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. 2002 Jun;128(3):444–452. doi: 10.1046/j.1365-2249.2002.01877.x

Human proteinase 3 can inhibits LPS-mediated TNF-alpha production through CD14 degradation: lack of influence of antineutrophil cytoplasmic antibodies

B A YARD 1, A-I WILLE 1, M HAAK 1, F J VAN DER WOUDE 1
PMCID: PMC1906262  PMID: 12067299

Abstract

The present study was conducted to investigate if proteinase-3 (PR3) is able to influence lipopolysaccharide (LPS) responses of monocytes via degradation of CD14 and if antineutrophil cytoplasmic antibodies (ANCA) may modify this process. Recombinant (r) CD14 and CD14 expressed on monocytes were investigated for PR3 mediated degradation by SDS-PAGE and FACS analysis, respectively. TNF-α production in whole blood was used to determine functional consequences of CD14 degradation. PR3 degraded rCD14 in a dose- and time-dependent fashion. Major degradation products were found with apparent molecular weight of 45, 25 and 10 kDa. Treatment of PR3 with PMSF completely abolished CD14 degradation. ANCA IgG did not inhibit CD14 degradation. In whole blood, addition of PR3 resulted in diminished CD14 expression on monocytes. In contrast, CD14 was increased in a subpopulation of cells that expressed major histocompatibility (MHC) class II and PR3, but lacked expression of CD64 and CD16. LPS mediated TNF-α production in whole blood was significantly inhibited when preincubated with PR3. This study demonstrates that PR3 can degrade rCD14 and that PR3 differentially affects CD14 expression in subsets of monocytes. ANCA IgG does not play a significant role herein.

Keywords: CD14, lipopolysaccharide, monocytes, proteinase-3, TNF-α

INTRODUCTION

Wegener’s granulomatosis is characterized by a prominent infiltrate of neutrophils and monocytes in the vessel wall [1]. Although this disease is strongly associated with the presence of antineutrophil cytoplasmic antibodies (ANCA) [2,3] the pathogenic role of these antibodies has not yet been completely delineated. Based on the staining pattern on ethanol-fixed granulocytes, ANCA can be divided into two categories that give either a cytoplasmic (C-ANCA) or a perinuclear (P-ANCA) staining. Whereas myeloperoxidase (MPO) is the major antigen that is recognized by P-ANCA IgG, C-ANCA IgG recognize mainly proteinase 3 (PR3) [4–7]. Both enzymes are expressed in the azurophilic granules of monocytes and neutrophils and they are believed to play a critical role, as effector enzymes in the destruction of microorganisms [8].

Monocytes and macrophages are of utmost importance in the first line of defence against pathogens. They not only function as phagocytic cells, they also express pattern recognition receptors that allow them to become activated and to produce an array of inflammatory mediators in the face of infection [9–12]. CD14, a 55-kDa glycoprotein, is one of these pattern recognition receptors, that is involved in the recognition of lipopolysaccharide (LPS) of Gram-negative bacteria [13]. The molecule is anchored by glycosylphosphatidylinositol (GPI) to the membrane of monocytes and macrophages. Upon activation of phosphatidylinsitol specific phospholipase C, CD14 is shed from the membrane and can be detected in serum as a soluble form (sCD14) [14]. sCD14 may also interact with LPS and facilitate the activation of endothelial cells by LPS [15–17].

Recently, it has been demonstrated that both human leucocyte elastase (HLE) and cathepsin G (CG) are able to reduce the expression of CD14 on monocytes through enzymatic degradation [18,19]. Although both enzymes belong to the class of serine proteinases, it is not known if other enzymes belonging to this class, such as PR3, are able to modulate CD14 expression on monocytes. Previously, we have described that ANCA IgG up-regulates the expression of CD14 in monocytes [20]. Since a fraction of PR3, can be expressed at the cell surface of monocytes, we reasoned that PR3 may function as a natural regulator of CD14 expression on these cells. ANCA could then interfere by binding to membrane associated PR3. If ANCA would thus prevent or slow down the turnover of CD14 on monocytes, this would not only result in an increased expression of CD14 but possibly also in an increased susceptibility towards LPS. This is in line with the concept that in vasculitis patients the mere presence of ANCA is not sufficient to develop disease, but that additional, most probable, infectious events are required [21,22].

To test the hypothesis that PR3 modulates CD14 expression and that this is inhibited by ANCA, the following questions were addressed in this paper. First, is PR3 able to degrade CD14 as recombinant protein and as native protein expressed on monocytes? Secondly, does ANCA interfere with this process? Thirdly, what is the functional consequence of CD14 degradation on monocytes?

MATERIALS AND METHODS

Isolation of azurophilic granules

Azurophilic granules were isolated from leucocytes according to the methods described by Borregaard [23] and modified by Leid et al.[24]. Briefly, leucocyte enriched blood was obtained from the bloodbank Mannheim (DRK). The blood was pooled and cells were resuspended in phosphate buffered saline (PBS). Leucocytes were collected by centrifugation for 15 min (3500 g) at 4°C. The sediment was incubated for 10 min with lysis buffer (167 mm NH4Cl, 10 mm KHCO3, 1 mm EDTA pH 7·4 (all from Sigma Aldrich, Deisenhofen, Germany)) to lyse the erythrocytes. Hereafter the leucocytes were collected by centrifugation and washed extensively with PBS. Cell concentrations were adjusted to 2 × 108 cells/ml in relaxation buffer (100 mm KCl, 3 mm NaCl, 1 mm ATP, 35 mm MgCl2, 10 mm Hepes pH 7·3 (Sigma Aldrich, Deisenhofen, Germany)). The cells were put into a nitrogen bomb (Parr Instruments Co., Moline, IL, USA) and pressurized for 20 min at 4°C using 350 pounds per square inch (p.s.i.). The azurophilic granules in the alpha fraction were isolated from the cell free supernatants by centrifugation on Percoll (Amersham Pharmacia Biotech, Freiburg, Germany) discontinuous gradients of 1·05 g/ml and 1·12 g/ml. The azurophilic granules were collected from the remaining Percoll by aspiration, resuspended and lysed in relaxation buffer containing 1% Triton X-100 (Sigma Aldrich, Deisenhofen, Germany). The mixtures were shaken well and incubated for 10 min on ice, centrifuged (3500 g), the supernatants collected and the pellets subjected to a second round of extraction. The supernatants of both extractions were pooled and used as starting material for isolation of PR3.

Isolation of PR3

The azurophilic granule extract was dialysed extensively against citrate buffer (80 mm Na2HPO4, 50 mm NaCl and adjusted to pH 7·0 with 80 mm citric acid), applied to a 30 × 1·5 cm Bio-rex 70 column (Bio-Rad laboratories, München, Germany), which had been equilibrated with citrate buffer. After all non-binding proteins had washed through the column, a linear gradient from 0 to 1 m NaCl in citrate buffer was applied. The initial fall-through contained almost exclusively PR3 as determined by SDS-PAGE, enzyme activity and ELISA. With increasing conductivity, MPO, CG and HLE were eluted from the column and could not be detected in the initial fall-through (ELISA). The PR3 positive fraction was dialysed overnight against distilled water and lyophilized. The lyophilized enzyme was resuspended in PBS and applied to a 60 × 2·5 Superdex 75 column (Amersham Pharmacia Biotech, Freiburg, Germany), equilibrated and run in 50 mm sodium phosphate containing 150 mm NaCl. The fractions were tested for PR3 activity and antigen using boc-alanine-o-nitrophenyl ester (boc-ala-ONP) (23) and ELISA, respectively.

Isolation of IgG

IgG from normal human sera (n = 6) or sera obtained from ANCA positive patients (C-ANCA n = 6, P-ANCA n = 6), all with active disease (Table 1), was isolated by affinity chromatography using Sepharose protein G columns according to the manufacturer’s recommendation (Amersham Pharmacia Biotech, Freiburg, Germany). All IgG fractions from ANCA positive patients were tested by indirect immunofluorescence and in ANCA ELISAs and were found to be positive in both tests (Table 1). The purity of the fractions was tested by SDS-PAGE followed by silver staining [24].

Table 1.

Patients’ sera used in this study

ELISA (U/ml)* IIF (titre)
Patient no. Anti-MPO Anti-PR3 P-ANCA C-ANCA Disease
1 −† 11 − 1:64 Wegener’s granulomatosis
2 − 52 − 1:128 Wegener’s granulomatosis
3 − 1100 − 1:512 Wegener’s granulomatosis
4 − − − 1:64 Wegener’s granulomatosis
5 − 124 − 1:64 RPGNc
6 − 65 − 1:256 Wegener’s granulomatosis
7 400 − 1:256 − Microscopic polyangiitis
8 68 − 1:128 − RPGN
9 110 − 1:128 − Microscopic polyangiitis
10 70 − 1:512 − RPGN
11 45 − 1:64 − RPGN
12 15 − 1:64 − Microscopic polyangiitis
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*

For both ELISAs a value of > 10 U/ml is used as cut-off

†

– = negative

c

RPGN: rapidly progressive glomerulonephropathy.

SDS-polyacrylamide gel electrophoresis and Western blotting

Degradation of rCD14 (Biometec, Greifswald, Germany) by PR3 was tested by Western blotting or by using radio-labelled 125I-rCD14 (Amersham Pharmacia Biotech, Freiburg, Germany). rCD14 (1 ng) was incubated in Tris buffer at 37°C with different concentrations of PR3 (0·05–1 μg) for various time-points, depending on the specific experiment. Hereafter, the reaction was stopped by adding sample buffer containing 5% (v/v) of 2-mercaptoethanol. In some experiments PR3 was inactivated by incubating with 1 μg of alpha 1 antitrypsin or 1 μm of PMSF for 60 min at 37°C. Purified IgG from healthy controls, C- and P-ANCA positive patients was tested for its effect on PR3 mediated degradation of CD14. IgG was used in a concentration of 100 μg/ml and was incubated with PR3 60 min prior to the addition of 125I-rCD14.

The samples were boiled for 5 min and applied on a 10% SDS gel. Electrophoresis was performed, essentially as described by Laemmli et al.[25]. The gels were either dried, if radio-labelled rCD14 was used, or in the case of unlabelled CD14, transferred to polyvinyl di-acetate filters (PVDF) by means of semidry blotting. The filters were incubated overnight in Tris-buffered saline (TBS) containing 5% milk powder. A polyclonal anti-CD14 antibody followed by goat antirabbit HRP conjugated IgG (both Santa-Cruz, Heidelberg, Germany) was added to the filter for at least 60 min. Hereafter, the filters were washed extensively and incubated with substrate solution (Amersham Pharmacia Biotech, Freiburg, Germany). Finally the filters were exposed to Kodak XAR-5 (Kodak Co., Buffalo NY, USA). When radio-labelled rCD14 had been used, the gels were exposed to the film directly after drying.

TNF-α production

Whole blood obtained from healthy volunteers was centrifuged and the plasma was collected. The sediment was washed extensively and finally resuspended in an approximately five times larger volume of RPMI 1640 medium. Hereafter the suspension was seeded in 5 ml Teflon hydrophobic Petriperm dishes (In Vitro Systems & Services, Göttingen, Germany) to avoid monocyte attachment. The cultures were treated with different concentrations of PR3 for 24 h. FACS analysis was performed hereafter to study phenotypical changes in the different leucocyte populations. In other experiments, washed whole blood was treated with PR3, as described above, and stimulated subsequently for 24h with different concentrations of LPS (0·1–5000 ng/ml). Before LPS stimulation, the cultures were supplemented with autologous plasma. Supernatants were collected and assessed for TNF-α production by using a commercial sandwich enzyme immunoassay according to the manufacturer’s instructions (R&D, Wiesbaden, Germany).

Flow cytometric analysis

At the end of the incubation period, the cells were harvested and centrifuged. One hundred μl of packed cells were incubated with different antibodies, which were conjugated either to FITC or RPE. The following monoclonal antibodies were used: CD14 (Tük4), CD64 (10·1), CD16 (DJ130c), MHC class II (C3/43) (all from DAKO, Hamburg, Germany). An isotype-matched idiotypic mouse monoclonal antibody, either FITC or RPE conjugated, was used as negative control in every experiment. The cells were incubated with monoclonal antibodies for 30 min at 4°C and hereafter washed extensively with PBS. Erythrocytes were lysed by incubating the sediment with 10 ml of 10% FACS lysing solution (Becton Dickinson, Heidelberg, Germany) for 10 min. The leucocytes were washed twice with PBS and resuspended in 600 μl of Cellwash (Becton Dickinson, Heidelberg, Germany). Flow cytometry was performed on FACScalibur and analysed using winmdi software.

Statistical analysis

Statistical significance was tested by employing anova. A P-value of P < 0·05 was considered to be significant.

RESULTS

Degradation of rCD14 by PR3

Degradation of CD14 by PR3 was first studied by incubating purified PR3 with rCD14. It was found that PR3 degraded rCD14 in a time- and dose-dependent manner (Fig. 1). Complete degradation was obtained using 1 μg of PR3 after 60 min. CD14 degradation was due to PR3 enzyme activity since PMSF-treated PR3 completely failed to degrade rCD14 (Fig. 2a). Moreover, preincubation of PR3 with alpha 1 antitrypsin (α1-AT) also inhibited the action of PR3 on rCD14 (Fig. 2b). Although degradation products could not be detected by Western blotting, degradation of radio-labelled rCD14 by PR3 revealed major degradation products with apparent molecular weights of ~45, ~25 and ~10 kDa. Both 45 and 25 kDa products were degraded further with time resulting in only one band of approximately 10 kDa (Fig. 3). To test if ANCA IgG was able to influence degradation of rCD14, PR3 was preincubated with control-, C- and P-ANCA IgG for 60 min before rCD14 was added. No influence of C-ANCA IgG, even when high concentrations were used, on PR3 mediated degradation of rCD14 was observed, suggesting that these IgG did not inhibit PR3 activity (Fig. 4a). Furthermore, when PR3 was preincubated with α1-AT in the presence or absence of control- or ANCA IgG, the inhibitory effect of α1-AT on degradation of rCD14 by PR3 was not influenced by C-ANCA IgG, demonstrating that the C-ANCA IgG used in this study, did not prevent the interaction of α1-AT with PR3 (Fig. 4b). To demonstrate that the C-ANCA IgG were able to inhibit PR3 enzyme activity when using other substrates, we made use of the conversion of boc-ala-ONP by PR3. When C-ANCA IgG were preincubated with PR3 for 60 min prior to the addition of boc-ala-ONP, three of six C-ANCA IgG fractions were able to inhibit the degradation hereof, while no P-ANCA- or control IgG did (Fig. 5a). Because α1-AT profoundly inhibited the conversion of boc-ala-ONP by PR3, it was tested if C-ANCA IgG could interfere with this process. To this end, PR3 was either incubated with control-, C-ANCA or P-ANCA IgG for 60 min prior to the addition of α1-AT and boc-ala-ONP. Inhibition of boc-ala-ONP conversion could not be prevented by these IgG (Fig. 5b).

Fig. 1.

Fig. 1

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PR3-mediated degradation of rCD14. Upper panel: rCD14 was incubated with various amounts of PR3 for 30 min at 37°C. Hereafter the reaction was stopped by adding sample buffer. Lower panel: 1 μg of PR3 was added to rCD14 for various time intervals before stopping the reaction by adding sample buffer. The samples were subjected to SDS-PAGE followed by Western blotting, as described in Material and methods. The results of a representative experiment (n = 4) are shown.

Fig. 2.

Fig. 2

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Degradation of rCD14 is dependent on PR3 enzyme activity. (a) PR3 was treated either with 1 μm of PMSF or not before rCD14 was added. PMSF treated (+) or untreated (–) PR3 were then tested for degradation of rCD14 for 60 min at 37°C. (b) PR3 was incubated either for 60 min with 1 μg of α1-AT (+) or not (–) before rCD14 was added. Hereafter the samples were incubated for 60 min and processed as described in Fig. 1. The results of a representative experiment (n = 3) are shown.

Fig. 3.

Fig. 3

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PR3-mediated degradation of 125I-rCD14. Radio-labelled rCD14 was incubated with either 1 μg of PR3 (+) or not (–) for 30 and 60 min at 37°C, respectively. Hereafter the reaction was stopped and the samples were processed as described. The results of a representative experiment (n = 3) are shown.

Fig. 4.

Fig. 4

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Influence of ANCA on PR3-mediated degradation of CD14. (a) PR3 was incubated either for 60 min with control (c), P-ANCA or C-ANCA IgG (100 μg) or no IgG (–) before radio-labelled rCD14 was added. In addition untreated rCD14 in the absence of IgG was included in these experiments. (b) PR3 was incubated either for 60 min with control (c), P-ANCA or C-ANCA IgG (100 μg) or not (–). Hereafter 1 μg of α1-AT was added to some of the samples (+) for 30 min before radio-labelled rCD14 was added. Degradation of rCD14 was performed during a period of 60 min at 37°C. The results of a representative experiment (n = 3) are shown.

Fig. 5.

Fig. 5

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Influence of ANCA and α1-AT on PR3 activity. (a) PR3 activity was measured using conversion of boc-ala-ONP as described [23] in the absence (▪) or presence of ANCA IgG (100 μg) (Inline graphic). The number of IgG fractions of each of the individual patients correspond to the number as indicated in Table 1. (b) The influence of ANCA IgG on α1-AT-mediated inhibition of PR3 activity was tested. Filled bar: PR3 activity in the absence of α1-AT; open bar: PR3 activity in the presence of α1-AT; hatched bars: PR3 activity in the presence of α1-AT and ANCA IgG. The results of a representative experiment (n = 3) are expressed as mean O.D 405 ± s.d. of triplicate samples. *P < 0·05 compared to PR3 activity in the absence of ANCA IgG or α1-AT (anova with Bonferoni adjustment for multiple testing).

Influence of PR3 on CD14 expression of monocytes

Since it was found that PR3 was able to degrade rC14, it was tested if this could also occur on monocytes. Because α1-AT, which is also present in serum, effectively inhibited degradation of rCD14, the influence of PR3 on CD14 expression on monocytes was tested under serum free conditions. To this end, whole blood was extensively washed with culture medium and subsequently incubated with PR3 for 24 h. Similar to the data obtained with rCD14 it was found that the expression of CD14 on monocytes was reduced. Interestingly, in a subset of mononuclear cells with a high forward scatter, CD14 expression was increased (Fig. 6a). No influence on CD14 expression in either population was observed when the assay was performed in the presence of serum or when PMSF treated PR3 was used (data not shown), suggesting that the influence was mediated via PR3 activity. Addition of ANCA IgG also did not influence PR3 mediated modulation of CD14 expression on these cells (data not shown).

Fig. 6.

Fig. 6

Fig. 6

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Influence of PR3 on CD14 expression on mononuclear cells. (a) Whole blood was washed extensively and incubated either in serum-free culture medium or serum-free culture medium supplemented with 1 μg of PR3 for 24 h. Hereafter the expression of CD14 on monocytes, that were gated in region 1 (R1) or region 2 (R2), respectively (upper panel), was measured by FACS. Lower panel, histograms show the expression of CD14 in both regions. Filled histogram: negative control (isotype matched irrelevant antibody); thin line: cells cultured in the absence of PR3; bold line: cells that were cultured in the presence of PR3. (b) Expression of PR3, MHC class II, CD16 and CD64, similar to (a), were determined in both regions. Filled histogram: negative control (isotype matched irrelevant antibody); thin line: cells cultured in the absence of PR3; bold line: cells that were cultured in the presence of PR3. The results of a representative experiment (n = 6) are shown.

To explore if other membrane antigens were influenced similarly by PR3, the expression of MHC class II, CD64, CD16 and PR3 was investigated in both populations (Fig. 6b). Whereas on monocytes the expression of CD64, CD16 and, to a lesser extent, the expression of PR3was reduced in the presence of PR3, the expression of MHC class II was strongly up-regulated on these cells. In the subpopulation of cells that showed increased CD14 expression after PR3 stimulation the expression of MHC class II and PR3 were also up-regulated; in contrast, CD64 and CD16 expression could not be detected in these cells, neither under unstimulated nor under PR3 stimulated conditions (Fig. 6b).

Influence of ANCA and PR3 on LPS mediated TNF-α production

It was then investigated if PR3 mediated down-regulation of CD14 was of functional relevance with respect to LPS stimulation. In whole blood, the production of TNF-α by LPS is completely dependent on the presence of CD14 positive cells and thus this assay is suitable to study functional consequences of CD14 down-regulation with TNF-α production as a read-out. Down-regulation of CD14 was obtained by culturing the cells for 24h under serum free conditions with 1 μg of PR3; however, for the subsequent stimulation with LPS, autologous serum was added to the cultures prior to LPS stimulation. It was found that LPS-induced TNF-α production was inhibited in PR3 treated whole blood over a large range of LPS concentrations (Fig. 7) and it thus seems that PR3 mediated down-regulation of CD14 is of functional relevance for monocytes.

Fig. 7.

Fig. 7

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Inhibition of LPS-mediated TNF-α production. Whole blood cells were washed extensively and incubated either in serum-free culture medium (open squares) or serum-free culture medium supplemented with 1 μg of PR3 (closed squares) for 24 h. Hereafter autologous serum was added and the cultures were stimulated with various concentrations of LPS for 24 h. TNF-α production was measured by ELISA as described. The results of a representative experiment (n = 3) are expressed as mean TNF-α production ± s.d. of triplicate wells.

DISCUSSION

Wegener’s granulomatosis (WG) is characterized by granulomatous inflammation in the upper and lower respiratory tract, systemic vasculitis affecting small blood vessels and pauci-immune necrotizing crescentic glomerulonephritis [1]. There is a close association between WG and ANCA that recognize PR3 as a target antigen [2,3]. The prevalence of PR3-ANCA in WG is about 80–90% [3]. In many patients titres of PR3-ANCA rise prior to relapse of WG, thus making these autoantibodies helpful in the diagnosis and follow-up of these patients. Although the aetiology of WG and pathophysiological role of ANCA is not known completely, clinical and experimental data suggest that ANCA may be involved in the pathogenesis of WG [3,26–29].

PR3, HLE and CG are three neutral serine proteases that are located in the azurophilic granules of neutrophils and monocytes. There is ample evidence for a role of PR3 in inflammatory responses. First, PR3 can degrade extracellular matrix proteins, e.g. elastin and collagen type IV [8], which may facilitate the migration of neutrophils to the site of inflammation. Secondly, PR3 may cleave IL-8 or TGFβ to a biologically more active form [30,31]. Apart from this, PR3 has been implicated in the inactivation of C1 inhibitor [24] in the cleavage of TNF-α and IL-1β [32,33] and may display microbicidal activity independently of its proteolytic activity [34].

Previously, we have demonstrated that ANCA IgG up-regulates the expression of CD14 on monocytes [20]. Because it was also shown that HLE can degrade CD14 [18], we speculated that PR3 might function as a natural regulator of CD14 expression and that ANCA IgG might interfere with this process. Indeed PR3, like HLE, is able to degrade rCD14 and down-regulate CD14 on monocytes. Although we did not establish that down-regulation in CD14 expression on monocytes was due to degradation, in experiments using rCD14 degradation in the presence of PR3 occurred in a time- and dose-dependent manner. Moreover, like degradation of rCD14, down-regulation of CD14 expression was dependent on the proteolytic activity of PR3 as it could be abolished completely by PMSF and α1-AT. However, it cannot be excluded completely that down-regulation in CD14 expression on monocytes also occurred at the transcriptional level. Most interestingly, it was found that in a subpopulation of mononuclear cells, the expression of CD14 was increased. These cells expressed MHC class II and PR3, but failed to express CD64 and CD16. Monocytes can be divided on the bases of CD14 expression into two subpopulations, i.e. CD14++/CD16– and CD14+/CD16+ cells [35]. In healthy controls the CD14+/CD16+ population accounts for approximately 8% of CD14 monocytes, but under certain inflammatory conditions this subpopulation is largely expanded and exhibits features of tissue macrophages [35–37]. They lack the expression of the chemokine receptor CCR2 and show a significantly higher surface expression of CCR5 [36].

Although CD14 was up-regulated in a subpopulation of mononuclear cells by PR3, pretreatment of blood cells with PR3 significantly abrogated TNF-α production in response to LPS. We did not study if the influence of PR3 on TNF-α production was on the RNA level or due to inhibition of TNF-α secretion. It can thus be argued that the inhibition of TNF-α production was due to PR3 mediated degradation of TNF-α, as has been reported previously [32,33]. Inasmuch as we could demonstrate that in the presence of serum, PR3 in itself neither influenced CD14 expression nor inhibited LPS mediated TNF-α production (data not shown), it is unlikely that degradation of TNF-α occurred, as stimulation of whole blood cells with LPS was performed in the presence of autologous serum. The rational to study the influence of PR3 treatment of whole blood cells on TNF-α production was, first, to demonstrate the functional consequence of CD14 degradation, and secondly, because TNF-α is used frequently as priming agent for neutrophils to become sensitive to ANCA stimulation.

In the present study we could not demonstrate that ANCA interfere with PR3 mediated degradation or down-regulation of CD14 and thus the ability of ANCA to up-regulate CD14 expression on monocytes [20] remains to be elucidated. At least for P-ANCA IgG it was not surprising that they did not influence CD14 degradation, as P-ANCA IgG recognize myeloperoxidase and these sera were completely negative in the anti-PR3 ELISA. It was, however, also found that C-ANCA IgG did not prevent degradation of rCD14 although three of six C-ANCA IgG were able to prevent PR3 activity using boc-ala-ONP as substrate. Because all C-ANCA IgG could bind to PR3, as tested by ELISA, and were positive in the indirect immunofluorescence, it can be excluded that the inability of C-ANCA IgG to bind to PR3 was underlying this observation. In previous reports it has been demonstrated that C-ANCA IgG recognize multiple epitopes and that in patients with WG C-ANCA IgG can inhibit PR3 activity, prevent the interaction with α1-AT or merely bind to PR3 without any obvious biological consequence. This is in concordance with our observations, in that only 50% of C-ANCA IgG were able to inhibit PR3 activity. It must be stressed, however, that inhibition of PR3 activity was dependent on the substrate that was used, as this was not observed with the degradation of CD14.

In conclusion, we have demonstrated that PR3 is able to degrade CD14 as recombinant protein under serum free con-ditions. The effects on the cell surface CD14 expression is dependent on the monocyte subset: CD14 is up-regulated on CD16– monocytes and down-regulated on CD16+ monocytes. The PR3-induced CD14 degradation on monocytes has an inhibitory effect on LPS induced TNF-α secretion. It seems that ANCA IgG, at least the fractions used in our study, do not have a fundamental role in this process.

Acknowledgments

This study was supported by a grant of the German kidney foundation.

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