A CYTOCHEMICAL ELECTRON MICROSCOPIC STUDY OF RABBIT HETEROPHILIC LEUKOCYTES DORING PHAGOCYTOSIS

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1 Nagoya J. med. Sci. 33: , 1971 A CYTOCHEMICAL ELECTRON MICROSCOPIC STUDY OF RABBIT HETEROPHILIC LEUKOCYTES DORING PHAGOCYTOSIS ACID AND ALKALINE PHOSPHATASE REACTIONS MoRIHIKO NnMr 1st Department of Internal Medicine, Nagoya University School of Medicine (Director: Asst. Prof Itsuro Sobue) ABSTRACT The present electron microscopic study was attempted to demonstrate a de tailed localization of acid and alkaline phosphatase within the phagocytosing rabbit heterophils and further to clarify the difference in time course between the appearance of acid phosphatase and of alkaline phosphatase reaction products in the phagocytic vacuoles. As material, rabbit bone marrow cell suspension and zymosan for the phagocytized particle were used. Alkaline phosphatase reaction was seen in relatively small granules (i.e. specific granules), and acid phosphatase reaction in large granules (i.e. azurophilic granules). Alkaline phosphatase fused into the phagocytic vacuoles earlier than acid phosphatase. These distinctly different patterns of enzyme fusion might be dependent on the qualitative heterogeneity of granules and also on the quantitative difference between granules with acid phosphatase and th ose with alkaline phosphatase of mature heterophils which show most active phagocytosis among ce!ls with var ying maturation. INTRODUCTION Many electron microscopic studies on phagocytosis have contributed to the elucidation of the nature of cytoplasmic granules and have indicated that these granules play a role in the process of phagocytosis I J-sJ. Cohn et at. 7 J and Ohta s J have demonstrated biochemically that non-specific acid and alkaline phosphatase and a variety of other hydrolytic enzymes are localizad in the granule fraction of leukocytes. Electron microscopic localization of non-specific acid and alkaline phosphatase has been accomplished in the heterophils of the rabbit 9 l. Acid phosphatase was demonstrated in primary granules and tertiary granules. In addition, alkaline phosphatase was evidenced in secondary granules 9 J Ill zsj. ~ ~ "i' $ Received for publication November, 24,

2 288 M. NIIMI Biochemical analysis of phagocytosing leukocytes was also reported by others ); a variety of hydrolytic enzymes are released from the granule fraction accompanied by an increase in enzyme activities in the supernate 8 J 12 J 13 ). Moreover, cinematographic studies demonstrated granulolysis around phagocytosed particles, implicating these granules to be a source of catabolic enzymes 29 J. This phenomenon was confimed by cytochemical method 16 ) 17 ) demonstrating that alkaline and acid phosphatase, which are associated specifically with leukocyte granules, fuse into the vacuoles of these cells. The present study presents the time course relationship between vacuoles with alkaline phosphatase activity and those with acid phosphatase activity during phagocytosis. 1) Preparation for phagocytosis MATERIALS AND METHODS Cells: Leukocytes were obtained by aspiration from rabbit tibial bone marrbw. They were suspended in phosphate buffered saline in a final concentration of 40 x 10 3 cells per mm 3 Zymosan (Tokyo Kasei Kogyo K.K.) was boiled, washed three times in saline, and used in a final concentration of 200 x 10 3 particles per mm 3 Rabbit serum: Serum was obtained by venipuncture from the same rabbit. Incubation for phagocytosis 5 ): 4 vol. of cell suspension, 1 vol. of zymosan suspension and 1 vol. of rabbit serum were mixed, and the incubation mixture was rotated slowly at 37 C in plastic centrifuge tubes for 1, 3, 5, 15, 30 or 60 minutes. 2) Preparation for cytochemistry Prejixation: After these procedures, the cells were recovered by centrifugation and fixed 60 minutes in Sodium Cacodylate-HCl buffered 1.5% Glutaral dehyde 18 J with I% sucrose at 4 C, then washed three times in Cacodylate buffer. Incubation: Acid phosphatase reaction was carried out by the Barka and Anderson modification 23 ) of the Gomori medium 24 ) at 37 C for 60 minutes. Alkaline phosphatase reaction was carried out in the Gomori medium 24 ) at 37 C for 60 minutes. After incubation, the materials were immersed for 5 minutes in 2% lead nitrate. When these incubation times were over, the materials were washed three times in Acetate-V eronal buffer with 7% sucrose. Post fixation: Following the cytochemical procedures, the materials were then postfixed 60 minutes in buffered 1% Os04 solution 19 ) prior to ethanol dehydration and embedding in Epon 20 ). Microscopy: Sections were prepared with an LKB ultratome ( Laboratorie

3 AN ELECTRON MICROSCOPIC STUDY OF PHAGOCYTOSIS 289 och Kemikaliska Produkter, Sweden), and a JEM electron microscope was used for all studies without staining procedures with lead 21 l or uranyl 22 l, unless otherwise indicated. 3) Control As one control the materials were fixed 60 minutes in Cacodylate buffered 1.5% Glutaraldehyde, followed by 60 minutes fixation in 1% OsO. solution with omission of the cytochemical procedures. As another control, similar cytochemical procedures were performed in the absence of substrate in the incubation medium. RESULTS In the control sections, after one minute incubation the zymosan particles began to be engulfed by protruding pseudopods of heterophils, and interdigitation of heterophils creates vacuoles, though phagocytosis is not completed (Figs. 1 and Fig. 2). After three minutes the particles were completely ingested by heterophils (Fig. 3 and Fig. 4) and numerous granules of phagocytosing mature cells were still dispersed within the cytoplasm (Fig. 3). Zymosan were seen as a dense core with a stippled-looking capsule 5 l. The diameter of the zymosan ranged from 2p to 3p. In favorable sections, several granules are seen fixed in the processes of invading phagocytic vacuoles when fusion has taken place between the granule membrane and the membrane lining the vacuole. In such vacuoles, a continuous space surrounded the ingested particles. (Fig. 5 and Fig. 6). In the sections cytochemically treated, a number of aggregates of numerous microparticles of high electron density were observed. These deposits with high density were considered to indicate the presence of activity of alkaline or acid phosphatase 27 l 25 l 26 l. These positive cytochemical findings were confirmed by the absence of reaction products in sections made from leukocytes incubated without substrate (Figs. 7, 8, 9 ), or in sections without cytochemical procedures (Figs. 1-6). Also, zymosan was free of reaction products when it was extracellularly located. Therefore, such deposits could be identified as the reaction products of these enzymes. In the cytoplasm, alkaline phosphatase reactions were observed in relatively small granules ( p.) and acid phosphatase reactions in large granules ( pl. (Figs. 10 and 16). Within three minutes, marked deposition of alkaline phosphatase reaction was noted in almost all phagocytic vacuoles (Figs. 10, 11, 12l. In the case of five minutes incubation, the reaction products in phagocytic vacuoles became

4 290 M. NIIMI abundunt (Fig. 13 ). On the other hand, the acid phosphatase reaction products of phagocytic vacuoles were not observed within three minutes (Fig. 15). At five minutes, the reaction was noted in only a few vacuoles (Fig. 16). In some sections, acid phosphatase reactive granules were observed entering the phagocytic vacuoles (Fig. 17). At the end of thirty minutes both acid and alkaline phosphatase reactive granules of the cytoplasm completely disappeared, whereas phagocytic vacuoles showed strongly positive reactions (Figs. 14 and 18). DISCUSSION This study was aimed to demonstrate the cytochemical pattern underlying the degranulation process of rabbit heterophils during phagocytosis. Horn et a!. I?) reported that non specific phosphatases are discharged from cytoplasmic granules of rabbit heterophils into the phagocytic vacuoles with the maintenance of membrane continuity. Hydrolytic enzymes are prevented from gaining access into the cytoplasm proper. This mechanism has been termed "exoplasmosis" (by Zucker-Franklin), by which particulate matter is eliminated to the outside of the cell as well as to the discharge of substances into intracellular vacuoles which, in reality, also represent extracellular spaces 5 ). In this study, the author was able to observe granule like materials localized inside the clear space of the phagocytic vacuoles. These findings con firmed the morphologic evidences reported by others 3 ) 4 ) 5 l. Also, acid phosphatase activity could be identified in these granule-like materials. The findings presented here might afford evidence that non-specific phosphatases are transferred from cytoplasmic granules of rabbit heterophils to newly formed phagocytic vacuoles 17 ). According to some reports 30 ) 31 ), rabbit heterophilic granules could be separated into three types, i.e. primary, secondary and tertiary granules 31). The primary and secondary granules were conventionally designated azurophilic and specific granules, respectively 30 l. Primary granules are present exclusively in early heterophils (promyelocytes) ; primary and secondary granules in intermediate heterophils (myelocytes, metamyelocytes and bands) ; and tertiary granules on top of primary and secondary granules in late heterophi!s (fully mature segmented forms). These granule types differ not only in the time of their appearance during cell development, but also in their enzyme compositions, as revealed by cytochemical methods 9 ) 10 ) ll) zs). Primary granules contain acid phosphatase 9 ) 11 l 28 l, acid mucosubstance 32 ) and strongly basic proteins 32 ) 33 l ; secondary granules contain alkaline phosphatase sj 11 ) 28 ) ; and tertiary granules acid phosphatase 9 l. In accord with these findings, the author recognized the same heterogeneity of the granules, i.e. in phosphatase reaction large granules

5 AN ELECTRON MICROSCOPIC STUDY OF PHAGOCYTOSIS 291 reacted in acid medium and small ones in alkaline medium. Further Horn et al. 17 > reported that phosphatase reaction products could be observed in phagocytic vacuoles. In this study, successive observations on the course of time during phagocytosis were made. Within three minutes, alkaline phosphatase reaction was noted in almost all phagocytic vacuoles, providing evidence consistent with the fusion of such granules with phagocytic vacuoles, as suggested above l7j. At five minutes, the reaction products of the phagocytic vacuoles became numerous. However, some delay was noted in the appearance of acid phosphatase reaction products in phagocytic vacuoles, namely between 5-15 minutes incubation. By 30 minutes all the phosphatase reactive granules disappeared, but reaction products were still strongly deposited in phagocytic vacuoles. It is likely that the pattern of phosphatase activity in phagocytic vacuoles substantiates indirect evidence cited above 12 > 29 > which suggest transfer of hydrolytic enzymes from leukocyte granules to the site of ingested particles. lt is interesting that the transfer of alkaline phosphatase during phagocytosis from the cytoplasmic granules of rabbit heterophils into phagocytic vacuoles takes place earlier than of acid phosphatase. This discrepancy of phosphatase reaction in phagocytic vacuoles in time course may depend upon the heterogeneity in behavior of granules during phagocytosis, which was shown by biochemical methods 8 > 12 >; such as the ready release of alkaline phosphatase when compared with acid phosphatase from granules. Moreover, it might be due to the presence of fewer acid phosphatase reactive granules than alkaline phosphatase reactive granules in mature heterophils which play the main role in phagocytosis. In fact, Bainton et aeo> n > has reported the quantitative changes in rabbit heterophilic granules during cell development by light and electron microscopic methods, concluding that azurophilic granules decrease and specific granules increase in number according to leukocyte maturation. CONCLUSION I) Zymosan, as seen by the electron microscope, was engulfed or ingested completely by rabbit heterophils at the end of three minutes. 2) Granules of heterophils were seen invading phagocytic vacuoles. 3\ By cytochemical electron microscopic study, acid phosphatase reaction was positive in the larger granules ( p ), and alkaline phosphatase reaction in the smaller ones ( pl. 4) According to time course of phagocytosis, alkaline phosphatase reaction products fused into phagocytic vacuoles earlier than acid phosphatase reaction products. This might be due to the qualitative heterogeneity of granules and also to the difference in quantity between alkaline phosphatase reactive granules

6 292 M. NIIMI and acid phosphatase reactive granules of mature heteropnils which play the principal role in phagocytosis. ACKNOWLEDGEMENT The author wishes to express his sincere gratitude to Asst. Professor Itsuro Sobue for his critical review of this manuscript and to Dr. Hiroshi Ohta for his invaluable advice and encouragement throughout this study. BIBLIOGRAPHY 1) Goodman, J. R., Moore, R. E. and Barker, R. F., Electron microscopic study of phagocytosis of Staphylococcus by human leukocytes. II. Virulent and non-virulent Staphylococci, ]. Bact., 72, 736, ) Goodman, J. R. and Moore, R. E., Electron microscopic study of phagocytosis of Staphylococcus by human leukocytes, ]. Bact., 71, 547, ) Essner, E., An electron microscopic study of erythrophagocytosis, ]. Biophys. Biochem. Cytol., 7, 329, ) Lockwood, W. R. and Allison, F., Electron micrographic studies of phagocytic cells. I. Morphologic changes of the cytoplasm and granules of rabbit granulocytes associated with ingestion of rough pneumococcus, Brit. ]. Exp. Path., 44, 593, ) Zucker-Franklin, D. and Hirsch, J. G., Electron microscope studies on the degranulation of rabbit peritoneal leukocytes during phagocytosis, ]. Exp. Med., 120, 569, ) Kishigami, Y., Phase microscopic and electron microscopic studies of bacterial phagocytosis by leukocytes. II. Behavior of leukocytes, especially their granules, following phagocytosis, Acta Haemat. ]ap., 31, 984, 1968 (in Japanese). 7) Cohn, Z. A. and Hirsch, ]. G., The isolation and properties of the specific cytoplasmic granules of rabbit polymorphonuclear leukocytes, ]. Exp. Med., 112, 983, ) Ohta, H., A biochemical study on the neutrophil granules isolated in a pure state from leukocyte homogenate, Acta Haemat. ]ap., 27, 555, ) Wetzel, B. K., Spicer, S. S. and Horn, R. G., Fine structural localization of acid and alkaline phosphatases in cells of rabbit blood and bone marrow, ]. Histochem. Cytochem., 15, 311, ) Bainton, D. F. and Farquhar, M. G., Differences in enzyme content of azurophil and specific granules of PMN leukocytes. I. Histochemical staining of bone marrow smears, ]. Cell Biol., 39, 286, ) Bainton, D. F. and Farquhar, M. G., Differences in enzyme content of azurophil and specific granules of PMN leukocytes. II. Cytochemistry and electron microscopy of bone marrow cells, ]. Cell Biol., 39, 299, ) Cohn, Z. A. and Hirsch, ]. G., The influence of phagocytosis on the intracellular distribution of granule-associated components,]. Exp. Med., 112, 1015, ) Hirsch, ]. G. and Cohn, Z. A., Degranulation of polymorphonuclear leukocytes following phagocytosis of microorganisms, ]. Exp. Med., 112, 1005, ) Bode!, P. and Malawista, S. E., Phgocytosis by human blood leukocytes during suppression of glycolysis, Exp. Cell Res., 56, 15, ) Crowder, ]. G., Martin, R. R. and White, A., Release of histamin and lysosomal enzymes by human leukocytes during phagocytosis of Staphylococci,]. Lab. Clin. Med., 74, 436, ) Axline, S, G. and Cohn, Z. A., In vitro induction of lysosomal enzymes by phagocytosis,

7 AN ELECTRON MICROSCOPIC STUDY OF PHAGOCYTOSIS 293 }. Exp. Med., 131, 1239, ) Horn, R. G., Spicer, S. S. and Wetzel, B. K., Phagocytosis of bacteria by heterophil leuzocytes. Acid and alkaline phosphatase cytochemistry, Amer. ]. Path., 45, 327, ) Sabatini, D. D., Bensch, K. and Barrnett, R. ]., Cytochemistry and Electron Microscopy. The preservation of cellular ultrastructure and enzymatic activity by aldehyde fixation, ]. Cell Biol., 17, 19, ) Pease, D. C., An electron microscopic study of red bone marrow, Blood, 11, 501, ) Luft, ]. H., Improvements in epoxy resin embedding methods, ]. Biophys. Biochem. Cytol., 9, 409, ) Millonig, G., A modified procedure for lead staining of thin section, ]. Biophys. Biochem. Cytol., 11, 736, ) Watson, M. L., Staining of tissue sections for electron microscopy with heavy metals, ]. Biochem. Cytol., 4, 475, ) Barka, T. and Anderson, P. ]., Histochemistry: Theory, Practice and Bibliography, Roeber Medical Division, New York, 1963, p ) Gomori, G., Microscopic Histochemistry: Principles aud Practice, University of Chicago Press, Chcago, 1952, p ) Goldfischer, S., Essner, E. and Novikoff, A. B., The localization of phosphatase activities at the level of ultrastructure, ]. Histochem. Cytochem., 12, 72, ) Brades, D., Observation on the apparent mode of formation of "pure" lysosomes, ]. Ultrastruct. Res., 12, 63, ) MOlbert, E. R. G., Duspiva, F. and Deimling, 0. H., The demonstration of alkaline phosphatase in electron microscope, ]. Biophys. Biochem. Cytol., 7, 387, ) Miller, F., Electron microscopic cytochemistry of leukocyte granules. In Sixth International Congress for Electron Microscopy, 2, Kyoto, Japan, R. Uyeda, editor. Maruzen Co. Ltd., Tokyo, 71, ) Hirsch, ]. G., Cinemicrophotographic observations on granule lysis in polymorphonuclear leukocytes during phagocytosis, }. Exp. Med., 116, 827, ) Bainton, D. F. and Farquhar, M. G., Origin of granules in polymorphonuclear leukocytes. Two types derived from opposite faces of the Golgi complex in developing granules, ]. Cell Biol., 28, 277, ) Wetzel, B. K., Horn, R. G. and Spicer, S. S., Fine structural studies on the development of heterophil, eosinophil and basophil granulocytes in rabbits, Lab. Invest., 16, 349, ) Horn, R. G. and Spicer, S. S., Sulfated mucopolysaccharide and basic protein in certain granules of rabbit leukocytes, Lab. Invest., 13, 1, )- Dunn, W. B., Hardin, ]. H. and Spicer, S. S., Ultrastructual localization of myeloperoxidase in human neutrophil and rabbit heterophil, and eosinophil leukocytes, Blood, 32, 935, EXPLANATION OF PLATES Abbreviations used n: nucleus pv: phagocytic vacuole z: zymosan particle Each scale indicates 1 p. pg: primary granule sg: secondary granule

8 294 M. NIIMI FIG. 1. Control section without cytochemical procedures. At one minute incubation zymosan particles began to be engulfed by protrusions of pseudopods of heterophils. Double-stained with uranyl acetate and lead citrate. FIG. 2. Same section showing interdigitation of heterophils creating vacuoles, though phagocytosis is not completed after one minute incubation. Double'stained with uranyl acetate and lead citrate. FIGS. 3 and 4. Control section without cytochemical procedures. At three minutes zymosan particles were completely ingested by mature heterophils and numerous granules were still dispersed. Double-stained with uranyl acetate and lead citrate. FIG. 5. Control section without cytochemical procedures. At three minutes zymosan particles were ingested with granule-like materials (arrow) in the phagocytic vacuoles. Double-stained with uranyl acetate and lead citrate. FIG. 6. Higher magnification of Fig. 5. Two granule-like materials (arrow) seen fixed in the process of entering the phagocytic vacuoles. FIG. 7. Control section treated in acid phosphatase medium in the absence of substrate,.8-glycerosphate. At five minutes no reactive deposits were observed. Unstained. FIGS. 8 and 9. Control section treated in alkaline phosphatase medium in the absence of.8-gl ycerophosphate. At three minutes, no reactive deposits were observed. Unstained. FIG. 10. Alkaline phosphatase preparation. Unstained. At three minutes reaction products observed in secondary granules and in the phagocytic vacuoles. Such a figure was observed in almost all phagocytosing cells. FIGS. 11 and 12. Higher magnification of Fig. 10. Alkaline phosphatase reaction products observed in relatively small granules, indicating primary granules to be not reactive. In phagocytic vacuoles, the reaction products were markedly deposited. FIG. 13. Alkaline phosphatase preparation. Unstained. At five minutes the phagocytic vacuoles fused with each other. The reaction products were scarce in cytoplasm and abundunt in phagocytic vacuoles. FIG. 14. Alkaline phosphatase preparation. Unstained. At 30 minutes, the reactive granules completely disappeared, whereas phagocytic vacuoles showed strongly positive reactions. FIG. 15. Acid phosphatase preparation. Unstained. Within three minutes no reaction products of phagocytic vacuoles were observed, whereas positive reactions were observed only in granules. FIG. 16. Acid phosphatase preparation. Unstained. At five minutes reaction products noted in only a few phagocytic vacuoles, and also in large granules, namely primary granulles. In smaller granules no reactions were observed. FIG. 17. Higher magnification of Fig. 16. Acid phosphatase reaction products observed in primary granules which are larger in size. Such a granule was also observed in phagocytic vacuoles (arrow). FIG. 18. Acid phosphatase preparation. Unstained. At 30 minutes reaction products disappeared in the cytoplasm, and predominant in phagocytic vacuoles around zymosan.

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