Cells often acquire resistance to the antiproliferative agents methotrexate (MTX)

Studies have demonstrated significant heterogeneity in neutrophil granule morphology and physical density. This study evaluated the heterogeneity morphometrically, morphologically, cytochemically, and biochemically. Intact human peripheral blood neutrophils collected from normal volunteers and a patient with Chédiak-Higashi syndrome (CHS) and isolated normal neutrophil granules were processed for ultrastructural morphology and peroxidase staining. Intact cells, nuclei, and granule profiles were analyzed by computer-assisted planimetry. Peroxidase-positive granules (PPG) represented about 40% of normal neutrophil granules and covered the entire spectrum of granule size. PPG in the least-dense fractions of isolated granules were significantly smaller than in higher-density fractions. PPG in low- and intermediate-density fractions differed from high-density fraction by moderate to strong vicinal glycol staining with Thiéry's periodate-thiocarbohydrazide-silver proteinate method. Differing ratios of % beta-glucuronidase/% myeloperoxidase (MPO) across granule fractions indicated PPG heterogeneity. Morphometric analysis of neutrophils treated with 1 microM calcium ionophore A23187 did not show significant differences in PPG size or number. Biochemically analyzed MPO in these cells was preserved, although the number of peroxidase-negative granules (PNG) and levels of vitamin B12-binding protein were markedly decreased. In CHS, about 20% of granules were PPG. Analysis of CHS neutrophils revealed the persistence of microgranules similar to normals. PNG number and volume fractions of PPG and TG were not different from normals. Complex heterogeneity of normal PPG was quantitated using morphometry and appeared preserved in ionophore-treated cells but was uniquely modified in CHS.

Rabbit heterophil and human neutrophil primary granules contain sulfated glycosaminoglycans (GAGs) and acid phosphatase, which can be readily stained in immature but not mature lysosomes. To determine whether this loss of staining represents masking of reactive components or removal of these components, we examined rabbit heterophils to see if high-iron diamine (HID)-reactive sulfate and acid phosphatase staining reappears in phagocytic vacuoles. Rabbit heterophils, obtained by peritoneal hivage, were incubated in vitro with latex beads or Pseudomonas aeruginosa for 15-60 mm Pre-embedment HID staining was enhanced in thin sections of unosmicated specimens with thiocarbohydrazide and silver proteinate (TCH-SP). Phagocytosis oflatex beads or bacteria was progressively more prominent with time. Primary granules that were degranulated or in the process of degranulating into phagocytic vacuoles demonstrated intense sulfate staining (L). Heterophils incubated 60 minutes with-.. latex particles. N, nuclei. Bar = 1 pun.

A microgranule fraction, isolated from human neutrophils by using a novel high-resolution Percoll density gradient system contained granules with the lowest density and diameter when compared with 12 other isopycnic granule fractions. Ultrastructurally, from 34% to 50% of the microgranules showed homogeneous diaminobenzidine (DAB) staining under conditions for localizing peroxidase reactivity. The presence of myeloperoxidase (MPO) was further confirmed by biochemical and spectral analysis and immunodiffusion methods. Periodate-thiocarbohydrazide- silver proteinate (PA-TCH-SP) intensely stained vicinal glycols in the matrix of greater than 97% microgranules in contrast to the weak or absent staining seen in larger primary granules. Directly sampled segmented neutrophils contained small DAB- and PA-TCH-SP-positive granules, which often appeared in clusters. These DAB-positive microgranules selectively remained within the cells after stimulation of exocytosis with the calcium ionophore A23187. The enriched DAB- positive microgranule fraction recovered from A23187-treated cells also contained lysozyme and beta-glucuronidase but lacked vitamin B12 binding protein activity. A similar small, DAB- and PA-TCH-SP-positive granule type was also identified in normal promyelocytes and was the predominant or only granule type observed in leukemic or preleukemic myeloid cells from four patients. This study demonstrates a unique subpopulation of MPO-containing microgranules in normal and leukemic human myeloid cells that are distinguished from (other) primary granules by their extremely low density, small size, content of complex carbohydrates, and resistance to secretion.