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American Journal of Pathology, Vol 109, 343-358, Copyright © 1982 by American Society for Investigative Pathology

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Ultrastructural localization of lactoferrin and iron-binding protein in human neutrophils and rabbit heterophils

RT Parmley, M Takagi, JC Barton, LA Boxer and RL Austin

Lactoferrin in marrow and blood granulocytes from rabbits and humans was stained with an immunoferritin method. Iron-binding protein(s) was localized by the staining of granulocytes with acid ferrocyanide after saturation of the iron-binding protein with iron. The latter was most readily accomplished by treatment of the glutaraldehyde-fixed cell suspension with 1% saponin, followed by treatment with an iron- nitrilotriacetate (Fe-NTA 3mM:4mM) solution, adjusted to pH 7.0 with NaHCO3. The affinity of purified lactoferrin and transferrin for radioiron after such treatment was minimally diminished. Both immunoferritin and iron-binding methods heavily stained osmiophiliuc (phospholipid-containing) mature primary granules in late promyelocytes, myelocytes, and polymorphonuclear cells. Early promyelocytes containing abundant immature primary granules lacked immunoferritin or iron staining. Trypsin digestion of rabbit marrow cells considerably diminished the cytochemically demonstrable iron- binding capability of the mature primary granules. Specimens sequentially stained for peroxidase and immunostained for lactoferrin or cytochemically stained for iron-binding protein confirmed that lactoferrin and iron-binding protein were in peroxidase-positive primary granules. Some peroxidase positive granules appeared to lack staining for lactoferrin and iron-binding proteining protein, and all secondary granules uniformly lacked staining. Treatment of human neutrophils with phorbol myristate acetate demonstrated early release of granules containing iron-binding protein with subsequent agglutination of neutrophils and attachment of iron-binding protein to the cell surface. In summary, this study distinguishes at least two subpopulations of primary granules and identifies lactoferrin and an iron-binding protein(s) in a subpopulation of peroxidase-positive primary granules in rabbit heterophils and human neutrophils.