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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 7 (April 1), 1999:
pp. 2173-2185
Metalloproteinases Are Involved in Lipopolysaccharide- and Tumor
Necrosis Factor- -Mediated Regulation of CXCR1 and CXCR2
Chemokine Receptor Expression
By
Masud H. Khandaker,
Gordon Mitchell,
Luoling Xu,
Joseph D. Andrews,
Rajkumari Singh,
Harry Leung,
Joaquín Madrenas,
Stephen S.G. Ferguson,
Ross D. Feldman, and
David J. Kelvin
From the Departments of Microbiology and Immunology, Medicine,
Pharmacology and Toxicology, and Zoology, The University of Western
Ontario and the London Health Sciences Centre, London; and the
Laboratory of Molecular Immunology and Inflammation, John P. Robarts
Research Institute, London, Ontario, Canada.
 |
ABSTRACT |
The neutrophil-specific G-protein-coupled chemokine receptors,
CXCR1 and CXCR2, bind with high affinity to the potent chemoattractant interleukin-8 (IL-8). The mechanisms of IL-8 receptor regulation are
not well defined, although previous studies have suggested a process of
ligand-promoted internalization as a putative regulatory pathway.
Herein, we provide evidence for two distinct processes of CXCR1 and
CXCR2 regulation. Confocal microscopy data showed a redistribution of
CXCR1 expression from the cell surface of neutrophils to internal
compartments after stimulation with IL-8, whereas stimulation with
bacterial lipopolysaccharide (LPS) or tumor necrosis factor-
(TNF-) did not induce CXCR1 internalization but instead mediated a
significant loss of membrane-proximal CXCR1 staining intensity. To
investigate whether proteolytic cleavage was the mechanism responsible
for LPS- and TNF--induced downmodulation of IL-8 receptors, we
tested a panel of proteinase inhibitors. The downmodulation of CXCR1
and CXCR2 by LPS and TNF- was most dramatically inhibited by
metalloproteinase inhibitors; 1,10-phenanthroline and EDTA
significantly attenuated LPS- and TNF--induced loss of CXCR1 and
CXCR2 cell surface expression. Metalloproteinase inhibitors also
blocked the release of CXCR1 cleavage fragments into the cell
supernatants of LPS- and TNF--stimulated neutrophils. In addition,
while treatment of neutrophils with LPS and TNF- inhibited IL-8
receptor-mediated calcium mobilization and IL-8-directed neutrophil
chemotaxis, both 1,10-phenanthroline and EDTA blocked these inhibitory
processes. In contrast, metalloproteinase inhibitors did not affect
IL-8-mediated downmodulation of CXCR1 and CXCR2 cell surface
expression or receptor signaling. Thus, these findings may provide
further insight into the mechanisms of leukocyte regulation during
immunologic and inflammatory responses.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
THE RESPONSE OF LEUKOCYTES to
chemoattractants is a central phenomenon in the inflammatory and
immunologic response. Chemokines are a large family of small
proinflammatory peptides now thought to regulate the activation and
migration of leukocytes to sites of inflammation and
infection.1-5 Neutrophils are generally acted upon by CXC
or  chemokines such as interleukin-8 (IL-8), while CC or  chemokines exhibit activity on multiple leukocyte populations including
monocytes, T lymphocytes, basophils, and eosinophils.1,3
Chemokines exert an effect by interacting with a superfamily of
heptahelical, rhodopsin-like, G-protein-coupled
receptors.3,6 Neutrophils express two chemokine receptors
for IL-8, CXCR1 (IL-8RA) and CXCR2 (IL-8RB). CXCR1 binds selectively to
IL-8 and GCP-2 with high affinity,7-9 while CXCR2 binds
with high affinity to IL-8 and to other CXC chemokines, including
neutrophil-activating peptide-2 and melanoma growth-stimulating
activator.10,11
IL-8-directed neutrophil activation and migration has been shown to be
regulated by the internalization and subsequent reexpression of CXCR1
and CXCR2.7,12,13 However, the expression of CXCR1 and
CXCR2 can also be regulated by other immunomodulators such as bacterial
lipopolysaccharide (LPS) and the proinflammatory cytokine tumor
necrosis factor- (TNF-).14 We have recently observed
that LPS-mediated downmodulation of both CXCR1 and CXCR2 occurs through
a previously unidentified ligand-independent, tyrosine kinase-dependent pathway that may be used in TNF--induced
downmodulation but is mechanistically distinct from IL-8-mediated
internalization of CXCR1 and CXCR2.15
Bacterial endotoxin has gained interest because of its implication in
the clinical syndrome of Gram-negative bacterial septic shock.16-18 Bacterial endotoxin can initiate a
pathophysiologic cascade characterized by an increased expression of
adhesion molecules and the release of cytokines including TNF-,
chemotactic recruitment of lymphoid cells, release of reactive oxygen
species, multiple organ failure, and persistence of
bacteremia.19-22 Thus, the significance of LPS-mediated
downregulation of CXCR1 and CXCR2 may be to serve as a mechanism of
immune evasion used by bacteria whereby alteration of chemokine
receptor expression interferes with the migration of neutrophils to
sites of bacterial infection. However, the precise mechanism of
receptor downregulation has not yet been determined.
Various studies have recently implicated the activity of
metalloproteinases and serine proteinases in the cleavage of cell surface molecules. Studies by Bazil and Strominger23 using
inhibitors of metalloproteinases (1,10-phenanthroline) and serine
proteinases (TLCK and 3,4-dichloroisocoumarin) demonstrated that CD43,
CD44, and CD16 are enzymatically cleaved from the surface of phorbol myristate acetate (PMA) stimulated leukocytes. Reports by various groups have shown that hydroxamic acid-based metalloproteinase inhibitors can attenuate the proteolytic release of
TNF-24-27 and L-selectin28-30 from the
surface of leukocytes. Thus, in an effort to determine the mechanism of
LPS-, TNF--, and IL-8-induced downmodulation of the neutrophil
chemokine receptors CXCR1 and CXCR2, we investigated the role of
proteinases in this process.
Herein, we provide the first evidence to suggest that the proteolytic
activity of metalloproteinases is involved in LPS- and TNF-- but
not IL-8-induced downmodulation of CXCR1 and CXCR2 chemokine receptor
expression. Metalloproteinases have not been previously described to
play a role in chemokine receptor expression, and thus, these data may
provide novel insight into the mechanism of chemokine receptor
regulation in various inflammatory diseases.
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MATERIALS AND METHODS |
Reagents.
Escherichia coli LPS (055:B5) was purchased from Difco
Laboratories (Detroit, MI). IL-8 and TNF- were purchased from Pepro Tech Inc (Rocky Hill, NJ). EDTA, EGTA, 1,10-phenanthroline, TLCK (N-p-tosyl-L-lysine chloromethyl
ketone), 3,4-dichloroisocoumarin, leupeptin, aprotinin,
1-antitrypsin, bestatin, phosphoramidon, trypan blue,
and propidium iodide were purchased from Sigma Chemical Co (St Louis,
MO). Pepstatin A was purchased from Calbiochem (La Jolla, CA).
FITC-conjugated anti-CXCR1 and PE-conjugated anti-CXCR2 antibodies
(Abs) were purchased from Pharmingen (San Diego, CA).
Isolation of leukocytes.
Peripheral blood leukocytes enriched for mononuclear cells or
granulocytes were obtained from healthy donors. Granulocytes were
purified by dextran sedimentation followed by Ficoll gradient centrifugation and hypotonic lysis of red blood cells.
Polymorphonuclear leukocytes (PMNs) were collected, washed in
phosphate-buffered saline (PBS), and resuspended at 5 × 106/mL in RPMI 1640 supplemented with 10% fetal calf serum
(unless otherwise indicated). The purity of PMN preparations was judged to be greater than 95% by morphologic criteria; the remaining cells
were typically lymphocytes.
Confocal microscopy.
Isolated neutrophils were incubated in 24-well tissue culture plates
(Nunc Plastics, Roskilde, Denmark) at 37°C for 1 hour in the absence
or presence of IL-8 (500 ng/mL), LPS (100 ng/mL), or TNF- (50 ng/mL). The cells were then washed twice with PBS, resuspended in 100 µL PBS, and fixed in an equal volume of 4% paraformaldehyde for 30 minutes at room temperature. After washing and resuspending the cells
in 100 µL PBS, the cells were permeabilized with an equal volume of
cold 0.1% Triton X-100 for 2 minutes on ice. Cells were again washed
twice and resuspended in 100 µL cold PBS and then incubated with an
optimal concentration of FITC-conjugated anti-CXCR1 Ab. Epifluorescence
was observed with a Zeiss Photomicroscope II (Zeiss,
Thornwood, NY) using an FITC filter. Confocal microscopy and image
reconstruction was performed using a BioRad (Richmond, CA) MRC 600 confocal argon/krypton laser-scanning microscope. Preparations were
photographed on Kodak (Eastman Kodak, Rochester, NY) Tri X Pan 35-mm
film. Luminosity analysis was performed using SigmaScan
Pro software (Chicago, IL).
Measurement of CXCR1 and CXCR2 surface expression.
Isolated neutrophils were preincubated with various inhibitors (as
indicated in the Figures) at 37°C for 30 minutes followed by LPS,
IL-8, or TNF- stimulation. The cells were washed twice with PBS and
then incubated with optimal concentrations of FITC-conjugated anti-CXCR1 or PE-conjugated anti-CXCR2 Abs for 1 hour at 4°C. They
were then washed with PBS and resuspended at 5 × 106/mL
for analysis on a FACScan flow cytometer (Becton Dickinson, San Jose, CA). LYSYS software was used to
acquire samples. CELLQUEST software (San Jose, CA) was
used to analyze electronically gated populations of live cells.
Calculation of percent inhibition of CXCR1 and CXCR2 downmodulation.
The calculation of percent inhibition of receptor downmodulation was
performed as previously described by Bazil and Strominger23 as follows: (mean fluorescence intensity [MFI] of cells treated with
inhibitors plus LPS/TNF-/IL-8  MFI of LPS/TNF-/IL-8-treated cells)/(MFI of untreated control cells MFI of
LPS/TNF-/IL-8-treated cells). Cells incubated in media alone were
used as the control.
Immunoblotting analysis.
Purified peripheral blood PMNs (10 × 106/mL)
resuspended in RPMI (10% fetal calf serum) were preincubated with
1,10-phenanthroline (0.5 mmol/L) at 37°C for 30 minutes followed by
stimulation with LPS (100 ng/mL) or TNF- (50 ng/mL) for 1 hour at
37°C. Cells were then centrifuged at 750g for 5 seconds, and
1 mL supernatant was removed. An additional centrifugation step removed
any remaining cells. The isolated supernatants were then heated to
100°C for 5 minutes. Twenty microliters of each supernatant sample
was added to sample buffer (8% sodium dodecyl sulfate [SDS], 8%
2-mercaptoethanol, 250 mmol/L Tris, pH 6.8, 40% glycerol, and 2%
bromphenol blue) and loaded onto 10% SDS-polyacrylamide gels. The
proteins were separated and transferred electrophoretically to
polyvinylidene fluoride (PVDF) membranes (Millipore Corp, Bedford, MA).
PVDF membranes were immunoblotted with polyclonal Abs to
the carboxy-terminal regions of CXCR1 and CXCR2 (Santa
Cruz Biotechnologies, Santa Cruz, CA). Signal detection was performed
using enhanced chemiluminescence reagents (Amersham, Cleveland, OH).
Measurement of [Ca2+]i.
[Ca2+]i in Indo-1AM-loaded cells was
monitored using a dual-wavelength fluorimeter (model RF-M2004; Photon
Technology International, Indianapolis, IN). Human PMNs
(5 × 106/mL) were preincubated with various proteinase
inhibitors (as indicated in the Figures) for 30 minutes at 37°C
followed by addition of LPS (100 ng/mL), TNF- (50 ng/mL), or IL-8
(500 ng/mL) for a further 1 hour at 37°C in medium containing 5 µmol/L Indo-1AM (Molecular Probes Inc, Eugene, OR). The cells were
then washed once with RPMI and resuspended in Hanks balanced salt
solution containing Ca2+ (1 mmol/L).
[Ca2+]i in Indo-1AM-loaded cells was
monitored with the excitation wavelength at 355 nm and emission
wavelength at 405 and 485 nm to detect bound and free Indo-1, respectively.
Neutrophil chemotaxis assay.
Neutrophil migration was evaluated using a 48-well microchamber
technique.31 A 25-µL aliquot of IL-8 (50 ng/mL) diluted in chemotaxis medium (RPMI 1640 containing 1 mg/mL BSA and 25 mmol/L
HEPES) was placed in the lower wells of the chamber (Neuroprobe, Cabin
John, MD), and a 50-µL cell suspension (1.5 × 106) in
the same medium was placed in the upper well. The upper and lower wells
were separated by a 5-µm pore size polycarbonate filter (Nucleopore,
Pleasanton, CA). After incubation at 37°C for 90 minutes, the filter
was removed, fixed, and stained with Diff-Quik (Harleco, Gibbstown,
NJ). The number of migrating cells in three high-powered fields
(400×) was counted after coding the samples. The results are
expressed as the mean number of migrating cells (mean ± SEM) per
high-power field in the area.
Cell viability.
Trypan blue dye-exclusion assays were performed on all PMN cultures to
assess cell viability following treatment with LPS, TNF-, or IL-8.
In addition, cells were stained with propidium iodide (50 µg/mL)
prior to analysis on a FACScan flow cytometer. Positively stained cells
for propidium iodide indicated the dead cell population.
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RESULTS |
Distribution of CXCR1 chemokine receptors using confocal microscopy.
Previous studies13,32 using radiolabeled
[125I]IL-8 binding assays have suggested that IL-8
receptors are rapidly internalized upon IL-8 binding, although visual
evidence in human PMNs has thus far not been reported. We have recently
observed that LPS and TNF- stimulations induce a rapid loss of CXCR1
and CXCR2 cell surface expression in human PMNs by a unique mechanism
distinct from IL-8-mediated internalization and dependent on tyrosine
kinase activation.15 To visually illustrate that the
mechanism of LPS- and TNF--mediated downregulation of IL-8
receptors differed from that of IL-8-induced internalization, we used
confocal microscopy techniques. A representative photograph of CXCR1
distribution in untreated neutrophils is shown in Fig 1A. CXCR1
fluorescence was rapidly redistributed from membrane-proximal regions
to internal cytoplasmic regions upon IL-8 stimulation, substantiating
previously observed data suggesting IL-8-induced internalization of
IL-8 receptors. However, CXCR1 fluorescence was not redistributed into internal regions of the cell upon LPS and TNF- stimulation; instead, CXCR1 fluorescence intensity was dramatically decreased at the cell
surface (Fig 1A). Figure 1B represents the
average intensity of CXCR1 staining on the membrane versus within the
cytoplasm for multiple cells. Again, the data indicate the presence of
CXCR1 internalized within the cytoplasm of IL-8- but not LPS- or
TNF--stimulated neutrophils. Thus, these studies further suggest
that the mechanism of LPS- and TNF--induced downmodulation of IL-8
receptors is not via receptor internalization as shown for
IL-8-mediated regulation.
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| Fig 1.
Distribution of CXCR1 expression on IL-8-, LPS-, and
TNF--treated neutrophils. Purified peripheral blood PMNs were
incubated for 1 hour at 37°C in media alone (RPMI/10% fetal calf
serum) or stimulated with IL-8 (500 ng/mL), LPS (100 ng/mL), or TNF-
(50 ng/mL). Cells were then stained with FITC-conjugated Ab to CXCR1
and examined by confocal microscopy using an oil immersion lens at
600× magnification. (A) Cellular distribution of maximal CXCR1
fluorescence for each treatment is shown at left, and transmission
light microscopy of the same cell is shown at right, with size bars
representing 10 µm. (B) Mean membrane v cytoplasm CXCR1
staining intensity is plotted for n = 15 (±SEM) cells per
treatment group. *Statistical significance (P < .05) using
1-way ANOVA for membrane luminosity of control untreated group
v treated groups. **Statistically significant increase
(P < .05; one-way ANOVA) in cytoplasm luminosity v
membrane luminosity of IL-8-treated cells.
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Inhibition of LPS- and TNF-- but not IL-8-induced
downmodulation of CXCR1 and CXCR2 expression by the metalloproteinase
inhibitors 1,10-phenanthroline and EDTA.
To further elucidate the mechanism of CXCR1 and CXCR2 cell surface
downmodulation by LPS and TNF-, we investigated whether proteinases
are involved in receptor downmodulation. As previously demonstrated,
LPS, TNF-, and IL-8 all induced a rapid decrease in
immunofluorescent staining of cell surface CXCR1 and CXCR2 on human
neutrophils (Fig 2). The metalloproteinase inhibitors 1,10-phenanthroline and EDTA markedly attenuated LPS- and
TNF--mediated loss of CXCR1 and CXCR2 expression. However, they had
no effect on IL-8-induced loss of CXCR1 and CXCR2 expression (Fig
2). In addition, the histogram distribution of CXCR1 and CXCR2 expression in
neutrophils treated with metalloproteinase inhibitors plus LPS or
TNF- resembled the histogram distribution of untreated neutrophils,
again indicating an inhibition of downmodulation by metalloproteinase
inhibitors. The inhibition of LPS- and TNF--induced CXCR1 and CXCR2
downmodulation by 1,10-phenanthroline and EDTA was dose dependent as
shown in Fig 4. The doses that have been reported to be most effective
at inhibiting enzymatic biological activity33 were also the
doses most effective at inhibiting CXCR1 and CXCR2 downmodulation by
LPS and TNF-. These data suggest that the activation of
metalloproteinases is important for LPS- and TNF--induced
downmodulation of CXCR1 and CXCR2 chemokine receptor expression.
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| Fig 2.
Effect of the metalloproteinase inhibitors
1,10-phenanthroline and EDTA on LPS-, TNF--, and IL-8-induced
downmodulation of CXCR1 and CXCR2. Purified peripheral blood
PMNs were preincubated with (A,B) 1,10-phenanthroline (Phen, 0.5 mmol/L) or (C,D) EDTA (5 mmol/L) for 30 minutes in media (RPMI/10%
fetal calf serum) followed by the addition of LPS (100 ng/mL), TNF-
(50 ng/mL), or IL-8 (500 ng/mL) for 1 hour at 37°C. CXCR1 and CXCR2
expression was measured cytofluorometrically. The x-axis indicates
fluorescence intensity measured on log10 scale, and the
y-axis indicates event counts per channel on a linear scale. MFI values
for individual histograms are indicated above each histogram.
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Aminopeptidase inhibitor bestatin does not inhibit LPS- and
TNF--induced downmodulation of CXCR1 or CXCR2
expression.
Previous reports by Bhattacharya et al14 indicated that a
Ca2+-dependent aminopeptidase was responsible for
LPS-induced proteolysis of IL-8 receptors. This was based on the
observation that the aminopeptidase inhibitor bestatin could
significantly attenuate the loss of IL-8 [125I] binding
in LPS-treated neutrophils. However, we found that bestatin had no
effect on the LPS-induced loss of cell surface CXCR1 and CXCR2 even at
biologically optimal doses. TNF--induced downregulation of CXCR1
and CXCR2 also was not affected by bestatin treatment (Figs 3 and
4). In contrast, bestatin
pretreatment moderately augmented IL-8-mediated downmodulation by
28.2% ± 8.2% for CXCR1 expression and 16.7% ± 7.1% for CXCR2
expression (Table 1). These observations suggest that the enzymatic
activity of aminopeptidases is likely not required for LPS-
or TNF--induced loss of CXCR1 and CXCR2 cell surface
expression, but may play a role in IL-8-induced internalization of CXCR1 and CXCR2.
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| Fig 3.
Effect of the aminopeptidase inhibitor bestatin on LPS-,
TNF--, and IL-8-induced downmodulation of CXCR1 and CXCR2.
Purified peripheral blood PMNs were preincubated with bestatin (100 µmol/L) for 30 minutes in media (RPMI/10% fetal calf serum) followed
by the addition of LPS (100 ng/mL), TNF- (50 ng/mL), or IL-8 (500 ng/mL) for 1 hour at 37°C. (A) CXCR1 and (B) CXCR2 expression was
measured cytofluorometrically.
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| Fig 4.
Dose-response of proteinase inhibitors on CXCR1 and CXCR2
induced downmodulation. Purified peripheral blood PMNs were
preincubated with various concentrations of (A) 1,10-phenanthroline (B)
EDTA, or (C) bestatin for 30 minutes followed by the addition of LPS
(100 ng/mL) ( ), TNF- (50 ng/mL) ( ), or IL-8 (500 ng/mL) ( )
for 1 hour at 37°C. The x-axis indicates inhibitor concentration
(µg/mL). The y-axis indicates percent inhibition of downmodulation.
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1,10-Phenanthroline blocks LPS- and TNF--stimulated
release of CXCR1 cleavage products.
To determine if the activation of metalloproteinases by LPS and TNF-
stimulation resulted in the liberation of CXCR1 or CXCR2 cleavage
products, we performed an immunoblotting analysis. Proteins isolated
from neutrophil supernatants were separated and electrophoretically transferred to PVDF membranes. We detected cleavage fragments in cell
supernatants of LPS- and TNF--stimulated neutrophils when membranes
were immunoblotted with Abs to the carboxy-terminal region of CXCR1,
but not CXCR2. These CXCR1 cleavage products migrated with an apparent
molecular weight of 30 to 40 kD and 20 to 25 kD (Fig
5). The metalloproteinase inhibitor
1,10-phenanthroline blocked the liberation of CXCR1 cleavage products
in LPS and TNF--stimulated neutrophils (Fig 5). These data suggest
that the loss of cell surface CXCR1 was due to proteolytic cleavage and
receptor release from the cell membrane into the extracellular
environment. These experiments do not rule out the possibility that
CXCR2 also undergoes similar cleavage, as we are currently attempting
to find Abs that identify CXCR2 cleavage products.
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| Fig 5.
1,10-Phenanthroline blocks LPS- and TNF--stimulated
release of CXCR1 cleavage products. Purified peripheral blood PMNs were
preincubated with 1,10-phenanthroline (0.5 mmol/L) for 30 minutes in
media (RPMI/10% fetal calf serum) followed by the addition of LPS (100 ng/mL) or TNF- (50 ng/mL) for 1 hour at 37°C. Cell supernatants
were isolated, and the proteins were assayed on a 10%
SDS-polyacryamide gel and electrophoretically transferred to PVDF
membranes. The PVDF membrane shown was immunoblotted with polyclonal Ab
recognizing the carboxy-terminal amino acids 341-359 of the CXCR1
molecule.
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Effect of other proteinase inhibitors on LPS-,
TNF--, and IL-8-mediated downregulation of CXCR1 and
CXCR2 expression.
To determine whether other classes of proteinases play a role in CXCR1
and CXCR2 regulation, we examined the effect of a range of proteinase
inhibitors on LPS-, TNF--, and IL-8-mediated downmodulation of
CXCR1 and CXCR2. They include serine proteinase inhibitors (3,4-dichloroisocoumarin, TLCK, leupeptin, and aprotinin), an aspartic
acid proteinase inhibitor (pepstatin A), a neutral endopeptidase inhibitor (phosphoramidon), and a selective inhibitor of elastase and
cathepsin G (1-antitrypsin). Our data indicate that the
serine proteinase inhibitors 3,4-dichloroisocoumarin and TLCK could
partially inhibit both CXCR1 and CXCR2 downmodulation induced by LPS
and TNF- (Table 1). Neither leupeptin
nor aprotinin treatment had any effect on LPS- or TNF--stimulated
loss of IL-8 receptor expression. This observation would lead us to
believe that only a specific subset of serine proteinases may be
involved in the regulation of CXCR1 and CXCR2 chemokine receptor
expression. In contrast, IL-8-mediated downregulation of both CXCR1
and CXCR2 was not substantially affected by pretreatment of neutrophils
with any of the serine proteinase inhibitors tested. These data suggest
that serine proteinase activity is not required for IL-8-induced
downregulation of CXCR1 and CXCR2.
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Table 1.
Effect of Proteinase Inhibitors on LPS-, TNF--, and
IL-8-Mediated Downmodulation of CXCR1 and CXCR2 Expression
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Inhibitors of neutral endopeptidase, a membrane-associated
metalloproteinase,34 and the azurophilic serine proteinases
elastase and cathepsin G35 did not markedly affect LPS-,
TNF--, or IL-8-mediated downregulation of CXCR1 and CXCR2
expression. While pepstatin A did not significantly inhibit LPS- or
TNF--induced downmodulation of CXCR1 and CXCR2 expression, the
extent of IL-8-induced downmodulation was moderately augmented by
pepstatin A pretreatment, suggesting the possible involvement of some
aspartic acid proteinases in ligand-induced internalization of CXCR1
and CXCR2 (Table 1).
1,10-Phenanthroline and EDTA but not bestatin restore G-protein
signaling and migration in LPS- and TNF--treated
neutrophils.
We have previously demonstrated that endotoxin treatment of neutrophils
results in inhibition of IL-8-induced neutrophil
chemotaxis,36 suggesting that CXCR1 and CXCR2 receptor
downmodulation is causally linked to the development of IL-8
hyporesponsiveness. In an attempt to determine if metalloproteinases or
aminopeptidases are involved in the downmodulation of functional IL-8
receptors, we used two functional assays of neutrophil responsiveness
to IL-8, Ca2+ mobilization and neutrophil chemotaxis.
[Ca2+]i was measured upon IL-8 stimulation of
neutrophils. Pretreatment of neutrophils with LPS, TNF-, or IL-8
resulted in an IL-8-hyporesponsive state, wherein cells did not show
an increase in [Ca2+]i when stimulated with
IL-8 (Fig 6A to C). However, pretreatment with the metalloproteinase inhibitors 1,10-phenanthroline and EDTA
restored IL-8-stimulated Ca2+ mobilization in LPS (Fig 6A)
and TNF- (Fig 6B) but not IL-8 (Fig 6C) pretreated cells. In
contrast, the aminopeptidase inhibitor bestatin had no effect on
IL-8-mediated enhancement of calcium levels with the any of the
treatments tested. Although IL-8 receptors were downmodulated by LPS,
TNF-, and IL-8, receptors for chemotactic peptide (fMLP) remained
functional (Fig 6D). These observations suggest that neutrophils were
still viable following LPS, TNF-, or IL-8 stimulation and indicate
specificity for receptor downmodulation.
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| Fig 6.
Measurement of functional IL-8 receptors by
Ca2+ mobilization. Purified peripheral blood PMNs
suspended in Indo-1AM medium were preincubated with 1,10-phenanthroline
(Phen, 0.5 mmol/L), EDTA (5 mmol/L), or bestatin (100 µmol/L) for 30 minutes at 37°C followed by the addition of LPS (100 ng/mL), TNF-
(50 ng/mL), or IL-8 (500 ng/mL) for a further 1 hour at 37°C. (A-C)
IL-8 (50 ng/mL) or (D) fMLP (5 × 10 7 mol/L) was
added to cells and Ca2+ flux was measured.
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In corroboration with the Ca2+ mobilization studies,
neutrophil migration data indicated that while IL-8-directed
neutrophil chemotaxis was significantly inhibited by LPS, TNF-, and
IL-8 pretreatment, the metalloproteinase inhibitors 1,10-phenanthroline and EDTA restored neutrophil migration in LPS- and TNF-- but not
IL-8-treated cells (Fig 7). Furthermore,
bestatin did not restore the migratory capacity of LPS-, TNF--, or
IL-8-treated neutrophils. However, among the inhibitors tested,
bestatin treatment alone consistently augmented neutrophil chemotaxis
to IL-8, suggesting a role of aminopeptidase activity in leukocyte
migration. The results of the Ca2+ mobilization studies and
chemotaxis assays together with the analyses of CXCR1 and CXCR2
chemokine receptor levels and localization collectively support the
hypothesis that LPS and TNF- mediate a distinct previously
undescribed pathway for CXCR1 and CXCR2 downmodulation involving the
proteolytic activity of metalloproteinases.
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| Fig 7.
Effect of proteinase inhibitors on IL-8-directed
neutrophil chemotaxis. Purified peripheral blood PMNs were untreated
() or preincubated with 1,10-phenanthroline (0.5 mmol/L,  ), EDTA
(5 mmol/L,  ), or bestatin (100 µmol/L,  ) for 30 minutes at
37°C followed by the addition of LPS (100 ng/mL), TNF- (50 ng/mL),
or IL-8 (500 ng/mL) for 1 hour at 37°C. The migration assay was then
performed. The data represent a single experiment from 4 performed.
Results are the mean ± SEM migrated cells counted from three
high-powered fields (400×) obtained in three replicates. *Statistical
significance (P < .05) using one-way ANOVA for control
untreated v treated groups.
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Mechanism of LPS-, TNF--, and IL-8-induced
downregulation of CXCR1 and CXCR2 is not due to cell death.
To examine whether cell death was the cause of CXCR downmodulation and
release into the extracellular environment, we performed cell viability
assays. Trypan blue dye-exclusion assays demonstrated that greater
than 95% of neutrophils were viable after 1 hour of treatment with
LPS, TNF-, or IL-8. Furthermore, there were no significant
differences in the cell viability of control unstimulated neutrophils
versus LPS-, TNF--, or IL-8-stimulated neutrophils for up to 24 hours poststimulation (data not shown). To further confirm the
viability of neutrophils following stimulation, propidium iodide
fluorescent staining for nucleic acids was used. Positive staining for
propidium iodide indicates cell death. Figure
8 shows that there was no difference in the
propidium iodide mean fluorescence intensity for unstimulated
neutrophils versus LPS-, TNF--, and IL-8-stimulated neutrophils,
although CXCR1 mean fluorescence intensity was significantly reduced in
these stimulated cells. While cell death can lead to a loss of CXCR
expression, as observed in dexamethasone-treated cells (Fig 8), it is
clear that cell death is not the mechanism of LPS-, TNF--, or
IL-8-induced downmodulation of CXCR1 and CXCR2.
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| Fig 8.
LPS-, TNF--, and IL-8-induced CXCR downregulation is
not due to cell death. Purified peripheral blood PMNs were incubated
for 3 hours at 37°C in media alone (RPMI/10% fetal calf serum) or
stimulated with LPS (100 ng/mL), TNF- (50 ng/mL), IL-8 (500 ng/mL),
or dexamethasone (100 mmol/L). Propidium iodide staining and CXCR1
staining was measured using two-color parameter flow cytometry. Data
are represented as contour plots with the x-axis indicating CXCR1
fluorescence intensity measured on a log10 scale and the
y-axis indicating propidium iodide fluorescence intensity measured on a
log10 scale. MFI values for CXCR1 staining are indicated in
the bottom right panel of each contour plot. MFI values for propidium
iodide staining are indicated in the top right panel of each contour
plot. Similar data were observed for CXCR2 expression.
|
|
| |
DISCUSSION |
In the present study, we investigated the mechanism of regulation of
the neutrophil-specific chemokine receptors CXCR1 and CXCR2 by the
immunomodulatory agents LPS, TNF-, and IL-8. We have recently
described two separate pathways of CXCR1 and CXCR2 downmodulation: (1)
a tyrosine kinase-dependent pathway induced by LPS and TNF- and (2)
a tyrosine kinase-independent pathway induced by IL-8.15
Using confocal microscopy techniques to visualize CXCR1 chemokine
receptor distribution in stimulated neutrophils, we observed that CXCR1
localizes to internal regions of the cell after stimulation with IL-8.
In contrast, the receptor localization of CXCR1 following LPS or
TNF- stimulation was qualitatively different from that observed with
IL-8: CXCR1 was predominantly localized to cell membrane-proximal
regions of the neutrophils. In addition, the membrane intensity of
CXCR1 expression was dramatically reduced, indicating a net loss of
surface receptors, possibly due to nascent degradation of
membrane-bound CXCR1 chemokine receptors.
We reasoned that since metalloproteinase inhibitors have been shown to
attenuate LPS-induced responses, such as production and cleavage of
TNF-,26,27,37 and tyrosine kinase inhibitors have been
shown to abrogate metalloproteinase activation,38-42 it was
possible that the tyrosine kinase-dependent pathway of CXCR1 and CXCR2
downmodulation initiated by LPS and TNF- stimulation involved the
proteolytic degradation of these receptors. The regulation of cell
surface expression of various integral membrane molecules has been
previously shown to involve enzymatic cleavage by various classes of
proteinases. Stimulation of leukocytes by MoAbs, cytokines, chemotactic
peptides, and PMA has been shown to induce proteolytic cleavage of IL-6
receptors,43 CD14,44 CD16,23,45
CD43,23 CD44,23 CD62L
(L-selectin),28,30 and TNF.35 The participation of metalloproteinases in the cleavage of membrane receptors has been
previously investigated through the use of 1,10-phenanthroline, a
chelator of the heavy metal ion Zn2+, and EDTA, a divalent
cation chelator, which are known inhibitors of
metalloproteinases.46 The findings presented here implicate the involvement of metalloproteinases in LPS- and TNF-- but not IL-8-induced downmodulation of CXCR1 and CXCR2. This is based on four
major observations. First, the metalloproteinase inhibitors 1,10-phenanthroline and EDTA significantly attenuated LPS- and TNF-- but not IL-8-induced loss of CXCR1 and CXCR2 cell surface expression (Fig 2 and Table 1). Second, LPS- and TNF--induced release of CXCR1 cleavage products into the cell supernatant could be
blocked by 1,10-phenanthroline treatment (Fig 5). Third, calcium mobilization studies showed that metalloproteinase inhibitors could
restore IL-8 receptor-mediated responses in LPS- and TNF--treated cells, but had no effect on IL-8-treated cells (Fig 6). Finally, metalloproteinase inhibitors reversed LPS- and TNF-- but not IL-8-induced suppression of neutrophil chemotaxis upon subsequent IL-8
administration (Fig 7). Preliminary data have also shown that
proteinase secretion is probably not the mechanism by which receptor
levels are reduced following LPS and TNF- stimulation (data not
shown). Thus, the enzymatic activity of an intracellular metalloproteinase(s) is likely required for LPS- and TNF--induced cleavage of the neutrophil chemokine receptors CXCR1 and CXCR2.
Previous reports have described the molecular weight of IL-8 receptors
to be in the range of 58 to 67 kD47; however, the predicted
molecular weight of IL-8 receptors based on the 359-amino acid
sequence is approximately 40 kD.7 This discrepancy in the
molecular size of IL-8 receptors can be accounted for by five potential
glycosylation sites: two in the N-terminal extracellular region
and three more potential glycosylation sites in the third extracellular
loop between amino acids 181 and 196.7,48 We observed that
LPS and TNF- stimulation of neutrophils induced the release of CXCR1
cleavage fragments containing the carboxy-terminal region of the
receptor. We detected the presence of a single distinct liberated
species that migrated with an apparent molecular weight of 20 to 25 kD,
as well as what appeared to be several CXCR1 cleavage fragments of
close apparent molecular weight in the range of 30 to 40 kD (Fig 5).
One possible explanation that could account for the observed CXCR1
cleavage products is that there are cleavage sites directly preceding
and following the glycosylation sites from amino acids 181 to 196 of
the CXCR1 molecule. Cleavage at these sites would produce a distinct
nonglycosylated species of approximately 20 kD and several glycosylated
species of varying but similar molecular weight in the range of 30 to
40 kD. Alternatively, previous studies have suggested that the
dissemination of membrane-associated molecules upon LPS stimulation of
monocytes may be due to membrane vesiculation.49-51 We have
preliminary data suggesting that a similar phenomenon may occur in
neutrophils whereby LPS and TNF- stimulate the release of vesicles
or microparticles containing fragments of the CXCR1 molecule into the
external environment. Our data also suggest that cell lysis and death
likely does not account for the release of CXCR1 cleavage fragments
into the extracellular environment, since trypan blue and propidium
iodide dye-exclusion assays indicated that LPS and TNF- stimulation
of neutrophils did not induce cell death (Fig 8), confirming similar
observations reported by others.52 The release of CXCR1
cleavage products could be blocked by 1,10-phenanthroline, further
suggesting the involvement of metalloproteinases in LPS- and
TNF--stimulated cleavage of IL-8 receptors.
Binding of intact functional IL-8 receptors by IL-8 triggers G-protein
signaling, Ca2+ mobilization, chemotaxis, granule
exocytosis, and respiratory burst.53-56 Calcium flux
studies demonstrated that 1,10-phenanthroline and EDTA restored
functionally active IL-8 receptors in LPS- and TNF--treated cells
(Fig 6). Likewise, metalloproteinase inhibitors significantly reversed
LPS- and TNF--mediated inhibition of neutrophil chemotaxis in
response to IL-8 (Fig 7). Thus, these data provide evidence to suggest
not only that metalloproteinase inhibitors preserve cell surface
expression of CXCR1 and CXCR2 in LPS- and TNF--treated neutrophils
but also that these preserved receptors are functionally responsive to
IL-8 stimulation. While we have found that IL-8 stimulates comparable
levels of CXCR1 and CXCR2 downmodulation, as well as comparable levels
of inhibition of calcium mobilization and neutrophil migration, to
those observed with LPS and TNF- treatment, it is clear from our
studies that the action of metalloproteinases is not involved in
ligand-dependent regulation of CXCR1 and CXCR2.
Previous reports by Bhattacharya et al14 have suggested the
possibility of the involvement of an aminopeptidase in proteolytic cleavage of IL-8 receptors induced by serum-activated LPS (SA-LPS). In
these studies, bestatin, a strong competitive inhibitor of aminopeptidases including the membrane glycoprotein
CD13,57-59 was shown to significantly inhibit
SA-LPS-induced loss of [125I]IL-8 binding to
neutrophils, suggesting that the enzyme involved in the downregulation
of IL-8 receptors is an aminopeptidase. However, studies by Kanayama et
al59 demonstrated that aminopeptidases degrade IL-8, since
treatment of neutrophils with aminopeptidases markedly decreased the
chemotactic activity of IL-8 and cleaved IL-8 from an 8-kD to a 6-kD
molecule. Since Baldwin et al60 reported that the amino
terminus of IL-8 was important for binding to IL-8 receptors and
Kanayama et al found that aminopeptidase treatment liberated the
N-terminal amino acids of IL-8, it is likely that
aminopeptidases change the receptor binding site of IL-8 through
proteolytic cleavage of the amino-terminal end of IL-8. These studies
by Kanayama et al could explain the loss of IL-8 binding induced by
SA-LPS observed by Bhattacharya et al. Since LPS has been shown to
increase the expression of aminopeptidases,59 thereby
augmenting the degradation of IL-8, treatment with bestatin could
inhibit aminopeptidase activity, thus inhibiting IL-8 degradation and
increasing [125I]IL-8 binding. To investigate whether
aminopeptidases are involved in CXCR1 and CXCR2 cleavage, we pretreated
LPS- and TNF--stimulated neutrophils with bestatin and observed
CXCR1 and CXCR2 cell surface expression. We found that bestatin had no
effect on LPS- and TNF--induced downregulation of CXCR1 and CXCR2
cell surface expression at any concentration tested (Figs 3 and 4).
However, bestatin did moderately augment IL-8-mediated downmodulation
of CXCR1 and CXCR2 (Fig 4 and Table 1). Calcium mobilization studies
also indicated that bestatin treatment did not restore IL-8
receptor-mediated signaling in neutrophils prestimulated with LPS and
TNF-. In addition, while chemotaxis studies indicated that bestatin
slightly restored the IL-8-induced chemotactic response of LPS- and
TNF--stimulated neutrophils, this could be the result of inhibition
of aminopeptidase-mediated cleavage of IL-8, since bestatin treatment
alone significantly augmented neutrophil chemotaxis (Fig 7). Thus, our
data indicate that bestatin does not prevent the loss of functional
cell surface CXCR1 and CXCR2 chemokine receptors induced by LPS and
TNF- treatment, but instead inhibits the proteolytic cleavage of
IL-8 by inactivating aminopeptidase activity, thereby increasing the
availability of ligand to bind to IL-8 receptors.
There are several candidate enzymes that may be involved in the
cleavage of CXCR1 and CXCR2. Two zinc-dependent matrix
metalloproteinases, collagenase and gelatinase, are expressed in
neutrophils in an inactive form and require serine proteinases for
activation.61-63 Recent studies have shown that both
collagenase and gelatinase are activated by LPS
stimulation64,65 and inhibited by tyrosine kinase
inhibitors.38-40,42 Nonmatrix metalloproteinases including members of the ADAM family such as TNF- converting enzyme (TACE) and
HuADAM10 are also candidate enzymes that may play a role in CXCR1 and
CXCR2 chemokine receptor downmodulation. Both TACE and HuADAM10 are
involved in TNF- cleavage,24,25,66 and HuADAM10 activity
was inhibited by 1,10-phenanthroline and EDTA.66 The third
major family of metalloproteinases includes integral membrane glycoproteins such as CD10 (neutral endopeptidase) and CD13
(aminopeptidase N), which behave as zinc-dependent metalloproteinases
and are abundant on the cell surface of neutrophils.34 Our
findings indicate that these enzymes are not involved in CXCR1 and
CXCR2 proteolysis, since their selective inhibitors did not prevent LPS- and TNF--induced downmodulation of IL-8 receptors.
In conclusion, we have demonstrated the presence of a novel pathway of
CXCR1 and CXCR2 chemokine receptor regulation mediated by LPS and
TNF- through the activation of one or more zinc-dependent proteinases. Additionally, our studies indicate that the activation of
proteinases is not involved in IL-8-mediated regulation of CXCR1 and
CXCR2. Of relevance to these findings with neutrophils, we have
evidence that LPS-stimulated activation of serine proteinases can
mediate the downmodulation of CCR2 chemokine receptor expression on
monocytes.67 Thus, it is conceivable that activation of
proteinases that can markedly and rapidly alter chemokine receptor
expression independently of ligand represents a mechanism by which the
chemotactic activity of neutrophils is reduced under conditions of high
exposure to inflammatory stimuli, thereby preventing their continued
migration and departure from the site.
| |
ACKNOWLEDGMENT |
The authors thank Anne Leaist, Dr Rahbar Rahimpour, and Luan Chau for
excellent technical assistance and Dr Bruce Gill for a critical review
of the manuscript.
| |
FOOTNOTES |
Submitted June 15, 1998; accepted November 19, 1998.
Supported by grants from the Medical Research Council of Canada,
Medical Research Council-Juvenile Diabetes Foundation International, and Heart and Stroke Foundation of Canada.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to David J. Kelvin, PhD, John P. Robarts Research Institute, University of Western Ontario, London,
Ontario, Canada, N6G 2V4; E-mail: kelvin{at}rri.on.ca.
| |
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downmodulation of CC chemokine receptor expression. (submitted)
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