Neutrophil granules: heterogeneity of their contents reflects targeting by timing

Once it was all so simple: circulating neutrophils were biosynthetically effete cells trained to generate hydrogen peroxide and discharge preformed granules during phagocytosis of foreign particles or organisms. Granules were histochemically, and simply, classified into two nicely defined categories according to their peroxidase content, a scheme that paralleled the temporal relationship of their development in bone marrow; the peroxidase-positive, azurophilic, or primary granules arise before the peroxidase-negative, specific, or secondary granules. Although both types of granules were membrane-bound organelles, the contents of the azurophilic granule more closely resembled those of a lysosome, containing a variety of acid hydrolases, than did the matrix of secondary granules. In response to stimuli each granule population exocytosed in an orchestrated and sequential order, the secondary granules preceding the azurophilic granules [reviewed in ref. 1]. However, as in other areas of investigation, the striking increase in the breadth and depth of understanding of the cell biology of phagocytic cells during the past two decades has added a new dimension of complexity to this orderly picture. Starting in the mid 1980s, studies from varied viewpoints challenged this simple scheme for classifying granules. Histochemical examination of cultured human leukemia cells revealed the presence of morphologically distinct species of azurophilic granules [2]; peroxidase-negative granules were structurally and functionally heterogeneous [3]; the lysosomelike azurophilic granules lacked lysosome-associated membrane protein 1 [4], a hallmark of true lysosomes; and biochemical analyses of subcellular fractions separated by gradient density centrifugation suggested that there existed remarkable heterogeneity among the granule subsets within neutrophils, identifying as many as 13 distinct populations [5, 6]. Given such heterogeneity among the contents of granules, the signals necessary to segregate nascent granule proteins into the correct intracellular compartment seemed extraordinarily complex and stimulated the search for the ‘‘sorting’’ mechanisms responsible for such precise and discriminating targeting. Throughout this period the Borregaard laboratory, among others, has made substantial contributions to our understanding of myeloid granule classification and biogenesis. To determine whether the contents within each granule possessed signals targeting them for a particular population of granules, cDNA encoding the secondary granule protein neutrophil gelatinaseassociated lipocalin (NGAL) was transfected into HL-60 cells, a cultured human leukemia cell line devoid of secondary granules or secondary granule proteins. In undifferentiated HL-60 cells, transfected NGAL concentrated in the azurophilic granule [7], whereas in differentiated HL-60 cells the transfected protein entered the constitutive secretory pathway [8]. These observations provided the basis for the ‘‘targeting by timing’’ hypothesis of granule protein sorting, whereby the contents of particular granules reflect the repertoire of granule protein mRNA expressed at a given time during differentiation rather than specific sorting signals intrinsic to the granule proteins. In this issue, Cowland and Borregaard describe studies of the regulation of granule protein mRNA by normal human bone marrow cells and provide additional data supporting their sorting hypothesis. Although these studies quantitate mRNA and not granule protein biosynthesis per se, the results are free from the caveats inherent to studies using cultured cell lines derived from patients with myeloid leukemias and likely reflect events occurring in normal bone marrow. The ease and lucidity with which the results are described may disguise the effort and rigor with which the studies were performed and the importance of the findings as they relate to granule protein sorting and its relation to normal myeloid differentiation. For the 11 matrix proteins and 4 granule-associated membrane proteins studied, the heterogeneity of granule constituents reflects the array of granule protein mRNA expressed at any given point in time. Human neutrophil defensin (HNP-3) deviates from the proposed paradigm, as mRNA for HNP-3, an azurophilic granule protein, mirrors the profile of lactoferrin, a specific granule constituent, rather than those of myeloperoxidase or other azurophilic granule markers. Although not explained by the authors, such a finding is not wholly unanticipated because