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1
*
Department of Hematology and Oncology, Clinical Sciences for Pathological Organs, Graduate School of Medicine, and
College of Medical Technology, Kyoto University, Sakyo-ku, Kyoto;
National Institute of Infectious Diseases, Tokyo; and
§
Department of Pharmacology, Cancer Research Institute, Kanazawa University, Kanazawa, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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is implicated in the initiation of cytokine cascades in
various inflammatory settings. To assess the interactions of multiple
cytokines at the level of inflammatory effector cells, we examined the
effects of TNF- on the expression of two IL-8Rs (CXCR1 and CXCR2) on
polymorphonuclear leukocytes (PMNs). TNF- decreased the surface
expression of CXCR2 in a dose- and time-dependent manner. In contrast,
CXCR1 expression was not affected by TNF-. The release of CXCR2 into
the supernatant of TNF--treated PMNs was detected by immunoblotting
and immuno-slot-blot analyses, suggesting that the down-regulation of
CXCR2 was caused mainly by shedding from the cell surface. The CXCR2
down-regulation was inhibited by PMSF and aprotinin, supporting the
hypothesis that the shedding was mediated by serine protease(s). The
intracellular Ca2+ mobilization and chemotaxis in
response to IL-8 were suppressed by the pretreatment of PMNs with
TNF-, indicating that the decrease in CXCR2 was reflected in the
decreased functional responses to IL-8. In contrast, the
O2- release, which is mediated by CXCR1, was not
suppressed by TNF-. The treatment of whole blood with TNF- also
caused a significant reduction in CXCR2 and markedly suppressed
intracellular Ca2+ mobilization and chemotaxis in response
to IL-8, while enhancing the O2- release. These
findings suggest that TNF- down-regulates CXCR2 expression on PMNs
and modulates IL-8-induced biologic responses, leading to the
intravascular retention of PMNs with an enhanced production of reactive
oxygen metabolites. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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TNF- plays a pivotal role in the initiation of inflammatory
responses in various pathophysiologic settings (3, 4, 5, 6, 7). Produced
predominantly by macrophages, TNF- exerts stimulatory effects on PMN
functions such as phagocytosis (8), adhesion, degranulation (9), and
the production of reactive oxygen species (10, 11, 12). Accumulating
evidence suggests that TNF- is at the apex of a proinflammatory
cytokine cascade involving TNF-, IL-1ß, granulocyte-macrophage
colony-stimulating factor (GM-CSF), granulocyte colony-stimulating
factor (G-CSF), and IL-8 in inflammatory diseases including rheumatoid
arthritis (6, 7). However, it is not yet understood how PMN functions
are affected by the presence of multiple cytokines.
IL-8 has also been implicated as an important mediator of PMN activation in inflammatory diseases such as rheumatoid arthritis (6, 13, 14), Behçet’s disease (4), ulcerative colitis (15), idiopathic pulmonary fibrosis (16), adult respiratory distress syndrome (3), and systemic inflammatory response syndrome (17). IL-8 belongs to the C-X-C branch of the chemokine family, which also includes growth regulated gene (GRO), neutrophil-activating peptide-2 (NAP-2), and epithelial neutrophil-activating factor, 78 amino acids (ENA-78) (1, 18, 19, 20). The different effects of these C-X-C chemokines on PMN functions can be explained by their selective interactions with two high affinity receptors, CXCR1 and CXCR2 (also known as IL-8RA and IL-8RB, respectively) (21). IL-8 binds to both CXCR1 and CXCR2 (22, 23), while GRO, NAP-2, and ENA-78 bind only to CXCR2 with high affinity (24, 25).
Previous studies have examined the effects of chemoattractants such as
FMLP, LPS, and IL-8 on the functional responses through the IL-8Rs
(CXCR1 and CXCR2) of PMNs (24, 25). PMNs undergo rapid IL-8R
desensitization upon stimulation with IL-8, C5a, or FMLP (26, 27).
Intracellular Ca2+ mobilization and the chemotaxis of PMNs
induced by IL-8 are inhibited by pretreatment with C5a and FMLP (27, 28). However, the effects of nonchemotactic stimuli such as TNF-,
IL-1ß, G-CSF, and GM-CSF on the expression of IL-8R and the
functional responses to IL-8 remain unknown.
In this study, we found that TNF- down-regulates the expression of
CXCR2 on PMNs. Soluble CXCR2 was detected in the supernatant of
TNF--treated PMNs, indicating that the receptor is shed from PMNs.
TNF--pretreatment of PMNs caused decreased IL-8-induced
intracellular Ca2+ mobilization and chemotaxis, whereas the
superoxide production induced by IL-8 was increased. These results are
discussed to support the view that TNF- plays a pivotal role in
inflammatory diseases by causing intravascular retention of PMNs with
an enhanced production of reactive oxygen metabolites.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recombinant human TNF-, rIL-1ß, rGM-CSF, and rG-CSF were
kindly provided by Dainippon Pharmaceutical (Osaka, Japan), Otsuka
Pharmaceutical (Tokushima, Japan), Schering-Plough (Osaka), and Kirin
Brewery (Tokyo, Japan), respectively. Recombinant human IL-8 was
expressed and purified as described (29, 30). Bestatin (Ubenimex) was
kindly provided by Nippon Kayaku (Tokyo). Polyclonal Abs against human
anti-IL-8R, type A (anti-CXCR1 Ab) and type B (anti-CXCR2
Ab), were obtained from New Zealand White rabbits immunized with
glutathione-S-transferase-fused protein with the
extracellular domains of human CXCR1 and CXCR2 as described
(31).
Isolation of PMNs
Human PMNs were routinely isolated to >96% purity from citrated venous blood of healthy volunteers by dextran sedimentation followed by gradient centrifugation on Percoll (Pharmacia, Uppsala, Sweden) as previously described (32). The PMN viability was >98% in all experiments, as measured by trypan blue exclusion.
FACS analysis of CXCR1 and CXCR2 expression
Isolated PMNs (5 x 106/ml) were
pretreated with TNF-, IL-1 ß, GM-CSF, or G-CSF in serum-free HBSS
at 37°C in air using polypropylene microfuge tubes. In other
experiments, whole blood was pretreated with these proinflammatory
cytokines for 30 min at 37°C before PMNs were isolated. PMNs
pretreated with or without various proinflammatory cytokines were
washed twice and resuspended at 1 x 106/ml with HBSS,
blocked with human AB serum for 30 min at 4°C, and washed with cold
FACS buffer (PBS containing 0.1% azide and 0.1% BSA). Then, 1 x
106 cells were incubated with F(ab')2 fractions
of anti-CXCR1 Ab or anti-CXCR2 Ab (5 µg/ml) for 30 min at
4°C and washed twice with FACS buffer. FITC-labeled goat
anti-rabbit IgG Ab (diluted 1:100) (Zymed Laboratories, San
Francisco, CA) was added, followed by incubation for 30 min at 4°C.
After the cells were washed twice and filtered through a nylon mesh,
flow cytometric analysis was performed using FACScan flow cytometer
(Becton Dickinson, San Jose, CA) and Lysis II software (Becton
Dickinson). Data collected from 10,000 cells are presented as either
histograms or mean fluorescence intensity (MFI) values.
Western blotting and immuno-slot-blot assay
Western blotting.
PMNs (4 x 107/ml) in which CXCR2 had been
labeled by pretreatment with 5 µg/ml anti-CXCR2 Ab for 30 min
were incubated for 30 min at 37°C with or without TNF- (100 U/ml).
Supernatants obtained by centrifugation for 10 min at 250 x
g were electrophoresed in 12.5% SDS-polyacrylamide gels,
transferred to nitrocellulose membranes (Amersham, Buckinghamshire,
U.K.), and stained with horseradish (HRP)-conjugated anti-rabbit Ig
(Organon Teknika, Durham, NC). The anti-CXCR2 Ab label released
from PMNs with CXCR2 was visualized by enhanced chemiluminescence (ECL;
Amersham).
Immuno-slot-blot assay. Slot-blots were prepared using a Hybri-Slot 24-well filtration manifold apparatus (Life Technologies, Rockville, MD) (33, 34). ECL membranes, premoistened in PBS, were inserted into the filtration manifold, and 200-µl aliquots per slot of various dilutions of conditioned supernatants were applied to the ECL membranes by vacuum filtration. The membranes were then incubated with anti-CXCR2 Ab as a primary Ab and HRP-conjugated goat affinity-purified Ab to rabbit IgG as a secondary Ab. The blotted CXCR2 was visualized by ECL.
Measurement of intracellular free Ca2+ transients in PMNs
PMNs (1 x 107/ml) were incubated with 2
µM fluo-3/AM (Molecular Probes, Eugene, OR) for 20 min at 37°C in
polypropylene tubes (35), followed by the addition of TNF-, IL-1ß,
GM-CSF, or G-CSF for pretreatment. The cells were then washed twice
with PBS and resuspended in HBSS at 5 x 106/ml. The
assay was started by adding fluo-3-loaded PMNs to a thermostatted
cuvette for 2 min at 37°C. Then, IL-8 or FMLP was added, and the
fluorescence emission was monitored at 530 nm with excitation at 505 nm
by a fluorescence spectrophotometer (F-3000, Hitachi, Tokyo, Japan).
The intracellular free calcium concentration
([Ca2+]i) was calculated from the
fluorescence intensities (F) using the equation:
[Ca2+]i = Kd (F
- Fmin)/(Fmax - F). The calcium dissociation
constant of fluo-3 in the cytoplasm of PMNs
(Kd) was assumed to be 864 nmol/L at
37°C (36). Fmax was determined by lysing the PMNs with
digitonin (30 µM). Fmin was determined by adding 5 mM
EGTA (pH 7.4) to the PMN lysate. The Fmax and
Fmin values were not significantly different between
nontreated and cytokine-treated PMNs.
Chemotaxis assay
PMN chemotaxis was assayed with the use of a chemotaxis chamber (NeuroProbe, Cabin John, MD) (37, 38). The lower wells were filled with 34 µl of IL-8 (100 nM), FMLP (100 nM), or HBSS and covered with a polyvinyl pyrolidon-free polycarbonate filter (pore diameter, 3 µm; NeuroProbe), and then 100 µl of a suspension containing 5 x 105 PMNs per ml was added to each upper well. After incubation under 90% humidity and in 5% CO2 for 20 min at 37°C, the filter was removed, and the cells remaining on the upper surface of the filter were gently wiped off with PBS-wetted paper. The cells retained in the filter were fixed and stained with Diff-Quik (International Reagents, Kobe, Japan). The number of migrated cells was estimated spectrophotometrically at 600 nm with a micro-ELISA plate reader.
The resulting data are presented as the chemotactic index. The value of the chemotactic index was obtained by subtracting the estimated number of migrated cells in the presence of medium alone from the number of migrated cells in the presence of the test sample.
O2- release
The reaction mixture containing 120 µM cytochrome c, PMNs (1 x 106/ml), and 10 nM IL-8 or 100 nM FMLP in a total volume of 0.5 ml was incubated for 15 min at 37°C. The supernatant from the reaction mixture was obtained by centrifugation at 700 x g for 20 min at 4°C (39). The release of O2- from the PMNs was calculated by subtracting the absorbance change at 550 nm in the presence of 30 µg/ml superoxide dismutase (SOD) from that in its absence, and the results are expressed as nanomoles of superoxide/1 x 106 cells, which was calculated using an absorbance coefficient of 2.1 x 104 M-1 cm-1 (40).
Statistical analysis
Statistical analyses of differences in all of the data were done with Student’s t test using the software program Microsoft Excel, version 7.0 for Windows 95. The value of p was adjusted for multiple comparisons according to the Bonferroni correction.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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on the expressions of CXCR1 and CXCR2 on PMNs
To examine the effects of various proinflammatory cytokines,
including TNF-, on the expression of CXCR1 and CXCR2 on PMNs, we
performed a flow cytometric analysis using Abs against CXCR1 and CXCR2.
TNF- significantly decreased the expression of CXCR2 on the PMNs, in
a dose-dependent manner. A decrease was detected at 1 U/ml, and the
maximal effect was obtained at 100 U/ml (Fig. 1
a). The effect of
TNF- was time dependent up to 30 min (Fig. 1b). In
contrast to CXCR2, the expression of CXCR1 was not affected by the
treatment with TNF- up to 100 U/ml (Fig. 1, c and
d). Other proinflammatory cytokines (GM-CSF, G-CSF,
and IL-1ß) did not significantly affect the expression of CXCR1 or
CXCR2 (Fig. 1, c and d). Since TNF-
could induce the release of O2- from PMNs during
incubation for 30 min, we examined the effect of SOD and catalase on
the decrease in CXCR2 expression. The quenchers of reactive oxygen
species did not affect the down-regulation of CXCR2, indicating that
TNF--induced release of O2- or
H2O2 is not involved in the decrease in CXCR2
induced by TNF- (data not shown).
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To delineate whether incorporation into the cytoplasm is
responsible for the decrease in CXCR2 expression, we examined the
effects of cytochalasin B and colchicine, reagents that can block
receptor incorporation by acting on actin microfilaments and
microtubules, respectively. These agents did not affect the
down-regulation of CXCR2 by TNF- (the MFI value of CXCR2 on PMNs
pretreated with TNF- in the presence of 5 µg/ml cytochalasin
B = 267 ± 33; in the presence of 50 µM colchicine =
269 ± 32; without cytochalasin B or colchicine = 258 ±
46; n = 3), which indicated that the decrease in CXCR2
was not dependent on intracytoplasmic incorporation. Next, we examined
the possibility that the receptor is released from the surface of PMNs
to the extracellular milieu. Western blotting with anti-CXCR2 Ab
and HRP-conjugated anti-rabbit IgG did not reveal CXCR2 in the
supernatant of TNF--treated PMNs (data not shown). However, we could
not exclude the possibility that anti-CXCR2 Ab was unable to react
with CXCR2 due to degradation of the receptor after shedding. We
therefore labeled CXCR2 on PMNs with anti-CXCR2 Ab before treating
the PMNs with TNF-. This was done to allow receptor shedding to be
detected upon the release of anti-CXCR2 Ab to the supernatant. As
shown in Figure 2a,
anti-CXCR2 Ab was detected as a band with a molecular mass of about
50 kDa in the supernatant from TNF--treated PMNs. The release of
anti-CXCR2 Ab from the PMNs incubated without TNF- was
significantly lower (Fig. 2a). We next performed an
immuno-slot-blot assay to concentrate the supernatant so that a small
amount of released CXCR2 could be detected. This analysis showed that
at least twice the amount of CXCR2 was released from the
TNF--treated PMNs compared with the untreated cells (Fig. 2b). Similarly, the immuno-slot-blot analysis of
supernatants from PMNs prelabeled with anti-CXCR2 Ab before TNF-
treatment showed that twice the amount of the label was released upon
TNF- treatment (Fig. 2c). FACS analyses of PMNs
treated with TNF- under the same conditions as the immuno-slot-blot
assay showed a decrease in CXCR2 expression of approximately twice that
of the PMNs incubated without TNF-. The percentage of decrease in
MFI on treatment with and without TNF- was 
20 and 10%,
respectively (n = 3; with the MFI value upon
treatment without TNF- for 30 min at 4°C regarded as 100%). These
findings indicated that CXCR2 was down-regulated by TNF- as a result
of shedding from the surface of PMNs. To explore the possibility that
proteolytic cleavage is involved in the release of CXCR2 from PMNs, we
examined the effect of protease inhibitors. Interestingly, serine
protease inhibitors such as PMSF and aprotinin inhibited the decrease
in expression of CXCR2 on PMNs pretreated with TNF- (Table I
). The percentage of inhibition by 200
µM PMSF, 20 µM PMSF, and 1 µg/ml aprotinin was 89, 46, and 43%,
respectively. EDTA, an inhibitor of metalloproteases, and bestatin, an
inhibitor of aminoproteases, did not have any effect on the
down-regulation of CXCR2. These results strongly suggest that serine
protease(s) are involved in the shedding of CXCR2 induced by
TNF-.
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on the increase in
[Ca2+]i of PMNs
We next examined whether the TNF--induced decrease in CXCR2 on
the surface of PMNs affects the intracellular Ca2+
transients of PMNs triggered by IL-8. When PMNs were pretreated with
TNF- for 30 min at 37°C, the increase in
[Ca2+]i induced by IL-8 was inhibited in a
dose- and time-dependent manner. The increase in
[Ca2+]i was significantly inhibited by
TNF- at 10 U/ml, and maximal inhibition was obtained at 100 U/ml
(Fig. 3a). The
inhibitory effect was parallel with the time incubated with TNF-,
and the maximal effect was obtained at >20 min of incubation (Fig. 3b). This was not due to a cytotoxic effect of
TNF- on the PMNs, since the PMN viability measured by trypan blue
exclusion was unaffected up to 45 min of incubation. In contrast to
IL-8, the FMLP-induced increase in [Ca2+]i
was not affected by the pretreatment of PMNs with TNF- at 100 U/ml
(data not shown). Other proinflammatory cytokines that did not modulate
the expression of CXCR1 and CXCR2, such as G-CSF, GM-CSF, and IL-1ß,
did not have any effect on the increase in
[Ca2+]i in response to IL-8 (Fig. 4). Pretreatment of PMNs with GM-CSF or
G-CSF at 1, 10, or 100 ng/ml or with IL-1ß at 1, 10, 100 U/ml for 30
min at 37°C did not have any effect on the IL-8-induced increase in
[Ca2+]i (data not shown). These findings
indicate that TNF- inhibits the calcium mobilization of PMNs induced
by IL-8 but not that induced by FMLP, and suggest that the
down-regulation of CXCR2 in TNF--pretreated PMNs is reflected in the
decline in the [Ca2+]i response to
IL-8.
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on the chemotactic response to IL-8
The effects of TNF- on chemotaxis mediated through CXCR1 and
CXCR2 were also examined. As shown in Figure 5, the chemotactic response of the PMNs
to IL-8 was inhibited by pretreatment with TNF-. Since SOD and
catalase did not have any effect on PMN chemotaxis (data not shown), it
is unlikely that oxygen radicals released in response to TNF- are
involved in the inhibition of PMN chemotaxis by TNF-. This result is
consistent with the decrease in CXCR2 induced by pretreatment with
TNF-.
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on O2- release from PMNs
stimulated by IL-8
When PMNs were incubated with 100 U/ml of TNF- for 30 min and
then washed, they released a small amount of O2- (Fig. 6). PMNs stimulated by IL-8 alone
released a minimal amount of O2-, whereas
TNF--pretreated PMNs released a considerable amount of
O2- in response to IL-8. These findings indicate that
TNF- primes PMNs for enhanced release of O2- in
response to IL-8. TNF- also augmented the release of
O2- in response to FMLP. These findings, in line with
previous observations (41, 42), exclude the possibility that the
inhibition of the IL-8-induced [Ca2+]i
transient by TNF- is due to a toxic effect of TNF- on PMNs. These
results demonstrated that IL-8-induced production of reactive oxygen
species that is mediated by CXCR1 was intact, in contrast to the
[Ca2+]i transient and chemotactic response to
IL-8, which are mediated by CXCR2 (43, 44, 45, 46).
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on PMNs when pretreated in whole blood
Our findings described above suggested that PMNs exposed to
TNF- do not mount an effective chemotactic response to IL-8 and
remain in the bloodstream with an increased ability to produce
O2-. Thus, we investigated the effects of TNF-
added to whole blood on the PMN surface expression of CXCR1 and CXCR2
and on the IL-8-induced functional responses of PMNs. The expression of
CXCR2 on the PMNs isolated from whole blood pretreated with 100 U/ml
TNF- was significantly decreased compared with that on the PMNs from
whole blood incubated without TNF- (the MFI value of CXCR2 on PMNs
from whole blood treated with TNF- = 332 ± 43; without TNF-
= 141 ± 27; n = 3). The IL-8-induced
[Ca2+]i transient and chemotaxis in the PMNs
isolated from whole blood pretreated with TNF- were also
significantly suppressed (Fig. 7,
a and b). In contrast to the pretreatment
of isolated PMNs by TNF-, the chemotactic response to FMLP was
inhibited by 40% by the pretreatment of PMNs in whole blood (Fig. 7b). The PMNs pretreated with TNF- in whole blood
released an increased amount of O2- without or with
stimulation with FMLP or IL-8 (Fig. 7c). Overall, the
effects of TNF- on the expression of CXCR2, the
[Ca2+]i transient chemotaxis, and
O2- release were more prominent with TNF-
pretreatment in whole blood than with the TNF- pretreatment of
isolated PMNs.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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down-regulates
the expression of CXCR2 (IL-8RB) on the surface of PMNs.
GM-CSF, G-CSF, and IL-1ß, which are also categorized as
proinflammatory cytokines, did not significantly affect the
expression of CXCR1 or CXCR2. The reduced expression of CXCR2 seemed to
be explained, at least in part, by the shedding of this receptor
from the surface of TNF--treated PMNs, since an increased release of
CXCR2 from PMNs was detected by the immuno-slot-blot analysis of the
supernatant and by the immunoblot analysis of the fate of
anti-CXCR2 Ab attached to PMNs before TNF- treatment. In
contrast to homologous desensitization by IL-8 (47), the role of the
incorporation of CXCR2 into the cytoplasm seemed to be minimal, since
cytochalasin B did not affect TNF--induced CXCR2
down-regulation. We could not exclude the possibility that CXCR2 was
modified in TNF--treated PMNs so that it could not react with the Ab
to CXCR2. However, the semiquantitative measurements suggest that
shedding accounts for most of the decrease in CXCR2. The amount of
CXCR2 shed into the supernatant of the TNF--treated PMNs was twice
that of the PMNs treated without TNF-, which was compatible with the
20% decrease in CXCR2 expression on TNF--treated PMNs and the 10%
decrease on PMNs treated without TNF-.
TNF--induced down-regulation of CXCR2 was inhibited by PMSF and
aprotinin, inhibitors of serine proteases, whereas EDTA, an inhibitor
of metalloprotease, and bestatin, an inhibitor for aminoprotease, had
no effect. In contrast, it has been demonstrated that EDTA and bestatin
prevent the decay of IL-8R on PMNs following up-regulation by treatment
with serum-activated LPS (24), and that FMLP causes a substantial loss
of CXCR2 from the PMN surface by inducing intracellular incorporation
(48). These differences may be explained by TNF- signaling pathways
in PMNs that are distinct from those of serum-activated LPS or FMLP.
Our finding that CXCR2 is released from TNF--treated PMNs is
consistent with the involvement of protease(s) that mediate the
shedding of membrane proteins from the cell surface, referred to as
"secretases" or "sheddases" (49). Our inhibitor studies
suggested that serine protease(s) play critical role(s) in the shedding
of CXCR2 from PMNs induced by TNF-. Recent reports indicate that
serine-protease inhibitors may inhibit the trafficking of substrate
proteins to the site of sheddase action rather than the sheddase
activity itself (50). Further studies are needed to determine whether
serine protease(s) directly catalyze the TNF--induced shedding of
CXCR2.
We also found that TNF- modulates the functional responses of PMNs
to IL-8. TNF- inhibited IL-8-stimulated chemotaxis and the
[Ca2+]i response, but not
O2- release. PMNs bear two kinds of IL-8R, CXCR1 and
CXCR2, and these two receptors mediate different functional responses
to IL-8. Changes in [Ca2+]i, the release of
granule enzymes, and chemotaxis in response to IL-8 are mediated
through both receptors. In contrast, O2- release, the
priming for an enhanced FMLP-induced O2- release, and
the activation of phospholipase D in response to IL-8 depend
exclusively on CXCR1 (43, 44, 45, 46). Our finding that TNF- down-regulates
CXCR2 but not CXCR1 is in line with the inhibition of the
[Ca2+]i transient and the chemotaxis mediated
by both receptors, and with the unaffected priming effect on
O2- release mediated exclusively by CXCR1. In contrast
to IL-8, the inhibitory effects of TNF- on the FMLP-stimulated
chemotaxis and [Ca2+]i response were minimal,
if any. This argues against the possibility that TNF- causes a
nonspecific suppression of the chemotactic and
[Ca2+]i responses of PMNs. Although
additional effects of TNF- on the signal transduction downstream of
CXCR2 could not be excluded, our findings indicated that the
TNF--induced shedding of CXCR2 is reflected in the depressed
functional responses of PMNs mediated by CXCR2.
Our present findings suggest that TNF-, in addition to inducing
cytokine cascades, can modulate the functional responses of
inflammatory cells to downstream cytokine(s) such as IL-8. Sheddases,
by down-regulating cytokine receptors, can play critical roles in the
interactions of multiple cytokines at the level of effector cells. Such
cytokine interactions may have pathophysiologic implications. The PMNs
isolated from whole blood treated with TNF- showed a more marked
reduction in CXCR2 expression, IL-8-induced [Ca2+]i
transients, and chemotaxis. This augmented suppression may be caused by
interaction(s) with other types of leukocytes and platelets (19, 51, 52, 53). Although further study is necessary to elucidate the
underlying mechanisms, this result strongly supports the view that
TNF- can have pronounced effects on PMN functions in whole blood. If
CXCR2 has a role in the initiation of PMN migration from a distant
site, as suggested by Chuntharapai et al. (25), TNF- in the
bloodstream may inhibit the chemotactic response to IL-8 through the
shedding of CXCR2, which results in intravascular retention of PMNs
with enhanced reactive oxidant production mediated by CXCR1. The
long-lasting accumulation of PMNs releasing reactive oxidants should,
in turn, result in an oxidative injury to endothelial cells. This may
be an underlying process in acute inflammatory response diseases such
as adult respiratory distress syndrome and systemic inflammatory
response syndrome (17, 51, 54, 55, 56, 57).
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. M. Sasada, College of Medical Technology, Kyoto University, 53 Shogoin Kawaharacho, Sakyo-ku, Kyoto, 606, Japan. E-mail address:
3 Abbreviations used in this paper: PMNs, polymorphonuclear leukocytes; IL-8RA, IL-8R type A; IL-8RB, IL-8R type B; GM-CSF, granulocyte-macrophage colony-stimulating factor; G-CSF, granulocyte colony-stimulating factor; SOD, superoxide dismutase; HRP, horseradish peroxidase; MFI, mean fluorescence intensity; ECL, enhanced chemiluminescence.
Received for publication August 15, 1997. Accepted for publication December 30, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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 G.-H. Fan, L. A. Lapierre, J. R. Goldenring, and A. Richmond Differential regulation of CXCR2 trafficking by Rab GTPases Blood, March 15, 2003; 101(6): 2115 - 2124. [Abstract] [Full Text] [PDF]
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 M J Leckie, G R Jenkins, J Khan, S J Smith, C Walker, P J Barnes, and T T Hansel Sputum T lymphocytes in asthma, COPD and healthy subjects have the phenotype of activated intraepithelial T cells (CD69+ CD103+) Thorax, January 1, 2003; 58(1): 23 - 29. [Abstract] [Full Text] [PDF]
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T. Doroshenko, Y. Chaly, V. Savitskiy, O. Maslakova, A. Portyanko, I. Gorudko, and N. N. Voitenok Phagocytosing neutrophils down-regulate the expression of chemokine receptors CXCR1 and CXCR2 Blood, September 18, 2002; 100(7): 2668 - 2671. [Abstract] [Full Text] [PDF]
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G.-H. Fan, W. Yang, J. Sai, and A. Richmond Hsc/Hsp70 Interacting Protein (Hip) Associates with CXCR2 and Regulates the Receptor Signaling and Trafficking J. Biol. Chem., February 15, 2002; 277(8): 6590 - 6597. [Abstract] [Full Text] [PDF]
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 A. D. Chishti, J. H. Dark, P. Kesteven, H. Powell, C. Snowden, B. K. Shenton, J. A. Kirby, and S. V. Baudouin Expression of chemokine receptors CXCR1 and CXCR2 during cardiopulmonary bypass J. Thorac. Cardiovasc. Surg., December 1, 2001; 122(6): 1162 - 1166. [Abstract] [Full Text] [PDF]
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 T. W. Hepburn, T. K. Hart, V. L. Horton, T. S. Sellers, L. P. Tobia, J. J. Urbanski, W. Shi, and C. B. Davis Pharmacokinetics and Tissue Distribution of SB-251353, a Novel Human CXC Chemokine, after Intravenous Administration to Mice J. Pharmacol. Exp. Ther., September 1, 2001; 298(3): 886 - 893. [Abstract] [Full Text]
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 A. J. Mackarel, K. J. Russell, C. M. Ryan, S. J. Hislip, J. C. Rendall, M. X. FitzGerald, and C. M. O'Connor CD18 Dependency of Transendothelial Neutrophil Migration Differs During Acute Pulmonary Inflammation J. Immunol., September 1, 2001; 167(5): 2839 - 2846. [Abstract] [Full Text] [PDF]
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S. Yousefi, P. R Cooper, S. L Potter, B. Mueck, and G. Jarai Cloning and expression analysis of a novel G-protein-coupled receptor selectively expressed on granulocytes J. Leukoc. Biol., June 1, 2001; 69(6): 1045 - 1052. [Abstract] [Full Text] [PDF]
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S. Yamashiro, H. Kamohara, J.-M. Wang, D. Yang, W.-H. Gong, and T. Yoshimura Phenotypic and functional change of cytokine-activated neutrophils: inflammatory neutrophils are heterogeneous and enhance adaptive immune responses J. Leukoc. Biol., May 1, 2001; 69(5): 698 - 704. [Abstract] [Full Text]
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R. Gillitzer and M. Goebeler Chemokines in cutaneous wound healing J. Leukoc. Biol., April 1, 2001; 69(4): 513 - 521. [Abstract] [Full Text]
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S. S. Cheng, J. J. Lai, N. W. Lukacs, and S. L. Kunkel Granulocyte-Macrophage Colony Stimulating Factor Up-Regulates CCR1 in Human Neutrophils J. Immunol., January 15, 2001; 166(2): 1178 - 1184. [Abstract] [Full Text] [PDF]
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A. Zaslaver, R. Feniger-Barish, and A. Ben-Baruch Actin Filaments Are Involved in the Regulation of Trafficking of Two Closely Related Chemokine Receptors, CXCR1 and CXCR2 J. Immunol., January 15, 2001; 166(2): 1272 - 1284. [Abstract] [Full Text] [PDF]
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C. Albanesi, C. Scarponi, S. Sebastiani, A. Cavani, M. Federici, O. De Pita, P. Puddu, and G. Girolomoni IL-4 Enhances Keratinocyte Expression of CXCR3 Agonistic Chemokines J. Immunol., August 1, 2000; 165(3): 1395 - 1402. [Abstract] [Full Text] [PDF]
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A. Ludwig, J. E. Ehlert, H.-D. Flad, and E. Brandt Identification of Distinct Surface-Expressed and Intracellular CXC-Chemokine Receptor 2 Glycoforms in Neutrophils: N-Glycosylation Is Essential for Maintenance of Receptor Surface Expression J. Immunol., July 15, 2000; 165(2): 1044 - 1052. [Abstract] [Full Text] [PDF]
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N. T. Luu, G. E. Rainger, and G. B. Nash Differential Ability of Exogenous Chemotactic Agents to Disrupt Transendothelial Migration of Flowing Neutrophils J. Immunol., June 1, 2000; 164(11): 5961 - 5969. [Abstract] [Full Text] [PDF]
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 M. A. Williams, C. M. Cave, G. Quaid, and J. S. Solomkin Chemokine Regulation of Neutrophil Function in Surgical Inflammation Arch Surg, December 1, 1999; 134(12): 1360 - 1366. [Abstract] [Full Text] [PDF]
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C. J. Cummings, T. R. Martin, C. W. Frevert, J. M. Quan, V. A. Wong, S. M. Mongovin, T. R. Hagen, K. P. Steinberg, and R. B. Goodman Expression and Function of the Chemokine Receptors CXCR1 and CXCR2 in Sepsis J. Immunol., February 15, 1999; 162(4): 2341 - 2346. [Abstract] [Full Text] [PDF]
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K. Yamashita, A. Takahashi, S. Kobayashi, H. Hirata, P. W. Mesner Jr, S. H. Kaufmann, S. Yonehara, K. Yamamoto, T. Uchiyama, and M. Sasada Caspases Mediate Tumor Necrosis Factor-alpha -Induced Neutrophil Apoptosis and Downregulation of Reactive Oxygen Production Blood, January 15, 1999; 93(2): 674 - 685. [Abstract] [Full Text] [PDF]
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