Kidney International, Vol. 43 (1993), pp. &—Y3

Heterogeneity of antibodies in Goodpasture syndrome reactingwith type IV collagen

NICHOLAS A. KEFALIDES, NOBUKO OHNO, and CURTIS B. WILSON

The Connective Tissue Research Institute and Department of Medicine, University of Pennsylvania and The University City Science Center,Philadelphia, Pennsylvania, and The Department of Immunology, Scripps Research Institution, La Jolla, California, USA

Heterogeneity of antibodies in Goodpasture syndrome reacting withtype IV collagen. Sera from patients with antiglomerular basementmembrane (anti-GBM) antibodies associated with Goodpasture syn-drome (GP) or glomerulonephritis were tested by ELISA and electroim-munoblot against whole basement membrane collagen (type IV) iso-lated from bovine anterior lens capsule (ALC) and bacterial collagenaseresistant domains of the collagen molecule, that is, the NC-I and 7-Sdomains isolated from either ALC or bovine and human glomerularbasement membrane (GBM).1 Reactivity was high with the NC-Idomain by both the ELISA and the electroimmunoblot techniques.Some of the anti-GBM sera reacted with both the NC-I and 7-S domainsof both human and bovine type IV collagen. At a time when thepatients' sera reacted weakly with a collagenase digest of human GBMusing a radioimmunoassay, the reactivity with the NC-I domain wasalso low, but some of the sera continued to react with the 7-S domain.The data suggest that there may be heterogeneity in the nature ofautoantibodies with respect to collagen type IV domain reactivity in thesera of patients with anti-GBM antibody disease.

Anti-glomerular basement membrane (anti-GBM) antibodyproduction in humans causes glomerulonephritis. When thiscondition is associated with episodic pulmonary hemorrhage itis referred to as Goodpasture syndrome (GP) [1]. The antibod-ies in these patients react with basement membranes in avariety of tissues including glomerular basement membrane(GBM), tubular and alveolar basement membranes, as well asthose of choroid plexus, intestine and lens capsule [1—31. Ourcurrent knowledge of the degree of heterogeneity of theseautoantibodies and their respective epitopes is incomplete. Aradioimmunoassay developed some years ago by Wilson [4] hasbeen used routinely to detect anti-GBM antibodies that reactwith an immunoaffinity purified fraction of collagenase-solubi-lized human GBM [2]. In recent years a number of studies[5—13] have demonstrated that sera from patients with anti-GBM antibodies react with intact procollagen type IV mole-cules of basement membranes, specifically with components ofthe NC-i domain of type IV collagen from GBM and anteriorlens capsule (ALC). The immunoreactivity is directed at the

'Abbreviations are: ELISA, enzyme-linked immunosorbent assay;PBS, phosphate-buffered saline; PBS-T, PBS-Tween 20 buffer; ALC,anterior lens capsule; GBM, glomerular basement membrane; NC-I,non-collagenous domain at the carboxyl terminus of type IV collagen;7-S, collagenous domain at the amino terminus of type IV collagen.

© 1993 by the International Society of Nephrology

monomeric (Mr 28,000) and dimeric (M 56,000) components ofNC-I and to some degree at the trimeric (Mr 84,000) compo-nent. The nature and location of the epitope which reacts morestrongly with sera from patients with GP has been investigatedby Saus et al [14], Gunwar et al [12], and Morrison, Germinoand Reeders [15]. Their findings suggest that the epitope islocated in the NC-i domain of the a3(IV) chain of basementmembrane coliagen and involves a peptide sequence comprisingamino acids 198 to 233. Fractionation of the bacterial collage-nase digest of GBM or ALC yields fractions in which either theNC-i or the 7-S region of the collagen type IV predominates.Earlier studies from our laboratory indicated that, although seraof the majority of patients with anti-GBM antibodies studiedreacted with the NC-i domain, a small number reacted with the7-S domain as well [9], suggesting that some heterogeneity ofantibodies among OP patients may be present. Using theenzyme-linked immunosorbent assay (ELISA), we demon-strated various degrees of reactivity between epitopes on typeIV collagen from ALC and human anti-GBM antibodies [9].Historically, ALC has provided an abundant source of base-ment membrane free of contaminating interstitial connectivetissue components. To relate the reactivity of the anti-GBMantibody radioimmunoassay with antigenicity of known sub-units of GBM, we used coilagenase-resistant fragments ofbovine ALC to inhibit the reaction of GBM subunits withpatients' sera. Initiai studies in our laboratory had demon-strated that a gel filtration fraction of solubilized bovine ALCthat contained the NC-i domain of type IV collagen was mostefficient in interfering with the anti-human GBM radioimmuno-assay reaction (unpublished data). In the current study, wereport on the comparative reactivity of human anti-GBM anti-bodies with (a) a fraction of collagenase solubilized humanGBM, purified by immunoaffinity chromatography, and with (b)bovine and human ALC fractions corresponding to the NC-iand 7-S domains of type IV collagen. The data show that serafrom patients with GP react by immunoblot and ELISA pre-dominantly with the NC-i domain of bovine type IV collagenfrom ALC. There were four patients whose sera reacted withboth the NC-i and 7-S domains, but more strongly with thelatter. The serum of one patient reacted only with the 7-Sdomain. It is suggested that ALC from heterologous species canbe used as a source of antigenic components capable of reactingwith human GBM autoantibodies. The data also indicate thatthere may be heterogeneity in the nature of autoantibodies

85

86 Kefalides et al: Auto-antibodies to BM collagen

reacting with type IV collagen epitopes in sera of patients withGP.

Methods

ReagentsHuman sera used in this study were obtained from patients

with glomernephritis or OP at a time when their antibody titersagainst GBM were high and again when the titers were low orabsent as measured by the radioimmunoassay of Wilson [4].Control sera were obtained from healthy adults and frompatients with rheumatoid arthritis, lung cancer and membra-nous glomerulonephritis. Normal rabbit serum was obtainedfrom Cappel Laboratories, Malvern, Pennsylvania, USA (5012-1380),

Rabbit antisera against the NC-i domain of BM collagen(type IV)

The NC-i domain was prepared from bovine ALC by digest-ing with bacterial coilagenase as described below. Antiseraagainst the NC-i domain were prepared in rabbits by injecting100 g subcutaneously every other week for 11 weeks. Theimmunogen was emulsified with an equal volume (1 ml) ofcomplete Freund's adjuvant. The rabbits were bled prior toinoculation and bled out 1.5 weeks after the last injection [16].

Preparation of antigensBM collagen (type IV). Native undegraded type IV collagen

was extracted from bovine ALC with 0.5 M acetic acid, andpurified on a DEAE-cellulose column as described by Brinkeret al [161. Rotary shadowing electron microscopy revealedprocoilagen-like molecules having a helical domain, a carboxylterminal, globular domain, and tetramers of such moleculesoverlapping at their amino termini to form the 7-S domain [16].

Bovine and human GBM. GBM was prepared from bovineand human kidneys. Bovine kidneys were obtained from thelocal abattoire, Human kidneys were obtained from cadavers ofindividuals who died without known renal disease. For thepreparation of GBM, renal cortices were isolated and processedas described by Freytag, Ohno and Hudson [17].

Bacterial collagenase-resistant fractions of BM collagen.The insoluble ALC that remained after extraction with 0.5 Macetic acid or intact GBM were subjected to bacterial collage-nase digestion [18]. Five hundred mg of lyophilized tissue wassuspended in 18 ml of 0.1 NI Tris-0.4 M NaCl, pH 7.4 buffercontaining protease inhibitors (N-ethylmaleimide, 3 mM;PMSF, 1 mM) and CaCl2, 6 m. Bacterial collagenase (CLSPA-Worthington Biochemicals, Freehold, New Jersey, USA) wasadded at an enzyme/protein ratio of 1: 100, and the digestionwas allowed to proceed at 37°C for 20 hours. At the end of thisperiod, 1 ml of 100 mi EDTA was added, and the solubilizedprotein was separated by centrifugation at 18,000 g for one hourat 4°C. The supernatant was dialyzed exhaustively againstdistilled water at 4°C and lyophilized. For gel filtration, 40 mg ofthe lyophilized digest was dissolved overnight at 4°C in a 2 mlsolution of 1 M CaCl2 containing 50 mrvi Tris, pH 8.5, and 40 p.gpepstatin (P-4265; Sigma Chemical Co., St. Louis, Missouri,USA). The solution was warmed for 30 minutes at 45°C, andthen centrifuged at 18,000 g for 30 minutes at 22°C. The pelletwas discarded and the supernatant was placed on a 6% agarose

A-S m column (1.6 cm x 145 cm). Proteins were eluted with abuffer containing 1 M CaCl2, 50 mii Tris-HCI, pH 7.5, at roomtemperature at a flow rate of 14.8 ml/hr (Fig. 1) [191. Thefractions were dialyzed at 4°C against water and lyophilized.

Radioimmunoassay. Anti-GBM antibodies were detected us-ing a double antibody radioimmunoassay which employs animmunopurified fraction of collagenase solubilized human GBMas an antigen. The immunoafilnity column was prepared with ahigh-titer human anti-GBM antibody. Radiolabeled human IgGwas included to assess the degree of immunoglobulin precipi-tation, and Na22 was included as an entrapment control. Theassay procedure and calculations are as described by Wilson[4].

ELISA. Antibodies against type IV collagen and its fractions,in patients with anti-GBM antibodies, were detected by ELISA.The assay was performed essentially according to previouslydescribed methods [16]. Native type IV collagen was coated inVoller's buffer (0.15 M Na2CO3, 0.35 M NaHCO3, 0.02% NaN3)at a concentration of 600 ng/well. The noncollagenous NC-idomain of type IV collagen (Fig. 1, peak V) was coated at 1200ng/well and the 7-S domain (Fig. 1. Peak III) was coated at 300ng/well.

Polyacrylamide gel elect rophoresis (SDS-PAGE). SDS-PAGE was performed on 5% or 9% polyacrylamide gels asdescribed by Laemmli [20].

Immunoblot. To further confirm the presence of antibodies totype IV collagen and its NC-i and 7-S domains in the sera ofpatients with anti-GBM antibodies, immunoblots were per-formed as described previously [18].

4.0

3.0

E

2.0

1.0

Fraction number

Fig. 1. Gel filtration of bacterial collagenase digest of anterior lenscapsule ('ALc on a 6% agrose A-5m column. Proteins were eluted witha buffer containing I M CaCI2 in 50 mM Tris-HCI, pH 7.5. Peak I is thevoid volume.

VT

e

30 50 70 90 110

Kefa/ides et al: Auto-antibodies to BM collagen 87

Table 1. Amino acid and sugar compositions of ALC type IVcollagen and its collagenase-resistant fractions

ALC typecollagen

CollagenaPeak III

se digestPeak V

Amino acidaCysteic acid3-Hydroxyproline 12.08 4.83 —Methionine sulfoxide — 2.26 1.70Hydroxyproline 82.55 96.28 TraceAspartic 40.51 39.32 59.73Threonine 31.78 35.04 64.80Serine 51.85 32.50 87.30Glutamic 105.42 89.03 93.33Proline 67.34 66.96 76.45Glycine 263.97 316.24 100.54Alanine 44.38 25.76 94.57Cystine (reduced) 9.48 32.08 34.36Valine 30.72 50.04 53.25Methionine 13.69 5.08 15.45Isoleucine 28.93 27.86 45.04Leucine 56,15 42.66 73.20Tyrosine 17.30 17.35 36.63Phenylalanine 33.93 17.76 43.28Hydroxylysine 35.27 41.41 4.48Lysine 21.16 11.13 30.24Histidine 15.48 12.24 36.14Arginine 38.02 41.09 57.10

Sugars pg/mg proteinMannose 6.84 34.07 1.85Fucose 1.46 11.093 1.66Galactose 30.77 44.40 2.57Glucose 33.90 36.37 2.51

Fractions were isolated after bacterial collagenase digestion of ALCand gel filtration.

a Residues per thousand

Immunodetection was performed using a protocol from Vec-tor Laboratories (Burlingame, California, USA). In the case oftype IV collagen, a nitrocellulose membrane was incubatedwith each of the following: rabbit anti-type IV collagen anti-serum, at a dilution of 1: 100; normal whole rabbit serum, at adilution of 1: 100; anti-GBM antibody positive serum, at adilution of 1: 10; and control human serum, at a dilution of 1: 10.

Amino acid analysis. Amino acid analysis of whole type IVcollagen and its NC-l and 7-S domains was carried out asdescribed previously [19].

Rotary shadowing. Rotary shadowing electron microscopywas performed on fractions of the collagenase digest of ALCand GBM according to the method of Shotton, Burke andBranton [21] as modified by Kuhn et al [22]. Photographs weretaken with a Hitachi model HU 1 iF electron microscope.Magnification was calibrated with carbon replica grating (No.1002, E.F. Fullam, Inc., Schenectady, New York, USA).

Results

Characterization of antigenic components of BMBM collagen. Table I shows that the purified ALC extract

has a high 3- and 4-hydroxyproline content, whereas its alaninecontent is low, consistent with previous analyses of type IVcollagen [16]. The equimolar amounts of glucose and galactoseas well as the low content of mannose and fucose furtherconfirm the nature of the material as type IV collagen.

Bacterial collagenase-resistant fractions of BM. The bacte-rial collagenase-resistant fractions of ALC were resolved intoseven fractions on an agarose A-5m column (Fig. 1). Aminoacid analysis of peaks I and II revealed the noncollagenousnature of the fractions, (data not shown). The analyses of peaksIII and V are shown in Table 1. Peak V had a noncollagenousamino acid composition. Peak III was collagenous. The 4-hy-droxyproline, hydroxylysine, and glycine content was muchhigher than that seen in the intact type IV collagen, whereas thealanine content was significantly lower. The cysteine contentwas high, indicative of high disulfide cross linking within the 7-Sdomain. The carbohydrate content was characterized by ahigher amount of galactose and a significantly higher content ofmannose and fucose, suggesting the presence of mannosecontaining heteropolysaccharides.

Figure 2A shows a rotary shadowing electron micrograph ofthe material eluting in peak V; it is composed of globulardomains characteristic of the NC-i domain. Globular compo-nents were the only structures noted in peak V. Peak III wascomposed entirely of rectangular pieces characteristic of the7-S domain of type IV collagen (Fig. 2B). Similar results wereobtained with peaks V and III from the collagenase digest ofbovine and human GBM (data not shown).

The bacterial collagenase-resistant fractions which wereeluted on agarose A-Sm were further characterized by SDS-PAGE (data not shown). Under reduced conditions peak Vcontained peptides that migrated with an M = 28,000 and56,000 suggesting a monomer-dimer relationship. Evidence fora trimer with a Mr = 84,000 was suggested by a slow movingband in peak V. Peak III which was composed of the 7-Sdomain contained peptides migrating with an Mr between52,000 and greater than 100,000.

Immunologic studies of BM componentsType IV collagen. The native type IV collagen was tested by

ELISA against sera from patients with GP. Figure 3 shows ahigh degree of reactivity with four patient sera at dilutionsranging from 1:20 to 1:160.

Collagenase resistant fractions of type IV collagen. Whenthe collagenase resistant fractions of ALC were tested byELISA against the anti-GBM positive sera, high reactivitieswere noted with dilutions as high as 1:320. Figure 4 shows theELISA reaction of peak V which contains the NC-l domain. Itreacted strongly with the same two sera (numbers 1 and 7) thathad the highest reactivity with intact type IV collagen (Fig. 3).Peak III (Fig. 5), which contains the 7-S domain, reactedstrongly against four anti-GBM antibody positive sera at adilution of 1:40, but only sera 1 and 7 gave strong reactions atdilutions of 1: 160.

An attempt was made to correlate the reactivity of sera fromGP patients with the immunopurified human GBM collagenasedigest, as measured by the radioimmunoassay of Wilson [4],and with peak V from bovine ALC as measured by ELISA.Sera from the same patients were taken during the time ofanti-GBM antibody production and after the anti-GBM anti-body was no longer detectable by the radioimmunoassay (lessthan 1% binding).

Figure 6A shows that when the sera had no binding (solidcircles) by the anti-GBM antibody radioimmunoassay there waslittle or no reactivity with peak V by ELISA. Only when the

88 Kefalides et at: Auto-antibodies to BM collagen

Fig. 2. Electron microscopic i'izualization of material fro,n peaks V (A) and III (B) of the gel filtration shown in Figure 1. Note the globularparticles (NC-i domain) in A and the rectangular particles (7-S domain) in B. Bar = 100 nM.

binding by radioimmunoassay rose above 15% was there asimilar increase in the reactivity to peak V by ELISA reaching2 to 2.3 O.D. units. This indicated a positive although lesssensitive correlation with the presence of anti-GBM antibodyusing the heterologous basement membrane antigen. The reac-tivity of the sera from patients with cancer, with rheumatoidarthritis, with membranous glomerulopathy, and from normalindividuals was low to absent with both methods of assay.

A similar correlation was attempted with peak III whichcontains the 7-S domain of ALC type IV collagen. A fewanti-GBM antibody positive sera showing a high percent ofbinding to the human GBM collagenase digest by radioimmu-noassay also demonstrated a high reactivity to this fraction byELISA. Some GP patient sera that were negative by theradioimmunoassay were also non-reactive by ELISA, whereas,others showed moderate reactivity with peak III (Fig. 6B). On

20 40 80 160

Serum dilutionFig. 3. An ELISA study with type IV collagen isolated from ALC.Antigen at 600 nglwell. The reactivity of sera from four patients [(x—x)#1, (—) #2, ( •) #4, (O—O) #71 with anti-GBM antibod-ies and from one normal (S S N.S.) is shown.

the other hand, some UP sera which demonstrated a highpercent of binding to the human GBM collagenase digest byradioimmunoassay showed low reactivities with the 7-S domainby ELISA. Sera from two patients with rheumatoid arthritis,from four with cancer, from one with membranous glomerul-opathy and from three normal individuals reacted with peak IIIby ELISA but had a negative reaction by the radioimmunoas-say. These data indicate that patients with a variety of disor-ders, including GP, may produce antibodies against more thanone domain of type IV collagen; however, the specificity ofreaction in GP is higher with peak V (NC-i domain).

Immunobiot analyses

Sera from patients anti-GBM antibodies were tested byimmunoblot against peaks III and V as well as against theunfractionated bacterial collagenase digest of bovine ALC andof bovine and human GBM.

Figure 7 shows the reaction of two anti-GBM antibodypositive sera (patients #3 and 1, respectively) in panels A andB. Lane 1 in panel A depicts the reaction of one patient's serumwith the whole unfractionated collagenase digest of ALC andlane 2, the reaction with peak V (NC-i domain) of the fraction-ated collagenase digest, (electrophoresis carried out undernon-reducing conditions). Lane 3 shows the reactivity of the GPserum with the same peak V and lane 4 with peak III (7-Sdomain) under reducing conditions. Although both peaks arereactive, the reaction is markedly diminished under reducingconditions. Panel B shows the reactivity of another anti-GBM

2.4

2.2

2.0

1.8

1.6

E 1.4

J1.21.0

0.8

0.6

0.4

0.2

Serum dilution

Fig. 4. An ELISA study with peak V (NC-I domain) of the collagenasedigest of ALC fractionated on agarose A-5m (Fig. 1). The reactivity ofsera from two patients [(x—x) #1, (O—O) #71 with anti-GBMantibodies and from one normal (5 5) is shown. Antigen at i200ng/well.

20 40 80 160

Serum dilutionFig. 5. An ELISA study with peak III (7-S domain) of the collagenasedigest of ALC fractionated on agarose A-5m (Fig. 1). The reactivity ofsera from four patients (x—x) #1, (L—) #2, ( •) #4,(O—O) #7] with antiGBM antibodies and from one normal (S 5)is shown. Antigen at 300 ng/well.

Kefalides et al: Auto-antibodies to BM collagen 89

4.0

3.5

3.0

2.5

2.0

1.5

1.0

0.5

E0

20 40 80 160 320 640

2.2

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1.6

1.4

1.2

1.0

0.8

0.6

0.4

0.2

E0

>..(I)C')Ce

-o

Co

CCe

c,)CC

Co

0.0. units bovine peak Ill ELISA

Fig. 6. Reaction of GP sera with the NC-i(A) and 7-S domains (B). Sera from patientsduring antibody production and after theantibody was no longer detectable (less than1% binding) by radioimmunoassay are shownas solid circles. The open squares representserum from patients with cancer. The solidtriangles are sera from patients withrheumatoid arthritis. The open hexagons are

2.5 sera from patients with membranousglomerulopathy and the closed squares aresera from normal individuals.

digests, comigrating with fractions from peak III reacts with theGP serum, differences in reactivity between peaks III and V areagain noted. Panel B shows the reaction of the same fractions(lanes in B are mirror image of lanes in A) with a normal serum.

Discussion

Initial studies by Wieslander et al [5, 6] and by Butkowski etal [7], suggested that the GP antigen of the GBM localized in thenon-collagenous regions of type IV collagen. They furthersuggested that the GP epitope was contained in a non-collage-nous polypeptide fragment, released by digestion with bacterialcollagenase, which exists as a monomer (Mr = 26,000) and adimer (M = 50,000). These subunits were designated M2 andD2, respectively. Later studies by Butkowski et al [8] and Sauset al [14] suggested that M2 and D2 are not components of theal or a2 chains of the type IV collagen but rather arise from anovel basement membrane collagen chain. A cDNA clonerepresenting the a3(IV) chain of basement membrane collagenhas been reported recently [15] and evidence has been pre-sented that the NC- 1 domain of this chain contains an epitopethat reacts with GP sera [13].

90 Kefalides et a!: Auto-antibodies to BM collagen

60

50

40

30

20

10

0

A

.•. .• •.U •

.•I

..._ I I

S.

0.5 1 1.5 2 2.5

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60

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B I

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I •• IVII

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antibody positive serum with the same fractions as in panel A.Note the differences in the degree of reactivity between the serain panels A and B. Note also the strong reaction with peak III(7-S domain) even under reduced conditions (panel B, lane 4).Lane 1 in Panel B shows reactivity with high molecular weightcomponent only, suggesting reaction with the 7-S domain. Noreaction was noted with a normal serum (panel C). Panel Dshows an immunoblot with a purified sample of peak V and aspecific rabbit antiserum.

Figure 8 shows the comparative reactivity of an anti-GBMantibody positive serum with material from peaks III and V ofthe collagenase digest of bovine and human basement mem-branes. In panel A there is a strong reaction with peak V frombovine GBM and ALC (lanes 1 and 4, respectively) as well aswith whole collagenase digest from human GBM (lane 2) andfrom bovine ALC (lane 3). The reactivity of peak III (underreducing conditions) from bovine ALC and GBM is shown inlanes 5 and 6, respectively. Lanes 7 and 8 show respectively thereactivity of unfractionated collagenase digest from bovineALC and human GBM (under reducing conditions). Althoughmaterial from both the human and bovine whole collagenase

!1I-a

I:

Kefalides et al: Auto-antibodies to BM collagen 91

A B C D

84K 84K

56K ' 56K

28K 28K

12 34 12 34 12 34Fig. 7. Electroimmunoblot of sera from patients (#1 and #3 respectively) with anti-GBM antibodies (A and B) and from a normal (C). Lanes 1and 2 in panels A, B and C under non-reduced conditions. Lanes 3 and 4 in all panels, and panel D under reduced conditions. Lanes 1 in panelsA and B, unfractionated collagenase digest of ALC. Lanes 2 and 3 in panels A and B, peak V (NC-i domain) from Figure 1. Lanes 4 in panels Aand B, peak III (7-S domain) from Figure 1. Panel D shows the reactivity of Peak V with an antiserum against it prepared in rabbits.

A B

84 K

56 K

28 K

1234 5678 8765 4321Fig. 8. Electroimmunoblot of a serum from a patient with anti-GBM antibodies (A) and from a normal (B). Lanes 1—4 in both panels are undernon-reduced conditions. Lanes 5—8 in both panels are under reduced conditions. Lanes 1 and 4, material from peak V of bovine GBM and bovineALC, respectively. Lanes 2 and 3, material from whole collagenase digest of human GBM and bovine ALC. Lanes 5 and 6, material from peakIII (7-S domain) of bovine ALC and bovine GBM, respectively. Lanes 7 and 8, material from whole collagenase digest of bovine ALC and humanGBM, respectively.

The data presented here demonstrate that patients with lagenous and non-collagenous domains of the basement mem-anti-GBM antibodies associated with Goodpasture syndrome or brane collagen (type IV). Unpublished data from our laboratoryglomerulonephritis alone carry antibodies directed against col- demonstrate that a number of the anti-GBM antibody positive

5-,-

S

•S..I

92 Kefalides et a!: Auto-antibodies to BM collagen

sera also reacted by ELISA with mouse and human laminin. Asimilar reaction with lamjnin was noted with sera from patientswith poststreptococcal glomeruionephritis [18]. Anti-GBM an-tibodies are capable of reacting not only with the humanbasement membrane components but with bovine as well. TheNC-i and 7-S domains of type IV collagen are capable ofreacting with sera containing anti-GBM antibodies from pa-tients with GP. The reactivity of these sera with the 7-S domaincannot be due to contamination of the latter by material fromthe NC-i domain, since Peak V under reduced conditionsshows little reactivity with the GP serum, whereas peak III,representing the 7-S domain, reacts quite strongly (Fig. 7B).

Reactivity of the anti-GBM antibody positive sera with theintact type IV molecule and with both the NC-i and 7-Sdomains was demonstrated by ELISA (Figs. 3 to 5) as well asby immunobiot (Figs. 7 and 8). However, when a comparisonwas attempted between the reactivity of these domains mea-sured by ELISA and by a specific radioimmunoassay for thehuman GBM antigen, the specificity of the reaction was greaterfor the NC-i domain than for the 7-S domain. Although fouranti-GBM antibody positive sera showed a high reactivity withthe 7-S domain by ELISA, the majority of anti-GBM antibodypositive sera demonstrated a low reactivity when the reactivityagainst the NC-i domain was high by radioimmunoassay.Furthermore, sera from patients with disorders, such as rheu-matoid arthritis, cancer or membranous glomeruionephritisreacted with the 7-S domain by ELISA but showed a negativereaction by the radioimmunoassay of Wilson [4] (Fig. 6 A andB).

Antibodies against other basement membrane components,such as laminin [181 or heparan sulfate proteoglycan [23], mayarise as a secondary non-specific response or it is possible thatmore than one epitope is responsible in the production ofnephritogenic antibodies. In this respect, it can be stated that abacterial or viral infection leads to a tissue injury, which in turnmay damage basement membranes resulting in the productionof antibodies directed against a number of tissue components.In patients with anti-GBM antibody disease, through someunknown mechanism, antibodies are produced against a minorcomponent in vascular basement membranes, a process thatcan lead to severe tissue damage. The rarity of this syndromesuggests: (1) that the antigenic epitope(s) or their accessibilitymay vary, possibly as a result of mutation in the structuralgene(s) responsible for the synthesis of basement membranecollagen; (2) another collagen molecule kept at very lowamounts in normal basement membranes may be produced inhigher yields in patients destined to develop GP 114], and (3)that GP patients who develop anti-GBM antibodies may havean abnormality in their immune system which allows specificantibody producing cells to produce autoantibodies.

To pinpoint more precisely the epitopes that give rise toanti-GBM antibodies in humans, we must employ additionalapproaches [24]. One approach may come from the applicationof information obtained through recombinant DNA studies ofthe genes responsible for the expression of proteins in basementmembranes. In a number of laboratories cDNA clones havebeen isolated for the NC-i domain of the human al (IV) and a2(IV) procollagen chains [25—3 1] and more recently for thea5(IV), s3(IV) [15], and a4(IV) chain [321.

Recently, we have identified antigenic determinants in the

NC-i domain of type IV collagen that are reactive with anti-GBM antibody positive sera by using synthetic peptides corre-sponding to segments of the human al(IV), a2(IV) and a3(IV)procoliagen chains [33 and 34, in this issue].

Acknowledgments

This work was supported in part by NIH Grants AR 20553, HL-29492, AR-07490 and DK-20043 from the U.S. Public Health Service.This is publication No. 6087-1MM of the Department of Immunology,Scripps Clinic and Research Foundation, La Jolla, California 92037. Weare grateful to Ms. Adrienne Webb for typing the manuscript.

Reprint requests to Dr. Nicholas A. Kefalides, Connective TissueResearch Institute, 3624 Market Street, Philadelphia, Pennsylvania19104, USA.

References

1. WILSON CB: Anti-GBM glomerulonephritis, in Pathology of Gb-merular Disease. Contemporary Issues in Surgical Pathology,edited by ROSEN S, ROTH LM, New York, Churchill Livingstone,1983, p. 171

2. WILSON CB, DIXON FJ: The renal response to immunologic injury,in The Kidney, edited by BRENNER BM, RECTOR FC JR. Philadel-phia, WB Saunders Co, 1986, p. 800

3. GLASSOCK RJ, COHEN AH, ALDER SG, WARD HJ: Secondaryglomerular disease, in The Kidney, edited by BRENNER BM,RECTOR FC JR, Philadelphia, WB Saunders Co, 1986, p. 1014

4. WILSON CB: Radioimmunoassay for anti-glomerular basementmembrane antibodies, in Manual of Clinical Immunology (2nd ed),edited by ROSE NR, FRIEDMAN H, Washington, American Societyfor Microbiology, 1980, p. 376

5. WIESLANDER J, BARR JF, BUTKOWSKI RJ, EDWARDS SJ, BYGRENP, HEINEGARD D, HUDSON BG: Goodpasture antigen of the gb-merular basement membrane. Localization to non-collagenous re-gions of type IV collagen. Proc Nat! Acad Sci USA 81:3838—3842,1984

6. WIESLANDERJ, BYGREN P, HEINEGARD D: Isolation of the specificglomerular basement membrane antigen involved in Goodpasturesyndrome. Proc Natl Acad Sci USA 81:1544—1548, 1984

7. Buricowsiu RJ, WIESLANDER J, WIsDOM J, BARR JF, NOELKENME, HUDSON BG: Properties of the globular domain of type IVcollagen and its relationship to the Goodpasture antigen. J BiolChem 260:3739—3747, 1985

8. BUTKOWSKI RJ, LANGEVELD JP, WIESLANDER I, HAMILTON J,HUDSON BG: Localization of the Goodpasture epitope to a novelchain of basement membrane collagen. J Biol Chern 262:7874—7877,1987

9. KEFALIDES NA, OHNO N, WILSON CB: Antigenic components ofbovine lens capsule that cross-react with serum from GoodpastureSyndrome. (abstract) Fed Proc 43:779, 1984

10. WICK G, VON DER MARK H, DIETRICH H, TIMPL R: Globulardomain of basement membrane collagen induces autoimmune pul-monary lesions in mice resembling human Goodpasture disease.Lab Invest 55:308—317, 1986

11. PUSEY CD, DASH A, KERSHAw MJ, MORGAN A, REILLY A, REESAJ, LOCKWOOD CM: A single autoantigen in Goodpasture's syn-drome identified by a monoclonal antibody to human gbomerularbasement membrane. Lab Invest 56:23—31, 1987

12. GUNWAR S, SAUS J, NOELKEN ME, HUDSON BG: Gbomerularbasement membrane identification of a fourth chain, 4, of type IVcollagen. J Rio! Chem 265:5466—5469, 1990

13. GUNWAR S, BALLESTER F, KALLURI R, THIONEDA J, CHONKOAM, EDWARDS SJ, NOELKEN ME, HUDSON BG: Gbomerularbasement membrane identification of dimeric subunits of the non-collagenous domain (hexamer) of collagen IV and the Goodpastureantigen. JBiol Chem 266:15318—15324, 1991

14. SAUS J, WIESLANDER J, LANGEVELD JPM, QUINONES S, HUDSONBG: Identification of the Goodpasture antigen as the a3(IV) chainof collagen IV. J Biol Chem 263:13374—13380, 1988

Kefalides et at: Auto-antibodies to BM collagen 93

15. MORRISON KE, GERMINO GG, REEDERS ST: Use of polymerasechain reaction to clone and sequence a cDNA encoding the bovinea3 chain of type IV collagen. J Biol Chem 266:34—39, 1991

16. BRINKER JM, PEGG MT, HOWARD PS, KEFALIDES NA: Immuno-chemical characterization of type IV procollagen from anterior lenscapsule. Collagen Ret Res 5:233—2244, 1985

17. FREYTAG JW, O-tro M, HUDSON BG: Large scale preparation ofbovine renal glomerular basement membrane in the presence ofprotease inhibitors. Prepar Biochem 8:215—226, 1978

18. KEFALIDES NA, PEGG MT, OHNO N, PooN-KING T, ZABRISKI J,FILLIT H: Antibodies to basement membrane collagen and tolaminin are present in sera from patients with poststreptococcalglomerulonephritis. J Exp Med 163:588—602, 1986

19. DEHM P, KEFALIDES NA: The collagenous component of lensbasement membrane: The isolation and characterization of ana-chain size collagenous peptide and its relationship to newlysynthesized lens components. J Biol Chem 253:6680-6686, 1978

20. LAEMMLI UK: Cleavage of structural proteins during the assemblyof the head of bacteriophage T4. Nature (Lond) 27:680—685, 1970

21. SHOTTON DM, BURKE BE, BRANTON D: The molecular structureof human erythrocyte spectrin. Biophysical and electron micro-scopic studies. J Mo! Biol 131:303—329, 1979

22. KUHN K, WIEDMANN H, TIMPL R, RISTELI J, DIERINGER H, VossT, GLANVILLE RW: Macromolecular structure of basement mem-brane collagens. Identification of 7-S collagen as a cross-linkingdomain of type IV collagen. FEBS Lett 125:123—128, 1981

23. FILLIT H, DAMLE SP, GREGORY JD, WOLIN C, POON-KING T,ZABRI5KIE J: Sera from patients with poststreptococcal glomerulo-nephritis contain antibodies to glomerular heparan sulfate proteo-glycan. J Exp Med 161:277—289, 1985

24. KEFALIDES NA: The Goodpasture antigen and basement mem-branes: The search must go on. Lab Invest 56:1—3, 1987

25. BRINKER JM, GUDAS U, L0IDL HR, WANG S-Y, ROSENBLOOM J,KEFALIDES NA, MYERS JC: Restricted homology between humanal type IV and other procollagen chains. Proc Nat! Acad Sci USA82:3649—3659, 1985

26. KURKINEN M, BARLOW D, HELFMAN DM, WILLIAMS JG, HOGAN

BLM: Isolation of cDNA clones for basal lamina components:Type IV procollagen. Nucleic Acids Res 11:6199—6205, 1983

27. OBERBAUMER I, LAURENT M, SCHWARZ U, SAKURAI Y, YAMADAY, VOGELI G, Voss T, SIEBOLD B, GLANVILLE RW, KUHN K:Amino acid sequence of the non-collagenous globular domain(NC-l) of the a-l(IV) chain of basement membrane collagen asderived from complementary DNA. Eur J Biochem 147:217—224,1985

28. PIHLAJANIEMI T, TRYGGVASON K, MYERS JC, KURKINEN M, LEBOR, CI-IEUNG M-C, PROCKOP DJ, BOYD CD: cDNA clones coding forthe pro-a-l(IV) chain of human type IV procollagen reveal anunusual homology of amino acid sequences in two halves of thecarboxyl-terminal domain. J Biol Chem 260:7681—7687, 1985

29. MYERS JC, JONES TA, POHJOLAINEN ER, KADRI AS, GODDARDAD, SHEER D, SOLOMON E, PIHLAJANIEMI T: Molecular cloning ofa5(IV) collagen and assignment of the gene to the region of the Xchromosome containing the Alport syndrome locus. Am J HumGen 46:1024—1033, 1990

30. HOSTIKKA SL, EDDY RU, BYERS MG, HOYHTYA M, SHOWS TB,TRYGOVASON K: Identification of a distinct type IV collagen achain with restricted kidney distribution and assignment of its geneto the locus of X chromosome-linked Alport syndrome. Proc Nat!AcadSci USA 87:1606—1610, 1990

31. PIHLAJANIEMI T, POHJOLAINEN ER, MYERS JC: Complete primarystructure of the triple helical region and the carboxy terminaldomain of a new type IV collagen chain, a5(IV). J Biol Chem265:13758—13766, 1990

32. MARIYAMA M, KALLURI R, HUDSON BG, REEDERS ST: The a4(IV)chain of basement membrane collagen: Isolation of cDNAs encod-ing bovine a4(IV) and comparison with other type IV collagens. JBiol Chem 267:1253—1258, 1992

33. KEFALIDES NA, ABRAMS WR, OHNO N, WILSON CB, ROSEN-BLOOM J: Identification of antigenic determinants in the NC-Idomain of type IV collagen reactive with Goodpasture sera. (ab-Stract) FASEB J 2:A629, 1987

34. KEFALIDES NA, OHNO N, WILSON CB, FILLIT H, ZABRISKI J,ROSENBLOOM J: Identification of antigenic domains in type IVcollagen by use of synthetic peptides. Kidney Ini 43:94—100, 1993