(MPO) and Proteinase 3 (PR3) in Patients with Systemic Vasculitis

Clin Exp Immunol 1996; 103:253–258

T cell responses to myeloperoxidase (MPO) and proteinase 3 (PR3) in patients with systemic vasculitis

M. E. GRIFFITH, A. COULTHART & C. D. PUSEY Renal Unit, Department of Medicine, Royal Postgraduate Medical School, Hammersmith Hospital, London, UK

(Accepted for publication 26 October 1995)

SUMMARY T cell-mediated immune responses are likely to be important in the pathogenesis of systemic vasculitis. However, identifying the T cells involved has proved difficult, and there are conflicting reports regarding T cell proliferation in response to different autoantigens. Perinuclear (P) and cytoplasmic (C) anti-neutrophil cytoplasmic antibodies (ANCA) are closely associated with systemic vasculitis, and are generally specific for MPO or PR3, respectively. We studied the proliferative responses to MPO and PR3 of peripheral blood mononuclear cells from patients with P- ANCA or C-ANCA specific for these antigens by ELISA. These responses were compared with those of normal controls, and of disease controls with P- or C-ANCA not specific for MPO or PR3.

The patient group as a whole showed signiﬁcant T cell proliferation in response to the autoantigens

compared with controls (P 0 005). Cells from nine of 13 P-ANCA-positive, anti-MPO-positive

X patients proliferated in response to MPO, compared with ﬁve of 16 controls (P 0 04). Cells from

ﬁve of eight C-ANCA-positive, anti-PR3-positive patients proliferated in response to PR3, compared

X with two of 11 controls (P 0 05). These experiments demonstrate that patients with P-ANCA or C- ANCA possess T cells which respond to MPO or PR3, respectively. As in other autoimmune diseases, responses to both antigens were also seen in a proportion of healthy controls. Further analysis of these responses will be important in understanding the pathogenesis of systemic vasculitis and in designing speciﬁc immunotherapy.

Keywords T cells systemic vasculitis proteinase 3 myeloperoxidase anti-neutrophil cytoplasmic antibodies

INTRODUCTION WG, although they are also found in MP [2]. The major target antigen of C-ANCA is PR3 [4,5], a neutrophil granule enzyme Microscopic polyangiitis (MP) and Wegener’s granulomatosis with bacteriocidal properties, which is identical to myeloblastin, (WG) are systemic vasculitides characterized by pauci-immune a leucocyte growth factor [6]. focal segmental and necrotizing glomerulonephritis, together The presence and titre of ANCA have been shown to with involvement of a variety of other organs. There is correlate with disease extent and activity, and a rise in titre increasing evidence that an autoimmune process underlies generally predicts relapse [7]. In vitro, ANCA are able to induce the development of systemic vasculitis. Two types of anti- neutrophil degranulation [8], and may both enhance neutrophil neutrophil cytoplasmic antibody (ANCA) are seen in these cytotoxicity and also have direct cytotoxic effects on endo- disorders, perinuclear (P)-ANCA and cytoplasmic (C)-ANCA, thelial cells [9,10]. T cell involvement in the disease process as deﬁned by their appearance on indirect immunoﬂuorescence is likely, not only in providing help for autoantibody production, of normal human neutrophils [1]. P-ANCA are found in a wider but also by direct cytotoxic mechanisms. Pathological lesions spectrum of disorders than C-ANCA, and have a greater variety

in WG are characterized by granuloma formation in the of target autoantigens [2]. When P-ANCA are present in MP, the

lungs, upper respiratory tract and elsewhere, and both CD4 target autoantigen is usually MPO [3], a neutrophil granule and CD8 T cells have been identified in the glomeruli and enzyme involved in host defence. C-ANCA predominate in interstitium in pauci-immune rapidly progressive glomerulonephritis (RPGN) [11]. Attempts to identify the T cells involved Correspondence: Dr M. E. Griffith, Renal Unit, Royal Postgraduate have been hindered by difficulties in obtaining sufficiently Medical School, Hammersmith Hospital, Du Cane Road, London W12 pure antigen preparations, and previous studies have given

0NN, UK. conﬂicting results [12–15]. 5 1996 Blackwell Science 253 254 M. E. Grifﬁth, A. Coulthart & C. D. Pusey

Table 1. Diagnoses of patients with anti-neutrophil cytoplasmic antibodies

(ANCA) speciﬁc for MPO or PR3

P-ANCA C-ANCA

Diagnosis MPO PR3

Microscopic polyangiitis 7 2 Wegener’s granulomatosis 0 6 Idiopathic rapidly progressive nephritis 3 0 Goodpasture’s disease 3 0

In this study, we examined responses of peripheral blood mononuclear cells (PBMC) to puriﬁed preparations of MPO or Fig. 1. A. SDS–PAGE of the PR3-containing fraction from a high PR3 in patients with P-ANCA or C-ANCA speciﬁc for these performance liquid chromatography (HPLC) separation, indicating its antigens purity. B. Western blot with anti-PR3 serum from a patient with Wegener’s granulomatosis (WG), demonstrating antigenicity.

PATIENTS AND METHODS Patients Preparation of antigens Blood or buffy coat (collected during plasma exchange) was Purified MPO was purchased from Calbiochem (Nottingham), obtained from 21 patients, 13 of whom had P-ANCA specific for UK) and its purity checked by SDS–PAGE. PR3 was purified MPO, and eight of whom had C-ANCA specific for using the method described by Gaskin et al. [18]. Briefly, whole PR3. Eighteen of these patients had systemic vasculitis, with blood was mixed with 3% dextran and the erythrocytes allowed diagnoses made according to criteria defined by the Chapel to sediment. The supernatant was resuspended on Lymphoprep Hill consensus conference (Table 1) [16]. Three patients with (Nycomed) and the lymphocytes were separated by density P-ANCA also had Goodpasture’s disease, with anti-glomerular gradient centrifugation. The resulting pellet was washed with basement membrane (anti-GBM) antibodies as detected by distilled water to remove any remaining erythrocytes by

hypotonic lysis, and the neutrophils then suspended in relaxa- ELISA specific for 3(IV)NCI [17]. Seven other vasculitis patients were selected as controls (Table 2), four with P- tion buffer before nitrogen bomb cavitation on ice. The alpha ANCA and three with C-ANCA which were not specific granules were then separated on a Percoll (Sigma Aldrich and for MPO or PR3. Cells from 12 normal controls were tested Co., Poole, UK) gradient, and disrupted by multiple freeze–thaw against MPO, and from eight against PR3. cycles followed by sonication. The granule contents were separated on a C4 reverse phase high performance liquid chromatography (HPLC) column (Alltech, Carnforth, UK), with an Isolation of PBMC acetonitrile gradient. The resulting fractions were freeze-dried, The PBMC were separated from blood or buffy coat by and those containing PR3 were identified by SDS–PAGE and density gradient centrifugation to Lymphoprep (Nycomed, Bir- Western blotting against sera containing C-ANCA specific for mingham, UK). Cells were resuspended in freezing PR3 by ELISA (Fig. 1). The fractions selected were confirmed to be PR3 by N-terminal amino acid sequencing. The PR3 was medium, consisting of 40% RPMI 1640 (GIBCO, Paisley, UK), with 10% dimethylsulphoxide (BDH, Dagenham, UK) and resuspended in PBS and the concentration of protein estimated by 50% fetal calf serum (FCS; M. B. Meldrum, Bourne End, UK), comparison of optical density with bovine serum albumin (BSA) and stored in liquid nitrogen. Before use, the cells were rapidly solutions of known concentration. thawed, washed in PBS pH 7.2, and their viability checked by staining with ethidium bromide and acridine orange. Assays for ANCA The method used for detection of ANCA by indirect immuno- fluorescence was based on that described by Wiik [19,20]. Table 2. Diagnoses of controls with anti-neutrophil cytoplasmic antibodies The ELISAs used for measurement of antibodies to MPO, (ANCA) not specific for MPO or PR3 PR3, and GBM were performed as previously described by our laboratory [17,18,21]. The antigens used in the MPO and

PR3 ELISAs were identical to those used in the prolifera-

P-ANCA C-ANCA tion assays.

ÿ Diagnosis MPOÿ PR3

Microscopic polyangiitis 1 0 T cell proliferation assays

Wegener’s granulomatosis 0 3 The isolated PBMC were cultured at 2 10 cells/well in ﬂat-

Idiopathic rapidly progressive nephritis 1 0 bottomed 96-well plates (GIBCO), suspended in 200 "l of RPMI Cryoglobulinaemia 1 0 1640 medium (GIBCO) containing 10% AB human serum (North

Scleroderma 1 0 West London Transfusion Centre, Edgware, UK), 100 U/ml

penicillin, 100 "g/ml streptomycin (GIBCO) and 4 mM glutamine 5 1996 Blackwell Science Ltd, Clinical and Experimental Immunology, 103:253–258 T cell responses in vasculitis 255

Fig. 2. Proliferative responses of peripheral blood mononuclear cells (PBMC) from patients and controls to MPO and PR3. g, Controls with P-ANCA or C-ANCA not speciﬁc for MPO or PR3, respectively.

Fig. 3. Proliferation of peripheral blood mononuclear cells (PBMC) from

h patients and controls to MPO is dose-dependent. g, Patient; , control. Error bars show s.e.m. (GIBCO). All assays were performed in triplicate, and included

control wells with no antigen and wells containing phytohae- magglutinin (PHA; Sigma Aldrich) at 2 "g/ml, as a non-specific T cell mitogen. Specific antigen was added to the wells in between those who responded to the specific autoantigens and

" concentrations varying from 0 1 "g/ml to 30 g/ml. Cells from those who did not.

patients with MPO-specific ANCA were tested against MPO, and The optimum autoantigen concentration was 10 "g/ml for those with PR3-specific ANCA were tested against PR3. The MPO, and 20 "g/ml for PR3. PBMC from patients and controls three patients with P-ANCA not specific for MPO were also that responded to the antigens did so in a dose-dependent tested against MPO, and those with C-ANCA not specific for manner (Fig. 3). Proliferation was seen at all three time points, PR3 were tested against PR3. Twelve normal controls were and was usually maximal at 7 or 10 days (Fig. 4). All batches of tested against MPO, and eight against PR3. Proliferative cells tested were viable, as shown by their response to PHA responses were measured at 5, 7 and 10 days. Tritiated thymi- (mean SI 78). There was no difference in the response to PHA

dine (Amersham, Little Chalfont, UK) was added to the wells at between those who responded to MPO or PR3 and those who did 1 "Ci/well, and 16 h later the cells were harvested onto glassﬁbre not. ﬁlters using an automated cell harvester (Pharmacia, Milton Cells from nine of 13 P-ANCA-positive, anti-MPO-positive Keynes, UK). Thymidine incorporation was measured using an patients proliferated in response to MPO, with a median SI of

. automated counter (Pharmacia). Results were expressed as 4 7, compared with ﬁve of 16 controls (ﬁve of 12 normal

either ct/min or as stimulation indices (SI: calculated as the ratio controls and nil of four ANCA-positive, anti-MPO-negative

. X of ct/min with antigen to ct/min with medium alone). An SI of patients), with a median SI of 1 7(P004). The highest SI 2.5 or more was regarded as positive. in the ANCA-positive, anti-MPO-positive group was 21.3, whereas that of controls was 9.5. Eight of the nine anti-MPO- positive patients who responded had cells collected during their Statistical analysis first presentation with active vasculitis, and all had started The Wilcoxon rank sum test was used to test for significant therapy with prednisolone and cyclophosphamide within the differences in proliferation between the study groups and previous 72 h. Patient 9 (SI 7.1) was clinically in remission controls. Analysis of variance was used to test for differences when the cells were taken and not on any treatment, although in responses to PHA, and background proliferation counts, she remained P-ANCA-positive and has since relapsed. Of the between those who responded to MPO or PR3 and those who four anti-MPO-positive patients who did not respond, three were did not. also positive for anti-GBM antibodies, and clinically were diagnosed as having Goodpasture’s disease. Cells from patients with Goodpasture’s were collected during their first presentation RESULTS with active disease, and all had started prednisolone and cyclo- T cell proliferation was observed to both MPO and PR3 in a phosphamide within the previous 72 h. The fourth non-responder proportion of patients and controls (Fig. 2). Overall, cells from had MP that was clinically in remission, and was on low dose 14 of 21 patients proliferated in response to their specific prednisolone.

autoantigen (median SI 4.63), which was signiﬁcantly greater Cells from ﬁve of eight C-ANCA-positive, anti-PR3-positive

. X than the seven of 27 controls (median SI 1 7, P 0 005). The patients showed signiﬁcant proliferation in response to PR3, the

mean background proliferation was 257 ct/min, and did not differ median SI being 3.8, compared with two of 11 controls (two of 5 1996 Blackwell Science Ltd, Clinical and Experimental Immunology, 103:253–258 256 M. E. Grifﬁth, A. Coulthart & C. D. Pusey

is also increasing evidence for the pathogenicity of ANCA [27], the production of which is likely to depend on T cell help. Furthermore, success in obtaining remission in patients with intractable vasculitis has been achieved by treatment with a combination of MoAbs to CD4 and CD52 [28]. T cell proliferation in response to specific B cell autoantigens has been demonstrated in other autoimmune diseases, such as myasthenia gravis [29], autoimmune thyroiditis [30,31], and Goodpasture’s disease [32]. We have now demonstrated significant T cell proliferation to MPO and PR3 in patients with ANCA specific for these antigens. Proliferation was also seen in cells from some healthy controls, a phenomenon observed in several other autoimune diseases [32–34]. Further analysis is required to ascertain whether these autoreactive T cells are present at a lower precursor frequency in controls than in patients, as shown in myasthenia gravis and multiple sclerosis [33,34]. Previous studies of T cell responses to MPO and PR3 in patients with vasculitis have been hampered by difficulties with antigen preparation, and results have been inconsistent. An early study failed to demonstrate proliferation to a crude acid

Fig. 4. Proliferation of peripheral blood mononuclear cells (PBMC) from extract of neutrophil granules, which was not separated into

h l

one patient to PR3 at three different time points: g, day 5; , day 7; , day individual components [12]. Convincing T cell proliferation has 10. Error bars show s.e.m. been observed in response to puriﬁed PR3, but not to MPO. Using boiled inactivated enzymes, Brouwer et al. demonstrated signiﬁcant proliferation to PR3, but obtained a response to

eight normal controls and nil of three C-ANCA-positive, anti- MPO in only one of ﬁve patients [14]. More recently, Ballieux

. X

PR3-negative controls), with a median SI of 1 3(P005). Of et al. [15] demonstrated proliferation to native PR3, but only at

X the five anti-PR3-positive patients who responded, three had a low level (maximum SI `3 5); however, their method of cells collected during their first presentation with active vascu- purification involved detergents which, they suggest, may litis, and had started treatment with prednisolone and cyclopho- have disrupted intra- and intermolecular bonds of PR3 and sphamide within the previous 72 h. Two patients were clinically resulted in alteration of antigen recognition and processing. in remission; one was taking prednisolone and azathioprine (SI Some neutrophil enzymes may be mitogenic and stimulate 4.6), and the other was not on treatment, but has always lymphocytes non-specifically [35]. However, as reported by remained C-ANCA-positive (SI 3.0). The three anti-PR3-positive the other group using native purified antigens [15], we did patients who did not respond also had cells collected during not observe this in our experiments. their first presentation with active vasculitis. Two had started All patients in the present study had their ANCA specificity prednisolone and cyclophosphamide in the previous 72 h, and determined by ELISA to ensure that the correct B cell auto- one had cells taken before starting any treatment (SI 1.7). The antigen was being investigated. Controls with ANCA not specific highest SI obtained in the C-ANCA positive, anti-PR3-positive for MPO or PR3 were also tested, to ensure that ANCA group was 18.6, whereas that of controls was 3.7. positivity itself was not affecting the results. Many (13/21) of Four individuals that were retested showed consistent our patients were undergoing plasma exchange at the time of responses. (e.g. maximum SIs were 9.2 and 8.4 in one patient, obtaining their PBMC, and may have had earlier or more severe and 2.8 and 3.1 in another). In view of the possible effects disease than patients in other studies; the higher proliferative of enzymatically active MPO and PR3 on T cell proliferation, responses obtained may therefore reflect an increased population three subjects were also tested with MPO or PR3 which was of autoreactive cells. boiled for 15 min to remove enzymatic activity [14]; the The isolation of T cell lines and clones in other autoim- responses obtained were the same as those with unheated mune diseases has been facilitated by the use of recombinant antigen. antigens [33]. The production of recombinant MPO has already been reported [36], and production of an antigenic form of recombinant PR3 is currently underway in our laboratory. These reagents should allow us to establish autoreactive lines and DISCUSSION clones from proliferating T cell populations, which will The role of T cells in many other autoimmune diseases is allow further investigation of the specificity and characteristics now well established [22], and evidence for their involve- of autoreactive T cells in systemic vasculitis. The character- ment in systemic vasculitis is increasing. The level of soluble ization of T cell responses at a molecular level has led to the IL-2 receptor, which is a marker of T cell activation, has been development of new immunotherapeutic approaches, for found to rise at times of active vasculitis [23,24]. More example, the use of tolerogenic peptide analogues based on indirect evidence for T cell involvement comes from reports key autoantigenic peptides, or immunization with peptides of associations between HLA class II genes and systemic based on the sequence of the relevant T cell receptors [37].

vasculitis, although there is as yet no consensus [25,26]. There These approaches have been shown to prevent or treat 5 1996 Blackwell Science Ltd, Clinical and Experimental Immunology, 103:253–258 T cell responses in vasculitis 257 autoimmune disease in a variety of animal models, and trials A hypothesis based on both humoral and cellular mechanisms. Neth in some human diseases are underway [38–40]. Systemic J Med 1990; 36:169–71. vasculitis is a relapsing disease with considerable morbidity 14 Brouwer E, Stegeman CA, Huitema MG, Limburg PC, Kallenberg and mortality, which is contributed to by the adverse effects CGM. T cell reactivity to proteinase 3 and myeloperoxidase in of non-speciﬁc immunosuppressive therapy required for dis- patients with Wegener’s granulomatosis (WG). Clin Exp Immunol 1994; 98:448–53. ease control. The development of speciﬁc immunotherapy 15 Ballieux BEPB, van der Burg SH, Hagen EC, van der Woude FJ, directed at autoreactive T cells in systemic vasculitis is there- Melief CJM, Daha MR. Cell mediated autoimmunity in patients fore of great potential importance. with Wegener’s granulomatosis (WG). Clin Exp Immunol 1995; 100:186–93. 16 Jennette JC, Falk RJ, Andrassy K et al. Nomenclature of systemic vasculitides. Proposal of an international consensus conference. ACKNOWLEDMENTS Arthritis Rheum 1994; 37:187–92. 17 Katbamna I, Turner N, Ross CN, Pusey CD. Development and M.E.G. is a Wellcome Trust Research Training Fellow. We are grateful to application of an ELISA for Goodpasture’s disease based on sheep

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