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The cytoskeleton of human polymorphonuclear leukocytesPhagocytosis and degranulation.

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THE ANATOMICAL RECORD 203:317-327 (1982)
The Cytoskeleton of Human Polymorphonuclear Leukocytes:
Phagocytosis and Degranulation
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'./
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;
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
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.
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
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
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 [1975], Buckley
[1975], Wolosewick and Porter [1976].) 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
[1981]).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 [1968]).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.
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
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
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
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
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-
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.
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
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.
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.
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.
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-
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.
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.
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
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leukocytesphagocytosis, cytoskeleton, polymorphonuclears, degranulation, human
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