The cytoskeleton of human polymorphonuclear leukocytesPhagocytosis and degranulation.код для вставкиСкачать
THE ANATOMICAL RECORD 203:317-327 (1982) The Cytoskeleton of Human Polymorphonuclear Leukocytes: Phagocytosis and Degranulation MAKK I. RYDEK. ItlCHAKD NIk:DE:HMAN. ,\\I) EDWARD J . 'I'AGGAKI' Department of Periotlontology I ) 4014. Univprsity (if ('uliforniu. School of Uentistr.v. 707 Purnassus Auenue, Sun Francisco. CA 94149 lM.1 K..E.J.?:) and Research Seruice, Vptrruns Administration Medicul ('enter atid H u r i u r d School of'Denta1 Medicine. Boston. M A 02132 fH.h'./ ABSTRACT Current evidence indicates that polymorphonuclear leukocyte (PMN)chemotaxis and phagocytosis are effected by an actin-myosin contractile system. However, the structural relationship of the contractile cytoskeleton to cell motility is still in question. In addition, while evidence suggests that microtubules are responsible for orientation during chemotaxis, the role of microtubules in degranulation is unresolved. To determine the organizational relationship between these cytoskeletal elements and phagocytosis, we examined whole-mount preparations of PMNs engulfing bacteria. These preparations were examined in the transmission electron microscope (EM)and photographed as stereo pairs. Two important observations were made. First, there was an increased density of cytoskeletal elements in the pseudopod surrounding bacteria. Second, microtubule elements were intimately associated with lysosomal granules, vesicles, and phagosomes. Lysosomal granules and vesicles aligned along microtubules and clustered around phagosomes. This suggests that the microtubules may provide a tracking mechanism whereby lysosomes are specifically parceled out to phagocytic vacuoles. These results also suggest that phagocytosis and degranulation may involve different effector mechanisms. The polymorphonuclear leukocyte (PMN) is a primary effector cell of the cellular host defense system. I t s function a s an effector cell can be characterized by major temporal events: chemotaxis, phagocytosis, and degranulation (Klebanoff and Clark, 1978). All of these events are motile functions and are related to the intracellular network of microfilaments, intermediate filaments, and microtubules. The importance of cytoskeletal elements in various PMN functions has been supported by a large volume of experimental evidence. Specifically, the role of microtubules in PMN orientation (Malechet al., 1977)and degranulation (Goldstein e t al., 1973; Zurier e t al., 1974; Haffstein and Weissman, 1978; Phaire-Washington e t al., 1980a, b) has been demonstrated by stimulating or inhibiting their function. Similarly, the role of microfilaments in chemotaxis and phagocytosis is supported by biochemical (Senda, 1976; Stossel e t al., 1980), pharmacological (Goldstein e t al., 1975; Weissman e t al., 1975; Hoffstein and Weissman 1978; Stossel e t al., 1980), and ultrastructural evidence (Berlin and Oliver 1978; Senda, 1976; 0003-276X/82/2033-0317$03.50 ( 1982 ALAN R. LISS. INC. Stossel et al., 1980). However, a major question in our understanding of all of these motile phenomena is their functional relationship with the structural cytoskeleton. Previous structural studies of sectioned material offered a limited view of the total cytoskeleton (Malech e t al., 1977; Berlin and Oliver 1978; Hoffstein and Weissman 1978). To examine the structure-function relationship between the cytoskeleton and phagocytosisldegranulation, we have employed wholemount preparations. In this preparation, PMNs are applied to cultures of nondividing bacteria. The PMNs are well spread and the cytoskeletal elements clearly visible in the transmission electron microscope. A series of stereoscopic views of the phagocytosing PMNs indicate that there is an increased density of microfilaments in the pseudopod and around the phagocytic vacuole. The micrographs also display granules and vesicles aligned along microtubules and clustering 318 M.I. RYDER, R. NIEDERMAN. AND E.J. TAGGART around phagosomes. These results may provide structural information on these motile events that have been hypothesized. In addition, they may provide evidence in support of early concepts for microfilament (Allison and Davies, 1974) and microtubule function (Malwista and Bodel, 1967)during phagocytosis and degranulation. MATERIALS AND METHODS A suspension of Streptococcus mutans serotype D in 0.1 M phosphate-buffered saline supplemented with 0.125 M sucrose, 0.029 M sodium fluoride, and 0.2 mgiml thimerosal, was adjusted to a concentration giving an optical absorbance of 1.0 (as determined on a Beckman DB-G spectrophotometer, operated at a wavelength of 540 nm). Several drops of this suspension were placed on formvar-coated 200-mesh copper or nickel grids, which were in turn attached to a Parafilm surface. The bacterial/sucrose suspension was incubated on the film-coated grids for 4-6 h at 37°C in a CO, incubator. Following this incubation, the unattached bacteria were washed off with several rinses of phosphate-buffered saline. Several drops of whole human blood were then placed onto the film surface via the finger poke technique (Zigmond, 1978) and allowed to clot for periods of 30 min at 37°C.' The blood clot was washed off with 37°C stabilization buffer (Taylor, 1976) (30 mM KC1, 1 mM MgC12,2 mM CaCl,, 5 mM EGTA, 5 mM PIPES, pH 7.0), thus leaving a complex of bacteria and PMNs attached to the formvar film surface. During the process of washing off the clot, a portion of the PMNs were horizontally sheared, leaving the bottom portion of the cell attached to the film surface, similar to the technique employed by Boyles and Bainton (1979). Preparation of this PMN-bacteria complex for electron microscopic observation was by one of three methods: 1) The PMN-bacteria complex attached to the film-coated grids was fixed for 10 rnin with 1.5% glutaraldehyde in stabilization buffer at 37"C, post-fixed for 3 min with 1%osmium tetroxide in stablilization buffer (pH 7.0) at 4"C, followed by three rinses of deionized distilled water, and then by stain- 'This finger poke technique of Zigmond yields greater than 9OYo P M N s adherent t o the film surface. The remainder of adherent cells consist of a mixed population of monocytes and lymphocytes. Whole P M N s can be distinguished from these other cells on the EM level by their numerous heterogeneous granules and by their multilobulated nuclei. ing in 1%aqueous uranyl acetate for 5 min at room temperature. The coated grids with attached cells were then dehydrated in ethanol, and critical-point dried from liquid COz in a Bomar SPC-900 apparatus. (Methods derived from Buckley and Porter , Buckley , Wolosewick and Porter .) 2) Some grids with the PMN-bacteria complex were treated with 0.5% Triton X-100 in stabilization buffer for 30-90 sec or 0.15% Triton X-100 in PHEM buffer for 90 sec (method derived from Schliwa and Blerkom ).This was followed by the fixation and critical-point drying as previously mentioned. 3) Some grids with the PMN-bacteria complex were initially fixed with a mixture of 0.5% osmium tetroxide and 1%gluteraldehyde in stabilization buffer for 3 min at 4°C (method derived from Hirsch and Fedorko ).This was followed by the staining, dehydration, and critical-point drying as previously mentioned. The grids with whole cells as prepared by one of these three methods were scanned and photographed on a Philips 300 Electron Microscope at 80 KV or a JEOL lOOC electron microscope at 100 KV. Stereoscopic photographic pairs were obtained through the tilting stages on these microscopes. Measurements on filament diameters were made by projecting the electron micrograph negatives in a lantern slide projector to 100 times their original magnification, and then making measurements with a millimeter rule. Alternatively, other grids were vacuum sputter coated with a 20 nM layer of gold in a Technics Hummer Sputter Coater for observations by scanning electron microscopy. These grids were photographed at 20 kV on a Cambridge S-150 SEM with a tilting stage. RESULTS The best yields of attached phagocytosing PMNs were obtained after 20 to 30 min of incubation with bacteria. The PMNs at these time periods typically displayed a triangular morphology when viewed in the light microscope or the scanning electron microscope (SEM)(Fig. 1).The nucleus was located at the narrow rounded rear of the cell, and a broad fan-shaped front extended toward bacterial masses. Small cytoplasmic processes or microvilli were seen to extend beyond the leading edge. In most of the PMNs observed, these microvilli appeared to extend under the bacterial masses. The general cytoskeletal organization of the fan-shaped front could best be observed in 319 PMN PHAGOCYTOSIS sheared cells (Fig. 2) and in Triton extracted cells (Fig. 3a,b). Two subclasses of filaments could be discerned: 15-25 nm thick elements and random meshwork of 5-12 nm filaments. The 15-25 nm elements appeared to radiate from the nucleus towards the front periphery. Numerous granules and small- and mediumsized vesicles appeared in intimate association with these 15-25-nm elements. In many cells extracted with Triton in PHEM buffer, these elements resolved into two parallel dense lines (a characteristic of microtubule structure). The internal cytoskeleton involved in the initial bacterial engulfment could best be observed at the thin peripheral areas of whole critical-point dried cells. Transmission electron microscopic (TEM) observations of these areas during initial engulfment revealed several basic patterns of filament organization. First, t h e cytoplasmic processes surrounding the bacteria contained an extensive three-dimensional meshwork of filaments ranging in diameter from approximately 5-25 nm. Second, a marked increase in the density of this meshwork was observed in the pseudopods (Figs. 4,5) as well as within portions of the PMN cytoplasm that were in the process of engulfing bacteria (Fig. 6). Stereo pairs indicate that this is a real increase in density, and not an artifact of increased cytoplasmic thickness (Figs. 8,9). The 15-25-nm subclass of larger (microtubules) elements could be discerned among the complex cortical meshwork of filaments. These elements appeared to have several patterns of organization. In areas of the cortical cytoplasm not associated with bacterial engulfment, these thicker elements were parallel to the cytoplasmic membrane. In areas of the cortical cytoplasm associated with bacterial engulfment, these thicker elements followed one of two courses: some elements extended into the pseudopod engaged in phagocytosis (Fig. 7);others were seen in association with the bacteria (Figs. lO,ll), or converging towards the bacteria (Fig. 12). Many granules and vesicles were seen in intimate association with the 15-25-nm elements (microtubules) throughout the phagocytic sequence (Figs. 2; 3a,b; 10; l l ) , whereas other granules and vesicles did not appear in association with these elements. (Someunassociated granules and vesicles in sheared PMNs may have been “washed out” during the shearing process.) That some of these granules and vesicles do, in fact, align along these elements can be appreciated in the stereo pairs (especial- ly Figs. 2, 10, 11).Of particular interest was the observation that the granules were seen associated with elements that converge on the phagocytic vacuole. Degranulation could best be observed in more central areas of the PMN. This was most easily seen in PMNs whose tops had been partially sheared off, or in PMNs extracted with Triton X-100. Although some of the cytoskeletal network had been washed out in processing, distinct phagocytic vacuoles, lysosomal granules, vesicles, and the 15-25-nm elements could be easily seen (Figs. 10, 11, 12). In these cells, the 15-25-nm elements (microtubules) appeared long and continuous. Many extended directly toward the phagocytic vacuole. In these micrographs, the granules and vesicles clustered around the phagosome. Two questions may be raised regarding potential artifacts: 1) Are PMN vesicles real or are they a blebbing artifact following glutaraldehyde fixation (Hasty and Hay, 1978)? PMNs were fixed simultaneously in glutaraldehyde and osmium tetroxide (Hirsch and Fedorko, 1968)to control for this problem. With the glutaraldehyde fixation, vesicles were still observed (Fig. 13). Thus, these vesicles probably do exist as part of the PMN. 2) Do granules and vesicles cluster around phagocytic vacuoles in sheared cells as a result of aggregation during shearing? This clustering is observed in whole unsheared cells (Fig. 14). Since it is unlikely that the vesicles and granules could translocate through the fixed, dense cytoskeletal network during cell processing, it seems reasonable to assume that this granule clustering also exists as a real phenomenon. DISCUSSION PMNs were allowed to settle, chemotax, phagocytose, and degranulate on an EM grid previously coated with a dilute solution of bacteria. TEM observations of the actively migrating and phagocytosing PMN cytoskeleton revealed two important organizational arrays. First, microfilaments occur in and constitute a major portion of the phagocytosing pseudopodial cytoskeleton. Second, granules, vesicles and phagosomes form intimate linear association with microtubule like elements. An increased density of elements 5-25 nm in diameter was observed in pseudopods engaged in phagocytosis. These elements appeared to be parallel or perpendicular to the cell membrane of the pseudopod. Given the 320 M.l. KYDER, K. NIEDEKMAN, A N D E.J. TAAGGAK'I' Fig. 1. SEM of the typical appearance of PMNs in this system. It displays broad anterior front to the left and a short rounded tail to the right. At the upper left portion of the front, cytoplasmic processes can be seen to extend around several chains of Streptococcus mutans. X 4,400. 20 kV. high concentration of actin (Davies and sis (Hoffstein and Weissman, 1978) of objects Stossel, 1977; Hartwig, 1977; Berlin and that are not directly attached to the cytoskeleOliver, 1978) and its associated proteins ton. The results reported here can be used as (Davies and Stossel, 1977; Hartwig, 1977; support for either interpretation. The imporValerius et al; 1981) in pseudopodial exten- tant point, however, is that filamentous elesions, one suspects a portion of these elements ments constitute a major portion of the are indeed filamentous actin. (Other elements pseudopodial cytoplasm. The second striking observation is the alignin this 5-25-nm range may include intermediate filaments and microtubules.) Two ment of granules along microtubule-like functional interpretations can be offered for elements. Previous studies on PMN chemothis observation. First, this condensation of taxis demonstrated the orientation of microfilaments represents direct points of attach- tubules parallel to the direction of the cell ment of the PMN cytoplasmic membrane to migration (Malech et al., 1977)' whereas the bacteria. A similar condensation of the studies of thin-sectioned PMNs have demoncytoskeletal network has been reported at the strated an intimate association of microattachment sites of PMNs to glass surfaces tubules with granules and vesicles (Hoffstein (Boyles and Bainton, 1979). to opsonized yeast et al., 1977; Hoffstein and Weissman, 1978). particles (Boyles and Bainton, 1981). and in These data, in conjunction with data demonphagocytosing macrophages (Reaven and Ax- strating an increase in microtubules upon line, 1973). Second, these condensations are phagocytic stimulation (Goldstein e t al. 1975; areas of increased polymerization and aggrega- Burchill et al., 1978; Hoffstein et al., 1976; tion of actin, myosin, and actin-bindingprotein Oliver et al., 1976; Weissman et al., 1975), led (Davies and Stossel, 1977; Hartwig, 1977; investigators to conclude that microtubules Valerius et al, 1981).These contractile proteins are required for enzyme secretion. Other data may move the membrane to effect phagocyto- contest this notion. A variety of pharmocologi- PMN PHAGOCYTOSIS 321 Fig. 2. TEM of a sheared triangular PMN front. the dense cytoskeletal network, numerous 15-25-nm Numerous granules ( g ) and various size vesicles (v) are apelements ( 1 ) can be seen to radiate from the nuclear area. b) parent. Towards the periphery a t the right. many of these A t higher magnification, these 15-25-nm elements often apgranules and vesicles can be seen in intimate association pear a s two parallel lines ( 6 ), characteristic of microwith 15-25-nm elements ( 1 ) (N. nucleus). Stereo pair: f6" tubule structure. Remnants of extracted granules (g)can be tilt. X 10.000. 80 k V . seen in intimate association with these microbutules IN. Fig. 3. a) TEM of a Triton-extracted P M N front. Amid nucleus. a) X 13,800. b) x 67,000. 100 kV. 322 M.I. RYDER. R. NIEDERMAN, AND E.J. TAGGART Fig. 4. TEM of a cell process from a whole (unsheared, unextracted) PMN. A dense cytoskeletal network of elements ranging in diameter from 5 t o 25 nm can he observed within this cell process. In the region of the PMN nearest to the bacterium (b). these elements have a dense matlike organization ( 8 ). X 43,000. 80 kV. Fig. 6. The cell periphery of a whole PMN phagocytosing a chain of two bacteria (b). An increased density in filament organization ( 0 ) can he ohserved around t h e infolding membrane of the PMN around the bacteria. X 19.500. 80 kV. Fig. 5. A whole PMN in the initial phases of phagocytosis. An increase in the density of the cytoskeleton ( 8 ) can he observed in t h a t portion of the cytoplasm tha t is folding over the bacteria (b). A convergent pattern of filaments upon the bacteria can he discerned( t). x 11,200. 80 kV. Fig. 7. A peripheral area from a whole PMN t h a t has ingested a bacterium. Some cytoskeletal elements. ( 1 ) are directed towards the bacterium. Also note the clustering of granules (g)and vesicles (v) around t h e bacterium. X 24,000 80 kV. PMN PHAGOCYTOSIS Fig. 8. A higher magnification stereo pair of Figure 4. Note the dense matlike organization of PMN filaments nearest t o the bacteria ( 4 ). Also note the bundle of filaments inserting into the cell process (1). Stereo pair: f 6" tilt. X 65,000. 80 kV. 323 Fig. 9. A higher magnification stereo pair of Figure 5 . Again, note the dense matlike organization of filaments nearest the bacteria ( 4 I. Some filaments and/or bundles of filaments ( 1 ) can be seen t o converge upon t h e bacteria, while other filaments ( 1 1 ) can be seen t o insert into the cell process Stereo pair: ? 6" tilt. X 22,000. 80 kV. P M N PHAGOCYTOSIS Fig. 12. A Triton-extracted whole P M N . The membrane of the phagocytic vacuole 1s absent, A convergent patternof 15-25-nm elements ( I ) upon the intetnalized bacterium (bl can be discerned. Stereo pair: 2 6" tilt. X 45.000. 80 kV. Fig. 13. A PMN initially fixed in a 0.5% osmium tetrox- 325 ide-l.O% glutaraldehyde mixture. Note the presence of numerous vesicles (arrows). X 38.500.100 kV. Fig. 14. Note the clustering (arrows) of granules arid vesicles around the bacterium (h) in this whole (unsheared. unextracted) PMN. X 38.000.80 kV. 326 M.I. RYDER. R. NIEDERMAN, AND E.J. TAGGART cal agents that destabilize microtubules do not inhibit secretion (Zurier et al., 1974;Weissman et al., 1975; Oliver et al., 1976; Burchill et al., 1978; Hoffstein and Weissman, 1978). The evidence presented here may provide a resolution for this apparent conflict. The key observations are: the convergence of microtubules toward phagosomes and the alignment of granules along microtubules. Therefore, the microtubules could provide a tracking mechanism. Several functional interpretations can be offered for this possible microtubule tracking mechanism: 1)Granules from the center of the cell may move along microtubules towards the peripherally located phagocytic vacuole; 2 ) microtubules aid in the movement of the peripheral phagocytic vacuole towards the granule-rich center, as proposed by Hoffstein et al., (1977);3) the phagocytic vacuole is carried into the microtubule granule network via another mechanism, such as an actinmyosin contractile system. Degranulation could then occur via either passive collisionsof the vacuole with the microtubule-associated granules, or via granule transport along the microtubule towards the vacuole. The origin and function of the numerous vesicles of various size found in these PMNs is unclear. One possibility is that they may be empty phagosomes formed as a result of a generalized phagocytic stimulus of the S. mutans. A second possibility is that many of these vesicles could be the result of plasma membranes recycling from either phagosomes or depleted lysomal granules. Indeed, the high degree of association of these vesicles with microtubules suggests that microtubules may aid in both the cellular organization and the transport of these vesicles. The techniques derived in this study may have further application in clinical studies of PMN dysfunction. For example, in ChediakHigashi syndrome, conflicting data exist as to whether there is an actual decrease in the number of microtubules in the PMN and monocyte (e.g., White and Clawson, 1979; Boxer et al., 1979). Quantitation in these studies was done on thin-sectioned material around the centriolar region. Thus, only a very limited view of the cytoskeleton was examined. With criticalpoint-dried whole cells and sheared cells, one can visualize a more complete cytoskeletal organization for quantitation. ACKNOWLEDGMENTS This work was supported by the Department of Periodontology,- university of California, San Francisco; the Veterans Administration; and the Alice and Julius Kantor Charitable Foundation. We thank Sandra Hughes, Donna Kantarges, and Clare Smith for assistance with the manuscript. We also thank Drs. Ernest Newbrun, Paul Goldsmith, John Long, and Mr. Gerry Morgan for their generosity and assistance. LITERATURE CITED Allison, A.C., and P. Davies (1974) Mechanisms of endocytosis and exocytosis. Symp. SOC.Exp. Biol., 28: 419-444. Berlin, R.D., and J.M. Olive (1978) Analogous ultrastructure and surface properties during capping and phagocytosis in leukocytes. J. Cell Biol., 77:789-804. Boxer, L.A., D.F. Albertini, R.L. Baehner, and J.M. Oliver (1979) Impaired microtubule assembly and polymorphonuclear leukocyte function in the Chediak-Higashi syndrome correctable by ascorbic acid. Br. J. Haematol., 93,207-213. Boyles, J.. D.F. 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