Distinct granule populations in human neutrophils and lysosomal organelles identified by immuno-electron microscopy

• Published in: Journal of immunological methods 1999-12, Vol.232 (1), p.153-168
• Main Author: Bainton, Dorothy F
• Format: Article
• Language: English
• Subjects: Antigens, CD - metabolism; Biomarkers; Cytoplasmic Granules - chemistry; Cytoplasmic Granules - classification; Cytoplasmic Granules - enzymology; Cytoplasmic Granules - ultrastructure; Double-labeling; Gelatinases - analysis; Granules; Humans; Hydrolases - analysis; Immunohistochemistry; Lysosomal Membrane Proteins; Lysosomes; Lysosomes - chemistry; Lysosomes - enzymology; Mannosephosphates - analysis; Marker proteins; Membrane Glycoproteins - metabolism; Membrane Proteins; Microscopy, Immunoelectron - methods; Neutrophils - chemistry; Neutrophils - enzymology; Neutrophils - metabolism; Neutrophils - ultrastructure; Peroxidase - analysis; Proteins - analysis; Receptor, IGF Type 2 - analysis
• ISSN: 0022-1759; 1872-7905
• DOI: 10.1016/S0022-1759(99)00173-8

In this paper, we illustrate the fine structural localization of distinct marker proteins in the organelles of human neutrophils and outline our preferred methods for processing ultrathin cryosections for use with immunoelectron microscopy. Previous work has determined the subcellular localization of certain marker proteins within intact polymorphonuclear neutrophilic leukocytes (PMN) and PMN fractions. These are as follows: myeloperoxidase (MPO) for azurophilic granules, lactoferrin for specific/secondary granules, gelatinase for gelatinase/tertiary granules, albumin for the secretory vesicles, and HLA class I and L-selectin for the plasma membrane. In addition to analyzing the heterogeneity of the PMN granule populations, new information on the lysosomal system of this cell is reviewed and extended by the localization of the lysosome-associated membrane proteins (LAMPs) and the cation-independent mannose 6-phosphate receptor (CI-M6PR). LAMPs were absent in all identified granule populations, but were found in the membranes of vesicles, multivesicular bodies (MVB), and multilaminar compartments (MLC). We show here that MVB contain CI-M6PR whereas MLC do not. Furthermore, since MLC contain LAMPs but not the receptor, they probably correspond to the late endosome. By current criteria, the true lysosomes of the resting PMN are MVB and MLC. Finally, although azurophil granules contain acid hydrolases their membranes do not contain LAMPs and they cannot be classified as lysosomes, but rather are more similar to regulated secretory granules.