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| 2007 JASN IMPACT FACTOR 7.111 | HOME AUTHOR INFO EDITORIAL BOARD SUBSCRIBE FEEDBACK ALERTS HELP | |||
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| 2007 JASN IMPACT FACTOR 7.111 | HOME AUTHOR INFO EDITORIAL BOARD SUBSCRIBE FEEDBACK ALERTS HELP | |||
| CURRENT ISSUE | ARCHIVES | JASN Express | ONLINE SUBMISSION | |
Franz Volhard Clinic and Max Delbrück Center for Molecular Medicine, Medical Faculty of the Charité, Humboldt University of Berlin, Berlin, Germany.
Correspondence to Dr. Ralph Kettritz, Division of Nephrology, Franz Volhard Clinic, Wiltbergstrasse 50, 13122 Berlin, Germany. Phone: 49-30-9417-2202; Fax: 49-30-9417-2206; E-mail: kettritz{at}fvk-berlin.de
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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(TNF-)
in vitro. TNF- priming results in translocation of ANCA
antigens to the cell surface, where they are recognized by the antibodies. The
signaling mechanisms involved in TNF- priming and subsequent
ANCA-induced activation were investigated. TNF--primed neutrophils were
stimulated with monoclonal antibodies (MAb) to human myeloperoxidase (MPO) and
proteinase 3 (PR3), and with preparations of human ANCA (three patients with
PR3-ANCA and two patients with MPO-ANCA). Respiratory burst was measured with
superoxide dismutase-inhibitable ferricytochrome C reduction and using
dihydro-rhodamine-1,2,3. Phosphorylation of p38 mitogen-activated protein
kinase (p38-MAPK) and the extracellular signal-regulated kinase (ERK) were
assessed by immunoblotting. ANCA-antigen translocation was studied by flow
cytometry. The tyrosine phosphorylation inhibitor genistein, but not
calphostin or staurosporin, resulted in a significant dose-dependent
superoxide generation inhibition (11.6 ± 1.7 nmol to 2.1 ± 0.5
for PR3-ANCA, and 16.0 ± 2.8 to 3.3 ± 1.3 for MPO-ANCA). The
p38-MAPK inhibitor (SB202190) and the ERK inhibitor (PD98059) diminished
PR3-ANCA-mediated superoxide production dose dependently (11.6 ± 1.7
nmol O2- to 1.9 ± 0.6 with 50 µM SB202190 and
4.0 ± 0.6 with 50 µM PD098059, respectively). For MPO-ANCA, the
results were similar (16.0 ± 2.8 nmol to 0.9 ± 1.0 nmol with
SB202190 and 6.4 ± 2.4 nmol with PD98059, respectively). Western blot
showed phosphorylation of both p38-MAPK and ERK during TNF- priming.
The p38-MAPK inhibitor and the ERK inhibitor showed the strongest effect on
respiratory burst when added before TNF- priming, further supporting an
important role for both signaling pathways in the priming process. Flow
cytometry showed that p38-MAPK inhibition decreased the translocation of PR3
(by 93 ± 2%) and of MPO (by 64 ± 2%). In contrast, no such
effect was seen when ERK was inhibited. Thus, p38-MAPK and ERK are important
for the TNF--mediated priming of neutrophils enabling subsequent
ANCA-induced respiratory burst. However, both pathways show differential
effects, whereby p38-MAPK controls the translocation of ANCA antigens to the
cell surface. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(TNF-) (priming) results in the translocation of target
antigens from the cytoplasm to the extracellular membrane, where the antigens
become accessible to circulating antibodies
(7,8).
Circulating neutrophils that express PR3 were detected in patients with active
Wegener's granulomatosis (9).
Human ANCA can activate TNF--treated neutrophils in vitro,
triggering the release of reactive oxygen species, toxic granule components,
cytokines, and leukotrienes
(7,10,11,12,13,14,15,16).
ANCA-activated neutrophils adhere to and damage endothelial cells
(17,18,19).
This cytotoxic effect is at least in part mediated by PR3 and elastase
(20) and can initiate
endothelial cell apoptosis
(21,22).
The early presence of neutrophils within vasculitic lesions has been shown in
patients (23). The importance
of neutrophils for the development of early vasculitic changes has been
demonstrated in animals (24).
However, little is known about the intracellular events that control
ANCA-mediated activation of TNF--primed neutrophils. Several soluble
and particulate stimuli result in increased tyrosine phosphorylation of
cellular proteins, and the importance of this signaling event to neutrophil
functions, including generation of reactive oxygen species, has been
demonstrated
(25,26,27).
Radford et al. (28)
reported that activation of cytokine-primed neutrophils by ANCA IgG results in
tyrosine phosphorylation of multiple proteins, including p39, p41, and p46 kD
bands, respectively. The data that we present here indicate a functional role
for tyrosine phosphorylation in ANCA activation of neutrophils. We explored
this issue in more detail and focused on mitogen-activated protein kinases
(MAPK). MAPK are activated via phosphorylation of threonine and tyrosine
residues by upstream dual-specificity kinases and provide potent inflammatory
signaling pathways
(29,30).
Furthermore, the activation of MAPK activity in neutrophils can be inhibited
by genistein
(31,32,33).
Therefore, we tested the hypothesis that the p38-MAPK and the extracellular
signal-regulated kinase (ERK) are intimately involved in activation of human
neutrophils by ANCA. |
Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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was obtained
from Genzyme (Rüsselsheim, Germany). Genistein,
SB202190, PD98059, staurosporin, and calphostin C were purchased from
Calbiochem (Bad Soden, Germany). Formyl-methionyl-leucyl-phenylalanine (FMLP),
daidzein, bovine erythrocyte superoxide dismutase (SOD; 2500 to 7000 U/mg
protein), ferricytochrome C, cytochalasin B, and phorbol myristate acetate
(PMA) were from Sigma. Dihydro-rhodamine-1,2,3 (DHR) was from Molecular Probes
(Eugene, OR). The polyclonal rabbit antibodies to tyrosine phosphorylated
p38-MAPK and ERK were acquired from New England Biolabs, Inc. (Beverly, MA),
horseradish peroxidase-labeled donkey anti-rabbit IgG was from Amersham
(Braunschweig, Germany), and FITC-conjugated F(ab)2-fragment of
goat anti-mouse IgG was from Dako (Hamburg, Germany). The 96-well microtiter
plates were from TPP-Company (Munich, Germany). Endotoxin-free reagents and
plastic disposables were used in all experiments.
Isolation of Human Neutrophils
Polymorphonuclear leukocytes (PMN) of healthy human volunteers were
isolated from heparinized whole blood by red blood cell sedimentation with
plasma gel, followed by Ficoll-Hypaque density gradient centrifugation.
Erythrocytes were lysed by incubation with hypotonic saline for 15 s. PMN were
spun down (200 g, 7 min) and reconstituted in HBSS with calcium and magnesium
(HBSS++). PMN (10 µl) in suspension were incubated with 40 µl
of trypan blue for 5 min at room temperature. Cells were counted in duplicate
using a hemocytometer and considered viable if able to exclude trypan blue.
The cell viability was detected in every cell preparation and found to be
greater than 99%. The percentage of PMN in the suspension was greater than 95%
by a Wright-Giemsa staining and by light microscopy.
Preparation of Immunoglobulins
Human IgG was prepared from Wegener's granulomatosis (three PR3-ANCA) and
microscopic polyangiitis (two MPO-ANCA) patients and from three healthy
controls. Samples were obtained from freshly drawn blood and kept at
-20°C. Plasma was filtered through a 0.2-µm syringe filter (Gelman
Sciences, Ann Arbor, MI) and applied to a protein G affinity column
(Pharmacia, Uppsala, Sweden). Bound immunoglobulins were eluted with 0.1 M
glycine-HCl buffer, pH 2.75 (elution buffer). After the antibodies emerged the
column, the pH was immediately adjusted to pH 7.0 using 1 M Tris-HCl, pH 9.0.
A mouse monoclonal anti-PR3 antibody (IgG1 
) previously used in our
laboratory (13) was
concentrated from tissue culture supernatant by using dia-flo ultrafilter YM
100 (Amicon, Beverly, MA) and purified by protein G column chromatography. A
mouse monoclonal to MPO (MPO-7, IgG1k) and an isotype-matched control (IgG1
) were purchased from Dako.
Superoxide Generation Assay
Superoxide was measured using the assay of SOD-inhibitable reduction of
ferricytochrome C as described by Pick and Mizel
(34). Briefly, neutrophils
were pretreated with 5 µg/ml cytochalasin B for 15 min at 4°C. Cells (1
x 106) were primed with 2 ng/ml TNF- for 15 min at
37°C before stimuli or buffer control was added. The final concentrations
were 5 µg/ml for the MAb to MPO, 15 µg/ml for the MAb to PR3, 75
µg/ml for purified IgG preparations, 1 µM FMLP, and 50 ng/ml PMA. All
experiments were set up in duplicates. When indicated, cells were preincubated
with inhibitory compounds or buffer control for 30 min on ice before cells
were primed with 2 ng/ml TNF-. The mixture was incubated in 96-well
plates at 37°C for up to 120 min, and the absorption of samples with and
without 300 U/ml SOD was scanned repetitively at 550 nm using a Microplate
Autoreader. No activating effect was seen when human and mouse control
antibodies were used or when cells were primed with 2 ng/ml TNF-. The
baseline activity of TNF--treated neutrophils was determined in every
experiment and was factored for each condition. In each independent experiment
using human ANCA-IgG preparations, three different PR3-ANCA and two different
MPO-ANCA preparations were tested.
Measurement of Respiratory Burst by Oxidation of DHR to
Rhodamine
The generation of reactive oxygen radicals was additionally assessed using
DHR. This method is based on the fact that reactive oxygen radicals cause an
oxidation of the nonfluorescence DHR to the green fluorescence rhodamine. In
brief, neutrophils (1 x 107/ml HBSS) were placed in
polypropylene tubes and kept for 5 min at 37°C in a water bath. Cells were
loaded with DHR (1 µM) for 10 min at 37°C. After 15 min of priming with
2 ng/ml TNF-, cells were divided and 5 x 105 cells
were incubated with the stimuli in a total assay volume of 100 µl. When
indicated, cells were preincubated with inhibitory compounds or buffer control
for 30 min on ice before the priming. After 45 min, the reactions were stopped
by adding 900 µl ice-cold PBS/1% bovine serum albumin. Samples were kept on
ice and analyzed using a FACScan (Becton Dickinson, Heidelberg, Germany).
Neutrophils were identified in the scatter diagram, and data were collected
from 10,000 cells per sample. The shift of green fluorescence in the FL-1 mode
was determined. For each condition, the mean fluorescence intensity (MFI,
representing the amount of generated reactive oxygen radicals) is
reported.
Western Blot Analysis for Phosphorylated p38-MAPK and ERK
PMN were stimulated with TNF- 2 ng/ml or buffer for 15 min followed
by stimulation with the MAb to MPO, isotype control, and buffer control,
respectively. Samples were harvested at the indicated time points, and cell
lysates were prepared by resuspending 2 x 106 cells in 20
µl of ice-cold lysing solution (20 mM Tris-Hcl [pH 8.0] containing 138 mM
NaCl, 1% Triton X-100, 2 mM ethylenediaminetetraacetate, 10% glycerol, 0.2 mM
sodium orthovanadate, 1 mM PMSF, 10 µg/ml aprotinin, 10 µg/ml leupeptin,
0.1 mM quercetin, 5 mM Iodoacetamide). Samples were stored for 20 min on ice
and centrifuged at 12,000 x g for 5 min at 4°C. Supernatant
was recovered and protein concentration was estimated by bicinchoninic acid
protein assay (Pierce, Munich, Germany). Samples were incubated for 5 min at
95°C in loading buffer (250 mM Tris-HCl [pH 6.8] with 4% sodium dodecyl
sulfate, 20% glycerol, 0.01% bromphenol blue, 6% ß-mercaptoethanol) and
25 µg of protein per lane were loaded on 10% sodium dodecyl
sulfate-polyacrylamide gel, electrophoresed, and blotted onto polyvinylidene
difluoride membrane by semidry technique. Membrane was blocked in 5% skim
milk/0.05% Tween/PBS overnight at 4°C. Phosphotyrosine was detected using
specific polyclonal rabbit antibodies to phospho-p38 and phospho-ERK1/2,
respectively (1:1,000 dilution) and a horseradish peroxidase-labeled donkey
anti-rabbit IgG (1:1,000). Blot was developed by incubation in a
chemiluminescence substrate (ECL, Amersham) and exposed to a x-ray film.
Densitometry of phospho-p38 and phospho-ERK1/2 was performed with scanned
x-ray films and the NIH image program.
Assessment of ANCA-Antigen Expression by Flow Cytometry
Flow cytometry was used to evaluate the effect of genistein, the p38-MAPK
inhibitor (SB202190), and the ERK-inhibitor (PD98059) on PR3 and MPO
expression on neutrophils. Immunostaining was performed as described
previously (13). Cells were
preincubated with 50 µM of the inhibitors or buffer control for 30 min on
ice, followed by treatment with 2 ng/ml TNF- or buffer control for 15
min at 37°C. Cells were pelleted at 200 x g for 5 min at
4°C and resuspended in 1 ml ice-cold PBS/0.5% paraformaldehyde. After the
cells were washed in HBSS without Ca2+/Mg2+, they were
incubated with dilutions of MAb to PR3, MPO, or an isotype control followed by
a secondary FITC-conjugated F(ab)2-fragment of goat anti-mouse IgG.
Flow cytometry was performed on the same day using a FACScan, and 10,000
events per sample were collected.
Statistical Analysis
Results are given as mean ± SEM. Comparisons were made by t
test or ANOVA as appropriate.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and activated by ANCA. Neutrophils were
preincubated with signal transduction inhibitors before the priming with
TNF- and the subsequent stimulation with ANCA. Using the assay of
SOD-inhibitable reduction of ferricytochrome C, our results demonstrate that
genistein decreased superoxide production by 30 min, whereas staurosporin and
calphostin did not (Figure 1A,
n = 3). Increasing concentrations of genistein (10-7 to
10-4 M) had a progressively greater effect
(Figure 1B). We next tested the
effect of the inhibitors in experiments using human ANCA as stimulators.
Figure 2 indicates that 50
µM genistein also inhibited the respiratory burst when neutrophils were
activated with human PR3- and MPO-ANCA, respectively. In each of the five
experiments using human PR3 ANCA, ANCA preparations from three different
patients were tested, and in each experiment using human MPO-ANCA, ANCA
preparations from two different patients were tested. Cells activated with
PR3-ANCA released 11.6 ± 1.7 nmol
O2-/106 cells per 60 min. Cells that were
preincubated with 50 µM genistein released only 2.1 ± 0.5 nmol
(P < 0.001). These values for MPO-ANCA without genistein were 16.0
± 2.8 nmol. Preincubation with genistein resulted in a release of 3.3
± 1.3 nmol (P < 0.001). To exclude the possibility that
genistein resulted in toxic effects that compromised the respiratory burst, we
determined the generation of superoxide in PMA-treated cells in the absence
and in the presence of 50 µM genistein. Neutrophils were incubated with 50
µM genistein or buffer control for 30 min on ice before the stimulation
with 50 ng/ml PMA. At 60 min, cells that were pretreated with buffer control
generated 47.7 ± 6.0 nmol O2-/106
cells, and pretreatment with genistein resulted in 45.7 ± 5.0 nmol
(n = 3). These data indicate that genistein used at 50 µM did not
affect the functional activity to generate superoxide. In addition, we
excluded that the observed effect of genistein on ANCA-stimulated respiratory
burst was nonspecific by showing that daidzein, an inactive analog of
genistein, did not inhibit respiratory burst. After 60 min, cells that were
pretreated with buffer control and stimulated with the MAb to MPO (5 µg/ml)
generated 27.8 ± 2.2 nmol O2-. Pretreatment with
genistein decreased this amount to 11.3 ± 3.2 nmol, whereas daidzein
resulted in 30.5 ± 2.5 nmol (n = 3). As demonstrated for the
activation with the MAb to MPO, staurosporin and calphostin also had no effect
on neutrophil activation by human ANCA (data not shown), whereas staurosporin
inhibited the superoxide generation in response to PMA (at 60 min from 41.6
± 5.6 nmol O2-/106 cells to 4.5
± 3.0 nmol; n = 3), and calphostin decreased superoxide
release in neutrophils stimulated with FMLP (at 60 min from 18.1 ± 4.6
nmol to 5.9 ± 2.8 nmol; n = 3), demonstrating that both
inhibitors were functioning. These data indicate that tyrosine kinase
activation plays a role in the respiratory burst of human neutrophils primed
with TNF- and activated by ANCA.
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Because tyrosine kinase activation can result in stimulation of
MAPK-dependent pathways, we examined the role of p38-MAPK and ERK in
ANCA-mediated superoxide release of human neutrophils. Using the MAb to MPO,
we titrated a dose-response curve for the effect of pretreatment of
neutrophils with the specific p38-MAPK inhibitor (SB202190) and the ERK
inhibitor (PD98059). Figure 3
demonstrates that SB202190 inhibits already at lower concentrations of 1
µM, whereas the inhibitory effect of PD98059 required higher concentrations
of 50 µM. Both compounds strongly inhibit at 50 µM and were selected for
the experiments with human ANCA and the MAb to MPO and PR3, respectively. In
each of the six experiments using human PR3-ANCA, ANCA preparations from three
different patients were tested, and in each of the six experiments using human
MPO-ANCA, ANCA preparations from two different patients were tested. For
clarity, although tested in a continuous superoxide assay, the data are given
for the representative 60-min time point of activation
(Figure 4). Both components
resulted in a significant inhibition of the respiratory burst. Activating
neutrophils with MPO-ANCA resulted in 16.0 ± 2.8 nmol
O2-/106 cells, whereas preincubation with
SB202190 decreased this amount to 0.9 ± 1.0 nmol (P <
0.001) and preincubation with PD98059 to 6.4 ± 2.4 nmol (P
< 0.01). PR3-ANCA stimulated 11.6 ± 1.7 nmol
O2-, and this amount was decreased by SB202190 to 1.9
± 0.6 nmol O2- (P < 0.001) and by
PD98059 to 4.0 ± 0.6 nmol O2- (P <
0.01). These results indicate that p38-MAPK and ERK are important for the
respiratory burst of human neutrophils primed with TNF- and activated
by ANCA.
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Using the dihydrorhodamine oxidation test, we studied the effect of
genistein, SB202190, and PD98059 on ANCA-mediated respiratory burst in human
neutrophils by a second independent method. The MFI was 11 ± 1 in
untreated cells and 20 ± 1 in cells primed with 2 ng/ml TNF-.
The MFI value increased to 578 ± 139 in TNF--primed neutrophils
activated with the MAb to MPO. This number was decreased to 18 ± 4 by
50 µM genistein, to 18 ± 5 by 50 µM SB202190, and to 267 ±
89 by 50 µM PD98059 (n = 3). A typical experiment is shown in
Figure 5. These results confirm
the assay of SOD-inhibitable reduction of ferricytochrome C, showing that all
three inhibitors decreased the respiratory burst in response to ANCA.
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Phosphorylation of p38-MAPK and ERK in TNF--Primed Neutrophils
Activated by ANCA
We performed Western blot analysis to study tyrosine phosphorylation of
p38-MAPK and ERK, respectively. Using antibodies that detect only the
phosphorylated forms of both kinases, we analyzed the effect of TNF-
priming as well as the effect of the subsequent ANCA stimulation.
Figure 6 shows representative
immunoblots of phosphorylated p38-MAPK and phosphorylated ERK
(Figure 6A) and the
corresponding densitometric analysis
(Figure 6, B through E). Minimal phosphorylation was seen at 0 time, whereas a significantly increased
phosphorylation was observed after priming with TNF-, reaching a
maximum at 5 min. The data show no significant additional effect of the MAb to
MPO on the phosphorylation of neither p38-MAPK nor ERK. With the use of
antibodies that recognize the nonphosphorylated forms of both kinases, no
change in expression was observed (data not shown). These data suggest an
important role for both the p38-MAPK and ERK pathways, during
TNF--mediated priming of human neutrophils.
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We determined the effect of genistein and the specific inhibitors of ERK
and p38-MAPK on the tyrosine phosphorylation of ERK and p38-MAPK
(Figure 7). Daidzein, an
inactive analog of genistein, was used as control. PMN were preincubated with
50 µM of the inhibitors, primed with 2 ng/ml TNF- and after 15 min
activated with 5 µg/ml of the MAb to MPO. On the basis of the kinetics of
tyrosine phosphorylation seen in Figure
6, samples were analyzed after 5 min of priming with TNF-
and after 5 min of stimulation with the MAb to MPO, respectively. Our data
indicate that genistein and PD98059 but not daidzein inhibited phosphorylation
of ERK, whereas none of the inhibitors diminished the phosphorylation of
p38-MAPK.
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Time-Dependent Inhibition of Superoxide by Genistein, the p38-MAPK
Inhibitor, and the ERK Inhibitor
To analyze further the role of p38-MAPK and ERK during TNF- priming
and during the subsequent ANCA stimulation, we studied the effect of the
p38-MAPK inhibitor, the ERK inhibitor, and genistein when added at different
time points. Figure 8 shows
that the effect of all three inhibitory compounds is time dependent. Adding
the p38-MAPK inhibitor SB202190 and genistein 15 min before TNF- and
within in the 15-min time period of TNF- priming resulted in a strong
decrease of superoxide release. Both substances demonstrated a similar degree
of inhibition. The inhibiting effect of the ERK inhibitor PD98059 was observed
only when the component was added before TNF-. All three inhibitors did
not decrease superoxide release significantly when added 15 min after the MAb
to MPO. These experiments provide further support for an important role of
tyrosine kinases and particularly for the p38-MAPK and ERK pathway during
TNF- priming.
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Effect of the p38-MAPK Inhibitor and the ERK Inhibitor on
Translocation of ANCA Antigens
We finally investigated a possible mechanism by which the p38-MAPK and the
ERK pathway may control ANCA-stimulated respiratory burst in
TNF--primed neutrophils. We explored the hypothesis that p38-MAPK and
ERK control the TNF--mediated translocation of ANCA antigens from the
intracellular granules to the cell surface. Using flow cytometry, we showed in
eight parallel experiments that inhibiting p38-MAPK with SB202190 (50 µM)
resulted in a decreased TNF--induced translocation of PR3. Untreated
cells showed an MFI for PR3 membrane expression of 31.4 ± 4.4. That
number was increased to 66.7 ± 8.6 by 2 ng/ml TNF-. The
TNF- effect was almost abrogated by SB202190 (34.1 ± 6.4;
P < 0.01). MPO membrane expression increased from 4.8 ± 1.1
in untreated cells to 14.3 ± 2.8 after TNF- treatment and was
diminished to 8.2 ± 2.2 by SB202190 (P < 0.05). In
contrast, no such inhibitory effect was seen when the ERK pathway was
inhibited (50 µM PD98059). A representative FACS analysis for PR3 membrane
expression is shown in Figure
9A with the corresponding statistics in
Figure 9B. In addition, we
assessed the effect of 50 µM genistein on the TNF--induced increase
in expression of PR3 and MPO (n = 5). The PR3 expression in untreated
cells was 27.5 ± 6.1 MFI, and genistein decreased the
TNF--induced increase of PR3 expression from 69.2 ± 14.0 MFI to
46.4 ± 9.5 MFI (not significant). The MPO expression was 4.2 ±
0.8 MFI for untreated cells, and genistein decreased the TNF--induced
MPO expression from 11.3 ± 2.4 to 7.3 ± 1.9 (P <
0.05). Together, these experiments indicate that tyrosine phosphorylation and
p38-MAPK control the TNF--mediated translocation of ANCA antigens to
the cell surface.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-pretreated human neutrophils can be activated by ANCA in
vitro. The results of this study show that tyrosine phosphorylation plays
an important role in this process. Tyrosine kinase activation can result in
stimulation of mitogenic signal transduction pathways, including p38-MAPK and
ERK. Our data indicate that inhibiting either of these two pathways results in
decreased respiratory burst. Furthermore, our data demonstrate that both
signaling pathways are crucial for ANCA activation by controlling
TNF--mediated priming. We show that both kinases act by different
mechanisms. P38-MAPK controls the translocation of ANCA antigens to the cell
surface. ERK does not seem to be specifically involved in this
translocation.
Several groups have shown that MAb to human ANCA antigens and human ANCA
itself can activate TNF--primed neutrophils and cause the release of
reactive oxygen species, toxic-granule components, cytokines, and leukotrienes
(7,10,11,12,13,14,15,16,35,36).
These effector functions may play an important role in inducing inflammation
in ANCA-associated vasculitis. Several investigations were performed to
determine which part of the ANCA molecule is important for the neutrophil
activation, e.g., F(ab')2 fragments versus
Fc part
(10,11,12,13).
However, the underlying intracellular signaling events in ANCA-induced
activation have not been elucidated in detail. An earlier report indicated
that activation of cytokine-primed neutrophils by ANCA IgG results in tyrosine
phosphorylation of multiple proteins, including p39, p41, and p46 kD bands,
respectively (28). Our
observation that genistein abrogates superoxide generation in ANCA-activated
TNF--primed neutrophils is in agreement with these findings. In
addition, our results indicate a functional significance of tyrosine
phosphorylation in this activation process.
Neutrophils that are challenged with various stimuli including ultraviolet
irradiation, growth factors, and cytokines respond with an activation of the
MAPK cascade. MAPK are activated via phosphorylation of threonine and tyrosine
residues by upstream dual-specificity kinases and phosphorylate substrates on
serine or threonine adjacent to proline residues
(29,30).
Three MAPK pathways have been described; however, only p38-MAPK and ERK play a
role in human neutrophils. No stimulus resulting in activation of Jun
N-terminal kinases/stress-activated protein kinase in neutrophils has been
reported. With the use of in vitro kinase activity assays, it was
shown that bacterial phagocytosis, granulocyte-macrophage colony-forming unit,
and TNF- stimulate ERK and p38-MAPK activation in human neutrophils.
This effect was inhibited by the specific inhibitors PD98059 and SB202190 and
by the tyrosine phosphorylation inhibitor genistein
(31,32,33).
In contrast, cross-linking of the Fc
II-receptor resulted in a
genistein-independent activation of MAPK
(37). Carefully established
dose-response experiments for the specific inhibitors PD98059 and SB202190
show a good dose-response curve from 0.1 µM up to 200 µM with a maximal
effect observed at approximately 50 to 100 µM
(38,39,40,41).
Here, we demonstrate that genistein and, more important, the specific ERK and
p38-MAPK inhibitors PD98059 and SB202190 decreased ANCA-induced respiratory
burst in TNF--primed neutrophils. These findings indicate an important
role of both MAPK pathways for the activation of neutrophils by ANCA.
Conceivably, the p39, p41, and p46 kD bands observed by Radford et
al. 28) in fact reflect
p38-MAPK and ERK (p42/44).
We found a rapid tyrosine phosphorylation of ERK and p38-MAPK in response
to small priming concentrations of TNF- peaking at 5 min and an
additional, although not significant, increase during the subsequent ANCA
stimulation. The strongest effect of the p38-MAPK and ERK inhibitors, as well
as of the tyrosine kinase inhibitor genistein, on superoxide generation
occurred when the inhibitory compounds were added before TNF- priming.
These data indicate the significance for tyrosine kinases and, in particular,
both MAPK pathways in TNF--mediated neutrophil priming rather than in
the consecutive ANCA stimulation. Other investigators also reported that
TNF- phosphorylates p38-MAPK and ERK observing the highest
phosphorylation or kinase activities after 10 min of stimulation
(33,42).
ERK activation by TNF- was also demonstrated by Rafiee et al.
(32). However, there is some
controversy regarding this issue. Other investigators reported that
TNF- activates only p38-MAPK
(43,44).
We showed that PD98059, the specific inhibitor of the ERK pathway, blocked
tyrosine phosphorylation of ERK, whereas the specific inhibitor of the
p38-MAPK pathway, SB202190, did not prevent tyrosine phosphorylation of
p38-MAPK. This finding is in agreement with reports from the literature;
others demonstrated that PD98059 inhibits the upstream MAPK/ERK kinase
preventing the phosphorylation and therefore the activation of ERK, whereas
SB202190 inhibits the p38-MAPK pathway by competing with ATP for the
ATP-binding site on p38-MAPK without affecting phosphorylation of p38-MAPK
itself
(41,43,45).
It is interesting that our data also indicate that genistein prevented
phosphorylation of ERK but not of p38-MAPK.
We demonstrated that p38-MAPK and ERK inhibit TNF--mediated
neutrophil priming via different mechanisms. P38-MAPK inhibition but not ERK
inhibition abrogated TNF--mediated translocation of ANCA antigens from
cytoplasmic granules to the cell surface. Several investigators showed that
treatment of resting neutrophils with TNF- results in increased
expression of PR3 and MPO. This process enables an interaction of ANCA with
the expressed target antigens, resulting in a full-blown neutrophil
activation. Witko-Sarsat et al.
(46) showed that within the
normal population, the percentage of neutrophils that express PR3 is variable
and that a high PR3 expression is significantly more frequent in patients with
ANCA-associated vasculitis. Additional activation resulted in a further
increase in the amount of PR3 expressed. Conceivably, high membrane
PR3-expressing neutrophils could favor the occurrence or the progression of
vasculitic inflammation. Thus, understanding the mechanisms that control PR3
and MPO expression is important. To our knowledge, our report is the first
indicating that the TNF--induced translocation of ANCA antigens from
cytoplasmic granules to the cell surface is controlled by p38-MAPK.
Our data demonstrate a similar effect of genistein and the p38-MAPK
inhibitor SB202190 on the respiratory burst to ANCA. The strongest inhibition
occurred when given before and during TNF- priming, whereas the ERK
inhibitor PD98059 affected only superoxide generation when given before
TNF-. We showed that genistein and SB202190 reduced respiratory burst
activity in the entire population of cells, whereas PD98059 decreased the
activity in only a portion of the neutrophils. In addition, SB202190 and
genistein but not PD98059 decreased TNF--induced ANCA-antigen
translocation. Together, these data support our hypothesis that tyrosine
phosphorylation and activation of p38-MAPK are important early steps in the
translocation of ANCA antigens and activation of neutrophils by ANCA, whereas
ERK seems to act by a different mechanism that will be explored in future
studies.
In summary, our data indicate that tyrosine phosphorylation and, in
particular, MAPK play an important role in the activation of human neutrophils
by ANCA. Inhibiting either of these pathways results in decreased respiratory
burst, mainly by abrogating TNF--mediated priming. Both kinases induce
different mechanisms, whereby p38-MAPK but not ERK controls the translocation
of ANCA antigens to the cell surface. Pharmacologic blockade of p38-MAPK and
ERK may limit inflammatory damage caused by ANCA-activated PMN.
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, controls; 
, samples that were pretreated
with 50 µM genistein. In each of the five independent experiments using
human PR3-ANCA, ANCA preparations from three different patients were tested,
and in each experiment using human MPO-ANCA, ANCA preparations from two
different patients were tested. Superoxide release was measured using a
continuous assay of SOD-inhibitable reduction of ferricytochrome C.
Immunoglobulins from healthy human controls (human Control) and a murine
IgG1
,
P < 0.01).
-,
TNF-
--, isotype control
buffer; (C) -
