Immunology 1992 76 617-620
Production of human monoclonal antibodies to myeloperoxidase M. R. EHRENSTEIN, B. LEAKER, D. ISENBERG & G. CAMBRIDGE Departments of Rheumatology Research, Immunology and Medicine, University College and Middlesex Hospital Medical School (UCMSM), London
Accepted for publication 21 July 1992
SUMMARY Two mouse-human heterohybridomas secreting human antibodies to myeloperoxidase (MPO) were derived from the peripheral blood of a patient who developed microscopic polyarteritis as the result of long-term treatment with hydralazine. Forty-five immunoglobulin-secreting lines were obtained from the fusion of patient lymphocytes with the CB-F7 heteromyeloma cell line. Of these, two antibodies, one IgG and one IgM, bound to myeloperoxidase in solid phase ELISA and gave a perinuclear staining pattern on ethanol-fixed human neutrophil cytospin preparations. The staining patterns were similar to those seen with serum from the patient. Antigen-inhibition studies revealed that the affinity of the IgG monoclonal antibody was 28 times higher (k = 1-4 x I0-) than the IgM antibody (k = 5 x 10-5). Cross-inhibition studies further suggested that the two monocloal antibodies recognized the same epitope on MPO. Of the other secreting cell lines, none produced antibody which reacted with the panel of autoantigens used for testing. Neither mononuclear antibody reacted with this panel indicating that they were not simply polyreactive natural autoantibodies. These are the first human monoclonal antibodies to native myeloperoxidase to be reported. The association between the presence of serum autoantibodies to neutrophil cytoplasmic components (ANCA) and vasculitic diseases is now clearly established. Using indirect immunofluorescence, two main staining patterns have been described on ethanol-fixed human neutrophils; a granular, cytoplasmic staining distribution known as 'classical' (c-ANCA) and a perinuclear distribution (p-ANCA).' Most antigens recognized by ANCA have been identified as neutrophil primary granule enzymes. Serine protease 3 (SP3) is the major antigen recognized by c-ANCA-containing sera.2 The p-ANCA staining pattern is in the majority of cases attributable to the presence of antimyeloperoxidase (MPO) antibodies in sera, although autoantibodies to other neutrophil granule enzymes such as elastase, lactoferrin and cathepsin G have also been identified. In patients with renal impairment, p-ANCA are strongly associated with
pauci-immune crescentic nephritis and in multi-system disease with microscopic polyarteritis and polyarteritis nodosa.3 These autoantibodies have proved useful both as a diagnostic aid and in the clinical monitoring of disease activity.4 Our objective was to produce and analyse human monoclonal anti-MPO antibodies. Prolonged treatment with hydralazine, an anti-hypertension drug, has been shown to cause nephritis associated with the appearance of circulating antibodies to MPO and a lupus-like syndrome in which circulating anti-nuclear and MPO antibodies are present.5'6 Nassberger and co-workers6 found that Correspondence: Dr G. Cambridge, Dept. of Immunology, University College and Middlesex Hospital Medical School (UCMSM), London WlP 9PG, U.K.
patients with hydralazine-induced renal vasculitis were ANCA negative but had anti-MPO antibodies. In this study, the generation is described of two human monoclonal antibodies which react with MPO and give an immunofluorescence pattern similar to that given by ANCA-positive sera, from a patient with hydralazine-induced vasculitis. For the generation of human monoclonal antibodies peripheral blood lymphocytes were used from a patient who presented with pulmonary haemorrhage, rash and rapidly progressive renal failure. A renal biopsy showed crescentic nephritis with no immunoglobulin or complement deposition detectable by the immunoperoxidase technique. In addition, the patient's serum gave a positive immunofluorescence test for ANCA (p-ANCA) pattern and both IgG and IgM autoantibodies to MPO by ELISA. The mononuclear cells from the peripheral blood were separated by Lymphoprep (Nycomed, Norway) gradient centrifugation. After washing twice with RPMI-1640 they were directly fused with the mouse-human heteromyeloma cell line CB-F7 (a kind gift of S. Jahn [Institute of Medical Immunology, Humbolt, Germany]-an HAT-sensitive ouabain-resistant non-secreting cell line) in the presence of PEG 1500 (Boerhringer Mannheim, Mannheim, Germany), according to the method of Grunow et al.7 Cells were seeded in flat-bottomed microtitre plates (Nunc, Roskilde, Denmark) at a concentration of 105 cells/well in a volume of 100 p1 of RPMI-1640 supplemented with 20% foetal calf serum (FCS) and cultured at 370 in 5% CG2 in air. After 24 hr, 100 pl/well of double-concentrated HAT medium (0-2 mm hypoxyxanthine, 0-8 mM aminopterin and 32 mm thymidine) was added to each well. After 18 days incubation, supernatants were tested for immunoglobulin pro-
M. R. Ehrenstein et al.
duction by a capture ELISA.8 Forty-five immunoglobulin secreting cell lines were produced as the result of the fusion of lymphocytes from the patient with the CB-F7 cell line. The fusion frequency was calculated to be two growing hybridoma cells/105 lymphocytes. Twenty-seven of these hybridomas producedIgM and 18 produced IgG antibodies. These supernatants containing immunoglobulin
screened for reactivity
with MPO and other autoantigens by direct binding ELISA (see below). Five primary cultures secreted immunoglobulin reacting with MPO in concentrations ranging from 2 to 5 cells/48 hr. Two of these, one IgGK and one IgMA, survived
two rounds of cloning by limiting dilution and
continuous culture for 3 months. Cells were plated at 0 3, 1, 5 cells/well in 96-well plates. Plates in which cells grew in more than one in every three wells were discarded. Antibodies
MPO, prepared from human neutrophils
according to the method of Mathieson et al.,9 SP3, isolated after the method of Goldschmeding et al.,2 and lactoferrin (from human milk; Sigma, Poole, U.K.), were detected using direct binding ELISA. Briefly, antigens were coated onto Maxisorb ELISA plates (Nunc) at a concentration of1 jg/ml in 0-1 M bicarbonate buffer (pH 9 6) at 37°. After blocking the plates with 2% casein for1 hr at 370, undiluted culture supernatants (sera diluted 1:1000) were incubated for1 hr at 37°. Antibodies were detected using an alkaline-phosphatase-conjugated goat anti-human IgM or IgG (Sigma) which produced a coloured product with p-nitrophenyl phosphate. The wells were read using a Dynatech MR 4000 ELISA reader set at 405 nm. Plates were read when the optical density (OD) of the positive control wells exceeded 1-0. Culture supernatants were regarded as positive when theOD of test wells exceeded those of uncoated wells by at least1-0. None of the monoclonal antibodies tested bound to the uncoated side of the plate above a phosphatebuffered saline (PBS) control. The reactivities of the supernatants were also assessed against a panel of other autoantigens including single-stranded (ss) and double-stranded (ds) DNA, cardiolipin and histones using methods as previously described.'°0" Rheumatoid factor activity was determined by ELISA as previously described.8 To assess binding of the two monoclonal anti-MPO antibodies in the fluid phase, they were preincubated with varying amounts of myeloperoxidase (0-100 gg/ml). After incubation for 1 hr at 370, the mixtures were transferred to ELISA plates coated with I pg/ml MPO and binding to MPO was detected as described above. Data were expressed as percentage binding to the solid phase MPO compared to the binding in the absence of inhibitor (%OD). Affinities were calculated using a method adapted to calculate the dissociation constant in an ELISA system'2 assuming the molecular weight of MPO is 140,000. ANCA were measured by standard techniques using ethanol-fixed human neutrophils as substrate.'3 Neat supernatants of sera diluted to 1/50 in PBS were incubated with cytospin preparations and antibody binding detected using fluoresceinconjugated sheep anti-human IgM and IgG (Wellcome Diagnostics, Beckenham, U.K.). Anti-nuclear antibodies were detected using an immunofluorescence method using commercially available Hep-2 cells as substrate (Biodiagnostics, U.K.). The two monoclonal anti-MPO antibodies bound MPO in solid phase ELISA and were inhibited by MPO in the fluid phase (Fig. 1). The Kd value for the IgM antibody was 5-2 x 10-I while the value for the IgG antibody was 1-4 x 10-7. Both antibodies
0 1 00
1. Inhibition of the
antibodies to solid phase binding to solid phase
binding of the two monoclonal anti-MPO % in the fluid phase. % by compared to that without inhibiting
Figure 2. Staining pattern given by the monoclonal IgG (a) and IgM (b) antibodies on ethanol-fixed neutrophils using immunofluorescence. gave a perinuclear staining pattern on ethanol-fixed neutrophils which was similar to the staining pattern seen with the serum of the patient from which the monoclonals were derived (Fig. 2).
Inhibition experiments were performed to determine whether the two antibodies bound to the same or different epitope(s) (Fig. 3). The antibodies were added to MPO-coated ELISA plates either alone or in 50:50 combination at a total concentration of 1 Mg/ml, which had previously been determined as the saturating concentration of both antibodies in this
Human monoclonal MPO antibodies 100.8
0-8- ~ ~ ~ ~ ~ ~ ~ ~ ~. . . ._...... G
.. ..----:- .....
Figure 3. Competition ELISA for MPG using the two anti-MPG antibodies. The effect on the binding by one antibody (G) by the other (M) was assessed by using anti-IgM and anti-IgG antibody conjugates. The two antibodies were added to MPG-coated ELISA plates either alone or in 50: 50 combination at a total concentration of 1 gg/ml, which had previously been determined as the saturating concentration of both antibodies in this system.
system. The ELISA was repeated with both anti-IgG and antiIgM conjugates. Both antibodies were able to inhibit each other's binding approximately in proportion to their affinity, implying that they are binding to the same epitope or two epitopes in close proximity.'4 Similar studies examining 18 mouse monoclonals against MPG have identified three distinct epitopes.'5 In addition, the IgG antibody at a concentration of 5 ig/ml inhibited the binding of 1gM anti-MPG antibodies in the patient's serum (1 in 1000) to MPG by 36% (data not shown). These antibodies were not polyreactive as they did not bind to Hep-2 cells or a range of other autoantigens (cardiolipin, histones, or DNA) or to other neutrophil granule constituents including lactoferrin and SP3. None of the other immunoglobulin-secreting hybridomas derived as a result of this fusion produced antibodies reacting with any of the autoantigens tested. Antibodies to myeloperoxidase belong to a family of human autoantibodies reacting with neutrophil granule enzymes which are present in sera from patients with vasculitis.' It was possible to generate an 1gM and IgG antibody from the same patient with active vasculitis disease. As far as we are aware this is the first description of human monoclonal anti-MPG antibodies. In addition, anti-MPG antibodies occur in a small minority of patients with idiopathic lupus and in most patients with hydralazine-induced lupus.5 Whereas in the lupus-like disease other autoantibodies occur, such as those directed against DNA and histones, in hydralizine-induced vasculitis there is a different range of autoantibody specificities.6 This was reflected both by the absence of antibodies against other autoantigens produced from the hybridomas and in the lack of cross-reactivity of the two anti-MPG antibodies. Cross-reactivity of human monoclonal antibodies, usually, although not exclusively associated with those of the 1gM isotype, is often used to criticize the relevance of these monoclonal antibodies to circulating autoantibodies.'6"'7 Although hydralazine is able to induce a lupuslike disease, as well as a vasculitic syndrome as in the patient in this study, the patient had no features of lupus clinically or serologically and the hybridoma-derived autoantibodies showed no evidence of polyreactivity. This suggests that there is a difference in the pathogenesis of these two conditions produced by hydralazine at least in terms of the antibodies produced, and that the two mooclonal anti-MPG antibodies
described reflect those found in the serum of the patient. In addition the patient did not have hypergammaglobulinaemia indicating that polyclonal activation was unlikely to be the cause of the production of anti-MPO antibodies. The mechanism leading to the production of anti-MPO antibodies may be different from that leading to autoantibodies such as anti-DNA antibodies characteristic of systemic lupus erythematosus (SLE). Studying the structure and specificity of the monoclonals may provide clues as to the nature of the stimulus producing these antibodies in a primarily T-cell-mediated disease. Vasculitic diseases associated with circulating antibodies to MPO share a number of common features at sites of vascular inflammation, in particular the absence of immune complexes and antibody binding to tissues. It has however been suggested that they may play a role in the pathogenesis of vasculitis. Immunoglobulin purified from ANCA containing human sera can activate neutrophils and monocytes to produce reactive oxygen species and cause degranulation.'8 Due to the probable diversity of epitopes recognized by anti-MPO antibodies in human sera, detailed analysis of possible pathogenic antibodies has not been possible. Monoclonal antibody production using hybridoma technology is essential to dissect further the role of these antibodies in the disease process. We are at present sequencing these antibodies and identifying the idiotypes which they carry. REFERENCES 1. FALK R.J. & JENNETFE J.C. (1988) Anti-neutrophil cytoplasmic autoantibodies with specificity for myeloperoxidase in patients with systemic vasculitis and idiopathic necrotising and crescentic glomerulonephritis. N. Engl. J. Med. 318,1651. 2. GOLDSCHMEDING R, VAN DER SCHOOT C.E., TEN BOKKEL HUINICK
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