Clin. exp. Immunol. (1992) 89, 274-278
Anti-myeloperoxidase autoantibodies react with native but not denatured myeloperoxidase R. J. FALK, M. BECKER, R. TERRELL & J. C. JENNETTE* Departments of Medicine and Pathology*, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
(Acceptedfor publication 30 April 1992)
SUMMARY We wondered whether anti-myeloperoxidase (MPO) autoantibodies (MPO-ANCA) found in patients with systemic vasculitis react with a conformational epitope or epitopes on the MPO molecule. Sera from 15 human MPO-ANCA, and a polyclonal and a monoclonal anti-MPO antibodies were reacted with MPO in native and denatured states. Human MPO-ANCA and mouse monoclonal anti-MPO reacted with native MPO, and a 120-kD band representing the MPO hologenzyme, but not with denatured MPO fragments; however, MPO-ANCA and mouse anti-MPO did not demonstrate competitive inhibition of binding to MPO. Polyclonal rabbit anti-MPO reacted with both native and denatured MPO. All MPO-ANCA tested showed the same patterns of reactivity with native and denatured MPO in dot blot and Western blot analyses. Both polyclonal and monoclonal anti-MPO antibodies inhibited MPO's protein iodination by over 90%, whereas MPOANCA IgGs, normal IgGs and disease control IgGs did not. These data suggest that (i) MPO-ANCA interact with a conformational epitope on the MPO molecule; and (ii) MPO-ANCA from different patients have similar reactivity with native versus denatured MPO.
Keywords autoimmunity/autoantibodies vasculitis ANCA
INTRODUCTION
Anti-neutrophil cytoplasmic autoantibodies (ANCA) have been found in the circulation of patients with the most common types of systemic necrotizing vasculitis, including microscopic polyarteritis nodosa, Wegener's granulomatosis, and ChurgStrauss syndrome [1-8]. There are two ANCA subtypes defined by indirect immunofluorescence microscopy staining patterns. Cytoplasmic pattern ANCA (CANCA), which are usually specific for proteinase-3 [9-14], and perinuclear pattern ANCA (P-ANCA), which are usually specific for myeloperoxidase (MPG-ANCA) [1,3,13]. Both types of autoantibodies are capable of activating cytokine-primed neutrophils in vitro inducing a respiratory burst and release of granule constituents [14]. Therefore, ANCA may be directly involved in the pathogenesis of necrotizing vascular inflammation, We wondered whether MPO-ANCA from different patients reacted with the same or different portions of the myeloperoxidase molecule, and whether this reactivity was directed at a
PATIENTS AND METHODS Patients and sera Sera containing MPO-ANCA were obtained from patients with pauci-immune necrotizing and crescentic glomerulonephritis with and without extra-renal vascular inflammation. Five of 11 patients had pulmonary capillaritis with alveolar haemorrhage; five had vasculitis involving the upper respiratory tract, nerves, or skin; and five patients had renal-limited disease. P-ANCA were identified using indirect immunofluorescence microscopy and alcohol-fixed normal human neutrophils as described previously [2,17]. Patients with P-ANCA reactivity were tested by ELISA for reactivity with neutrophil cytoplasm extract and reactivity with purified MPO using previously described methof the P-ANCA used in this study had anti-MPO 'ods [1] All sp[1]. All ofte P-ANCAu in this s y a anti-MPG the anti-MPO ELISA of greater than 3 s.d. above the obtained with 13 normal control sera.
mean
Dot blots NircluoesipwredtdwthMG ihwihanMPO antibody reactivity was to be analysed. Myeloperoxidase was elther purchased from Calbiochem (San Diego, CA) or isolated from purified azurophilic granules using a modification of the procedure of Matheson [18]. Reactivity of MPG-ANCA and anti-MPG antibodies was evaluated after the MPG had been exposed to the following conditions: denaturation with 2%
epitope structure epitope primary structure or at at aa primary [15] or epitope [151 conformational epitope
[16].rmational
Correspondence: Ronald J. Falk, MD, Department of Medicine, Division of Nephrology, CD No. 7155, 3034 Old Clinic Bldg, Chapel Hill, NC 27599, USA.
274
Anti-myeloperoxidase autoantibodies SDS, reduction with 2-6% beta-mercaptoethanol (B-ME), combined chemical denaturation and reduction, and thermal denaturation at 1000C for 2 min. One hundred nanograms of MPO was placed in each dot. The strips were washed in 150 mM NaCl, 50 mm Tris pH 7-2 (TBS), blocked with 5% non-fat dry milk for 2 h, and incubated with MPO-ANCA, polyclonal anti-c MPO, or monoclonal anti-MPO. The strips were washed and exposed to the appropriate secondary antibodies and alkaline phosphatase substrate. For staining of immunoblots, human MPO-ANCA + serum was used at a dilution of 1: 40. Polyclonal rabbit anti-human MPG (Dako, Carpinteria, CA) was used at a dilution of 1:2000, and mouse monoclonal anti-human MPO (Dako) was used at 1:500. Appropriate secondary antibodies) included goat anti-rabbit IgG alkaline phosphatase conjugate
and 0 45 mMr 5-bromo-4-chloro-3-indolyl-phosphate (Sigma) in 100 mBuTris, 100 mn sodium chloride, 5 m magnesium chloride buffer, pH 95. Western blots
Myeloperoxidase was run on 10% or 13% polyacrylamide electrophoresis with a 4% stacking gel. Before evaluating reactivity with antibodies, MPG was exposed to the following conditions: denaturing conditions with 2% SDS; reducing conditions with 2-6% B-ME; combined denaturing and reducing conditions; and thermal denaturation by heating to 1000C for 2 mmi. pM nitrocellulose (Schleicher and TransferoofrthesMPGwtoh a 4 Schuell, Keene, NH) was performed using a Mini-transblot cell (Biorad, Rockville Center, NY) with 10% methanol in Trisglycine buffer pH 86 at 250 mA for I h [19]. Transfer of protein was assessed by staining the nitrocellulose strips with amido black. The nitrocellulose strips were washed in distilled water and TBS, and blocked in 5% non-fat dry milk for 2 h The strips were incubated in the different primary anti-MPG antibodies overnight, washed in 5% non-fat dry milk three times, and incubated again in secondary antibody for 2 h. The reaction product was developed with alkaline-phosphatase developing
buffer.
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acidic buffered (2); denatured and boiled (3); denatured, reduced and boiled (4); denatured and reduced, but not boiled (5); and denatured but not boiled (6). Polyclonal rabbit anti-MPG reacted with MP under all conditions (lane A). Mouse monoclonal anti-MPG recognized MPG in its native state, but not when it was thermally denatured (lane B). Seven MPg oANCA sera tested also reacted to MP in its native state, but not when it was thermally denatured (lanes C-I). Normal human sera did not react with MPG (lane J). affected by a non-specific protein effect so that the amount of IgG added was rigorously standardized. The following reagents were added in sequence: MPG, H2G2, MPG-ANCA IgG or polyclonal or monoclonal anti-MPG antibodies, and Nad25I. The mixture was rotated for 30 mm at room temperature and the reaction stopped with cold 5 6 mu sodium thiosulphate (Fisher, Fair Lawnf NJ), followed by the addition of 20% trichloroacetic acid (TCA) (EM, Cherry Hill, NJ) to precipitate the protein. The pellet was repetitively washed with 10% TCA, centrifuged and then protein bound radioactivity was counted. The addition of Na azide served as the positive control for each assay while the negative controls included assays performed without H2e2 and without MPG. The monoclonal anti-MPG, polyclonal antiMPG, MPG ANCA IgG, C-ANCA IgG and normal IgG were used at a final concentration 2t04 of ,ug/ml, g/ml, 1 mg/ 0o76 2 0t ml or 0 025 mg/ml. Results were expressed as a percentage of inhibition compared to the inhibition observed with Na azide.
Functional activity oflmyeloperoxidase In order to assess whether MPo-ANCA could block MPG function, protein halogenation by MPG in the presence of MPG-ANCA was studied. Modification of a standard MPo RESULTS protein halogenation assay [20,21] was designed as a cell-free Dot blot analysis system. In this assay, 100 mU of MPG (Calbiochem), 0 1 mo MPG-ANCA sera were tested on dot blots for reactivity of the Na'251 (New England Nuclear, Irvine, CA), and 0-1 mM human autoantibodies with MPG in its native form, treated with acid denatured with SDS and/or reduced with B-ME, and hydrogen peroxide (H2e2) (Sigma) were mixed in Hanks buffered saline solution with calcium and magnesium thermally denatured. For purposes of comparison, polyclonal rabbit anti MPG aswell as amonoclonal mouse anti-MPG were (HBSS++~) (137 mMs NaCJ, 137 mMs KCl, 352 mMs Na2HPG4, 441 mM KH2PG4, with 12d 9 moi CaCl2.H2G) andma 4 tested. The polyclonal rabbit anti-MPG reacted with MPG So4.7H2. Todefine themaximum degree ofinhibition,assays under all the conditions (Fig. 1, lane A). In contrast, the were run with and without 10 0 mo sodium azide (Sigma). The monoclonal anti-MPG did not react with thermally denatured reaction was carried out in borosilicate glass tubes to which MPn but reacted with MPG in its native state, as well as when it HBSSy + and heat-inactivated sera were added and incubated at was partially denatured with SDS or reduced with B-ME (Fig. 1, 37C. Because of the peroxidase activity of sera, purified IgG was prepared as previously described [19]. The assay was also
lane B)u Seven MPo-ANCA reacted in a fashion exactly the same as the MoAb, i.e. by not reacting with thermally denatured
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Fig. 2. By Western blot analysis, myeloperoxidase anti-neutrophil cytoplasmic autoantibodies (MPG-ANCA) reacted with approximately 120-kD band of SDS-treated MPG (15 representative samples).
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3 Fig 3. By Western blot analysis, polyclonal rabbit anti-myeloperoxidase (MPG) reacted with all of the subunits of MPG obtained under thermally denaturing and reducing conditions (lane 1). In contrast, the monoclonal anti-MPG antibody did not react with any of the MPG subunits (lane 2), nor did any of the eleven MPG anti-neutrophil cytoplasmic autoantibodies (ANCA) (one representative sample, lane 3). 2
MPGbutwit MP initsnatve tat denaturedith SDS ( ig 1,lnsC *
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Western blot analysis By Western blot analysis 15 MPG ANCA reacted with an approximately 120-kD band derived from SDS-treated MPG, but not with thermally denatured MPG. The degree of reactivity varied between samples (Fig. 2). The polyclonal rabbit antiMPG antibody also reacted with an approximately 120-kD band, and with all of the bands denived from thermally denatured MPG (Fig. 3). Monoclonal mouse anti-MPG did not react with thermally denatured MPG (Fig. 3, lane 2), nor did any of the MPG-ANCA (Fig. 3, lane 3). None of the control sera reacted with the 120-kD band, nor any of the larger molecular
weight fragments of MPG; however, several control sera did react with undefined material that migrated at approximately 14 kD (Fig. 3).
~12
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Fig. 4. By Western blot analysis, human myeloperoxidase anti-neutrophil cytoplasmic autoantibodies (MPG ANCA) (lanes 3, 4) and mouse monoclonal anti-MPG antibodies (lanes 1, 2) reacted in a very similar fashion with MPG treated with SDS (lanes 1, 3), and MPG treated with SDS and beta-mercaptoethanol (B ME) (lanes 2, 4). None of the MPG was thermally denatured. Normal serum (lane 5) was not reactive with the SDS- and B-ME-treated MPG.
Human MPG-ANCA and mouse monoclonal anti-MPG reacted in a similar fashion on Western blots (Fig. 4). Both reacted with partially denatured MPG, and with MPG that was reduced but not thermally denatured. A doublet was observed that was recognized by both monoclonal anti-MPG and MPGANCA (Fig. 4, lanes 2, 4).
Inhibition of myeloperoxidase function We determined whether MPG ANCA were capable of inhibiting MPG functional activity by measuring MPG-induced protein iodination in a cell-free assay. Purified immunoglobulins were prepared from MPG ANCA, C-ANCA, normal, and disease control sera. The polyclonal anti-MPG antibody inhibited MPG-induced protein iodination to the same extent as that observed for sodium azide (92% ± lO-2 (mean ± 2 s.d.) n = 10) (Fig. 5). Monoclonal anti MPG was also able to inhibit protein iodination 9[5% +7 4 (mean+s.d.) (n =3). In contrast, seven MPG-ANCA IgG, eight C ANCA IgG, seven normal IgG and seven disease control IgG all inhibited the assay to a
277
Anti-myeloperoxidase autoantibodies 100 750
50 0
25
0 Polyclonal MPO n = 10
p-ANCA IgG n=7
c-ANCA IgG n: 6
Control IgG n=8
Fig. 5. Purified IgG preparations of myeloperoxidase anti-neutrophil cytoplasmic autoantibodies (MPO-ANCA), C-ANCA, disease control and normal human sera were tested for the ability to inhibit MPO function. Polyclonal and monoclonal anti-MPO antibodies inhibited MPO iodination ofprotein by 92%. In contrast, only 25-30% inhibition of MPO activity was seen with MPO-ANCA IgG, C-ANCA IgG, disease control IgG, and normal IgG. There were no differences in the inhibition of MPO by MPO-ANCA IgG when compared with other IgGs.
similar degree. The per cent inhibition was not statistically different in any of these groups. While there was some degree of inhibition with these MPO-ANCA IgG, none of the patients was significantly different from that observed for the control of IgG patients.
DISCUSSION ANCA have been found in the circulation of patients with a spectrum of disease ranging from systemic vasculitis (e.g. microscopic polyarteritis nodosa, Wegener's granulomatosis, and Churg-Strauss syndrome) to patients with renal-limited disease (i.e. 'idiopathic' crescentic glomerulonephritis) [4]. These diseases are distinct from immune complex mediated or anti-glomerular basement membrane antibody-mediated diseases by virtue of the absence of immunoglobulin and complement deposition in the injured vessels [3]. There are two well described ANCA subtypes. One reacts with the cytoplasm of alcohol-fixed neutrophils (C-ANCA), and is specific for a 29-kD serine protease, usually designated proteinase 3 [9-12,22]. The second ANCA subtype (P-ANCA) produces perinuclear staining of alcohol-fixed neutrophils. In patients with vasculitis and glomerulonephritis, approximately 90% of P-ANCA are specific for MPO, although a very few patients also have P-ANCA that react with elastase [12,13] or lactoferrin [23]. The P-ANCA pattern observed by indirect immunofluorescence microscopy is an artifact of the permeabilization of neutrophils during substrate preparation that results in the diffusion of primary granule constituents to the nucleus where they are retained by ionic forces [17]. C-ANCA and P-ANCA, including MPO-ANCA, are capable of activating neutrophils in vitro. The activation process results in the production of reactive oxygen species, as well as degranulation [14]. This process is facilitated by cytokines, such as tumour necrosis factor, which induce translocation of ANCA antigens (e.g. MPO) to the cell membrane, thus allowing for interaction with ANCA [14].
In this study, we sought to learn more about the interaction of human MPO-ANCA with MPO. MPO-ANCA reacted with native MPO on dot blots, and with a 120-kD band on Western blots. This 120-kD band is the heterodimer of MPO [24]. MPOANCA did not react with any of the bands that correspond to the subunits of MPO obtained by thermal denaturation [25]. All 15 MPO-ANCA that were tested showed the same patterns of reactivity when analysed by dot blot and Western blot. These results suggest that MPO-ANCA react with a conformational antigen or antigens found on the native molecule. This antigen(s) is resistant to mild denaturation with SDS or reduction with B-ME, but is destroyed by greater denaturation (e.g. thermal denaturation). It is not surprising that MPOANCA appear to react with a conformational antigen, since most determinants recognized by autoantibodies are directed at epitopes on molecules that are in the appropriate conformational state [15]. Because extracellular MPO may serve as a feedback inhibitor of neutrophil activation [26], we sought to determine whether MPO inhibited MPO function. No inhibition by MPOANCA of MPO-induced protein iodination was observed, although the polyclonal anti-MPO antibody, as well as the mouse monoclonal anti-MPO antibody, were able to inhibit MPO function. These studies suggest that the activation by MPO-ANCA of neutrophils is not dependent on the inhibition of a negative feedback loop. In conclusion, MPO-ANCA react with MPO conformationdependent epitope, and MPO-ANCA binding to this epitope does not inhibit MPO function. ACKNOWLEDGMENTS This study was supported in part by grants from the National Institutes of Health (KD40208), the Thomas R. Arthur Trust Fund, and was presented at the American Society of Nephrology 1990.
REFERENCES 1 Falk RJ, Jennette JC. Anti-neutrophil cytoplasmic autoantibodies with specificity for myeloperoxidase in patients with systemic vasculitis and idiopathic necrotizing and crescentic glomerulonephritis. N Eng J Med 1988; 318:1651-7. 2 van der Woude FJ, Rasmussen N, Lobatto S et al. Antibodies against neutrophils and monocytes: tool for diagnosis and marker of disease activity in Wegener's granulomatosis. Lancet 1985; 1:425-9. 3 Jennette JC, Wilkman AS, Falk RJ. Anti-neutrophil cytoplasmic autoantibody-associated glomerulonephritis and vasculitis. Am J Pathol 1989; 135:921-30. 4 Jennette JC, Falk RJ. Antineutrophil cytoplasmic autoantibodies and associated diseases: A review. Am J Kidney Dis 1990; 15:517-29. 5 Falk RJ, Hogan S. Carey TS, Jennette JC, Glomerular Disease Collaborative Network. The clinical course of patients with antineutrophil cytoplasmic autoantibody (ANCA) associated glomerulonephritis and systemic vasculitis. Ann Int Med 1990; 113:656-63. 6 Falk RJ. ANCA associated renal disease. Kidney Int 1990; 38:9981010. 7 Cohen-Tervaert JW, van der Woude FJ, Fauci AS et al. Association between active Wegener's granulomatosis and anticytoplasmic antibodies. Arch Intern Med 1989; 149:2461-5. 8 Nolle B, Specks U, Ludemann J, Rohrbach MS, DeRemee RA, Gross WL. Anticytoplasmic autoantibodies: their immunodiagnostic value in Wegener's granulomatosis. Ann Int Med 1989; 11:28-40. 9 Ludemann J, Utecht B, Gross WL. Anti-neutrophil cytoplasm
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antibodies in Wegener's granulomatosis recognize an elastinolytic enzyme. J Exp Med 1990; 171:357-62. Niles JL, McCluskey RT, Ahmad MF, Arnaout MA. Wegener's granulomatosis autoantigen is a novel neutrophil serine proteinase. Blood 1989; 74:1888-93. Jennette JC, Hoidal JH, Falk RJ. Specificity of anti-neutrophil cytoplasmic autoantibodies for proteinase 3. Blood 1990; 78:2263-4. Goldschmeding R, van der Schoot CE, ten Bokkel Huinink D et al. Wegener's granulomatosis autoantibodies identify a novel diisopropylfluorophosphate-binding protein in the lysosomes of normal human neutrophils. J Clin Invest 1989; 84:1577-87. Cohen-Tervaert JW, Goldschmeding R, Elema JD et al. Autoantibodies against myeloid lysosomal enzymes in crescentic glomerulonephritis. Kidney Int 1990; 37:799-806. Falk RJ, Terrell RS, Charles LA, Jennette JC. Anti-neutrophil cytoplasmic autoantibodies induce neutrophils to degranulate and produce oxygen radicals in vitro. Proc Natl Acad Sci USA 1990; 87:4115-9. Benjamin DC, Berzofsky JA, East IJ et al. The antigenic structure of proteins: a reappraisal. Annu Rev Immunol 1984; 2:67-101. Laver WG, Air GM, Webster RG, Smith-Gill SJ. Epitopes on protein antigens: misconceptions and realities. Cell 1990; 61:553-6. Charles LA, Falk RJ, Jennette JC. Reactivity of anti-neutrophil cytoplasmic autoantibodies with HL-60 cells. Clin Immunol Immunopathol 1989; 53:243-53.
18 Matheson NR, Wong PS, Travis J. Isolation and properties of human neutrophil myeloperoxidase. Biochem 1981; 20:325-30. 19 Finn AL, Tsai LM, Falk RJ. Monoclonal antibodies to the apical chloride channel in necturus gallbladder inhibit the chloride conductance. Proc Natl Acad Sci USA 1989; 86:7649-52. 20 Root RK, Stossel TP. Myeloperoxidase-mediated iodination by granulocytes-intracellular site of operation and some regulating factors. J Clin Invest 1974; 53:1207-15. 21 Klebanoff SJ, Clark RA. Iodination by human polymorphonuclear leukocytes: a re-evaluation. J Lab Clin Med 1977; 89:675-86. 22 Jenne DE, Tschopp J, Ludemann J et al. Wegener's autoantigen decoded (Letter). Nature 1990; 346:520. 23 Lesavre P. Chen N, Nusbaum P et al. Anti-neutrophil cytoplasm antibodies (ANCA) with antilactoferrin activity in vasculitis (Abstract). Kidney Int 1990; 37:442. 24 Andrews PC, Krinsky NI. The reductive cleavage of myeloperoxidase in half, producing enzymatically active hemi-myeloperoxidase. J Biol Chem 1981; 256:4211-8. 25 Nauseef WM, Malech HL. Analysis of the peptide subunits of human neutrophil myeloperoxidase. Blood 1986; 67:1504-7. 26 Jandl RC, Andre-Schwartz J, Borges-Du-Bois L et al. Termination of the respiratory burst in human neutrophils. J Clin Invest 1978; 61:1176-85.
Anti-myeloperoxidase autoantibodies react with native but not denatured myeloperoxidase R. J. FALK, M. BECKER, R. TERRELL & J. C. JENNETTE* Departments of Medicine and Pathology*, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
(Acceptedfor publication 30 April 1992)
SUMMARY We wondered whether anti-myeloperoxidase (MPO) autoantibodies (MPO-ANCA) found in patients with systemic vasculitis react with a conformational epitope or epitopes on the MPO molecule. Sera from 15 human MPO-ANCA, and a polyclonal and a monoclonal anti-MPO antibodies were reacted with MPO in native and denatured states. Human MPO-ANCA and mouse monoclonal anti-MPO reacted with native MPO, and a 120-kD band representing the MPO hologenzyme, but not with denatured MPO fragments; however, MPO-ANCA and mouse anti-MPO did not demonstrate competitive inhibition of binding to MPO. Polyclonal rabbit anti-MPO reacted with both native and denatured MPO. All MPO-ANCA tested showed the same patterns of reactivity with native and denatured MPO in dot blot and Western blot analyses. Both polyclonal and monoclonal anti-MPO antibodies inhibited MPO's protein iodination by over 90%, whereas MPOANCA IgGs, normal IgGs and disease control IgGs did not. These data suggest that (i) MPO-ANCA interact with a conformational epitope on the MPO molecule; and (ii) MPO-ANCA from different patients have similar reactivity with native versus denatured MPO.
Keywords autoimmunity/autoantibodies vasculitis ANCA
INTRODUCTION
Anti-neutrophil cytoplasmic autoantibodies (ANCA) have been found in the circulation of patients with the most common types of systemic necrotizing vasculitis, including microscopic polyarteritis nodosa, Wegener's granulomatosis, and ChurgStrauss syndrome [1-8]. There are two ANCA subtypes defined by indirect immunofluorescence microscopy staining patterns. Cytoplasmic pattern ANCA (CANCA), which are usually specific for proteinase-3 [9-14], and perinuclear pattern ANCA (P-ANCA), which are usually specific for myeloperoxidase (MPG-ANCA) [1,3,13]. Both types of autoantibodies are capable of activating cytokine-primed neutrophils in vitro inducing a respiratory burst and release of granule constituents [14]. Therefore, ANCA may be directly involved in the pathogenesis of necrotizing vascular inflammation, We wondered whether MPO-ANCA from different patients reacted with the same or different portions of the myeloperoxidase molecule, and whether this reactivity was directed at a
PATIENTS AND METHODS Patients and sera Sera containing MPO-ANCA were obtained from patients with pauci-immune necrotizing and crescentic glomerulonephritis with and without extra-renal vascular inflammation. Five of 11 patients had pulmonary capillaritis with alveolar haemorrhage; five had vasculitis involving the upper respiratory tract, nerves, or skin; and five patients had renal-limited disease. P-ANCA were identified using indirect immunofluorescence microscopy and alcohol-fixed normal human neutrophils as described previously [2,17]. Patients with P-ANCA reactivity were tested by ELISA for reactivity with neutrophil cytoplasm extract and reactivity with purified MPO using previously described methof the P-ANCA used in this study had anti-MPO 'ods [1] All sp[1]. All ofte P-ANCAu in this s y a anti-MPG the anti-MPO ELISA of greater than 3 s.d. above the obtained with 13 normal control sera.
mean
Dot blots NircluoesipwredtdwthMG ihwihanMPO antibody reactivity was to be analysed. Myeloperoxidase was elther purchased from Calbiochem (San Diego, CA) or isolated from purified azurophilic granules using a modification of the procedure of Matheson [18]. Reactivity of MPG-ANCA and anti-MPG antibodies was evaluated after the MPG had been exposed to the following conditions: denaturation with 2%
epitope structure epitope primary structure or at at aa primary [15] or epitope [151 conformational epitope
[16].rmational
Correspondence: Ronald J. Falk, MD, Department of Medicine, Division of Nephrology, CD No. 7155, 3034 Old Clinic Bldg, Chapel Hill, NC 27599, USA.
274
Anti-myeloperoxidase autoantibodies SDS, reduction with 2-6% beta-mercaptoethanol (B-ME), combined chemical denaturation and reduction, and thermal denaturation at 1000C for 2 min. One hundred nanograms of MPO was placed in each dot. The strips were washed in 150 mM NaCl, 50 mm Tris pH 7-2 (TBS), blocked with 5% non-fat dry milk for 2 h, and incubated with MPO-ANCA, polyclonal anti-c MPO, or monoclonal anti-MPO. The strips were washed and exposed to the appropriate secondary antibodies and alkaline phosphatase substrate. For staining of immunoblots, human MPO-ANCA + serum was used at a dilution of 1: 40. Polyclonal rabbit anti-human MPG (Dako, Carpinteria, CA) was used at a dilution of 1:2000, and mouse monoclonal anti-human MPO (Dako) was used at 1:500. Appropriate secondary antibodies) included goat anti-rabbit IgG alkaline phosphatase conjugate
and 0 45 mMr 5-bromo-4-chloro-3-indolyl-phosphate (Sigma) in 100 mBuTris, 100 mn sodium chloride, 5 m magnesium chloride buffer, pH 95. Western blots
Myeloperoxidase was run on 10% or 13% polyacrylamide electrophoresis with a 4% stacking gel. Before evaluating reactivity with antibodies, MPG was exposed to the following conditions: denaturing conditions with 2% SDS; reducing conditions with 2-6% B-ME; combined denaturing and reducing conditions; and thermal denaturation by heating to 1000C for 2 mmi. pM nitrocellulose (Schleicher and TransferoofrthesMPGwtoh a 4 Schuell, Keene, NH) was performed using a Mini-transblot cell (Biorad, Rockville Center, NY) with 10% methanol in Trisglycine buffer pH 86 at 250 mA for I h [19]. Transfer of protein was assessed by staining the nitrocellulose strips with amido black. The nitrocellulose strips were washed in distilled water and TBS, and blocked in 5% non-fat dry milk for 2 h The strips were incubated in the different primary anti-MPG antibodies overnight, washed in 5% non-fat dry milk three times, and incubated again in secondary antibody for 2 h. The reaction product was developed with alkaline-phosphatase developing
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acidic buffered (2); denatured and boiled (3); denatured, reduced and boiled (4); denatured and reduced, but not boiled (5); and denatured but not boiled (6). Polyclonal rabbit anti-MPG reacted with MP under all conditions (lane A). Mouse monoclonal anti-MPG recognized MPG in its native state, but not when it was thermally denatured (lane B). Seven MPg oANCA sera tested also reacted to MP in its native state, but not when it was thermally denatured (lanes C-I). Normal human sera did not react with MPG (lane J). affected by a non-specific protein effect so that the amount of IgG added was rigorously standardized. The following reagents were added in sequence: MPG, H2G2, MPG-ANCA IgG or polyclonal or monoclonal anti-MPG antibodies, and Nad25I. The mixture was rotated for 30 mm at room temperature and the reaction stopped with cold 5 6 mu sodium thiosulphate (Fisher, Fair Lawnf NJ), followed by the addition of 20% trichloroacetic acid (TCA) (EM, Cherry Hill, NJ) to precipitate the protein. The pellet was repetitively washed with 10% TCA, centrifuged and then protein bound radioactivity was counted. The addition of Na azide served as the positive control for each assay while the negative controls included assays performed without H2e2 and without MPG. The monoclonal anti-MPG, polyclonal antiMPG, MPG ANCA IgG, C-ANCA IgG and normal IgG were used at a final concentration 2t04 of ,ug/ml, g/ml, 1 mg/ 0o76 2 0t ml or 0 025 mg/ml. Results were expressed as a percentage of inhibition compared to the inhibition observed with Na azide.
Functional activity oflmyeloperoxidase In order to assess whether MPo-ANCA could block MPG function, protein halogenation by MPG in the presence of MPG-ANCA was studied. Modification of a standard MPo RESULTS protein halogenation assay [20,21] was designed as a cell-free Dot blot analysis system. In this assay, 100 mU of MPG (Calbiochem), 0 1 mo MPG-ANCA sera were tested on dot blots for reactivity of the Na'251 (New England Nuclear, Irvine, CA), and 0-1 mM human autoantibodies with MPG in its native form, treated with acid denatured with SDS and/or reduced with B-ME, and hydrogen peroxide (H2e2) (Sigma) were mixed in Hanks buffered saline solution with calcium and magnesium thermally denatured. For purposes of comparison, polyclonal rabbit anti MPG aswell as amonoclonal mouse anti-MPG were (HBSS++~) (137 mMs NaCJ, 137 mMs KCl, 352 mMs Na2HPG4, 441 mM KH2PG4, with 12d 9 moi CaCl2.H2G) andma 4 tested. The polyclonal rabbit anti-MPG reacted with MPG So4.7H2. Todefine themaximum degree ofinhibition,assays under all the conditions (Fig. 1, lane A). In contrast, the were run with and without 10 0 mo sodium azide (Sigma). The monoclonal anti-MPG did not react with thermally denatured reaction was carried out in borosilicate glass tubes to which MPn but reacted with MPG in its native state, as well as when it HBSSy + and heat-inactivated sera were added and incubated at was partially denatured with SDS or reduced with B-ME (Fig. 1, 37C. Because of the peroxidase activity of sera, purified IgG was prepared as previously described [19]. The assay was also
lane B)u Seven MPo-ANCA reacted in a fashion exactly the same as the MoAb, i.e. by not reacting with thermally denatured
R. S. J. Falketal.
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Fig. 2. By Western blot analysis, myeloperoxidase anti-neutrophil cytoplasmic autoantibodies (MPG-ANCA) reacted with approximately 120-kD band of SDS-treated MPG (15 representative samples).
-0 2003
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3 Fig 3. By Western blot analysis, polyclonal rabbit anti-myeloperoxidase (MPG) reacted with all of the subunits of MPG obtained under thermally denaturing and reducing conditions (lane 1). In contrast, the monoclonal anti-MPG antibody did not react with any of the MPG subunits (lane 2), nor did any of the eleven MPG anti-neutrophil cytoplasmic autoantibodies (ANCA) (one representative sample, lane 3). 2
MPGbutwit MP initsnatve tat denaturedith SDS ( ig 1,lnsC *
an whn prtill
Western blot analysis By Western blot analysis 15 MPG ANCA reacted with an approximately 120-kD band derived from SDS-treated MPG, but not with thermally denatured MPG. The degree of reactivity varied between samples (Fig. 2). The polyclonal rabbit antiMPG antibody also reacted with an approximately 120-kD band, and with all of the bands denived from thermally denatured MPG (Fig. 3). Monoclonal mouse anti-MPG did not react with thermally denatured MPG (Fig. 3, lane 2), nor did any of the MPG-ANCA (Fig. 3, lane 3). None of the control sera reacted with the 120-kD band, nor any of the larger molecular
weight fragments of MPG; however, several control sera did react with undefined material that migrated at approximately 14 kD (Fig. 3).
~12
4
Fig. 4. By Western blot analysis, human myeloperoxidase anti-neutrophil cytoplasmic autoantibodies (MPG ANCA) (lanes 3, 4) and mouse monoclonal anti-MPG antibodies (lanes 1, 2) reacted in a very similar fashion with MPG treated with SDS (lanes 1, 3), and MPG treated with SDS and beta-mercaptoethanol (B ME) (lanes 2, 4). None of the MPG was thermally denatured. Normal serum (lane 5) was not reactive with the SDS- and B-ME-treated MPG.
Human MPG-ANCA and mouse monoclonal anti-MPG reacted in a similar fashion on Western blots (Fig. 4). Both reacted with partially denatured MPG, and with MPG that was reduced but not thermally denatured. A doublet was observed that was recognized by both monoclonal anti-MPG and MPGANCA (Fig. 4, lanes 2, 4).
Inhibition of myeloperoxidase function We determined whether MPG ANCA were capable of inhibiting MPG functional activity by measuring MPG-induced protein iodination in a cell-free assay. Purified immunoglobulins were prepared from MPG ANCA, C-ANCA, normal, and disease control sera. The polyclonal anti-MPG antibody inhibited MPG-induced protein iodination to the same extent as that observed for sodium azide (92% ± lO-2 (mean ± 2 s.d.) n = 10) (Fig. 5). Monoclonal anti MPG was also able to inhibit protein iodination 9[5% +7 4 (mean+s.d.) (n =3). In contrast, seven MPG-ANCA IgG, eight C ANCA IgG, seven normal IgG and seven disease control IgG all inhibited the assay to a
277
Anti-myeloperoxidase autoantibodies 100 750
50 0
25
0 Polyclonal MPO n = 10
p-ANCA IgG n=7
c-ANCA IgG n: 6
Control IgG n=8
Fig. 5. Purified IgG preparations of myeloperoxidase anti-neutrophil cytoplasmic autoantibodies (MPO-ANCA), C-ANCA, disease control and normal human sera were tested for the ability to inhibit MPO function. Polyclonal and monoclonal anti-MPO antibodies inhibited MPO iodination ofprotein by 92%. In contrast, only 25-30% inhibition of MPO activity was seen with MPO-ANCA IgG, C-ANCA IgG, disease control IgG, and normal IgG. There were no differences in the inhibition of MPO by MPO-ANCA IgG when compared with other IgGs.
similar degree. The per cent inhibition was not statistically different in any of these groups. While there was some degree of inhibition with these MPO-ANCA IgG, none of the patients was significantly different from that observed for the control of IgG patients.
DISCUSSION ANCA have been found in the circulation of patients with a spectrum of disease ranging from systemic vasculitis (e.g. microscopic polyarteritis nodosa, Wegener's granulomatosis, and Churg-Strauss syndrome) to patients with renal-limited disease (i.e. 'idiopathic' crescentic glomerulonephritis) [4]. These diseases are distinct from immune complex mediated or anti-glomerular basement membrane antibody-mediated diseases by virtue of the absence of immunoglobulin and complement deposition in the injured vessels [3]. There are two well described ANCA subtypes. One reacts with the cytoplasm of alcohol-fixed neutrophils (C-ANCA), and is specific for a 29-kD serine protease, usually designated proteinase 3 [9-12,22]. The second ANCA subtype (P-ANCA) produces perinuclear staining of alcohol-fixed neutrophils. In patients with vasculitis and glomerulonephritis, approximately 90% of P-ANCA are specific for MPO, although a very few patients also have P-ANCA that react with elastase [12,13] or lactoferrin [23]. The P-ANCA pattern observed by indirect immunofluorescence microscopy is an artifact of the permeabilization of neutrophils during substrate preparation that results in the diffusion of primary granule constituents to the nucleus where they are retained by ionic forces [17]. C-ANCA and P-ANCA, including MPO-ANCA, are capable of activating neutrophils in vitro. The activation process results in the production of reactive oxygen species, as well as degranulation [14]. This process is facilitated by cytokines, such as tumour necrosis factor, which induce translocation of ANCA antigens (e.g. MPO) to the cell membrane, thus allowing for interaction with ANCA [14].
In this study, we sought to learn more about the interaction of human MPO-ANCA with MPO. MPO-ANCA reacted with native MPO on dot blots, and with a 120-kD band on Western blots. This 120-kD band is the heterodimer of MPO [24]. MPOANCA did not react with any of the bands that correspond to the subunits of MPO obtained by thermal denaturation [25]. All 15 MPO-ANCA that were tested showed the same patterns of reactivity when analysed by dot blot and Western blot. These results suggest that MPO-ANCA react with a conformational antigen or antigens found on the native molecule. This antigen(s) is resistant to mild denaturation with SDS or reduction with B-ME, but is destroyed by greater denaturation (e.g. thermal denaturation). It is not surprising that MPOANCA appear to react with a conformational antigen, since most determinants recognized by autoantibodies are directed at epitopes on molecules that are in the appropriate conformational state [15]. Because extracellular MPO may serve as a feedback inhibitor of neutrophil activation [26], we sought to determine whether MPO inhibited MPO function. No inhibition by MPOANCA of MPO-induced protein iodination was observed, although the polyclonal anti-MPO antibody, as well as the mouse monoclonal anti-MPO antibody, were able to inhibit MPO function. These studies suggest that the activation by MPO-ANCA of neutrophils is not dependent on the inhibition of a negative feedback loop. In conclusion, MPO-ANCA react with MPO conformationdependent epitope, and MPO-ANCA binding to this epitope does not inhibit MPO function. ACKNOWLEDGMENTS This study was supported in part by grants from the National Institutes of Health (KD40208), the Thomas R. Arthur Trust Fund, and was presented at the American Society of Nephrology 1990.
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