Abstract
Proteinase 3 (PR3) is a pleiotropic and destructive serine protease and it is also a major target for autoantibodies in systemic small vessel vasculitis. We have shown recently that patients in stable remission have increased circulating levels of PR3, independent of autoantibody titre, inflammation, neutrophil degranulation and renal function. Here we explore the possibility of increased PR3 gene transcription. RNA was purified from peripheral blood monocytes from vasculitis patients and controls. Specific mRNA was measured by TaqMan real-time polymerase chain reaction (PCR). The monocyte-like cell lines THP-1 and U937 and human peripheral blod monocytes from healthy controls were stimulated with cytokines and lipopolysaccharide (LPS) for different time periods. PR3 protein was measured in plasma with enzyme-linked immunosorbent assay (ELISA). The median result for PR3 mRNA was 9·6 (1·8–680) for 22 patients, compared to 1 (0·1–2·8) for the 15 healthy controls. Elastase expression was also significantly increased, whereas myeloperoxidase and interleukin-8 were not. Stimulation of monocytes with tumour necrosis factor (TNF)-α, interferon (IFN)-γ or LPS did not result in any increase of PR3 or elastase transcription, whereas interleukin (IL)-8 transcription was increased 10-fold. Circulating monocytes from patients with systemic vasculitis display increased PR3 gene transcription compared to healthy controls and patients with sytemic lupus erythematosus (SLE). This may be important for the development of vasculitis. Our results do not favour a role for cytokines, antineutrophil cytoplasmic antibodies (ANCA) or immunosuppressive medication in the upregulation of PR3 transcription in vasculitis.
Keywords: ANCA, monocytes, proteinase 3, real time PCR, systemic vasculitis
Introduction
Wegener's granulomatosis and microscopic polyangiitis are systemic small vessel vasculitides of unknown aetiology. Due to the strong association with autoantibodies reacting with granule constituents of neutrophils and moncoytes, they are often referred to as ANCA-associated small vessel vasculitis (AASV; ANCA: antineutrophil cytoplasmic antibodies). The major ANCA target antigens are myeloperoxidase (MPO) and proteinase 3 (PR3) [1].
PR3 is a 29-kDa serine protease stored in granules of neutrophils and monocytes. It is also present on the surface of primed or apoptotic human neutrophils in a bioactive form [2]. Extracellularly, PR3 is inhibited through binding to serine protease inhibitors (SERPINs), the most abundant in plasma being alpha-1-antitrypsin (α1-AT). The substrates of PR3 include several extracellular matrix components, and the protease has been shown to produce emphysema in hamsters when installed intratraceally [3]. Besides its proteolytic effects, PR3 has influence on the proliferation of granulopoietic progenitor cells [4] and it can induce apoptosis in endothelial cells [5]. PR3 is also involved in cytokine activation, chemokine activity amplification and processing of cytokine binding proteins, indicating a regulatory role in inflammatory processes [6].
Few genetic polymorphisms have been associated reproducibly with AASV. An exception is the PiZ deficiency allele of α1-AT that is associated with an increased risk to develop AASV. This association has been reported in studies from France, Austria, Holland, United States, England and Sweden [7]. In our cohort we also found the PiZ allele to be linked to disease extension and a worse prognosis. These findings suggest increased protease activity to be part of the pathogenesis.
We have recently published findings of raised circulating levels of PR3 in AASV patients in remission or with chronic smouldering disease activity [8]. These high levels were not an effect of general inflammation, as there was no correlation between PR3 and C-reactive protein (CRP) or interleukin (IL)-6; nor was there any correlation with clinical disease activity, using Birmingham Vasculitis Activity Score (BVAS). We could also rule out decreased renal function, using relevant disease controls and cystatin C as a marker of glomerular filtration of endogenous polypeptides. Neutrophil gelatinase-associated lipocalin (NGAL) and soluble tumour necrosis factor (TNF) receptor 1 (sTNFr1) were measured as markers of neutrophil degranulation and monocyte activation, respectively. None of them showed any correlation with PR3, indicating increased circulating PR3 levels being independent of leucocyte activation. ANCA level and ANCA specificity had no significant influence. Remaining possible explanations for the increased circulating levels include selective leakage from granules, defects in the liver or reticulo-endothelial uptake of PR3/SERPIN complexes or factors leading to increased PR3 production. The latter possibility was supported by the results from Dr J. J. Yang et al. earlier this year, reporting up-regulation of the PR3 gene in leucocytes [9].
The monocytes play a central part in the scheme of inflammation and constitute a relatively homogeneous cell population with a versatile transcription apparatus. We chose to study PR3 expression in monocytes in order to investigate the possibility of up-regulated PR3 gene transcription. Peripheral blood monocytes were isolated from patients with AASV in different stages of disease activity, healthy blood donors (HBD) and patients suffering from systemic lupus erythematosus (SLE). As we found markedly increased levels among AASV patients we also studied other granular proteins such as human leucocyte elastase (HLE) and myeloperoxidase (MPO). To explore the possibility that the PR3 up-regulation was an effect of inflammation in general we also measured IL-8 mRNA and performed in vitro stimulation experiments using the monocyte like cell lines U937 and THP-1 [10] as well as human peripheral blood monocytes (PBMC) from healthy controls.
Methods
Patients
Twenty-two patients with AASV, according to the Chapel Hill Consensus Conference definitions, none of which were on dialysis or suffered from any bacterial or viral infections or cancer, were consecutively included in this study (Table 1). Based on clinical observations performed by their regular physicians at the Department of Nephrology, Lund University Hospital, their status at the time of sampling was classified either as remission, chronic smouldering activity or relapse. Clinical status, BVAS, relapse tendency as well as the development of any severe organ damage due to vasculitic complications were registered. The clinical evaluation was conducted without access to the results of our analyses. The patients were grouped according to ANCA specificity (PR3 or MPO). Nine patients were MPO-positive and 12 patients were PR3-positive. Patient 22 had a diagnosis of Wegener's granulomatosis (restricted to the upper airways), but had never shown any positive results in ANCA analysis and is thus classified as ‘seronegative’.
Table 1. Patients with antineutrophil cytoplasmic antibodies (ANCA)-associated small vessel vasculitis.
| Patient | Age | Sex | Status | Spec. | Titre | BVAS | CRP | WBC | Immunosuppressive treatment at the day of sampling |
|---|---|---|---|---|---|---|---|---|---|
| 1 | 70 | F | G | MPO | 580 | 2 | 18 | 7·6 | MTX12·5; P 2·5 |
| 2 | 55 | M | R | MPO | >320 | 10 | <5 | 8·4 | CP 150; P 80 |
| 3 | 60 | M | R | MPO | >320 | 24 | 13 | 6·9 | None |
| 4 | 62 | M | R | MPO | 184 | 13 | 74 | 7·3 | None |
| 5 | 52 | F | R | MPO | 147 | 15 | <5 | 5·8 | None |
| 6 | 83 | F | Q | MPO | 41 | 0 | <5 | 5·2 | AZ 100; P 7·5 |
| 7 | 84 | F | Q | MPO | 37 | 0 | 12 | 4·6 | P 5 |
| 8 | 52 | F | R | MPO | 25 | 8 | <5 | 8·0 | CP 150; P 60 |
| 9 | 68 | F | Q | MPO | 0 | 0 | <5 | 7·6 | MMF 500; P 7·5 |
| 10 | 67 | F | Q | PR3 | 85 | 0 | <5 | 4·7 | AZA 100 |
| 11 | 59 | F | Q | PR3 | 74 | 1 | <5 | 6·1 | CyA 150; P 2·5 |
| 12 | 68 | F | G | PR3 | 37 | 2 | 9 | 5·9 | MTX 12·5; P 10 |
| 13 | 51 | F | Q | PR3 | 23 | 0 | 13 | 7·3 | CP 75; P 7·5 |
| 14 | 26 | M | G | PR3 | 17 | 3 | <10 | 3·6 | MTX 22·5; P 10 |
| 15 | 48 | M | Q | PR3 | 0 | 0 | <5 | 5·8 | CP 100; P 7·5 |
| 16 | 68 | M | Q | PR3 | 0 | 0 | <9 | 6·0 | AZA 100; P 7 |
| 17 | 45 | M | Q | PR3 | 0 | 1 | 11 | 8·7 | MTX 20; P 10 |
| 18 | 57 | M | Q | PR3 | 0 | 0 | <5 | 8·6 | CP 125; P 20 |
| 19 | 74 | M | Q | PR3 | 0 | 0 | 6 | 6·3 | MTX 10; P 5 |
| 20 | 65 | M | G | PR3 | n.d. | 3 | 16 | 7·9 | AZ 100; P 10 |
| 21 | 61 | M | G | PR3 | n.d. | 3 | 10 | 6·4 | CyA 200; MMF 1000 |
| 22 | 28 | M | Q | Seroneg | n.d. | 0 | <5 | 5·9 | None |
M = male; F = female. G = grumbling activity; Q = quiescent disease/remission; R = relapse; Spec = ANCA specificity; MPO = antibodies to myeloperoxidase (MPO-ANCA) at diagnosis; PR3 = antibodies to proteinase 3 (PR3-ANCA) at diagnosis; titre = results of ANCA determinations at the day of sampling [enzyme-linked immunosorbent assay (ELISA) units]; n.d. = not done; CRP = C-reactive protein (mg/l); WBC = white blood cell count (×109/l); MTX = methotrexate mg/week; P = prednisolone mg/day; CP = cyclophosphamide mg/day; AZ = azathioprine mg/day; CyA = cyclosporin A mg/day; MMF = mycophenolatmophetil mg/day.
Our control groups comprised 15 HBD and 18 patients suffering from SLE. The majority of the patients with SLE were on corticosteroid therapy; four patients had doses of >15 mg/day (Table 2). The disease activity of the SLE patients is presented in Table 2 (SLEDAI: SLE disease activity index).
Table 2. Control group, patients with systemic lupus erythematosus (SLE).
| Patient | Age | Sex | SLEDAI | Immunosuppressive treatment at the day of sampling | PR3 expression by real time PCR |
|---|---|---|---|---|---|
| 1 | 25 | F | 4 | P 30; CP, pulse therapy | 266 |
| 2 | 60 | F | 4 | P 6 | 22 |
| 3 | 28 | F | 0 | P 5; AZ 50 | 0·4 |
| 4 | 59 | F | 5 | AZ 25 | 0·1 |
| 5 | 51 | F | 2 | P 5; AZ 100 | 0·5 |
| 6 | 35 | F | 2 | P 7·5; AZ 150 | 1·8 |
| 7 | 30 | F | 3 | P 10; AZ 50 | 1·0 |
| 8 | 59 | F | 0 | None | 0·2 |
| 9 | 28 | F | 0 | None | 0·2 |
| 10 | 51 | F | 0 | P 8·75 | 2·8 |
| 11 | 27 | M | 9 | P 30; AZ 150 | 482 |
| 12 | 42 | F | 0 | P 5 | 0·9 |
| 13 | 59 | F | 2 | None | 0·7 |
| 14 | 21 | F | 1 | P 10; MMF 500 | 2·4 |
| 15 | 25 | M | 8 | P 20 | 46 |
| 16 | 57 | F | 10 | P 20; MMF 500 | 216 |
| 17 | 60 | F | 1 | P 10; AZ 50 | 0·9 |
| 18 | 24 | F | 0 | P 2·5; MMF 2000 | 3·4 |
M = male; F = female. SLEDAI = SLE disease activity index. P = prednisolone mg/day; CP = cyclophosphamide mg/day; AZ = azathioprine mg/day; MMF = mycophenolatmophetil mg/day; PCR = polymerase chain reaction; PR3 = proteinase 3. The real time data are corrected according to the 2–ΔΔCT formula and then expressed in relation to the median value of the healthy controls.
The studies were performed after approval from the Ethical committee at Lund University and written informed consent of the patients.
Blood samples
Venous blood, 48 ml, from each subject was obtained in ethylene diamine tetra-acetic acid (EDTA) tubes. The blood was put on ice and 32 ml was transferred immediately for monocyte purification. The remaining 16 ml were centrifuged within 1 h; plasma was aspirated carefully and stored at −20°C until assayed.
Enzyme-linked immunosorbent assay (ELISA)
PR3
ELISA was performed using monoclonal antibodies as capture antibodies, as described earlier [8].
MPO
A microtitre plate (Nunc immunoplate) was coated overnight with 100 µl/well of a monoclonal anti-MPO antibody, 2B11 [11], 1 µg/ml in coating buffer (0·01 M Na2CO3, 0·04 M NaHCO3, 0·02% NaN3, pH 9·5–9·7) at 4°C. The plate was blocked with coating buffer, containing 2% bovine serum albumin (BSA) for 30 min in room temperature and washed with 0·9% NaCl, 0·05% Tween 20 three times. All subsequent incubations were performed in 100 µl volumes at room temperature on a rocking table and followed by washing three times. Plasma samples diluted to 1/50, 1/100 and 1/200 in sample buffer [phosphate buffered saline (PBS) 7·3–7·4, 0·05% Tween 20, 0·2% BSA] were added and the plate incubated for 1 h. After washing MPO was detected by 1-h incubation with rabbit anti-MPO (Dako, Glostrup, Denmark) diluted to 1/2000 in sample buffer. Washing was followed by addition of the conjugate (alkaline phosphatase-labelled swine anti-rabbit IgG, Dako), diluted to 1/1000 in sample buffer and 1-h incubation. P-nitrophenyl-phosphate disodium (Sigma-Aldrich Corp., St Louis, MO, USA) 1 mg/ml in substrate buffer (1·0 M diethanolamine, pH 9·8, 0·5 m M MgCl2, 0·02 NaN3) was used as substrate and optical densities were read at 405 nm. A standard curve was produced by incubation of a twofold dilution series of MPO (Wieslab AB, Lund, Sweden), starting with 10 ng/ml.
IL-6, IL-8
A quantitative sandwich enzyme immunoassay from R&D Systems (Abingdon, UK), where a monoclonal antibody specific for either IL-6 or IL-8 had been precoated onto a microplate, was used.
Cystatin C, CRP, WBC
The Clinical Chemical Laboratory at Lund University Hospital, Lund, Sweden, performed analyses on a Hitachi 917 Pluto. Kits from Roche Diagnostics (Basel, Switzerland) and Dako were used.
ANCA
Wieslab AB, Lund, Sweden, performed analyses of PR3-ANCA and MPO-ANCA using routine methods [12].
Cell separation
Peripheral blood monocytes were isolated by means of a monocyte isolation technique based on the OptiPrep density-gradient medium (Axis-Shield PoC AS, Oslo, Norway) [13]. The method was carried out at 4°C, during sterile conditions, using sterile solutions. Briefly, OptiPrep working solution (WS) was added to whole blood. A centrifugation gradient was created by mixing WS and solution B [Dulbecco's modified Eagle's medium (DMEM) medium supplemented with 10% serum; Invitrogen, Carlsbad, CA, USA]. Five ml blood was pipetted into a 15 ml test tube, after which 5 ml gradient was added carefully and finally 0·5 ml solution B on top (in order to avoid banding of the cells at a liquid/air interface). During the following centrifugation (700 g, 30 min, 4°C, no brake during deceleration) the monocytes float to the top of the gradient layer. After collection, the cells were gently diluted with 2 vol solution B, harvested by centrifugation and resuspended in solution B. Twenty-five µl cell suspension was then mixed with Türk's solution (methyl-violet) and counted in a Bürker chamber. A cell smear from each sample was also stained with May—Grünewald for differential counting. The monocyte purity was 85–95%, with single contaminating lymphocytes.
RNA extraction
Total RNA was extracted with RNeasy Mini kit (Qiagen, VWR International, West Chester, PA, USA) using the supplied protocol. High purity and good integrity were determined in two ways: first by optical density, 260/280 nm spectrophotometric ratios and then by the Agilent 2100 Bioanalyser, using the RNA 6000 Nano Assay reagent kit (Agilent Technologies, Palo Alto, CA, USA). After adding a gel-dye mix together with the RNA sample (25–500 ng) to the RNA 6000 Nano Chip channel system, the bioanalyser uses electrophoretic and electro-osmotic forces to drive fluids through capillaries to produce a virtual gel image and an electropherogram. In the electropherogram RNA of good quality shows up with clear 18 S and 28 S rRNA peaks and a flat baseline, whereas in the gel you see the corresponding sharp bands − the larger ribosomal band being more intense. Only RNA samples that met these criteria were accepted for further analyses.
Quantitative PCR assay
Total RNA was transcribed into cDNA, using the TaqMan Reverse Transcription Reagents kit (Applied Biosystems, Foster City, CA, USA), according to the manufacturer's recommendations. In short, random hexamers were used as template and put into the mastermix together with MultiScribe reverse transcriptase, RNase inhibitor, dNTPs, 5·5 m M MgCl2 and reverese transcription buffer. Five hundred ng total RNA was added to each 50-µl reaction and put into the thermocycler, set at 25°/10 min, 48°/30 min and 95°/5 min. For determination of gene expression, quantitative PCR assays were performed on an ABI PRISM 7000 Sequence Detector (Applied Biosystems) with TaqMan Universal Master Mix UNG, using the standard conditions determined by the company. After UNG incubation for 2 min at 50° and AmpliTaq Gold activation for 10 min at 95°, 40 cycles were run with denaturing temperature 95° (15 s) and annealing/extension temperature 60° (1 min). Assay on Demand, a unique combination of forward and reverse primers and fluorescent MGB-probes designed by the company, was used for each target gene. β-actin expression levels were used for normalization. cDNA corresponding to 10 ng RNA was used per 25 µl reaction and each reaction was performed in triplicate. The level of expression was calculated based upon the PCR cycle number (Ct) at which the exponential growth in fluorescence from the probe passes a certain threshold value. Relative expression was determined by the difference in the Ct values for the target genes after normalization to RNA input level, using β-actin Ct values. Relative quantification was determined by standard 2(–ΔΔCt) calculations [14]. Data are presented in relation to the median value of the HBD, set as 1.
In vitro stimulation assay
Stimulation of cell-lines U937/THP1 cells
The monocyte-like cell lines THP-1 and U937-4, a subclone of U937, were stimulated with cytokines and lipopolysaccharide (LPS) for different time periods [10]. The cells were cultured in suspension in RPMI-1640 with 10% fetal calf serum (FCS) (Invitrogen, Carlsbad, CA, USA) and exposed to interferon (IFN)-γ or IFN-α 100 U/ml (Boehringer, Ingelheim, Germany), LPS 10 µg/ml (Sigma-Aldrich Corp.), TNF-α 20 ng/ml (ICN Biomedicals, Aurora, Ohio, USA) and LPS 10 µg/ml plus TNF-α 20 ng/ml. The cells were harvested after stimulation and incubation for 6 and 20 h, respectively. Total RNA was extracted using the Trizol LS reagent (Invitrogen). Real time data are presented in relation to cells that have been incubated for 6 versus 20 h, without stimuli, set as 1.
Stimulation of human PBMC from healthy controls
The cells were purified using Optiprep as described above and 106 cells/well were cultured in RPMI-1640 with 10% FCS (Invitrogen), 10 ng/ml IL-4 and 20 ng/ml granulocyte macrophage colony-stimulating factor (GM-CSF) for 1 h. The cells were then exposed to IFN-γ 100 U/ml (Boehringer), LPS 10 µg/ml (Sigma-Aldrich Corp.) and TNF-α 10 ng/ml (ICN Biomedicals), respectively, and harvested after 6 h. Total RNA was extracted using the RNeasy Mini kit. Real time data are expressed in relation to non-stimulated cells, set as 1.
Statistical analysis
All statistics were performed in statview 5·01. Due to non-normally distributed parameters, the non-parametric Spearman's rank correlation test was used for correlation analysis in order to reduce the impact of outliers. Analysis of variance was performed using the non-parametric Kruskal—Wallis test and Mann—Whitney U-test.
Results
PR3 and MPO levels in plasma
Compared to HBD, we found increased circulating levels of the PR3 protein in patients with AASV, regardless of ANCA specificity (P < 0·0001) (Table 3). MPO levels were also found to be higher in the patients with AASV compared to HBD (P < 0·05). The SLE patients had higher levels of PR3 in plasma than the HBD (non-significant), but not to the same extent as in AASV, whereas MPO levels were similar to AASV. IL-6 and IL-8 were measured in plasma as inflammatory markers, showing similar inflammatory activity in the two disease groups.
Table 3. Proteinase 3 (PR3) and myeloperoxidase (MPO) protein levels in plasma.
| PR3 (µg/l) | MPO (µg/l) | IL-6 (ng/l) | IL-8 (ng/l) | |
|---|---|---|---|---|
| AASV | 560 (110–3940) | 74 (14–120) | 3·2 (1·6–8·6) | 8·3 (2·4–18·9) |
| MPO-pos | 560 (380–1770) | 68 (14–103) | 3·9 (1·8–7·8) | 7·2 (2·4–18·9) |
| PR3-pos | 570 (110–3940) | 74 (45–120) | 3·1 (1·6–8·6) | 9·1 (7·7–17·1) |
| HBD | 350 (110–580) | 15 (12–18) | 1·0 (0·3–6·1) | 4·7 (2·4–8·8) |
| SLE | 435 (138–959) | 69 (49–90) | 3·0 (1·2–8·7) | 5·7 (3·7–12·0) |
AASV = all patients with ANCA-associated vasculitis; MPO-pos = patients with MPO-ANCA at diagnosis; PR3-pos = patients with PR3-ANCA at diagnosis; HBD = healthy blood donors; SLE = disease controls with systemic lupus erythematosus; IL: interleukin. Data are presented as median (range).
mRNA levels in monocytes
Compared to both HBD (P < 0·0001) and SLE patients (P = 0·01), we found a 10-fold increase of PR3 expression in monocytes from patients with ANCA-associated vasculitis (Table 4). As shown in Fig. 1, all but two of the ANCA-positive patients had a PR3 expression greater than the maximum HBD expression. Four of our patients had no ongoing immunosuppressive treatment, whereas the others had different combinations of corticosteroids and other immunosuppressants. Four of the SLE patients had high PR3 expression (46–482). These patients all had relatively high doses of corticosteroids, at similar levels to AASV patients 2 and 5, and they were all active in their disease (Tables 1, 2). The other patients had low doses of corticosteroids. A fourfold increase of HLE expression was seen among patients with AASV and a threefold increase among SLE patients (P < 0·05 when compared to HBD). MPO and bactericidal permeability-increasing protein (BPI) expression did not differ from that in HBD for patients with AASV. IL-6 and IL-8 were measured in plasma as inflammatory markers (Table 3). On the RNA-level, IL-6 expression was very low, and data were therefore unreliable and not shown. This observation indirectly contradicts an unspecific activation of the cells during the purification process. IL-8 expression was increased in the SLE group but not in the AASV group, indicating a difference in inflammatory mobilization, as shown in Table 4.
Table 4. Monocyte expression profile, relative mRNA levels.
| PR3 | HLE | MPO | BPI | IL-8 | |
|---|---|---|---|---|---|
| AASV, n = 22 | 9·6 (1·8–680) | 4·0 (0·6–52) | 2·1 (0·4–14) | 1·4 (0·3–12) | 0·9 (0·03–13) |
| MPO-pos, n = 9 | 15·4 (5·4–70) | 5·7 (0·6–12) | 2·1 (0·8–8·1) | 1·4 (0·7–12) | 1·3 (0·2–13) |
| PR3-pos, n = 12 | 8·1 (1·8–680) | 2·5 (0·9–52) | 1·6 (0·4–14) | 1·5 (0·3–7·6) | 0·3 (0·03–6·4) |
| ANCA-neg, n = 1 | 0·8 | 0·6 | 0·3 | 0·3 | 2·0 |
| HBD, n = 15 | 1·0 (0·1–2·8) | 1·0 (0·2–4·8) | 1·0 (0·4–8·1) | 1·0 (0·3–23) | 1·0 (0–42) |
| SLE, n = 18 | 3·1(0·1–480) | 1·4 (0·4–540) | 5·7 (1·0–150) | 1·2 (0·3–34) | 5·2 (0·2–33) |
AASV = all patients with antineutrophil cytoplasmic antibodies (ANCA)-associated vasculitis; MPO-pos = patients with myeloperoxidase (MPO)-ANCA at diagnosis; PR3-pos = patients with proteinase 3 (PR3)-ANCA at diagnosis; HBD = healthy blood donors; SLE = disease controls with systemic lupus erythematosus. The real time data are corrected according to the 2–ΔΔCT formula and then expressed in relation to the median value of the healthy controls. Results are shown as median (range).
Fig. 1.
Monocyte proteinase 3 (PR3) mRNA levels among 22 patients with antineutrophil cytoplasmic antibodies (ANCA)-associated small vessel vasculitis. Patient enumeration is the same as in Table 1. Patients 1–9 are myeloperoxidase (MPO)-ANCA positive, 10–21 are PR3-ANCA-positive and patient 22 is seronegative. Levels are normalized using β-actin as housekeeping gene and the median value of our 15 healthy controls was set as 1·0 (range 0·1–2·8). The difference between ANCA-positive patients and healthy controls is highly significant, while the difference between MPO-ANCA and PR3-ANCA is not significant. X = outlier reaching 680.
In patients with AASV, plasma levels of PR3 tended to reach higher levels in patients with greater expression, but the correlation was not statistically significant (Fig. 2). No significant correlation was seen between plasma levels of CRP, IL-6 or IL-8 and PR3 expression. The correlation coefficients were −0·01, 0 and 0·4, respectively. PR3-ANCA titres showed negative correlation with PR3 expression (ρ−0·7, P < 0·05), whereas the opposite was seen with white blood cell counts (WBC, ρ 0·6, P < 0·05). There was no correlation between PR3 expression and the monocyte counts.
Fig. 2.
Correlation between plasma proteinase 3 (PR3) concentrations and monocyte PR3 mRNA levels among 18 of 22 patients with antineutrophil cytoplasmic antibodies (ANCA)-associated small vessel vasculitis. Plasma levels are measured in µg/l using enzyme-linked immunosorbent assay (ELISA). mRNA levels are normalized using β-actin as housekeeping gene and the median value of the healthy controls is set to 1. The correlation coefficient is 0·3 (not significant) when using the Spearman's rank correlation test.
In the ANCA patients PR3 expression covaried to some extent with HLE, and to a minor extent with MPO and BPI. The SLE patients, however, exhibited an apparent covariation between all four measured granular proteins (Table 5).
Table 5. Covariation of the relative mRNA levels of some monocyte genes.
| IL-8 | BPI | MPO | HLE | |
|---|---|---|---|---|
| PR3, AASV | 0·2 n.s. | 0·5* | 0·6* | 0·7** |
| PR3, SLE | −0·4 n.s. | 0·8** | 0·8** | 0·9** |
| HLE, AASV | 0·5* | 0·7* | 0·7* | |
| MPO, AASV | 0·4* | 0·6* | ||
| BPI, AASV | 0·5* |
AASV = antineutrophil cytoplasmic antibodies (ANCA)-associated systemic vasculitis; SLE, systemic lupus erythematosus; MPO = myeloperoxidase; HLE = human leucocyte elastase; PR3 = proteinase 3; IL = interleukin. Correlations are expressed as Rho;
P < 0·05;
P < 0·001; n.s. P >0·05.
In vitro stimulation of monocyte-like cell lines and PBMC
Stimulation of the monocyte like cell lines U937 and THP1 and human PBMC from healthy controls with various cytokines and LPS resulted in activation, as a substantial up-regulation of the IL-6 and IL-8 mRNA levels was seen. This, however, was not accompanied by any increase in the mRNA for the granule constituents PR3 or HLE, as shown in Tables 6,7.
Table 6. Cytokine stimulation of PR3, HLE and IL-8 expression in THP1 and U937 cell lines.
| PR3 | HLE | IL-8 | IL-6 | |
|---|---|---|---|---|
| THP1, 6 h | ||||
| unstimulated | 1 | 1 | 1 | 1 |
| IFN-γ | 1·5 | 1·3 | – | – |
| TNF-α | 0·8 | 0·6 | 10·5 | 3·0 |
| LPS + TNF-α | 0·8 | 0·7 | 4·0 | 2·8 |
| THP1, 21 h | ||||
| unstimulated | 1 | 1 | – | – |
| IFN-γ | 0·5 | 0·5 | – | – |
| TNF-α | 0·3 | 0·3 | – | – |
| LPS | 0·6 | 0·8 | 19·3* | – |
| IFN-α | 0·3 | |||
| U937, 6 h | ||||
| unstimulated | 1 | – | – | 1 |
| IFN-γ | 0·7 | – | – | 24·3 |
| TNF-α | 0·8 | – | – | 4·7 |
| LPS | 0·9 | – | – | 2·5 |
| U937, 21 h | ||||
| unstimulated | 1 | – | – | – |
| TNF-α | 0·1 | – | – | – |
| LPS | 0·3 | – | – | – |
| IFN-α | 0·3 | – | – | – |
| Mono, 21 h | ||||
| unstimulated | 1 | – | – | – |
| IFN-α | 1·1 | – | – | – |
The cells were incubated for 6 and 21 h, respectively. The real time reverse transcription-polymerase chain reaction (RT-PCR) data are corrected according to the 2–ΔΔCT formula and expressed in relation to unstimulated cells, incubated for the same length of time.
Unstimulated THP1 incubated 6 h set as 1 in this experiment. – = not done; IFN-γ= interferon gamma 100 U/ml; TNF-α= tumour necrosis factor alpha 20 ng/ml; LPS = lipopolysaccharide 10 µg/ml. Mono = human monocytes; IFN-α= interferon alpha, 1000 U/ml.
Table 7. Cytokine stimulation of PR3, human leucocyte elastase (HLE) and interleukin expression in healthy peripheral blood monocytes.
| PR3 | HLE | IL-8 | IL-6 | |
|---|---|---|---|---|
| Culture medium | 0·4 | 0·3 | 2·3 × 104 | 0·4 × 104 |
| LPS | 0·2 | 0·2 | 10 × 104 | 1·2 × 104 |
| TNF-α | 0·3 | 0·2 | 3·2 × 104 | 0·2 × 104 |
| IFN-γ | 0·2 | 0·1 | 3·5 × 104 | 0·4 × 104 |
The cells were incubated with stimuli for six hours. Data are presented as mean values of peripheral blood mononuclear cells (PBMC) from three separate donors, in relation to the corresponding freshly drawn, non-stimulated samples. Culture medium = RPMI-1640 with 10% fetal calf serum (FCS), 10 ng/ml interleukin (IL)-4 and 20 ng/ml granulocyte macrophage-colony stimulating factor (GM-CSF); LPS = lipopolysaccaride, 10 µg/ml; TNF-α = tumour necrosis factor alpha, 10 µg/ml; IFN-γ = interferon gamma, 100 µ/ml; PR3 = proteinase 3.
Discussion
This study demonstrates a strong relative increase in mRNA levels for PR3 in monocytes from patients with AASV compared to both HBD and disease controls. The median result for PR3 mRNA was a 10-fold increase, and the separation between HBD and AASV was remarkable. The correlation between plasma PR3 concentration and monocyte PR3 mRNA was positive but weak, and failed to reach statistic significance when using a non-parametric test. This is, however, not surprising considering that there are several steps between mRNA levels and circulating protein. High levels of PR3 mRNA in circulating monocytes do not necessarily mean an increased total amount of PR3. PR3 is also produced by neutrophils, which are more abundant, and our data concern normalized RNA and thus indicate PR3 production per cell rather than the total production in the body. Furthermore, autoantibodies against PR3 might influence the half-life of PR3 in the circulation and/or the measurement of PR3 in plasma. In the present study higher levels of PR3-ANCA were associated with lower levels of circulating PR3 (data not shown). There was also a negative correlation between ANCA levels and PR3 mRNA in the monocytes, which argue against a direct causative effect of the autoantibodies on the PR3 mRNA production. An in vitro study by Yang and coworkers did not find PR3 among genes up-regulated in neutrophils treated with ANCA-IgG [15].
PR3 is normally transcribed during myelopoiesis and is supposed to be turned off in mature leucocytes [16]. Constitutive expression of PR3 is a feature of many haematopoietic malignancies where a differentiation block have prevented the maturation and kept the cells in a proliferative state [17]. An alternative name for PR3 is myeloblastin, which was first described as a substance that maintained the proliferative capacity of myeloblasts. Anti-sense treatment blocking PR3 transcription led to growth arrest and differentiation of promyelocytic leukaemia cells [18].
Recently there have been several reports showing that PR3 production can occur in more mature cells. Zhou et al. have published a study showing de novo synthesis of PR3 by circulating mononuclear cells that were cultured and stimulated with TNF-α. This study was performed using mononuclear cells from healthy donors and the percentage of monocytes/lymphocytes was 40/60, leaving a potential lymphocyte influence on the results [19]. Another study, by Just et al., showed an up-regulation of PR3 mRNA expression in circulating monocytes, but not in neutrophils, in cystic fibrosis patients, correlating with pulmonary exacerbation [20]. Brockman et al. found increased PR3 expression in macrophages at inflammatory sites in lung tissue from patients with WG [21]. Earlier this year Yang et al. demonstrated increased PR3 transcription in circulating leucocytes from patients with AASV compared with HBD and SLE patients, correlating with disease activity [9]. These reports indicate that our results might be a result of cytokine action caused by general inflammation. There are several findings in our study that argue against this notion: (1) there was no correlation between circulating IL-6, IL-8 or CRP-levels and PR3 expression; (2) there was no significant IL-6 or IL-8 up-regulation on the mRNA level in the monocytes; (3) there was no general up-regulation of granular proteins; and (4) in vitro stimulation of monocyte-like cell lines and healthy PBMC with TNF-α, IFN-γ or LPS for 6 or 20 h did not result in any increase of PR3 production, whereas the IL-8 expression was highly up-regulated.
In addition to the increased PR3 levels we also found a minor increase in HLE mRNA levels when compared to HBD, but we found no significant alteration of MPO or BPI. These results differ from those of Dr Yang, which indicated a more general up-regulation of granular proteins [9]. What we did see was a strong covariation of the transcription of the granular enzymes in the SLE patients, thus there were both quantitative and qualitative differences between SLE and AASV monocyte gene transcription in the present study. There are reports that granulocyte colony stimulating factor (G-CSF) can up-regulate PR3 transcription [22]. Indeed we found a positive correlation between total white blood cell count and monocyte PR3 mRNA. The G-CSF was shown to stimulate through the G-CSF receptor and the transcription factor PU.1. Because PU.1 response elements are present in the vast majority of promoters for granule constituents, high G-CSF levels are unlikely to be responsible for the qualitative difference between AASV and SLE patients.
Another concern is that of immunosuppressive drugs. This was a major reason for choosing SLE patients as disease controls. Fifteen of 22 patients with AASV had low doses of corticosteroids and three patients had high doses (exceeding 15 mg per day). The latter three had high PR3 expression, but otherwise there was no correlation with the corticosteroid dose. Four of the SLE patients also had high doses of corticosteroids and they exhibited high expression of all measured granular mRNAs, indicating that above a certain dose corticosteroids could influence PR3 expression. Three of our AASV patients were treatment naive, but still demonstrated considerably raised PR3 expression.
A skewed distribution of a polymorphism in the promoter region of the PR3 gene, involving a transcription factor binding site, have recently been described [23]. Because the difference in allele frequency between patients and controls was relatively small, it is very unlikely that this polymorphism could explain our present findings. This notion has been further supported by the findings in our recently published study, showing that the −564 A/G polymorphism does not increase PR3 promotor activity [24].
If the increased PR3 levels in AASV are not caused by inflammation, medication, autoantibodies or germline genetic variants, what could then be the cause? Our study, as well as the work of Dr Yang et al. [9], has demonstrated up-regulated PR3 gene transcription in AASV. This would imply an increased PR3 production. A highly speculative hypothesis would be that a somatic mutation occurs in a stem cell leading to very late maturation block with increased PR3 production as a major feature. This is a bold hypothesis in need of extensive work in order to be thoroughly evaluated.
In conclusion, circulating monocytes from patients with systemic vasculitis display up-regulated transcription of the PR3 gene, implying increased PR3 production and a potential source of the increased circulating levels described earlier [8]. Considering the great toxic potential of PR3, this may be of importance for the development of vasculitis. The origin of this potentially increased production remains obscure; however, our results do not favour an influence of cytokines, ANCA or immunosuppressive medication. All these aspects, however, require further study.
Acknowledgments
This study was supported by grants from the Swedish Scientific Council (project 71X-09487 and 74X-13489), the foundations of the Royal Physiographic Society and Thelma Zoega, the Foundation for Strategic Research, Riksförbundet för Njursjuka, Sweden. We wish to thank Nermina Jaganjac, Åsa Pettersson (Department of Nephrology) and Carina Strand (Department of Oncology) for skilful laboratory assistance.
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