| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Institut National de la Santé et de la Recherche Médicale Unité 479 and Service d’Immunologie et d’Hématologie, Paris, France
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
, IL-1, IL-8, MIP-1, MIP-1
, and IL-12, whereas it
potentiates LPS-elicited secretion of IL-1 receptor antagonist
(8, 9, 10, 11, 12, 13). IL-10 also down-regulates other PMN effector
functions, such as phagocytic and bactericidal activities, Ab-dependent
cellular cytotoxicity, and platelet-activating factor and ROS
production (14, 15, 16, 17).
IL-10 activities are mediated by a high-affinity cell surface receptor
(IL-10R) that is structurally related to IFN receptors
(18, 19, 20). IL-10R is a complex of a ligand-binding chain
(IL-R1 or ) and a recently identified IL-10R2 (CRFB4/CRF-2) molecule
whose linking to the ligand-binding chain results in a functional
IL-10R (21, 22, 23). IL-10R1 has been found at the surface of
a variety of cells, including several human lymphoid and myeloid cell
lines (24, 25, 26, 27, 28). IL-10 binding to the PMN surface has
recently been reported, albeit to a lesser extent than to monocytes and
lymphocytes (29). However, no information is available on
the regulation of IL-10 binding at the surface of mature PMN.
To further define the pro- and anti-inflammatory balance at an
inflammatory site, we investigated IL-10 binding to the PMN surface
during treatment with proinflammatory mediators (LPS, TNF-, GM-CSF,
IL-1, IL-8) that have different effects on PMN functions (8, 10, 30, 31, 32, 33, 34). We observed differential modulation of IL-10 binding to
PMN by these proinflammatory agents, which was related to differential
mobilization of the subset of specific granules, which was found to
stock IL-10 receptors.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The reagents and sources were as follows: recombinant human
TNF- (rhTNF-; 105 U/ml), IL-1
(105 U/ml), and IL-8 (77 aa) produced by
endothelial cells (Genzyme, Cambridge, MA); GM-CSF (1.2 x
105 ng/ml; Schering-Plough, Kenilworth, NJ); LPS
endotoxin from Escherichia coli (O55:B5), PMA,
diisopropylfluorophosphate (DFP), fMLP, primaquine, and a protease
inhibitor mixture (Sigma, St. Louis, MO); pentoxifylline (PTX; Hoechst,
Paris-La-Défense, France); rhIL-10 (5 µg/ml), biotinylated
rhIL-10, FITC-avidin, and mouse biotinylated anti-human IL-10R
polyclonal Ab (R&D Systems, Abingdon, U.K.); rat anti-human IL-10R
mAb (3F9) (a gift from Kevin Moore, DNAX Research Institute, Palo Alto,
CA); rabbit anti-human IL-10R polyclonal Ab (Santa Cruz
Biotechnology, Santa Cruz, CA); peroxidase-conjugated swine
anti-goat Ig (BioSource International, Camarillo, CA); Dako LSAB
alkaline phosphatase kit (Dakopatts, Glostrup, Denmark);
purified monoclonal mouse anti-human CD11b; FITC-streptavidin
(Immunotech, Marseille, France); FCS (Life Technologies Laboratories,
Grand Island, NY), and hydroethidine (HE; Fluka, Buchs, Switzerland).
Stock solutions of HE (15 mg/ml) and fMLP (10-2
mol/L) were prepared in acetonitrile and DMSO, respectively, and stored
at -20°C. The solutions were diluted in PBS (Pharmacia Fine
Chemicals, Uppsala, Sweden) immediately before use. All of the
recombinant cytokines were used as recommended by the
manufacturers.
Study of IL-10 binding to the PMN surface
IL-10 binding to the PMN surface was measured by means of flow
cytometry in whole blood, to minimize procedure-related changes in
surface receptor expression (35). Whole blood (1 ml) from
healthy donors was either kept on ice or incubated with TNF-
(0.1–1000 U/ml), GM-CSF (0.1–500 U/ml), IL-1 (0.1–500 U/ml), IL-8
(0.1–50 ng/ml), LPS (0.1–5 µg/ml), PMA (100 ng/ml), or PBS for
5–30 min (depending the stimulus) at 37°C with constant agitation.
In some experiments, samples were preincubated with PTX (1 µM to 10
mM), primaquine (250 µg/ml), or a protease inhibitor mixture (10
µg/ml) for 5 min before adding TNF-. IL-10 binding to the PMN
surface was determined by using a biotinylated IL-10, with FITC-avidin
revelation according to the manufacturer guidelines. Briefly, samples
(100 µl) were incubated with biotinylated IL-10 for 60 min at 4°C.
Nonspecific binding was determined by incubating samples with a
negative control reagent consisting of a soybean trypsin inhibitor
biotinylated to the same extent as the cytokine (5 µg/ml). The cells
were then directly treated with FITC-avidin for 30 min at 4°C. The
specificity of the reaction was checked by preincubating the cytokine
reagent for 15 min with a blocking anti-hIL-10 Ab. After
erythrocyte lysis with FACS lysing solution (BD Biosciences, Mountain
View, CA) and one wash in PBS, cells were resuspended in washing buffer
and kept on ice until flow cytometry.
Determination of intracellular IL-10R expression by flow cytometry
Erythrocytes were lysed with FACS lysing solution. Leukocytes were washed twice with PBS containing 2% FCS. Paraformaldehyde (0.25%) was then added while vortexing, and samples were incubated in the dark for 15 min at room temperature. After one wash with PBS, the leukocytes were incubated with ice-cold PBS-70% methanol in the dark for 60 min at 4°C to permeabilize the membranes as previously described (36). After one wash in PBS, the samples were incubated with a mouse biotinylated anti-human IL-10R Ab (R&D Systems) for 30 min at 4°C and then with FITC-streptavidin for 30 min at 4°C. After one wash with ice-cold PBS containing 2% FCS, cells were resuspended in 1% paraformaldehyde-PBS and kept on ice until flow cytometry. Nonspecific Ab binding was determined on cells incubated with the same concentration of an irrelevant biotinylated Ab of the same isotype. The positive control used an anti-CD11b Ab.
O2-° production by PMN
O2-° production was measured by using a flow cytometric assay derived from the HE oxidation technique described by Rothe and Valet (37). Whole-blood samples (1 ml) were preincubated for 15 min with HE (1500 ng/ml) in a water bath with gentle horizontal agitation at 37°C. (HE diffuses into the cells and, during the PMN oxidative burst, nonfluorescent intracellular HE is oxidized by O2-° to highly fluorescent ethidium that is trapped in the nucleus by intercalation into DNA, leading to an enhancement of fluorescence.) Samples were then incubated with TNF (100 U/ml) for various times (0, 5, 10, 15, 30, 45, or 60 min); PBS or IL-10 (30 ng/ml) was then added for 35 min, followed by fMLP (10-6 mol/L) for 5 min. The reaction was stopped and RBC were lysed with FACS solution (BD Biosciences). After one wash (400 x g for 5 min), white cells were suspended in 1% paraformaldehyde-PBS and kept on ice until flow cytometry on the same day.
Flow cytometry
We used a Becton Dickinson FACScan (BD Immunocytometry Systems, San Jose, CA) with a 15-mW 488-nm argon laser. Forward and side scatter were used to identify the granulocyte population and to gate out other cells and debris. The purity of the gated cells was assessed by using FITC- or PE-conjugated anti-CD3, CD45, CD14, and CD15 Abs (BD Biosciences). Ten thousand events were counted per sample, and the fluorescence pulses were amplified by 4-decade logarithmic amplifiers. The green fluorescence of FITC-avidin was recorded from 515 to 545. The orange fluorescence of ethidium was recorded from 549 to 601 nm (575 ± 26). All the results were obtained with a constant photomultiplier gain value. The data were analyzed with LYSIS II software (BD Biosciences, San Jose, CA) and the mean fluorescence intensity (MFI) was used to quantitate the responses. The effect of IL-10 on TNF priming of the PMN oxidative burst was calculated as the percentage inhibition, as follows: (MFI of the TNF-preincubated sample - MFI of the TNF-preincubated sample treated with IL-10)/MFI of the TNF-preincubated sample x 100%.
Immunocytochemical staining of intracellular IL-10R
Smears of unstimulated blood from healthy donors were air-dried for 24 h and incubated in cold acetone/methanol (1:1) at 4°C for 10 min to fix and permeabilize the membranes. Nonspecific staining was blocked by a 5-min incubation with the blocking reagent from the Dako LSAB kit. The smears were then incubated with a rat anti-human IL-10R mAb (25 µg/ml; DNAX) for 30 min, followed by sequential 10-min incubation steps with a biotinylated goat anti-rat Ig Ab and alkaline phosphatase-labeled streptavidin, as recommended by the manufacturer (Dako LSAB Alkaline Phosphatase kit). Staining was revealed with freshly prepared substrate-chromogen solution. Counterstaining was then performed with hematoxylin and ammonia water. Positive staining developed as a fuchsia-colored reaction product. The negative control consisted of incubating smears with an irrelevant Ab of the same isotype, and the positive control with an anti-CD11b mAb.
Subcellular fractionation of isolated PMN
Human PMN were purified in sterile conditions by 2% dextran sedimentation and centrifugation on Ficoll-Paque cushions (38). Contaminating erythrocytes were removed by hypotonic lysis, and the purified neutrophils (100 x 106/ml) were suspended in PBS and treated with DFP (2.7 mM for 15 min at 4°C), then washed and resuspended in 5 ml of ice-cold relaxation buffer [100 mmol/L KCl, 3 mmol/L NaCl, 1 mmol/L ATPNa2, 3.5 mmol/L MgCl2, 10 mmol/L PIPES, pH 7.2], until subcellular fractionation. To study translocation under stimulation, DFP-treated PMN (10 x 106/ml) were incubated in Hank’s buffer supplemented with 0.05% BSA, in the absence or presence of PMA (100 ng/ml for 10 min at 37°C) or TNF (100 U/ml for 30 min at 37°C). The reaction was stopped by adding ice-cold PBS and subsequent centrifugation at 400 x g for 10 min at 4°C. Then PMN were resuspended in ice-cold relaxation buffer supplemented with antiproteases. Subcellular fractionation was performed as previously described (39). Briefly, PMN were pressurized with N2 for 20 min at 450 psi with constant stirring in a nitrogen bomb. The cavitas was then collected dropwise into EGTA, sufficient for a final concentration of 1.25 mmol/L. Nuclei and unbroken cells were pelleted by centrifugation of the cavitas at 400 x g for 10 min. The supernatant was decanted, loaded at the top of a two-layer Percoll gradient (1.05/1.12 g/ml) precooled to 4°C, and spun at 4°C for 30 min at 40,000 x g. This resulted in a gradient with three visible bands (from the bottom: a band containing azurophil granules, a band containing specific and gelatinase granules, and a band containing plasma membranes and secretory vesicles). The cytosol remained above the upper band, on top of the Percoll gradient. The different fractions were then collected. The purity of the specific and azurophilic granule fractions was assessed by measuring their respective markers, lactoferrin and myeloperoxidase, in each fraction (azurophil granules, specific granules, and membranes) and in the total cavitas using ELISA methods (R&D Systems).
Electrophoresis and blotting
A cellular equivalent of total cavitas and of each subcellular fraction (cytosol, membranes, specific and azurophilic granules) was added to 2x Laemmli sample buffer, and proteins were electrophoresed in 9% SDS-polyacrylamide gels. Proteins were transferred to nitrocellulose membranes (Protran BA83; Schleicher & Schuell, Keene, NH) at 100 V for 1 h in 25 mM Tris, pH 8.3, 192 mM glycine, and 20% methanol buffer. The membranes were first blocked overnight at 4°C in TBS-Tween (25 mM Tris-HCl, pH 7.6, 137 mM NaCl, and 0.2% Tween 20) containing 7.5% BSA, and then incubated (60 min, 37°C) with a rabbit anti-human IL-10R polyclonal Ab (final concentration, 0.2 µg/ml; Santa Cruz Biotechnology). The membranes were then washed and incubated for 45 min at room temperature in TBS-Tween with a HRP-conjugated swine anti-rabbit Ig Ab (1/10,000 dilution). The immunoblots were developed using a chemiluminescence method (ECL; Amersham Life Sciences, Arlington Heights, IL) following the manufacturer guidelines. Mononuclear cells (lymphocytes and monocytes) obtained after separation of peripheral blood on Ficoll-Paque were loaded and blotted in parallel and served as a positive control.
Quantification of lactoferrin release after PMN stimulation
Whole blood collected in sterile lithium heparinate tubes was
either kept on ice or incubated at 37°C for 30 min with TNF- (100
U/ml), GM-CSF (100 U/ml), IL-1 (100 U/ml), IL-8 (50 ng/ml), or LPS
(5 µg/ml), or for 5 min with PMA (100 ng/ml). Control samples were
incubated with PBS. Samples were then centrifuged at 1500 x
g for 15 min at 4°C. Plasma samples were stored at
-70°C for no longer than 15 days before the assay. Lactoferrin in
plasma was assayed in duplicate by using ELISA methods (R&D Systems)
with a detection limit of 1 ng/ml. PMN counts in whole blood were
obtained using an automated hemocytometer (H1; Bayer, Elkhart,
IN). The measurement of lactoferrin in total cavitas of isolated
PMN (as previously described) permitted us to evaluate the percentage
of release from the intracellular pool to the extracellular medium.
Statistical analysis
All results are expressed as means ± SEM. Means were compared using Student’s t test, and p values of 0.05 or less were considered significant.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
After 30 min of incubation at 4°C, IL-10 barely bound to resting
whole-blood PMN. The MFI of the sample incubated with
biotinylated-rhIL-10 and revealed with FITC-avidin was moderately,
although significantly, increased as compared with samples incubated
with the biotinylated control reagent and FITC-avidin (8.3 ± 2.5
vs 5.2 ± 1.2, respectively; n = 10,
p = 0.0003). In contrast, incubation of whole blood
with 100 U/ml TNF- for 30 min at 37°C strongly increased IL-10
binding to the PMN surface, with a MFI 
6-fold higher than that of
the sample incubated in the same conditions with PBS (Table I
). A slightly increased value of IL-10
binding was observed in the sample incubated with PBS alone at 37°C
(10.75 ± 1.1) as compared with the values obtained at 4°C. The
effect of TNF was concentration-dependent (Table I). Moreover, when a
blocking anti-hIL-10 Ab was added before incubation with TNF, IL-10
binding to the PMN surface was suppressed (MFI: 4.6 ± 0.4),
supporting the specificity of the reaction. A kinetic study showed that
IL-10 binding induced by TNF began as early as 10 min after TNF
treatment and increased in a time-dependent manner, with maximum
expression at 30 min (Table II). IL-10
binding decreased 45 and 60 min after TNF treatment. This diminution
was reversed by sample preincubation with primaquine, an endocytosis
inhibitor (40) (Table II), whereas protease inhibitors had
no effect (data not shown), suggesting an internalization of IL-10
receptors.
|
|
, incubation of whole
blood with predetermined optimal concentrations of LPS (5 µg/ml for
30 min) or GM-CSF (100 U/ml for 30 min) increased IL-10 binding to the
PMN surface. This increase in IL-10 binding was in the same range as
that observed with the PKC activator PMA (100 ng/ml for 5 min), a
potent stimulus of numerous PMN functions. Indeed, the stimulation
index (SI) (ratio of the MFI of agonist-treated cells to that of
PBS-treated cells) was 4–5 in the presence of TNF, GM-CSF, LPS, or
PMA. In contrast, in our conditions of stimulation, IL-1 (100 U/ml for
30 min) and IL-8 (50 ng/ml for 30 min) did not significantly modify
specific IL-10 binding to the PMN surface, with a SI always <2 (Table III); other times and higher concentrations had no effect either (data
not shown). Moreover, IL-1 and IL-8 did not down-regulate TNF-induced
IL-10 binding to PMN (data not shown).
|
The TNF-induced increase in IL-10 binding to the PMN surface was observed very rapidly after addition of the reagent and was maximal after 30 min of incubation. Furthermore, the kinetics of release was similar in the presence of GM-CSF and LPS at optimal concentrations (data not shown). As PMA is also a potent inducer of human neutrophil degranulation (41), these results suggested the translocation, to the PMN surface, of intracellular IL-10R. Thus we investigated whether the degranulation process after stimulation contributed to the increased IL-10R expression.
We tested the effect of PTX, a methylxanthine derivative known to
inhibit neutrophil degranulation (42, 43), on
IL-10 binding after TNF stimulation. Pretreatment of whole blood with
PTX induced concentration-dependent inhibition of the TNF-induced
increase in IL-10 binding (Table IV). The
effect of TNF was completely reversed by 10 mM PTX.
|
Resting human PMN contain an intracellular pool of IL-10R
To investigate the existence of an intracellular pool of IL-10R in
resting human PMN, we performed intracellular immunostaining after PMN
permeabilization, using both flow cytometry and immunocytochemical
staining. Flow cytometry, performed after methanol permeabilization,
revealed a pool of IL-10R in PMN. The MFI of the sample incubated with
a biotinylated mouse anti-IL-10R mAb (R&D Systems) and then with
FITC-streptavidin was significantly higher than that of the control
(biotinylated isotypic control and FITC-streptavidin) (45 ± 2 vs
25 ± 1, respectively). Furthermore, the presence of intracellular
IL-10R in PMN was confirmed by immunocytochemistry: a rat
anti-human IL-10R mAb (DNAX) yielded positive staining inside
unstimulated PMN, whereas no staining was observed with the negative
isotype control (Fig. 1). Nevertheless,
the staining was weaker than that observed with the anti-CD11b Ab
used as a positive control (data not shown).
|
To further localize the human PMN intracellular IL-10R pool,
subcellular fractionation of human PMN was performed. The purity of
fractionation was assessed by measuring the reference markers of
specific and azurophilic granules, lactoferrin and myeloperoxidase,
respectively, in each fraction (azurophil granules, specific/gelatinase
granules, membranes) and in the total cavitas, using ELISA methods (R&D
Systems). As shown in Table V, the
measurements showed >95% lactoferrin in the specific/gelatinase
fraction and >95% myeloperoxidase in the azurophil fraction, the
other fractions containing <5% of each marker. Total cavitas,
membranes, cytosol, specific/gelatinase, and azurophilic granule
fractions were immunoblotted. Fig. 2
shows the presence of a band of 90 kDa (migrating between the 77-
and 103-kDa markers) in the specific/gelatinase granule fraction as
well as in the total PMN cavitas. In contrast, no significant amounts
of IL-10R were observed in the cytosol or in the azurophilic granules.
Small amounts of IL-10R were found in the membranes (<5% of the
specific granule content). The immunoreactive band detected in the
total PMN cavitas, specific/gelatinase granules, and membranes migrated
to the same region as that obtained with a cavitas of mononuclear cells
used as a positive control. Stimulation with PMA (100 ng/ml, 10 min at
37°C) induced a 60% increase of the 90-kDa band located in the
membrane fraction as compared with unstimulated cells (Fig. 3) with no detectable decrease in the
intensity of the specific granule fraction. The amount of translocation
from the specific/gelatinase granules to the membranes after
stimulation was 10% of the total IL-10R content (as measured by
scanning of the autoradiography) (Fig. 3). Similar results were
obtained with TNF-stimulated PMN (data not shown).
|
|
|
To explain the difference in the induction of IL-10 binding by the
various proinflammatory agents and to confirm their differential
mobilizing effects of specific granules to the cell surface, we
assayed, in parallel to IL-10-binding, the extracellular release of
lactoferrin (a marker of specific granules; Ref. 44). As
shown in Table III
, significant amounts of lactoferrin were released
after 30 min of stimulation with TNF-, GM-CSF, and LPS as compared
with the PBS control, whereas stimulation with IL-1 and IL-8 did not
induce significant lactoferrin release. The effects of TNF, GM-CSF, and
LPS on lactoferrin release were similar to that of PMA, which is known
to induce strong degranulation, in particular of specific granules.
Variations of SI paralleled that of IL-10 binding using the different
agonists. In addition, the amount of lactoferrin released into the
extracellular medium after TNF, GM-CSF, LPS, and PMA was 10% of the
total content of PMN as calculated from the lactoferrin content
measured in the total cavitas of isolated PMN and PMN counts in 1 ml of
whole blood (mean ± SEM = 30,200 ± 2545 ng in 4.2
± 0.5 x 106 PMN).
TNF-induced up-regulation of IL-10 binding to the PMN surface was associated with increased inhibitory effect of IL-10 on PMN oxidative burst
To assess the functional importance of the TNF-induced
up-regulation of IL-10R, we studied the effect of IL-10 on
O2-° production by PMN in
response to formyl peptides after TNF preincubation. IL-10 was added
after various times of TNF stimulation. Our results showed that the
inhibitory effect of IL-10 increased from 5% when IL-10 was added
simultaneously with TNF to a maximum of 38 and 36% when IL-10 was
added 15 and 30 min, respectively, after TNF treatment and thereafter
decreased (Table VI). This IL-10
inhibitory effect on O2-°
production paralleled the kinetic of increased IL-10 binding at the PMN
surface (Table II).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This study was performed with whole-blood PMN to minimize cell activation related to isolation procedure (35). The low-level binding of IL-10 to the surface of resting PMN, which confirms data published by Bovolenta et al. (29), may explain the rarity of data on the direct effect of IL-10 on PMN functions (17), whereas IL-10 is widely reported to modulate LPS- and TNF-stimulated functions such as proinflammatory cytokine down-regulation. We clearly show that IL-10 binding to the PMN surface is strongly and very rapidly (10 min) up-regulated by TNF, GM-CSF, LPS, and PMA. The diminution of IL-10 binding observed after 45 and 60 min of TNF treatment was reversed by primaquine, an endocytosis inhibitor (40), suggesting an internalization of IL-10 receptors.
This rapid up-regulation of IL-10 binding to the PMN surface, together
with the observation that cycloheximide did not inhibit TNF-induced
IL-10 binding, strongly suggested TNF-induced translocation of a
pre-existing pool of IL-10R instead of de novo synthesis of the
receptor by PMN, at least at the early times studied here. This
intracellular pool of IL-10R was detected in permeabilized neutrophils
by both flow cytometry and immunocytochemistry, using two different
Abs. It is noteworthy that these Abs did not reveal IL-10R expression
at the PMN surface by flow cytometry, even after optimal stimulation.
These results are in keeping with the literature and with a low density
of IL-10R expression on hemopoietic cells (19, 20, 24, 26, 27, 28). The presence of IL-10R in PMN was confirmed by Western
blot analyses using another anti-human IL-10R1 Ab on total extract
of PMN disrupted by cavitation, showing a band near the 90 kDa
standard. The apparent molecular mass of 90 kDa is in keeping with
previous data obtained with cell lines expressing rhIL-10R, and with
mononuclear cells (20, 26).
After subcellular fractionation, Western blotting also showed the same
band in the specific granule fraction, but not in cytosol or in the
azurophilic granules. A band of <5% the intensity of that observed in
specific granules was detected in the membrane fraction of resting PMN.
The 60% increase in IL-10R observed in the membrane fraction after
stimulation of PMN by PMA is in accordance with data concerning rap
proteins, predominantly associated with specific granules, which
increase in similar amounts as IL-10R in the plasma membranes following
PMA stimulation (45). The low level of translocation from
the specific granules to the cell surface (10% of the total specific
granule IL-10R pool) is in accordance with previous reports concerning
proinflammatory mediator-induced degranulation of other molecules
located in specific granules, i.e., lactoferrin and vitamin
B-12-binding protein, in contrast to observations for molecules in the
more easily mobilizable granules such as secretory vesicles (46, 47). This low level of degranulation of specific granules was
confirmed here with TNF, GM-CSF, and LPS, which all induced a release
of 10% of the total lactoferrin content in our experimental
conditions (Table III).
To further understand the differences between TNF, GM-CSF, and LPS on the one hand and IL-1 and IL-8 in contrast in the enhancement of IL-10 binding to the PMN surface, we measured in parallel, in the same whole-blood conditions, the extracellular release of lactoferrin. We observed the same differential effects of the proinflammatory mediators on lactoferrin release as on IL-10 binding, with parallel variations in stimulation indices. These data suggest that differences in specific granule mobilization could explain the differences in IL-10 binding.
That TNF, GM-CSF, LPS, and PMA induced increased IL-10 binding is related to translocation from the specific granules to the membranes is further supported by the observation that PTX, which inhibits degranulation, completely reversed the effect of TNF on IL-10 binding to the PMN surface. In addition, we have previously shown that PMA- and TNF-induced vascular endothelial growth factor release from specific granules is inhibited by PTX (42). Lastly, PTX also reduced TNF-induced CD11b up-regulation at the PMN surface (43).
A low level of translocation of different molecules in PMN has been shown to trigger a biological effect, e.g., <10% translocation of p47phox is sufficient to induce NADPH oxidase activity with no detectable decrease in the cytosolic fraction (48). It appears that the low level of IL-10R translocation can indeed account for the increased IL-10 binding and the biological effect of the cytokine. Indeed, we demonstrated that IL-10 significantly inhibited PMN oxidative burst in response to formyl peptides when IL-10 was added 15 or 30 min after TNF treatment. This kinetic pattern matched that of the increase in IL-10 binding during TNF incubation, suggesting that TNF-induced up-regulation of IL-10 binding to the PMN surface may play an important role in the regulation of inflammatory reactions by inhibiting neutrophil activation and thereby limiting tissue injury.
In addition to their role as phagocytic and killer cells, PMN can produce and respond to numerous cytokines that regulate the immune processes. PMN potentiate inflammatory responses by releasing proinflammatory cytokines such as IL-8 and TNF, and generate ROS in response to various stimuli. Some proinflammatory cytokines have been found to prime or activate PMN production of ROS. Thus PMN are a first line of defense against pathogens, and participate in the regulation of inflammatory responses. However, excessive or inappropriate PMN responses can lead to vascular or tissular injury. In acute settings, IL-10 production plays a major role in establishing an anti-inflammatory balance, by down-regulating the inflammatory response and thereby limiting tissue damage (49). Our results demonstrating that functional IL-10R may be rapidly up-regulated by stimuli such as TNF, GM-CSF, and LPS, which are potent inflammatory mediators, points to a potential in vivo role of IL-10R in down-regulating PMN activities and thereby preventing inappropriate inflammatory responses.
In conclusion, this study shows that PMN contain an intracellular pool of IL-10-receptors localized in specific granules. We also observed increased IL-10 binding to the PMN surface after stimulation with LPS and the proinflammatory cytokines TNF and GM-CSF, but not with IL-1 and IL-8. This differential enhancement of IL-10 binding was related to differential mobilization of the relevant specific granules by these agents. Differential modulation of IL-10 binding may play a key role in optimal regulation of inflammatory responses.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Marie-Anne Gougerot-Pocidalo, Laboratoire d’Immunologie et d’Hématologie, Centre Hospitalier Universitaire Xavier Bichat, 46 rue Henri Huchard, 75877 Paris Cedex 18, France.
3 Abbreviations used in this paper: PMN, polymorphonuclear neutrophil(s); ROS, reactive oxygen species; rhTNF-, recombinant human TNF-; DFP, diisopropylfluorophosphate; PTX, pentoxifylline; HE, hydroethidine; MFI, mean fluorescence intensity; SI, stimulation index.
Received for publication August 21, 2000. Accepted for publication February 5, 2001.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
and IL-1 in mediating the production of IL-8 triggered by lipopolysaccharide. J. Exp. Med. 178:2207. to induce IL-1ra production by human neutrophils. Cytokine 8:147.[Medline]
interferon and interleukin-10 on generation of oxidizing species by human granulocytes. Inflamm. Res. 45:313.[Medline]
RI/CD64) gene and stat protein binding to the IFN- response region (GRR) are regulated differentially in human neutrophils and monocytes by IL-10. J. Immunol. 160:911. in whole blood: identification of two PMN subpopulations in response to formyl-peptides. Blood 82:633.RI initiate separate signalling pathways involved in endocytosis and phagocytosis. EMBO J. 14:432.[Medline]
This article has been cited by other articles:
|
|
 |
|
  J. S. Neves, S. A. C. Perez, L. A. Spencer, R. C. N. Melo, L. Reynolds, I. Ghiran, S. Mahmudi-Azer, S. O. Odemuyiwa, A. M. Dvorak, R. Moqbel, et al. Eosinophil granules function extracellularly as receptor-mediated secretory organelles PNAS, November 25, 2008; 105(47): 18478 - 18483. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Tamassia, F. Calzetti, N. Menestrina, M. Rossato, F. Bazzoni, L. Gottin, and M. A. Cassatella Circulating neutrophils of septic patients constitutively express IL-10R1 and are promptly responsive to IL-10 Int. Immunol., April 1, 2008; 20(4): 535 - 541. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Hoarau, B. Gerard, E. Lescanne, D. Henry, S. Francois, J.-J. Lacapere, J. El Benna, P. M.-C. Dang, B. Grandchamp, Y. Lebranchu, et al. TLR9 Activation Induces Normal Neutrophil Responses in a Child with IRAK-4 Deficiency: Involvement of the Direct PI3K Pathway J. Immunol., October 1, 2007; 179(7): 4754 - 4765. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. R. Jog, M. J. Rane, G. Lominadze, G. C. Luerman, R. A. Ward, and K. R. McLeish The actin cytoskeleton regulates exocytosis of all neutrophil granule subsets Am J Physiol Cell Physiol, May 1, 2007; 292(5): C1690 - C1700. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K.-J. Li, M.-C. Lu, S.-C. Hsieh, C.-H. Wu, H.-S. Yu, C.-Y. Tsai, and C.-L. Yu Release of surface-expressed lactoferrin from polymorphonuclear neutrophils after contact with CD4+T cells and its modulation on Th1/Th2 cytokine production J. Leukoc. Biol., August 1, 2006; 80(2): 350 - 358. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. M.-C. Dang, C. Elbim, J.-C. Marie, M. Chiandotto, M.-A. Gougerot-Pocidalo, and J. El-Benna Anti-inflammatory effect of interleukin-10 on human neutrophil respiratory burst involves inhibition of GM-CSF-induced p47PHOX phosphorylation through a decrease in ERK1/2 activity FASEB J, July 1, 2006; 20(9): 1504 - 1506. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. A. Spencer, R. C. N. Melo, S. A. C. Perez, S. P. Bafford, A. M. Dvorak, and P. F. Weller Cytokine receptor-mediated trafficking of preformed IL-4 in eosinophils identifies an innate immune mechanism of cytokine secretion PNAS, February 28, 2006; 103(9): 3333 - 3338. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Francois, J. El Benna, P. M. C. Dang, E. Pedruzzi, M.-A. Gougerot-Pocidalo, and C. Elbim Inhibition of Neutrophil Apoptosis by TLR Agonists in Whole Blood: Involvement of the Phosphoinositide 3-Kinase/Akt and NF-{kappa}B Signaling Pathways, Leading to Increased Levels of Mcl-1, A1, and Phosphorylated Bad J. Immunol., March 15, 2005; 174(6): 3633 - 3642. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Elbim, C. Guichard, P. M. C. Dang, M. Fay, E. Pedruzzi, H. Demur, C. Pouzet, J. El Benna, and M.-A. Gougerot-Pocidalo Interleukin-18 Primes the Oxidative Burst of Neutrophils in Response to Formyl-Peptides: Role of Cytochrome b558 Translocation and N-Formyl Peptide Receptor Endocytosis Clin. Vaccine Immunol., March 1, 2005; 12(3): 436 - 446. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J.-D. Ji, I. Tassiulas, K.-H. Park-Min, A. Aydin, I. Mecklenbrauker, A. Tarakhovsky, L. Pricop, J. E. Salmon, and L. B. Ivashkiv Inhibition of Interleukin 10 Signaling after Fc Receptor Ligation and during Rheumatoid Arthritis J. Exp. Med., June 2, 2003; 197(11): 1573 - 1583. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Crepaldi, L. Silveri, F. Calzetti, C. Pinardi, and M. A. Cassatella Molecular basis of the synergistic production of IL-1 receptor antagonist by human neutrophils stimulated with IL-4 and IL-10 Int. Immunol., October 1, 2002; 14(10): 1145 - 1153. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
