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Cell Motility and the Cytoskeleton 38:215–228 (1997)
Video-Rate Dynamics of Exocytotic Events
Associated With Phagocytosis in Neutrophils
Etsuko Suzaki,1 * Hideyuki Kobayashi,1 Yuka Kodama,1 Tsutomu Masujima,1
and Susumu Terakawa2
1Institute
of Pharmaceutical Sciences, Hiroshima University School of Medicine,
Hiroshima, Japan
2Photon Medical Research Center, Hamamatsu University School of Medicine,
Hamamatsu, Japan
Exocytotic responses associated with phagocytosis were investigated in a single
neutrophil with a special reference to their dynamic properties and their spatiotemporal relationships with ionic and chemical responses during phagocytosis. The
real-time sequence of phagocytosis-exocytosis was directly visualized by videoenhanced contrast differential interference contrast (VEC-DIC) microscopy. The
actual release of contents from such a granule was proven by examining a cell
loaded with quinacrine with a dual imaging system that allowed us to observe DIC
and fluorescence images simultaneously at a high magnification. During the
process of phagosome formation in a neutrophil engulfing an opsonized zymosan,
the exocytotic response was observed first in a granule located near the cell surface
initially attached to the zymosan, and then in other granules sequentially along
pseudopodia surrounding the zymosan. When the phagocytosis was induced in a
medium containing luminol, a chemiluminescence due to active oxidants was
detected exclusively in the region of phagosome, suggesting that exocytosis took
place on the phagosomal membrane and not on the plasma membrane. Changes in
cytosolic free calcium concentration ([Ca21]1) were further measured using fura-2
under the dual imaging system. [Ca21]i transients were more closely related to the
extension of pseudopodia for engulfing zymosan and not directly to the exocytosis.
These findings lead to a conclusion that exocytosis associated with phagocytosis is
initiated by attachment of the cell membrane to the invading organism and
mediated by local activation of the phagosomal membrane. Cell Motil. Cytoskeleton 38:215–228, 1997. r 1997 Wiley-Liss, Inc.
Key words: video microscopy; chemiluminescence; cytosolic free calcium; rabbit neutrophil
INTRODUCTION
Phagocytosis is one of the most important functions
of the neutrophil to eliminate invading microorganisms.
This function involves a series of subcellular activities
taking place in a highly coordinated manner, i.e., an
attachment of the cell membrane to a microorganism,
ingestion of the organism with extended pseudopodia,
formation of a phagosome, rearrangement of secretory
granules, and release of granular contents into the forming phagosome by exocytosis. Various intracellular signals with molecular and ionic mediators underlie these
responses in order to maintain their orchestration. As the
r 1997 Wiley-Liss, Inc.
exocytotic response is one of the smallest structural
changes associated with the phagocytosis, only cinemicrophotographic [Hirsch and Cohn, 1960; Hirsch, 1962] and
electron micrographic [Zucker-Franklin and Hirsch, 1964]
studies of this response have been done to reveal fine
Contract grant sponsor: Grant-in-Aid for Scientific Research from the
Ministry of Education, Science and Culture, Japan
*Correspondence to: Etsuko Suzaki, Department of Anatomy, Hiroshima University School of Medicine, 1-2-3 Kasumi, Minami-ku,
Hiroshima 734, Japan. e-mail: etchan@mcai.med.hiroshima-u.ac.jp.
Received 17 December 1996; accepted 23 June 1997
216
Suzaki et al.
aspects and some dynamic nature of exocytosis. It was an
early histochemical study that suggested sequential degranulation of specific and azurophilic granules in a time
range of 0.5 to 10 min [Bainton, 1973], while a later
immunocytochemical study suggested a release of bactericidal substances within 5 s after microorganism attachment [Pryzwansky et al., 1979]. Measurement of the
membrane capacitance recently provided some insights
into dynamics of exocytosis in a single neutrophil [Nüsse
and Lindau, 1988]. However, this technique may not
easily be applied to the exocytotic response during
phagocytosis, because the membranous change for the
phagocytosis itself is large and long lasting. Therefore,
spatial and temporal relationships between exocytotic
responses and other signaling activities involved in
phagocytosis have remained obscure. To obtain exact
knowledge about these processes, a more accurate and
continuing measurement with a higher time resolution
would be necessary. An intracellular Ca21 signal, for
example, is reported to appear rapidly when a neutrophil
attaches to microorganisms and then disappear oscillatory
or monotonically [Jaconi et al., 1990]. If so, how is the
phagosome formation regulated, and how is the exocytosis initiated? Does exocytosis also start as early as the
microorganism attachment or later than the microorganism ingestion? How is the timing of exocytosis regulated
to maximize the bactericidal effects in phagosome? In
order to obtain clues to answer these questions, it is
necessary to determine sequential relationships between
differing activities involved in phagocytosis.
The differential interference contrast (DIC) microscopy combined with contrast enhancement [Allen et al.,
1981] by an advanced image-processing technique [Weiss
and Galfe, 1992] has improved spatial and temporal
resolutions significantly. Thus, it became a powerful tool
for real-time observation of exocytosis in various secretory cells [Terakawa et al., 1991, 1993, 1995a,b; Terakawa and Suzuki, 1991; Kamijo et al., 1993; Sakurai and
Terakawa, 1995]. In the present study, we revisited the
problems related to dynamic nature of exocytosis in the
neutrophil, employing the video-enhanced DIC microscopy. Particular attention was paid to spatial distribution
and temporal sequence of exocytosis in reference to a
zymosan-induced phagosome formation. The release of
active substances from the granules has been visualized
and quantified using a fluorescent probe [Kolber and
Henkart, 1988; Kawasaki et al., 1991] and a chemiluminescent probe [Yanai, 1979; DeChatelet et al., 1982].
Accordingly, we employed a dual imaging system that
allowed simultaneous visualization of a fluorescence
image and a DIC image [Foskett and Melvin, 1989]. We
improved resolution of the system to a submicron level,
and correlated spatiotemporal patterns of granule dis-
charge as well as an intracellular Ca21 signal with the
DIC images of exocytosis. Here, we describe properties
of phagocytosis-exocytosis sequence observed by these
video-rated imaging techniques and present evidence that
active substances are released from granules to the
phagosome with a maximum efficiency for the host
defense mechanism. Furthermore, we present a clue to
the intracellular signal other than [Ca21]i that may
specifically regulate exocytotic responses associated with
phagocytosis.
MATERIALS AND METHODS
Isolation of Neutrophils
Neutrophils were isolated from the peripheral blood
of the rabbit using a combined dextran/Ficoll-Conray
sedimentation procedure [Yanai, 1979]. Contaminating
erythrocytes were removed by hypotonic lysis, and
neutrophils were finally suspended in a density of 2.5 3
106/ml in Ca21-free Hanks’ balanced salt solution
(HBSS(-)).
Zymosans
Zymosan A from Saccharomyces cerevisiae (Sigma
Chemical Co., St. Louis, MO) was opsonized as previously described [Wardlaw and Pillemer 1956]. Briefly,
zymosan particles (40 mg) suspended in veronal buffer
(20 mM, pH 7.4) were boiled, incubated with fresh rabbit
serum at 37°C for 30 min, washed twice with Hanks’
balanced salt solution (HBSS), and resuspended in the
same buffer. The opsonized zymosans (44 µg/ml) in
HBSS were placed in a chamber and settled on a
silicone-coated coverslip for 10 min. The excess amount
of solution was removed immediately before use.
Observations of Phagocytosis and Exocytosis
The neutrophil was prewarmed at 37°C for 3 min
and placed in HBSS in a Flexiperm-Disc (Heraeus
Biotechnology, Hanau, Germany) of which the bottom
was made of a silicone-coated coverslip. Opsonized
zymosans were then added to the neutrophil preparation
at a concentration of 150 µg/ml. They were observed
under inverted microscopes (Axiovert 35 and 135-TV,
Zeiss, Oberkochen, Germany) equipped with 3100 DIC
objective lenses and 32.5 insertion lenses. Images were
obtained with a CCD camera (SSC-M350, Sony, Tokyo).
The contrast of video image was enhanced by about
5-fold with a digital image processor (PIP-4000, ADS,
Osaka). The process of phagocytosis was monitored and
recorded with a videotape recorder (AG-7735, Panasonic,
Tokyo). Phagocytosis and exocytosis of a neutrophil were
investigated in detail from the recorded images. The
exocytotic process of a single granule was analyzed in
detail from the change in light intensity of a small spot in
Phagocytosis-Exocytosis Sequence in Neutrophils
the recorded DIC images where exocytosis of a granule
occurred. For this calculation, a series of images were
digitized at an 8-bit resolution and stored in the frame
memories. For further analyzing exocytosis, the recorded
images were processed through the sequential subtraction
(time differentiation) mode, (ARGUS-100 and ARGUS20, Hamamatsu Photonics, Hamamatsu [Terakawa et al.,
1991]) by which the rapid exocytosis was selectively
distinguished from the slower granule movements or
other static configurations. The time differentiation processing was done by subtracting an image from the
previous image consecutively; i.e., image 2 was subtracted from image 1, and image 3 was subtracted from
image 2, and so on. Resultant images were continuously
displayed in real time with the contrast enhanced by about
2-fold. The exocytotic responses were extracted as bright
and dark spots, and were counted for constructing a
frequency histogram of a single cell. The sites of exocytosis were traced also from this differential display of the
video record.
Simultaneous Observations of DIC and Quinacrine
Fluorescence Images
Neutrophils were incubated with quinacrine (20
µM) in HBSS at 37°C for 10 min. After the incubation,
the cells were placed on the coverslip and were observed
under an inverted microscope (Axiovert 35, Zeiss)
equipped with a 3100 DIC objective lens and a 32.5
insertion lens. The cells were maintained on the microscope stage at 32–36°C by continuous perfusion with an
oxygenated and warmed HBSS. Both DIC and fluorescence images were obtained with a dual imaging system
[Foskett and Melvin, 1989]. For the DIC image, a long
wavelength of 650 nm was used. This light was separated
from the fluorescence by a dichroic mirror (half-reflection
wavelength 5 580 nm), detected by an intensified CCD
camera (WV-150, Panasonic), and the image was enhanced with an image processor (PIP-4000, ADS). For
the fluorescence image, a combination of a blue excitation filter (bandpass at 470 nm), a dichroic mirror
(half-reflection wavelength 5 490 nm), and a short cut
filter (half-pass at 520 nm) were used. The fluorescence
image was detected with another intensified CCD camera
(DAS-512, Imagista, Tokyo) equipped with a frame
memory by which fluorescence intensity was accumulated for 66 ms. The fluorescence image was then
enhanced 2- to 3-fold with an analog amplifier of video
signal (410, Suncomm Technical, Tokyo). Both DIC and
fluorescence images during the phagocytosis-exocytosis
sequence were recorded simultaneously with two video
tape recorders (AG-7735 and AG-7500, Panasonic). For
analyzing exocytosis further, the recorded images were
processed through the sequential subtraction (time differentiation) mode [Terakawa et al., 1991].
217
Chemiluminescence Imaging
Neutrophils were settled on a coverslip in a Flexiperm-Disc placed on an inverted microscope (DiaphotTMD, Nikon, Tokyo) equipped with a 3100 objective
lens and an incubation box (37°C). After 5 min of
prewarming, a reaction medium containing zymosans
(300 µg/ml) and luminol (10 µg/ml, Nakarai Chemical
Co., Tokyo) in HBSS was added to the chamber. The
neutrophils were observed initially in the brightfield
mode. As soon as the initiation of the phagocytosis was
confirmed, all illuminations were turned off, and a
chemiluminescence was recorded. The chemiluminescence derived from active oxidants was detected with a
photon-counting camera (C2400-20, Hamamatsu Photonics) coupled with an image processor (ARGUS-100,
Hamamatsu Photonics). Two-dimensional images of chemiluminescence were accumulated for 2 min in a frame
memory. Brightfield images obtained before and after the
chemiluminescence measurement were also recorded.
The chemiluminescence image superimposed on the
brightfield image was taken to indicate the loci of oxidant
production.
Simultaneous Observations of DIC and Fura-2
Fluorescence Images
Neutrophils were prewarmed at 37°C for 10 min
and incubated with fura-2/AM (2 µM) in HBSS (-) which
contained 0.5% bovine serum albumin (BSA) for 30 min.
After the incubation, the cells were placed on a zymosanattached coverslip in a chamber. The cells were maintained on the microscope stage at 32–36°C by continuous
perfusion with an oxygenated and warmed HBSS, and
were observed using the dual imaging system. For
estimating intracellular Ca21 concentration ([Ca21]i),
fluorescence images were taken with an intensified CCD
camera (DAS-512, Imagista) at an emission wavelength
of 510 nm and alternative excitations at 380 nm (yielding
a [Ca21]i-dependent fluorescence) and 360 nm (yielding a
[Ca21]i-independent fluorescence). Interference filters of
a 10-nm bandwidth were used to select the wavelengths.
These images were accumulated for 0.5 s alternately and
continuously during the phagocytosis-exocytosis sequence, and were further processed by an image processor (ARGUS-100, Hamamatsu Photonics) to calculate the
ratio value of the fluorescence intensities (I360 nm/I380 nm).
The DIC image was obtained simultaneously with these
fluorescence images by illuminating at 650 nm (bandwidth of interference filter 5 40 nm) and separating the
light with a dichroic filter (half-reflection wavelength 5 570 nm). Another intensified CCD camera (WV150, Panasonic) was used for detecting the DIC image.
218
Suzaki et al.
RESULTS
Phagosome Formation and Exocytotic Responses
Under the video microscope with the proper contrast enhancement, a whole sequence of the phagocytosisexocytosis response was clearly visible (Fig. 1). Neutrophils attached easily to the bottom of the microscopic chamber made of a coverslip, and moved slowly in various
directions. They spread and retracted pseudopodia or lamellipodia repeatedly, and transported granules back and forth
intracellularly. Zymosans, when added to the chamber,
sank to the bottom, but did not attach to it. Instead, they
fluctuated in a Brownian movement near the bottom. A
glass-attached neutrophil approached them in 20–30 s,
and formed a contact with them using a short hand-like
structure. An exact moment of the firm attachment to a
zymosan could be accurately determined by its abrupt
cessation of the Brownian movement. After a certain lag
period following the capture of zymosan, the neutrophil
extended new pseudopodia around the zymosan and
engulfed it into an intracellular space, i.e., a phagosome
(Fig. 1). By changing the focal plane up and down, it was
confirmed that the zymosan was totally surrounded by the
cytoplasm of the neutrophil. The lag period from the
neutrophil-zymosan attachment to the emergence of
pseudopodia was 34.1 6 3.4 s (mean 6 S.E.M., n 5 30)
and the time from the latter to a complete closure of a
phagosome was 36.4 6 3.2 s (n 5 30, Fig. 2).
During the phagosome formation, a rather rapid
transport of granules was observed from the tail to the
head of a neutrophil, i.e., in the direction towards the
captured zymosan. The nucleus of the neutrophil also
moved together with the granules to some extent toward
the phagosome. Soon after these morphological changes,
many of the granules located around the phagosome
stopped moving and then abruptly disappeared one by
one. This was a very characteristic response which gave
an impression of ‘‘popping’’ or ‘‘flickering’’ of granules,
in a resemblance to a burst of bubbles. We referred to this
response as ‘‘popping.’’ The images obtained through the
sequential subtraction mode selectively distinguished the
popping of a granule from other configurations. We refer
to this response as exocytotic response. The clarity of
these images was high enough to determine the sequential
relationships of the exocytotic responses with other
responses (Fig. 2).
In many cases, the initial exocytotic responses were
observed while the pseudopodia still extended to engulf
the zymosan. Later, similar responses continued also after
the complete closure of the phagosome. These responses
were release of granular contents into the phagosome
which was isolated partially at the beginning and then
completely from the extracellular space. The percentage
of enclosure of a zymosan into the phagosome was
calculated as a ratio of the pseudopodial length to the
circumference of zymosan. This percentage, measured at
the moment of the first exocytotic response, varied from
27% to 100% and was 69% 6 21% (mean 6 S.D.,
n 5 24) on average. Timewise, the first exocytotic response occurred at 59.1 6 4.3 s (n 5 30) after the capture
of a zymosan. Subsequently, many exocytotic responses
followed and continued for a period of 38.1 6 3.2 s
(mean 6 S.E.M., n 5 30; Fig. 2). Thus, it took 97.2 6
5.5 s (n 5 30) for a whole sequence of phagocytosisexocytosis responses to complete.
Visualization of Exocytotic Discharge With a
Fluorescent Probe
Quinacrine-loaded neutrophils were observed using
the dual imaging system. In order to avoid a heat
absorption or a photodynamic action, we started illuminating the cell with an excitation light after observing the
initial sign of the phagocytosis in the DIC image. In the
fluorescence image, the cells showed many bright granules of 0.4–1.0 µm in diameter. Many of these granules
surrounded the zymosan which appeared dark as a result
of negative staining. Exactly when a granule showed
‘‘popping’’ in the DIC image and in the time differential
image, a bright granule abruptly disappeared at the same
site in the fluorescence image (Fig. 3). The disappearance
was so quick and discrete that it was easy to distinguish
from the fading of fluorescence due to photobleaching.
These observations indicated that the popping of a
granule in the DIC image was in fact the image of
exocytosis accompanied with the discharge of granular
contents. The rate of fluorescence change indicated that
about 66 ms were necessary for a single granule to
discharge its fluorescent content.
Dynamics of Exocytosis
Dynamic aspects of exocytosis were further analyzed at a time resolution of the video rate. One of the
major factors that alters the light intensity of a small spot
in the DIC image is a change in refractive index resulting
from a change in density of substances in that spot.
Fig. 1. Video micrographs of a neutrophil showing a whole sequence
of phagocytosis and exocytosis. a: Onset of phagocytosis. A neutrophil
(N) attached to a zymosan (Z) using a hand-like structure (H) for more
than 30 s. b: Initiation of phagosome formation. Pseudopodia started to
extend after a lag period. The front line of pseudopodia lay between the
arrows. The image was taken 10 s after a. c: Phagosome formation.
Pseudopodia extended (arrows) further around the zymosan. The
image was taken 23 s after a. d: Completion of phagosome formation.
Tips of pseudopodia fused together to form a phagosome. The image
was taken 46 s after a. Nc, nucleus. e–h: High magnification images of
the same neutrophil at the stage shown in d. A granule undergoing
exocytosis is indicated by arrows. The interval between images was 33
ms. Inset: One of the time differential images made by sequential
subtraction of g from f. A small area including the granule indicated by
the arrow is shown at the same magnification. The exocytosed granule
was selectively extracted.
Phagocytosis-Exocytosis Sequence in Neutrophils
Figure 1.
219
220
Suzaki et al.
Fig. 2. Time course of the phagocytosis-exocytosis sequence. Relationships between a neutrophil and a
zymosan in three stages of the phagocytosis are schematically drawn on the bottom: left, attachment to a
zymosan; middle, extension of pseudopodia; and right, the completion of phagosome formation. Average
periods (n 5 30) of the lag time, phagosome formation, and exocytosis are shown on the top with their
onset time relative to each other. Time zero indicates the time of attachment of the neutrophil to a zymosan.
Therefore, the observed change in light intensity would
reflect the process of discharge of the granule contents
and flattening of the omega-shaped membrane. Figure
4 shows representative time courses of light intensity
change obtained from four granules that underwent
exocytosis. The light intensity changed from a stable
level to a sharp peak within 33–66 ms and then to the
original level in about 200 ms. These kinetics of exocytotic response measured in a single granule were faster
than those observed in some exocrine cells [Terakawa et
al., 1993, 1995a; Kamijo et al., 1993].
Site of Exocytotic Responses
For counting the frequency and determining the
exact site of individual exocytotic responses, the images
processed through the sequential subtraction (time differentiation) mode were analyzed in detail. Through this
processing, a secretory granule showed abrupt and rapid
change in light intensity at the exocytotic moment and
eventually disappeared (Figs. 1, inset; and 3c). The
frequency of such discrete changes of individual granules
was counted in a neutrophil as a measure of their
exocytotic activity (Fig. 5). This activity reached a peak
5–20 s after the appearance of the first exocytotic
response. Granules ranging from 14 to 95 in number
(36 6 17 granules, mean 6 S.D.; n 5 30) underwent
exocytosis during a single sequence of phagocytosis.
Occasionally, a neutrophil captured 2, 3 or 4 zymosans
sequentially at different portions, forming several phagosomes at some intervals. In such cases, the exocytotic
activity was always higher during the first phagosome
formation than the later.
In the neutrophil that engulfed a zymosan, exocytosis occurred only at the periphery of the phagosome and
never on the cell surface directly facing the medium. This
was in sharp contrast to the exocytotic responses induced
by application of chemical stimulants like concanavalin A
and cytochalasin E. These stimulants induced similar
popping responses of granules on the surface of a
neutrophil without any preferred loci (data not shown).
Phagocytosis-Exocytosis Sequence in Neutrophils
Fig. 3. Video micrographs of DIC and fluorescence images simultaneously obtained at the moment of an exocytotic response in a
neutrophil. The cell was previously loaded with quinacrine. a:
Fluorescence image. a1: A whole view of a neutrophil engulfing two
zymosans (asterisks) at both ends. a2–a5: Sequential video images
enlarged from the area indicated by the white frame in a1. b: DIC
image. b1: A whole view of the same neutrophil. Asterisks indicate
zymosans. b2–b5: Sequential video images enlarged from the area
221
indicated by the black frame in b1. c: Differential image made from
the DIC image. Image c1–2 was made by subtracting image b2 from
the previous image, image c2–3 by subtracting b3 from image b2, and
so on. A bright spot in a quinacrine-loaded neutrophil abruptly
disappeared (white arrows in a) when a granule showed an abrupt
popping response in DIC image (black arrows in b and c). The
interval between each micrograph was 66 ms.
delayed, the first exocytosis was observed at about 3 µm
apart from this area. As the pseudopodia extended to
engulf the zymosan, the region active for exocytosis
shifted from this proximal area to the distal area along the
periphery of the phagosome (Fig. 7). Although many
granules were accumulated around a phagosome, only a
fraction of them underwent exocytosis at its periphery.
Luminol-Dependent Chemiluminescence
Fig. 4. Time course of a single exocytotic response observed during
the phagocytosis. The relative changes in light intensity of a single
granule were measured from the video records and plotted at a time
resolution of video rate. Four representatives are shown. The decrease
in light intensity reflects extrusion of granule contents, and the
recovery phase reflects retrieval of the omega-figured membrane.
When a neutrophil initially attached to a zymosan, a
small portion of the cell membrane, about 2 µm in length,
served as an attachment site. The first exocytosis always
appeared near this place where the neutrophil first
recognized the foreign object. Figure 6 shows the localization relationship between the site of initial attachment and
the site of the first exocytosis. Mostly, the first exocytosis
occurred within 1 µm of the initial attachment site. In
some cases in which the initiation of exocytosis was
When phagocytosis of a neutrophil was induced by
a zymosan in a medium containing luminol, a significant
chemiluminescence was observed. About 30 photons
were counted from a single cell during a 2-min period of
its highest activity. This period roughly paralleled with
that of exocytosis. Luminol-dependent chemiluminescence activities measured by photon-counting imaging
were certainly localized to a narrow region in the cell
(Fig. 8). Superposition of this chemiluminescence image
on the brightfield image revealed that chemiluminescence
activity was high in the region of the phagosome. There
was a tendency that the highest chemiluminescence
appeared along the periphery of the zymosan.
[Ca21]i Measurement With Fura-2
Illumination of fura-2-loaded cells with the UVlight tended to suppress their exocytotic responses. Because of this, continuous observation of fura-2 fluorescence in
the neutrophil through the whole phagocytosis-exocytosis
222
Suzaki et al.
Fig. 5. Frequency of exocytosis during the phagocytosis of a single zymosan in a neutrophil. The number
of exocytosis in every 5 s was counted in 30 neutrophils after processing the video with the time
differentiation mode (see Figs. 1, inset; and 3c). The time 0 is set at the first appearance of exocytosis.
sequence was very difficult. In practice, we started the fluorescence illumination when extension of pseudopodia for
engulfing the zymosan was recognized in the DIC image.
Relationship between the exocytosis and [Ca21]i increase
was thus analyzed from simultaneous video recordings.
Figure 9 shows DIC images obtained simultaneously with [Ca21]i images during formation of a
zymosan-engulfing phagosome. A rapid increase in [Ca21]i
was observed in the whole cytoplasm when pseudopodia
started to extend. [Ca21]i was not particularly high in the
region where the exocytotic response was frequent (in
more than 10 cells). The time course of [Ca21]i changes
was calculated from the fluorescence images (Fig. 10).
The high [Ca21]i level was maintained for about 30 s and
then gradually decreased to the basal level (Fig. 10). The
rising phase of the [Ca21]i response occurred simultaneously with the initiation of phagosome formation but
not with that of exocytosis. In a neutrophil shown in
Figures 9 and 10, 37 exocytosis were observed in the DIC
image at the late stage of the [Ca21]i response which was
already gradually decreasing and subsiding (Fig. 10). On
the contrary, a control cell which did not show phagocytosis maintained almost the same [Ca21]i level (Fig. 10).
DISCUSSION
Phagocytosis
Many studies have already shown phase-contrast
and fluorescence pictures of neutrophils undergoing
Fig. 6. Locational relationship between the initial attachment site to
a zymosan and the site of the first exocytosis occurrence during
the phagocytosis in a neutrophil. The first exocytosis in 15 neutrophils was traced (open circles) after aligning both the attachment site
on the pseudopodium (the hand structure) and the position of zymosan.
Phagocytosis-Exocytosis Sequence in Neutrophils
Fig. 7. Localization of exocytosis during phagocytosis of a neutrophil. Top: Site of exocytosis in reference to the location of zymosan
traced sequentially from the video images. Small circles indicate the
site where each exocytosis was observed. Thirty-four exocytotic
responses were observed in this example. Every 10 responses are
sequentially grouped depending on their relative time of response (in
phagocytosis [Hirsch, 1962; Pryzwansky et al., 1979;
Sawyer et al., 1985; Jaconi et al., 1990]. These pictures
have been widely accepted as the images of particle
ingestion. It was demonstrated by these pictures that a
neutrophil extended pseudopodia around a particle [Pry-
223
seconds), and are represented by different symbols. Bottom: Distance of the responding site measured from the site of the first
exocytosis. The ordinate represents the order of responses, and the
abscissa represents the distance with directions (right and left in
reference with the longitudinal axis of neutrophil).
zwansky et al., 1979], and that the particle was isolated
from outer medium and was engulfed within a neutrophil
[Jaconi et al., 1990]. Our DIC and fluorescence images
were fully consistent with these observations. By changing the focal plane up and down, it was confirmed that the
224
Suzaki et al.
Fig. 8. Luminol-dependent chemiluminescence observed in neutrophils during phagocytosis of zymosans.
a: Brightfield image of neutrophils. Zymosans ingested by neutrophils are indicated by arrows, and a
lymphocyte by an asterisk. b: Photon counting image of the same neutrophils immersed in luminolcontaining solution. c: Brightfield image and photon counting image superimposed. White dots represent
detection of single photons derived from active oxidants. The brightness of the dots is proportional to the
number of photons detected.
cytoplasm of a neutrophil was present all around a
zymosan. In the fluorescence image, the quinacrineloaded granules surrounded the dark zymosan area.
Therefore, we conclude that the observed images really
reflect the ingestion of particles by phagocytosis as
shown by the early studies.
The video (VEC-DIC) microscopy allowed us to
observe exocytotic events associated with phagocytosis
at the highest possible magnification at a time resolution
of the video rate (33 ms in NTSC format). By using this
technique, we have succeeded in visualizing ‘‘popping’’
responses of the granules, and concluded that these
popping responses are exocytosis in the neutrophil (in the
sense that granule contents are released transmembranously to a large fluid space) because of the following: (1)
the responses involved decrease in refractive index, and
thus decrease in density inside the granule (Figs. 1 and 4);
(2) they were associated with a loss of dye from the
granules as evidenced by the fluorescence microscopy
(Fig. 3); (3) they were clearly distinguishable from the
translocational movement of granules in neutrophils
(Kawai et al., submitted for publication); (4) they were
extremely rapid in comparison to the phagocytotic movement (Fig. 4); (5) after such responses, granules were not
recovered by readjustment of the microscope focus
(indicating degranulation); (6) they were induced only
when phagocytosis took place and were never observed
without stimulation; (7) they were similar to responses
observed in neutrophils stimulated with concanavalin A
and cytochalasin E (our unpublished observation); and
(8) they were similar to exocytotic responses found in
other secretory cells, namely chromaffin cells [Terakawa
et al., 1991, 1993, 1995b], pancreatic b-cells [Sakurai and
Terakawa, 1995], colonic goblet cells [Terakawa and
Suzuki, 1991], salivary acinar cells [Segawa et al., 1991],
nasal epithelial goblet cells [Kamijo et al., 1993], and
gastric mucosal cells [Terakawa et al., 1995a]. Our
technique therefore attained a spatiotemporal resolution
high enough to study exocytosis associated with the phagocytosis, and thus provided an insight into the signaling
pathway for such exocytosis in the neutrophil. Spatiotemporal observations described in the Results suggest that
attachment of the cell membrane to invading organisms
may send some signals for initiation of exocytosis.
Efficient Sequence of the Granule Fusion
A previous study showed that the two types of
granules in neutrophils discharge sequentially, specific
granules fusing with the phagosome before azurophils
[Bainton, 1973]. Others reported that they fuse at random
[Zucker-Franklin and Hirsch, 1964; Pryzwansky et al.,
1979]. As the sequence of granule discharge roughly
parallels the change in pH, thereby providing optimal
conditions for coordinated activity of granule contents,
the former view is attractive to have been widely
accepted. However, both views have not yet been evidenced substantially. In our observation, both relatively
small and relatively large granules seemed to undergo
exocytosis randomly. Although we could not distinguish
specific granules from azurophilic granules precisely in
the DIC image, a wide range of the granule size suggests
random exocytotic sequence of the two classes of gran-
Phagocytosis-Exocytosis Sequence in Neutrophils
225
Fig. 10. Time courses of [Ca21]i transient in neutrophils with (filled
circle) and [Ca21]i without (open circle) phagocytosis. The ordinate
represents fluorescence ratio (I360 nm/I380 nm), and the abscissa experimental time with the origin at the moment of pseuodpodial extension. Data
shown with filled circles were measured from the whole area of the
fura-2-loaded neutrophil shown in Figure 9. The phagosome was
formed during the period indicated by the open bar, and exocytosis was
observed during the period indicated by the filled bar. From the data
obtained by ordinary fluorescence ratiometry without DIC imaging, the
resting level of [Ca21]i was estimated to be 270 nM, and the peak value
of [Ca21]i to be 600 nM.
Fig. 9. Sequential video micrographs of a neutrophil showing its shape
and [Ca21]i transient during phagocytosis of a zymosan observed by the
simultaneous imaging system. a–f; DIC images (upper) and coincidental fluorescence ratio (I360 nm/I380 nm) images (lower) observed after
loading the neutrophil with fura-2. The brightness in the ratio images
indicates an increase in [Ca21]i. Pseudopodia started extending at time
zero. Images were taken at 5 s (a), 25 s (b), 45 s (c), 55 s (d), 75 s (e),
and 85 s (f) after the pseudopodial extension. The arrow in d indicates
the site of fusion of extended pseudopodia. Exocytotic responses were
observed in the period including d and e.
ules. Even random exocytosis may be able to achieve
adequate pH conditions for each granule content to
function, as mature neutrophils contain much smaller
population of azurophils than specific granules.
It should be emphasized that a considerable lag
period exists after attachment of a zymosan and before
actual initiation of exocytosis. This lag period, commonly
found in phagosome-lysosome fusion in macrophages
[Zimmerli et al., 1996, see below], is probably very
important in phagocytosis. Since a neutrophil attaches to
a zymosan at any part of the cell membrane during
random movements, it probably reorients cytosolic organelles including granules and the nucleus to positions most
proper for the defense reaction. Exocytosis, occurring
with this latency, continues only for the late 38-s period
during the whole phagocytosis process that lasts for 97 s
(Fig. 2). Oxygen radicals are released to phagosome
during this period (Fig. 8); see also Suzuki et al. [1993].
The first exocytosis takes place when the zymosan is
engulfed by about 70% into the phagosome. These are
very effective tactics for the bactericidal action, because
the secretions from the granules can challenge the foreign
object at the earliest timing without decreasing their
concentration in a narrow space between the phagosomal
membrane and the object. The long lag time makes this
possible. Although the first exocytosis occurs before a
complete closure of the phagosome, this exocytosis
seems to be properly regulated to occur at an optimal time
in a localized region close to the bottom of phagosome
where the possibility for the secretions to leak outside
(i.e., regurgitation) is minimum.
Spatial Regulation
In addition to the temporal regulation, exocytosis
was also regulated spatially. The first exocytosis appears
to be restricted to the place where a neutrophil first
recognized the foreign object (Fig. 6). Furthermore, only
the granules located at the periphery of a phagosome
proceed to exocytosis. The direction of secretion is also
restricted. The photon-counting imaging provides evidence that the secretions are simultaneous with release of
active oxidants, and suggests that they are released into
the phagosomal space (Fig. 8). These exocytotic responses are different from those induced by agonists such
226
Suzaki et al.
as cytochalasin B [Zurier et al., 1973], formyl-methionylleucyl-phenylalanine (fMLP) [Lew et al., 1987], and
leukotriene B4 (LTB4) [Showell et al., 1982; Lew et al.,
1987] in the sense that secretions never took place on the
plasma membrane but on the phagosomal membrane in spite
of the identical property of the both membranes. When two or
more zymosans were ingested sequentially at different parts
of a neutrophil, exocytotic responses appeared in groups
separated spatially and temporally. Thus, a highly organized regulatory mechanism is underlying the initiation
of this class of exocytosis. Probably, immune receptors or
some adhesion molecules on ingesting pseudopodia [Griffin et al., 1976] recognize the foreign object in an ordered
manner, and play a key role in sending internal signals for
exocytosis with a site-dependent proper delay time.
Although there are many granules around a phagosome,
the location of granules which undergo actual exocytosis
is limited to the peri-phagosomal region closer to the cell
body and the nucleus. These are also advantageous for the
effective bactericidal action.
Nature of Granule Fusion
During phagocytosis, a series of exocytotic responses is limited only to the phagosomal membrane and
no additional exocytotic response occurs at the other part
of the cell membrane. Some of these responses occurring
in the early stage of phagosome formation are indeed
exocytosis as the phagosomal space is still open to the
extracellular space and granule contents may be regurgitated to extracellular medium (Fig. 11). However, the
same responses occurring in the later phase are not
exocytosis but rather lysosomal fusion with the phagosome, because its membrane is completely closed within
the cell. Since it is not clear whether or not the content of
granules involved in these responses is really lysozyme,
we propose to name these responses, simply based on
morphological observation, as ‘‘encytosis’’ (from Greek
en meaning inward). This change in the nature of the
granule fusion, well-defined by the present technique, is
very characteristic of the membrane fusion during phagocytosis in neutrophils.
Signal for Exocytosis
The time difference between the initiation of phagosome formation and of exocytosis was 25.0 s on the
average. This value corresponds well with the time
difference between the completion of phagosome formation and that of exocytosis which averaged out to 26.7 s.
In addition, the localization where exocytosis occurs
diffuses more distal from the site of the first exocytotic
occurrence as the pseudopodial extension proceeds. These
facts suggest that the formation of phagosome is closely
related to the exocytosis occurrence, and that about 25 s
are needed to cause the activations of key molecules for
the membrane fusion after receiving the stimulus. These
findings also suggest that the attachment to a zymosan
itself is not an immediate cause of exocytosis, but
chemical reactions are involved before directly triggering
exocytosis in a localized area. As far as this final step of
stimulation-secretion sequence in neutrophils is concerned, the temporal and spatial coordination of exocytotic events should be taken into consideration in order to
understand their molecular mechanism.
Degranulation in response to activation of receptors
for chemoattractants such as fMLP and LTB4 has been
shown to be a very sensitive [Ca21]i-dependent process in
human neutrophils [Lew et al., 1987]. On the contrary,
recent studies on a wide range of secretory cell types
indicate that [Ca21]i-independent exocytosis also occurs.
Especially in those related to phagosome-lysosome fusion, the fusion between granules and the plasma membrane is mediated by a multifactorial signaling system
involving [Ca21]i increases, protein kinase C activation
[Di Virgilio et al., 1984], and GTP binding to its
regulatory proteins [Barrowman et al., 1986]. What is the
signaling system for exocytosis on the phagosomal
membrane? [Ca21]i elevation was shown to be a necessary and sensitive signal that triggers the phagosomegranule fusion event [Jaconi et al., 1990], and shown to be
a localized response in the cytosol surrounding the
phagosome during phagocytosis [Sawyer et al., 1985].
However, we found that [Ca21]i increases in the whole
cytoplasm and the initiation of [Ca21]i increase corresponds well with the timing of the pseudopodial extension, which agrees well with the finding by Theler et al.
[1995]. Our findings indicate that [Ca21]i elevation is
closely related to the cytoskeletal activation for phagosome formation [Downey et al., 1990], but not to the
machinery determining the timing and localization of the
exocytosis. It is noteworthy here that phagosomelysosome fusion in macrophages is [Ca21]i-independent
[Zimmerli et al., 1996]. Since [Ca21]i is not completely
decreased to the basal level during the exocytosis, it may
be necessary as an environmental factor to cause phagosome-granule fusion, but not as an immediate regulatory
factor leading to exocytosis. There must be more closely
related molecules for the phagosome-granule fusion
event other than [Ca21]i.
We infer that attachment of the membrane to
foreign objects activates some membrane receptors, giving rise to a molecular signal immediately beneath the
membrane. Opsonized zymosans bear IgG and C3b
complements on their cell wall. Neutrophils possess
membrane receptors for Fc portion of immunoglobulin
and for C3b complements. During phagocytosis, therefore, a zymosan is gradually wrapped in a neutrophil by a
zip-up mechanism based on binding of these ligands to
receptors. It is highly possible that the membrane recep-
Phagocytosis-Exocytosis Sequence in Neutrophils
227
Fig. 11. Phagocytosis-exocytosis sequence in the neutrophils. A neutrophil (red) is activated by attachment to a zymosan (green). It engulfs the
zymosan by extending pseudopodia. Granules translocate near the zymosan (white arrows). The first exocytotic response (red granule) occurs
at the bottom of phagosome when pseudopodia are still extending (black arrows) and the phagosomal space is open to the outside. This
response as well as other few responses (yellow granules) may cause secretion to the environment by regurgitation (dashed red arrow). The
following exocytotic responses occur in order of numbers indicated. Some granules (blue ones) fuse with the phagosomal membrane after
complete closure of the phagosome. Thus, this response should be referred to as lysosomal fusion or encytosis (see text), although the
molecular process must be the same as the one underlying the first exocytosis.
228
Suzaki et al.
tors of neutrophils are thus activated sequentially, causing
a sequential exocytosis along the phagosomal membrane.
ACKNOWLEDGMENTS
E.S. is a recipient of a JSPS Fellowship for Japanese
Junior Scientists. This work was partly supported by
Grant-in-Aid for Scientific Research from the Ministry of
Education, Science and Culture, Japan.
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