Membrane changes in polymorphonuclear leukocytes during Ionophore (A23187)-induced lysosomal release

EXPERIMENTAL

AND MOLECULAR

PATHOLOGY

30, 420-433

(1979)

Membrane Changes in Polymorphonuclear Leukocytes lonophore (A23187Mnduced Lysosomal Release L. MOORE,’

PATRICK

Pathology

Received

SAMUEL

Department, Charleston,

November

L. BANK,~ PHILIP

HARVEY

AND

14, 1977;

SANNES,

S. SPICER

Medical South and

L.

during

University Carolina in revised

of South

Carolina,

29401

form November

10,

1978

Micromolar concentrations of the divalent ionophore A23187 and millimolar concentrations of extraccllular calcium caused the rapid exocytosis of lysosomes from rabbit polymorphonuclear leukocytes. In control experiments the larger granules remained within the leukocytes while another class of smaller granules fused with the plasma membrane. Whenever membrane fusion and exocytosis occurred intramembranous particle-free regions developed within the plasma membrane at the sites of fusion. Neither a massive aggregation of intramembranous particles (IMPS), nor the formation of highly symmetrical rosette patterns of IMPS was seen in either experimental or control preparations.

INTRODUCTION Several lines of evidence suggest that calcium and/or magnesium play important roles in many polymorphonuclear (PMN) leukocyte functions, including phagocytosis (Bryant, 1969), adhesion (Allison et al., 1963), and chemotaxis (Becker et al., 1972). However a clear understanding of the biological roles of these ions is not yet available because of the difficulty involved in measuring intracellular free-ion concentrations. Nonetheless, several recent investigations suggest that calcium is involved in regulating cytoplasmic movements, exocytosis, and secretion. These investigations demonstrate: (1) that calcium is required for the in vitro “activation” of amoeboid movement (Taylor et al., 1973; Condeelis et al., 1976; Moore, 1975), (2) that calcium solates actin gels (Condeelis and Taylor, 1977), (3) that chemotactic factors alter leukocyte membrane potentials (Gallin et al., 1977), (4) that radioactive calcium kB enters leukocytes during chemotactic stimulation (Naccache et al., 1977), and finally (5) that the ionophore A23187 and calcium stimulate secretory events in a wide variety of cells (Cochrane et al., 1974; Murer et al., 1975) including PMN leukocytes (Becker et al., 1972; Goldstein et al., 1974). This paper focuses on the effects of calcium and the ionophore A23187 on the membrane structure of leukocytes. The ionophore A23187 is a lipid soluble antibiotic produced by cultures of *Dr. Moore’s current address and the address to which correspondence Dept. of Internal Medicine, LCI-609, Yale University Medical School, New ‘Reprint requests should be sent to South Carolina, c/o Dr. Bank. 420 0014-4800/79/030420-14$02.00/0 Copyright All rights

@ 1979 by Academic Press, of reproduction in any form

Inc. reserved.

should be sent: Haven, CT 06510.

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Streptomyces chartruensis. It complexes with both calcimll and magnesium (Reed et al, 1972; Pfeiffer et al., 1974; Pressman et al., 1975), and, as mentioned

above, in the presence of calcium, it stimulates a number of biological processes especially those involving secretion. Recent biochemical experiments on human PMN leukocytes demonstrate that A23187 and calcium cause the release of l!sosomal enzymes into the extracellular medium without disrupting these cells (Becker et OZ., 1972; Goldstein et al., 1974). This unusual change in the distribution of lysosomal enzymes from inside to outside the leukocyte ma)- be analogous to the escape of lysosomal enzyme into the extracellular medium during phagocytosis (Hirsch, 1962; Wright and Malawistn, 1972), and certain pathological conditions (Henson, 1971). Because of the potential similarity between these pathological events and ionophore and calcium induced events, we decided to use A23187 and calcium as a model system for observing the ultrastructural changes that occur in leukocyte membranes during lysoson~nl release. It seems likely that an understanding of the cellular changes which accompan\ ionophore-induced esocytosis will prove useful in understanding the many short term membrane-membrane interactions that occur during such leukocyte pr:;cesses as membrane fusion, secretion, and phagocytosis. The specific aim of this and a companion investigation (Sannes et al., 1977) is to investigate the structural changes that occur in leukocytes treated with the divalent ionophore A23187 and calcium. Together these studies show that calcium and ionophore treatments produce structural changes within the plasma membrane of polymorphonuclear (PM/IN) leukocytes and within the leukocyte itself. The membrane changes demonstrated in this paper show that intramembranous particle free (IMP-free) regions develop at the sites of fusion between the lysosomal and plasma membranes. Neither a massive aggregation of IMPS (Elgaester et al., 1976; Pinto du Silva et al., 1971), nor the formation of highly symmetrical rosettes (Satir et al., 1973) was seen in either ionophore-calcium treated PMN leukocytes, or control cells. In addition, evidence is presented that a class of small cytophasmic granules, presumably tertiary granules (Wetzel et al., 1967; Wetzel, 1970; Murata and Spicer, 1973) fuse with the plasma membrane even in the absence of added calcium and ionophore. Thus the movement, fusion, and release of these granules appears to be structurally and perhaps mechanistically distinct from that of the larger granules residing in the same cell. MATERIALS

,4ND METHODS

Polymorphonuclear leukocytes were obtained from glycogen-induced peritoneal exudates of New Zealand rabbits according to the method of Hirsch (Hirsch, 1956). The details of the exact procedure used are described elsewhere (Moore et al., 1976; Moore et al., 1978). The exudates consisted of 95 to 99% PMN leukocytes. The final cell concentration was adjusted to 3 x loo cells per milliliter; the resulting suspension was divided into 15.0 ml aliquoits and centrifuged for 3 min at 500 g. Eacll aliquoit was then resuspended in one of the test media given in Table I. All incubations were carried out for three minutes at 37”C, unless otherwise indicated. Cells were incubated in test tubes for freezefracture preparations; but were allowed to attach to and incubate on glass coverslips for scanning electron microscopic ( SEA4) preparations.

MOORE

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TABLE

Eagle’s minim&+ * essential media 1. 2. 3. 4. 5.

Eagle’s Eagle’s Eagle’s Eagle’s Eagle’s

HEPES bufferedb,e saline 1. 25 mM HEPES, 150 mM NaCl 2. 25 mM HEPES, 150 mM NaCl 3. 25 rn2lf HEPES, 150 rnM NaCl 4. 25 mill HEPES, 150 mM NaCl 5. 25 mM HEPES, 150 mill NaCl

Added ca2

ET AL. I

Ionophorc~ A23187

Release of’ large granules

Release of’ small granules

+ -

+ + + + +

3.0 mJl 3.0 rnilf -

>O.l(;l, > 0.1 ‘j;, > 0.1 ‘,:L. -

3.0 mill

>O.lc,I

+

+

-

>O.l ‘,;I

-

+

-

>0.17;,

-

+

-

-

-

+

3.0 rnAf

-

-

+

QMEM-Minimum Essential Medium (Eagle’s) with Hank’s Salts F-12 (already contains 1.26 mM CaC12, 0.396 mdd MgSO+ 5.5 mllf glucose) Grand Island Biological Company, New York. * The pH of all media was 7.2 to 7.4; osmolarity of all media was 280 to 300 mOs. c Ionophore A23187 was obtained from Dr. Robert Hamill, Eli Lily Company, Indianapolis, Indiana. d Dimethyl sulfoxide, a solvent for the ionophore never cxcbccded concentrations of 0.19s (vol/vol) in any of the media used. 6 HEPES, 4-(2-hydroxy ethyl)-1-Pipcrazinc ethanc-sulfonic acid, Polysrienccs, Incorporated, Warrington, PA. f Release of granules was judged to occur when large or small granules were seen outside the cells in SEM preparations, when IMP-free areas were present in freeze-etched cells, and when diminished populations of lysosomes were seen in thin sections of cells from the same populations.

In our initial experiments we found that if cells were incubated for time intervals similar to those normally used in biochemical assays of lysosomal enzyme release, exocytosis was already complete. We interpreted this to mean that the enzyme assaysmeasured not only time required to release the enzyme but also the time required to solubilize and/or activate these enzymes. In order to study the rapid membrane events associated with lysosomal release, it was necessary to determine the time at which most of the cells were in the process of exocytosis. This was done as indicated below. Rabbit PMN leukocytes were treated with 1 pA4 ionophore A23187 and 3 mM extracellular calcium for 1, 3, 8, and 15 min at 37”C, and the sequence of structural events characterized. Briefly the results indicated that most of the different stages of exocytosis were seen in the three minute treatments. After incubation in the appropriate test media all cells were fixed with 2% glutaraldehyde in 0.1 M sodium cacodylate, pH 7.2, 380 milliosmols at 37°C or room temperature for 1 hr. For SEM preparations, leukocytes were also fixed for 1 hr in 2% osmium tetroxide in 0.1 &f sodium cacodylate, pH 7.2 at room tem-

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FIG. 1. Scanning electron micrograph of rabbit PMN leukocyte incubated for 3 min in Eagle’s medium containing all components except the ionophore A23187. Bar = 2.0 pm.

x5000. perature and treated with thiocarbohydrazide and osmium tetroxide to increase their electrical conductivity as previously described (Moore et al., 1976; Moore et al., 1978). Subsequently, they were dehydrated in ethanol, critically point dried (Anderson, 1951; Cohen, 1974), coated with a thin layer of gold (ca. 10.0 nm) and viewed in a Coates and Welter Model NO. 106 Field Emission Scanning Electron Microscope. Glutaraldehyde fixed cells that were to be prepared for freeze-fracture techniques were centrifuged and resuspended in 30% aqueous glycerol. After equilibration in glycerol a dense cell suspensionwas loaded into gold specimen holders and freeze-fractured in a modified (Bank et al., 1976) Denton freeze-etch device according to the recommendations of Steere (Steere, 1969). The freezing, replication, and digestion procedures used were presented in a previous study (Moore et al., 1975; Moore et al., 1978). Freeze fracture replicas were observed in a Hitachi HU-12 Transmission Electron Microscope operated at 75 kV and calibrated with a replica of a cross-ruled optical diffraction grating.

FIG. 2. Scanning electron micrograph of PMN leukocyte incubated for 3 min in Eagle’s medium containing 3 mM calcium and 1 pM ionophore A23187. A change in cell shape and lysosomal release are evident (arrow). Bar = 2.0 pm. x5000.

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FIG. 3. Scanning electron micrograph of two leukocytes treated for 8 min with and calcium. Massive release of lysosomal granules is evident, and cell disruption become apparent. Bar = 2.0 pm. X2500.

ionophore begins to

RESULTS Initially ionophore-calcium treated cells were compared with control cells for structural evidence of lysosomal release. Normally leukocytes are somewhat elongate in shape, and have a relatively smooth cell surface which is interrupted only occasionally by thin membrane-bound protrusions known as lamellapodia or ruffles. Usually lysosomal granules are separated from the plasma membrane by a thin filamentous layer about 100 nm thick. During ionophore-calcium treatments these structural features change in a time dependent fashion. One minute treatments with one micromolar ionophore A23187 in the presence of three millimolar extracellular calcium resulted in little morphological alteration in cell shape or the release of detectable lysosomal material. After 3 min a change in cell shape, a reduction in the size of the membrane bound extensions, and an increase in lysosomal release was seen in ionophore and calcium treated cells. (Compare Figs. 1 and 2). In addition both thin section evidence (Sannes et al., 1977, Fig. la) and SEM evidence (Sannes et al., 1977, Fig. 2) showed that material was exocytosed outside the leukocyte. After 8 min of treatment nearly all cells had released their granular contents (Fig. 3). This was also demonstrated

FIG. 4. The extracellular fracture face (EF) of the plasmamembrane treated leukocytes has circular regions about 100 nm in diameter x75,000. membranous particles (arrow). Rar = 0.1 finI.

which

of ionophore-calcium are free of intra-

MEMBRAKE

FIG. regions

CHANCES

5. The protoplasmic fracture fact which are free of intrameml)ranous

IN

42.3

LEIIKWZYTES

(PF) of the plasma particles (arrow).

membrane Bar = 0.1

also pm.

has circular x75,000.

in thin sections in our prevkms paper (Sannes et al., 1977, Fig. 1). After 15 min no further Iysosomal release was observed, but increasing numbers of cells had concavities on their cell surfaces suggestive of cell damage. In addition to SEM observations in vitro observations of cell pellets showed a marked reduction in the size of cellular pellets exposed to calcium and ionophore for long periods of time (> 15 min). Both of these observations suggest that cell damage occurs with longer incubations probably as a result of the increased amounts of lysosomal enzyme present in the extracellular medium. Since the three minute treatments with 1 phi A23187 and 3 rnnrl calcium contained the masimum number of esocytic events, and little or no evidence of cell damage, this incubation time was selected for study with freeze fracture techniques. Freeze fracture results indicated that calcium and the ionophore-induced lysosomal release is accompanied by structural changes within the plasma membrane. These membrane changes appeared to be associated with membrane fusion. Normally the thin filamentous layer prevents Iysosomes from contacting the plasma membrane of leukocytes. Yet in ionophore treated cells freeze fracture replicas reveal a close contact with the plasma membrane of the larger

Similar FIG. 6. ionophore treated x 75,000. m.

circular leukocytes

regions were in a HEPES

observed buffered

in freeze fractured saline solution (See

images arrow).

of calciumBar = 0.1

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FIG. 7. The larger intramembranous particle-free the fusion of the larger lysosomal granules ( L) with pm. X75,000.

AL.

regions (arrow) appear to be caused the plasma membrane (PM). Bar =

by 0.1

granule membranes. This close contact is also seen in thin sectioned preparations (Sannes et al., 1977, Figs. 7-9) and is also correlated with the appearance of larger IMP-free regions as described below. In ionophore-calcium treated PMN leukocytes both fracture faces of the plasma membrane had small intramembranous particle-free (IMP-free) regions which were 60 to 150 nm (longest dimension) and either a circular or somewhat irregularly circular shape (Figs. 4 and 5). Similar results were obtained with two different buffer systems (Table I; Figs. 4-6). These IMP-free regions appeared to be located at the sites where lysosomes fused with the plasma membrane ( Fig. 7 ) . In control preparations, leukocytes also had IMP-free regions. However, these IMP-free regions differed from those in ionophore and calcium treated leukocytes. Control IMP-free regions were smaller 60 * 20 nm (diameter) and more circular in shape than those in ionophore-calcium treated leukocytes (Compare Fig. 4 and 5 with Fig. 10). In addition the ‘control IMP-free regions were similar in diameter to a class of small granules in the cytoplasm. These small granules were seen within the submembranous filamentous region, in contact with the plasma membrane,

FIG. 8. The smaller IMP-free the smaller, presumably tertiary membrane ruffle prior to fusion

regions appeared to be caused by the fusion granules. Here the small granules can be with the plasma membrane. Bar = 0.1 pm.

and release seen within x20,000.

of a

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FIG 9. The small granules appear to move through the cytoplasm to fuse with the plasma membrane. The larger lystrsomcs (L) are retained in these untreated cells (HEPES buffered saline with no added calcium, ionophore, or D51SO). Bar = 0.1 ym. X25,000.

and in the extracellular medium (Figs. 10 and 11). Large IMP-free regions were not observed in control preparations processed in the same way at the same time, thus we do not believe these to be artifactual. Figs. 8-11 represent, what we currently believe, is the sequence of events that occurs during small granule membrane fusion and release. We present this only speculatively being fully aware of the pitfalls of describing a kinetic process from static images. At present, we think that these granules move through the submembranous region (Figs. 8 and 9), contact the plasma membrane (Fig. 11 ), fuse with it, producing IMPfree regions within the plasma membrane (Fig. 10) and release their contents into the extracellular medium (Fig. 9). It is interesting to note that the larger granules are retained in control cells (Fig. 9) even though small granules are released (Figs. S-11 ). This stands in striking contrast to the ionophore-calcium treated leukocytes which release both their larger and smaller (Tnblc I; Fig. 7). I n addition to their size difference, large and small granules &I appear to differ with respect to IMPS. The smaller granules appear to lack IMPS whereas larger granules possess them (Moore et (II., 1978).

FIG. 10. Small IMP-free regions were observed in all control cells. These regions tended to be smaller in diameter (60 5 20 nm) and more uniform in size than those in ionophorex75,000. calcium treated cells. (Compare with Fig. 5.) Bar = 0.1 pm.

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FIG. 11. A higher magnification of Fig. 9 shows the contact and fusion of the small granules with the plasma membrane (arrow). Compare the relative size of the granules in this figure Bar x 0.1 pm. X75,000. with that in Fig. 7, an ionophore-calcium treated leukocyte.

Although membrane fusion occurred in a large variety of conditions in this study (both with and neither a massive aggregation of intramembranous symmetrical rosette pattern of IMPS was observed

number of cells, under a wide without ionophore treatment), particles (IMPS ) nor a highly in our preparations.

DISCUSSION This report shows that millimolar concentrations of extracellular calcium and micromolar concentrations of the divalent ionophore A23187 produce structural changes in PMN leukocyte membranes. It is clear that the major ultrastructural change within the plasma membrane is the development of an IMP-free region at the site of lysosomal fusion. Beyond this finding, we have identified another type of membrane fusion involving a class of small granules which differs from ionophore-induced membrane fusion. Since small granule release occurs in the absence of ionophore and calcium treatment, it may also be mechanistically distinct from ionophore-induced lysosomal release. During phagocytosis we (Moore et al., 1978) have observed similar structural changes within leukocyte membranes. For example, in both ionophore treated and phagocytising cells, IMP-free regions are present at the sites of mlysosomal membrane fusion. In addition IMPS do not aggregate into large masses as they do in some experiments with erythrocyte ghosts (Elgaester et al., 1976; Pinto da Silva et al, 1971). Furthermore, highly symmetrical rosette patterns of IMPS similar to those seen in protozoan mucocyst secretion (Satir et al., 1973) are not seen. Thus on the basis of these structural criteria, membrane fusion in calcium and ionophore treated leukocytes appears to be similar to that seen during phagocytosis in the same cells. Because of these structural similarities we believe that the calcium and ionophore treatments trigger a natural cellular response which is not an artifact of the ionophore treatment. As mentioned above leukocyte membrane fusion differs from protozoan membrane fusion in that rosettes appear to be absent. There are a variety of interpretations which might explain this result. For example, there are obvious experimental and genetic differences between these two studies. In addition it is possible that IMPS aggregate and dissaggregate so rapidly that they might not

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be seen in our preparations. However, in the present report and in a number of recent reports on mammalian (Burwen et al., 1977; Heuser et al., 1977; Lawson et al., 1977; Orci et al., 1977) and on plant membrane fusion (Weiss et al., 1977), membrane fusion is accompanied by the formation of IMP-free regions but not highly symmetrical rosette formations. Although it is clear that rosettes are present in protozoan cells (Satir et al., 1973) convincing evidence that rosettes are present in more highly evolved cells is lacking. Thus, at present, it appears that the formation of IMP-free regions within fusing membranes is the most universal feature of membrane fusion, The precise mechanism(s) by which calcium and the divalent ionophorc A23187 act on living cell is not completely understood. However, biochemical evidence suggests that this lipid soluble ionophore complexes with calcium and magnesium and carries these ions across biological membranes (Reed et al., 1972; Pfeiffer et al., 1974). Recent experiments employing the calcium sensitive protein, aquorin, demonstrate that A23187 produces a measurable rise in the free calcium levels in living cells (Ridgeway ef al.2 1976). Direct experimental evidence showing that A23187 affects the intracellular free ion concentrations of other divalent ions such as magnesium is lacking. Yet, the substitution of magnesium for calcium in ionophore experiments does not produce the same biological effect as calcium (Cochrane et a!., 1974). At present, then, the available evidence suggests that changes in the intracellular calcium levels promote ionophore-induced Iysosomal release. It is important to realize that calcium and the divalent ionophore A23187 treatments affect a variety of cytoplasmic sites in addition to membranes. For example, we have observed transient structures on the cytoplasmic surface of the plasma membrane at the sites of Iysosomal fusion (Sannes et al., 1977). In addition, in the present study we have shown that cell shape changes occur. That is, membrane ruffles are gradually lost during these treatments as the cells round LIP, and the usually smooth contours of the leukocyte take on a much less smooth appearance when the lysosomes approach the surface. All these observations suggest that calcium and A23187 affect a variety of sites within leukocytes. Since calcium is known to bring about an interaction between actin and myosin during muscle contraction ( Ebashi et al., 1968), and to “activate” the contractile systems of amoebae Taylor et al., 1973), and since leukocytes also contain an actomyosin contractile system (Senda et al., 1969; Stossel and Pollard, 1973), it seemslikely that the alteration in cell shape, and the fusion and re!ease of lysosomes are due to a calcium activation of the contractile elements and perhaps, membranes in non-muscle cells. At present, there are at least three way-s in which this “activation” might occur: through a dissolution of an actin gel caused by the addition of calcium ions ( Condeelis and Taylor, 1977), through a calcium activated actomyosin interaction similar to that observed in amoebae Taylor et al., 1973), or through the dissolution of microtubules caused by high calcium concentrations ( Weisenberg, 1972; Poste and Nicholson, 1976). It should be apparent that these mechanisms are not mutually exclusive, in fact, all three might occur when intracellular free calcium ion concentrations rise. The freeze fracture observations showing that a class of small granules similar in size to previously reported tertiary granules suggests that these small granules may be tertiary granules. Our observations that these granules can undergo differential release might resolve someof the controversy about the existence of these

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small granules in rabbit leukocytes (see Murata and Spicer 1973). Assuming these granules represent tertiary granules, and as our results suggest they can be exocytosed, then, their numbers within the cell at any one time could be reduced relative to the other granules. Consequently cell fractionation or histochemical techniques might not always detect large numbers of these granules. In contrast to cell fractionation, and histochemical techniques, freeze fracturing readily detects the presence and release of these small granules. This is because freeze fracturing tends to expose large areas of membrane surface thereby increasing the chances of observing these spatially limited exocytic events. For this reason we believe that small granule exocytosis is more readily detected with freeze fracture techniques. The new finding that a class of small granules penetrate the submembranous, filamentous network (Allison et al., 1973)) or cortical gel (Hartwig and Stossel, 1975) and fuse with the plasma membrane is interesting because it clearly demonstrates the differential release of large and small granules in mammalian leukocytes. Furthermore the data suggeststhat the release of these small granules is not subject to the same constraints as that of the larger granules. Thus, if the cortical gel is a barrier to leukocyte secretion, our data suggests that it is an incomplete and selective barrier. This selective release could be particularly important for leukocytes especially during chemotaxis and during blood banking storage. Wright and Gallin (1977) have recently shown a differential release of chemotactic activators and inactivators in human PMN leukocytes using cell fractionation techniques. It is conceivable that the differential release we have observed in rabbit leukocytes is analogous to that observed in human PMN leukocytes. In addition, the heretofore unrecognized small granule release in PMN’s could be responsible for the lability of PMN leukocytes during long term storage. The exact mechanism by which this differential release occurs is not clear. However, there are several structural differences between the large and small granules which might account for their differential release, of course, there is a size difference. The small granules are smaller and more uniform in diameter than the larger granules. We suspect that this size difference helps the smaller granules ,penetrate the filamentous network which excludes the larger granules. In addition to their small size, the small granules also appear to lack intramembranous particles (IMPS), while the larger granules possessthem (Moore et al., 1978). This apparent structural difference suggests that additional mechanisms such as the composition of intracellular membrane and/or surface charge might regulate granule release. Obviously much further work needs to be done to confirm differences in their storage content ( Murata et al., 1973), to elucidate the composition of their membranes, to determine mechanism( s ) of their release and the possible pathological roles of these small granules. At this point, however, the ultrastructural evidence is compelling: there is another, structurally distinguishable type of membrane fusion and release in PMN leukocytes. Furthermore the mechanism of release of these small granules may differ from that of the larger granules. ACKNOWLEDGMENTS This work Smith, Kline,

was supported by NIH and French Laboratories.

grants AM-10956 The final revisions

and AM-11028 and a grant from of this manuscript were completed

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while the author (PLM) was supported by NIH training grant AM 07107 at Yale University Medical School. The authors wish to thank James Scoggins III, Jane Farrington, and Dorthy Noe for excellent technical assistance and Norma Bendt (MUSC) and Betsy Tartagni (YUMS) for secretarial assistance. We also wish to express our appreciation to Dr. G. R. Hennigar, Professor and Chairman of the Pathology Department, whose support and enthusiastic encouragement made this research possible. A preliminary report of this work has been accepted for presentation at the 35th Annual Meeting of the Electron Microscopy Society of America, Boston, Massachusetts, 1977.

REFERENCES ALLISON, A. C. (1973). The role of microfilaments and microtubules in cell movement, endocytosis, and exocytosis. In “Locomotion of Tissue cells.” A Ciba Symposium, No. 15. Elsevier Publishing Co., N.Y. 109. ALLISON, F. JR., LANCASTER, G., and CROSTHWAITHE, J. L. (1963). Studies on the pathogenesis of acute inflammation. V. An assessment of the factors that influence in vitro phagocytic and adhesive properties of leukocytes obtained from rabbit pertoneal exudates. Am. J. Pathology 43, 775-795. ANDERSON, T. F. ( 1951). Techniques for the preservation of three dimensional structure for electron microscopy. Trans. N.Y. Acad. Sci. 13, 130-134. BANK, H. L., and ROBERTSON, J. D. (1976). A simple electrode for metallic replication. J. Microscopy 106, 343349. BECKER, E. L., and SHOWELL, H. (1972). The effect of Ca+’ and Mg” on the chemotactic responsiveness and spontaneous motility of rabbit polymorphonuclear leukocytes. 2. Immunitiitsforsch. 143, 466476. BRYANT, R. E., (1969). The effect of divalent cation depletion on phagocytes of staphlococci. Yale J. Biol. Med. 41, 303-310. BURWEN, S. J., and SATIR, B. H. (1977). Plasma membrane folds on mast cell surface and their relationship to secretory activity. J. Cell Biol. 74, 690-698. COCHRANE, D. E., and DOUGLAS, W. W. ( 1974). Calcium-induced extrusion of secretory granules ( exocytosis ) in mast cells exposed to 48/80 or the divalent ionophores A23187 or X-537A. Proc. Nat. Acad. Sci. USA 71, 408412. COHEN, A. L. ( 1974). Critical point drying. In “Principles and Techniques in Scanning Electron Microscopy,” pp. 44-112. Van Nostrand ReinhoId, N.Y. CONDEELIS, J. S., and TAYLOR, D. L. (1977). Th e contractile basis of amoeboid movement. V. The control of gelation, solation, and contraction in extracts from Dictyostelium discoideum. J. Cell Biol. 74, 901-927. CONDEELIS, J. S., TAYLOR, D. L., MOORE, P. L., and ALLEN, R. D. (1976). The mechanochemical basis of amoeboid movement. II. The effect of low divalent ion concentration on filament stability in amoeba cytoplasm. Exp. Cell Res. 101, 134-142. EBASHI, S., and ENDO, M. ( 1968). Calcium ion and muscle contraction. Prog. Biophys. Mol. Biol. 18, 123-183. ELGAESTER, A., SHOTTEN, D. M., and BRANTON, D. (1976). Intramembraneous particle aggregation in erythrocyte ghosts. II. The influence of spectrin aggregation. Biochimica et Biophysics Acta 426, 101-122. ESTENSEN, R. D., REUSCH, M. E., EPSTEIN, M. L., and HILL, H. (1976). Role of calcium and magnesium in some neutrophil functions as indicated by ionophore A23187. Infection and Immunity 13, 146-151. GALLIN, E. K., and GALLIN, J. I. ( 1977). Interaction of chemotactic factors with human macrophages. Induction of transmembrane potentials. J. Cell Biol. 75, 277-289. GOLDSTEIN, I .M., HORN, J. K., KAPLAN, H. B., and WEISSMAN, G. ( 1974). Calcium-induced lysozyme secretion from human polymorphonuclear leukocytes. Biochem. and Biophys. Res. Cwzm. 60, 807-812. HA~TWI~, J. H., and STOSSEL, T. P. (1975). Isolation and properties of actin, myosin, and a new actin binding protein in rabbit alveolar macrophages. J. Cell BioZ. 250, 5696-5705. HENSON, P. M. ( 1971). The immunological release of constituents from neutrophil leukocytes.

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