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Introduction
Inflammation is a multiple-step process initiated by the adhesion of leukocytes to the activated endothelial cells. The
recruitment of leukocytes from the flowing bloodstream is a rapid event that requires special mechanisms to establish the
contact of leukocyte with endothelium. Selectins represent a class of cell adhesion molecules that are specialized for this
purpose[1]. P-selectin glycoprotein ligand 1 (PSGL-1), the best characterized ligand of the selectins, has been demonstrated
to mediate the adhesion of leukocytes to all three selectins
in vivo[2-5].
PSGL-1 is a highly extended mucin expressed on microvilli of almost all types of leukocytes. In addition to its direct role
in the capture of leukocytes from the bloodstream, PSGL-1 has also been demonstrated to function as a signal-transduced
receptor and initiate a series of intracellular signal events during the activation of leukocytes, including activation of
β2-integrin[6-8], tyrosine-protein
phosphorylation[9,10], secretion of
cytokines[9] and transcriptional
activation[11]. Our previous work suggests that PSGL-1-mediated signals induce the alteration of F-actin-based cytoskeleton in neutro-phils, and
non-receptor tyrosine kinase c-Abl may be involved in the
process[12]. A recent study showed that PSGL-1 stimulated
lymphocyte function-associated antigen (LFA)-1-mediated Th1 cell binding to intercellular adhesion molecule-1
(ICAM-1)[13].
In the present work, we demonstrated that the engagement of PSGL-1 with mAb or P-selectin induced the increased
expression of β2-integrin and enhanced the adhesion of neutrophils to the recombinant ICAM-1. TS1/18, the monoclonal
antibody against CD18, impaired the adhesion of the
PSGL-1-engaged neutrophils to the ICAM-1, suggesting the
contribution of β2-integrins to the adhesion of neutrophils to ICAM-1 is caused by the engagement of PSGL-1. Moreover,
cytochalasin B (an inhibitor for the assembly of F-actin-based cytoskeleton) largely blocked the increased expression of
β2-integrin and the adhesion of PSGL-1-engaged neutrophils to ICAM-1, which implies an important role of the F-actin-based cytoskeleton.
Our combined results suggest that the PSGL-1-transduced signal can enhance
β2-integrin-involved adhesion of neutrophils
to the recombinant ICAM-1 through the increase of
β2-integrin expression, and this process depends on the dynamics of the
F-actin-based cytoskeleton.
Materials and methods
Recombinant proteins, antibodies and
reagents The recombinant human P-selectin (ADP3) and ICAM-1 (ADP4) were
purchased from R&D Systems (Minneapolis, MN, USA).
PL2 (the non-blocking mAb against PSGL-1, mouse
IgG1, sc-18856) was purchased from Santa Cruz Biotechnology (Santa
Cruz, CA, USA). W6/32 (the anti-HLA-A,B,C mAb, mouse
IgG1, 14-9983) was purchased from Ebioscience (San Diego, CA,
USA). 9E1 (the mAb recognizing the extra-cellular domain of recombinant human P-selectin, mouse
IgG1, BBA30) was purchased from R&D Systems. TS1/18 (the mAb against CD18, which can block the ligation of
β2-integrin with ICAM-1, mouse
IgG1, MA1810) was purchased from Pierce Biotechnology (Rockford, IL, USA). Fluorescein isothiocynate
(FITC)-conjugated β2-integrins antibody IB4 was purchased from Ancell Corporation (Bayport, MN, USA) (167-040). Dextran T-500
used for neutrophil isolation was purchased from Pharmacia (Uppsala, Sweden). Cytochalasin B (an inhibitor for assembly
of F-actin) was purchased from Sigma (Saint Louis, MO, USA).
Neutrophil isolation Neutrophils were isolated by standard procedures after sedimentation of erythrocytes by dextran
T-500 and centrifugation of leukocytes over Ficoll-Hypaque gradients as described in our previous
work[12]. After lysis of contaminating erythrocytes with hypotonic saline (0.17 mol/L Tris, 0.16 mol/L
NH4Cl, pH 7.2), the
neutrophils were washed and re-suspended in cold PBS containing 0.1% bovine serum albumin (BSA). The isolated neutrophils were adjusted at a
concentration of 0.5×106 cells/mL and stored at 4 ºC until use. More than 95% of the isolated cells were polymorphonuclear
leukocytes, and viability was determined to be 98% by Trypan blue exclusion.
Engagement of PSGL-1 Preceding the interaction with
ICAM-1 monolayer, the isolated neutrophils were ligated
with PL2, an mAb against PSGL-1 (or control antibody
W6/32) at a concentration of 10 µg/mL at 4 ºC for 20 min. In other experiments, the
isolated neutrophils were subjected to flow on the P-selectin/ICAM-1 monolayer to encounter and engage with P-selectin.
Monolayer preparation To prepare the ICAM-1 monolayer, the petri dishes were coated with 3 µg/mL recombinant
ICAM-1 overnight and then blocked with 1% BSA at
37 ºC for 2 h, and in control groups, recombinant ICAM-1 was substituted with BSA. In other experiments, the petri dishes
were simultaneously coated with 3 µg/mL of each recombinant P-selectin and ICAM-1 overnight and then blocked with 1%
BSA at 37 ºC for 2 h.
Cell adhesion assay The adhesion of PSGL-1-engaged neutrophils to the recombinant ICAM-1 monolayer was detected
by using a parallel-plate flow chamber (GlycoTech, Rockville, MD, USA). Briefly, the neutrophils were washed twice and
re-suspended in RPMI 1640 medium. At a shear stress of 1.2
dyn/cm2, cells were infused over the ICAM-1 (or
P-selectin/ICAM-1) monolayer via a syringe
pump[14, 15]. Interactions of neutrophils with monolayer were allowed by stopping the flow at
different times, after which the flow was carried out again at a shear stress of 1.2
dyn/cm2. The adherent cells could then be
counted by using an inverted microscope (Olympus Optical, Tokyo, Japan) equipped with a camera (Panasonic, Yokohama,
Japan) connected to a VCR and a TV monitor. Each data represented the percentage of the number of remaining cells visible
when flow was re-infused to that of all halted cells after flow was stopped in 10 fields of view under a ×10 objective.
For antibody blocking experiments, neutrophils (or antibody-engaged neutrophils) were treated with TS1/18 at a
concentration of 10 µg/mL at 4 ºC for 15 min preceding being infused on the monolayer. In other experiments the
P-selectin/ICAM-1 monolayer was blocked with 9E1, the mAb recognizing the extra-cellular domain of recombinant human P-selectin, at a
concentration of 10 µg/mL at 4 ºC for 15 min to block the engagement of PSGL-1 with P-selectin.
For inhibition experiments, the isolated neutrophils were treated with 20 µmol/L cytochalasin B at 4 ºC for 30 min preceding
the engagement of PSGL-1 and the interaction with the ICAM-1 (or P-selectin/ICAM-1) monolayer. The control cells were
treated with the equivalent volume of dimethyl sulfoxide (DMSO).
Fluorescence assay To examine the expression of
β2-integrins on the surface of neutrophils, cells
(1×106/mL) were treated with (or without) PL2 (10 µg/mL) at 37 ºC for 30 min, then washed with PBS twice before being fixed with 4% paraformaldehyde
for 20 min at room temperature. The expression of CD18 was visualized by staining with FITC-conjugated
β2-integrins antibody IB4 (10 µg/mL) for 30 min. The levels of surface expression of
β2-integrins were detected with Fluorescence Reader
Gemini EM (Molecular Device Corporation, Sunnyvale, CA, USA). The well was set with an equal number of cells, without
antibody treatment and FITC-labeled CD18 antibody IB4 staining as blank, the data of all sample wells were normalized by the
value of blank. The data indicate the OD values of the detector¡¯s absorbance of the fluorescence that samples emit, and each
data represents the mean±SD of three independent experiments and the significant difference from
thecontrol was determined by ANOVA
(P<0.01).
Results
Engagement of PSGL-1 enhanced the adhesion of neutrophils to recombinant
ICAM-1 We set up an in vitro flow system
to investigate the contribution of signals transduced by PSGL-1 to the activation of neutrophils and the mechanism by which
the interaction of PSGL-1 with P-selectin enhances the adhesion of neutrophils to ICAM-1 expressed on the surface of
activated endothelial cells during inflammation.
First, recombinant ICAM-1 was coated as a monolayer on the petri dish bottom. After engagement with (or without)
antibodies, the neutrophils were infused in the flow system at a shear stress of 1.2
dyn/cm2. The adhesion of neutrophils to
the recombinant ICAM-1 was allowed for different times. The percentage of adherent cells for those engaged with PL2 was
significantly higher compared with that of resting, Mo-IgG-treated or W6/32-treated neutrophils (Figure 1A). The extreme
lack of adhesion for those differently treated neutrophils to BSA-coated petri dishes implied the specificity of the interaction
of engagement-activated neutrophils to the ICAM-1 monolayer (Figure 1B).
Second, the isolated neutrophils were infused by flow on the petri dish bottom, which was coated with P-selectin,
ICAM-1, or both P-selectin and ICAM-1 as a monolayer. The neutrophils were infused in the flow system and the adhesion of
neutrophils to the monolayer was allowed for 10 min. As shown in Figure 1C, the percentage of neutrophils adherent to
P-selectin/ICAM-1 monolayer was significantly increased compared with that of neutrophils adherent to ICAM-1 or P-selectin
monolayer alone, which suggested that the presence of P-selectin enhances the interaction of neutrophils with the
recombinant ICAM-1 (Figure 1C).
β2-integrins were involved in the adhesion of PSGL-1-engaged neutrophils to recombinant ICAM-1
The leukocyte-specific β2-integrins regulate the migration of leukocytes and the initiation of immune responses through binding to
ICAM-1, -2 or -3[16, 17]. In order to investigate the role of
β2-integrins in the adhesion of PSGL-1-engaged neutrophils to the
recombinant ICAM-1 monolayer, we performed antibody-blocking experiments by using antibodies TS1/18 and 9E1,
respectively, against CD18 and P-selectin (both antibodies recognize the extra-cellular ligand-binding domains of CD18 and
P-selectin). The results showed that the increased percentage of adherent neutrophils induced by PL2
antibody-cross-linking was abolished by the incubation of neutrophils with TS1/18, the mAb against CD18 (Figure 2A). Moreover, if the
P-selectin/ICAM-1 monolayer and neutrophils were respectively pre-incubated with 9E1 and TS1/18 alone, or pre-incubated
with both 9E1 and TS1/18 prior to the neutrophils being infused in the flow system, the percentage of the adherent
neutrophils decreased in different degrees compared with that of the control. Especially when the neutrophils interacted with the
monolayer were blocked with both 9E1 and TS1/18, the percentage of the adherent neutrophils dropped more than 70%
compared with that of the control (from 51.36% to 15.08%) (Figure 2B). The results from the antibody-blocking experiments
suggested that β2-integrins were involved in the increased adhesion of neutrophils to the recombinant ICAM-1 caused by
the engagement of PSGL-1.
To further understand the contribution of
β2-integrins to the adhesion of activated neutrophils to the recombinant ICAM-
1, we investigated the alteration of β2-integrin expression. The neutrophils were incubated with PL2 or control IgG at 37
ºC for 30 min, and then fixed and labeled with FITC-conjugated antibody IB4. The result showed that the engagement of
PSGL-1 induced an increased expression of
β2-integrins on the surface of activated neutrophils
(Figure 3).
β2-integrin-involved adhesion of neutrophils to recombinant ICAM-1 depends on the dynamics of
F-actin-basedcytoskeleton Because the cytoskeleton often plays an important role in the transduction of extra-cellular signals, we further explored
the significance of the dynamics of the cytoskeleton to the PSGL-1-initiated and
β2-integrin-involved
adhesion of neutrophils to the recombinant ICAM-1. We pre-incubated the isolated neutrophils with cytochalasin B, the
cells were then engaged with mAb and infused to the ICAM-1 monolayer, or the inhibitor-treated cells were infused to the
P-selectin/ICAM-1 monolayer. Consequently, the adhesion of PSGL-1-engaged neutrophils to recombinant ICAM-1 was
significantly suppressed, suggesting that the PSGL-1-initiated adhesion of neutrophils to the recombinant ICAM-1 depend
on the dynamics of the cytoskeleton (Figure 4A and 4B). The exposure of neutrophils to cytochalasin B also resulted in a
dramatic blocking of the increased expression of
β2-integrins (Figure 5), implying the significance of the dynamics of the
F-actin-based cytoskeleton to the β2-integrin-mediated adhesion of neutrophils to ICAM-1.
Discussion
During the rolling adhesion of leukocytes on the activated endothelial cells, PSGL-1 functions not only as an anchor to
arrest the leukocytes through the interaction with P-selectin, but also as a signal receptor to transduce the extra-cellular
signals and participate in the activation of
leukocytes[18, 19]. ICAM-1 is an Ig-like cell adhesion molecule expressed by several
cell types, including leukocytes and endothelial cells after stimulation
with cytokines such as IL-1, TNF-α or
IFN-γ[20]. Within the vascular system, ICAM-1 expressed on the surface of activated endothelial cells interacts with integrins expressed by
leukocytes to achieve the firm adhesion of leukocytes on the endothelium.
CD18 integrins are leukocyte-specific, and it is still controversial as to what mechanism CD18 integrins rely on to enhance
the adhesion of activated leukocytes to ICAM-1. It may be the extroversion of the
β2-integrin from the intracellular
vesicles[21], increased affinity of
β2-integrin due to the conformational change of extra-cellular structure,
de novo synthesis or simply the cluster of the molecules on the surface of
leukocytes[17].
In the present study, we addressed the implication of leukocyte-specific
β2-integrins in the adhesion of neutrophils to
endothelial cells induced by the engagement of
PSGL-1 with P-selectin during inflammation. We established an
in vitro adhesion system by using a
parallel-plate flow chamber. The incubation with TS1/18 abolished the increased adhesion of neutrophils
caused by the engagement of PSGL-1. Meanwhile, fluorescence assay revealed that the engagement of PSGL-1 led to an
increase of β2-integrin expression on the activated neutrophils, which suggested the involvement of CD18 integrins in the
adhesion process. Additionally, the pre-incubation of neutrophils with cytochalasin B not only significantly decreased the
percentage of the adherent cells, but also blocked an increase in the level of CD18 on the PSGL-1-engaged neutrophils, which
suggested that the dynamics of cytoskeleton is essential for the
β2-integrin-mediated adhesion of PSGL-1-engaged
neutrophils to ICAM-1.
It is well known that PSGL-1-transduced signals can enhance
β2-integrin-mediated adhesion of activated neutrophils to
ICAM-1. Our present study revealed that increased expression of
β2-integrin contributed to this adhesion enhancement. Our
previous study demonstrated engagement of PSGL-1 with antibodies led to the association of PSGL-1 with F-actin-based
cytoskeleton[12], and the present study further elaborated that PSGL-1-transduced signals depended on the dynamic of
F-actin-based cytoskeleton to increase the expression of CD18 integrin and
β2-integrin-mediated adhesion.
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