(Journal of Leukocyte Biology. 2000;68:650-654.)
© 2000
by Society for Leukocyte Biology
Clustering on the forward surfaces of migrating neutrophils of a novel GPI-anchored protein that may regulate neutrophil adherence and migration
Yukiko Nakamura-Sato*,
Katsunori Sasaki
,
Hiroshi Watanabe
,
Yoshihiko Araki* and
Fujiro Sendo*
* Department of Immunology and Parasitology, Yamagata University, School of Medicine;
Department of Anatomy, Shinshu University, School of Medicine, Matsumoto; and
Department of Nursing, Yamagata University, School of Medicine, Yamagata, Japan
Correspondence: Fujiro Sendo, Department of Immunology and Parasitology, Yamagata University, School of Medicine, 2-2-2, Iida-Nishi, Yamagata 990-9585, Japan. E-mail: fsendo{at}med.id.yamagata-u.ac.jp
 |
ABSTRACT
|
|---|
We previously reported a novel glycosylphosphatidylinositol
(GPI)-anchored glycoprotein (tentatively designated GPI-80) on human
leukocytes that may be involved in the regulation of neutrophil
adherence and migration. In this study, we examined by immuno-
and scanning electron microscopy, the distribution of GPI-80 on
neutrophil surfaces. GPI-80 was diffusely distributed on the surface of
resting neutrophils and on the peripheral areas of adherent cells after
stimulation with N-formyl-methionyl-leucyl-phenylalanine.
After longer stimulation (60 min), GPI-80 decreased in number and was
again diffusely distributed on the surfaces of round neutrophils. Few
GPI-80 were detected on the ventral surfaces of adherent neutrophils.
Clusters of GPI-80 were detected on the forward surfaces of neutrophils
transmigrating through pores of nitrocellulose membranes. These results
may give a morphological background of possible role of GPI-80 for
neutrophil extravasation.
Key Words: integrin • electron microscopy • localization
|
INTRODUCTION
|
|---|
Extravasation of peripheral blood leukocytes is an essential
process that enables the cells to function in inflammatory and immune
responses. However, little is known about the mechanisms that involve
rapid cycles of leukocyte adhesion to and de-adhesion from the
endothelium concurrent with their locomotion through the endothelium.
To study this problem, we developed a mAb, designated 3H9, by screening
for inhibition of human neutrophil adherence to plastic plates
[1
]. This mAb enhances early phase ß2
integrin-dependent neutrophil adherence and inhibits the late-phase
responses, and also has similar effects on in vitro
transendothelial migration of neutrophils [2
].
Furthermore, in the phagokinetic track assay, 3H9 induces neutrophil
locomotion [3
]. We performed molecular cloning of the
molecule reacting with 3H9 and showed that it is a novel
glycosylphosphatidylinositol (GPI)-anchored glycoprotein (tentatively
designated GPI-80) that may modulate ß2
integrin-dependent neutrophil adhesion and transendothelial migration
[2
].
It is interesting that GPI-80 is highly homologous with Vanin-1, which
has been reported to be localized in perivascular tissue of the mouse
thymus and to be involved in prethymic cell homing to the thymus
[4
]. Both GPI-80 and Vanin-1 have approximately 40%
homology with human biotinidase [5
], suggesting that
there is a superfamily of biotinidases that may be involved in
leukocyte trafficking.
In the previous study using a confocal fluorescence microscopy, we
found that (1) GPI-80 was localized on pseudopodia and adherent
portions of polarized neutrophils, (2) that it was associated with
CD11b/CD18 in resting neutrophils and moved to lamellipodia upon
activation of these cells while CD11b/CD18 became localized on uropods,
and (3) that this dissociation is dependent on extracellular calcium
[Sendo, unpublished results].
In this study, we tried to obtain morphological information relating
our previous experimental results on GPI-80 and we used backscatter
electron images obtained by high-resolution, field emission scanning
electron microscopy (FESEM). We examined the topographical distribution
of GPI-80 on the surface of human neutrophils.
|
MATERIALS AND METHODS
|
|---|
Culture media and reagents
Cells were incubated in RPMI 1640 medium (GIBCO, Grand Island,
NY) supplemented with 10% heat-inactivated fetal bovine serum (FBS;
ICN Biomedicals, Osaka, Japan). The reagents used were purchased from
the following companies:
N-formyl-methionyl-leucyl-phenylalanine (fMLP) and
fibrinogen, Sigma Chemical, St. Louis, MO; Ficoll-Paque, Pharmacia Fine
Chemicals, Uppsala, Sweden; heparin sodium injection-N, Shimizu
Pharmaceutical, Shimizu, Japan; Dextran 200, ethanol, butyl alcohol,
WAKO Pure Chemical, Osaka, Japan; sodium cacodylate, TAAB, Berks, UK;
and glutaraldehyde, Nissin EM, Tokyo, Japan. University of Wisconsin
(UW) solution (100 mM lactobionate, 30 mM raffinose, 25 mM
KH2PO4, 5 mM MgSO4, 3 mM
glutathione, 5 mM adenosine, 1 mM allopurinol, 105 mM KOH, 20 mM NaOH),
which had been introduced in organ preservation for transplantation in
1986 [6
], was used in the preparation of tissues for
electron microscopy to suppress swelling and bleb formation of cells
[7
, 8
]. All other reagents were of the
highest grade commercially available.
Antibodies
Our previously described anti-GPI-80 monoclonal antibody (mAb),
3H9 (IgG1) [1
], was used in this study. A mAb, TCY-3,
against Trypanosoma cruzi antigen, which does not
cross-react with human neutrophils was used as an IgG1 control mAb. In
the first experiments shown in Figure 1
, we used 15-nm colloidal gold particles conjugated with sheep
anti-mouse IgG purchased from E-Y Laboratories (SanMateo, CA). In later
experiments we used 10-nm particles (Funakoshi, Tokyo, Japan) because
we found that labeling with 10-nm colloidal gold particles was more
efficient than with 15-nm ones.
View larger version (118K):
[in this window]
[in a new window]
|
Figure 1. Localization of GPI-80 on the apical cell surface. (A–C) low
magnification of neutrophils observed by BSE imaging. Immediately after
stimulation by fMLP, neutrophils had a round shape and random
distribution of GPI-80 on the cell surface (A, 0 min). In the early
phase of fMLP activation, cells displayed spreading margins on the
substratum, and GPI-80 accumulation on their peripheral surfaces
(arrows, B, 15 min). At 90 min of incubation, cells had a round shape
with few projections on their surfaces, and the distribution of GPI-80
was diffuse and decreased in number (C, 90 min). (D–I) At high
magnification, GPI-80 was distributed diffusely in some parts of the
specimen (E), but in other parts in a clustered form (G and I,
arrowheads). (D, F, and H) SE images. (E, G, and I) backscatter images
of D, F, and H, respectively. Bars, 1.0 µm (A–C), 0.3 µm (D and
E), and 0.1 µm (F–I).
|
|
Isolation of human neutrophils
Heparinized venous blood obtained from healthy volunteers was
mixed with one-quarter volume of phosphate-buffered saline (PBS; pH
7.4) containing 3% Dextran 200 and allowed to stand at room
temperature for approximately 60 min for erythrocytes to sediment. The
leukocyte-rich supernatant was centrifuged at 400 g for 5
min at room temperature, the pellet was washed with PBS, and
centrifuged on Ficoll-Paque density gradient at 400 g for 30
min at room temperature. The granulocyte-rich erythrocyte layer was
treated with hypotonic shock and centrifuged at 300 g for 5
min. The pellet was collected and used as a source of neutrophils. It
showed more than 96% purity by Giemsa staining.
Electron microscopy
For scanning electron microscopy (SEM) to examine the
immunolocalization of GPI-80 on the apical surface of neutrophils,
cells were seeded on glass coverslips in a 48-well flat-bottom culture
plate (Falcon). For the observation of the ventral surface, cells were
seeded onto a combination of two attached membranes. The upper ones
were 10 µm thick PC MEMB 110615 membranes having multiple holes 10
µm in diameter (Nomura Micro-Science, Kanagawa, Japan) and the lower
ones (PC MEMB 110606) had no holes. The lower membrane was incubated
with 0.2% fibrinogen in the plate (Falcon) to coat the surface with
fibrinogen at 37°C for 2 h. After washing three times with PBS,
the upper membrane was overlaid on the treated lower membrane, and they
were again treated with fibrinogen at 37°C for 2 h and then
washed with PBS. After addition of 2 x 106/mL
neutrophils, the plates were centrifuged (200 g) for 3 min
at 4°C and incubated at 37°C for 15 min in 5% CO2 in
air. We chose 15 min of incubation because many neutrophils adhered at
this time. After the incubation, the two membranes were separated and
the upper one was put upside down. Both membranes were washed twice
with UW solution and incubated with 1% FBS/UW for 5 min at room
temperature. We used the Boyden chamber technique [9
],
as modified by Zigmond and Hirsch [10
], to observe the
ventral surface of migrating neutrophils. The two compartments of the
chamber (Nomura Micro-Science) were separated by a cellulose acetate
filter with a pore size of 8 µm (Poretics, Livermore, CA).
Neutrophils (2 x 106/mL, 200 µL) in the upper
chamber were incubated for 15 min at 37°C, with 10-6 M
fMLP in the lower compartment, and then the filters were stripped off.
After blocking, the samples were incubated with 100 µL of 3H9 (10
µg/mL) for 30 min at 4°C and were then washed twice with UW
solution. They were labeled with immunogold by incubation at 4°C with
colloidal gold conjugated with goat anti-mouse IgG (diluted 1:10 in UW
solution) for 30 min and were then washed twice with UW solution. Then
the specimens were treated with 2.5% glutaraldehyde containing sodium
cacodylate buffer (pH 7.4, 0.1 M), rinsed several times with the same
buffer, and treated with 1% osmium tetroxide in the same buffer. They
were dehydrated in graded ethanol, immersed in isoamylacetate, critical
point-dried with CO2, and coated with carbon using a CC-40F
carbon coater (Meiwa Shouji, Tokyo, Japan). The cells were observed in
FESEM by high-resolution backscatter electron (BSE) imaging using a YAG
detector as well as secondary electron (SE) imaging. To quantify the
number of gold particles, we counted them on five cells in randomly
chosen 15 squares (2.25 cm2) using 8,000-fold magnified
photographs.
|
RESULTS
|
|---|
Distribution of GPI-80 on the surfaces of fMLP-stimulated
neutrophils incubated in the culture plates
Apical surfaces
Very early after fMLP stimulation (1 min), which induces
neutrophil activation, cells showing a spherical shape bore small
numbers of short protrusions. At low magnification, GPI-80 was
diffusely distributed on the surfaces of the cells (Fig. 1A
). With further incubation (15 min), many long protrusions
appeared on the apical surfaces with increased spreading. GPI-80 was
mainly detected on the peripheral surfaces of spreading cells (Fig. 1B)
. The amount of GPI-80 on apical surfaces at 1 and 15 min did not
differ significantly. At approximately 90 min of incubation, cells
changed to a round shape with decreasing numbers of protrusions and
GPI-80 was again distributed diffusely (Fig. 1C)
. When we counted the
number of gold particles as described in Materials and Methods, at 90
min of incubation GPI-80 was significantly reduced (140 ±
9.0/2.25 cm2 in 8,000-fold magnification) compared to the
very early stage of fMLP stimulation (412 ±
11.4/2.25-cm2 in 8,000-fold magnification).
When the distribution of GPI-80 on the surfaces was determined more
precisely with high magnification, in some parts of the specimen (Fig. 1E)
GPI-80 was distributed diffusely, but in other parts in a clustered
form (Fig. 1G
and 1I)
.
Ventral surfaces
We examined the distribution of GPI-80 on the ventral surfaces of
cells adhering to the lower membranes after stripping off the upper
membranes as described in Materials and Methods. Many flattened large
projections were observed in some parts of the specimens, suggesting
that these areas are the ventral surfaces that adhered to the lower
membranes (Fig. 2A
). On the adhering surfaces very few GPI-80 were detected (Fig. 2B
and 2C)
. The reason for the scanty number of GPI-80 on these surfaces
could not be ascribed to being ripped off when the two membranes were
separated because no GPI-80 was detected on the surfaces of the lower
membranes after being stripped from the upper membranes, and numerous
CD18 were detected on the ventral surfaces of adhering cells (data not
shown). On the other hand, GPI-80 in relatively small number was
diffusely detected on the surfaces of small round projections, which
may not have adhered to the lower membranes (Fig. 2D
and 2E) .
View larger version (77K):
[in this window]
[in a new window]
|
Figure 2. Localization of GPI-80 on the ventral cell surface. A typical ventral
surface is shown in A. (B and D) higher magnifications of framed areas
in A. (B and C) few GPI-80 were detected on the flattened surfaces of
large projections, suggesting a tight cell-substratum contact area of
the neutrophil (arrowheads). (D and E) on the small round projections,
relatively small numbers of GPI-80 were detected (arrowheads). (A, B,
and D) SE images. (C and E) BSE images of B and D, respectively. Bars,
1.0 µm (A), and 0.2 µm (B–E).
|
|
Clustered distribution of GPI-80 on the forward surfaces of
neutrophils transmigrating through a Boyden chamber
We next examined the distribution of GPI-80 on the surfaces of
neutrophils that passed through holes of a Boyden chamber. Many GPI-80
were detected on the forward surfaces of the migrating portions of
neutrophils, resulting in a clustered distribution of GPI-80 on the
migrating portions under low magnification (Fig. 3A
, D-G
, and I-L
). On the other
hand, few GPI-80 were detected on the surfaces of the adherent portions
of neutrophils still remaining in the upper chambers (Fig. 3B
and 3C)
. At higher magnification, many clustered GPI-80 were
detected on the forward surfaces of transmigrating neutrophils (Fig. 3E
3G 3J
and 3L)
.
View larger version (114K):
[in this window]
[in a new window]
|
Figure 3. Clustered distribution of GPI-80 on the forward surfaces of migrating
neutrophils. (A–G) features of a migrating neutrophil observed from
the apical sides of the upper chamber. (A) a complete picture of a
migrating neutrophil. (B and D) higher magnifications of framed areas
in A. Few GPI-80 are observed on the surface of the adherent portions
in B and C. (E) BSE imaging of D. Clusters of GPI-80 are observable
(arrowheads). (F) higher magnifications of framed areas in D. (G) BSE
imaging of F. Clusters of GPI-80 are observed on the forward surfaces
of a migrating neutrophil (arrowheads). (H–L) features of a migrating
neutrophil observed from the ventral sides of the lower chamber. (H) a
tip of a migrating neutrophil. (I and K) higher magnifications of
framed areas in H and I, respectively. J and L are BSE imaging of I and
K, respectively. Clusters of GPI-80 are observed on the forward
surfaces of a migrating neutrophil (arrowheads). A, B, D, F, H, I, and
K are SE images. C, E, G, J, and L are BSE images of B, D, F, I, and K,
respectively. Bars, 10 µm (A), 0.3 µm (B, C, D, E, H, I, and J),
and 0.1 µm (F, G, K, and L).
|
|
|
DISCUSSION
|
|---|
In this study we have demonstrated the expression of a
novel GPI-anchored protein on the surfaces of human neutrophils,
tentatively designated GPI-80, that may regulate ß2
integrin-mediated adhesion and transendothelial migration of human
leukocytes [2
]. The most characteristic feature of our
findings is the clustered expression of this GPI-anchored protein on
the forward surfaces of migrating neutrophils (Fig. 3)
. Because
the number of gold particles on the surfaces of the adherent portions
of neutrophils that remained in the upper chamber was reduced compared
with that observed on the apical surfaces of fMLP-stimulated adhering
neutrophils after 15 min of incubation (Fig. 1B)
, GPI-80 may form
clusters on the surfaces of migrating neutrophils, although it is still
not clear whether or not the total amount of GPI-80 increased during
migration. This result indirectly suggests that this GPI-anchored
protein is involved in migration of neutrophils, although its actual
role in leukocyte migration is still unknown. In our literature survey,
we found no electron microscope studies showing that cell adhesion
molecules or their regulating molecules, such as integrins or the
urokinase-type plasminogen activator receptor (uPAR), that may be
involved in leukocyte migration, are localized on the surfaces of
migrating leukocytes in a manner such as shown in the present study.
Therefore, at present we are unable to discuss our result that the
GPI-80 is clustered on the forward surfaces of migrating neutrophils in
terms of the relationships between GPI-80 and integrins or the uPAR.
However, it has been well documented that changes in expression or new
epitope appearance of integrins on leukocytes occurs depending on their
activation state [11
12
13
]. Furthermore, localization of
GPI-anchored proteins such as uPAR and lipopolysaccharide (LPS)/LPS
binding protein receptor (CD14) on leukocyte surfaces that may be
involved in regulation of leukocyte migration changes with activation
of the cells [14
]. With this in mind, it seems highly
possible that molecules involved in leukocyte migration may cluster on
the forward surfaces of migrating leukocytes as shown in the present
study of GPI-80, or they disappear from these areas. Further
immuno-electron microscopy studies on the localization of other
molecules involved in leukocyte migration may clarify the significance
of our results on the characteristic localization of GPI-80.
Sparse distribution of GPI-80 on ventral surfaces adhering to ligands
on the membranes was an unexpected result, since in a previous study we
demonstrated that 3H9 (a mAb to GPI-80) modulates ligand binding
avidity of ß2 integrin, which should be abundantly
localized on the surfaces of leukocytes adhering to ligands. Sparse
distribution of GPI-80 on the ventral adhering surfaces and clustering
one on the forward surfaces of migrating neutrophils may suggest that
GPI-80 is not actually involved in modulation of cell adherence but
rather only of cell locomotion. However, we cannot be sure of this,
because the experimental procedure we used to observe GPI-80 expression
on ventral surfaces in the present study is not the ideal condition for
this purpose. As described in Materials and Methods, the upper
membranes on which neutrophils were placed were 10 µm thick. Under
this condition, we actually investigated the ventral surfaces of
neutrophils that migrated through the hole of the upper membranes in
distances of at least 10 µm, but not those of neutrophils adhering to
the surface of the upper membranes. To this end, we will need revised
techniques.
In conclusion, the present result showing a clustered distribution of
GPI-80 on the forward surfaces of migrating neutrophils may suggest not
only a role for this novel GPI-anchored protein in leukocyte migration
but also a certain role for GPI-anchored proteins in general. Molecules
in this group are easily able to form clusters because of their high
lateral mobility when such changes are required for certain biological
activities of cell membranes.
Received August 10, 1999;
revised June 22, 2000;
accepted June 23, 2000.
|
REFERENCES
|
|---|
-
Ohtake, K., Takei, H., Watanabe, T., Sato, Y., Yamashita, T., Sudo, K., Kuroki, M., Chihara, J., Sendo, F. (1997) A monoclonal antibody modulates neutrophil adherence while enhancing cell motility Microbiol. Immunol. 41,67-72[Medline]
-
Suzuki, K., Watanabe, T., Sakurai, S., Ohtake, K., Kinoshita, T., Araki, A., Fujita, T., Takei, H., Takeda, Y., Sato, Y., Yamashita, T., Araki, Y., Sendo, F. (1999) Regulation of neutrophil extravasation by a novel glycosylphosphatidyl inositol (GPI)-anchored protein on leukocytes J. Immunol. 162,4277-4284[Abstract/Free Full Text]
-
Suzuki, H., Takei, H., Ohtake, K., Watanabe, T., Sendo, F. (1997) External calcium-dependent, F-actin-independent and pertussis toxin-insensitive novel neutrophil locomotion induced by a mAb Int. Immunol. 9,763-769[Abstract/Free Full Text]
-
Aurrand-Lions, M., Galland, F., Bazin, H., Zakharyev, V. M., Imhof, B. A., Naquet, P. (1996) Vanin-1, a novel GPI-linked perivascular molecule involved in thymus homing Immunity 5,391-405[Medline]
-
Cole, H., Reynolds, T. R., Lockyer, J. M., Buck, G. A., Denson, T., Spence, J. E., Hymes, J., Wolf, B. (1994) Human serum biotinidase: cDNA cloning, sequence, and characterization J. Biol. Chem. 269,6566-6570[Abstract/Free Full Text]
-
Wahlberg, J., Southard, J. H., Belzer, F. O. (1986) Development of a cold storage solution for pancreas preservation Cryobiology 23,477-482[Medline]
-
Okouch, Y., Sasaki, K., Tamaki, T. (1994) Ultrastructural changes in hepatocytes, sinusoidal endothelial cells and macrophages in hypothermic preservation of the rat liver with University of Wisconsin solution Virchows Arch 422,477-484
-
Sasaki, K., Okouchi, Y., Pabst, R., Rothkotter, H. J. (1993) Three dimensional detection of the expression of intercellular adhesion molecule-1 (ICAM-1) in the high endothelial venule (HEV) of the rat lymph node Microsc. Res. Tech. 25,264-265[Medline]
-
Boyden, S. V. (1962) The chemotactic effect of mixtures of antibody and antigen on polymorphonuclear leukocytes J. Exp. Med. 115,453[Abstract]
-
Zigmond, S. H., Hirsch, J. G. (1973) Leukocyte locomotion and chemotaxis: new methods for evaluation and demonstration of a cell-derived chemotactic factor J. Exp. Med. 137,387-410[Abstract]
-
Richard, O. H. (1992) Integrins: versatility, modulation, and signaling in cell adhesion [Review] Cell 69,11-25[Medline]
-
Diamond, M. S., Springer, T. A. (1994) The dynamic regulation of integrin adhesiveness [Review] Curr. Biol. 4,506-517[Medline]
-
Yamada, K. M., Miyamoto, S. (1995) Integrin transmembrane signaling and cytoskeletal control Curr. Opin. Cell Biol. 7,681-689[Medline]
-
Petty, H. R., Todd, R. F., III (1996) Integrins as promiscuous signal transduction devices Immunol. Today 17,209[Medline]
This article has been cited by other articles:
|
|
|
|
 
C. J. G. de Almeida, L. B. Chiarini, J. P. da Silva, P. M. R. e Silva, M. A. Martins, and R. Linden
The cellular prion protein modulates phagocytosis and inflammatory response
J. Leukoc. Biol.,
February 1, 2005;
77(2):
238 - 246.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Funaro, E. Ortolan, B. Ferranti, L. Gargiulo, R. Notaro, L. Luzzatto, and F. Malavasi
CD157 is an important mediator of neutrophil adhesion and migration
Blood,
December 15, 2004;
104(13):
4269 - 4278.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. Yoshitake, Y. Takeda, T. Nitto, and F. Sendo
Cross-linking of GPI-80, a possible regulatory molecule of cell adhesion, induces up-regulation of CD11b/CD18 expression on neutrophil surfaces and shedding of L-selectin
J. Leukoc. Biol.,
February 1, 2002;
71(2):
205 - 211.
[Abstract]
[Full Text]
[PDF]
|
|
|
TOP
ABSTRACT
INTRODUCTION
