Journal of Leukocyte Biology Myeloid cells, immune suppression, tumor immunology
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(Journal of Leukocyte Biology. 2002;71:996-1004.)
© 2002 by Society for Leukocyte Biology

Wegener’s granulomatosis: antiproteinase 3 antibodies induce monocyte cytokine and prostanoid release—role of autocrine cell activation

Katja Hattar*, Annette Bickenbach*, Elena Csernok{dagger}, Simone Rosseau*, Ulrich Grandel*, Werner Seeger*, Friedrich Grimminger* and Ulf Sibelius*

* Department of Internal Medicine, Justus-Liebig-University, Giessen, Germany; and
{dagger} Department of Rheumatology, Medical University of Lübeck, Germany

Correspondence: Dr. Ulf Sibelius, Department of Internal Medicine, Justus-Liebig-University Giessen, D-35385 Giessen, Germany. E-mail: ulf.sibelius{at}innere.med.uni-giessen.de


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Antineutrophil cytoplasmic antibodies (ANCA) targeting proteinase 3 [PR3; cytoplasmic ANCA (c-ANCA)], a leukocyte serine protease, are highly specific for Wegener’s Granulomatosis (WG). A pathogenetic role for c-ANCA has been proposed as a result of their ability of activating neutrophils, whereas their interaction with monocytes is less well characterized. We investigated the influence of monoclonal anti-PR3 antibodies (anti-PR3) and c-ANCA from WG sera on monocyte cytokine and prostanoid release. We found that PR3 was expressed on the surface of isolated monocytes. Anti-PR3 challenge provoked a pronounced release of cytokines with early appearance of tumor necrosis factor {alpha} (TNF-{alpha}) and interleukin (IL)-1ß and delayed release of IL-6, IL-8, and thromboxane A2 (TxA2). The secretory response was reproduced by c-ANCA but not by human and murine control IgG and anti-CD14 antibodies. Because F(ab)2 fragments of anti-PR3 were ineffective, coligation of Fc gamma receptors (Fc{gamma}R) was apparently mandatory for monocyte activation. Using soluble receptors for TNF-{alpha} and IL-1ß and a Tx receptor antagonist, we noted that the "early" cytokines functioned as inducers of TxA2, which then activated IL-8 release. In contrast, IL-6 formation was an independent event. We concluded that anti-PR3 antibodies are potent inducers of monocyte cytokine and prostanoid release, and TNF-{alpha}, IL-1ß, and TxA2 function as facilitators of the secretory response. These mechanisms may contribute to inflammatory tissue injury in WG.

Key Words: autoimmunity • macrophages • lipid mediators • inflammation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The pathogenesis of Wegener’s Granulomatosis (WG), a systemic vasculitis most frequently affecting the lung and kidneys, is still poorly understood. Granuloma formation and endothelial necrosis with leukocyte accumulation in and around vessel walls represent the predominant histological findings [1 , 2 ]. Proinflammatory cytokines, such as tumor necrosis factor {alpha} (TNF-{alpha}), interleukin (IL)-1ß, and IL-8, seem to play a central role during the development of vascular lesions, as they are found to be elevated systemically and locally at inflammatory sites in WG [3 4 5 6 ]. Moreover, a pathogenetic role was attributed recently to IL-8 in antineutrophil cytoplasmic antibody (ANCA)-associated glomerulonephritis [4 ]. Monocytes and resident macrophages are the major cellular source of proinflammatory cytokines. Increased TNF-{alpha} expression of circulating mononuclear cells in WG has been demonstrated [7 ], although the mechanisms stimulating monokine synthesis in WG remain unknown.

The diagnosis of WG has profited largely from the detection of ANCA [8 ]. Cytoplasmic ANCA (c-ANCA) targeting proteinase 3 (PR3), a leukocyte serine protease localized within the granules of polymorphonuclear leukocytes (PMN) and monocytes [9 , 10 ], possess a high sensitivity and nearly 95% specifity for WG [11 , 12 ]. As the c-ANCA titer correlates well with the disease activity in some studies [8 , 13 , 14 ], a pathogenetic role for these autoantibodies has been proposed. Moreover, in vitro, the interaction of ANCA with neutrophils results in activation of PMN degranulation, superoxide secretion, release of lipid mediators, and stimulation of neutrophil-related endothelial cytotoxicity [15 16 17 18 ]. Thus, neutrophils are suggested to be major effector cells in the development of inflammatory lesions in active WG.

In contrast, the role of the monocyte has not yet been evaluated to the same extent. Monocytes are an integral part of granulomas and glomerular crescents in active WG [1 , 2 , 19 ]. Moreover, the extent of monocyte activation, as assesed by soluble products of monocyte activation such as neopterin and IL-6, was found to correlate with the disease activity in WG [20 ]. Recently, it has been reported that the interaction of PR3-ANCA with TNF-{alpha}-primed mononuclear cells stimulates IL-8 release by cross-linking Fc gamma receptors (Fc{gamma}R) and PR3 expressed on the monocyte cell surface [21 ]. Moreover, these autoantibodies induce monocyte chemoattractant protein-1 release from mononuclear cells [22 ]. Against this background, we now investigated the effect of monoclonal anti-PR3 antibodies and c-ANCA originating from WG sera on cytokine and prostanoid release from highly purified monocytes in the absence of a preceding priming maneuver. Our interest was centered on the monocyte-derived "classic" proinflammatory cytokines TNF-{alpha}, IL-1ß, IL-6, and IL-8, on monocyte prostanoid generation, and on a putative role of TNF-{alpha}, IL-1ß, and thromboxane (Tx) A2 as autocrine or paracrine facilitators of full monocyte activation, as suggested from non-ANCA-related stimulation of mononuclear cells [23 24 25 26 ].


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Ficoll-Paque was purchased from Pharmacia (Upsala, Sweden), fetal calf serum (FCS) from Greiner (Frickenhausen, Germany), and all other media and supplements from Gibco (Eggenstein, Germany), unless otherwise indicated.

All antibodies used for cytokine enzyme-linked immunosorbent assays (ELISA) as well as recombinant cytokines were purchased from R&D Systems (Wiesbaden, Germany): monoclonal antibodies (mAb) against TNF-{alpha} (mAb 610), IL-1ß (mAb 601), IL-6 (mAb 206), and IL-8 (mAb 208); biotinylated anti-human antibodies against TNF-{alpha} (BAF 210), IL-1ß (BAF 201), IL-6 (BAF 260), and IL-8 (BAF 208); recombinant human IL-1ß (201-LB-005), TNF-{alpha} (210-TA), IL-6 (206-IL-010), and IL-8 (208-TA-010); as well as recombinant human IL-1 soluble receptor type II (IL-1sR) and recombinant human soluble TNF receptor type II (TNFsR). Peroxidase-conjugated streptavidin [horseradish peroxidase (HRP)] was purchased from Zymed Laboratories (San Fransisco, CA), phycoerythrin-(PE)-conjugated goat anti-mouse immunoglobulin G (IgG) was from Dako (Hamburg, Germany), and pooled human IgG (Octagam) was from Octapharma (Langenfeld, Germany). Murine monoclonal CD-14 antibodies (MY-4) were purchased from Coulter Immunotech (Hamburg, Germany), and isotype-control mouse IgG1 was from Sigma Chemical Co. (Deisenhofen, Germany). The TxA2 receptor antagonist daltroban was obtained from Boehringer Mannheim (Mannheim, Germany), and cycloheximide, polymyxin B (PMB), and cytochalasin B (CCB) were purchased from Sigma Chemical Co. Protein G sepharose columns and PD-10 desalting columns were from Amersham Pharmacia Biotech (Uppsala, Sweden). The kinetic-OLC limulus amebocyte cell lysate test for the detection of endotoxin was from Chromogenix (Mölndal, Sweden). ELISA kits for the detection of TxB2 were from Cayman Laboratories (Ann Arbor, MI), and cell culture plasticware was purchased from Falcon (Mannheim, Germany).

Anti-PR3 antibodies
Murine mAb targeting PR3 were prepared by hybridoma technology, as described previously [10 ]. The clone WGM2 (IgG1) was chosen for further experiments. F(ab)2 fragments were generated by digestion with pepsin in 0.1 NaAc for 16 h at 37°C. After dialysis against phosphate-buffered saline (PBS), purity was checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. ANCA-IgG was isolated from monospecific anti-PR3-positive serum (c-ANCA titer>1:160) obtained from patients with active WG, and normal IgG was isolated from healthy volunteers. IgG fractions were prepared using commercially available protein G sepharose columns according to the manufacturer’s instructions. In brief, serum samples were diluted with an equal volume of binding buffer (20 mM sodium phosphate) and applied on the column, which had been equilibrated previously with 10 vol binding buffer. Subsequently, the column was washed with binding buffer and than eluted with 1 M acetic acid, pH 2.7. The IgG fractions were buffer exchanged using PD-10 desalting columns, and IgG concentration was measured according to standard procedures. PR3 specificity of the mAb- and serum-derived antibodies as well as the capacity of the F(ab)2 fragments to bind PR3 were assessed by antigen specific ELISA. Using the kinetic-OLC limulus amebocyte cell lysate test, endotoxin contamination of the murine and human anti-PR3 antibodies was below 15 pg/ml.

Isolation of monocytes
Monocytes were isolated by countercurrent centrifugal elutriation from leukocyte-enriched buffy coats kindly provided by the local blood bank. Initially, peripheral blood mononuclear cells (PBMC) were separated by density-gradient centrifugation on Ficoll-Paque gradients. Then, monocytes were purified by elutriation in a Beckmann centrifugal elutriator (JE-5.0 elutriation system). Purity was determined by fluorescence-associated cell sorting, and only fractions containing >=90% monocytes, <1% granulocytes, and <10% lymphocytes were used for experiments. Cell viability was always >97% as assessed by the trypan blue-dye exclusion test.

Flow cytometry
To determine surface expression of PR3, flow cytometry was performed. In brief, elutriated monocytes (5x106/ml) were resuspended in RPMI supplemented with 2% FCS, 1% penicillin-streptomycin, and 1% glutamin and were incubated with TNF-{alpha} (2 ng/ml) or sham-incubated for 30 min. Cells were pelleted at 4°C and resuspended in PBS containing 0.1% bovine serum albumin and 0.02% sodium acid. Then, 2 x 105 cells were distributed to each well of flexible round-bottom microtiter plates and were washed. Before adding mAb, 20 µl pooled human Ig (100 µg/ml) was added to block monocyte FcIgG receptors. Next, murine monoclonal anti-PR3 antibodies (10 µg/ml) or mouse control IgG (10µg/ml) were added and were incubated for 30 min at 4°C. After three washes, the secondary antibody, a PE-conjugated goat anti-mouse IgG (50 µg/ml), was added and again incubated for 30 min at 4°C. After three final washes, cells were resuspended in PBS and kept on ice until flow cytometric analysis. Flow cytometry was performed in a FACScan (Becton-Dickinson, Mountain View, CA) using forward and orthogonal light scatter to select viable cells. CellQuest® research software (Becton-Dickinson) was used to analyze the generated data.

Cell culture and stimulation
Elutriated monocytes were resuspended in RPMI supplemented with 2% FCS, 1% penicillin-streptomycin and 1% glutamin, plated in 24-well tissue culture plates at 106/ml (500 µl/well), and incubated at 37°C in a 5% CO2-humidified atmosphere. Monocytes were cultured with media alone, with murine monoclonal anti-PR3 antibodies (1 µg/ml) or with purified PR3-ANCA (1 µg/ml) originating from WG serum. Mouse IgG1 isotype control (1 µg/ml) and murine antibodies targeting CD14 (MY-4; 1 µg/ml) were used as control antibodies.

In experiments designed to investigate the effects of TNF-{alpha}, IL-1ß, or Tx, TNFsR (0.25 µg/ml), IL1sR (2.5 µg/ml), or the Tx receptor antagonist daltroban (10 µM) was applied 10 min before anti-PR3 challenge. When addressing the effect of PMB (10 µg/ml), this agent was co-applied with antibody challenge, whereas cyclohexemide (1 µg/ml) and CCB (5 µg/ml) were admixed 30 min before stimulation. After 4, 8, 16, or 24 h of incubation, cell supernatants were harvested, debris was removed by centrifugation, and samples were stored at -20°C until further processing.

Cytokine ELISA
Release of TNF-{alpha}, IL-1ß, IL-6, and IL-8 was determined in a direct sandwich ELISA. In brief, immunoassay plates were coated with mouse monoclonal anti-human TNF-{alpha}, IL-1ß, IL-6, or IL-8 antibodies at a concentration of 4 µg/ml. After a blocking period, samples were added. Recombinant human TNF-{alpha}, IL-1ß, IL-6, and IL-8 were used for standard titration curves. To sandwich the antigen, biotinylated antibodies were applied at the following concentrations: 400 ng/ml polyclonal anti-TNF-{alpha}, 200 ng/ml anti-IL-1ß, 50 ng/ml anti-IL-6, or 40 ng/ml anti-IL-8. Next, plates were incubated with HRP-conjugated streptavidin followed by addition of substrate (H2O2 and ABTS). Adsorbance was measured at 450 nm in a microplate reader using SLT LabInstruments software (Creilsheim, Germany) to analyze the generated data. IL-8 and IL-6 ELISA were sensitive to 15 pg/ml, IL-1ß, and TNF-{alpha} to 7 pg/ml. The assays did not exhibit detectable cross-reactivity with TNFsR, IL-1sR, the Tx receptor antagonist, or the anti-PR3 antibodies. TxB2 was quantified in a commercial ELISA system.

Statistics
For statistical comparison, one-way analysis of variance was performed, followed by Tukey’s honestly significant difference test when appropriate. A level of P < 0.05 was considered to be significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Isolated monocytes express surface PR3
To determine monocyte surface expression of PR3, a prerequisite for specific ANCA-targeting monocytes was subjected to flow cytometry after the elutriation procedure. Flow cytometry demonstrated that ~80% of isolated monocytes were positive for surface PR3 (Fig. 1A ), as determined by staining with anti-PR3 (10 µg/ml) versus control IgG (10 µg/ml). In contrast, when analyzed for PR3 surface expression in a whole blood assay, monocytes were only weakly positive for the c-ANCA target antigen (Fig. 1B) . PR3 surface expression on isolated cells could be enhanced further by monocyte priming with TNF-{alpha} (2 ng/ml, 30 min; Fig. 1A ). To avoid any effect of such priming procedures on the monocyte-secretory functions, the following experiments addressing the effect of anti-PR3 on monocyte cytokine and prostanoid generation were performed in the absence of TNF priming.



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  		Figure 1. (A) Surface expression of PR3 on isolated monocytes. Isolated monocytes (5x106/ml) were sham-incubated or primed with TNF-{alpha} (2 ng/ml) for 30 min. Following incubation with isotype control IgG (10 µg/ml) or anti-PR3 (10 µg/ml), a PE-labeled goat anti-mouse IgG (50 µg/ml) was added, and flow cytometry was performed. Note the strong staining for PR3 vs. control IgG. PR3 surface expression was enhanced further by TNF-{alpha}. Representative data of five flow cytometric studies are given. (B) Surface expression of PR3 on whole blood monocytes. Monocytes (5x106/ml) of the same donor were stained with anti-PR3 (10 µg/ml) in a whole blood assay or after the elutriation procedure. After incubation with a PE-labeled goat anti-mouse IgG (50 µg/ml), flow cytometry was performed. Note the weak staining for anti-PR3 in whole blood vs. elutriated monocytes. Representative data of three flow cytometric studies are given.

 
Anti-PR3 antibodies induce a time-dependent activation of the monocyte-cytokine cascade
Isolated monocytes (106/ml) were treated with anti-PR3 (1 µg/ml) or isotype-matched control IgG (1 µg/ml) or were sham-incubated for various time periods. Upon anti-PR3 challenge, but not in response to control IgG, a pronounced, time-dependent activation of cytokine release was noted, and impressive quantities of TNF-{alpha} (Fig. 2A ), IL-1ß (Fig. 2B) , IL-6 (Fig. 2C) , and IL-8 (Fig. 2D) were recovered from the cell supernatants. However, kinetics of release did differ between these cytokines: Whereas TNF-{alpha} and IL-1ß levels peaked after 8 h, plateauing of IL-6 and IL-8 required 16 h.



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  		Figure 2. Time course of cytokine release in response to anti-PR3. Isolated monocytes (106/ml) were challenged with anti-PR3 (1 µg/ml) or control IgG (1 µg/ml), or sham incubation (control) was performed. At indicated time points, cell supernatants were harvested, and release of TNF-{alpha} (A), IL-1ß (B), IL-6 (C), and IL-8 (D) was quantified by ELISA. Means ± SEM of at least six independent experiments each are given. *, Values differ significantly from controls.

 
Activation of the cytokine cascade can be reproduced by c-ANCA
Aiming to reproduce the prosecretory effect of the murine mAb, three different ANCA-IgG preparations, derived from patients with monospecific anti-PR3-positive WG, were used (100 µg/ml). In response to ANCA-IgG, but not to normal IgG from healthy donors, pronounced activation of TNF-{alpha}, IL-1ß, IL-6, and IL-8 generation was noted, with similar kinetics, but overall lower quantities as compared with monocyte activation with murine monoclonal anti-PR3 (Fig. 3A 3B 3C 3D ).



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  		Figure 3. Time course of cytokine release in response to human c-ANCA. Isolated monocytes (106/ml) were challenged with IgG preparations from three patients with anti-PR3-positive WG (ANCA-IgG1–3; 100 µg/ml) or IgG isolated from healthy donors (IgGc, 100 µg/ml), or sham incubation (control) was performed. At indicated time points, cell supernatants were harvested, and release of TNF-{alpha} (A), IL-1ß (B), IL-6 (C), and IL-8 (D) was quantified by ELISA. Data represent means ± SEM of two independent experiments with each value performed in triplicates. *, Values differ significantly from controls.

 
The anti-PR3-induced cytokine release is specific, requires de novo protein synthesis, and is dependent on ligation of Fc{gamma}R
As depicted for IL-8 release after 16 h (Fig. 4 ), the anti-PR3-induced secretory response may not be evoked by some contamination with endotoxin, as it was unaffected by PMB (10 µg/ml), and equal amounts of PMB abrogated IL-8 release elicited by lipopolysaccharide (LPS) concentrations up to 100 ng/ml (LPS: 193.74±22.6 ng/ml IL-8; LPS plus PMB: 4.5±0.8 ng/ml IL-8 after 16 h of incubation). Further, anti-PR3-induced monokine release was not dependent on phagocytic events, as disruption of the actin cytoskeleton by CCB (5 µg/ml) did not affect the monokine response. As demonstrated by the inhibitory effect of cycloheximide (1 µg/ml), cytokine secretion demanded de novo protein synthesis. F(ab)2 fragments of the murine monoclonal anti-PR3 antibody (1 µg/ml) failed to stimulate monokine release. As a further murine IgG targeting a constitutively expressed monocyte surface antigen, the anti-CD14 antibody MY-4 (1 µg/ml) was investigated for its ability to induce monocyte cytokine formation, but no substantial IL-8 formation was noted upon anti-CD14 exposure.



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  		Figure 4. Dependence of anti-PR3-induced monokine release on de novo protein synthesis and Fc{gamma}R targeting. Isolated monocytes (106/ml) were challenged with anti-PR3 (1 µg/ml) in the absence or presence of PMB (10 µg/ml), CCB (5 µg/ml), or cycloheximide (1 µg/ml). Alternatively, incubation with F(ab)2 fragments (1 µg/ml) of the murine anti-PR3 antibody or anti-CD14 antibodies (MY-4, 1 µg/ml) was performed. After 16 h of incubation, IL-8 was quantified in the cell supernatant. Means ± SEM of at least four independent experiments each are given. *, Values differ significantly from anti-PR3-stimulated cells.

 
Anti-PR3 antibodies activate monocyte-prostanoid generation
The anti-PR3-induced release of proinflammatory cytokines was accompanied by an activation of monocyte cyclooxygenase metabolism resulting in the formation of TxA2, the predominant eicosanoid metabolite released by monocytes [27 ] (Fig. 5 ). Generation of TxA2 was quantified by accumulation of its stable metabolite, TxB2, in the cell supernatant of anti-PR3-treated cells. The release of Tx was evident within 4 h upon anti-PR3 challenge, with a subsequent steady accumulation. As expected, TxA2 generation was abrogated completely in the presence of indomethacin (10 µM; unpublished results), and control IgG was ineffective to provoke monocyte-prostanoid formation.



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  		Figure 5. Time course of Tx generation in response to anti-PR3. Isolated monocytes (106/ml) were challenged with anti-PR3 (1 µg/ml) or control IgG (1 µg/ml), or sham incubation (control) was performed. At indicated time points, cell supernatants were harvested, and release of TxB2, the stable metabolite of TxA2, was quantified by ELISA. Means ± SEM of at least four independent experiments each are given. *, Values differ significantly from controls.

 
Autocrine role of TNF-{alpha} and IL-1ß in mediating prostanoid and IL-8 release triggered by anti-PR3
As TNF-{alpha} and IL-1ß are capable of triggering phospholipase A2 activation [28 ] and IL-8 release secondarily in LPS-stimulated monocytes [24 ], their role in anti-PR3-induced mediator generation was investigated. To block the bioactivity of both mediators, soluble receptors for TNF-{alpha} (0.25 µg/ml) and IL-1ß (2.5 µg/ml) were applied prior to anti-PR3 challenge. In the presence of TNFsR, the anti-PR3-induced TxA2 synthesis was reduced to 57.8 ± 8.6% after 8 h, to 52.4 ± 3.3% after 16 h, and to 50.9 ± 4.1% after 24 h (Fig. 6 , left), while application of sole IL-1sR blocked TxA2 generation to a minor extent. When both soluble receptors were co-applied, the TxA2 release in response to anti-PR3 was diminished to 53.9 ± 5.6% after 8 h, to 46.6 ± 6.0% after 16 h, and to 42.8 ± 5.9% after 24 h of incubation.



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  		Figure 6. Autocrine role of TNF-{alpha}, IL-1ß, and Tx in mediating anti-PR3-induced IL-8 release. Isolated monocytes were challenged with anti-PR3 (1 µg/ml) in the absence or presence of TNFsR (0.25 µg/ml), IL-1sR (2.5 µg/ml), or both (TNFsR/IL-1sR). Additionally, blockade of Tx receptors with daltroban (10 µM) was performed. After 8, 16, and 24 h, release of TxB2 and IL-8 was quantified and is expressed as percentage of anti-PR3-induced mediator release (100%). Means ± SEM of at least four independent experiments each are given. *, Values differ significantly from anti-PR3-induced mediator release.

 
Correspondingly, the anti-PR3-induced IL-8 release was diminished significantly in the presence of TNFsR (to 66.6±10.1% after 8 h, to 54.3±6.24% after 16 h, and to 55.7±7.4% after 24 h; Fig. 6 , right). Blocking the biologic activity of IL-1ß reduced the anti-PR3-elicited IL-8 release to 88.7 ± 22.9% after 8 h, to 73.6 ± 6.9% after 16 h, and to 68.3 ± 7.2% after 24 h of incubation. Combined application of both soluble receptors resulted in a slightly further reduction of IL-8 release as compared with mono-TNFsR application. However, formation of IL-6 in response to anti-PR3 was unchanged in the presence of both soluble cytokine receptors (data not given in detail).

As the kinetics of prostanoid release preceded those of IL-8 formation, a putative role of TxA2 in triggering IL-8 release was investigated further. To inhibit the biological effects of TxA2, the TxA2 receptor antagonist daltroban (10 µM) was applied prior to anti-PR3 challenge, and IL-8 synthesis was quantified after the various incubation periods. At all time points, the anti-PR3-induced IL-8 release was diminished dramatically in the presence of daltroban (to 49.1±7.4% after 8 h, to 48.9±5.6% after 16 h, and to 52.1±4.1% after 24 h of incubation). In contrast, the anti-PR3-elicited formation of TNF-{alpha}, IL-1ß, and IL-6 was not affected by the Tx receptor antagonist (unpublished results). Combined neutralization of TNF-{alpha}, IL-1ß, and Tx did not result in further inhibition of monocyte IL-8 release (to 50.3±6.6% after 24 h of incubation).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the current study, using highly purified human monocytes, monoclonal as well as WG serum-derived anti-PR3 antibodies were found to be potent inducers of the generation of the "classic" proinflammatory monocyte-derived cytokines TNF-{alpha}, IL-1ß, IL-6, and IL-8, accompanied by marked Tx synthesis. Interestingly, preceding cytokine priming was not mandatory for this mononuclear response to anti-PR3. TNF-{alpha}, IL-1ß, and TxA2 were found to be operative as paracrine or autocrine facilitators in the process of anti-PR3-elicited monocyte activation. In addition to anti-PR3 binding, ligation of monocyte Fc{gamma}R is apparently necessary for induction of the signaling cascade, as anti-PR3 F(ab)2 fragments were found to be entirely ineffective.

The presence of PR3 in the granules and on the plasma membrane of monocytes has been demonstrated previously by in vivo and in vitro studies [9 , 10 , 21 , 29 , 30 ]. When using anti-PR3 for provocation of monocyte IL-8 release, preceding pulse priming of the monocytes with TNF-{alpha} was recently found to be a prerequisite for PR3 surface expression and antibody-related cytokine release [21 ]. Aiming to avoid such TNF priming in order to address a putative role of endogenous TNF-{alpha} generation in the sequence of anti-PR3-elicited monocyte activation we presently noted impressive PR3 surface expression on the elutriated monocytes, in the absence of additional priming, together with the capability of anti-PR3 antibodies to provoke cell activation. That isolated monocytes interact with PR3-ANCA in the absence of additional priming maneuvers is well in line with earlier investigations [22 , 31 ], but contrasts the recent study of Ralston et al. [21 ]. This discrepancy with the findings of Ralston et al. [21 ] may not be fully resolved at present; however differences in the techniques of cell isolation (sole isolation of peripheral blood mononuclear cells by polysucrose/sodium diatrizoate density-gradient centrifuation in the previous investigation vs. purification of monocytes by countercurrent centrifugal elutriation in the present study), or varying antigen recognition by the different monoclonal anti-PR3 antibodies used (CLB 12.8 vs. WGM2 in the present study) may be relevant in this respect. Clearly, the elutriation procedure per se represents some kind of a priming maneuver, as only elutriated cells, but not whole blood monocytes, displayed strong surface PR3 expression in the present study. In accordance with Ralston et al. [21 ], further up-regulation of monocyte surface PR3 in response to TNF-{alpha} was also noted in the current investigation, but was not used in the further experimental protocols.

The exposure of the purified monocytes to murine monoclonal anti-PR3 antibodies and three different c-ANCA preparations originating from WG sera indeed provoked a pronounced activation of the mononuclear cytokine cascade with the release of TNF-{alpha} and IL-1ß preceding the formation of IL-6 and IL-8. Although similar as to the kinetics, the magnitude of the cytokine response to human c-ANCA, also varying between the different IgG preparations, was lower overall when compared with activation by murine monoclonal anti-PR antibodies. These differences may be explained by variances in antigen-epitope recognition between mAb and the different polyclonal antibody preparations or due to differences in FcR engagement and subsequent signaling between mouse and human IgG subtypes.

In parallel with the cytokine response, substantial amounts of TxA2, the predominant eicosanoid metabolite of monocytes, were liberated from anti-PR3-stimulated cells. Importantly, human and murine control IgG did not provoke monocyte activation in all assays currently used.

In line with the kinetics of appearance, the use of specific, soluble TNF-{alpha} and IL-1ß receptors clearly indicated that the majority of monocyte IL-8 generation was not triggered directly by the anti-PR3 antibodies, but may be attributed to a para-/autocrine function of these "early" cytokines with TNF-{alpha} representing the more effective agent: the IL-8 response was suppressed to {approx}50% in the presence of TNFsR and to 45–50% in the presence of TNFsR and IL-1sR. In contrast, IL-6 release upon anti-PR3 challenge did not depend on an auto-/paracrine function of TNF-{alpha} and IL-1ß. These findings are well in line with previous data in LPS-stimulated monocytes, in which a secondary triggering of IL-8 but not IL-6 formation via early induction of TNF-{alpha} and IL-1ß was similarly noted [24 ]. It is interesting that formation of the main anti-PR3-induced prostanoid, TxA2, was also suppressed by capturing the early cytokines TNF-{alpha} and IL-1ß via soluble receptors, and the extent of blockage of the prostanoid response corresponded well to the degree of inhibition of IL-8 formation under these conditions. Moreover, TxA2 itself is apparently involved in the signaling cascade, resulting in IL-8 generation in response to anti-PR3 challenge as documented by the inhibitory capacity of the specific Tx receptor antagonist daltroban. This finding is in line with the recent observation in zymosan-activated monocytes describing that Tx liberated in response to the particular stimulus is operative in triggering secondary cytokine generation [26 ]. Hence, anti-PR3-elicited IL-8 release is suggested to proceed largely via early synthesis of TNF-{alpha} and IL-1ß, secondary triggering of Tx generation, and stimulation of IL-8 synthesis via an auto-/paracrine function of this prostanoid. Thus, such a role of Tx in a proinflammatory cascade exceeds the vasoconstrictor functions hitherto recognized as the predominant feature of this arachidonic acid metabolite. As combined blocking of TNF-{alpha}, IL-1ß, and Tx did not result in any further inhibition of IL-8 generation, some quantities of this chemokine are apparently liberated directly after autoantibody stimulation. In contrast to IL-8, IL-6 formation, although also representing a late issue in response to anti-PR3 challenge, is totally independent of TNF-{alpha}/IL-1ß synthesis and mononuclear Tx formation.

The molecular mechanisms by which the anti-PR3 antibodies trigger the sequence of cytokine and Tx release are less obvious. Unspecific, complement-dependent monocyte activation may be ruled out, as the experiments were performed in the absence of exogenous complement components. Phagocytic events cannot be involved either, as disruption of the actin cytoskeleton by CCB did not affect monocyte activation by anti-PR3. Importantly, isotype-matched murine control IgG or pooled human IgG did not cause cytokine or lipid mediator release from monocytes in any of the assays used. Therefore, sole Fc{gamma}R ligation may not explain PR3-ANCA-related monocyte activation. However, binding of PR3 by F(ab)2 could not reproduce the inflammatory cell response, suggesting that targeting PR3 and Fc{gamma}R engagement was underlying the anti-PR3-induced monocyte activation. This is well in line with previous observations in neutrophils [32 ] and monocytes [21 ]. Monocyte activation as a result of co-ligation of Fc{gamma}R and distinct monocyte surface antigens has been described [33 , 34 ]; however, coligation of any monocyte surface molecule with Fc{gamma}R does not necessarily induce cell activation as shown previously [33 , 34 ] and presently by the lack of monocyte activation in response to CD14 ligation by the murine monoclonal IgG MY-4. These differences may be a result of the spatial distribution of the respective antigens on the monocyte surface or because of varying downstream signaling pathways following antigen ligation. Clearly, the signaling events initiated by coligation of PR3 and Fc{gamma}R in monocytes require further investigation.

In conclusion, the present study identifies PR3-ANCA as potent inducers of cytokine and Tx release in human monocytes. Apparently, coligation of PR3 and Fc{gamma}R is mandatory to provoke this response, and TNF-{alpha}, IL-1ß, and TxA2 are operative as paracrine or autocrine facilitators in the sequence of anti-PR3-elicited monocyte activation. Thus, targeting the mononuclear cells by PR3-ANCA may account for the local and systemic cytokine appearance in WG [3 4 5 6 ], thereby essentially contributing to the maintenance of the inflammatory process in this enigmatic disease.


   ACKNOWLEDGEMENTS
 
This work was supported by the Deutsche Forschungsgemeinschaft (GR 534).

Received September 22, 2000; revised February 2, 2002; accepted February 4, 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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