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Vth Medical Clinic (Nephrology, Endocrinology), University-Clinic Mannheim, Medical Faculty of the University of Heidelberg, Germany.
Correspondence to Dr. Rainer Nowack, Vth Medical Clinic (Nephrology, Endocrinology), University-Clinic Mannheim, Theodor-Kutzer-Ufer 1-3, 68167 Mannheim, Germany. Phone: 49-8382-5577; Fax: 49-8382-24091; E-mail: rnowack{at}t-online.de
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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receptor crosslinking. Upregulation of CD14 and
CD18 on monocytes by ANCA suggests a pathogenetic role of ANCA monocyte
interactions in systemic vasculitis. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Small vessel vasculitides are associated with antineutrophil cytoplasmic autoantibodies (ANCA). ANCA are specific markers of Wegener's granulomatosis (WG), microscopic polyangiitis (MPA), idiopathic crescentic necrotizing glomerulonephritis (NCGN), and Churg-Strauss syndrome (CSS) (4), and they were found to correlate with disease activity (5,6), although this remains controversial (7,8). The target antigens are constituents of the primary and secondary granules in the cytoplasm of neutrophils and monocytes. By indirect immunofluorescence (IIF) on ethanol-fixed granulocytes, sera from WG patients yield the cytoplasmic ANCA (C-ANCA) pattern with proteinase 3 (Pr 3) as the main target antigen (9,10,11), whereas in MPA and NCGN, a perinuclear ANCA (P-ANCA) pattern is usually found by IIF, with myeloperoxidase (MPO) as the main target antigen (12).
These target antigens can be expressed on the cell surface of neutrophils
and monocytes, and an interaction of ANCA with them is considered to be of
pathogenetic relevance in SV. Evidence for a pathogenetic role of ANCA comes
from ex vivo experiments in which incubation of neutrophils with IgG
from ANCA-positive patients caused a degranulation and release of toxic
reactive oxygen species (13),
upregulation of adhesion molecules, and cytokine release
(14,15,16).
These effects of ANCA on neutrophils can be observed only after prestimulation
of cells with tumor necrosis factor 
(TNF-; priming). Priming
increases surface expression of ANCA target antigens and thereby accessibility
for an interaction with ANCA.
Although monocytes are presumed to have an important role in ANCA-mediated disease, the interaction of ANCA with monocytes has been less well studied. In vivo, circulating monocytes are activated in SV as illustrated by upregulation of adhesion molecules and by enhanced generation of reactive oxygen species, respectively (17,18). Recently, ANCA IgG was shown to stimulate the production of monocyte chemoattractant protein 1 (MCP-1) and Il-8 in primed and unprimed monocytes (19,20).
In the present study, the effect of C- and P-ANCA from patients with WG, MPA, and NCGN on the phenotype of isolated monocytes and of monocytes from freshly drawn blood from healthy volunteers is investigated.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Purification and Modification of IgG
Sera were filtered through a sterile acetate membrane of 0.45-µm pore
width. (Nalgene Co., Rochester, MN). IgG were purified using a HiTrap protein
G affinity chromatography column according to the manufacturer's instructions
(Pharmacia Biotech AB, Uppsala, Sweden). After elution of the IgG, they were
dialysed against 0.1 M sodium acetate buffer. Protein concentration was
measured by Coomassie protein microtiter assay (Pierce Chemical Co.,
Rocheford, IL), and the purity of the IgG was confirmed by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis on a 10% sodium dodecyl
sulfate-polyacrylamide gel, using 10 µg of protein per lane. Before
incubation experiments, ANCA positivity was checked by ELISA and IIF again.
IgG were aliquoted and kept at -20°.
F(ab)2 fragments were obtained by pepsin digestion (pepsin A from porcine stomach mucosa, 4500 U/mg; Sigma, St. Louis, MO). Pepsin, 0.2 mg, was added to 10 mg of isolated IgG and kept in 0.1 M sodium acetate buffer overnight at pH 4.2 and 37°C. Digestion was stopped by adjusting the pH to 7.4. Samples were applied to a HiTrap affinity chromatography column to eliminate undigested IgG, and the fall-through was dialysed against 0.1 M sodium acetate buffer. Protein concentration was measured, and 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis was done to check for purity. To compare the binding affinity of IgG isolated from ANCA-positive sera and their corresponding F(ab)2 fragments, a modification of the Wieslab ANCA-ELISA für Pr-3 and MPO (Wieslab Ideon Research Park) was performed, using an alkaline phosphatase conjugated anti-F(ab)2 fragment-specific antibody (Jackson Immuno Research, Dianova, Hamburg, Germany) in a 1:1250 dilution.
For depletion of anti-Pr 3-specific and anti-MPO-specific antibodies, purified Pr 3 (a gift from Prof. Andrassy, Heidelberg) and MPO (Call Biochem, Bad Soden, Germany), respectively, were coupled with a N-hydroxysuccinimide activated HiTrap Sepharose column (Pharmacia Biotech AB). Complete IgG, isolated from C-ANCA- and P-ANCA-positive sera, were applied to the column, and the fall-through, cleared from anti-Pr 3 IgG and anti-MPO, respectively, was collected. The fall-through was dialysed and the protein concentration was measured. The material was checked for negativity to react with Pr 3 and MPO, respectively, by ELISA and IIF.
Monoclonal murine IgG1 antibodies against Pr 3 and MPO were purchased (Wieslab AB, Lund, Sweden). Monoclonal antibody (MAb) against two different epitopes of Pr 3 were used (B, C) (21), as well as one MAb directed against human MPO (22). These MAb were also used to prove surface expression of Pr 3 and MPO on monocytes by fluorescence-activated cell sorter (FACS) or microscopy.
Isolation of Monocytes and Incubation Protocol
For incubation, either isolated monocytes or freshly prepared whole blood
from healthy volunteers was used. Monocytes were isolated using Ficoll
gradient centrifugation and harvested after adherence to glass. Freshly
harvested whole blood was incubated in a 1:5 dilution with RPMI 1640 (Life
Technologies, Paisley, Scotland). Control IgG or patient IgG was added in a
concentration of 100 µg/ml; in experiments with F(ab)2
fragments, these were added to yield a concentration of 50 µg/ml and MAb
were used in a concentration of 30 µg/ml. After addition of IgG, the cells
were kept in dishes (petriPerm50 hydrophobic; Heraeus Instruments GmBH,
Osterode, Germany) for 18 h at 37°C under sterile conditions.
In some experiments, either polymyxin B (Sigma) was added to the cell suspension in a concentration of 1 µg/ml or cycloheximide (Sigma) was added to the medium in a concentration of 5 µg/ml.
Flow Cytometry
After incubation, cells were recovered by gentle pipetting, placed in 12
75-mm polystyrene tubes (Becton Dickinson Labware, Heidelberg, Germany), and
double-stained for CD14/CD18 and CD14/MH-CII (in some experiments) with
monoclonal mouse-anti-human IgG2a and IgG1 antibodies, R-phycoerythrin and
fluorescein isothiocyanate conjugated (DAKO A/S Glostrup, Denmark) according
to the manufacturer's instructions. One sample of every condition was
incubated with an irrelevant antibody (Simultest control IgG2a/IgG1, Becton
Dickinson, Immunocytometry Systems, San Jose, CA) and used as a negative
control. After labeling, erythrocytes were lysed by a lysing solution (FACS
Brand Lysing Solution, Becton Dickinson), and remaining cells were washed
twice with phosphate-buffered saline and resuspended in cell wash (Becton
Dickinson 38 Co., Erermbodegem, Belgium). Analyses were performed on a FACScan
(Becton Dickinson, Immunocytometry Systems) with an argon laser used at 488 nm
and the Lysis II software system (Hewlett Packard, Palo Alto, CA). Monocytes
were gated by forward/sideways scatter for their granularity and size
identity. A total of 10,000 monocytes were counted. Files were saved and
evaluated with the WinMDI software, version 2.1. Mean fluorescence intensity
in the patients group and the control group were compared.
Semiquantitative Reverse Transcription-PCR
For PCR analysis, peripheral blood mononuclear cells were isolated by
Ficoll Hypaque density gradient centrifugation. One µg of total RNA
(Trizol, Life Technologies BRL), isolated from ANCA- or control IgG-stimulated
peripheral blood mononuclear cells
(23), was reversed transcribed
into cDNA by oligo-dT priming and M-MLV reverse transcriptase (Life
Technologies BRL) in a total volume of 20 µl. PCR was subsequently
performed using 2 µl of cDNA and the following primers for CD14: reverse
5'-CGTTCGCCCAGTCCAGGAT-3' and forward
5'-ACGGTCAAGGCTCTCCGC-3'; for glyceraldehyde phosphate
dehydrogenase (GAPDH); reverse 3'-ATCCACAGTCTTCTGGGTGG-5' and
forward 3'-GTCTTCACCACCATGGAGAA-5'. Amplification was performed in
a 50-µl reaction mixture containing 50 mM KCl, 10 mM Tris-HCl (pH 8.4), 2
mM MgCl2, 0.06 mg/ml BSA, 0.25 mM dNTP, l U of Taq
polymerase, and 50 pmol of each primer using a Perkin Elmer 480 thermocycler
(Weiterstadt, Germany). PCR profiles were as follows: 94°C for 1 min,
62°C for 1 min, and 72°C for 2 min (27 cycles) followed by final
extension at 72°C for 7 min and cooling at 4°C. PCR products were
analyzed on a 1% agarose gel containing ethidium bromide. Intensity of the
bands was quantified by SCION imaging version 2.0 (Frederick, MD). Results
were expressed as CD14/GAPDH ratio
(24).
Statistical Analysis
T test for paired samples was used to calculate the equality of
mean comparisons using the Stata Statistical Software for MS Windows 95
(release 5.0, Stata Corporation, TX). Significance was defined according to a
P value of less than 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Effect of Complete IgG from ANCA-Positive Patients on CD14 and CD18
Expression
Incubation of freshly drawn whole blood with IgG from C-ANCA-positive
patients and from P-ANCA-positive patients resulted in a significant increase
in CD14 and CD18 but not in MHCII expression in monocytes, as compared with
control IgG (Table 2,
Figure 1). Similarly, the
incubation of isolated monocytes with ANCA IgG of both specificities caused an
upregulation of CD14 and CD18 but not of MHCII when compared with control IgG:
for CD14, the upregulation by C-ANCA IgG was 50.3% (SD: 20) and for P-ANCA
IgG, 61% (SD: 17), respectively, in six experiments each. The corresponding
results for CD18 were 23% (SD: 10) for C-ANCA and 15% (SD: 7) for P-ANCA.
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Coincubation of control and C- or P-ANCA IgG with polymyxin B (1 µg/ml) had no effect on the increase of CD14/CD18 expression, tested in three experiments each, using different C-ANCA IgG and P-ANCA IgG. The upregulation of CD14 by ANCA was 69% (SD: 14) in the presence of polymyxin B and 70% (SD: 9) in the absence of polymyxin B. The data for CD18 were 28% (SD: 11) versus 21% (SD: 7), respectively. Coincubation with cycloheximide (5 µg/ml) led to a reduction of CD14 upregulation by C-ANCA IgG to 42% (SD: 8) as compared with 69% (SD: 13) without cycloheximide in four experiments with four different C-ANCA IgG.
Time Response of CD14 Upregulation on Monocytes
Mean fluorescence intensity for CD14 on monocytes from freshly drawn blood
was measured at different time points between 2 and 24 h after the start of
incubation with IgG in three experiments each, for C-ANCA IgG and P-ANCA IgG.
The difference of CD14 expression between control IgG and ANCA IgG was first
detectable after 6 h of incubation and reached a peak after 10 h for C-ANCA
IgG and after 14 h for P-ANCA IgG. The difference in CD14 expression was
maintained up to 24 h (Figure
2).
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Effect of F(ab)2 Fragments from ANCA-Positive Patients on
CD14 and CD18
Incubation of monocytes from freshly drawn blood with F(ab)2
fragments from C-ANCA- and P-ANCA-positive IgG caused an upregulation of CD14
and CD18 when compared with F(ab)2 fragments from control IgG. The
upregulation of CD14 but not of CD18 was significantly lower for
F(ab)2 fragments compared with whole IgG (Tables
2 and
3). In additional experiments
with a modified ELISA, less antigen binding of F(ab)2 fragments of
Pr3- and MPO-ANCA in the experimental concentration (50 µg/ml) than with
the corresponding concentration of whole IgG (100 µg/ml) was found. Antigen
binding as indicated by ELISA was reduced to 30%.
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Effect of C-ANCA IgG Depleted from Anti-Pr 3 Antibodies and P-ANCA
IgG Depleted from Anti-MPO Antibodies on CD14 Expression
Incubation of monocytes from freshly drawn blood with C-ANCA IgG depleted
from anti-Pr 3 antibodies versus control IgG significantly reduced
the upregulation of CD14 as compared with the corresponding complete C-ANCA
IgG. P-ANCA IgG depleted from anti-MPO antibodies also caused significantly
less upregulation of CD14 than did the complete P-ANCA IgG
(Table 4,
Figure 3).
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Effect of MAb Against Pr 3 and MPO
Incubation of monocytes from freshly drawn blood with MAb against two
different epitopes of Pr 3 resulted in a significant increase of CD14 when
compared with human control IgG and even more markedly when compared with a
murine monoclonal isotype-control against CD8
(Table 5). Incubation with MAb
against MPO also led to a strong upregulation of CD14 when compared with human
control IgG or a murine monoclonal isotype control.
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Effect of Intact C-ANCA IgG on mRNA of CD14
After a 10-h incubation of monocytes with C-ANCA IgG (n = 4) and
P-ANCA IgG (n = 4), the ratio of CD14/GAPDH mRNA was slightly but
significantly (P < 0.05) increased as compared with monocytes
incubated with control IgG (Figure
4).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Incubation of monocytes from healthy volunteers with IgG purified from ANCA-positive sera caused an upregulation of CD14 and CD18 but not of MHCII antigens. The same results were obtained using isolated monocytes.
In each experiment, one IgG out of 12 ANCA sera from patients with active disease was compared with one IgG coming from 6 healthy control subjects. A considerable variation in the upregulation of CD14 and CD18 was observed, which was attributed to the heterogeneity of the different ANCA IgG preparations. However, in all experiments, the upregulation after ANCA incubation was present.
CD14 is one of the receptors for lipopolysaccharide (LPS) and is known to be upregulated by LPS complexed with its binding protein (26). Therefore LPS contamination of ANCA IgG causing CD14 upregulation has to be considered. Evidence against such a notion is provided. The difference between ANCA IgG and control IgG was still present when the cells and IgG were coincubated with polymyxin B, a cationic peptide that neutralizes LPS, and part of the experiments were performed with isolated monocytes in conditions free of human serum and lacking LPS-binding proteins that are required to mediate the LPS effects. This makes LPS contamination rather than a specific effect of ANCA IgG unlikely.
Moreover, the experiments suggest that upregulation of CD14 on monocytes is mainly the consequence of antigen-specific interactions of ANCA. The surface expression of ANCA target antigens on monocytes had been demonstrated beforehand by FACS analysis. After anti-Pr 3 antibodies were removed from C-ANCA IgG and anti-MPO antibodies were removed from P-ANCA IgG by immunoadsorption, the difference between ANCA IgG and control IgG with respect to CD14 and CD18 was strongly reduced, although not completely abolished. That upregulation was still detectable points to an additional role of antibodies with other specificities within the ANCA IgG for this effect. MAb against Pr 3 and MPO when compared with an isotype control also resulted in upregulation of CD14, even more than polyclonal ANCA IgG. Ralston et al. (20) also incubated monocytes with an MAb against Pr 3 in addition to IgG isolated from ANCA-positive patients and found a markedly increased production of IL-8.
There is a continuing debate on the importance of Fc-receptor involvement for the ANCA-associated effects in neutrophils. When using F(ab)2 fragments of ANCA IgG that lack the Fc fragment, some investigators found the same level of toxic oxygen species release as compared with complete IgG (13,27), whereas others have found no or reduced effects when the Fc receptor was not engaged (20,28,29).
In the experiments presented here, incubation of monocytes with F(ab)2 fragments from P- or C-ANCA IgG as compared with F(ab)2 fragments from normal IgG still caused a significant upregulation of CD14 and CD18, although this was reduced for CD14. Because F(ab)2 fragments maintain the antigen-binding sites, the results suggest that the antigen-binding sites of ANCA are primarily responsible for the observed effects. However, F(ab)2 fragments should be given in concentrations that are capable of binding as much antigen as the undigested IgG. The concentrations used in these experiments had been derived from literature but proved, in fact, to have only one third of the antigen-binding capacity of undigested IgG. This could explain the reduced upregulation of CD14 rather than lack of crosslinking of Fc receptors. The results contrast with other data showing that IL-8 release from primed monocytes was only induced by intact monoclonal anti-Pr 3 but not by F(ab)2 fragments of that monoclonal (20). It therefore seems that F(ab)2 fragments of ANCA IgG are sufficient to increase surface expression of certain proteins, such as CD14, whereas for cytokine synthesis and release, complete IgG is required.
It is not known how signal transduction is initiated after binding of ANCA to their antigens. Cycloheximide was able to reduce CD14 upregulation, and after 10 h of ANCA incubation the mRNA for CD14 was slightly increased. So, it seems that the effect is not due to mobilization of intracellular pools of CD14 molecules to the cell surface but the result of increased transcription and de novo synthesis of protein.
it is of note that upregulation of CD14 and CD18 by ANCA was observed in
unprimed monocytes. Not only was a preincubation with proinflammatory
cytokines such as TNF- withheld, but also the stimulatory effect of
monocyte isolation was avoided. In viable neutrophils, priming is required for
further activation by ANCA
(13,27,29).
In apoptotic neutrophils, however. ANCA react with their antigens exposed on
the surface (30). The
importance of priming in neutrophils was mainly explained as rendering ANCA
antigens accessible for ANCA on the cell surface. In monocytes, there also
seems to be a role for priming with TNF-, as it was shown to increase
Pr 3 surface expression (20)
in experiments in which ANCA induced IL-8 production. The investigators,
however, did not study unprimed monocytes. Conversely, Casselman et
al. (19) found MCP-1 to
be released from unprimed monocytes in response to incubation with IgG from
ANCA-positive patients. It can be concluded with caution that to be stimulated
by ANCA, monocytes do not depend on priming in the same way that neutrophils
do.
What could be the functional consequences of CD14/CD18 upregulation in
response to the ANCA-monocyte interactions? CD14 is one of several receptors
for LPS (31), and LPS that is
bound to LPS-binding protein reacts with CD14. More expression of CD14 on the
surface could increase the interaction with this complex, and this is known to
induce cytokine production and upregulation of adhesion molecules
(32). CD14 participates in the
TNF- release of macrophages and is therefore integrated in the
proinflammatory cascade that will eventually result in tissue damage
(32). From other experiments,
it is known that CD14 is further upregulated after binding of the
LPS/LPS-binding protein complex and that concomitantly CD18/CD11a is
upregulated and its avidity for the ligand intercellular adhesion molecule-1
is increased (33). CD14
thereby contributes to the adherence of monocytes to cytokine prestimulated
endothelial cells (34).
Activated monocytes will then synthesize and secrete proinflammatory
cytokines, which will further trap inflammatory cells at these site, start to
proliferate locally, and participate in granulomata formation and cause
necrosis and crescent formation in the glomeruli
(35,36).
Twenty years ago, it was shown for the first time in a series of elegant
experiments using a model with exchange of marrow transplantation between
Chediak-Higashi mice and syngeneic partners that marrow-derived monocytes
contribute to mesangial cell hypercellularity after deposition of immune
complexes (37). Since that
time, the concept of ANCA-associated disease has evolved and intraglomerular
monocytes in human ANCA-associated diseases have also been demonstrated
(2,3).
Our study suggests that ANCA may play an additional pathogenic role by direct
induction of phenotypic changes in unprimed monocytes. The pathophysiologic
consequences of the observed phenotypic changes of monocytes should be
investigated further.
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Acknowledgments |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RIIa. J Immunol153
: 1271-1280,1994[Abstract]
RII-dependent process. Clin Exp Immunol98
: 270-278,1994[Medline]
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