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REGULAR ARTICLES |
*
Division of Nephrology, Department of Internal Medicine, University
Hospital Groningen, Groningen, The Netherlands.
Division of Clinical Immunology, Department of Internal Medicine,
University Hospital Groningen, Groningen, The Netherlands.
Department of Pathology, University Hospital Groningen, Groningen, The
Netherlands.
Correspondence to Dr. Casper F. M. Franssen, Department of Internal Medicine, Division of Nephrology, University Hospital Groningen, Hanzeplein 1, 9713 GZ Groningen, The Netherlands. Phone: 31 50 3612621; Fax: 31 50 3619310; E-mail: m.j.s.graler{at}int.azg.nl
 |
Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-primed neutrophils compared
with IgG fractions from anti-MPO-positive patients, as assessed by
ferricytochrome c reduction (P < 0.05) and dihydrorhodamine 123
oxidation (P < 0.01). In addition, IgG fractions from
anti-PR3-positive patients generated more neutrophil degranulation of
ß-glucuronidase (P < 0.01) than IgG fractions from
anti-MPO-positive patients. In conclusion, IgG fractions from
anti-PR3-positive patients with NCGN are more potent activators of the
respiratory burst and degranulation in vitro than IgG fractions from
anti-MPO-positive patients. These observations may be relevant in view of the
clinical differences between anti-PR3- and anti-MPO-positive patients with
NCGN. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Neutrophils are important effector cells of tissue damage in
ANCA-associated vasculitis and NCGN
(7). Activated neutrophils,
releasing lytic enzymes and oxygen radicals, are present in affected glomeruli
and in the renal interstitium of patients with ANCA-associated NCGN
(8). The number of activated
intraglomerular neutrophils correlates with the severity of renal tissue
damage as reflected in serum creatinine levels
(8). Several studies have shown
that ANCA are capable of activating neutrophils in vitro. Both
anti-PR3 and anti-MPO can activate tumor necrosis factor-
(TNF-)-primed neutrophils, leading to the production of reactive oxygen
metabolites and the release of lysosomal enzymes, including the ANCA antigens
themselves
(8,9,10,11,12).
In addition, ANCA can stimulate neutrophil cytotoxicity toward activated
endothelial cells in culture
(13,14).
These in vitro studies support the hypothesis that ANCA-mediated
neutrophil activation plays an important role in the pathophysiology of tissue
damage in patents with ANCA-associated vasculitis and/or NCGN
(15,16).
Until now, no studies have been published that have systematically compared the neutrophil-activating capacity of IgG fractions from consecutive anti-PR3- and anti-MPO-positive patients with ANCA-associated vasculitis or NCGN. Because of the observed differences in clinical and histopathologic disease activity, we questioned whether IgG fractions from anti-PR3-positive patients are capable of inducing a more pronounced activation of neutrophils in vitro than IgG fractions from anti-MPO-positive patients. Therefore, we measured the superoxide production and the release of granule constituents from healthy donor neutrophils upon stimulation with IgG fractions isolated from anti-PR3- and anti-MPO-positive patients with active NCGN. In addition, we evaluated whether the capacity of the IgG fractions to activate neutrophils was related to clinical, laboratory, or histopathologic parameters of vasculitic disease activity.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Extrarenal vasculitic disease was categorized as follows (17). Ear, nose, and/or throat: nasal mucosal ulceration, serous otitis media, sinusitis, tracheal stenosis; Lung: pulmonary infiltrates, coin lesions, alveolar hemorrhage; Skin: palpable purpura, ulcers, or nodules; Nervous system: mononeuritis multiplex, peripheral neuropathy; Musculoskeletal tract: arthralgia, arthritis, polymyalgia; Eyes: (epi)scleritis, keratitis, uveitis, retinal vasculitis; Heart: pericarditis, myocardial infarction, cardiomyopathy; Gastrointestinal tract: bowel perforation, aneurysms by abdominal angiography.
The Birmingham vasculitis activity score at the time of plasma collection for the isolation of the IgG fraction was retrospectively calculated (18). The score could range from 0 to 63. The control group consisted of 16 healthy volunteers, eight men and eight women, ages 22 to 48 yr (median, 34 yr).
Histopathologic Studies
All kidney biopsies were evaluated by the same pathologist (Dr. Tiebosch),
who was unaware of the ANCA specificity. Numbers of normal glomeruli and
numbers of glomeruli with global sclerosis were counted. Percentages of
glomeruli with necrosis and cellular and fibrous crescents were calculated by
dividing the number of affected glomeruli by the total number of nonsclerosed
glomeruli. Glomerular leukocyte influx and interstitial lesions, such as
interstitial inflammation, interstitial fibrosis, and tubular atrophy, were
graded semiquantitatively on a scale of 0 to 3 (absent, mild, moderate, and
severe, respectively). In addition, each biopsy was scored according to an
activity and chronicity index as reported previously
(19). The maximal possible
scores for the activity index and the chronicity index were 24 and 18,
respectively.
Plasma Samples and IgG Isolation
Plasma samples were obtained from freshly drawn blood and stored at
-20°C until isolation of IgG. Purified IgG fractions were prepared using a
protein G column (Mab Trap G II; Pharmacia Biotech, Uppsala, Sweden). Before
their use in the activation experiments, the IgG fractions were centrifuged in
an Eppendorf centrifuge for 15 min at 14,000 x g to remove
aggregates that might be present. None of the IgG fractions contained
endotoxin as determined by the limulus amoebocyte assay (Coatest, Endosafe,
Charleston, SC).
Detection of ANCA
IgG fractions were tested for ANCA by indirect immunofluorescence (IIF) as
described previously (20). IgG
fractions were serially diluted from 1:20 to 1:640. Two observers
independently read the slides. IgG fractions were tested for the presence of
anti-PR3, anti-MPO, and anti-elastase antibodies by enzyme-linked
immunosorbent assay as described previously
(2).
Isolation and Priming of Neutrophils
All experiments were performed twice using two different neutrophil donors.
The first neutrophil donor was a 45-yr-old healthy male volunteer. The second
neutrophil donor was a 25-yr-old healthy female volunteer. Peripheral blood
was collected from these donors into a Vacutainer tube containing 0.34 M
ethylenediaminetetra-acetic acid (EDTA) as anticoagulant. Peripheral blood was
diluted 1:1 in NaCl 0.9%, and polymorphonuclear granulocytes were isolated by
density gradient centrifugation (1000 x g for 20 min) on a
Lymphoprep density gradient (Nycomed Pharma, Oslo, Norway). Contaminating
erythrocytes were lysed with erythrocyte lysing buffer (155 mM
NH4Cl, 10 mM KHCO3, and 0.1 mM Na2EDTA
· H2O) for 5 min on ice. Next, cells were centrifuged at 600
x g for 5 min. This step was repeated once. Cells were then
washed twice in ice-cold phosphate-buffered saline, pH 7.4, and pelleted at
200 x g for 10 min. Finally, the neutrophils were suspended in
Hanks' balanced salt solution (Life Technologies, Paisley, Scotland, United
Kingdom) containing calcium and magnesium. Before the activation experiments,
the neutrophils were gradually warmed to 37°C. All buffers and media used
in the isolation procedure contained less than 5 pg/ml endotoxin as determined
by the limulus amoebocyte assay. To ascertain expression of PR3 and MPO on the
neutrophil surface
(9,10),
neutrophils (1 x 106/ml) were primed with 10 ng/ml
recombinant human TNF- (Boeringher Ingelheim, Heidelberg, Germany) for
15 min at 37°C. Before the activation experiments, the neutrophils were
treated with 5 µg/ml cytochalasin B (Serva, Heidelberg, Germany) for 5 min
at 37°C.
Superoxide Release Measured by the Ferricytochrome C Reduction
Test
Superoxide release by neutrophils was determined by measuring the
superoxide dismutase (SOD)-inhibitable reduction of ferricytochrome c
according to the method of Pick and Mizel
(21) with minor modifications
(22). In short, freshly
isolated primed neutrophils at a final concentration of 0.8 x
106/ml were incubated in 96-well microtiter plates (F-form; Greiner
BV, Alphen aan den Rijn, The Netherlands) with ferricytochrome c (C7752; Sigma
Chemical Co., St. Louis, MO) at a final concentration of 0.856 mg/ml, either
with SOD (S9636; Sigma Chemical Co.) at a final concentration of 13.16 U/ml or
with an equal volume Hanks' balanced salt solution, and stimulus. As stimulus,
we used the purified IgG fractions at a final concentration of 200 µg/ml.
N-formylmethionylleucylphenylalanine (fMLP; F3506, Sigma Chemical
Co., final concentration 0.67 µmol/L) served as a positive control
stimulus. The plates were incubated at 37°C for a total period of 120 min.
During these 120 min, the plates were scanned repetitively at 550 nm using an
automated microplate reader (Thermomax; Molecular Devices, Menlo Park, CA).
Between the readings, the plates were kept at 37°C. The superoxide
production was expressed as the difference in optical density (OD) 550 nm
(
OD 550) between the ferricytochrome c reduction test in the absence
and in the presence of SOD. Each test was performed in quadruplicate. Mean
values are reported.
Oxygen Radical Production Measured by Oxidation of Dihydrorhodamine
123 to Rhodamine
The release of reactive oxygen radicals by neutrophils was additionally
determined by measuring the oxidation of dihydrorhodamine 123 to rhodamine
(23). Freshly isolated primed
healthy donor neutrophils (final concentration 0.8 x 106/ml)
were placed in 96-well plates (flat-bottom polystyrene Black Cliniplate;
Labsystems Oy, Helsinki, Finland) with IgG fractions (final concentration 400
µg/ml) or with fMLP (final concentration 1.34 µmol/L) as stimulus and
with DHR (D632; Molecular Probes, Eugene, OR) at a final concentration of 0.16
mmol/L. During the following incubation period of 120 min at 37°C,
rhodamine production was measured fluorometrically (excitation and emission
wavelength 485 and 538 nm, respectively) by repetitive scanning using a
fluorimeter (Titertek Multiscan; Eflab Oy, Helsinki, Finland). Results are
expressed as fluorescence units (U). Each test was performed in triplicate.
Mean values are reported.
Degranulation Assays
Freshly isolated primed healthy donor neutrophils (final concentration 0.8
x 106/ml) were incubated in a 96-well microtiter plate
(U-form, Greiner) with the IgG fractions (final concentration 400 µg/ml) or
fMLP (final concentration 1.34 µmol/L) for 120 min at 37°C. At the end
of this incubation period, cell-free supernatants were collected for the
determination of ß-glucuronidase and lactoferrin.
ß-Glucuronidase activity of the supernatant was assessed by the cleavage of P-nitrophenolate from P-nitrophenyl-ß-glucuronide (Sigma, N1627), which can be measured spectrophotometrically at a wavelength of 405 nm (24). The assay was performed using 96-well microtiter plates (Greiner, F-form). Each well contained 50 µl of a 0.01 M solution of P-nitrophenyl-ß-glucuronide in 0.1 M NaAC, pH 4.0, which was mixed with 50 µl of the cell-free supernatant. After an 18-h incubation period in the dark at 37°C, the reaction was stopped by adding 100 µl of a 0.4 M solution of glycine buffer, pH 10, to each well. Finally, the plates were scanned at 405 nm with a microtiter plate reader. Nonstimulated primed neutrophils provided a baseline, whereas the total neutrophil ß-glucuronidase content was obtained by incubating the same amount of neutrophils with 1% Triton X-100 (Sigma). Results are expressed as percentage of the total ß-glucuronidase content released per 8 x 105 cells/ml.
The lactoferrin content of the supernatant was measured as described previously (25). Briefly, Hycult plates (Uden, The Netherlands) were coated with a F(ab')2 rabbit anti-human lactoferrin polyclonal antibody (Jackson Laboratory, West Grove, PA) overnight at room temperature at a dilution of 1:750, then washed and incubated with serial (twofold) dilutions of the samples, starting at a dilution of 1:25, for 1 h at 37°C. After washing, a rabbit anti-human lactoferrin polyclonal antibody conjugated with horseradish peroxidase (Jackson) was incubated for 30 min at 37°C at a dilution of 1:500. Finally, O-phenylenediamine (Sigma) substrate was incubated for 15 min. The color reaction was stopped with 100 µl per well of 2N H2SO4. OD values were measured at 492 nm. The standard consisted of a supernatant from neutrophils activated with calcium ionophore and was in the range 0.8 to 100 ng/ml lactoferrin.
Statistical Analyses
Differences in age, laboratory parameters, number of affected extarenal
organs, Birmingham vasculitis activity scores, renal biopsy characteristics,
OD 550 nm values at 120 min (ferricytochrome c reduction), fluorescence
units at 120 min (dihydrorhodamine 123 oxidation), and the release of
ß-glucuronidase and lactoferrin between groups were tested with the
Mann-Whitney U test. Differences in the male-to-female ratio and
organ involvement between groups were tested with the 
2 test
with Yates continuity correction. The level of significance used was 0.05. All
reported P values are two-sided. The normal range of the test results
was considered to be the mean ± 2 SD from the healthy control group.
Correlations between parameters were tested with the Spearman rank test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Table 4 shows the renal biopsy characteristics by autoantibody group. Patients with anti-PR3 tended to have a higher degree of glomerular leukocyte influx (P = 0.07) and a higher percentage of glomeruli with necrosis (P = 0.07) compared to patients with anti-MPO. Anti-PR3-positive patients had a higher renal activity index (P = 0.04) than anti-MPO-positive patients. The renal chronicity index did not differ significantly between both antibody groups.
|
IgG Fractions
The IgG concentration in the IgG fractions from anti-PR3- and
anti-MPO-positive patients was comparable (median IgG concentration [range]:
8.1 mg/ml [4.0 to 12.2] and 8.2 mg/ml [4.0 to 12.5]; NS). IIF titers of the
IgG fractions (tested at 4 mg/ml) did not differ significantly between
patients with anti-PR3 and those with anti-MPO (median IIF titer [range]: 80
[40 to >640] and 160 [40 to >640], respectively; NS). IgG fractions from
the patients with anti-PR3 did not contain anti-MPO and/or anti-elastase, and
IgG fractions from the patients with anti-MPO did not contain anti-PR3 and/or
anti-elastase as determined by enzyme-linked immunosorbent assay. None of the
IgG fractions from healthy control subjects contained anti-PR3, anti-MPO, or
anti-elastase.
Superoxide Release Measured by the Ferricytochrome C Reduction
Test
Figure 1A shows the results
of the ferricytochrome c reduction test, using primed healthy donor
neutrophils. fMLP induced more superoxide release compared with IgG fractions
from either healthy control subjects or from ANCA-positive patients. In
addition, the fMLP-induced superoxide release followed a different time course
than the IgG-induced superoxide release. Superoxide release upon stimulation
with fMLP was most pronounced in the first 30 min and leveled off thereafter,
whereas IgG fractions from healthy control subjects and ANCA-positive patients
induced a gradual release of superoxide over time. The results obtained with
the two different neutrophil donors were comparable. Superoxide release upon
stimulation with IgG fractions from healthy control subjects and from
anti-MPO-positive patients did not differ (first and second neutrophil donor,
P = 0.79 and P = 0.16, respectively). In contrast, IgG
fractions from anti-PR3-positive patients elicited significantly more
superoxide release compared with IgG fractions from healthy control subjects
(P < 0.05 for both neutrophil donors) or IgG fractions from
anti-MPO-positive patients (first and second neutrophil donor, P <
0.05 and P < 0.01, respectively). The superoxide release after 120
min did not differ significantly between the anti-PR3 and anti-MPO group when
corrected for the difference in renal activity index between both antibody
groups.
|
Using neutrophils from the first neutrophil donor, nine of the 17 IgG fractions from anti-PR3 (53%) and three of the 14 IgG fractions from anti-MPO-positive patients (21%) induced more superoxide release than the mean superoxide production + 2 SD induced by IgG fractions from healthy control subjects. Using neutrophils from the second neutrophil donor, nine of the 17 IgG fractions from anti-PR3 (53%) and two of the 14 IgG fractions from anti-MPO-positive patients (29%) induced more superoxide release than the mean superoxide production + 2 SD induced by IgG fractions from healthy control subjects.
Oxygen Radical Production Measured by Oxidation of Dihydrorhodamine
123 to Rhodamine
Results of the dihydrorhodamine oxidation test using primed donor
neutrophils are presented in Figure
1B. The results obtained with the two different neutrophil donors
were comparable. IgG fractions from healthy control subjects and from
anti-MPO-positive patients did not induce any measurable rhodamine production.
In contrast, anti-PR3-positive IgG elicited more rhodamine production compared
with healthy control IgG fractions (first and second neutrophil donor,
P < 0.0001 and P < 0.01, respectively) or
anti-MPO-positive IgG fractions (first and second neutrophil donors,
P < 0.0001 and P < 0.01, respectively). Notably, the
amount of rhodamine produced upon stimulation with IgG fractions from
anti-PR3-positive patients was modest compared with the effect of fMLP.
Degranulation Assays
Results of the ß-glucuronidase degranulation assay using
TNF--primed donor neutrophils are shown in
Figure 2 and
Table 5. The results obtained
with neutrophils from the two different neutrophil donors were comparable. IgG
fractions from anti-PR3 ANCA-positive patients induced more
ß-glucuronidase release than IgG fractions from either healthy control
subjects or anti-MPO ANCA-positive patients.
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As shown in Table 5, IgG fractions from anti-PR3-positive patients induced more lactoferrin release compared with IgG fractions from healthy control subjects. This was true for both neutrophil donors. Using neutrophils from the first donor, lactoferrin release was higher upon stimulation with IgG fractions from anti-MPO-positive patients compared with IgG fractions from healthy control subjects, but the difference did not reach statistical significance (P = 0.10). Using neutrophils from the first neutrophil donor, lactoferrin release was comparable for IgG fractions from anti-PR3- and anti-MPO-positive patients (P = 0.33). Using neutrophils from the second neutrophil donor, IgG fractions from anti-PR3-positive patients induced more lactoferrin release compared with IgG fractions from anti-MPO-positive patients (P < 0.01).
Relation between Neutrophil-Activating Capacity and Parameters of
Vasculitis Activity
Table 6 and
Figure 3 show correlations
between the capacity of the IgG fractions from ANCA-positive patients to
induce superoxide release, expressed as the OD 550 nm at 120 min in the
ferricytochrome c reduction test (using neutrophils from the first neutrophil
donor), and parameters of vasculitic disease activity in these patients. For
the whole group of ANCA-positive patients (anti-PR3 and anti-MPO; n =
31), there was no significant correlation between the capacity of the IgG
fractions to induce superoxide release and the number of affected organs, the
Birmingham vasculitis activity scores, serum creatinine levels, or C-reactive
protein levels. For the whole group (n = 31) and for the
anti-MPO-positive patient group (n = 14), the neutrophil-activating
capacity of the IgG fractions correlated with the ANCA titer in the IgG
fractions. The capacity of the IgG fractions to induce superoxide release
correlated with the percentage of glomeruli with cellular crescents
(r = 0.42; P = 0.03) and with the percentage of glomeruli
with necrosis (r = 0.53; P = 0.005) and tended to correlate
with the extent of glomerular leukocyte influx (r = 0.36; P
= 0.07). Accordingly, the neutrophil activating capacity of the IgG fractions
correlated with the renal activity index (r = 0.47; P =
0.02). In addition, there was an inverse correlation between the capacity of
the IgG fractions to elicit superoxide release and the percentage of glomeruli
with fibrous crescents (r = -0.61; P = 0.0009), the extent
of interstitial fibrosis (r = -0.57; P = 0.002), and the
extent of tubular atrophy (r = -0.46; P = 0.02). In line
with these findings, the capacity of the IgG fractions to induce superoxide
release correlated inversely with the renal chronicity index (r =
-0.59; P = 0.002) in these patients.
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For anti-MPO-positive patients, there was a comparable positive correlation between the capacity of the IgG fractions to induce superoxide release and the percentage of glomeruli with necrosis (r = 0.75; P = 0.003) and with the renal activity index (r = 0.56; P = 0.049) (Figure 3), as well as a comparable inverse correlation with the extent of interstitial fibrosis (r = -0.79; P = 0.001), the extent of tubular atrophy (r = -0.61; P = 0.03), and the renal chronicity index (r = -0.87; P = 0.0001) (Figure 3). For anti-PR3-positive patients, the capacity of the IgG fractions to induce superoxide release did not correlate significantly with any of the renal biopsy characteristics.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-primed neutrophils leading to the
production and release of reactive oxygen radicals and the release of
lysosomal enzymes
(8,9,10,11,12).
In these studies, patient selection criteria differed widely and IgG fractions
from a relatively small number of patients were tested. This is the first
study that has systematically compared the in vitro
neutrophil-activating capacity of anti-PR3 and anti-MPO ANCA from a relatively
large group of consecutive patients with ANCA-associated NCGN. We found that
most, but not all, anti-PR3-positive IgG fractions from patients with active
NCGN were capable of inducing the respiratory burst and degranulation of
healthy donor neutrophils in vitro. In contrast, only a minority of
anti-MPO-positive IgG fractions from patients with active NCGN induced the
respiratory burst or degranulation to an extent greater than that observed
with IgG fractions from healthy control subjects. These observations may be
relevant in view of the clinical and histopathologic differences between
anti-PR3 and anti-MPO ANCA-positive patients with NCGN that we and others have
found, such as a faster deterioration of pretreatment renal function and more
active renal lesions (including necrosis and cellular crescents) in patients
with anti-PR3, and more chronic renal lesions such as sclerosis and fibrosis
in patients with anti-MPO ANCA
(3,4,5,6).
Also in the present study, we found that renal biopsies from patients with
anti-PR3 ANCA-associated NCGN had a higher renal activity index compared to
patients with anti-MPO ANCA-associated NCGN.
The mechanisms involved in ANCA-mediated neutrophil activation are not
fully clarified. Upon priming, PR3 and MPO translocate from the cytoplasmic
granules to the extracellular membrane, where these antigens become available
for interaction with anti-PR3 and anti-MPO ANCA
(9,10).
ANCA probably interact with neutrophils by recognizing and binding PR3 or MPO
through the Fab portion of the Ig molecule
(26). The actual activation of
neutrophils by ANCA is subsequently largely mediated by Fc
RIIa
receptors
(12,27,28).
In some studies, however, F(ab')2 fragments of the ANCA
antibodies also induced neutrophil activation, suggesting that
FcRIIa-independent processes may be involved as well
(9,11,26).
In the present study, we found that IgG fractions from anti-PR3 and
anti-MPO-positive patients differ in their capacity to activate neutrophils
in vitro. IgG fractions from anti-PR3 and anti-MPO-positive patients
had similar titers of ANCA. However, it is important to note that the specific
activity of each type of antibody in the IgG preparations is not known. There
are several possible explanations for the observed differences in
neutrophil-activating capacity by IgG fractions from anti-PR3- and
anti-MPO-positive patients. The receptor engagement by ANCA might differ
because of a quantitative difference between anti-PR3 and anti-MPO in the IgG
preparations. Another explanation may be that receptor engagement by anti-PR3
and anti-MPO differs due to differences in IgG subclass distribution and/or
affinities of anti-PR3 and anti-MPO for their respective antigens.
Alternatively, anti-PR3- and anti-MPO-mediated neutrophil stimulation may use
different signal transduction routes. Finally, quantitative differences in the
expression of PR3 and MPO on the neutrophil surface
(29) may result in a divergent
availability of interaction sites for anti-PR3 and anti-MPO, respectively.
We found no significant correlation between the in vitro capacity of the IgG fractions to induce neutrophil superoxide release and parameters of vasculitic disease activity such as serum creatinine levels, C-reactive protein levels, and Birmingham vasculitis activity scores. Previously, we also found no significant correlation between the in vitro capacity to activate neutrophils and the number of H2O2-producing neutrophils present within the glomeruli (8). In the present study, however, we found that the capacity of the IgG fractions to induce superoxide release was positively correlated with the severity of active lesions, such as the proportion of glomeruli with necrosis. Interestingly, we also found an inverse relation between the neutrophil-activating capacity of the IgG fractions and the extent of subacute or chronic renal lesions, such as the proportion of glomeruli with fibrous crescents and the severity of interstitial fibrosis and tubular atrophy in these patients. This indicates that patients with predominantly active renal lesions had ANCA with a higher capacity to activate neutrophils in vitro, whereas patients with predominantly chronic renal lesions had ANCA with a lower capacity to activate neutrophils in vitro. However, the relation between the capacity of the IgG fractions to induce superoxide release and certain renal histopathologic characteristics was found only in the total ANCA-positive patient group (both anti-PR3 and anti-MPO) and in the anti-MPO-positive patient group, but not in the anti-PR3-positive patient group alone. Therefore, other factors besides ANCA-mediated neutrophil activation are probably important for the in vivo outcome. Thus, extrapolation of in vitro results to the in vivo situation must be done with great caution. In vivo, cytokines and other factors are present, which may influence neutrophil chemotaxis and activation. Indeed, ANCA-induced production of oxygen radicals in vitro was markedly enhanced in the presence of extracellular arachidonic acid (30).
In conclusion, anti-PR3 are more potent in activating neutrophils than anti-MPO. We propose that this finding may partly explain the clinical and histopathologic differences found in patients with anti-PR3 ANCA compared to patients with anti-MPO ANCA.
|
Acknowledgments |
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This study was supported by Grant C94-1434 from the Dutch Kidney Foundation. W. J. Sluiter is acknowledged for his statistical advice.
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Footnotes |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Blood 86:3189
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G. A. Preston, C. S. Zarella, W. F. Pendergraft III, E. H. Rudolph, J. J. Yang, S. B. Sekura, J. C. Jennette, and R. J. Falk Novel Effects of Neutrophil-Derived Proteinase 3 and Elastase on the Vascular Endothelium Involve In Vivo Cleavage of NF-{kappa}B and Proapoptotic Changes in JNK, ERK, and p38 MAPK Signaling Pathways J. Am. Soc. Nephrol., December 1, 2002; 13(12): 2840 - 2849. [Abstract] [Full Text] [PDF]
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R. Kettritz, M. Choi, W. Butt, M. Rane, S. Rolle, F. C. Luft, and J. B. Klein Phosphatidylinositol 3-Kinase Controls Antineutrophil Cytoplasmic Antibodies--Induced Respiratory Burst in Human Neutrophils J. Am. Soc. Nephrol., July 1, 2002; 13(7): 1740 - 1749. [Abstract] [Full Text] [PDF]
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 U. Schonermarck, P. Lamprecht, E. Csernok, and W. L. Gross Prevalence and spectrum of rheumatic diseases associated with proteinase 3-antineutrophil cytoplasmic antibodies (ANCA) and myeloperoxidase-ANCA Rheumatology, February 1, 2001; 40(2): 178 - 184. [Abstract] [Full Text] [PDF]
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K. Hattar, U. Sibelius, A. Bickenbach, E. Csernok, W. Seeger, and F. Grimminger Subthreshold concentrations of anti-proteinase 3 antibodies (c-ANCA) specifically prime human neutrophils for fMLP-induced leukotriene synthesis and chemotaxis J. Leukoc. Biol., January 1, 2001; 69(1): 89 - 97. [Abstract] [Full Text]
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R. KETTRITZ, A. SCHREIBER, F. C. LUFT, and H. HALLER Role of Mitogen-Activated Protein Kinases in Activation of Human Neutrophils by Antineutrophil Cytoplasmic Antibodies J. Am. Soc. Nephrol., January 1, 2001; 12(1): 37 - 46. [Abstract] [Full Text]
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, n = 17), IgG fractions from anti-myeloperoxidase
(anti-MPO) ANCA-positive patients (
, n = 14), or IgG fractions
from healthy control subjects ([UNK], n = 16). Presented are mean
values ± SEM. The dashed line indicates the superoxide production
induced by N-formylmethionylleucylphenylalanine (fMLP). (B) Oxygen
radical release measured by oxidation of dihydrorhodamine 123 to rhodamine
using TNF-
, n = 14). See