Ultrastructural and biochemical characterization of glycosaminoglycans in HNK-1-positive large granular lymphocytes

Abstract

Natural killer (NK) cells are large granular lymphocytes (LGLs) that contain distinct lysosomal granules. The present study was undertaken to determine if these lysosomes contain glycosaminoglycans (GAGs) similar to those previously described in myeloid cells. Mononuclear cells from human blood were stained with HNK-1 fluoresceinated monoclonal antibody, and the NK cell population reactive with this antibody were isolated with a fluorescence-activated cell sorter (FACS). Specific staining of sulfated macromolecules with the cationic reagent, high iron diamine, was observed in the lysosomal granules of 90% of the HNK-1 positive cells. Staining in the same location was also observed in the unsorted LGLs, presumed to be NK cells, and intense staining of the cell surface was also a prominent feature of these cells. Surface staining was not evident in the majority of the FACS-separated NK cells. Digestion with chondroitinase ABC or treatment with nitrous acid reduced the staining in both locations; after sequential treatment with both chondroitinase and nitrous acid, little or no staining was seen. The presence of chondroitin sulfate (and/or dermatan sulfate) and heparan sulfate was also shown by the finding that incubation of the isolated NK cells with 35S-sulfate yielded cell-associated radiolabeled macromolecules with the characteristics of these two groups of GAGs. Of the labeled GAG pool, 60% was degraded by chondroitinase and 40% was susceptible to nitrous acid treatment. LGLs of a patient with Chediak-Higashi syndrome was also stained, and intracellular sulfate staining was clearly localized to the enlarged granules, supporting the conclusion that the lysosomes are the major site of intracellular accumulation of GAGs in normal NK cells.