calcimycin and Chediak-Higashi-Syndrome

Studies have demonstrated significant heterogeneity in neutrophil granule morphology and physical density. Using cytochemical methods to localize peroxidase and vicinal glycol containing complex carbohydrates we examined the heterogeneity of neutrophil granules from intact human neutrophil granules in 13 isolated granule density fractions, calcium ionophore A23187 treated neutrophils and neutrophils from patients with Chediak-Higashi Syndrome and Specific Granule Deficiency. At least four distinct populations of peroxidase positive granules (PPG) were identified based on peroxidase staining, vicinal glycol staining, morphology, beta-glucuronidase and defensin content, and physical density characteristics. The smallest (0.15 micron diameter) PPG was the least dense granule, had a unique peroxidase/beta-glucuronidase ratio, reacted intensely for vicinal glycols, resisted ionophore degranulation and was not consumed in giant granule formation in Chediak-Higashi Syndrome. The largest (0.3 micron average diameter) and most physically dense PPG was rich in defensins, stained weakly for vicinal glycols, and was absent in specific granule deficiency. These studies demonstrate and correlate morphologic, biochemical, functional, and pathologic differences in PPG populations.

Topics: Adult; Blood Bactericidal Activity; Blood Proteins; Calcimycin; Chediak-Higashi Syndrome; Child; Coloring Agents; Cytoplasmic Granules; Defensins; Glucosides; Glucuronidase; Glycols; Humans; Ionophores; Microscopy, Electron; Neutrophils; Peroxidase; Peroxidases; Pyrimidinones

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.

Topics: Calcimycin; Cells, Cultured; Chediak-Higashi Syndrome; Humans; Male; Microscopy, Electron; Neutrophils; Peroxidases

The degranulation response of peritoneal mast cells to calcium ionophore A23187 and compound 48/80 has been compared by scanning electron microscopy and transmission electron microscopy in normal C57 black mice and C57 beige mice with a genetic defect analogous to the human Chédiak-Higashi Syndrome (CHS). These methods reveal granule secretion in response to the degranulating agents in mast cells of both normal and beige mice. The observations indicate that beige peritoneal mast cells retain the capacity to exocytose their granules upon stimulation and suggest that the defect underlying the formation of the mega-inclusions is not attributable to impaired degranulation.

Topics: Animals; Ascitic Fluid; Calcimycin; Calcium; Chediak-Higashi Syndrome; Disease Models, Animal; Exocytosis; Female; Male; Mast Cells; Mice; Mice, Mutant Strains; Microscopy, Electron; Microscopy, Electron, Scanning; p-Methoxy-N-methylphenethylamine

Previous studies using membrane potential sensitive probes have provided evidence that chemotactic factors elicit membrane potential changes in normal human neutrophils (PMN). In addition to stimulation of PMN motility, chemotactic factors also stimulate degranulation and superoxide ion (O-2) generation and it has been suggested that alteration of membrane potential activates these events (Korchak, H. M., and G. Weissmann. 1978. Proc, Natl, Acad, Sci. U. S. A. 75: 3818--3822). To further define the inter-relationship of these functions, studies were done with two indirect probes of membrane potential, 3-3'-dipentyloxacarbocyanine and triphenylmethylphosphonium ion (TPMP+) using PMN from normal subjects, from patients with abnormal O-2 production (chronic granulomatous disease [CGD]), and from patients with defective degranulation and/or chemotaxis (Cheddiak-Higashi syndrome and patients with elevated immunoglobulin (Ig)E and recurrent staphylococcal infections). The stimuli used were the chemoattractant N-formyl-methionyl-leucyl-phenylalanine (f-Met-Leu-Phe) and the secretagogues ionophore A23187 and phorbol myristate acetate (PMA). The results obtained with 3-3'-dipentyloxacarbocyanine and TPMP+ were comparable. The apparent membrane potential changes elicited by f-Met-Leu-Phe and PMA in normal PMN were reduced or entirely absent in PMN obtained from patients with CGD but normal in PMN from other patients. PMN from patients with CGD had normal calculated resting membrane potentials and normal responses elicited by the potassium ionophore valinomycin. The responses to calcium ionophore A23187 were only slightly impaired. The abnormality of the elicited response of CGD cells of f-Met-Leu-Phe and PMA could not be attributed to the absence of O-2, hydroxyl radical, singlet oxygen, or hydrogen peroxide acting on the probes. Instead this abnormality appears to be associated with a dysfunction in the normal molecular mechanism(s) stimulated upon neutrophil activation. The data suggest chemoattractant alteration of membrane potential in normal PMN is related to activation of oxidative metabolism but the relationship to chemotaxis and degranulation remains to be established.

Topics: Adolescent; Adult; Calcimycin; Carbocyanines; Chediak-Higashi Syndrome; Chemotactic Factors; Chemotaxis, Leukocyte; Child; Child, Preschool; Female; Granulomatous Disease, Chronic; Humans; In Vitro Techniques; Male; Membrane Potentials; Neutrophils; Onium Compounds; Superoxides; Tetradecanoylphorbol Acetate; Tetraphenylborate; Trityl Compounds; Valinomycin

Blood leukocytes from a patient with Chediak-Higashi syndrome (CHS) were compared with normal cells for their capacity of extruding (exocytosis) the lysosomal enzyme myeloperoxidase during phagocytosis or after a treatment with the ionophore A23187 and Ca2+. A decreased rate and extent of exocytosis in phagocytizing CHS cells was observed also with the Ca2+ ionophore. This suggests that a defect in Ca2+ mobilization is not responsible for the impaired secretion of granule content. Isolated granules of CHS cells and of leukocytes were treated with the detergent Triton X-100. Since the solubilization of myeloperoxidase from the CHS granules was much lower than from the normal ones, we suggest that the former organelles have a more resistant membrane.

Topics: Calcimycin; Chediak-Higashi Syndrome; Child, Preschool; Exocytosis; Female; Humans; Leukocytes; Peroxidase; Phagocytosis; Polyethylene Glycols