Identification of a subpopulation of primary granules in human neutrophils based upon maturation and distribution. Study by transmission electron microscopy cytochemistry and high voltage electron microscopy of whole cell preparations.

Two main classes of primary granules have been identified in human neutrophils by peroxidase cytochemistry and electron microscopy, spherical granules and elongated granules, containing crystals with well-defined periodicity. In the present study, we report another subpopulation of primary granules, distinguishable from the other primary granules by their small size, strong peroxidase reactivity under selective incubation conditions with diaminobenzidine, stage of neutrophil maturation at which they appear, and distinctive morphological characteristics. Early promyelocytes, reacted with diaminobenzidine before fixation, demonstrate no reactivity in the large primary granules, but strong peroxidase activity in the endoplasmic reticulum and Golgi apparatus. At the end of the promyelocyte stage of maturation, when the endoplasmic reticulum decreases in peroxidase reactivity, small, round or elongated granules (100 to 200 nm) are observed, reacting strongly for peroxidase. These small granules persist during maturation. The data suggest that these small granules are packaged after the large primary granules and before the secondary granules. All three classes of primary granules exhibit peroxidase activity when fixed prior to incubation in diaminobenzidine at neutral or alkaline pH. Distinctive morphological characteristics of the small primary granules are observed in circulating neutrophils. The small granules are arranged in chains or clusters primarily at the cell margin or uropod in moving cells and are aligned along the axis of cell polarity. This granule association is more evident in adherent neutrophils, particularly after short incubation with phorbol myristate acetate. Chains consisting of as many as 17 small granules are observed. Stereo high voltage electron microscopy of whole-mount preparations of adherent neutrophils reveals chains of small granules apparently interconnected by microtrabeculae. Thus, the small granules observed in thin sections do not represent a transverse section of elongated granules, and the interconnection of granules by microtrabeculae may determine the arrangement of granules. These data support the existence of subpopulations of primary granules which contain distinct forms of myeloperoxidase.