Acta haemat. 57: 279-289 (1977)
Large Granules and Lysosomal Fusion in Human ChediakHigashi White Blood Cells L. A. R ozenszajn, E. B en D avid and S. B ar Sela Hematologic Laboratories, Meir Hospital, Kfar Saba and Department of Life Sciences, Bar-llan University, Ramat-Gan
Key Words. Acid phosphatase • Chediak-Higashi syndrome • Cytochemistry • Electron microscopy • Lysosomes • Peroxidase Abstract. The phenomenon of giant anomalous lysosome formation in human Chediak-Higashi syndrome leukocytes was analyzed. Ultrastructure findings com bined with cytochemical procedures for visualizing acid phosphatase and peroxidase activity showed giant anomalous granules in addition to normal, small and enlarged granules. Massive granules in lymphocytes had an appearance and structure differ ent from those found in other leukocytes. The giant granules seem to be a product of an active fusion between primary and secondary normal sized or enlarged lyso somes. This fusion occurs in polymorphonuclear neutrophils, eosinophils and in monocytes. No fusion was found in lymphocyte granules.
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The Chediak-Higashi syndrome (CH) is a rare disorder determined by one autosomal recessive gene and is lethal in the homozygous state [lj. This disorder occurs in man [2, 3] and animals [4-7]. It is clinically man ifested by pancytopenia, lymphoadenopathy, hepatosplenomegaly, photo phobia, partial albinism in most cases, and increased susceptibility to infection. Studies of leukocyte function have documented defective granu locyte chemotaxis [8, 9] and impaired intracellular destruction of phagocytized bacteria [10] as well as reduced quantities and abnormal distribu tion of lysosomal enzymes [11, 12], Cytologically, the prominent characteristic findings are large anomalous granules in granule-containing cells [13-15], However, leukocyte granule abnormalities are pathognomonic for this syndrome [1-3, 6, 16], The presence of acid phosphatase and peroxidase activity determined by cyto chemistry techniques led to the conclusion that these large granules repre
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sent lysosome varieties [1, 17]. Furthermore, studies in CH cells have in dicated a disturbance in their lipid metabolism [18, 19]. It has been sug gested, on the basis of ultrastructure of white blood cells of man and ani mals that the formation of abnormal granules may be due to a process of lysosomal fusion [17, 20]. In CH beige mouse hepatocytes, the giant lyso somes are formed from the Golgi endoplasmic reticulum lysosome (GERL) elements [21]. The present investigation is a study of the structure and the formation, through a process of fusion, of the anomalous granules in human CH leu kocytes. The identification of the fusing lysosomes, as well as the forma tion of the giant granules, was based on their enzymatic staining and structural analogies. Materials and Methods
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Peripheral blood huffy coat and bone marrow from a 4-year-old non-albino male were examined. Morphologic studies. Using the May-Griinwald-Giemsa stain (MGG), morphol ogic studies were performed on smears of peripheral blood, micro buffy coat and bone marrow cells. Phase-contrast microscopy was used to examine fresh prepara tions. Electron microscopy. Examinations were performed on buffy coat obtained from heparinized (200 1U/10 ml) peripheral blood and bone marrow cells, fixed in 2.5°/o glutaraldehyde in 0.2 m Na-cacodylate-HCl buffer (pH 7.2) containing 7°/o sucrose and kept at 4 °C for 2 h. The samples were washed with Na-cacodylate, postfixed in l°/o O s04 in cacodylate-HCl buffer, dehydrated in graded ethanols followed by pro pylene oxide and embedded in Epon 812 as described by L uft [22], Sections were cut with LKB ultratome, stained with uranyl acetate and lead citrate and examined with a Philips 300 electron microscope. Ultrastructural cytochemistry. Acid phosphatase activity was demonstrated as follows: peripheral blood and bone marrow cells were fixed in 1% glutaraldehyde in 0.2 m Na-cacodylate-HCl buffer (pH 7.2) for 15 min at 4 °C. The cells were then washed 3 times in the same cacodylate buffer. The fixed cells were suspended in Gomori incubation media [23] and maintained at 37 °C, pH 4.8, for 2 h. Following in cubation, the cells were washed three times in Na-cacodylate buffer and prepared for electron microscopy as described above. Staining with uranyl acetate and lead citrate was performed after testing the presence of the enzyme activity product on unstained sections. As a control for the specificity of the staining method, fixed cells were incubated with the incubation solution lacking the substrate /¡'-glycerophos phate. For demonstration of peroxidase activity the samples were incubated in Gram-Karnovsky medium containing 3,3'diaminobenzidine (DAB) as substrate for 15 min [24], Following incubation, the samples were treated as described above for acid phosphatase activity.
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Fig. 1. Electron micrograph of immature granulocyte from peripheral blood of a patient with the CH syndrome. Large number of normal-sized granules distributed in the cytoplasm (arrow) and surrounding a large granule (double arrow) character istic of this syndrome. Numerous mitochondria (M) can also be seen. N — Nucleus. X 12,000.
Results
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Morphological observations. In peripheral blood, MGG staining and phase microscopy revealed anomalous granules in granulocytes, mono cytes and lymphocytes. In the bone marrow, granulocyte precursors showed giant vacuoles in the cytoplasm as well as the typical inclusion bodies. Electron microscopy. Granulocytes and monocytes in CH blood cells showed, in addition to normal small granules, enlarged and giant ones
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Fig. 2. Eosinophilic polymorphonuclear, distinguished by its large-size granules and the presence of a crystalloid in most of them (arrows). An extremely large granule (double arrows) containing multiple crystalloids makes it seem likely that these giant granules are formed by fusion. Fusion between two, and between three eosinophilic granules are seen (arrowheads). X 12,000.
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(fig. 1-3). Scanning of many cells indicates that the latter are a product of an active fusion process of primary and secondary lysosomes. Giant anomalous granules were present in various stages of cell maturation, but appeared predominantly in young myeloid cells. The lysosomal nature of these granules was evidenced by their content of characteristic lysosomal enzymes, as manifested by ultracytochemical positive staining for peroxi dase and acid phosphatase reaction (fig. 5, 6). Most neutrophils and mon ocytes contained the anomalous lysosomes, presenting one or more giant granules surrounded by some others of normal size (fig. 1-3). Beside their
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different size, the granules were pleomorphic in nature and contained uni form or amorphous materials. Some of the giant lysosomes contained electron-lucent vacuoles which were interpreted as being lipid droplets, probably washed out during the preparation processes (fig. 6). Vacuolization in the cytoplasm of granulo cytes was a common finding and many of the granulocytes had large de stroyed areas which showed acid phosphatase and peroxidase activities (fig. 5). Eosinophil granules were also enlarged with various configura tions, indicating an active fusion and containing one or more crystaloids of unusual size and shape (fig. 2). Variation in the number and configuration of the fusing lysosomes was found: fusion was observed between lysosomes of the same or with differ-
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Fig. 3. Neutrophilie metamyelocyte showing common small lysosomes (arrows), large granules (double arrows) and giant fusing lysosomes (arrowhead). N = Nucle us; M = mitochondria. X 18,000.
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Fig. 4. Tubular structures in a small clear lymphocyte. Kidney-shaped nucleus, condensation of chromatin along the nuclear envelope. Few mitochondria (M), ribo somes and a giant organelle (arrowheads) containing tubule-filled structures (in set). The tubular elements are sectioned longitudinally and transversely. X 13,000. Inset X 40,000.
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ent sizes and shapes, between primary or secondary lysosomes, as well as between granules in different stages of lysosomal activity (fig. 3). The lip id-laden lysosomes were observed fusing with one or more granules form ing a complex of giant granules (fig. 6). On the other hand, no fusion was found in lymphocytes. Anomalous large granules in circulating lymphocytes showed cytoplasmic inclusions containing a mass of microtubular-like structures having two types of dif ferent diameters and arrangements. One type contained few microtubules, 300-350 A in diameter, scattered in the granules. The second type cons isted of a large mass of microtubules, 150-200 À in diameter, packed
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Fig. 5. Neutrophil polymorphonuclear from peripheral blood containing charac teristic cytoplasmic inclusions stained by enzymatic reaction product. The sites of acid phosphatase activity are indicated by deposits of lead phosphate. The cytoplasm is filled with numerous vacuoles (secondary lysosomes). Note enzymatic reaction in a giant lysosome (arrowhead) and acid phosphatase reaction product in wall area (ar rows) of the vacuoles (V), and within the cytoplasmic granules and containing heter ogenous material (double arrows). X 90,000.
tightly together and occupying most of the inclusion space (fig. 4). The di ameter of the microtubules in each inclusion was uniform. The different types of inclusions could be found located in the same cell.
Discussion
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The lysosomal nature of the enlarged and giant fusing granules present in leukocytes in CH was revealed by the cytochemical reactions indicating
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Fig. 6. Electron micrograph of an immature granulocyte from bone marrow stained by peroxidase reaction product, showing various stages of the fusing lyso somes. The peroxidase staining appears as an amorphous precipitate localized in anomalous lysosomes (arrows). Point of connection and fusion between lysosomes. Lipid droplets are nonreactive (L). X 12,600.
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acid phosphatase activity in addition to the peroxidase staining. The mas sive granules in lymphocytes had an appearance and structure different from those observed in other white blood cells. It seems that the large granules in the lymphocytes were formed without a fusion process and ap peared in their protein component to be different from those observed in other leukocytes. Single giant organelles filled with masses of tubular ele ments seem to appear in CH in a higher percentage of lymphocytes than those found in normal cases [25],
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Naturally occurring fusion of lysosomes is of great importance in the execution of the physiological role of lysosomes and their enzymatic con tent. Two kinds of formation of the anomalous large granules in CH were previously suggested. The first dealt with the formation of the anomalous lysosomes in liver cells in beige mouse, and indicated that hepatocytic giant lysosomes arise from GERL elements [21]. The second suggested that the giant lysosomes are formed through a process of fusion observed in CH mink and beige mouse leukocytes [20, 26]. The present study indi cates that giant granules resulting from lysosomal fusion can occur both in early or late stages of cell maturation, as well as between primary lyso somes and phagolysosomes or even between two secondary lysosomes. This finding is in contrast with the observation in the CH beige mouse leukocytes in which the giant granules were found to be the result of fus ing of lysosomes having a similar structural appearance [26]. The fact that in the presence of a surfactant normal leukocytes show an increased enzymatic activity similar to that found in CH would suggest that the CH has a defective lysosomal membrane structure [27]. A defi ciency in membrane stability of the cytoplasmic granules in CH was also indicated by ultrastructure studies [28], This change in structure would result in an increased permeability and eventual osmotic rupture. The ap pearance of giant lysosomal vacuoles in the early myeloid cells may also be explained by a process of digestion due to release of hydrolytic en zymes. A white blood cell turnover increase due to an intramedullary de struction and an increased serum muramidase level has been reported [29]. Membrane instability and abnormal active lysosomal fusion processes may be explained by postulating the presence of surface active molecules in CH cytoplasmic granules [30], This may be produced by a disturbed lipid metabolism, favoring the formation of globular micelles of the lipid particles, if compared with the bimolecular phospholipid leaflet configur ation of the lysosomal membrane.
References
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1 Sadan, N.; Y affe , D.; R ozenszajn , L. A.; A dar, H.; Soroker, B., and E frati, P.: Cytochemical and genetic studies in four cases of Chediak-Higashi-Steinbrink syndrome. Acta haemat. 34: 20-29 (1965). 2 C hédiak , M.: Nouvelle anomalie leucocytaire de caractère constitutionnel fami lial. Revue Hematol. 7.- 362-367 (1952).
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3 H igashi, O.: Congenital gigantism of peroxidase granules. First case ever report ed of qualitative abnormality of peroxidase. Tohoku J. exp. Med. 59: 315-320 (1954). 4 L eader, R. W.; P adgett, G. A., and G orham, J. R.: Studies of abnormal leuko cyte bodies in mink. Blood 22: 477-484 (1963). 5 P adgett , G. A.; R eignam, C. W.; G orham, J. R.; H enson , J. B., and O 'M ary, C. C.: Comparative studies of the Chediak-Higashi syndrome. Am. J. Path. 51: 553-572 (1967). 6 L utzner , M. A.; L ow rie , C. T., and T ordan, H. W.: Giant granules in leuko cytes of the beige mouse. J. Hercd. 58: 299-300 (1967). 7 T aylor, R. F. and F arrell, R. K.: Light and electron microscopy of perihperal blood neutrophils in a killer whale affected with Chediak-Higashi syndrome. Fed. Proc. Fed. Am. Socs exp. Biol. 32: 822 (1973). 8 C lark, R. A. and K imball, H. R.: Defective granulocyte chemotaxis in the Che diak-Higashi syndrome. J. clin. Invest. 50: 2645-2652 (1971). 9 G allin , J. L; K limerman, J. A.; P adgett , G. A , and Sheldon , M. W.: Defec tive mononuclear leukocyte chemotaxis in the Chediak-Higashi syndrome of hu mans, mink and cattle. Blood 45: 863-870 (1975). 10 R oot , R. K.; R osenthal, A. S., and Balestra, D. J.: Abnormal bactericidal metabolic and lysosomal functions of Chediak-Higashi syndrome leukocyte. J. clin. Invest. 51: 649-665 (1972). 11 K imball, H. R. and F ord , G. H.: Granulocyte lysosomal enzymes in the Chedi ak-Higashi syndrome (CHS). Am. Fed. clin. Res. 18: 407 (1970). 12 K imball, H. R.; F ord , G. H., and W olf , S. M.: Lysosomal enzymes in normal and Chediak-Higashi blood leukocytes. J. Lab. clin. Med. 86: 616-630 (1975). 13 L utzner , M A.; T ierney , J. H., and Benditt , E. P.: Giant granules and wide spread cytoplasmic inclusions in a genetic syndrome of Aleutian mink. An elec tron microscopic study. Lab. Invest. 14: 2063-2079 (1965). 14 E sner , E.; O liver , C., and H aïmes, H.: Fate of exogenous peroxidase in renal lysosomes of mice with Chediak-Higashi syndrome. Am. J. Path. 77: 407-416 (1974). 15 C hi , E. Y. and L agunoff , D.: Abnormal mast cell granules in the beige (CHS) mouse. J. Histochem. Cytochem. 23: 117-122 (1975). 16 Blume , R. S.; P adgett, G. A.; W olff , S. M., and Bennett , J. M.: Giant neutro phil granules in the Chediak-Higashi syndrome of man, mink, cattle and mice. Can. J. comp. Med. 33: 271-274 (1969). 17 W hite , J. G.: The Chediak-Higashi syndrome. A possible lysosomal disease. Blood 28: 143-156 (1966). 18 K ritzler , R. A.; T erner , J. Y.; L indenbaum , J.; M agidson, J.; W illiams, R.; P reising , R., and P hillips , G. B.: Chediak-Higashi syndrome. Cytologic and serum lipid observations in a case and family. Am. J. Med. 36: 583-594 (1964). 19 K anfer , J. N.; B lume, R. S.; Y ankee, R. A., and W olff , S. M.: Alteration of sphingolipid metabolism in leukocytes from patients with the Chediak-Higashi syndrome. New Engl. J. Med. 279: 410-413 (1968). 20 D avis, W. C.; Spicer , S. S.; G reene , W. B., and P adgett, G. A.: Ultrastructure of bone marrow granulocytes in normal mink and mink with the homology of
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25 26
27
28
29
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the Chediak-Higashi trait of humans. I. Origin of the abnormal granules present in the neutrophils of mink with CHS trait. Lab. Invest. 24: 303-317 (1971). E ssner, E. and O liver , C.: Lysosome formation in hepatocytes of mice with Chediak-Higashi syndrome. Lab. Invest. 30: 596-607 (1974). L uft , J. H.: Improvements in epoxyresin embedding methods. J. biophys. biochem. Cytol. 9: 409-^114 (1961). R ozenszajn, L. A. and E frati, P.: Cytochemical and phase-contrast observa tions on Gaucher cells. Acta haemat. 25: 43-48 (1961). G raham, R. C. and K arnovsky, M. J.: The early stages of absorption of injected horseradish peroxidase in the proximal tubules of the mouse kidney. Ultrastruc tural cytochemistry by a new technique. J. Histochem. Cytochem. 14: 291-302 (1966). H uhn , D.: Neue Organellen in peripheren Lymphozyten? Dt. med. Wschr. 93: 2099-2100 (1968). O liver , C. and E ssner , E.: Formation of anomalous lysosomes in monocytes, neutrophils and eosinophils from bone marrow of mice with Chediak-Higashi syndrome. Lab. Invest. 3 2 :17-27 (1975). R ozenszajn , L. A. and R adnay, J.: The lysosomal nature of the anomalous granules and chromosome aberrations in cultures of peripheral blood in Chedi ak-Higashi syndrome. Br. J. Haemat. 18: 683-689 (1970). E frati, P. and Danon, D.: Electron-microscopical study of bone marrow cells in a case of Chediak-Higashi-Steinbrink syndrome. Br. J. Haemat. 15: 173-184 (1968). Blume , R. S.; Bennett , J. M.; Y ankee, R. A., and W olff , S. M.: Defective granulocyte regulation in the Chediak-Higashi syndrome. New Engl. J. Med. 279: 1009-1015 (1968). D ingle , J. T.: Vacuoles, vesicles and lysosomes. Br. med. Bull. 24: 141-145 (1968).
Prof. L. A. R ozenszajn, Hematologic Laboratories, Meir Hospital, Kfar Saba (Is rael)
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Large Granules and Lysosomal Fusion in Human ChediakHigashi White Blood Cells L. A. R ozenszajn, E. B en D avid and S. B ar Sela Hematologic Laboratories, Meir Hospital, Kfar Saba and Department of Life Sciences, Bar-llan University, Ramat-Gan
Key Words. Acid phosphatase • Chediak-Higashi syndrome • Cytochemistry • Electron microscopy • Lysosomes • Peroxidase Abstract. The phenomenon of giant anomalous lysosome formation in human Chediak-Higashi syndrome leukocytes was analyzed. Ultrastructure findings com bined with cytochemical procedures for visualizing acid phosphatase and peroxidase activity showed giant anomalous granules in addition to normal, small and enlarged granules. Massive granules in lymphocytes had an appearance and structure differ ent from those found in other leukocytes. The giant granules seem to be a product of an active fusion between primary and secondary normal sized or enlarged lyso somes. This fusion occurs in polymorphonuclear neutrophils, eosinophils and in monocytes. No fusion was found in lymphocyte granules.
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The Chediak-Higashi syndrome (CH) is a rare disorder determined by one autosomal recessive gene and is lethal in the homozygous state [lj. This disorder occurs in man [2, 3] and animals [4-7]. It is clinically man ifested by pancytopenia, lymphoadenopathy, hepatosplenomegaly, photo phobia, partial albinism in most cases, and increased susceptibility to infection. Studies of leukocyte function have documented defective granu locyte chemotaxis [8, 9] and impaired intracellular destruction of phagocytized bacteria [10] as well as reduced quantities and abnormal distribu tion of lysosomal enzymes [11, 12], Cytologically, the prominent characteristic findings are large anomalous granules in granule-containing cells [13-15], However, leukocyte granule abnormalities are pathognomonic for this syndrome [1-3, 6, 16], The presence of acid phosphatase and peroxidase activity determined by cyto chemistry techniques led to the conclusion that these large granules repre
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sent lysosome varieties [1, 17]. Furthermore, studies in CH cells have in dicated a disturbance in their lipid metabolism [18, 19]. It has been sug gested, on the basis of ultrastructure of white blood cells of man and ani mals that the formation of abnormal granules may be due to a process of lysosomal fusion [17, 20]. In CH beige mouse hepatocytes, the giant lyso somes are formed from the Golgi endoplasmic reticulum lysosome (GERL) elements [21]. The present investigation is a study of the structure and the formation, through a process of fusion, of the anomalous granules in human CH leu kocytes. The identification of the fusing lysosomes, as well as the forma tion of the giant granules, was based on their enzymatic staining and structural analogies. Materials and Methods
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Peripheral blood huffy coat and bone marrow from a 4-year-old non-albino male were examined. Morphologic studies. Using the May-Griinwald-Giemsa stain (MGG), morphol ogic studies were performed on smears of peripheral blood, micro buffy coat and bone marrow cells. Phase-contrast microscopy was used to examine fresh prepara tions. Electron microscopy. Examinations were performed on buffy coat obtained from heparinized (200 1U/10 ml) peripheral blood and bone marrow cells, fixed in 2.5°/o glutaraldehyde in 0.2 m Na-cacodylate-HCl buffer (pH 7.2) containing 7°/o sucrose and kept at 4 °C for 2 h. The samples were washed with Na-cacodylate, postfixed in l°/o O s04 in cacodylate-HCl buffer, dehydrated in graded ethanols followed by pro pylene oxide and embedded in Epon 812 as described by L uft [22], Sections were cut with LKB ultratome, stained with uranyl acetate and lead citrate and examined with a Philips 300 electron microscope. Ultrastructural cytochemistry. Acid phosphatase activity was demonstrated as follows: peripheral blood and bone marrow cells were fixed in 1% glutaraldehyde in 0.2 m Na-cacodylate-HCl buffer (pH 7.2) for 15 min at 4 °C. The cells were then washed 3 times in the same cacodylate buffer. The fixed cells were suspended in Gomori incubation media [23] and maintained at 37 °C, pH 4.8, for 2 h. Following in cubation, the cells were washed three times in Na-cacodylate buffer and prepared for electron microscopy as described above. Staining with uranyl acetate and lead citrate was performed after testing the presence of the enzyme activity product on unstained sections. As a control for the specificity of the staining method, fixed cells were incubated with the incubation solution lacking the substrate /¡'-glycerophos phate. For demonstration of peroxidase activity the samples were incubated in Gram-Karnovsky medium containing 3,3'diaminobenzidine (DAB) as substrate for 15 min [24], Following incubation, the samples were treated as described above for acid phosphatase activity.
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Fig. 1. Electron micrograph of immature granulocyte from peripheral blood of a patient with the CH syndrome. Large number of normal-sized granules distributed in the cytoplasm (arrow) and surrounding a large granule (double arrow) character istic of this syndrome. Numerous mitochondria (M) can also be seen. N — Nucleus. X 12,000.
Results
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Morphological observations. In peripheral blood, MGG staining and phase microscopy revealed anomalous granules in granulocytes, mono cytes and lymphocytes. In the bone marrow, granulocyte precursors showed giant vacuoles in the cytoplasm as well as the typical inclusion bodies. Electron microscopy. Granulocytes and monocytes in CH blood cells showed, in addition to normal small granules, enlarged and giant ones
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Fig. 2. Eosinophilic polymorphonuclear, distinguished by its large-size granules and the presence of a crystalloid in most of them (arrows). An extremely large granule (double arrows) containing multiple crystalloids makes it seem likely that these giant granules are formed by fusion. Fusion between two, and between three eosinophilic granules are seen (arrowheads). X 12,000.
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(fig. 1-3). Scanning of many cells indicates that the latter are a product of an active fusion process of primary and secondary lysosomes. Giant anomalous granules were present in various stages of cell maturation, but appeared predominantly in young myeloid cells. The lysosomal nature of these granules was evidenced by their content of characteristic lysosomal enzymes, as manifested by ultracytochemical positive staining for peroxi dase and acid phosphatase reaction (fig. 5, 6). Most neutrophils and mon ocytes contained the anomalous lysosomes, presenting one or more giant granules surrounded by some others of normal size (fig. 1-3). Beside their
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different size, the granules were pleomorphic in nature and contained uni form or amorphous materials. Some of the giant lysosomes contained electron-lucent vacuoles which were interpreted as being lipid droplets, probably washed out during the preparation processes (fig. 6). Vacuolization in the cytoplasm of granulo cytes was a common finding and many of the granulocytes had large de stroyed areas which showed acid phosphatase and peroxidase activities (fig. 5). Eosinophil granules were also enlarged with various configura tions, indicating an active fusion and containing one or more crystaloids of unusual size and shape (fig. 2). Variation in the number and configuration of the fusing lysosomes was found: fusion was observed between lysosomes of the same or with differ-
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Fig. 3. Neutrophilie metamyelocyte showing common small lysosomes (arrows), large granules (double arrows) and giant fusing lysosomes (arrowhead). N = Nucle us; M = mitochondria. X 18,000.
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Fig. 4. Tubular structures in a small clear lymphocyte. Kidney-shaped nucleus, condensation of chromatin along the nuclear envelope. Few mitochondria (M), ribo somes and a giant organelle (arrowheads) containing tubule-filled structures (in set). The tubular elements are sectioned longitudinally and transversely. X 13,000. Inset X 40,000.
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ent sizes and shapes, between primary or secondary lysosomes, as well as between granules in different stages of lysosomal activity (fig. 3). The lip id-laden lysosomes were observed fusing with one or more granules form ing a complex of giant granules (fig. 6). On the other hand, no fusion was found in lymphocytes. Anomalous large granules in circulating lymphocytes showed cytoplasmic inclusions containing a mass of microtubular-like structures having two types of dif ferent diameters and arrangements. One type contained few microtubules, 300-350 A in diameter, scattered in the granules. The second type cons isted of a large mass of microtubules, 150-200 À in diameter, packed
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Fig. 5. Neutrophil polymorphonuclear from peripheral blood containing charac teristic cytoplasmic inclusions stained by enzymatic reaction product. The sites of acid phosphatase activity are indicated by deposits of lead phosphate. The cytoplasm is filled with numerous vacuoles (secondary lysosomes). Note enzymatic reaction in a giant lysosome (arrowhead) and acid phosphatase reaction product in wall area (ar rows) of the vacuoles (V), and within the cytoplasmic granules and containing heter ogenous material (double arrows). X 90,000.
tightly together and occupying most of the inclusion space (fig. 4). The di ameter of the microtubules in each inclusion was uniform. The different types of inclusions could be found located in the same cell.
Discussion
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The lysosomal nature of the enlarged and giant fusing granules present in leukocytes in CH was revealed by the cytochemical reactions indicating
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Fig. 6. Electron micrograph of an immature granulocyte from bone marrow stained by peroxidase reaction product, showing various stages of the fusing lyso somes. The peroxidase staining appears as an amorphous precipitate localized in anomalous lysosomes (arrows). Point of connection and fusion between lysosomes. Lipid droplets are nonreactive (L). X 12,600.
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acid phosphatase activity in addition to the peroxidase staining. The mas sive granules in lymphocytes had an appearance and structure different from those observed in other white blood cells. It seems that the large granules in the lymphocytes were formed without a fusion process and ap peared in their protein component to be different from those observed in other leukocytes. Single giant organelles filled with masses of tubular ele ments seem to appear in CH in a higher percentage of lymphocytes than those found in normal cases [25],
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Naturally occurring fusion of lysosomes is of great importance in the execution of the physiological role of lysosomes and their enzymatic con tent. Two kinds of formation of the anomalous large granules in CH were previously suggested. The first dealt with the formation of the anomalous lysosomes in liver cells in beige mouse, and indicated that hepatocytic giant lysosomes arise from GERL elements [21]. The second suggested that the giant lysosomes are formed through a process of fusion observed in CH mink and beige mouse leukocytes [20, 26]. The present study indi cates that giant granules resulting from lysosomal fusion can occur both in early or late stages of cell maturation, as well as between primary lyso somes and phagolysosomes or even between two secondary lysosomes. This finding is in contrast with the observation in the CH beige mouse leukocytes in which the giant granules were found to be the result of fus ing of lysosomes having a similar structural appearance [26]. The fact that in the presence of a surfactant normal leukocytes show an increased enzymatic activity similar to that found in CH would suggest that the CH has a defective lysosomal membrane structure [27]. A defi ciency in membrane stability of the cytoplasmic granules in CH was also indicated by ultrastructure studies [28], This change in structure would result in an increased permeability and eventual osmotic rupture. The ap pearance of giant lysosomal vacuoles in the early myeloid cells may also be explained by a process of digestion due to release of hydrolytic en zymes. A white blood cell turnover increase due to an intramedullary de struction and an increased serum muramidase level has been reported [29]. Membrane instability and abnormal active lysosomal fusion processes may be explained by postulating the presence of surface active molecules in CH cytoplasmic granules [30], This may be produced by a disturbed lipid metabolism, favoring the formation of globular micelles of the lipid particles, if compared with the bimolecular phospholipid leaflet configur ation of the lysosomal membrane.
References
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3 H igashi, O.: Congenital gigantism of peroxidase granules. First case ever report ed of qualitative abnormality of peroxidase. Tohoku J. exp. Med. 59: 315-320 (1954). 4 L eader, R. W.; P adgett, G. A., and G orham, J. R.: Studies of abnormal leuko cyte bodies in mink. Blood 22: 477-484 (1963). 5 P adgett , G. A.; R eignam, C. W.; G orham, J. R.; H enson , J. B., and O 'M ary, C. C.: Comparative studies of the Chediak-Higashi syndrome. Am. J. Path. 51: 553-572 (1967). 6 L utzner , M. A.; L ow rie , C. T., and T ordan, H. W.: Giant granules in leuko cytes of the beige mouse. J. Hercd. 58: 299-300 (1967). 7 T aylor, R. F. and F arrell, R. K.: Light and electron microscopy of perihperal blood neutrophils in a killer whale affected with Chediak-Higashi syndrome. Fed. Proc. Fed. Am. Socs exp. Biol. 32: 822 (1973). 8 C lark, R. A. and K imball, H. R.: Defective granulocyte chemotaxis in the Che diak-Higashi syndrome. J. clin. Invest. 50: 2645-2652 (1971). 9 G allin , J. L; K limerman, J. A.; P adgett , G. A , and Sheldon , M. W.: Defec tive mononuclear leukocyte chemotaxis in the Chediak-Higashi syndrome of hu mans, mink and cattle. Blood 45: 863-870 (1975). 10 R oot , R. K.; R osenthal, A. S., and Balestra, D. J.: Abnormal bactericidal metabolic and lysosomal functions of Chediak-Higashi syndrome leukocyte. J. clin. Invest. 51: 649-665 (1972). 11 K imball, H. R. and F ord , G. H.: Granulocyte lysosomal enzymes in the Chedi ak-Higashi syndrome (CHS). Am. Fed. clin. Res. 18: 407 (1970). 12 K imball, H. R.; F ord , G. H., and W olf , S. M.: Lysosomal enzymes in normal and Chediak-Higashi blood leukocytes. J. Lab. clin. Med. 86: 616-630 (1975). 13 L utzner , M A.; T ierney , J. H., and Benditt , E. P.: Giant granules and wide spread cytoplasmic inclusions in a genetic syndrome of Aleutian mink. An elec tron microscopic study. Lab. Invest. 14: 2063-2079 (1965). 14 E sner , E.; O liver , C., and H aïmes, H.: Fate of exogenous peroxidase in renal lysosomes of mice with Chediak-Higashi syndrome. Am. J. Path. 77: 407-416 (1974). 15 C hi , E. Y. and L agunoff , D.: Abnormal mast cell granules in the beige (CHS) mouse. J. Histochem. Cytochem. 23: 117-122 (1975). 16 Blume , R. S.; P adgett, G. A.; W olff , S. M., and Bennett , J. M.: Giant neutro phil granules in the Chediak-Higashi syndrome of man, mink, cattle and mice. Can. J. comp. Med. 33: 271-274 (1969). 17 W hite , J. G.: The Chediak-Higashi syndrome. A possible lysosomal disease. Blood 28: 143-156 (1966). 18 K ritzler , R. A.; T erner , J. Y.; L indenbaum , J.; M agidson, J.; W illiams, R.; P reising , R., and P hillips , G. B.: Chediak-Higashi syndrome. Cytologic and serum lipid observations in a case and family. Am. J. Med. 36: 583-594 (1964). 19 K anfer , J. N.; B lume, R. S.; Y ankee, R. A., and W olff , S. M.: Alteration of sphingolipid metabolism in leukocytes from patients with the Chediak-Higashi syndrome. New Engl. J. Med. 279: 410-413 (1968). 20 D avis, W. C.; Spicer , S. S.; G reene , W. B., and P adgett, G. A.: Ultrastructure of bone marrow granulocytes in normal mink and mink with the homology of
Lysosomes in Chediak-Higashi Leukocytes
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the Chediak-Higashi trait of humans. I. Origin of the abnormal granules present in the neutrophils of mink with CHS trait. Lab. Invest. 24: 303-317 (1971). E ssner, E. and O liver , C.: Lysosome formation in hepatocytes of mice with Chediak-Higashi syndrome. Lab. Invest. 30: 596-607 (1974). L uft , J. H.: Improvements in epoxyresin embedding methods. J. biophys. biochem. Cytol. 9: 409-^114 (1961). R ozenszajn, L. A. and E frati, P.: Cytochemical and phase-contrast observa tions on Gaucher cells. Acta haemat. 25: 43-48 (1961). G raham, R. C. and K arnovsky, M. J.: The early stages of absorption of injected horseradish peroxidase in the proximal tubules of the mouse kidney. Ultrastruc tural cytochemistry by a new technique. J. Histochem. Cytochem. 14: 291-302 (1966). H uhn , D.: Neue Organellen in peripheren Lymphozyten? Dt. med. Wschr. 93: 2099-2100 (1968). O liver , C. and E ssner , E.: Formation of anomalous lysosomes in monocytes, neutrophils and eosinophils from bone marrow of mice with Chediak-Higashi syndrome. Lab. Invest. 3 2 :17-27 (1975). R ozenszajn , L. A. and R adnay, J.: The lysosomal nature of the anomalous granules and chromosome aberrations in cultures of peripheral blood in Chedi ak-Higashi syndrome. Br. J. Haemat. 18: 683-689 (1970). E frati, P. and Danon, D.: Electron-microscopical study of bone marrow cells in a case of Chediak-Higashi-Steinbrink syndrome. Br. J. Haemat. 15: 173-184 (1968). Blume , R. S.; Bennett , J. M.; Y ankee, R. A., and W olff , S. M.: Defective granulocyte regulation in the Chediak-Higashi syndrome. New Engl. J. Med. 279: 1009-1015 (1968). D ingle , J. T.: Vacuoles, vesicles and lysosomes. Br. med. Bull. 24: 141-145 (1968).
Prof. L. A. R ozenszajn, Hematologic Laboratories, Meir Hospital, Kfar Saba (Is rael)
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