Natural anti-GBM antibodies from normal human sera recognize α3(IV)NC1 restrictively and recognize the same epitopes as anti-GBM antibodies from patients with anti-GBM disease

Natural anti-GBM antibodies from normal human sera recognize α3(IV)NC1 restrictively and recognize the same epitopes as anti-GBM antibodies from patients with anti-GBM disease

Clinical Immunology (2007) 124, 207–212 a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m w w w. e l s e v i e r. c o m / l o c a t e /...

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Clinical Immunology (2007) 124, 207–212

a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

w w w. e l s e v i e r. c o m / l o c a t e / y c l i m

Natural anti-GBM antibodies from normal human sera recognize α3(IV)NC1 restrictively and recognize the same epitopes as anti-GBM antibodies from patients with anti-GBM disease Rui Yang a , Zhao Cui a , Thomas Hellmark b , Marten Segelmark b , Ming-hui Zhao a,⁎, Hai-yan Wang a a

Renal Division, Department of Medicine, Peking University First Hospital, Institute of Nephrology, Peking University, Key Laboratory of Renal Disease, Ministry of Health of China, Beijing 100034, PR China b Department of Nephrology, Clinical Sciences, Lund University, SE-22184 Lund, Sweden Received 9 January 2007; accepted with revision 2 May 2007 Available online 6 June 2007

KEYWORDS Natural anti-GBM antibody; Antigen specificity; Epitope mapping

Abstract Anti-GBM disease is a rare autoimmune condition characterized by autoantibodies targeting the α3 chain non-collagen 1 domain of type IV collagen (α3(IV)NC1). Recently, we isolated IgG reacting with α3(IV)NC1 from normal healthy human sera. The current study examined the antigen and epitope specificity of these natural autoantibodies (NAA) using recombinant human α1, 3, 5(IV)NC1 and three constructs expressing, previously defined epitope regions designated EA, EB and S2, in the α1(IV)NC1 background. The NAA preparations reacted with recombinant human α3(IV) NC1 to the same extent as with purified bovine α(IV)NC1, but not with recombinant human α1 and α5 (IV)NC1. NAA preparations recognized the three chimeric proteins (EA, EB and S2) yielding similar absorbance values. We conclude that anti-GBM NAA recognize the same major epitopes as anti-GBM antibodies from patients with Goodpasture's disease. © 2007 Elsevier Inc. All rights reserved.

Introduction Anti-GBM disease, also called Goodpasture’s disease (GP), is a rare autoimmune disorder characterized by the presence of anti-glomerular basement membrane (GBM) antibodies in circulation or bound to the GBM of the patients. The central role of anti-GBM antibodies in the pathogenesis of anti-GBM

⁎ Corresponding author. Fax: +86 10 66551055. E-mail address: [email protected] (M. Zhao).

disease has been demonstrated by their ability to transfer the disease to monkeys or to human kidney allografts [1,2]. Basement membranes are assembled through interweaving of type IV collagen molecules with fibronectin, laminins, nidogen and sulfated proteoglycans. The type IV collagen of human GBM is composed of five α chains. The main target antigen of anti-GBM antibodies is the NC1 domain of the type IV collagen α3 chain [3,4]. However, sera from N 80% of patients with anti-GBM disease also react with other α(IV) NC1 [5]. Over the past two decades, extensive efforts have been focused on identifying the epitopes of the anti-GBM

1521-6616/$ – see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.clim.2007.05.001

208 antibodies. It has been shown that anti-GBM antibodies react with conformational epitopes of α3(IV) NC1, limiting the application of linear synthetic peptides for mapping. Instead strategies based on chimeric proteins have been applied [6–9]. Amino acids from α3(IV) sequences have been substituted at the corresponding positions into the homologous but non-reactive α1(IV) NC1 domain. Hellmark et al. [6] identified nine critical amino acid residues in the amino-terminal part of the α3(IV)NC1 sequence (positions 17, 18, 19, 21, 24, 27, 28, 31, 57) and produced a recombinant construct named S2 expressing these substitutions in the α1(IV) background. In other studies, two regions harboring conformational anti-GBM epitopes had been defined at residues 17–31 and 127–141 of the α3(IV) NC1 domain, which were named as EA and EB, respectively [7,8]. Natural antibodies refer to antibodies, circulating in healthy individuals, which have been produced in the absence of overt specific antigenic stimulation [10]. Most natural antibodies in healthy people are autoantibodies. The natural autoantibodies recognize a limited set of selfantigens that are highly conserved in evolution including nuclear structures, intracellular and cell surface components and extracellular molecules [11]. Recent evidence indicated that autoantigens could stimulate autoreactive B cells to grow and produce natural autoantibodies [12]. Natural autoantibodies are more polyreactive than immune antibodies, in the sense that they can often bind to different antigens [11]. The natural autoantibodies are proposed to have several functions, including binding to pathogens, defending against infection and helping remove senescent or altered molecules, cells and tumors [11]. Natural autoantibodies react with self-antigens that are also targets for autoantibodies in autoimmune disease [13]. The mechanisms of anti-GBM antibody production are not clear. Recently, we have identified natural autoantibodies against GBM in normal human sera [14]. The natural antiGBM antibodies also recognized α3(IV)NC1 but had lower titer and lower affinity to human α(IV)NC1 compared to the anti-GBM antibodies of patients with anti-GBM disease. It has been suggested that natural autoantibodies might be polyreactive [13]; therefore, we speculated that the natural anti-GBM antibodies might recognize multiple antigens of type IV collagen. The current study aimed to examine the antigen and epitope specificity of the natural anti-GBM autoantibodies.

Materials and methods Sera Normal human sera were obtained from five healthy blood donors, three males and two females, with an average age of 24 years ranging from 18 to 30 years. All five blood donor sera were negative for anti-GBM antibody in routine ELISA. Sera from 10 patients with anti-GBM disease, comparable in age and gender distribution, were collected at the Peking University First Hospital and preserved at − 20 °C. The patients with anti-GBM disease were diagnosed by renal pathology showing crescentic glomerulonephritis together with linear staining of the GBM by IgG using direct

R. Yang et al. immunofluorescence and by detection of circulating antiGBM antibodies by ELISA.

Preparation of human and bovine α(IV)NC1 Human and bovine α(IV)NC1 were prepared as described previously [15]. In brief, glomeruli were isolated by differential sieving, and cells of glomeruli were lysed in 5 mmol/l Tris–HCl, pH 7.4, containing 0.02% (w/v) sodium azide, and protease inhibitors, at 4 °C overnight. Crude GBM was suspended in 4% (w/v) deoxycholic acid, 0.02% sodium azide and protease inhibitors at 37 °C for 2.5 h. After extraction with detergent, GBM was resuspended in deoxyribonuclease I (50 KU/ml), 0.02% sodium azide at 37 °C for 1 h and then digested by bacterial collagenase I (Sigma, St. Louis, MO, USA) at 1:10 enzyme/protein ratio, in 50 mmol/l N-hydroxyethylpiperazine-N′-ethane sulfonic acid buffer pH 7.5, with 10 mmol/l CaCl2 and protease inhibitors at 37 °C for 20 h. After inactivating collagenase at 60 °C for 10 min, the supernatant was applied to a Resource Q ion exchange chromatography column (Amersham Pharmacia, Uppsala, Sweden) with 50 mmol/l Tris–HCl, pH 7.5 as starting buffer, and 50 mmol/l Tris–HCl containing 1 mol/l NaCl, pH 7.5 as elution buffer, by a gradient of 20 column volumes. NC1 domains, which did not bind to the column, were collected, concentrated and dialyzed against 0.01 mol/l phosphatebuffered saline (PBS).

Preparation of bovine α(IV)NC1 affinity column Purified bovine α(IV)NC1 10 mg was coupled to 3.5 g cyanogens bromide activated-Sepharose 4B (Amersham Pharmacia, Sweden) with 0.1 mol/l NaHCO3 and 0.5 mol/l NaCl, pH 8.3 as coupling buffer at room temperature for 2 h and was blocked with 0.2 mol/l glycine, pH 8.0 at room temperature for 2 h.

Isolation of natural anti-GBM antibodies The natural anti-GBM antibodies were isolated as described before [14]. In brief, IgG fractions were purified from 30 ml sera by protein G affinity chromatography (Amersham Pharmacia) from each of the five normal blood donors. The normal human IgG preparations were applied to the bovine α(IV)NC1 affinity column with 0.01 mol/l PBS, pH 7.4 as starting buffer and 0.05 mol/l glycine, 0.5 mol/l NaCl, pH 2.7 as elution buffer, at a flow rate of 1 ml/min at room temperature. Natural anti-GBM autoantibodies were eluted, neutralized to pH 7.0, concentrated and dialyzed against PBS. The natural anti-GBM antibodies were then applied to a column containing cyanogen bromide activated-Sepharose 4B gel only, to exclude non-specific binding. Natural antiGBM antibodies not binding to the column were washed out, concentrated and dialyzed against PBS.

Preparation of recombinant human α1, α3 and α5(IV)NC1 and chimeric proteins containing EA, EB and S2 Recombinant human α1, α3 and α5(IV)NC1 were produced as described earlier [6,9,16]. In brief, cDNA from the NC1

Specificity of natural anti-GBM antibodies

209

domain of human type IV collagen α1, α3 and α5 were ligated to a type X collagen triple-helix leader sequence and subcloned into the pcDNA3 vector. The constructs were then stably transfected into a human embryonic kidney cell line (HEK 293), and recombinant proteins were harvested and purified from the medium. Chimeric constructs containing different combinations of sequences from α1(IV) and α3 (IV) were produced by extension PCR technique. The construct containing the EA epitope [7] consists of the α1 (IV)NC1 sequence, but starting with 44 amino acids from α3 (IV)NC1 (aa 1–44). The EB construct consists of the α1(IV)NC1 sequence with 38 amino acids from α3(IV)NC1 (aa 130–167) spanning over the previously identified EB epitope [7]. Construct S2 was constructed in the α1(IV) background by changing nine amino acid residues to the corresponding amino acid [6]. The EA construct overlap eight of the nine substitutions found in the S2 construct.

Enzyme-linked immunosorbent assay Polystyrene microtiter plates (Nunc immunoplate, Roskilde Denmark) were coated with 100 μl of antigen in coating buffer (50 mM sodium carbonate, pH 9.6) over night at room temperature. All the antigens were coated at 0.5 μg/ml. The plates were then washed three times. One hundred microliters of human sera, diluted 1/100, or affinity-purified natural anti-GBM antibodies, diluted 1/10 (equivalent to a dilution of 1:1 of original sera) in PBS with 0.2% bovine serum albumin (PBS-BSA), were added to each well. The plates were incubated at room temperature for 1 h, and after washing, alkaline phosphatase-conjugated goat anti-human IgG (Fc specific, Sigma, St. Louis, MO, USA) diluted 1/20,000 was added. Incubation resumed for 1 h. P-nitrophenyl phosphate (1 mg/ml; (Sigma, St. Louis, MO, USA) in substrate buffer (1 M diethanolamine, 0.5 mM MgCl2, pH9.8) was used as substrate, and color development was measured spectro-

Table 1

photometrically at 405 nm. All assays were run in duplicate, and when standard errors greater than 10% were found, samples were reanalyzed. BSA was used as a non-specific control antigen, and absorbance values from anti-BSA ELISA were subtracted from results from NC1 and construct ELISAs. Twenty plasma from healthy blood donors, diluted 1/100, were used to build up a cut-off value using the mean + 2 SD.

Cross-reactivity of anti-GBM antibodies to EA and EB Plates were coated with EA or EB as described above. Sera from patients with anti-GBM disease and the preparations of affinity-purified natural anti-GBM antibodies were diluted in PBS-BSA buffer in order to obtain approximately the same absorbance after 1 h. The diluted sera or natural antibodies preparations were preincubated with chimeric EA or EB protein at increasing concentrations from 0.002 μg/ml to 2 μg/ml overnight at room temperature. The mixtures were subjected to ELISA using different coatings and bound human antibodies were detected with alkaline-phosphatase-conjugated secondary antibodies, as described above. Inhibition rates were calculated by the following formula: 1

absorbance value with inhibitor absorbance value without inhibitor

Results Antigen specificity Antigen specificity of the natural anti-GBM antibodies was detected by ELISA using purified recombinant human α1, α3 and α5(IV)NC1 as solid-phase ligands. All the 10 patients with anti-GBM disease were positive for α3(IV)NC1, and 7/10 patients were also positive to α1 and/or α5(IV)NC1. The

Antigen specificity of anti-GBM antibodies

Donor 1 Donor 2 Donor 3 Donor 4 Donor 5 Patient 1 Patient 2 Patient 3 Patient 4 Patient 5 Patient 6 Patient 7 Patient 8 Patient 9 Patient 10 Mean of normal sera + 2 SD

Purified bovine NC1

Recombinant human α1

Recombinant human α3

Recombinant human α5

1.614 0.538 3.077 1.226 1.132 2.576 2.019 2.348 1.346 3.162 3.008 2.022 2.82 3.499 2.821 0.113

0.125 0.000 0.327 0.000 0.030 0.003 0.002 0.094 0.046 0.114 0.327 0.397 0.830 1.461 0.476 0.181

1.329 0.356 2.956 1.036 0.946 1.282 1.312 2.630 2.619 2.781 2.685 1.766 2.761 2.655 2.781 0.064

0.049 0.009 0.056 0.125 0.041 0.092 0.045 0.008 0.025 0.073 0.240 0.072 0.123 0.575 0.024 0.055

Absorbance values after subtraction of values obtained in wells coated with BSA. Antibodies from donors were used at a concentration equivalent to a dilution of 1:1 of original sera. Antibodies from patients were diluted 1/100.

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R. Yang et al.

natural anti-GBM antibodies from all the five normal blood donors exhibited reactivity with human recombinant α3(IV) NC1 and bovine α(IV)NC1. However, the absorbance values of natural anti-GBM antibodies to α1 and α5(IV)NC1 were marginal (Table 1).

Epitope specificity and cross-reactivity of natural anti-GBM antibodies Epitope specificity of the natural anti-GBM antibodies was studied using three chimeric proteins expressing the S2, EA and EB epitopes. The natural anti-GBM antibodies from all the five healthy blood donors recognized all three chimeric proteins. While patient sera exhibited a diverse reaction pattern towards different epitopes, all five normal sera showed almost identical absorbance values in the three epitope ELISAs (Table 2). This finding urged us to check for possible cross-reactivity. We used EA and EB proteins in solution to inhibit antibodies reacting with EB and EA proteins in the solid phase of ELISA plates. Sera from both patients with anti-GBM-disease sera and the natural anti-GBM antibodies seemed to harbor two specific sets of antibodies against the EA and EB epitopes. That is, antibodies against the EA in solid phase could be inhibited by EA protein in solution but not by EB protein in solution. Correspondingly, antibodies against the EB could be inhibited only by EB but not by EA. (Fig. 1 and Table 3).

Discussion Recently, we successfully isolated natural anti-GBM antibodies from IgG fractions of normal human sera, their specificity to human GBM antigens was confirmed by Western blot analysis using human α(IV)NC1 as autoantigen [14].

Table 2

Epitope specificity anti-GBM antibodies

Donor 1 Donor 2 Donor 3 Donor 4 Donor 5 Patient 1 Patient 2 Patient 3 Patient 4 Patient 5 Patient 6 Patient 7 Patient 8 Patient 9 Patient 10 Mean of normal sera + 2 SD

α3

S2

EA

EB

1.329 0.356 2.956 1.036 0.946 1.282 1.312 2.63 2.619 2.781 2.685 1.766 2.761 2.655 2.781 0.064

1.566 0.596 2.706 1.27 1.089 2.478 0.878 1.763 0.986 2.986 3.182 0.957 2.802 2.938 2.872 0.065

1.750 0.498 2.86 1.495 1.519 2.316 0.241 1.861 0.513 3.101 2.919 1.009 2.609 3.129 2.971 0.080

1.520 0.458 2.753 1.149 1.173 0.413 0.915 0.312 1.464 3.05 2.943 1.12 2.321 3.041 2.99 0.125

Absorbance values after subtraction of values obtained in wells coated with BSA. Antibodies from donors were used at a concentration equivalent to a dilution of 1:1 of original sera. Antibodies from patients were diluted 1/100.

Figure 1 Cross-reactivity of Natural antibodies against antiGBM, measured using inhibition ELISA. Mean absorbance values in ELISA using the EA and EB proteins as coating and increasing amount of inhibitor in solution.

However, the fine antigen specificity of natural anti-GBM antibodies was not determined. In the present study, we examined the specificity of the natural anti-GBM antibodies by bovine α(IV)NC1 and several recombinant proteins, including α1, 3 and 5(IV)NC1. The absorbance value of the natural antibody to recombinant α3 (IV)NC1 was almost the same as to that of purified α(IV)NC1. However, the natural antibodies showed only with marginal binding to α1 and α5(IV)NC1, indicating a distinct restriction to α3(IV)NC1 as the major target antigen. In contrast, 4 sera of the patients only recognized α3(IV)NC1, while 6 sera of the patients also recognized α1 and α5(IV)NC1, in addition to α3 (IV)NC1. This result is consistent to the previous report that most GP patients have a response to the other α chains, in addition to α3(IV)NC1 [5,17]. This is a surprising finding considering that natural antibodies are supposed to be nonspecific. In the present study, the anti-GBM antibodies from patients seem to have a broader spectrum than natural antiGBM antibodies. Type four collagen is present in most multicellular organisms and is highly conserved in evolution. But α3 chain is distributed in restricted tissues comparing to α1 chain of type four collagen. [4] One recent study showed that the α3 chain emerged later than α1 chain in evolution [18]. The α1 chain is present from C. elegans to human,

Table 3 Cross-reactivity of anti-GBM antibodies measured by inhibition ELISA EB

ELISA coat

EA

Inhibitor in solution (0.2 μg/ml)

EA

EB

EB

EA

Donor 1 Donor 2 Donor 3 Donor 4 Donor 5 Patient 1 Patient 2 Patient 3 Patient 4 Patient 5

48% 35% 55% 52% 40% 59% 32% 67% 66% 36%

23% 22% 11% 11% 10% 14% 1% 6% 4% 0

34% 14% 32% 43% 43% 72% 41% 68% 23% 16%

8% 0% 0% 5% 26% 0 6% 5% 2% 4%

Specificity of natural anti-GBM antibodies while the α3 can be detected from zebrafish, and the epitopes EA and EB are emerged even later. The anti-GBM antibodies from patients cannot bind the α3 from zebrafish, which demonstrated the epitope EA and EB are essential for anti-GBM antibodies. The evolutionary change and restricted distribution of α3(IV) chain might be the reasons that the natural anti-GBM antibodies are restricted to α3 chain. In healthy individuals, natural anti-GBM antibodies could not induce immune response. Once the immune tolerance was broken, The α3(IV)NC1 became the pathogenetic antigen to induce disease. It has been reported that 13 amino acid residues of α3(IV)NC1 could induce severe glomerulonephritis in rat and elicit an Ab response to diverse native GBM proteins [19]. So the epitope spreading might be the reason that anti-GBM antibodies from patients have a broader spectrum. When studying the epitope specificity, we found the natural anti-GBM antibodies could recognize the three epitopes with comparable absorbance values. In patients with anti-GBM disease, the absorbance values of the antibodies were not consistent to each other; especially we found discrepancies between anti-EA and anti-EB antibodies. Both this finding and the findings regarding antigen specificity are consistent with the speculation that epitope spreading has occurred during the formation of disease causing autoantibodies. It has been reported that the EA and EB regions represent two separate and distinct epitopes that are held in close proximity to each other by the disulfide bonds yielding the possibility to generate an alloepitope named EAB recognized by alloantibodies found in transplanted patients with autosomal recessive Alport's syndrome [20]. In patients with antiGBM disease, the autoantibodies recognized separate EA and EB epitopes of α3NC1 but not the composite epitope EAB. Since the absorbance values of antibody targeting EA and EB were almost the same, we wondered whether the natural antibodies had cross-reaction to each other. In the inhibition ELISA, neither EA nor EB could inhibit each other. The result suggested that the natural anti-GBM antibodies targeting EA and EB might come from two different B cell clones, which is consistent to the anti-GBM antibodies in patients with antiGBM disease. The eventual presence of natural antibodies reacting with EAB was not tested in this study. It is hard to detect the natural anti-GBM antibodies from normal sera directly. We could only isolate the natural antiGBM antibodies from the IgG pool purified from normal sera. This can be explained by the low concentrations of the natural anti-GBM antibodies. Another speculation is that natural inhibitors to the natural anti-GBM antibodies might exist. It has been reported that autoantibodies to ribosomal P proteins (anti-P) were detectable in serum from healthy adults only after applying to affinity columns coated with ribosomes, which is because the inhibitory antibodies masked the anti-P [21,22]. A further study should focus on why the natural anti-GBM antibodies cannot induce immune response, which might provide a new clue to the Goodpasture's disease therapy, since the anti-GBM antibodies from patients could recognize the same antigen and epitope as natural anti-GBM antibodies. In conclusion, natural anti-GBM antibodies from normal sera recognize α3(IV)NC1 and the same epitopes as anti-GBM antibodies from patients with anti-GBM disease.

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Acknowledgments The technique support by Lena Gunnarsson and Ellinor Johnsson were greatly appreciated. The study was supported by a grant of the Chinese 985 project (985-2-033-39) and the Swedish Research Col (grants 71X-15152 and 73X-09487).

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