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Department of Internal Medicine,
Department of Periodontology, Osaka Dental University, and
Department of Microbiology, Kinki University School of Medicine, Osaka, Japan; Departments of
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Gastrological Surgery and
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Hematology and Oncology, Kyoto University Graduate School of Medicine, Kyoto, Japan; and
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Division of Cellular and Gene Therapies (HFM-518), Center for Biologics Evaluation Research, Food and Drug Administration, Bethesda, MD 20892
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and IFN-
, have no effect on
endothelial permeability (11, 12), suggesting that
cell-to-cell contact between NK or LAK cells and ECs is necessary to
induce VLS. The endothelium plays an important role in the recruitment
and emigration of circulating effector cells into sites of inflammation
and immune responses, and endothelial cells can be the primary target
of immunologic injury, which results in vasculopathy and organ
dysfunction (13, 14, 15, 16, 17). NK cells accumulate rapidly in
tissues under certain conditions, including viral infections,
administration of biologic response modifiers, and transplantation of
allografts or xenografts (16, 18). However, the mechanisms
underlying the recruitment and extravasation of NK cells as well as NK
cell-mediated EC damage under these conditions, including VLS and
transplantation rejection, are not well understood. Chemokines were first described as chemoattractant cytokines synthesized at sites of inflammation and are the major regulatory proteins for leukocyte recruitment and trafficking (19, 20, 21, 22, 23, 24). Fractalkine, a recently identified chemokine, is a transmembrane protein consisting of a Cys-X-X-X-Cys chemokine domain presented on top of an extended mucin-like stalk and is expressed on the surface of activated ECs (25, 26). We have recently identified a receptor for fractalkine (CX3CR1, V28), have demonstrated that it is expressed predominantly on NK cells as well as on CD8+ T cells and CD14+ monocytes, and have shown that soluble fractalkine (s-fractalkine) exhibits a potent chemoattractant activity for those cells (27). In the present study we further investigated the functional effects of fractalkine on NK cell activity and NK cell-mediated damage of ECs. Our results showed that fractalkine enhances vascular EC damage by NK cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Hybridomas producing mAbs against CD18 (TS1/18), CD11a (TS1/22), and CD3 (OKT3) were purchased from American Type Culture Collection (Manassas, VA), and mAbs were purified as previously described (28). Anti-CD14, anti-CD19 mAbs were purchased from Immunotech (Marseilles, France). FITC- or PE-conjugated Abs against CD3, CD14, CD16, CD21, and ICAM-1 were obtained from Becton Dickinson (Mountain View, CA). The monoclonal anti-secreted form of placental alkaline phosphatase (SEAP) Ab was purchased from Genzyme (Cambridge, MA). Monoclonal anti-CD106 (VCAM-1) Ab and anti-CD54 (ICAM-1) Ab were purchased from Ancell (Bayport, MN) and Serotec (Kidlington, U.K.), respectively. Anti-fractalkine mAbs were prepared as previously described (27). Affinity-purified Ab against CX3CR1 was provided by Dr. Yiyang Xia (Torrey Pines Biolabs, San Diego, CA). Recombinant IL-2 was provided by Shionogi (Osaka, Japan). Goat anti-mouse IgG-coupled magnetic beads (Biomag) were purchased from PerSeptive Biosystems (Framingham, MA), and calcein-AM was purchased from Wako Pure Chemical Industries (Osaka, Japan).
Production of recombinant proteins
Soluble fractalkine (s-fractalkine), fractalkine-SEAP, CX3C-SEAP, and mucin-SEAP fusion proteins were prepared using a baculovirus expression system as described previously (27). Briefly, to express fractalkine-SEAP, the DNA fragment encoding fractalkine was amplified from fractalkine cDNA by PCR using 5' SalI-fractalkine primer (+5'-CGCGTCGACTCAGCCATGGCTCCGATATCT-3') and 3' fractalkine-XbaI primer (-5'-CGCTCTAGAGGTGGCTGCCTGGGCGTCAGG-3')andweresub-cloned into pDREF-SEAP (His)6-Hyg vector as described previously (29). CX3C-SEAP was similarly generated using 5' SalI-fractalkine primer and 3' fractalkine-XbaI-2 primer (-5'-CGCTCTAGATAGGGCAGCAGCCTGGCGGTC-3'). For generation of mucin-SEAP, the DNA fragment encoding SalI-oncostatin M signal sequence XbaI-fractalkine lacking the chemokine domain NheI was amplified by three-step PCR using 5' SalI-OMC-fractalkine primer (+5'-CTGTTTCCATGCATGGCGAGCATGTCT3') and 3' fractalkine-NheI primer (-5'-CGCGCTAGCGGTGGCAGCCTGGGCGTCAGG-3'). After 3–4 days the supernatants were collected. For one-step affinity purification, supernatants were applied to 1 ml of Hisbond resin (Qiagen, Hilden, Germany). After washing, bound recombinant proteins were eluted with 100 mM imidazol. Soluble fractalkine with a tag of six histidine residues, (His)6, at its C terminus was prepared using a baculovirus expression system. The cDNA fragment encoding the extracellular domain of fractalkine was subcloned into the SalI-XbaI sites of the modified pFastBac1 baculovirus transfer vector (Life Technologies, Gaithersburg, MD) to express fractalkine as a soluble fusion protein with Ser-Arg-Ser-Ser-Gly-(His)6. The recombinant bacmids were generated in Escherichia coli DH10Bac and transfected into Spodoptera frugiperda Sf9 cells using Lipofectin (Life Technologies) to obtain the recombinant viruses. For expression of the recombinant fractalkine-(His)6, Trichoplusia in BTI-TN-5B1–4 cells were infected with the recombinant viruses at multiplicity of infection of 10–20. The culture supernatants collected 2 days after infection were applied to a 1 ml Hisbond resin (Qiagen, Valencia, CA). After washing, bound fractalkine-(His)6 was eluted with 100 mM imidazol. Protein concentration was determined by the BCA kit (Pierce, Rockford, IL). The concentration of each recombinant protein was determined by a sandwich-type ELISA as described previously (29).
Cells and cell culture
PBMCs were isolated from samples of venous blood from consenting healthy volunteers by Ficoll-Paque (Pharmacia Biotech, Uppsala, Sweden) density-gradient centrifugation. NK cells were isolated by negative selection using a mixture of anti-CD3, anti-CD14, and anti-CD19 mAbs and immunomagnetic beads (PerSeptive Diagnostics, Cambridge, MA) to deplete T cells, monocytes and B cells, respectively, as described previously (28, 30). NK cell populations used in all experiments were >85% pure as confirmed by flow cytometric analysis for the presence of CD16 and/or CD56. K562 cells were maintained in RPMI 1640 medium supplemented with 10% FCS, 2 mM L-glutamine, and penicillin. ECV304 cells were obtained from American Type Culture Collection4 and maintained in medium 199 supplemented with 10% FCS. For stable expression of membrane-bound fractalkine in ECV304 cells, the expression plasmid pCAGG-Neo-fractalkine-1 was transfected into ECV304 by Lipofectamine (Life Technologies). After selection with 800 µg/ml of G418 for 1–2 wk, drug-resistant cells were pooled as described previously (27). HUVECs were obtained from Iwaki (Chiba, Japan) and maintained in endothelial cell growth medium (10 ng/ml human epidermal growth factor, 1.0 µg/ml hydrocortisone, 50 µg/ml gentamicin, 50 µg/ml amphotericin B, 12 µg/ml bovine brain extract, and 2% FBS) as recommended by the manufacturer. For transient expression of membrane-bound fractalkine in HUVECs, the expression plasmid pCAGG-Neo-fractalkine-1 was transfected into HUVECs by Lipofectamine. Fractalkine- or vector-transfected cells were cultured for 16 h and used for NK cell-mediated cytotoxicity assays.
Adhesion assay
Each well of 16-well glass dishes was coated with 50 µl of anti-SEAP Ab (10 µg/ml) in 50 mM Tris-HCl, pH 9.5, at 4°C overnight. SEAP fusion proteins (10 nM) were added to the wells, and plates were incubated for 2 h at room temperature and washed extensively. Fresh NK cells (4000 cells/mm2) were added to each well in a final volume of 50 µl and incubated for 30 min at room temperature. After removing nonadherent cells, the remaining cells were fixed with 1% glutaraldehyde. All assays were performed in duplicate, and the percent adhesion of input cells was determined by counting cells in five x400 fields (0.07 mm2)/well (27).
To assess NK cell adhesion to ECV304 cells (control-ECV and FRK-ECV), cells were seeded at 2 x 104 cells/well in 48-well culture dishes and cultured overnight to form confluent monolayers. Freshly separated NK cells were fluorescently labeled by incubation with calcein-AM and added to each well (1–2 x 104 cells/well) in a final volume of 100 µl and incubated for 30 min at 37°C. After removal of nonadherent cells, fluorescence was measured using a Wallac 1420 ARVO fluoroscan (Pharmacia Biotech), using excitation and emission wavelengths of 496 and 520 nm, respectively. In blocking experiments using Abs or s-fractalkine, endothelial cells and NK cells were pretreated with saturating amounts of Abs or 10 nM of s-fractalkine for 30 min at room temperature before the assay.
Cytotoxicity and
N-benzyloxycarbonyl-L-lysine
thiobenzyl ester (BLTE) esterase assay
For cytotoxicity assay, target cells (K562, control-ECV, FRK-ECV, and HUVECs) were labeled with calcein-AM for 30 min at 37°C. Then target cells (5000/well) and effector cells were plated onto 96-well plates at the indicated E:T cell ratio and incubated for 4 h at 37°C in a humidified atmosphere containing 5% CO2. In some experiments, s-fractalkine was included in the assay medium at the indicated concentrations for the indicated time intervals. In blocking experiments using Abs or s-fractalkine, NK cells were pretreated with saturating amounts of Abs or 10 nM s-fractalkine for 30 min at room temperature before the assay. After incubation, supernatants were transferred to new wells, and fluorescence was measured using a Wallac 1420 ARVO fluoroscan. Assays were performed in triplicate, and data were expressed as the percent lysis or as lytic units 20% in 107 effector cells (1 lytic unit containing that number of effector cells lysing 20% of the targets) (31).
For the BLTE esterase assay, purified NK cells were suspended at 7 x 106 cells/ml in phenol red-free RPMI 1640 (IBL, Fujioka, Japan) containing 0.5% FCS and the indicated concentration of s-fractalkine and were added to flat-bottom microplate wells at a final volume of 200 µl. In some experiments, NK cells were pretreated with pertussis toxin (PTX) at 500 ng/ml for 1 h at 37°C before assays. Wells were incubated at 37°C for 4 h, and 100-µl aliquots were harvested from each well for BLTE esterase assay. The BLTE esterase assay was adapted to 200 µl for use in 96-well plates as previously described (28, 32). OD was read at 405 nm on an ELISA microplate reader (Iwaki, Osaka, Japan), and the percentage of BLTE esterase activity was calculated.
FACS analysis
For assessment of membrane markers by immunofluorescence, NK cells, ECV304 cells, or HUVECs were stained directly with FITC-labeled reagents or with an unlabeled Ab and FITC-goat (F(ab')2) anti-mouse IgG (Becton Dickinson). Expression of membrane-bound fractalkine on control-ECV, FRK-ECV, HUVECs, and fractalkine-transfected HUVECs was assessed by an indirect method using mAb 1D6 to the mucin domain of fractalkine as described previously (27). For analysis of CX3CR1 expression, NK cells were incubated with 10 nM fractalkine-SEAP or control SEAP on ice for 30 min in staining buffer (1% FCS and 0.02% sodium azide in PBS). After washing, cells were incubated with biotinylated anti-SEAP Ab, washed, and incubated with FITC-conjugated streptavidin. Cells were then analyzed on a FACSCalibur (Becton Dickinson).
Statistical analysis
All data were expressed as the mean ± SEM. Differences between groups were examined for statistical significance using Student’s t test for unpaired data and paired t test for paired data. A p value <0.05 denoted the presence of a statistically significant difference.
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Results |
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We have previously reported the expression of fractalkine
receptor, CX3CR1 (V28) mRNA mainly in
CD16+ NK cells and low levels were also detected
in CD8+ T cells and CD14+
monocytes, which correlated well with the pattern of surface expression
(27). To assess the surface expression of
CX3CR1, freshly isolated NK cells were incubated
with a fusion protein of fractalkine and SEAP (fractalkine-SEAP),
reacted with biotinylated anti-SEAP Ab and FITC-conjugated
streptavidin, and analyzed by flow cytometry. The mean fluorescent
channel of cells treated with control SEAP was 5.17, whereas that with
fractalkine-SEAP was 29.18 (Fig. 1
A), indicating that NK cells
express receptors for fractalkine.
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B, immobilized fractalkine-SEAP, but not truncated forms
(mucin-SEAP and CX3C-SEAP) or control SEAP,
supported the adhesion of NK cells. Because specific
anti-fractalkine-neutralizing mAbs are not available at present, we
exploited the inhibitory actions of s-fractalkine to dissect the
interaction of fractalkine and CX3CR1 in inducing
cell adhesion (27). The results demonstrated that
s-fractalkine efficiently inhibited the adhesion of NK cells to
immobilized fractalkine. These results indicate that
CX3CR1 on NK cells recognize the intact
fractalkine (i.e., CX3C domain plus mucin domain)
and that fractalkine functions as an adhesion molecule on NK cells
without the involvement of other adhesion molecules. Effects of s-fractalkine on NK cell cytotoxicity
Several chemokines, including MCP-1, -2, and -3 and RANTES, are
known to modulate NK cell activities (33, 34). Therefore,
we examined whether s-fractalkine enhances NK cell-mediated
cytotoxicity against K562 target cells. Soluble fractalkine (10 ng/ml)
or rIL-2 (2 nM) was included in a 4-h cytotoxicity assay against K562
cells at an E:T cell ratio of 20:1 for the indicated intervals.
Although the effect of s-fractalkine on NK cytotoxicity was modest
compared with that of IL-2, s-fractalkine significantly enhanced
NK cell-mediated K562 lysis in a time-dependent manner (Fig. 2A). Next, we examined
the effects of the s-fractalkine dose on NK cell-mediated cytotoxicity
in 10 different donors, and the results were expressed as 20% lytic
units. Fig. 2B shows that s-fractalkine enhances lysis of
K562 in a dose-dependent manner, with the maximum effect, approximately
a 25% increase in lysis, observed at a concentration of 10 nM
(p < 0.05). The effect of s-fractalkine on
NK cytotoxicity was also compared with those of MCP-1, RANTES,
as well as IL-2 (Fig. 2C). Because treatment of NK
cells with s-fractalkine induced no significant change in the
expression of LFA-1 or -ß, VLA-4 or -5, and CD2 (data not shown),
this enhancement of cytotoxicity was not due to an increase in other
surface adhesion receptors.
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One key mechanism by which NK cells lyse target cells is through
to be the granular exocytosis pathway, which primarily uses the lytic
mediators, perforin and granzymes, a family of serine esterase
(35, 36). To measure the effect of s-fractalkine on
granule exocytosis, freshly isolated NK cells were incubated in the
presence of s-fractalkine for 4 h, and supernatants were assayed
for esterase activity using BLTE as a substrate. Soluble fractalkine
enhanced NK cell granule exocytosis in a dose-dependent manner (Fig. 3A), indicating that the
increased lytic activity by NK cells induced by s-fractalkine might be
due at least in part to enhanced granular exocytosis. Next, we examined
the effect of s-fractalkine on granular exocytosis in NK cells from
nine different donors. As shown in Fig. 3B, s-fractalkine
enhanced granular exocytosis in all donors tested.
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C, enhancement of granular exocytosis induced by
s-fractalkine was completely prevented by PTX. Interaction between NK cells and fractalkine-expressing ECV cells
Because the membrane-bound form of fractalkine is reported to be
expressed on EC membranes following stimulation by TNF- or IL-1
(25), we hypothesized that fractalkine might be involved
in NK cell-mediated endothelial cell damage. To examine this
possibility, we used the endothelial cell line, ECV304 (ECV), to
establish a fractalkine-transfected subline (FRK-ECV) that
constitutively expressed membrane-bound fractalkine without significant
changes in the expression of other adhesion molecules (Fig. 4). We then used this subline to examine
whether NK cells adhere to membrane-bound fractalkine. As shown in Fig. 5, NK cells adhered to FRK-ECV more
efficiently than to control-ECV. Soluble fractalkine, used as a
competitor, markedly reduced the adhesion between NK cells and FRK-ECV
to the basal levels of adhesion between NK cells and control-ECV,
whereas mAbs against ICAM-1 and VCAM-1 only partially inhibited NK cell
adhesion to FRK-ECV. The enhanced adhesion of NK cells to FRK-ECV could
not be attributed to increased expression of surface receptors usually
thought to be involved in adhesion by NK cells, since there was no
significant difference in the expression of ICAM-1, VCAM-1, or class I
molecules between FRK-ECV and control ECV (Fig. 4). These findings
support the conclusion that physical interaction between the
membrane-bound fractalkine on ECs and CX3CR1 on
NK cells can directly mediate adhesion between ECs and NK cells
independent of integrins.
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Although endothelial cells are protected from proteolytic attacks
by extracellular matrix, heparan sulfate, or other glycosaminoglycans
under normal conditions (37), it has been reported that NK
cells or LAK cells show enhanced adhesion to the endothelium and
increased cytotoxicity against endothelial cells through the
interaction of LFA-1/ICAM-1 or VLA-4/VCAM-1 (38, 39, 40, 41). The
endothelium is a major site for chemokine action in leukocyte
activation and for transmigration of leukocytes through
vessels (14, 16, 42). Because fractalkine functions not
only as an adhesion molecule but also as a regulator for NK cell lytic
activity (
Figs. 1–3), we analyzed the sensitivity of FRK-ECV to lysis
by NK cells. As shown in Fig. 6A, FRK-ECV showed increased
susceptibility to NK cell-mediated cytolysis compared with control-ECV
in repeated tests using cells from eight different donors, although
some variability was observed among individuals. Moreover, we examined
whether the increased susceptibility of FRK-ECV to NK lysis was due to
the enhanced interaction between membrane-bound fractalkine and
CX3CR1 on NK cells. As shown in Fig. 6B, s-fractalkine, which efficiently blocked adhesion of NK
cells to FRK-ECV (Fig. 5), significantly inhibited NK cell-mediated
FRK-ECV lysis in all experiments in different donors.
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Finally, we examined the involvement of membrane-bound fractalkine
in NK cell-mediated endothelial cell damage in a more native setting
using primary HUVECs. Although treatment of HUVECs with
TNF-, IL-1, and IFN- markedly induced membrane-bound fractalkine
on the surface as reported by Bazan et al. (25),
expression of ICAM-1 as well as that of VCAM-1 were also strongly
enhanced on HUVECs. Therefore, to demonstrate the involvement
of fractalkine in primary endothelial cell damage by NK
cells, we exploited transfection of fractalkine cDNA in HUVECs for the
transient expression of membrane-bound fractalkine without
up-regulation of ICAM-1 and VCAM-1 expression (Fig. 7). We examined their susceptibility to
NK cell-mediated lysis and found that transfection of fractalkine cDNA
markedly increased the susceptibility of HUVECs to NK cell-mediated
cytolysis compared with vector transfectants. Moreover,
affinity-purified Ab against CX3CR1 strongly
decreased NK cell cytotoxicity against fractalkine-transfected HUVECs,
whereas no effect of anti-CX3CR1 Ab was
observed on cytolysis of control transfectants (Fig. 8).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A) and
adhered to intact fractalkine immobilized on glass slides or
membrane-bound fractalkine expressed on ECs, and that such adherence
did not require the participation of other adhesion molecules, such as
integrins (Figs. 1B and 5). Moreover, both types of adhesion
were effectively inhibited by s-fractalkine, used as a competitor to
dissect the interaction of fractalkine and CX3CR1
on NK cells (Figs. 1B and 5), indicating that fractalkine
functions as an adhesion molecule for NK cells. Our results are
consistent with the recent studies by Fong et al. (44) and
Haskell et al. (45) that fractalkine-mediated adhesion
under physiologic flow is integrin independent.
Because several cytokines (IL-2, IFN-, and IL-12) and CC-chemokines
(macrophage inflammatory protein-1; IFN--inducible protein-10;
MCP-1, -2, and -3; and RANTES) have been reported to enhance NK
cytotoxicity and granular exocytosis (33, 46, 47), we
examined the functional roles of fractalkine on NK cell activity.
Although the effects of fractalkine were modest compared with those of
rIL-2, s-fractalkine significantly enhanced NK cell-mediated
cytotoxicity against K562 target cells and granular exocytosis from NK
cells in a dose- and time-dependent manner (Figs. 2 and 3). At present,
the mechanisms of fractalkine-mediated enhancement of NK cell activity
are not clear. However, NK cell functions, such as cytotoxicity and
granular exocytosis, are mediated by activation of phospholipase C,
protein tyrosine kinases (PTKs), and phosphatidylinositol 3-kinase (PI
3-kinase) (28, 32), and it has been reported that the
chemokine receptors identified to date, including
CX3CR1, all manifest a seven-transmembrane, G
protein-linked architecture (20, 21, 24, 27, 48) and
transduce signals for PTKs and PI 3-kinase activation that lead to
increased adhesion and migration of the cells (49, 50, 51, 52).
One possible explanation for enhancement of NK cell cytotoxicity is G
protein-mediated activation of PTKs and PI 3-kinase by fractalkine,
despite the fact that fractalkine-mediated cell adhesion is independent
of G proteins (27, 45). In support of this idea, we found
that pertussis toxin, an inhibitor of Gi,
markedly inhibited the fractalkine-mediated granular exocytosis in NK
cells (Fig. 3C).
Systemic therapy with IL-2 or with adoptively transferred LAK cells has
exhibited significant systemic toxicity and multiorgan dysfunction in
human. These effects have been attributed to VLS, in which
proteinaceous vascular fluid leaks across the endothelial barrier into
the interstitium of various organs, including the lungs, liver, and
spleen, and also into the interstitial space of connective tissues
(6, 7, 8, 9, 10). Although IL-2 promotes the adhesion of NK cells
to ECs and enhances the susceptibility of ECs to NK cell- or LAK
cell-mediated cytolysis, several IL-2-inducible cytokines, including
TNF-, IL-1ß, and IFN- do not show any direct effect on
endothelial permeability (11, 12). Therefore, direct
cell-to-cell contact between NK or LAK cells and ECs is thought to be
necessary to cause VLS. Because fractalkine is expressed on EC
membranes after stimulation by TNF- or IL-1 and promotes strong
adhesion of NK cells to ECs, we postulate that fractalkine is likely to
be involved in NK cell-mediated vascular damage. However, the
cytokine-activated HUVECs express high levels of ICAM-1 and VCAM-1 as
well as exhibit de novo expression of fractalkine (Fig. 7), producing a
highly complex pattern of adhesion pathways between NK cells and
activated HUVECs. To assess the role of membrane-bound fractalkine in
the adhesion of NK cells to endothelial cells and NK cell-mediated
endothelial cell damage in a nonconfounded system, we have established
a fractalkine-transfected subline (FRK-ECV) that constitutively
expresses membrane-bound fractalkine without significant changes in the
expression of other adhesion molecules (Fig. 4). We have clearly
demonstrated that FRK-ECV expressing the membrane-bound fractalkine
showed increased adhesion of NK cells and susceptibility to NK
cell-mediated cytolysis compared with control-ECV (Figs. 5 and 6A). Moreover, s-fractalkine, which efficiently dissects the
interaction of membrane-bound fractalkine on ECs and
CX3CR1 on NK cells (Fig. 5), significantly
inhibited NK cell-mediated FRK-ECV lysis (Fig. 6B). Finally,
we also examined the involvement of membrane-bound fractalkine in NK
cell-mediated endothelial cell damage in a more native setting by
transfection of fractalkine cDNA in primary HUVECs (Fig. 7). The
results indicated that fractalkine-transfected HUVECs exhibited
increased susceptibility against NK cell-mediated cytolysis compared
with vector transfectants (Fig. 8). Although our data show that
fractalkine-mediated adhesion and susceptibility of endothelial cells
do not necessitate the participation of other adhesion molecules, it is
possible that cellular interactions stabilized through other adhesion
molecules, e.g., LFA-1/ICAM-1 and VLA-4/VCAM-1, which are induced on
ECs by TNF- and IL-1, may be involved in vascular damage in
vivo.
As predicted by the missing self theory of immunosurveillance,
initially proposed by Ljunggren and Karre (53), several
families of NK inhibitory receptors that recognize allelic forms of MHC
class I molecules generate signals that inhibit NK cell-mediated
cytotoxicity (3). Therefore, autologous endothelial cells
are considered to be protected from NK cell-mediated lytic attack by
expression of MHC class I molecules. However, viruses, bacteria and
their products, ischemia-reperfusion, or IL-2 administration directly
or indirectly activate ECs and induce cytokine production, such as
IL-1, TNF-, and IL-6 (13, 14, 15, 16), resulting in induction
of fractalkine as well as ICAM-1 and VCAM-1 on the EC membrane.
Although the mechanisms underlying endothelial damage in various
pathological states are not clear, NK cells may be able to damage the
vascular endothelium despite the presence of autologous MHC in these
conditions, where fractalkine on NK cells acts not only as an adhesion
molecule, but also as a regulator in cooperation with other adhesion
molecules and cytokines. Recently, Chen et al. (54) have
reported that a viral protein, vMIP-II, encoded by human herpesvirus 8,
has antagonistic activity for CC, CXC, and CX3C
chemokine receptors and anti-inflammatory activity in an
experimental glomerulonephritis in rat model system. Although
additional studies are needed, these findings suggest that the use of
fractalkine antagonists might be therapeutically useful for endothelial
injury in various pathological states, including acute inflammatory
reaction, graft-vs-host disease, and VLS.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Hisanori Umehara, 8-1 Kuzuha Hanazono-cho, Hirakata-shi, Osaka 573-1121, Japan.
3 Abbreviations used in this paper: LAK, lymphokine-activated killer; VLS, vascular leak syndrome; EC, endothelial cell; s-fractalkine, soluble fractalkine; SEAP, secreted form of placental alkaline phosphatase; FRK-ECV, fractalkine-transfected ECV304 cell; PTX, pertussis toxin; VLA, very late Ag; MCP, monocyte chemoattractant protein; PTK, protein tyrosine kinase; PI 3-kinase, phosphatidylinositol 3-kinase; BLTE, N-benzyloxycarbonyl-L-lysine thiobenzyl ester.
4 After completing the experiments using ECV304 cell lines and fractalkine-transfected ECV304 cells, American Type Culture Collection announced that ECV-304 should be considered a variant of human bladder cancer line T-24 derived by cross-contamination.
Received for publication August 25, 1999. Accepted for publication February 4, 2000.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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V. Faure, C. Cerini, P. Paul, Y. Berland, F. Dignat-George, and P. Brunet The uremic solute p-cresol decreases leukocyte transendothelial migration in vitro Int. Immunol., October 1, 2006; 18(10): 1453 - 1459. [Abstract] [Full Text] [PDF]
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S. Uehara, C. M. Chase, W. H. Kitchens, H. S. Rose, R. B. Colvin, P. S. Russell, and J. C. Madsen NK Cells Can Trigger Allograft Vasculopathy: The Role of Hybrid Resistance in Solid Organ Allografts J. Immunol., September 1, 2005; 175(5): 3424 - 3430. [Abstract] [Full Text] [PDF]
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A. Bielawska-Pohl, C. Crola, A. Caignard, C. Gaudin, D. Dus, C. Kieda, and S. Chouaib Human NK Cells Lyse Organ-Specific Endothelial Cells: Analysis of Adhesion and Cytotoxic Mechanisms J. Immunol., May 1, 2005; 174(9): 5573 - 5582. [Abstract] [Full Text] [PDF]
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E. Lavergne, J. Labreuche, M. Daoudi, P. Debre, F. Cambien, P. Deterre, P. Amarenco, C. Combadiere, and on Behalf of the GENIC Investigators Adverse Associations Between CX3CR1 Polymorphisms and Risk of Cardiovascular or Cerebrovascular Disease Arterioscler Thromb Vasc Biol, April 1, 2005; 25(4): 847 - 853. [Abstract] [Full Text] [PDF]
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T. Nanki, Y. Urasaki, T. Imai, M. Nishimura, K. Muramoto, T. Kubota, and N. Miyasaka Inhibition of Fractalkine Ameliorates Murine Collagen-Induced Arthritis J. Immunol., December 1, 2004; 173(11): 7010 - 7016. [Abstract] [Full Text] [PDF]
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S. Y. Ahn, C.-H. Cho, K.-G. Park, H. J. Lee, S. Lee, S. K. Park, I.-K. Lee, and G. Y. Koh Tumor Necrosis Factor-{alpha} Induces Fractalkine Expression Preferentially in Arterial Endothelial Cells and Mithramycin A Suppresses TNF-{alpha}-Induced Fractalkine Expression Am. J. Pathol., May 1, 2004; 164(5): 1663 - 1672. [Abstract] [Full Text] [PDF]
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A. Schafer, C. Schulz, M. Eigenthaler, D. Fraccarollo, A. Kobsar, M. Gawaz, G. Ertl, U. Walter, and J. Bauersachs Novel role of the membrane-bound chemokine fractalkine in platelet activation and adhesion Blood, January 15, 2004; 103(2): 407 - 412. [Abstract] [Full Text] [PDF]
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S. Vitale, A. Schmid-Alliana, V. Breuil, M. Pomeranz, M.-A. Millet, B. Rossi, and H. Schmid-Antomarchi Soluble Fractalkine Prevents Monocyte Chemoattractant Protein-1-Induced Monocyte Migration via Inhibition of Stress-Activated Protein Kinase 2/p38 and Matrix Metalloproteinase Activities J. Immunol., January 1, 2004; 172(1): 585 - 592. [Abstract] [Full Text] [PDF]
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H. Umehara, E. T. Bloom, T. Okazaki, Y. Nagano, O. Yoshie, and T. Imai Fractalkine in Vascular Biology: From Basic Research to Clinical Disease Arterioscler Thromb Vasc Biol, January 1, 2004; 24(1): 34 - 40. [Abstract] [Full Text] [PDF]
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J. Barlic, J. M. Sechler, and P. M. Murphy IL-15 and IL-2 oppositely regulate expression of the chemokine receptor CX3CR1 Blood, November 15, 2003; 102(10): 3494 - 3503. [Abstract] [Full Text] [PDF]
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J.-Q. Gao, Y. Tsuda, K. Katayama, T. Nakayama, Y. Hatanaka, Y. Tani, H. Mizuguchi, T. Hayakawa, O. Yoshie, Y. Tsutsumi, et al. Antitumor Effect by Interleukin-11 Receptor {alpha}-Locus Chemokine/CCL27, Introduced into Tumor Cells through a Recombinant Adenovirus Vector Cancer Res., August 1, 2003; 63(15): 4420 - 4425. [Abstract] [Full Text] [PDF]
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 E. R. Sherwood, C. Y. Lin, W. Tao, C. A. Hartmann, J. E. Dujon, A. J. French, and T. K. Varma {beta}2 Microglobulin Knockout Mice Are Resistant to Lethal Intraabdominal Sepsis Am. J. Respir. Crit. Care Med., June 15, 2003; 167(12): 1641 - 1649. [Abstract] [Full Text] [PDF]
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A. Obata-Onai, S.-i. Hashimoto, N. Onai, M. Kurachi, S. Nagai, K.-i. Shizuno, T. Nagahata, and K. Matsushima Comprehensive gene expression analysis of human NK cells and CD8+ T lymphocytes Int. Immunol., October 1, 2002; 14(10): 1085 - 1098. [Abstract] [Full Text] [PDF]
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Y. Hayakawa, K. Takeda, H. Yagita, M. J. Smyth, L. Van Kaer, K. Okumura, and I. Saiki IFN-gamma -mediated inhibition of tumor angiogenesis by natural killer T-cell ligand, alpha -galactosylceramide Blood, August 13, 2002; 100(5): 1728 - 1733. [Abstract] [Full Text] [PDF]
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D. L. Hodge, W. B. Schill, J. M. Wang, I. Blanca, D. A. Reynolds, J. R. Ortaldo, and H. A. Young IL-2 and IL-12 Alter NK Cell Responsiveness to IFN-{gamma}-Inducible Protein 10 by Down-Regulating CXCR3 Expression J. Immunol., June 15, 2002; 168(12): 6090 - 6098. [Abstract] [Full Text] [PDF]
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M. Nishimura, H. Umehara, T. Nakayama, O. Yoneda, K. Hieshima, M. Kakizaki, N. Dohmae, O. Yoshie, and T. Imai Dual Functions of Fractalkine/CX3C Ligand 1 in Trafficking of Perforin+/Granzyme B+ Cytotoxic Effector Lymphocytes That Are Defined by CX3CR1 Expression J. Immunol., June 15, 2002; 168(12): 6173 - 6180. [Abstract] [Full Text] [PDF]
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 Q. Zhang, K. Shimoya, Y. Ohta, R. Chin, K. Tenma, S. Isaka, H. Nakamura, M. Koyama, C. Azuma, and Y. Murata Detection of fractalkine in human seminal plasma and its role in infertile patients Hum. Reprod., June 1, 2002; 17(6): 1560 - 1564. [Abstract] [Full Text] [PDF]
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A. M. Fong, S. M. Alam, T. Imai, B. Haribabu, and D. D. Patel CX3CR1 Tyrosine Sulfation Enhances Fractalkine-induced Cell Adhesion J. Biol. Chem., May 24, 2002; 277(22): 19418 - 19423. [Abstract] [Full Text] [PDF]
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 K. Fujimoto, T. Imaizumi, H. Yoshida, S. Takanashi, K. Okumura, and K. Satoh Interferon-gamma Stimulates Fractalkine Expression in Human Bronchial Epithelial Cells and Regulates Mononuclear Cell Adherence Am. J. Respir. Cell Mol. Biol., August 1, 2001; 25(2): 233 - 238. [Abstract] [Full Text] [PDF]
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D. H. McDermott, J. P.J. Halcox, W. H. Schenke, M. A. Waclawiw, M. N. Merrell, N. Epstein, A. A. Quyyumi, and P. M. Murphy Association Between Polymorphism in the Chemokine Receptor CX3CR1 and Coronary Vascular Endothelial Dysfunction and Atherosclerosis Circ. Res., August 31, 2001; 89(5): 401 - 407. [Abstract] [Full Text] [PDF]
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25%. C, Comparison of
effect of s-fractalkine on NK cell cytotoxicity with those of MCP-1,
RANTES, and IL-2. Data are expressed as the mean ± SEM for
relative increase in 20% lytic units in 107 effector cells
and are representative of three independent experiments. *,
p < 0.05; **, p <
0.01.
