Autoimmunity in rapidly progressive glomerulonephritis

Kidney International, Vol. 35 (1989), pp. 929—937

Autoimmunity in rapidly progressive glomerulonephritis CHARLES D. PUSEY and C. MARTIN LOCKWOOD Department of Medicine, Royal Postgraduate Medical School, Hammersmith Hospital, Du Cane Road, London, and Department of Medicine, University of Cambridge Clinical School, Addenbrooke's Hospital, Hills Road, Cambridge, England, United Kingdom

antibody eluted from diseased kidneys could be used to induce nephritis in monkeys [10]. The glomerulonephritis of anti-GBM disease pursues a charfailure within weeks or months. Although RPGN may be acteristically rapid course, and light microscopy generally reassociated with a variety of systemic diseases, or develop on a veals a high proportion of crescents [11]. Although the mechabackground of primary glomerulonephritis, two distinct types nism of crescent formation is not fully understood, recent predominate in most nephrological practices. In one, which is ultrastructural studies suggest that necrosis of the GBM is the less common, there are autoantibodies to glomerular basement common underlying abnormality [12]. Pulmonary hemorrhage membrane (GBM) which may also react with lung basement occurs in 50 to 90% of patients in different series [4], and is membrane producing Goodpasture's syndrome, and in the much more common in those who smoke cigarettes [13]. The other there are autoantibodies to neutrophil cytoplasmic anti- prognosis of untreated patients was poor, and the improved gens and clinicopathological features of small vessel vasculitis. outlook following therapy with plasma exchange and immunoThe latter type usually occurs in the context of Wegener's suppressive drugs provides further evidence for the pathogegranulomatosis (WG) or microscopic polyarteritis (MP), al- nicity of anti-GBM antibodies [14]. Only exceptionally do though so-called idiopathic RPGN probably forms part of the patients who are dialysis-dependent at presentation recover same spectrum of disease. We shall consider separately what is worthwhile renal function despite adequate control of antiknown of autoimmunity to glomerular antigens in these two GBM antibody production. disorders, although it has recently been recognized that both Diagnosis and monitoring immune mechanisms may sometimes co-exist. The demonstration of linear deposits of IgG on the GBM by direct immunofluorescence (IF) of renal biopsy material has Autoimmunity to glomerular basement membrane in been the standard way of confirming the diagnosis since the late Goodpasture's syndrome 1950's, but there are now several reliable assays for detection of Goodpasture's syndrome is the term used to describe the circulating anti-GBM antibodies. These include radioimmucombination of severe glomerulonephritis and lung hemorrhage noassays (RIA) [15—17] and enzyme-linked immunosorbent [1, 2]. Various immunopathological processes, including sys- assays (ELISA) [18, 19], which are more sensitive than indirect temic vasculitis, are responsible for the clinical picture but now immunofluorescence techniques, and are almost completely the eponym is usually reserved for cases with detectable specific for anti-GBM disease [15]. These assays depend upon antibodies to glomerular basement membrane [3]. It is also the reactivity of autoantibodies with soluble glycoprotein reapparent that an identical form of antibody-mediated glomeru- leased from GBM by collagenase. lonephritis can occur with or without lung hemorrhage, and The assay has been of great diagnostic value, and has also thus the term "anti-GBM disease" is appropriate in either proved useful in the monitoring of treatment. Reduction in the circumstance [41. concentration of circulating antibody parallels clinical improveThe nephrotoxicity of heterologous antibodies raised to kid- ment, and once antibody has become undetectable immunosupney homogenates had been appreciated in experimental animals pressive treatment can generally be discontinued [20, 21]. Our since 1900 [5], but it was not until the 1950s that the GBM was RIA detects only binding of IgO to the GBM, whereas direct demonstrated to be the source of the nephritogenic antigen [6]. fluorescence may detect other antibody isotypes. Although The development of immunofluorescence microscopy allowed uncommon, binding of both IgA and 1gM anti-GBM antibodies the demonstration of linear deposition of immunoglobulin along has been recorded [14], and recent studies [22, 23] indicate that the GBM in nephrotoxic nephritis [7], and similar findings were the autoimmune response is often restricted to IgGi and IgG4 soon reported in renal tissue of patients with Goodpasture's subclasses. In one report [22], recurrence of an IgGi antibody syndrome [8, 9]. The pathogenicity of these autoantibodies was was associated with clinical disease, whereas that of an IgG4 subsequently demonstrated by transfer experiments in which antibody was not. Rapidly progressive glomerulonephritis (RPGN) is characterized histologically by focal necrotizing glomerular lesions with crescent formation, and clinically by the development of renal

© 1989 by the International Society of Nephrology

Production of monoclonal antibody (P1) to GBM The reactivity of human autoantibodies with collagenasedigested GBM in the RIA demonstrated that the autoantigen

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Pusey and Lockwood: Autoimmunity in RPGN

was present in this preparation and so attempts have been made

using this material to obtain a monoclonal antibody reactive

Table 1. Reactivity of monoclonal P1, Goodpasture eluate and Steblay eluate with human extraglomerular basement membranes

with the Goodpasture antigen (GA). Mice were immunized with Positive Kidney the collagenase digest of human GBM and spleen cells from a

responsive animal were fused with mouse myeloma NS1. Hybridomas were screened by ELISA for anti-GBM activity and the positives cloned by limiting dilution; reactivity of

Lung Choroid plexus

antibody from positive clones was confirmed by indirect IF on

Eye

normal human kidney. One such monoclonal (P1) reacted strongly with GBM in solid phase assays, and showed a similar

pattern of binding to GBM and tubular basement membrane (TBM) as did autoantibody eluted from patients' kidneys [24]. This reagent has been used subsequently in studies of the autoantigen in Goodpasture's syndrome [25, 26]. Distribution of the Goodpasture antigen

Direct IF of tissue from patients with anti-GBM disease

Breast Liver

distal tubule, loop of Henle and collecting duct alveolar basement membrane basement membrane of choroidal epithelium choroid, ciliary body, Bruch's membrane and lens capsule ducts hepatic arteries within portal

tract

Thyroid Pituitary Adrenal Negative Placenta Skin Spleen Small intestine Stomach Submandibular gland

follicular basement membrane

Cervix Endometnum Umbilical cord Prostate Renal and carotid arteries Pancreas

almost invariably demonstrates IgG deposition along the GBM, and less frequently there are deposits along certain (but not all) TBM [3, 4, 27]. Adequate demonstration of IgG on the alveolar basement membrane is technically more difficult, but both focal Note: Data are from reference [26]. Monoclonal P1 also bound to and diffuse linear deposits have been reported in affected lung membranes in the cochlea [25].

[3, 28, 29]. Rarely, IgG has been reported on choroid plexus basement membrane [26, 27, 301 and on basement membranes of the retinalchoroid of the eye [31]; binding in all these sites has been reported in a single patient [26]. There have been very few studies of the ultrastructural localization of antibodies to GA in

blay eluate, and P1 on normal human tissues. Further co-

localization studies have been performed to compare the distribution of GA with that of type IV collagen, using appropriate the GBM, because adequate fixation for electron microscopy combinations of Goodpasture and Steblay eluates and P1 totends to destroy antigenicity. One report, which has yet to be gether with a mouse monoclonal (PHM12), and a polyclonal confirmed, suggests that GA is present mainly in the lamina rara guinea-pig anti-serum to human type IV collagen. The various antibodies to the GA bound only where type IV collagen was interna [32]. The distribution of immunoreactive Goodpasture antigen in found, but the distribution of type IV was much more extennormal human tissue has been studied using monoclonal P1, sive. In the kidney, anti-type IV collagen antibodies bound to autoantibody eluted from patients' kidneys (GE) and antibody GBM, Bowman's capsule, all TBM, mesangial matrix and eluted from the kidneys of sheep with Steblay nephritis (SE) vascular basement membranes (Fig. 1). [25, 261. The pattern of binding was the same with all three These observations permit a number of conclusions to be reagents. There was strong linear binding to GBM and weaker, drawn about the autoimmune response in Goodpasture's synbut consistent, binding to Bowman's capsule. Only certain drome. Since the pattern of binding of several autoantibodies tubular basement membranes showed linear staining by IF, and was identical to that of P1 and SE, it seems reasonable that all these were shown to have the characteristics of distal tubules reacted with the same antigen. Furthermore, if the monoclonal when studied by immunoperoxidase techniques. There was antibody P1 recognizes one antigenic determinant, as should be linear binding to both alveolar and choroid plexus basement the case, then the same autoantigen must be present in the membranes, as expected, and also to basement membranes different organs affected in Goodpasture's syndrome, notably within the eye. Of additional interest, there was binding to sites kidney and lung. Factors other than the reactivity of the in the cochlea, thyroid, adrenal, pituitary, breast and liver, but autoantibody may therefore determine the clinical features of not in other organs studied (Table 1). the disease. One explanation, with some experimental support Previously [33], differences have been reported in the binding [36, 37], is that factors which increase alveolar capillary perpattern of anti-GBM antibodies eluted from the kidneys of meability may allow penetration of antibody to the basement different patients. In particular, eluates from cases of Good- membrane. The effects of cigarette smoking [13] and intercurpasture's syndrome bound more frequently to extraglomerular rent infection [381, which may provoke lung hemorrhage in sites than did those from patients with nephritis only. However, patients, could act, at least in part, by this mechanism. all four patients' eluates in our study bound in the same pattern The presence of immunoreactive GA in the lens capsule, as Steblay eluate and the monoclonal P1. We were also unable Bruch's membrane of the retina, and the cochlea provides to confirm, using all three types of antibody, a report [34] that further indirect evidence that a single autoantigen is involved in GA was detectable in skin and placenta after denaturation with Goodpasture's syndrome. This deduction is based on the fact 6 M urea at pH 3.5. In this connection, however, immunoblot- that patients with Alport's syndrome, a form of hereditary ting studies have demonstrated the presence of GA in collage- nephritis [39], may demonstrate: (i) lenticonus, macular flecks nase digests of placental basement membrane [351. and nerve deafness; (ii) impaired reactivity of GBM with Co-localization experiments, using double immunofluores- anti-GBM antibodies [40, 411. The second point has been cence, confirmed identical binding of Goodpasture eluate, Ste- confirmed by studies [42, 43] using P1 as well as GE to show

931

Pusey and Lockwood: Autoimmunity in RPGN

.

p

-.i.

'p.. •, ,

Fig. 1. Double immunofluorescence of kidney showing more widespread distribution of type IV collagen than of Goodpasture antigen, as

demonstrated by binding of(A) monoclonal P1 and (B) guinea-pig anti-type IV collagen. Photos are courtesy of Mr. S.J. Cashman.

that patients with Alport's syndrome have reduced or absent polyacrylamide gel, a number of components can be detected binding of anti-GBM antibody in the kidney; other organs have by standard protein stains. Separated GBM can be transferred not yet been examined. The pattern of organ involvement in to nitrocellulose sheets and overlaid with anti-GBM antibodies Alport's syndrome may therefore be due to an inherited abnor- (Western blotting) to identify the components containing GA. niality of a single molecule related to the Goodpasture antigen. Several studies of this type have been reported [25, 35, 5 1—53] Finally, there is increasing evidence that GA is contained using human autoantibodies. The results show that GA can be within the non-collagenous domain (NC1) of type IV collagen. detected in a small number of bands (not more than 6) between If this is the case, then the double immunofluorescence studies approximately 26 and 58 kD in molecular weight. These obsersuggest that GA is not an integral part of the type IV collagen vations cannot exclude the possibility that several autoantigens molecule, or (more likely) that type IV collagen is heterogenous are involved in Goodpasture's syndrome, and so this problem in its primary sequence or three-dimensional structure [26]. was addressed using the monoclonal P1 [25]. In these experiments, collagenase-solubilized GBM, as used Immunochemistry of the Goodpasture antigen in the RIA, was run in a 5 to 20% gradient SDS polyacrylamide Despite considerable advances in the understanding of the gel and transferred to nitrocellulose sheets. These were overlaid biochemistry of the GBM [44, 45], the precise molecular with monoclonal P1 or with sera from patients with anti-GBM structure of the GA remains unknown [461. However, there is disease, and the binding detected with appropriate radiolabelled good evidence that it is contained within one of the non- secondary reagents followed by autoradiography. Blocking collagenous domains of the type IV collagen molecule [47, 48]. studies were also performed, in which an excess of human Type IV collagen forms a network within GBM which is built up anti-GBM antibody was pre-incubated on the nitrocellulose from type IV monomers. Each of these consists of a rod-like sheets before addition of P1, or alternatively P1 was added triple helix with a globular domain (NC 1) at the C-terminal end. before human autoantibody. Sera from all 42 patients studied The triple helix is composed of parallel pro-collagen chains, bound to the same components of GBM, between 26 and 58 kD, probably two al(IV) and one a2(IV) molecules [451. Adjacent as did monoclonal P1. Furthermore, pre-incubation with sera triple helical molecules bind to each other at the globular from eight of eight patients blocked the subsequent binding of domain, so that a hexameric NC1 complex is produced after P1; densitometer scanning of the autoradiographs quantified collagenase digestion. The primary sequence of both al(IV) and inhibition of binding at 83 to 89%. Similarly, pre-incubation a2(IV) chains, including the NC! domain, is now known [49, with P1 blocked the subsequent binding of IgG from six of six patients' sera by 58 to 89%. 50]. The recognition by monoclonal P1 of the same six bands This information must be related to immunochemical studies of the GA. When collagenase-digested GBM is run on an SDS which were recognized by autoantibodies from different pa-

932

Pusey and Lockwood: Autoimmunity in RPGN

tients (with and without lung hemorrhage) confirms that a single antigen is the target of the autoimmune response, and that it is present on fragments of different molecular weight in denatured GBM. The blocking experiments demonstrate that the same (or

by collagenase and purified by ion exchange, gel filtration and reversed phase HPLC. A similar range of monomers and dimers was identified as from bovine material, and again only M2 was found to be reactive with human anti-GBM antibodies. It is, a very closely related) epitope is recognized by both P1 and however, difficult to reconcile this observation with the results several human autoantibodies; the autoimmune response is from other groups, which show that at least two components in therefore highly restricted in its specificity. In addition, this the monomeric size range are immunoreactive. Differences in observation validates the use of P1 in attempts to isolate the the preparation of antigen, reaction conditions, or characteristics of the antibodies used could be responsible. antigen by immunoaffinity chromatography.

There is, however, a report in which the heterogeneity of

Concluding remarks nephritogenic antigens was studied by 2-D gel electrophoresis of human GBM followed by Western blotting with patients' sera Goodpasture's syndrome is now one of the best characterized [54]. The authors found that staining patterns were similar with autoimmune diseases in man. Anti-GBM autoantibodies have all autoantibodies, which bound predominantly to cationic been shown to be pathogenic, although direct T cell-mediated components, but that certain sera (3 of 10) reacted more immunity may also be important and this possibility requires strongly with additional neutral components. Differences in further investigation. The detection of anti-GBM antibodies is charge, avidity or concentration of the antibodies could account of proven value in the diagnosis and monitoring of this disease, for this finding, as well as the postulated heterogeneity of the and has contributed to the development of effective though antigen. In addition, serum from a case of anti-GBM disease non-specific treatment. The mechanisms of induction of autoimfollowing transplantation bound in a different pattern, which munity have also been explored in this disorder, and the role of could be due to alloreactivity. A further study from the same both genetic and environmental factors has been clearly demgroup demonstrated broad similarities in the binding pattern of onstrated [4]. In particular, there is a strong association of the anti-GBM antibodies to components of GBM from different disease with major histocompatability class II antigen HLAspecies, although only human GBM contained a very cationic DR2 [59], and it is possible that various environmental stimuli monomer (M28) thought to represent the GA [55]. (such as infection or toxic agents) may expose or alter the GA There is general agreement that the GA is associated with the to render it immunogenic in susceptible individuals. AlternaNC1 domain of type IV collagen, and that it is unlikely to be tively, the disease could result from a breakdown in normal part of other recognised components of GBM. The work of immunoregulatory mechanisms. These possibilities are considWieslander and of Butkowski [47, 48, 56, 57] provides most ered in more detail elsewhere in this issue. The molecular basis evidence that GA is localized to the NC1 domain. They for the autoimmune response to GBM is still under investigademonstrated the reactivity of human anti-GBM antibodies tion, but both the restricting MHC class II gene product and the with non-collagenous polypeptides isolated from bovine GBM, autoantigen should soon be characterized. This level of underand noted the monomer-dimer relationship of these molecules standing should lead on to the development of specific immuin denaturing conditions. The monomeric form was chemically notherapeutic strategies for anti-GBM disease, which may be equivalent to collagenase-resistant products of type IV a widely applicable in human autoimmunity. chains, suggesting that monomeric and dimeric subunits of the

NC1 hexamer of type IV collagen contained the GA. Further resolution of these polypeptides using reversed phase HPLC and SDS-gel electrophoresis revealed that one set of monomeric and related dimeric components, termed M2 and D2, were immunoreactive whereas others were not. There was also cross reactivity of antibodies raised to type IV collagen a chains with M2, and vice versa. Physicochemical studies subsequently revealed that the NCI hexamer, although not itself reactive with anti-GBM antibodies,

could be dissociated by guanidine HCL or acetic acid to form monomers and dimers, and hence expose the antigenic sites [56]. The precise molecular structure of the bovine equivalent of GA, however, remains elusive. One recent study [57] reported amino-terminal amino-acid sequence data on various monomeric peptides from NC1 of bovine lens. Ml was found to

Autoimmune responses in the RPGN associated with systemic vasculitis

Systemic vasculitis is the term used to describe a variety of clinicopathological syndromes which have in common inflammation and necrosis of blood vessels at various sites throughout the body [60, 61]. The process may arise as a primary event and

be restricted to blood vessels or it may occur secondary to diseases such as rheumatoid arthritis, cryoglobulinemia or atrial myxoma. The primary vasculitic syndromes can be classified according to the size of vessel affected and presence or absence

of granulomata (Table 2) [62]. Renal involvement is most

commonly found in primary small vessel vasculitides, such as microscopic polyarteritis or Wegener's granulomatosis which, although perhaps indolent at first, can readily undergo a rapidly correspond to the non-collagenous segments of al and a2 progressive phase. Biopsy at this stage frequently shows a focal chains of type IV collagen; M2 (containing the GA) and M3 had segmental necrotizing glomerulonephritis with crescents, indisdistinctive sequences but each had Gly-X-Y triplets, suggesting tinguishable histologically from that found in anti-GBM disease. a collagen chain origin. The authors concluded that these However, the evidence that similar autoimmune mechanisms represented novel chains of basement membrane collagen, are operating in the development of RPGN associated with which may be part of type IV or a new species of collagen. systemic vasculitis is not substantial. Immune deposits in the Although the above studies were performed on bovine mate- glomeruli are not commonly found in systemic vasculitis rial, the same group has recently reported similar findings using whereas linear deposits of IgG are the rule in anti-GBM disease human GBM [58]. In these experiments GBM was solubilized [4, 60, 61]. Furthermore, anti-GBM autoantibody has been

Pusey and Lockwood: Autoimmunity in RPGN

Table 2. Classification of syste mic vasculitides Vessel Size

Large

Medium Small

With granulomata

Takayasu's arteritis Churg-Strauss disease Wegener' s granulomatosis

Without granulomata

Temporal arteritis Polyarteritis nodosa Henoch-SchOnlein purpura Microscopic polyarteritis Kawasaki disease

933

WG sera showed a coarsely granular appearance in the cytoplasm (accentuated nearer to the center of the cell where the vertical diameter was greater), but no binding to the multilobed nucleus was apparent. Binding also occurred to the cytoplasm of monocytes, but not lymphocytes. Radioimmunoassay [69, 70] and ELISA [71] solid phase assay systems have now been developed to allow objective detection and quantitation of AN CA. Our RIA uses as ligand an acid extract of sonicated normal human neutrophils. Binding of

autoantibodies in test sera is detected by a radio-labelled eluted from such renal tissue and its pathogenicity demon- antihuman globulin. This proved to be an order of magnitude strated by transfer experiments [101. Circulating immune complexes (putatively phlogistic if trapped in glomerular capillary walls) are detectable in some patients with systemic vasculitis but, unlike anti-GBM antibody, bear little relationship to the presence of immune deposits in the kidney. Perhaps the most

attractive evidence to support an autoimmune aetiology has come from studies of treatment. Striking responses to cytotoxic drugs, particularly cyclophosphamide, and steroids have been reported in patients with systemic vasculitis and nephritis, even

where renal disease is severe [61, 63]. Furthermore, plasma exchange has been found to confer additional benefit to drug treatment in patients with severe nephritis in a prospective controlled trial, thereby providing a clue that circulating humoral factors might be important in pathogenesis [64]. Much interest was provoked, therefore, when reports appeared that certain patients with systemic vasculitis had circulating autoantibodies to neutrophil cytoplasm antigens (ANCA) and that their levels fluctuated with disease activity [65].

more sensitive than indirect immunofluorescence. Modification of the assay to incorporate an inhibition stage

(by pre-incubation with soluble antigen or control protein) demonstrated specificity of the antibody binding activity. This was of particular use when results were close to background and possibly elevated due to non-specific stickiness of some

sera, for example in systemic lupus erythematosus (SLE). Substitution of class (and subclass) specific antisera allowed detection of different isotypes of autoantibody immunoglobulin, and led to definition of subgroups of patients restricted in their

autoantibody class; those restrictions, in turn, appeared to be associated with particular clinical features [72]. Use of ANCA assays for diagnosis

The use of assays for ANCA has shown that they can be

detected in patients with MP as well as those with WG; although the fine specificity for neutrophil antigens may be different [691. Other closely related vasculitides such as Churg-

Strauss syndrome [73, C.M. Lockwood and C.D. Pusey, unAntibodies to neutrophil cytoplasm antigens published observations) and idiopathic RPGN (C.M. Lockwood Antibodies specific for neutrophil cytoplasm were first de- and C.O.S. Savage, unpublished observations) have also been scribed in patients with diseases consistent with systemic characterized by presence of ANCA, whereas patients with vasculitis and necrotizing glomerulonephritis in the early 1980s granulomatous disorders, such as tuberculosis or sarcoid, or [66, 67]. However, their significance as diagnostic markers was inflammatory lung disease, such as fibrosing alveolitis, do not only appreciated when Van der Woude showed that they could possess these antibodies. Patients with primary glomerular be detected in a large number of patients with WG [65]. Since disease, such as membranous or mesangiocapillary glomerulotiters correlated with disease activity it appeared that levels nephritis, are also negative. Rarely, patients with rheumatoid might be useful in monitoring treatment. This led to the devel- arthritis or SLE give false positive results, which on further opment of a range of assays for these antibodies, so that their investigation using the RIA inhibition test, have shown that the specificity for vasculitis could be examined, and also to efforts binding was not specific [691. In an attempt to ascertain the value of ANCA in the diagnosis to try to identify the nature of the autoantigens. of WG or MP, we combined both indirect immunofluorescence Assays for anti-neutrophil cytoplasmic antibodies and radioimmunoassays to conduct a prospective double blind Indirect immunofluorescence tests for ANCA have now been study of their sensitivity and specificity [70]. Serum samples established in many centers, and a standardized technique was from 100 consecutive patients in renal units throughout the UK, agreed at an International Workshop on ANCA in Copenhagen, with a putative diagnosis of RPGN, were tested by indirect IF in January 1988 [68]. In brief, this involved separation of normal and RIA with inhibition studies. Clinical data were obtained by human neutrophils from heparinized blood by layering over a postal questionnaire and analyzed by a physician experienced 1.25% methyl cellulose 13% hypaque gradient. The cells were in the management of vasculitis, but who was unaware of the then washed with a neutral buffer containing protein (for laboratory findings. The patients were assigned to four groups. example, phosphate buffered saline/l% human serum albumin) These consisted of: (i) those whose diagnosis was untreated and attached to microscope slides in a cytocentrifuge. Fixation primary systemic vasculitis (either WG or MP); (ii) those with was by incubation for five minutes with prechilled absolute primary systemic vasculitis (WG or MP) but treated or inactive; ethanol at 4°C. Thereafter the slides could be stored for up to (iii) other forms of systemic vasculitis (such as, Henochone week at 4°C before use. Standard indirect IF methods were SchOnlein purpura); (iv) patients not thought to have primary then employed (30 to 60 mm at room temperature with test or systemic vasculitis. Twenty-two of 23 patients in the first group control sera diluted 1/16, followed by a similar incubation with were positive in both assays. Eight of 17 patients in the second fluoresceinated anti-human IgG to detect specific antibody group were positive in the RIA (five of these also being positive binding). The typical immunofluorescence pattern produced by by hF); presumably this reflects the effect of treatment in

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Pusey and Lockwood: Autoimmunity in RPGIV

reducing antibody levels. None of the six patients in the third group were positive. In the fourth group, 52 of 54 patients did not have ANCA, and of the two patients who were positive in both ANCA assays, one had idiopathic RPGN and the other had an undefined systemic illness with eosinophilia. Thus when results for both assays were combined, assays for ANCA had a specificity and a sensitivity of 96% for the diagnosis of WG and MP. Evidence is accumulating from long-term follow-up studies that these assays are useful for monitoring progress. In our own practice, none of the patients who have become ANCA negative and remained so have relapsed. Patients who have relapsed have all been ANCA positive, and in a number of them a rise in ANCA titers preceded the recurrence of clinical symptoms.

lyzed; 246 (28%) were positive for ANCA alone, 47 (5%) were

positive for anti-GBM antibodies alone, while 20 (2%) were positive for both ANCA and anti-GBM by RIA. The specificity of the RIA was confirmed by specific inhibition in all 20 patients

and 18 were also positive by indirect immunofluorescence. Furthermore, binding to neutrophil antigen in the RIA could be inhibited by soluble neutrophil cytoplasmic antigen but not by soluble GBM; similarly binding in the anti-GBM RIA could be inhibited by soluble GBM and not by soluble neutrophil cyto-

plasmic antigen. Additional studies with Western blotting showed that IgG in all 20 sera recognized GA. Clinical and pathological information on the 20 patients was obtained by questionnaire. The patients (11 male, 9 female)

ranged in age from 13 to 73 years. All had hematuria, and although renal function was normal in three, the remainder had

raised creatinines, nine of these 17 being dialysis dependent. Eighteen patients were biopsied and all showed crescentic change (mean of 70% glomeruli involved with crescents), with (a) Children. Crescentic glomerulonephritis is a rare cause of segmental necrosis in the capillary ioops. Granulomata were renal failure in children, and when severe (crescents in at least seen in three biopsies although vasculitis was only seen in one. 80% glomeruli) is usually associated with RPGN. Pediatric Immunofluorescence of the renal biopsy was available from 15 patients with RPGN have been screened for ANCA [74], and patients: 12 of 15 had linear IgG on the GBM and one had linear ANCA was identified in three of 10 cases of severe crescentic C3; the remaining two patients had no linear IgG but granular Subgroups of patients with anti-neutrophil cytoplasm antibodies

RPGN; one of these had clinicopathological evidence of Wegener's, while the other two had isolated idiopathic RPGN. (b) Pulmonary-renal syndrome associated with 1gM ANCA. The use of class-specific antisera in the RIA for ANCA identified a small group of patients characterized as having 1gM class ANCA (without IgG) at presentation. Eight such patients have

1gM and C3 only. Deposits of complement in vessel walls were noted in five patients. Evidence of pulmonary hemorrhage was

present in 13 (only two were habitual smokers). Other extrarenal symptoms were not prominent but sinusitis was present in 5 patients and arthritis and a rash each occurred in two patients. With treatment, three of the nine dialysis-dependent patients

been reported, all with pulmonary hemorrhage which was regained independent renal function, which is not uncommon in severe enough to require assisted ventilation in five patients. patients who have systemic vasculitis but is unusual in dialysisSeven of the eight also had renal involvement and four required dependent patients with anti-GBM disease [4, 14, 63]. Two dialysis. None of the patients had circulating anti-GBM anti- patients have relapsed (pulmonary in one, and pulmonary and bodies. Renal biopsy performed in six showed crescent formation in five patients; in the other patient renal tissue obtained later at autopsy showed normal glomeruli by light microscopy. Immunofluorescence examination of the biopsies showed that there were glomerular immunoglobulin deposits in four of the six; all four showed granular IgG and 1gM in capillary loops and two of four also had 1gM deposits in vessels. In the one patient whose renal tissue was available at autopsy, immunoglobulins

renal in the other), and on both occasions ANCA were positive but anti-GBM antibodies were not detectable.

Vasculitis associated with anti-GBM disease has been reported before, seemingly in a small number of patients and based on the histological appearances of biopsy material [4]. That up to one-third of patients with anti-GBM disease have evidence of ANCA suggests that vasculitis may play a more major role than was previously thought, although whether this

were eluted which showed specific binding in the RIA for is cause or consequence of anti-GBM antibody-mediated injury ANCA. Clinical involvement of other organs in these patients awaits further studies. was minimal, being confined to rash in two, arthralgia in two Specficities of ANCA: Search for autoantigens and sinusitis in one. Half the patients died, three of unconThe nature of the cytoplasmic antigens recognized in WG and trolled disease shortly after referral and one from a myocardial infarction which occurred two weeks after admission during MP has been examined by fractionating the neutrophil acid extract on a gel filtration column [69]. Material from normal convalescence. In three patients it was possible to obtain serial serum neutrophils reproducibly generated multiple components from samples during treatment. In all three the class of the autoan- 100 kD to 2 kD as measured by optical density of the fractions.

When these fractions were coated to microtiter plates, sera was not associated with evidence of disease activity. If these from WG bound to three different fractions of approximately 1gM autoantibodies are pathogenetically important, then the 100, 6 and 2 kD; sera from patients with MP appeared to bind use of plasma exchange as therapy is particularly appropriate as only to the 100 kD fragment. The 6 kD component was purified 1gM is predominantly restricted to the intravascular compart- and used to raise monoclonal antibodies, one of which, W8, was used for further studies. The monoclonal W8 bound to the same ment and unlike IgG has little extravascular distribution, (c) Patients with anti-GBM disease. A search for ANCA was three fractions as did WG sera, suggesting that these autoanticonducted prospectively in patients who had circulating anti- bodies were recognizing a single determinant which was carried GBM antibodies detectable by RIA [75]. Serum samples from on fractions of different molecular weight. The observation of raised concentrations of alkaline phospha890 consecutive patients suspected to have RPGN were anatibody immunoglobulin switched from 1gM to IgG, although this

935

Pusey and Lockwood; Autoimmunity in RPGN

tase in the sera from many patients with systemic vasculitis, growing experience suggests that substantial recovery of renal and the fact that this enzyme is a constituent of polymorpho- function can be obtained even in patients with severe renal nuclear leucocytes likely to be extractable by acid sonication, impairment at referral [63, 64]. How useful these laboratory raised the question whether this could be an autoantigen. Direct tests will be for monitoring patients during follow-up is as yet binding and competitive inhibition studies, using calf alkaline unclear, although in some anecdotal reports they have proved phosphatase, together with fluid phase immune complex forma- valuable. Whether ANCA are pathogenetically important is the tion between labelled alkaline phosphatase and vasculitic sera, subject of speculation. That they are found in virtually all have supported this hypothesis [691, and similar results were patients with WG or MP argues for the fact that their generation obtained using W8. This monoclonal was then used to examine is a mechanism fundamental to the development of these forms the location of the antigenic determinants in tissue sections. of systemic vasculitis. Better understanding of the pathogenesis Strong binding to vascular endothelial cells lining the lumen of of these disorders will be forthcoming when the nature of the human arteries and arterioles suggested that determinants cross antigenic target is determined at a molecular level. reactive with those in polymorph cytoplasm were present there.

Further evidence for cross reactivity with glomerular antigen Reprint requests to C.D. Pusey, M.R.C.P., Department of Medicine, has come from indirect immunofluorescence studies in which Royal Postgraduate Medical School, Hammersmith Hospital, Du Cane Road, London W12 ONN, England, United Kingdom. W8 or F(ab)2 fragments of strongly positive ANCA-containing sera showed binding to cultured glomerular cells with characReferences teristics of both epithelial and endothelial cells [761. Biochemical characterization of the neutrophil autoantigen 1. GOODPASTUREEW: The significance of certain pulmonary lesions in relation to etiology of influenza. Am J Med Sci 95:383—389, 1919 has been attempted using purified 6 kD component, which was

then reduced and yielded a 2 kD component that was still antigenic, thus far the smallest component recognized by Wegener's sera. Amino acid sequencing of this fragment showed it to be a decapeptide with strong amino acid sequence homology

with the published sequences of the "loop" region of human placental and liver alkaline phosphatase. Neutrophil alkaline phosphatase itself has not yet been sequenced. Other researchers working with different preparations have tried to localize the antigen to polymorph granules either alpha (primary) or beta (secondary). Rasmussen, Boregaard and Wiik [77] and Falk and Jennett [71] have isolated primary granules

and found that systemic vasculitis sera bound to these in ELISA. Goldschmeding Ct al [78] and Wieslander [68] have derived antigenic material from the alpha granules which, on SDS-PAGE or by immunoprecipitation, runs with a molecular weight of approximately 30 kD. By contrast, Gross, Ludemann and Schroder have suggested that the antigen can be obtained from neutrophils after treatment with phorbol ester, a chemical which specifically releases secondary granules [79]. Recently, Falk and Jennett have shown that antibodies in some vasculitic sera react with myeloperoxidase, another neutrophil enzyme, situated on the primary granules [71]. Sera from 27 of 35 patients with diseases ranging from idiopathic necrotizing glomerulonephritis (without extra-renal involvement) to systemic vasculitis typical of Wegener's or polyartentis reacted with purified myeloperoxidase used as an antigen in an ELISA or nitrocellulose dot-blot system. At present the precise nature of the autoantigenic determinant remains an enigma; it may be that patients with systemic vasculitis have anti-neutrophil autoantibodies of more than one specificity, or that the determinant recognized, such as the "loop" region, is common to different molecules. Concluding remarks Autoantibodies to components of neutrophil cytoplasm have come to be recognized as useful diagnostic markers for certain

patients whose RPGN is associated with systemic vasculitis. Thus, for the first time, there is a sensitive laboratory test with high specificity for these disorders which can alert physicians for the need for treatment. This is the more important since

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