protein isolated from human and guinea pig milk. Immunochemical quantitations showed that lactoferrin occurs in human neutrophilic leukocytes at the concentration of 3 µg per 106 cells. Tissue cultures from guinea pig bone marrow and spleen actively synthesized the protein, as shown both by net production of lactoferrin and incorporation of labeled amino acids into the protein. Immunohistochemical data indicate that lactoferrin first appears in myeloid cells at the stage of the promyelocyte.

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The hyposideremia of inflammation was found to be based on a three-step mechanism involving lactoferrin, the iron-binding protein from the specific granules of neutrophilic leukocytes. (a) Lactoferrin is Released from Neutrophils in an Iron-Free Form. When phagocytosis was induced in neutrophils by zymosan or bacteria, lactoferrin was recovered in the incubation medium together with other constituents of the specific granules, such as alkaline phosphatase and lysozyme. Lactoferrin extracted from leukocytes was able to bind the amount of iron corresponding to its theoretical iron-binding capacity. After injection of endotoxin into rats, lactoferrin was detected in various tissues where it was normally absent, or in the plasma when the reticuloendothelial system (RES) had previously been blocked by injections of India ink or aggregated albumin. (b) Lactoferrin is Able to Remove the Iron from Transferrin. Significant exchange of iron from transferrin to lactoferrin was observed in vitro only at a pH below 7.0 or in the presence of a high concentration of citrate. However, the fast elimination of lactoferrin in vivo, when saturated with iron, might account for the observed transfer of iron to endogenous or administered apolactoferrin. Intravenous injection of human apolactoferrin into rats caused a marked decrease of the plasma iron level. The kinetics of this process, as well as controls with other proteins, ruled out the possibility of a secondary inflammatory effect due to phlogogenic contaminants. (c) Fe-Lactoferrin is Taken-up by the RES. By immunofluorescence, lactoferrin was shown to be bound and ingested by monocytes. The rate of elimination of human Fe-lactoferrin injected into rats was particularly fast when compared to that of human apolactoferrin, succinylated Fe-lactoferrin, or other human proteins. Blockade of the RES slowed down the rate of clearance of Fe-lactoferrin and was also found to retard the elimination of endogenous rat lactoferrin released by endotoxin. These experiments suggest the existence of specific receptors for Fe-lactoferrin on the membrane of macrophages.

Lactoferrin has been identified in rabbit heterophil leukocytes on the basis of its immunological reactivity, electrophoretic mobility, acid-resistant iron-binding properties, and spectral characteristics. Leukocyte lactoferrin was found to be exclusively localized in the specific (secondary) granules, which have been resolved from other subcellular components by zonal differential centrifugation and by isopycnic equilibration.

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