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Granule release by polymorphonuclear leukocytes treated with the lonophore A23187.

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Granule Release by Polymorphonuclear Leukocytes
Treated with the lonophore A231 87
Department of Pathology, Medical Uniuersity of South Carolina, Charleston,
South Carolina 29401
Polymorphonuclear leukocytes (PMN's) incubated three t o
eight minutes a t 37OC in medium containing 1 x
M of the ionophore antibiotic A23187 released their cytoplasmic granules into the extracellular medium. Transmission electron microscopy of treated cells showed microfilament
bundles extending between adjacent granules within the cytoplasm and between
granules and the plasma membrane. Tiny dense projections (beads) 8-12 nm in
diameter were observed along segments of the cytoplasmic surface of the plasma
membrane with a periodicity of 20-30 nm. These beads were observed on the
plasma membrane only in the vicinity of intra- or extracytoplasmic granules.
The structural relationships of the beads with the plasma membrane microfilaments suggest they play a role in the process of ionophore-induced granule
release from polymorphonuclear leukocytes.
The ionophore antibiotic A23187, extracted
from cultures of Streptomyces chartreusensis,
has been shown to alter the permeability of
biological membranes to calcium (Reed and
Lardy, '72). Such phenomena as lymphocyte
mitogenesis (Hovi e t al., '76; Luckasen e t al.,
'74), salivary gland secretion (Prince et al.,
'731, leukocyte chemotaxis (Estensen et al.,
'76; Wilkinson '751, lymphocyte agglutination
(Poste and Nicholson, '76) and capping (Poste
and Nicholson, '76; Schreiner and Unanue,
'76) have been shown to be initiated, or otherwise affected by the changes in intracellular
calcium levels induced by this ionophore.
Recent biochemical evidence has indicated
that this ionophore enhanced secretion of the
specific granule enzyme, lysozyme, from human neutrophils, thus implicating calcium in
the induction of its release (Estensen et al.,
'76; Goldstein et al., '74). In the present
report, transmission and scanning electron
microscopy were employed t o assess the structural changes that occur during granule
release in rabbit PMN's exposed to the
ionophore A23187.
Polymorphonuclear leukocytes (PMNs)
were obtained from 1-2 kg New Zealand rabbits by intraperitoneal injection of 200 ml
ANAT. REC.. 189: 177-186.
sterile PBS (pH 7.2) containing 200 mg glycogen (Type 11, Sigma Chemical Co., St. Louis,
Missouri) as previously described (Moore et
al., '76). The final concentration of cells was
adjusted to 3 X lo6 celldm1 and the exudate
separated into 15 ml aliquots. The cells were
then centrifuged a t 500 g, (3 minutes), resuspended in one of the test or control media
(table 1) a t the original cell concentration,
and incubated a t 37OC for three to eight
minutes on coverslips for SEM (scanning electron microscopy) or in test tubes for TEM
(transmission electron microscopy). Cells for
both TEM and SEM were fixed one hour with
2% glutaraldehyde in 0.1 M Na-cacodylateHC1 buffer, pH 7.2, 380 f 2OmOsm, and
postfixed one hour in 2% osmium tetroxide in
pH 7.2 Na-cacodylate-HC1 buffer. For TEM,
cells were dehydrated either utilizing ethanol
or dimethoxypropane (Miiller and Jacks, '75)
and embedded in low viscosity resin (Spurr,
'69). Thin sections were cut on a Porter-Blum
MT-1 and stained with uranyl acetate and
lead citrate and examined with either a
Hitachi HS-8 or HU-12 electron microscope.
Cells for SEM were treated with thiocarbohydrazide and osmium tetroxide (Kelley et
Received Feb. 17. '77. Accepted Apr. 11, "77.
' Present address: Department of Biology, Yale University. New
Haven, Connecticut.
lncuhation media
1 Minimal Essential Medium (MEM)
2 25mM Hepes buffer
150 mM NaCl
3 MEM 3 mM CaCl,
4 Hepes + 5 mM CaC1,
6 Hepes +DMSO
7 MEM+3 mM CaCl,+DMSO
8 Hepes 5 rnM CaC1, DMSO
9 MEM+lX 1 0 - f i M A23187i~DMSO
10 Hepes + 1 X 10-6M A23167 + DMSO
11 Hepes+5mMCaCl,+lX 1Ow6M
A23167 + DMSO
Granule release
' Over half the cells show some degree of granule release Le.. cells
depleted of granules or large numbers of extracellular granules were
observed, fig. 1).
Eagles, F-12. Grand Island B i d Co.. New York (contains 1.26m M
Polysciences. Inc., Warrington. Pennsylvania.
DMSO. a solvent for the ionophore, never exceeded concentrations
of 0.lX (vol/voll in any of the media
al., '751, dehydrated, critically point dried,
and gold coated as previously described
(Moore et al. '76). Specimens were viewed
with a Coates and Welter model no. 106 Field
Emission Scanning Electron Microscope.
The addition of 1 X
M of A23187 to any
of the media containing calcium produced profound and unique morphological changes in
rabbit PMN leukocytes. After a 3-minute incubation a majority of the viable PMN's
released many primary, secondary and tertiary granules extracellularly as demonstrated
by fixation and examination with TEM and
SEM. In thin sections, some cells were devoid
of granules (fig. l), while others were observed
to have granules in various stages of release
(fig. 2). PMN's which were releasing granules
(exocytosis) often had irregular surface conformations and numerous small villar-like
projections. Generally, granules were observed in the vicinity of the plasma membrane, both inside and outside the cell. The
distribution of groups or bundles of microfilaments was confined to four locations: (1)
between adjacent granules with the cytoplasm (fig. 3); (2) between intracellular granules and the plasma membrane (fig. 4); (3) between extracellular granules and the plasma
membrane (fig. 5); (4) and between granules
on opposite side of the plasma membrane
(fig. 6).
Tiny dense projections, termed beads, were
found associated with segments of the inner
surface of the plasma membrane of PMN leukocytes in which granule release was observed
(figs. 7-11). These beads had dimensions of 81 2 nm, and were discrete, electron dense, and
spaced regularly a t 20-30 nm intervals. These
beads were often seen associated with
microfilaments (5-7 nm in diameter) which
appeared to be attached to cytoplasmic granules (figs. 7, 8). Plasma membrane associated
beads were also observed adjacent to granules
which were in intimate contact with the
plasma membrane. These contact areas were
characterized by a single dense lamina rather
than two opposing trilaminar structures.
Cells not releasing granules or those having
released all their granules (fig. 1) had no
plasma membrane associated beads.
Longitudinally sectioned microtubules (18
nm) were only rarely observed in cells treated with A23187. Microtubule-like structures
were occasionally seen associated with granule membranes in some PMN's (fig. 12).
Microfilament bundles were observed between adjacent granules, but never between
granules and plasma membrane in control
PMN's, or the few non-reactive cells treated
with ionophore. In addition, plasma membrane associated beads were not seen in control cells. In rabbit PMN's releasing granules,
beads were not always observed in cross sections of granules in proximity to the plasma
membrane. When present, however, the beads
were always in close association with a granule (intra-or extracellular) filament bundle,
or both (figs. 7-10).
PMNs treated with medium containing
ionophore but no calcium as well as those not
containing ionophore showed comparatively
little granule release as judged by the relative
number of both cytoplasmic granules remaining within the cells and extracellular granules (table 1).Increased extracellular calcium
alone did not result in appreciable granule
release. Similar results were obtained with
different buffer systems or low DMSO concentrations (<0.1%).
It has been proposed by other investigators
that release of lysozyme by human PMN leukocytes may be mediated by an increase in
intracellular calcium levels, and that these
levels can be readily manipulated with the
ionophore A23187 (Estensen et al., '76; Goldstein et al., '74). Goldstein et al. ('74) have
suggested that the specific release of only secondary (specific) granules may be indicative
of differences in membrane properties of the
various granule types. Estensen et al. ('761,
however, reported release of both primary and
secondary granule types. Our results with
rabbit PMN's show three granule types are
released by PMN's treated with ionophore a t
concentrations known to produce release of
lysozyme in human PMN's (Estensen et al.,
'76; Goldstein e t al., '74). The mechanism of
granule release, however, still remains enigmatic. In the normal functioning capacity of
the PMN lysosome, its limiting membrane
only fuses with those of phagocytic vacuoles
(Spicer and Hardin, '691, although extracellular release of lysosomal enzymes has been
shown to occur under certain conditions
(Goldstein e t al., '75, '76). These conditions
were believed to manipulate specific membrane fusion capacities. Prior t o extracellular
release of granules, a t least two major kinetic
events must take place: (1) the movement of
the granule to the plasma membrane and (2)
the fusion of granule and plasma membranes.
The ionophore-induced granule release from
rabbit PMN's provided a suitable model for
studying these events.
Microfilaments have been implicated in directly mediating phagocytosis and membrane
motility in macrophages (Axline and Reaven,
'74). Plasma membrane-associated actin has
been demonstrated in echinoderm sperm
(Tilney, '761, amoeba (Pollard and Korn, '711,
mammalian intestinal microvilli (Mooseker
and Tilney, '75) and mammalian lymphocytes
(Barker and Crumpton, '76). Previous observation of bundles of organized microfilaments
associated with rabbit PMN granules (Moore
e t al., '76) is confirmed here. In this study, the
results indicate attachment of a single bundle
of microfilaments to both granule and plasma
membrane. If these microfilaments belong to
a non-muscle actomyosin contractile system
such as those described by others (Boxer et al.,
'74; Pollard and Korn, '71; Stossel and
Pollard, '73;Taylor et al., '73; Tilney, '761, it
seems likely that these structures could be capable of moving granules to the plasma membrane and facilitating their release from rabbit PMN leukocytes.
On the other hand, a change in the plasma
membrane rather than, or in addition to, an
effect on contractile filaments would seem to
be entailed in the ionophore-induced capacity
of granule membranes to fuse with plasma
membrane. Perhaps an altered action potential on the plasma membrane as a result of increased cytoplasmic calcium levels could underlie the exocytosis of cytoplasmic granules.
The ionophore-altered plasmalemma appears
in this light t o have undergone changes comparable to those which occur when plasma
membrane is converted t o limiting membrane
of phagocytic vacuoles and which mediate
fusion of cytoplasmic granules with phagocytic vacuoles. This is supported by evidence
which indicates that A23187 reproduces the
stimulated oxidative activities characteristic
of phagocytosis (Schell-Frederick, '74).
The areas presumed to represent membrane
fusion events between the plasma membrane
and cytoplasmic granules were characterized
by single electron dense lamina rather than
simple adjacent trilaminar membranes. These
were similar t o those granule-plasmamembrane fusion regions described by Chi et al.
('76) in mast cell secretion. These specialized
regions, however, differed in that those in the
present study lacked the "fuzzy coating" described for mast cells (Chi et al., '76). In the
ionophore treated PMN, these areas also occasionally had beads in close proximity (fig. 9).
Microtubules have also been implicated in
playing a role in release of lysosomal enzymes
in human PMNs (Goldstein, "75) but their
precise function in this capacity is not clear.
The tubule-like structures observed in ionophore-treated rabbit PMNs were too sparse to
correlate with granule release.
The role of the newly described plasma
membrane associated beads in ionophoreinduced granule release is unknown a t present. Their size and periodicity are similar to
membrane-associated filaments in budding
yeast (Byers and Goetsch, '761, but no
filament ring structure was observed in the
ionophore-treated PMN's. Beads were recently described associated with an interface between Golgi complex and endoplasmic reticulum and were thought to be related to the
movement of transitional vesicles between
these two cytoplasmic elements (Locke and
Huie, '76). Similarly, in our study, the structural intimacy of the beads with the plasma
membrane a t the site where the granule approaches the plasma membrane suggests
these beads may play a role in the trans-
membrane passage of granules. Regardless of
their role in granule release, the existence of
the beads is transitory for they are not seen in
cells in which all or almost all the granules
had been released.
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Mitogenic properties of calcium ionophore, A23187. Proc.
The authors wish to express their gratitude
Natl. Acad. Sci. (U.S.A.), 71:5088-5090.
to Ms. Jane Farrington and Ms. Dot Noe for Moore, P. L., H. L. Bank, N. T. Brissie and S. S. Spicer 1976
Association of microfilament bundles with polymorphotheir technical assistance and to Ms. Dot
nuclear leukocyte lysosomes. J. Cell. Biol., 71: 659-666.
Smith for secretarial assistance. A special Mooseker, M. S., and L.G. Tilney 1975 Organization of a n
note of appreciation to Doctor Robert Hamill
actin-filament membrane complex. Filament polarity and
membrane attachment in the microvilli of intestinal epiof Eli Lilly Co., Indianapolis, Indiana for his
thelial cells. J. Cell. Biol., 67: 725-743.
generous gift of the ionophore A23187 and to Miiller,
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Thiocarbohydrazide-mediated osmium binding: a tech.
1 PMN leukocyte devoid of cytoplasmic granules after eight minutes treatment with
ionophore. Scale, 1 pm. X 8,750.
2 Scanning electron micrograph of PMN treated with ionophore showing numerous
granules (arrows) on the external surface of a PMN leukocyte. Scale, 1p m . X 12,000.
3 Cytoplasmic granules (G)with microfilaments between them (arrows). Scale, 0.2 pm.
X 57,500.
Microfilaments (arrows) are observed between a cytoplasmic granule (G) and the
plasma membrane (arrowheads) a t the left side of the micrograph. Scale, 0.2 pm.
x 75,000.
5 Microfilaments (arrows) between an extracellular granule (GIand the plasma membrane (arrowheads). Scale, 0.2 p m . X 77,500.
6 Microfilaments (arrow) between cytoplasmic granule (G) and extracellular granule
(G’),Scale, 0.2 p m . X 77,500.
P L Sannes. H. L. Bank, P L Moore and S S Spicer
7 Microfilament (arrows) between cytoplasmic granule (GI and plasma membrane associated bead (arrowhead). Scale, 0.25 pm. X 97,500.
8 Microfilament (small arrow) between cytoplasmic granule (G) and membrane associated bead (arrowhead). Scale, 0.25 pm. X 97,500.
9 Cytoplasmic granule (GI in contact with a thickened portion of the plasma membrane (arrowheads). Membrane associated beads (small arrows) are observed in close
association with t h e granule. Scale, 0.25 pm. X 97,500.
10 Cytoplasmic granule (GI in close proximity to a number of beads (arrows). Opposite
the beads is a cluster of extracellular granules. Scale, 0.2 pm. X 95,000.
11 A small cyl.oplasmic granule (G) in close proximity to a number of membrane associated beads (arrows). Scale, 0.2 pm. X 95,000.
12 Cytoplasmic granule ( G ) in close association with a tubule-like structure (arrow).
Scale, 0.2 pm. X 95,000.
P . L. Sannes, H. L. Bank, P. L. Moore and S. S. Spicer
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a23187, lonophore, release, polymorphonuclears, granules, leukocytes, treated
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