Granules of the human neutrophilic polymorphonuclear leukocyte.

neity simply that some proteins must be segregated to survive P inside the neutrophil, or for the neutrophil to survive, much OLYMORPHONUCLEAR leukocytes were discovered by Paul Ehrlich, when fixation and staining techniques as two-component glue has to be stored in two separate made it possible to identify the lobulated nucleus and the tubes, although both will be used at the same time? Whether granules that have given name to these cells and allowed for one or the other reason applies, both raise the question of their classification as eosinophils, basophils, and neutrophils. how the neutrophil can target proteins into different granule Neutrophilic granulation refers to staining by a mixture of subsets. basic and acid, ie, neutral, dyes, whereas the term specific The purpose of this review is to present the heterogeneity granules was used by Paul Ehrlich to distinguish true granof granules and discuss the functional importance of this ules from artifacts with a granular appearance. It was later and, furthermore, to address how the neutrophil controls the realized that two types of granules could be distinguished in structure and mobilization of these different granules. neutrophils on the basis of their affinity for dye: azurophil granules, which take up the basic dye azure A at the promyBASIC ASPECTS OF GRANULOGENESIS elocytic stage, due to their content of acid mucopolysacchaGranules start to form at the stage of neutrophil maturation ride,and specific granules, which do not. A clear distinction marked by transition from myeloblast to promyelocyte. between these two types of granules was established when From here on, formation of granule proteins continues even the peroxidase staining method for electron microscopy was up to the stage of segmented cells. adopted to identify myeloperoxidase (MPO), which is presIn general, granules are believed to be formed by aggregaent only in azurophil granules. This distinction was corrobtion of immature transport vesicles that bud off from the orated by the development of subcellular fractionation techtrans-Golgi network (TGN), in which sorting takes place niques for separation of the granules. It has become dogma between constitutively secreted proteins and proteins that that these two types of granules are fundamentally different. are routed into the regulated secretory pathway, ie, go to Specific granules have been characterized as secretory grangranules. The original study by Bainton et al showed ules that play important roles in initiating the inflammatory that such vesicles bud off from cis-Golgi to form storage response, whereas azurophil granules are often viewed as granules at the promyelocyte stage, but from the trans-Golgi lysosomes that are particularly active in the digestion of at the myelocyte stage, where specific granules are formed. phagocytosed material. This implies that the sorting apparatus (if existing) is localThis simplistic view of granules has been challenged by ized in the cis-Golgi in promyelocytes and moves to the results from subcellular fractionation experiments in which TGN in more mature cells. Unlikely as this may seem, it granule proteins were found in more than two peaks on agrees with the finding that MPO, a major protein of azurodensity gradients. In addition, the existence of a terphil granules, does not contain complex carbohydrate side tiary granule type, identified by its late appearance during chains, but it is contradicted by the finding that several myeloid maturation, was indicated by electron microscopy. other azurophil granule proteins, including elastase, cathepDuring the last 15 years, high-resolution subcellular fractionsin G, and proteinase 3, acquire complex oligosaccharide ation techniques, alone or in combination with immune-elecside chains. Refining of carbohydrate side chains from tron microscopy and flow cytometry, have shown a bewildersimple to complex is a feature of the intermediateand transing heterogeneity of the neutrophil’s granules and have Golgi stacks. furthermore identified an additional regulated exocytotic Several regulatory steps will be required if granules are storage organelle, the secretory vesicle. formed by fusion from smaller unit transport vesicles that A novel aspect of the physiology of granules was unravbud off from the Golgi and undergo homotypic fusion until eled by the discovery that these regulated storage organelles they achieve the size of a granule. It must be secured that (granules and secretory vesicles) are not just simple bags of such transport vesicles do not fuse with the plasma memproteolytic or bactericidal proteins that are kept in store until brane, as do transport vesicles that mediate constitutive seliberated either to the outside of the cell or to the phagocytic cretion. Furthermore, it must be secured that such transport vacuole, but are also important reservoirs of membrane provesicles do not fuse with already formed granules, because teins that become incorporated into the surface membrane this would lead to mixing of granule proteins destined for of the neutrophils when these organelles fuse with the plasma different types of granules. membrane and exocytose their content. In this way, granules and secretory vesicles may fundamentally change the From The Granulocyte Research Laboratory, The Finsen Center, ability of the neutrophil to interact with its environment. Department of Hematology, Rigshospitalet, Copenhagen University Realizing this, a number of important questions immediHospital, Copenhagen, Denmark. ately appear. Why this heterogeneity? Does it provide the Submitted November 6, 1996; accepted January 29, 1997. neutrophil with the ability to differentially exocytose (or, in Address reprint requests to Niels Borregaard, MD, PhD, Rigshoscase of membrane proteins, translocate) proteins stored in pitalet L-4042, 9 Blegdamsvej, DK-2100 Copenhagen, Denmark. different granules, and if so, how is this differential exoq 1997 by The American Society of Hematology. 0006-4971/97/8910-0049$3.00/0 cytosis controlled? Or, is the reason for the granule heteroge-