(Journal of Leukocyte Biology. 2002;71:205-211.)
© 2002
by Society for Leukocyte Biology
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
Hiroshi Yoshitake,
Yuji Takeda,
Takeaki Nitto and
Fujiro Sendo
Department of Immunology and Parasitology, 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
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ABSTRACT
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Previously, we described a novel glycosylphosphatidyl inositol
(GPI)-anchored glycoprotein (designated GPI-80) on human neutrophils
and monocytes that may regulate ß2 integrin-dependent
neutrophil adherence and migration. However, the mechanism regulating
ß2 integrin remains to be clarified. To study this, we
examined changes in ß2 integrin expression and function
caused by cross-linking GPI-80. GPI-80 cross-linking induced
up-regulation of CD11b/CD18 (Mac-1) expression on neutrophil surfaces
and shedding of L-selectin, which depends on tyrosine phosphorylation
and cytoskeleton remodeling. Furthermore, the cross-linking enhanced
fMLP-induced human neutrophil adherence. These results suggest that
GPI-80 may be a regulator of ß2 integrin in
neutrophils.
Key Words: integrin adherence uPAR DAF
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INTRODUCTION
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Neutrophils play important roles in inflammation and the immune
response, not only through their effector mechanisms using bioactive
molecules but also through modulation of inflammation and the immune
response by cytokines that they produce. For these activities,
neutrophil extravasation is essential. In this process, neutrophils
first bind loosely to endothelial cells through interaction between
L-selectin on the neutrophils or P-selectin on the endothelial cells
and their ligands, and then the neutrophils roll on the endothelium in
the direction of the bloodstream. Next, the neutrophils attach firmly
to the endothelial cells through interaction between ß2
integrins on the neutrophils and their ligands, and then the cells pass
through the basal membrane and reach the extravascular tissues. This
outline of neutrophil extravasation has been documented in the last 10
years [1
2
]. However, the precise molecular mechanisms
involved in sequential adhesion and deadhesion of neutrophils to the
endothelium have not yet been clarified.
During our study about the mechanisms of neutrophil extravasation, we
established a monoclonal antibody (mAb), 3H9, that modulates
ß2 integrin-dependent neutrophil adhesion
[3
]. We cloned the molecule recognized by 3H9,
identified it as a novel 80-kD glycosylphosphatidyl inositol
(GPI)-anchored protein, and designated it as GPI-80 [4
].
It is well known that GPI-anchored proteins transduce biochemical
signals into the cytoplasm, but the mechanisms are still obscure
because GPI-anchored proteins do not have intracellular domains.
Conversely, clustering of GPI-anchored protein on cell surfaces induces
intracellular signaling [5
6
7
]. Previously, we
demonstrated that cross-linking GPI-80 on neutrophil surfaces by the
mAb 3H9 induces intracellular tyrosine phosphorylation
[8
].
Similar to GPI-80, GPI-anchored proteins such as urokinase-type
plasminogen-activator receptor (uPAR, CD87), Fc
RIIIb (CD16b), and
CD14, regulate the functions of ß2 integrins on
neutrophil surfaces [9
]. Cross-linking neutrophil
surface uPAR or Fc
RIIIb by their antibodies induces an increase in
the expression of CD11b on the surfaces of the cells [10
,
11
]. In this study, we examined whether cross-linking
GPI-80, which possibly regulates ß2 integrin-dependent
neutrophil adhesion, modulates the expression of Mac-1 (CD11b/CD18) or
L-selectin on neutrophil surfaces as does cross-linking other
modulators of neutrophil function, uPAR, or Fc
RIIIb.
 |
MATERIALS AND METHODS
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Reagents
Used reagents were purchased from the following companies:
Dextran 200,000 and paraformaldehyde, Wako Pure Chemical Industries,
Osaka, Japan; Ficoll-Paque, Pharmacia Biotech, Uppsala, Sweden; Hanks
balanced saline solution (HBSS), Nissui Pharmaceutical Co., Tokyo,
Japan; and N-formyl-methionyl-leucyl-phenylalanine (fMLP),
genistein, and cytochalasin B, Sigma Chemical Co., St. Louis, MO.
Antibodies
The anti-GPI-80 mAb 3H9 [mouse immunoglobulin G (IgG1)] was
established and characterized as described previously
[3
]. The F(ab')2 fragment of 3H9
was prepared by the methods of Lamoyi and Nisonoff [12
].
Fluorescein isothiocyanate (FITC)-conjugated 3H9 F(ab')2
was prepared by a modified method described by Johnson et al.
[13
]. mAb TCY-3 (mouse IgG1), specific for
Trypanosoma cruzi, was used for control studies
[14
]. CBRM1/5 mAb (mouse IgG1, specific for a
subpopulation of neutrophils CD11b) was a gift from Dr. Timothy A.
Springer (Harvard Medical School, Boston, MA) [15
].
Antibodies were purchased from the following companies: FITC-conjugated
anti-CD11b mAb and FITC-conjugated goat anti-mouse IgG Fc
region-specific antibody F(ab')2 fragment, Immunotech A
Coulter Co., Marseille, France; FITC-conjugated anti-CD55 mAb and
r-phycoerythrin (RPE)-conjugated anti-CD18 mAb, PharMingen, San Diego,
CA; RPE-conjugated anti-CD11b mAb and RPE-conjugated anti-HLA class I
mAb, DAKO, Glostrup, Denmark; RPE-conjugated anti-Leu-8 (L-selectin)
mAb, Becton Dickinson Immunocytometry Systems, San Jose, CA; and rabbit
anti-mouse IgG (H+L) antibody F(ab')2 fragment and negative
control mouse IgG F(ab')2 fragment, Jackson
ImmunoResearch Laboratories, West Grove, PA.
Preparation of human neutrophils
Heparinized venous blood from healthy volunteers was sedimented
through Dextran 200,000. The leukocyte-rich supernatant (buffy coat)
was centrifuged at 450 x g for 5 min and washed with
phosphate-buffered saline (PBS). Neutrophils were isolated from the
buffy coat using Ficoll-Paque as described previously
[16
], and residual erythrocytes were lysed by hypotonic
shock. The isolated neutrophils were resuspended in HBSS. The isolated
neutrophils were >95% pure.
Cross-linking GPI-80 or decay accelerating factor (DAF) by their
antibodies
The isolated neutrophils were treated for 30 min at 4°C with
10 µg/ml 3H9 F(ab')2 or a 1/30 dilution of anti-CD55
(DAF) mAb in HBSS and then washed twice with cold HBSS. Next, the cells
were treated for 30 min at 4°C with 20 µg/ml rabbit anti-mouse IgG
F(ab')2 in HBSS and then at 37°C for various times. After
incubation, the cells were fixed for 30 min at 4°C with 2%
paraformaldehyde in PBS and then washed twice with PBS.
Labeling cells
The fixed cells were treated for 30 min at 4°C with FITC or
RPE-conjugated anti-CD11b, RPE-conjugated anti-CD18, RPE-conjugated
Leu-8, or HLA class I antibody in PBS, after which the cells were
washed twice with PBS. The CBRM1/5 mAb, bound to a subpopulation of
CD11b, was labeled as follows. The fixed cells were treated with 30
µg/ml control mouse IgG F(ab')2 fragment for 30 min at
4°C in PBS to block remaining binding sites of rabbit anti-mouse IgG
F(ab')2 used for cross-linking. After washing twice with
PBS, the cells were incubated for 30 min at 4°C in PBS containing 3
µg/ml CBRM1/5 mAb and 1.5 µg/ml FITC-conjugated goat anti-mouse IgG
Fc region-specific Ab F(ab')2 fragment in PBS and then
washed twice with PBS.
Flow cytometry
The expression of CD11b, CD18, L-selectin, and HLA class I on
the surfaces of the cells was measured with a FACSCalibur (Becton
Dickinson Immunocytometry Systems). Dead cells and debris were excluded
from the analysis by forward- and side-scatter gating.
Confocal laser microscopy
Isolated neutrophils were treated for 30 min at 4°C with
FITC-conjugated 3H9 F(ab')2 fragment or FITC-conjugated
anti-CD55 mAb in HBSS and then washed twice with cold HBSS. The cells
were treated for 30 min at 4°C with 20 µg/ml rabbit anti-mouse IgG
F(ab')2 in HBSS and then incubated for 30 min at 37°C or
4°C. After incubation, the cells were fixed for 30 min at 4°C with
2% paraformaldehyde in PBS and then washed with PBS. The fixed cells
were seeded on Vectabond (Vector Lab, Burlingame, CA)-coated
coverslips. The specimens were mounted in PBS and examined with a
confocal laser microscope (TCS, Lica, Hiderberg, Germany).
Neutrophil adherence assay
Details of this assay have been described elsewhere
[16
]. Briefly, Falcon 3072 plates (Becton Dickinson
Labware, Franklin Lakes, NJ) were coated for 2 h at 37°C with 50
µg/ml fibrinogen and then washed with PBS. Neutrophil suspensions in
200 µl (5x105 cells/well), which had been treated with
antibodies to GPI-80, DAF, or TCY-3 and then with anti-mouse IgG mAbs
or stimulated with 1 x 10-7 M fMLP suspensions
(5x105 cells/well) in HBSS, were added to
fibrinogen-coated plates and incubated at 37°C for various times. The
percentage of adherence was calculated using the following fomula:
{% adhesion=[(OD570 nm after GPI-80 or DAF cross-linking, or fMLP
stimulation-OD570 nm after TCY-3 cross-linking)/OD570 nm after TCY-3
cross-linking]x100}.
Statistical analysis
Analysis of the statistical significance of differences between
two mean values was performed using the Students t-tests.
P values < 0.05 were considered significant.
 |
RESULTS
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Enhancement of CD11b expression and down-modulation of L-selectin
on neutrophil surfaces by cross-linking GPI-80 with mAb 3H9
We examined the change in CD11b expression on neutrophil surfaces
after cross-linking GPI-80 with 3H9 and anti-mouse IgG antibody. As
shown in Figure 1 A
, cross-linking GPI-80 enhanced the cell-surface expression of
CD11b in a time-dependent manner for up to 5 min of incubation when it
reached a plateau. As shown by Borregaard et al. [17
],
neutrophil surface expression of CD11b is up-regulated by fMLP
treatment, and we found a similar pattern with GPI-80 cross-linking.
Neither treatment with 3H9 alone in the absence of cross-linking with
anti-mouse IgG antibody nor treatment with anti-mouse IgG antibody
alone changed the expression of CD11b. In addition, cross-linking
GPI-80 did not change the expression of HLA on neutrophil surfaces.

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Figure 1. Up-regulation of CD11b expression on neutrophil surfaces and shedding
of L-selectin by cross-linking GPI-80. After cross-linking GPI-80, the
cells were incubated for 0, 1, 3, 5, 15, or 30 min at 37°C. Then the
neutrophils were labeled with FITC-conjugated anti-CD11b mAb,
RPE-conjugated anti-L-selectin mAb, or anti-HLA mAb. (A) Changes in
CD11b expression. () CD11b expression after GPI-80 cross-linking
[CD11b XL(+)]. ( ) CD11b expression after treatment with 3H9 alone
[CD11b XL(-)]. ( ) CD11b expression after fMLP stimulation [CD11b
fMLP(+)]. ( ) HLA expression after GPI-80 cross-linking [HLA
XL(+)]. ( ) CD11b expression after treatment with anti-mouse IgG Ab
alone (CD11b a-mIg). (B) Changes in L-selectin expression. ()
L-selectin expression after GPI-80 cross-linking [L-s XL(+)]. ( )
L-selectin expression after treatment with 3H9 alone [L-s XL(-)].
( ) L-selectin expression after fMLP treatment [L-s fMLP(+)]. ( )
HLA expression after GPI-80 cross-linking [HLA XL(+)]. The data shown
are the increases in mean fluorescence intensity (MFI; i.e., MFI after
incubation/MFI before incubation) ± SE (A,
n=5; B, n=3). **, P < 0.01; *,
P < 0.05 compared with control without GPI-80
cross-linking.
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CD11b forms a heterodimer with CD18 on leukocyte surfaces
[18
]. Therefore, we then examined the expression of CD18
after GPI-80 cross-linking and found that it was enhanced like that of
CD11b (unpublished results).
Conversely, GPI-80 cross-linking decreased the expression of L-selectin
on neutrophil surfaces in a time-dependent manner for up to 5 min, at
which time it reached a plateau. As demonstrated by Borregaard et al.
[17
], treatment with fMLP decreases L-selectin
expression, and we found a similar pattern with GPI-80 cross-linking.
L-selectin expression did not change significantly by treatment with
3H9 alone (Fig. 1B)
.
We examined the effect of 3H9 concentration on the increase in CD11b
expression by GPI-80 cross-linking and found that it increased in a
concentration-dependent manner up to 10 µg/ml 3H9 when it reached a
plateau (Fig. 2
).

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Figure 2. Effect of 3H9 concentration on CD11b expression induced by
cross-linking GPI-80. After cross-linking GPI-80 with 0, 1, 2, 3, 5, 7,
10, 15, or 20 µg/ml 3H9 F(ab')2 fragments and rabbit
anti-mouse IgG F(ab')2 (20 µg/ml), the cells were
incubated for 15 min at 37°C. () CD11b expression after GPI-80
cross-linking [XL(+)]. ( ) CD11b expression after treatment with
3H9 alone [XL(-)]. The data shown are the increases in MFI
(i.e., MFI in the presence of 3H9/MFI in the absence of 3H9) ±
SE (n=3). *, P < 0.05 between
cross-linking and no cross-linking.
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Change in localization of GPI-80 on neutrophil surfaces after
cross-linking with 3H9
Using confocal laser microscopy, we examined the localization of
GPI-80 after being cross-linked. When neutrophils were treated with 3H9
alone, GPI-80 was broadly spread on entire neutrophil surfaces, before
incubation and after incubation for 30 min at 37°C (Fig. 3A
and B). Conversely, after GPI-80 was cross-linked by
the addition of antibodies to mouse Ig, it formed a patchy pattern
before incubation (Fig. 3C)
and a cap after incubation for 30 min at
37°C (Fig. 3D)
, similar to uPAR after being cross-linked
[19
].

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Figure 3. Change in localization of GPI-80 on the surface of neutrophils
after cross-linking GPI-80 by 3H9. GPI-80 on the cells was labeled with
FITC-conjugated 3H9 and observed by confocal laser microscopy. (A)
GPI-80 was not cross-linked, and the cells were not incubated. (B)
GPI-80 was not cross-linked, and the cells were incubated for 30 min
at 37°C. (C) GPI-80 was cross-linked, and the cells were not
incubated. (D) GPI-80 was cross-linked, and the cells were incubated
for 30 min at 37°C.
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The effect on cell-surface expression of CD11b by cross-linking
another GPI-anchored protein, DAF (CD55)
We determined whether cross-linking another cell-surface
GPI-anchored protein, DAF, affects cell-surface expression of CD11b to
ascertain if the observed phenomena may be ascribed to cross-linking
GPI-anchored proteins in general. Before doing this, we examined the
cell-surface localization of DAF after cross-linking it and incubating
at 37°C. Before cross-linking DAF, it was observed evenly on the
entire cell surface (Fig. 4 A
).

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Figure 4. Localization of DAF on neutrophil surfaces after cross-linking with a
relevant mAb. (A) No cross-linking. (B) Cross-linking without
incubation. (C) Cross-linking and incubation for 30 min at
37°C.
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We examined the change in the localization of DAF on cell surfaces
caused by its cross-linking. As shown in Figure 4C
, the result was the
same as that with GPI-80 in that cross-linking DAF followed by
incubation for 30 min at 37°C resulted in cap formation. These
results show that cross-linking GPI-80 and DAF induces similar
localization of the proteins, prompting us to examine the effect of DAF
cross-linking on the expression of CD11b on neutrophil surfaces.
Although, as already shown, cross-linking GPI-80 increased the
cell-surface expression of CD11b, cross-linking DAF had no effect on
CD11b expression (Fig. 5
), suggesting that cross-linking GPI-anchored proteins in general
does not induce enhanced expression of CD11b. To eliminate the
possibility that the results we obtained might be ascribed to a
difference in the amounts of CD55 and GPI-80 on neutrophil surfaces, we
compared them by flow cytometry and found that there was not a large
difference in their amounts on neutrophil surfaces (unpublished
results).
Effect of a tyrosine-kinase inhibitor, genistein, and an inhibitor
of actin polymerization, cytochalasin B, on the enhanced expression of
CD11b caused by cross-linking GPI-80
Previously, we showed that cross-linking GPI-80 induces tyrosine
phosphorylation of a 34-kD protein that is inhibited by cytochalasin B
[8
]. Therefore, we examined the effects of a tyrosine
phosphorylation inhibitor, genistein, and an inhibitor of actin
polymerization, cytochalasin B, on the enhanced expression of CD11b on
cell surfaces caused by GPI-80 cross-linking. As shown in Figure 6
, both agents inhibited the enhanced expression of CD11b in a
concentration-dependent manner, suggesting that tyrosine
phosphorylation and cytoskeleton remodeling are involved in the
enhancement of CD11b expression on neutrophil surfaces caused by GPI-80
cross-linking.

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Figure 6. Effects of genistein and cytochalasin B on the up-regulation of CD11b
expression induced by GPI-80 cross-linking. After treating with 3H9
F(ab')2 fragments, the cells were treated with 50 or 100
µg/ml genistein or 5 or 10 µg/ml cytochalasin B for 15 min at room
temperature. After GPI-80 cross-linked, the cells were incubated for 15
min at 37°C in HBSS containing genistein or cytochalasin B at the
indicated concentrations. The data are shown as %MFI [i.e., (MFI with
GPI-80 cross-linking in the presence of inhibitor-MFI without
GPI-80 cross-linking in the presence of inhibitor)/(MFI with
GPI-80 cross-linking in the absence of inhibitor-MFI without GPI-80
cross-linking in the absence of inhibitor)] x 100 ±
SE (A, n=4; B, n=3). **,
P < 0.01; *, P < 0.05 compared with
no inhibitors.
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Effect of GPI-80 cross-linking on ß2
integrin-dependent neutrophil adherence
It is well documented that enhanced expression of ß2
integrins on cell surfaces is not necessarily followed by increased
cell adhesion [20
, 21
] and that activation
epitopes of ß2 integrins are critically important for
ß2 integrin-dependent cell adhesion [15
].
Thus, we examined whether activation epitopes of CD11b were increased
by GPI-80 cross-linking. For this, we used CBRM1/5 mAb
[15
]. Figure 7
shows that this cross-linking up-regulated the expression of CD11b
recognized by CBRM1/5 on neutrophil surfaces, although the rate of
up-regulation was less than that induced by fMLP treatment.

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Figure 7. Up-regulation of CBRM1/5-recognized CD11b expression on the neutrophil
surfaces by cross-linking GPI-80. After cross-linking GPI-80, the cells
were incubated for 0, 1, 3, 5, 15, or 30 min at 37°C. Then the cells
were labeled with CBRM1/5 or TCY-3 and FITC-conjugated anti-mouse IgG
Fc region-specific Ab. () CBRM1/5 expression after GPI-80
cross-linking [XL(+)]. ( ) CBRM1/5 expression after treatment with
3H9 alone [XL(-)]. ( ) TCY-3 expression after GPI-80 cross-linking
[XL(+) TCY-3]. ( ) CBRM1/5 expression after fMLP stimulation
[fMLP(+)]. The data shown are the increases in MFI (i.e., MFI after
incubation/MFI before incubation) ± SE
(n=4). ***, P < 0.005; **,
P < 0.01; *, P < 0.05 compared with
no GPI-80 cross-linking.
|
|
Next, we determined whether GPI-80 cross-linking augments
ß2 integrin-dependent neutrophil adherence. As shown in
Figure 8
, fMLP treatment enhanced neutrophil adherence on a plate precoated
with fibrinogen, but GPI-80 cross-linking had no effect, suggesting
that GPI-80 cross-linking itself does not affect ß2
integrin-dependent adherence, although it enhances the expression of
the ß2 integrin-activation epitope.
Although it was shown that GPI-80 cross-linking itself does not affect
ß2 integrin-dependent adherence (Fig. 8)
, we examined the
priming effects of GPI-80 cross-linking on fMLP-induced human
neutrophil adherence. Figure 9
shows that cross-linking GPI-80 augmented fMLP-induced neutrophil
adherence at 30 min of incubation, whereas cross-linking another
GPI-anchored protein on human neutrophils, DAF, did not affect
adherence.

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Figure 9. Enhancement of fMLP-induced neutrophil adherence by GPI-80
cross-linking. After cross-linking GPI-80 or DAF, neutrophil
suspensions and 200 µl HBSS containing 1 x
10-9 M fMLP were added to fibrinogen-coated
plates, which were incubated at 37°C for 15, 30, 60, and 120 min.
() GPI-80 cross-linking. ( ) DAF cross-linking. The data shown are
% adherence [i.e., (OD values after GPI-80 or DAF cross-linking-OD
values of TCY-3 cross-linking)/OD values of TCY-3 cross-linking] x
100 ± SE (n=3). *, P <
0.05 compared with TCY-3 cross-linking.
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 |
DISCUSSION
|
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To elucidate the mechanisms by which a novel GPI-anchored protein
on neutrophils, GPI-80, regulates ß2 integrin-dependent
neutrophil adherence, we examined the effect of cross-linking GPI-80 on
the cell-surface expression of Mac-1 by human neutrophils. We found
that cross-linking GPI-80 enhances the expression of Mac-1 and
down-regulates that of L-selectin on neutrophils. These results
strengthen the possibility that GPI-80 regulates ß2
integrin-mediated cell adherence. Recently, we have demonstrated that
GPI-80 forms clusters on the forward surfaces of transmigrating
neutrophils [22
] and now show that cross-linking GPI-80
induces its cluster formation (Fig. 3)
. Some other GPI-anchored
proteins, such as uPAR (CD87), Fc
RIIIb (CD16b), and CD14, modulate
adherence and migration of neutrophils [9
], and
cross-linking uPAR or Fc
RIIIb with relevant antibodies enhances
Mac-1 expression on neutrophil surfaces [10
,
11
]. The present results, cross-linking GPI-80, mimic
those obtained with these other GPI-anchored proteins, which regulate
adherence. Although we have not yet identified the GPI-80 ligand,
cross-linking GPI-80, which results in its clustering (Fig. 3)
, may
mimic the physiological effect of the ligand binding to GPI-80, which
likely causes its clustering.
Conversely, with respect to the modulation of L-selectin shedding by
cross-linking GPI-anchored proteins that regulate adhesion, varying
results have been obtained. In this study and in the case of
cross-linking certain alleles (Fc
RIIaR131) of Fc
RIIa
[11
], cell-surface expression of L-selectin was
down-regulated. However, cross-linking another allele
(Fc
RIIaH131) of Fc
RIIa, Fc
RIIIb
[11
], or uPAR did not induce down-regulation of
L-selectin, although in the uPAR cross-linking experiment, expression
of L-selectin was down-regulated even by treatment with a control
antibody [10
], which was not observed in the other
cross-linking studies including the one described here. Although the
reason for these conflicting results remains to be clarified, each
GPI-anchored protein that regulates cell adhesion may work at different
stages of leukocyte extravasation.
Previously, it was shown that fMLP-induced degranulation of primary and
secondary granules is inhibited by genistein [23
]. As
shown in Figure 6A
, genistein inhibits the up-regulation of CD11b
expression on neutrophil surfaces caused by GPI-80 cross-linking,
suggesting that tyrosine phosphorylation is required for degranulation
following GPI-80 cross-linking. Conversely, in contrast to genistein,
cytochalasin B enhances fMLP-induced degranulation of primary and
secondary granules [23
]. However, as shown in Figure 6B
,
degranulation caused by cross-linking GPI-80 was inhibited by
cytochalasin B. It has been shown that cytoskeletal reorganization is
required for capping GPI-anchored proteins in lymphocytes
[24
]. Under these situations, cytochalasin B may affect
capping formation of GPI-80 that may be required for signal
transduction for Mac-1 expression enhancement.
Although not only was total expression of Mac-1 enhanced (Fig. 1) , but
expression of an activation epitope of CD11b (CBRM1/5) was also
increased by GPI-80 cross-linking (Fig. 7)
, and ß2
integrin-dependent neutrophil adherence was not affected (Fig. 8)
. This
suggests that cross-linking GPI-80 does not induce full activation of
neutrophils that are required for ß2 integrin-dependent
adherence of these cells and that expression of an activation epitope
recognized by CBRM1/5 is a required but not sufficient condition for
ß2 integrin-dependent cell adhesion. The finding that
cross-linking GPI-80 primes neutrophils for fMLP-induced adherence
(Fig. 9)
but does not itself induce adherence (Fig. 8)
supports this
notion. Our preliminary results showing that cross-linking GPI-80 did
not change the topographical localization of CD18 on neutrophil
surfaces (unpublished results) may further explain the condition of
neutrophils whose GPI-80 is cross-linked. We should note here that as
shown in our previous study, treatment of neutrophils with the mAb to
GPI-80, 3H9, modulates the fMLP-induced adherence of neutrophils
[3
], but 3H9 in the absence of additional neutrophil
stimulation does not induce adherence, indicating that GPI-80 is a
regulator of cell adhesion. Furthermore, in the present study,
cross-linking GPI-80 augmented fMLP-induced neutrophil adherence (Fig. 9)
, although by itself it did not induce adherence (Fig. 8)
. This
suggests that cross-linking GPI-80 has a priming activity on neutrophil
function(s).
GPI-anchored proteins on lymphocytes are localized in a
detergent-insoluble cell-membrane fraction [25
], which
is called a "microdomain" or "raft" containing an abundance of
the signaling molecules [26
, 27
]. Rafts are
considered to be involved in signal transduction into the cytoplasm
through cross-linking GPI-anchored proteins, although the precise
mechanisms remain to be clarified [28
]. By cross-linking
with relevant antibodies, GPI-anchored proteins become focused on one
point of the cell-surface membrane, forming a capping. Rafts are
translocated to this point, and signaling proteins in rafts are
phosphorylated [29
]. Although it is not known whether
rafts exist on neutrophil-surface membranes, cross-linking GPI-80 and
DAF on human neutrophils by relevant antibodies causes them to move to
the same point of cell-surface membranes and form a cap (Figs. 3
and 4)
. However, DAF cross-linking did not induce an increase in
cell-surface expression of Mac-1 (Fig. 5) in contrast to the effect of
GPI-80 cross-linking (Fig. 1A) , suggesting that the phosphorylation
induced by cross-linking GPI-anchored proteins cannot be explained by
the raft phenomena, but certain mechanisms by which specificity of each
GPI-anchored protein are determined.
In summary, the present result that cross-linking GPI-80 enhances Mac-1
expression on neutrophil surfaces and the adherence suggests that this
GPI-anchored protein is involved in the regulation of ß2
integrin-dependent neutrophil adherence and consequently its
transendothelial migration.
 |
ACKNOWLEDGEMENTS
|
|---|
We thank Dr. Timothy A. Springer, Harvard Medical School, for the
gift of the monoclonal antibody, CBRM1/5.
Received June 14, 2001;
revised September 18, 2001;
accepted September 19, 2001.
 |
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|
|---|
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Butcher, E. C. (1991) Leukocyte-endothelial cell recognition: three (or more) steps to specificity and diversity Cell 67,1033-1036[Medline]
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Infect. Immun.,
August 1, 2003;
71(8):
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[Abstract]
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[PDF]
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