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Restriction in V Gene Use and Antigen Selection in Anti-Myeloperoxidase Response in Mice

Hitendra S. Jethwa^*, Stephen H. Clarke, Yoshie Itoh-Lindstrom, Ronald J. Falk, J. Charles Jennette^* and Patrick H. Nachman

Departments of ^* Pathology and Laboratory Medicine, Microbiology and Immunology, and Medicine, University of North Carolina, Chapel Hill, NC 27599

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Anti-neutrophil cytoplasmic Abs, directed primarily toward myeloperoxidase (MPO) and proteinase 3, are detected in the majority of patients with distinct forms of small vessel vasculitides and pauci-immune necrotizing glomerulonephritis. However, the origin of these autoantibodies remains unknown. We studied the V region gene use in murine anti-MPO Abs derived from Spontaneous Crescentic Glomerulonephritis/Kinjoh mice. A total of 13 anti-MPO-producing hybridomas were generated from four unimmunized mice. Ten of the 13 hybridomas (corresponding to 3 of 4 clones) expressed V1C but differed in their use of V_H genes. The remaining three hybridomas expressed a V5 gene. Anti-MPO hybridomas from individual mice were derived from single clones as deduced by sequence similarity and splice-site identity. We found a statistically significant bias of amino acid replacement mutations to the complementarity-determining regions (CDR) in the V1C-expressing hybridomas. Intriguingly, all 10 V1C hybridomas share a lysine to glutamate mutation in the CDR1. To determine the effects of somatic V gene mutations on binding to MPO, we generated an anti-MPO Ab with an unmutated V1C L chain and compared its ability to bind MPO with its mutated counterpart. The mutated hybridoma-derived Ab has a 4.75-fold higher avidity for MPO than the unmutated Ab. These results suggest that: 1) the L chain plays a dominant role in determining Ab specificity to MPO, 2) the anti-MPO Ab response is oligoclonal, consistent with Ag selection, and 3) MPO is a driving Ag in the murine anti-MPO Ab response.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Anti-neutrophil cytoplasmic autoantibodies (ANCA)³ are frequently detected in sera from patients with systemic necrotizing vasculitis, including Wegener’s granulomatosis, microscopic polyangiitis, and Churg-Strauss syndrome, and patients with pauci-immune necrotizing crescentic glomerulonephritis (1). ANCA are directed toward constituents of the primary granules of neutrophils and the peroxidase-positive lysosomes of monocytes (1). By indirect immunofluorescence assay using neutrophils as substrates, ANCA that demonstrate a diffuse granular staining of cytoplasmic granules are called C-ANCA and those that show a perinuclear staining are referred to as P-ANCA (2). In patients with small vessel vasculitis, and/or glomerulonephritis, 90% of C-ANCA are specific for proteinase 3 (PR3), and 90% of P-ANCA are specific for myeloperoxidase (MPO) (3).

Since the first descriptions of ANCA by Davies et al. (4) in the early 1980’s, there have been great advances in our understanding of these autoantibodies and their possible role in the pathogenesis of small vessel vasculitis and glomerulonephritis. However, the origin and evolution of the autoimmune response to MPO and PR3 remain enigmatic.

In the original report of ANCA, it was suggested that the autoimmune response might be associated with infection with an endemic virus; however, patients with ANCA have been seen in other parts of the world where this infection does not occur (4, 5, 6, 7). It has also been suggested that environmental exposure to silica or chronic bacterial infections may be associated with the genesis of ANCA (8, 9). An association with the HLA DR13 and DR6 has also been suggested; however, the link remains equivocal (10). The relatively low frequency of the autoantibodies and their associated diseases suggests that multiple environmental and genetic factors may be required for the autoimmune response.

In analyzing the origin and evolution of an autoimmune response, it is important to ascertain the role of the autoantigen. The autoantigens may be innocent bystanders that are attacked by an autoimmune response that they have not influenced, or the autoantigens may participate in the initiation and/or modulation of the immune response. At least in some autoimmune responses, the autoantigens appear to play no part in the response and polyclonal activation is responsible for B cell activation and expansion (11). Conversely, studies of autoimmune responses to autoantigens, such as Sm, DNA, rheumatoid factor, and histone, suggest active participation of the Ag in the maturation of that response (12, 13, 14, 15).

The small vessel vasculitides associated with ANCA are uncommon diseases, and their occurrence appears to be random. It is our hypothesis that the low incidence of ANCA is due to multiple environmental, immunologic, and genetic factors, including the requirement for particular V region recombination (V(D)J) or specific somatic mutations, or both. Furthermore, we hypothesize that MPO is a driving Ag in the anti-MPO Ab response. To test this hypothesis, we studied the V region gene selection and rearrangement in spontaneous anti-MPO Abs derived from unimmunized SCG/Kj mice.

The SCG/Kj is a recombinant inbred strain that spontaneously develops vasculitis and crescentic glomerulonephritis and was established from (BXSB/Mp x MRL/Mp-lpr/lpr) F₁ hybrid mice by brother-sister mating of those mice whose parents had the highest frequency of glomerular crescent formation (16). Phenotypically, SCG/Kj mice exhibit an exhuberant lymphadenopathy and splenomegaly similar to that seen in MRL/Mp-lpr/lpr mice. When compared with MRL/lpr mice, SCG/Kj mice exhibit an earlier appearance and faster progression of glomerulonephritis, with fewer immune complex deposits in the glomerular tuft, and a lower frequency of circulating anti-DNA Abs. Sera from some of these mice contain anti-MPO Abs (17). The spontaneous (autoimmune) nature of the disease in these mice is an advantage in analyzing the autoimmune response and as a model for the human disease.

In this study, we provide evidence that suggests that the low incidence of anti-MPO Abs could be due to the requirement of particular Ig V region gene usage. We also determine the role of MPO in the murine anti-MPO Ab response by analyzing the extent and nature of Ig somatic hypermutation and determining the functional significance of any structural changes by measuring Ab affinity. We show that there is evidence of an Ag driven process in the anti-MPO Ab response in SCG/Kj mice and that MPO is a driving Ag in this autoantibody response.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mice

The SCG mice were obtained from a colony at the Animal Institute of the University of South Florida (Tampa, FL; kindly provided by Dr. Robert Good). All animal care and manipulation was in accordance with the guidelines of the University of North Carolina (Chapel Hill, NC).

Production of hybridomas

Hybridomas were generated by fusing isolated splenic mononuclear cells with the P3-X63Ag8.653 murine myeloma cell line as previously described (18). Hybridomas were screened for anti-MPO Ab production by ELISA. Cells from wells testing positive for anti-MPO Abs were rendered monoclonal by limiting dilution. To determine specificity, all MPO-ELISA-positive supernatants were also tested by ELISA using wells coated with PR3 (INOVA Diagnostics, San Diego, CA), BSA (Sigma, St. Louis, MO), or calf-thymus DNA (Sigma). The mAbs were purified using 50% v/v saturated ammonium sulfate and dialysis against a 1.5 M glycine, 3 M NaCl buffer following the method described by Brinkman et al. (19). The high salt conditions remove any Ag bound to the Ag binding sites. The mAbs were then further purified using a protein G column (Pharmacia, Piscataway, NJ). The class, subclass, and light chain type of the mAbs were determined using the Mouse Monoclonal Ab Isotyping Kit (Amersham, Little Chalfont, U.K.) following the manufacturer’s instructions.

Anti-MPO ELISA

Purified human myeloperoxidase (Calbiochem, San Diego, CA) was coated onto high-binding microtiter plates (Costar, Acton, MA) at 5 µg/ml in bicarbonate coating buffer (Sigma) overnight at 4°C. The plates were blocked for 1 h at room temperature with PBS containing 0.05% Tween and 0.2% normal donkey serum (Sigma). After washing with PBS/Tween, 100 µl of tissue culture supernatant was added and incubated for 1 h at room temperature. The plates were washed and 100 µl of alkaline phosphatase conjugated donkey anti-mouse Ig heavy and light (Jackson ImmunoResearch, West Grove, PA) was added for 1 h at room temperature. After washing, paranitrophenyl phosphate substrate (Sigma) was added for 1 h. The OD was measured at 405 nm wavelength. Any supernatant giving an OD₄₀₅ value at least twice that obtained with media alone was considered positive.

Indirect immunofluorescence staining

Murine WEHI 3BD⁺ myelomonocytic cells (kindly provided by Dr. A. C. Sartorelli, Yale University, New Haven, CT), which are known to express abundant murine MPO (20), were used to verify binding of the monoclonal anti-MPO Abs to murine MPO. WEHI 3BD⁺ cells were cytocentrifuged onto microscope slides, then fixed with ice-cold ethanol or with formaldehyde/ethanol. The slides were washed with PBS, and high-salt purified Ab from the hybridoma culture supernatants was added to each slide. The slides were incubated for 30 min, followed by two 10-min washes in PBS. Secondary Ab, goat anti-mouse IgG and IgM conjugated with FITC, was added to each slide (Jackson ImmunoResearch). The slides were incubated for an additional 20 min and washed again with PBS. After applying coverslips, the fluorescence was evaluated microscopically.

Expressed V region cloning and sequencing

Hybridomas testing positive for anti-MPO Abs were placed in RNA STAT-60 (Tel-Test, Friendswood, TX), and the total RNA was extracted with chloroform and precipitated with isopropanol. The mRNA was isolated using the Poly(A)Tract Isolation System (Promega, Madison, WI) following manufacturer’s instructions. The 5' Rapid Amplification of cDNA Ends system (5' RACE; Life Technologies, Gaithersburg, MD) was used to generate the Ig V region cDNAs following the manufacturer’s instructions. Briefly, the first strand cDNA was generated by reverse transcription using isotype-specific heavy and light chain constant region gene specific primers (GSP1) and was purified using the GlassMax DNA isolation system (Life Technologies). A poly(dCTP) tail was added to the purified cDNA using TdT. The tailed cDNA was then amplified by PCR using second, nested constant region gene-specific primers (GSP2) and poly(dGTP) anchor primers. The amplification was conducted for 35 cycles at 94°C for 1 min, 57°C for 0.5 min, and 72°C for 2 min. The GSP1 primers used were: 5'-TAACTGCTCACTGGATGGTGGGAAG-3' for the light chain, 5'-CAGATTCTTATCAGACAG-3' for the IgM heavy chain, and 5'-GGTCACCATGGAGTTA-3' for the IgG1 heavy chain. The nested GSP2s used were 5'-(CAU)₄ATGGATACAGTTGTTGCAGCATCAG-3' for the chain, 5'-(CAU)₄GCTCTCGCAGGAGACGAG-3' for the IgM chain, and 5'-(CAU)₄GGGCAGCAGATCCAGG-3' for the IgG1 chain. The amplified DNA was directionally cloned into the pAMP1 vector using the CLONEAMP System (Life Technologies). DH5 competent cells (Life Technologies) were transformed following the manufacturer’s recommendations. Plasmid DNA was obtained from ampicillin resistant colonies using the Qiagen (Chatsworth, CA) Plasmid Mini Kit and sequenced using an automated dye-chain termination DNA sequencer using M13 primers (UNC Sequencing Facility). The sequences obtained were analyzed using the DNASTAR software (DNASTAR, Madison, WI). The Basic Local Alignment Search Tool (BLAST) protocol was used to search the GenBank database to determine homology with V regions of other murine Abs that have been sequenced (21).

Germline V1C gene segment cloning and sequencing

Genomic DNA was obtained separately from the liver of three SCG/Kj mice by homogenization, phenol-chloroform extraction, and precipitation with an equal volume of isopropanol at -70°C overnight. The tubes were centrifuged at 13,000 x g for 30 min, and the pellets were washed with 70% ethanol. The pelleted DNA was resuspended in sterile deionized water. The V1C gene was amplified by PCR using leader exon and heptamer-spacer specific oligonucleotides that contain (CAU)₄ sequence. The amplification was conducted for 35 cycles at 94°C for 1 min, 57°C for 0.5 min, and 72°C for 2 min. The 5' leader exon primer (5'-(CAU)₄ATGAAGTTGCCTGTTAGG-3') was determined from the sequence of the V1C obtained from the hybridomas. The sequence for the 3' primer (5'-(CAU)₄AGGGTCTGTATCACTGTG-3') was obtained from published V1 family sequences (22). The PCR product was cloned, and three separate plasmid preparations from each liver DNA were sequenced as described above. The statistical significance of observed mutation(s) was calculated using the binomial distribution described by Shlomchik et al. (23).

Recombinant PCR

An unmutated, germline revertant V1C rearrangement was generated using the recombinant PCR method diagrammed in Fig. 1 (24). The PCR conditions were as described above. Briefly, framework 1 (FWR1) through the FWR3 of the germline V1C gene was amplified by PCR using primers specific for this region: 5' primer, 5'-TATAGAATTCCAGGTTCCACAGGTGATGTTTTGATGACCCAAACTC-3' and 3' primer, 5'-CTTGGTCCCAGCACCGAACGTGAACGGAACATGTGTAACTTGAAG-3'. A separate PCR overlapping the 3' end of the first and going through the FWR4 of the hybridoma V region obtained from genomic DNA was done. The overlapping 3'-5' region was generated by using a 5' primer for the second PCR that contained an overhang that did not bind to the hybridoma complementarity-determining region 3 (CDR3) but is complementary to the 3' end of the germline sequence. The primers used were: 5'primer, 5'-CTTCAAGTTACACATGTTCCGTTCACGTTCGGTGCTGGGACCAAG-3' and 3' primer, 5'-CAGTGAATTCTCTAGAAGACCACGCTACCTGCAGTCAGACCC-3'. The products were gel purified using the QIAEX DNA Gel Extraction Kit (Qiagen). The gel purified products were combined in a third recombinant PCR using a 5' primer complementary to the FWR1 of the germline gene and a 3' primer complementary to the end of the hybridoma V gene FWR. The products were fractionated by electrophoresis on 1% agarose gels and gel purified. The two primers in the final PCR reactions contained EcoRI sites, and the product was cloned into the pVSD/C cloning vector as described below.

View larger version (18K): [in this window] [in a new window]   FIGURE 1. Schematic representation of the recombinant PCR strategy used to generate a germline V1C-J5 gene segment.

The unmutated V1C Ab (germline revertant) construct was cloned first into the pVSD/C cloning vector, which contains the promoter and leader sequences necessary for expression, followed by subcloning into the pSV-Neo-C vector, which contains the constant region (25). The mutated V1C from the 8E2 anti-MPO Ab-producing hybridoma was also cloned in the same manner to generate a mutated control. Briefly, the PCR products were gel purified, digested with EcoRI (New England Biolabs, Beverly, MA) and ligated to linearized pVSD/C vector using the Rapid DNA Ligation Kit (Boehringer Mannheim, Indianapolis, IN). Following transformation of DH5 cells, plasmids that contained the insert in the correct orientation were digested with XbaI (New England Biolabs), then subcloned into the linearized pSV-Neo-C vector. The ligated product was used to transform DH5 cells, and several plasmid preparations were sequenced.

The same approach was used to generate the heavy chain construct. The hybridoma-derived V_H 3609 gene segment was amplified by PCR and cloned into the pVHSD/C vector, which contains the promoter, leader, and enhancer regions necessary for expression (25). The primers used were 5'-AAAGGTACCCTGTCACAGGTTACTCTGAAAGAGTCT-3' and 5'-GTATCTAGAAATCTATCTAAGCTGAATAGAAGAGAG-3' as the forward and reverse primers, respectively. The forward primer contained a KpnI site and the reverse primer a XbaI site to facilitate cloning into the pVHSD/C vector. The PCR product was gel purified, digested with KpnI and XbaI, and ligated into the shuttle vector after digestion with KpnI and XbaI (New England Biolabs). Again, several plasmid preparations were submitted for sequencing. The cassette containing the promoter, leader, insert, and enhancer was cut with NotI (New England Biolabs), and the gel purified fragment was subcloned into the pSV-Neo-Cµ expression vector (12).

Transfectoma generation

The germline revertant V1C and the mutated counterpart from hybridoma 8E2 expression constructs were linearized with SalI (New England Biolabs) and the VH3609 construct was linearized with SfiI (New England Biolabs). Each light chain was separately cotransfected with the heavy chain construct into P3-X63Ag8.653 myeloma cells. Briefly, 5 µg of each linearized constructs was added to 1 x 10⁷ cells in 0.5 ml serum-free DMEM medium, and the cells were electroporated using a Bio-Rad (Hercules, CA) Gene Pulser apparatus at a voltage of 0.22 kV and capacitance of 960 µF. Supernatant from wells with G418 (Geneticin; Life Technologies)-resistant cells were screened after 14 days for the production of IgM Abs and for MPO binding by ELISA.

Transfectoma Ab purification

The Abs from transfected cells were purified by saturated ammonium sulfate precipitation followed by affinity chromatography using a Hi-Trap NHS column (Pharmacia) coupled with goat anti-mouse IgM (Southern Biotechnology Associates, Birmingham, AL). The concentration of Ab was determined spectrophotometrically (Pierce Scientific, Rockford, IL). Tissue culture supernatants and purified Abs were tested for IgM/ Abs by ELISA. The MPO binding capacity of the supernatants from resistant cells producing IgM/ Abs and affinity-purified Ab was measured by ELISA.

Measurement of bound Ab and dissociation constant.

The amount of bound Ab was estimated following the method described by Underwood (26). Briefly, serial 2-fold dilutions of the purified transfected Abs were added to plates coated with either 5 µg/ml MPO or BSA (Sigma) and incubated for 2 h at room temperature. After incubation, the contents were transferred to fresh wells coated with 5 µg/ml MPO. The plates were washed and 100 µl of alkaline phosphatase conjugated goat anti-mouse IgM (Jackson ImmunoResearch) was added for 1 h at room temperature. After washing, p-nitrophenyl phosphate substrate (Sigma) was added and the OD was measured at 405 nm wavelength after 1 h. The binding curves from the BSA-incubated Abs were used as standards from which the concentration of free Ab remaining in the MPO-coated wells was estimated. The dissociation constant (K_d) was calculated graphically from three separate experiments using the GraphPad Prism software (GraphPad Software, San Diego, CA).

Statistical method

Confidence intervals for the observed frequency of V1C gene use among anti-MPO clones were calculated using a modified continuity correction for the Wald method, that takes into account the small sample size. (27)

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Hybridoma generation

Thirteen hybridomas producing anti-MPO Abs were generated from four unimmunized SCG/Kj mice that had spontaneous circulating anti-MPO Abs (Table I). The number of MPO specific hybridomas generated from each mouse ranged from 2 to 6.

To verify that the Abs produced by the SCG/Kj-derived hybridomas reacted to murine MPO (i.e., were truly autoantibodies), purified Abs from the anti-MPO producing hybridomas were tested in an indirect immunofluorescence assay using WEHI 3BD+ murine myelomonocytic cells as substrates. These Abs demonstrated a perinuclear staining pattern with ethanol fixation and cytoplasmic pattern when a cross-linking fixative, formalin, was used, which is the expected result for anti-MPO Abs (2, 3)

V region sequencing

The expressed V region genes from all the hybridomas were cloned and sequenced from isolated mRNA using constant region gene specific primers for the H and L chains and the 5' RACE system. The expressed H and L chain V genes of these hybridomas are listed in Table I. (GenBank accession numbers AF113096–AF113115 for V_H sequences and AF113228–AF113247 for V_L sequences). The identity of the V region genes used was determined by searching the GenBank database for homologies to known V region genes using the BLAST protocol (21). Ten of the 13 hybridomas, corresponding to 3 of 4 clones, express the V1C L chain gene and 3 express V5 (22, 28). All the hybridomas from mice 1, 2, and 3 use V1C, and the three hybridomas from mouse 4 use the V5.

Based on sequence similarity and splice-site identity of the expressed V region gene, the hybridomas derived from individual mice can be assigned to a single B cell clone. The hybridomas from mouse 1 use the J558 V_H gene segment rearranged to DSP2.2 and JH1 (29), and V1C rearranged to J1. These two hybridomas have 100% identity in the nucleotide sequence of the H and L chain, including the sequence at the V(D)J junction, and are assigned to clone A (Table I). The hybridomas from mouse 2 express a V_H 3609 gene rearranged to DQ52 and JH2 (30), and V1C rearranged to J5, and are assigned to clone B. The members of this clonal set were further subdivided into B.1 and B.2 subgroups based on seven nucleotide differences in V_H and six nucleotide differences in V chain variable region. The members of each subgroup have identical nucleotide sequence throughout the entire V_H and V exons. The hybridomas from mouse 3 are designated clone C. They express a S107 V_H gene segment rearranged to DFL16.1 and JH4 regions and V1C rearranged to J2. They have no differences in nucleotide sequence. The three hybridomas from mouse 4 express a 7183 V_H gene segment recombined to DSP2 and JH3, and V5 rearranged to J3. They differ by four base pairs in the H and four bases pairs in the L chain V region gene segments. They are designated clone D.

Germline comparison

To determine the extent of somatic mutations, the expressed V1C genes were compared with the germline gene sequences obtained from three separate SCG/KJ liver genomic DNA. The germline V gene was amplified by PCR using V1C leader exon and heptamer-spacer-specific oligonucleotides. The sequence of the leader exon was determined from the sequences obtained from the hybridoma expressed V1C. Because the heptamer-spacer sequences of the members of a family are similar, they can be used to amplify all members of a family (31). Three separate plasmid preparations with the cloned germline DNA were sequenced from each liver DNA. All nine sequences were identical.

When compared with the SCG/Kj liver DNA-derived germline V1C sequence, the anti-MPO hybridomas expressed a mutated V1C. The number of R mutations ranged from 1 to 12, and the number of S mutations ranged from 0 to 2 (Fig. 2, and Table II). The binomial distribution described by Shlomchik et al. (23), was used to calculate whether there was a statistically significant bias of R mutations to the CDRs. All the V1C clones had a statistically significant bias of R mutations to the CDRs (p = 0.0034, 0.0008, 0.0036, and 0.0458 for clones A, B.1, B.2, and C, respectively). Interestingly, all the V1C L chains, from three clones and three mice, have a lysine to glutamate replacement mutation in the CDR1 (Fig. 2).

View larger version (25K): [in this window] [in a new window]   FIGURE 2. Amino acid sequence of the coding regions of the V1C clones aligned with the liver DNA-derived germline V1C sequence. *, Residues identical with the germline. Framework regions (FR) and CDRs according to Kabat et al.

View this table: [in this window] [in a new window]   Table II. Summary of mutations in V1C

To determine the effects of somatic mutations on MPO binding, we generated a V1C germline revertant Ab and compared its ability to bind MPO with that of a somatically mutated Ab from one of the V1C expressing hybridomas. We used a recombinant PCR technique (Fig. 1) to rearrange an unmutated V1C gene, obtained from liver DNA, to the J5 expressed by hybridomas in clone B.2. B.2 was selected because it had the greatest number of somatic R mutations (24). The first set of PCR reactions were used to introduce homologous overlapping ends and yielded products of 348 and 359 base pairs for the V and the J regions, respectively. These products were purified and a second PCR reaction was used to generate the full-length germline revertant V1C and yielded the predicted 617-base pair fragment. The amino acid sequence of the in vitro generated revertant V1C is identical with the coding region of germline V1C sequence up to the V-J splice site, where the revertant sequence becomes identical with the FR IV (J5) of the hybridoma-derived sequence (Fig. 3).

View larger version (14K): [in this window] [in a new window]   FIGURE 3. Amino acid sequence of the in vitro generated germline revertant V1C aligned with the hybridoma counterpart V1C. *, Residues identical. Framework regions (FR) and CDRs according to Kabat et al.

View larger version (8K): [in this window] [in a new window]   FIGURE 4. IgM ELISA of serial 2-fold dilutions of purified transfected Abs with V1C in either germline or somatically mutated configuration.

View larger version (9K): [in this window] [in a new window]   FIGURE 5. MPO ELISA of serial 2-fold dilutions of purified transfected Abs with V1C in either germline or somatically mutated configuration.

View larger version (12K): [in this window] [in a new window]   FIGURE 6. Amount of purified transfected Ab bound to MPO by ELISA, with V1C in either germline or somatically mutated configuration. (Data from three separate experiments.)

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

ANCA-associated vasculitis is an uncommon disease, as is the presence of ANCA in the circulation. Patients with ANCA-small vessel vasculitis share Ids, suggesting the human ANCA response is oligoclonal (32, 33). We hypothesize that the low incidence of ANCA is due to multiple environmental, immunologic, and genetic factors, including the requirement for particular V region recombination (V(D)J) and/or Ag-driven somatic mutations.

Recently, a recombinant inbred strain of mice SCG/Kj was established. Mice of this strain spontaneously develop a rapidly progressive crescentic glomerulonephritis and necrotizing vasculitis (16). The strain was derived from (BXSB x MRL/Mp-lpr/lpr) F₁ hybrid mice by selective inbreeding of litter mates of mice with histopathologically highest frequency of glomerular crescents for over 20 generations. The disease in SCG/Kj mice resembles human ANCA-associated vasculitis as there are only slight fine granular deposits of Ig and complement along the glomerular basement membrane, and vasculitis involving small arteries and arterioles can be found in the spleen, ovary, uterus, heart, and stomach.

Serologically, the SCG/Kj mice have circulating anti-DNA Abs, albeit at a lower frequency than in the MRL mice (16). On the other hand, sera from these mice produce a P-ANCA staining pattern by indirect immunofluorescence assay using ethanol-fixed human or murine neutrophils as substrates, and a cytoplasmic pattern using paraformaldehyde-fixed substrates (17). The sera also react with both human and murine neutrophil extracts by ELISA. Thus, the SCG/Kj mice provide a model for studying the link between the spontaneous development of MPO-ANCA and vasculitis, and therefore are a valuable model to analyze the immunobiology of the anti-MPO autoimmune response.

To test the hypothesis that the development of ANCA is in part dependent on the requirement for particular Ig V region gene selection and recombination, we studied V region gene use and rearrangement in spontaneous anti-MPO B cells derived from SCG/Kj mice.

We generated 13 hybridomas from 4 unimmunized SCG/Kj mice, which produced Abs that react with MPO by ELISA but not with DNA, PR3, or BSA. In addition, purified Ab from these hybridomas were tested by indirect immunofluorescence microscopy using a murine myelomonocytic cell line (WEHI 3BD⁺) as substrate. When the substrate is fixed with ethanol, the cationic MPO is free to translocate to the nucleus and the staining pattern seen with anti-MPO Abs shows a perinuclear pattern. The 13 mAbs produced a perinuclear staining pattern using WEHI 3BD⁺ cells. This result is consistent with binding with murine MPO, and confirms the "autoimmune nature" of the mAbs, even though they were originally selected using commercially available human MPO (because of inavailability of purified murine MPO).

Oligoclonal response

The V gene use in these hybridomas was analyzed by cloning and sequencing the expressed V region genes. Within individual mice, the same J gene is recombined to the V1C genes and the splice-sites are identical. Similarly, although the V_H, D, and J_H gene use was diverse, all hyridomas derived from an individual mouse expressed the same V(D)J recombination and identical splice-sites. This indicates clonal relatedness and that the anti-MPO Ab response is oligoclonal because no more than one anti-MPO clone was obtained from each mouse. This "oligoclonal" response may represent immunodominant, or particularly expanded clones, whereas other, less expanded anti-MPO B cell clones or clones with lower affinity to MPO may have been overlooked by our hybridoma-based methodology. No other data regarding the clonality of anti-MPO Abs in mice are currently available; however, isoelectric focusing of sera from patients with ANCA-small vessel vasculitis and circulating anti-MPO autoantibodies revealed an oligoclonal anti-MPO response in humans (32).

L chain dominance

Three of the four anti-MPO clones (corresponding to 10 of 13 hybridomas) derived from four mice expressed the V1C gene suggesting a restriction in the light chain V region gene selection in anti-MPO Abs. This observed frequency of V1C gene use (75%) is significantly greater than expected (confidence intervals 40–99%) even if one takes into account the reported over-representation of the three functional genes of the V1 family in BALB/c mice, where they account for 13% of V gene use (34). None of seven anti-DNA Abs that we have generated from SCG/Kj mice use V1C, nor did we find a restriction in V gene use among them (data not shown). Although inconclusive, this finding further supports the suggestion that the observed restriction in the anti-MPO Abs is not due to generalized over-expression of this gene segment in these mice. The J genes used by the SCG-derived anti-MPO hybridomas are diverse. This restriction in L chain V gene use in the anti-MPO autoantibodies is in contradistinction to other autoantibodies such as anti-Sm or anti-DNA where no restriction in V gene use was found (13, 35). However, the fact that one of the four clones expresses a gene other that V1C is evidence that expression of that gene is not essential for conferring the Ab the ability to bind MPO.

The V_H genes used by the anti-MPO hybridomas are diverse, deriving from four different V_H families. Whether or not there is a restriction in V_H use will require a larger sample size. The D and J regions of the heavy chains are also diverse and result in diverse CDR3 sequences (36). Thus, anti-MPO Abs are encoded by a restricted set of V genes, suggesting that the L chain plays a dominant role in MPO binding.

Ag-driven response

Assessment of the role of MPO in the murine anti-MPO Ab response requires an analysis of the nature and extent of somatic mutation, as well as the demonstration of the relationship between increasing mutations and Ab affinity. We thus analyzed the extent and nature of somatic hypermutation in the V1C genes expressed by hybridomas derived from SCG/Kj mice. To calculate the significance of any bias, we used the binomial distribution described by Shlomchik et al. (23). This method takes into account the size of the CDRs and the number of R mutations to calculate a probability (p) value for the observed number of R mutations in the CDRs. We found a statistically significant bias of R mutations to the CDRs in the hybridoma-derived expressed sequences when compared with the liver DNA-derived germline sequence.

To demonstrate the functional effects of the observed somatic mutations, we used a recombinant PCR technique to generate an unmutated V1C rearrangement to J5. A construct containing this rearrangement was cotransfected with a construct containing a H chain rearrangement from clone B.2 into a myeloma cell line. To determine the binding constants for the two transfected Abs, we followed the method described by Underwood to measure the amount of bound Ab (26). Although the method used to determine the affinity of each Ab to MPO is not very accurate, it was used to primarily to compare the relative affinities of two Abs. We found that the activity per unit protein of the somatically mutated Ab was 5-fold higher than that of the germline counterpart, suggesting a higher affinity. The finding of a bias in R mutations to the CDRs and the demonstration of increased affinity to MPO associated with these somatic mutations provide strong evidence that MPO is a selecting Ag in the maturation of the anti-MPO immune response.

The observation that the germline V1C-expressing Ab binds MPO is not particularly surprising as other unmutated Abs have been shown to bind Ag. For example, unmutated murine and human anti-Sm Abs were shown to bind Sm (37, 38). Alternatively, the binding of MPO by the unmutated V1C-expressing Ab may be ascribable to the (mutated) heavy chain used for cotransfection or to the nonreverted L chain CDR3. Thus, although the light chain appears to play a dominant role in MPO specificity as evidenced by the biased V1C usage, the heavy chain may also contribute to MPO binding.

Interestingly, all the V1C expressing hybridomas share a lysine to glutamate replacement mutation in the CDR1. Considering that multiple identical germline sequences were obtained from three different liver DNA preparations, it is unlikely that a SCG/Kj germline gene with a glutamate at that position was overlooked. In addition, the mutation could not be derived from a common precursor because the hybridomas were derived from three different mice. In light of the cationic nature of MPO, it is intriguing to find a shared mutation among independently derived hybridomas that converts a positively charged residue to a negatively charged one. This raises the possibility that this mutation contributes significantly to the binding of MPO, although an Ab with an unmutated V1C L chain is still capable of binding MPO, albeit at lower avidity. The difference in the relative avidity of these Abs is also likely contributed by the other R mutations, as there are a total of 12 amino acid differences between the two transfected Abs. Similar increase in arginine residues in the CDRs have been observed in anti-Sm and anti-DNA Abs (12, 23, 37).

In conclusion, the results of this study indicate that: 1) the anti-MPO Ab response in SCG/Kj mice is restricted in V gene use, arguing that the L chain plays a dominant role in determining specificity for MPO, 2) the response is oligoclonal, 3) there is a statistically significant bias of amino acid replacement mutations to the Ag binding sites, and 4) there is an increase in the MPO binding capacity with increased somatic mutation in the L chain. The latter two findings provide strong evidence that MPO is a selecting Ag in the maturation of the anti-MPO Ab response in SCG/Kj mice.

	Acknowledgments

 
We thank Dr. Koken Kinjoh for his work on SCG/Kj mice.

	Footnotes

 
¹ This work was supported by National Institutes of Health Grant DK40208-09. P.H.N. is supported by NCRR Clinical Associate Physician Grant M01RR00046.

² Address correspondence and reprint requests to Dr. Patrick H. Nachman, Division of Nephrology and Hypertension, Campus Box 7155, University of North Carolina, Chapel Hill, NC 27599-7155.

³ Abbreviations used in this paper: ANCA, anti-neutrophil cytoplasmic autoantibodies; PR3, proteinase 3; MPO, myeloperoxidase; CDR, complementarity-determining region; FWR, framework.

Received for publication August 16, 1999. Accepted for publication July 12, 2000.

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