Induction of anti-DNA antibody with DNApeptide complexes
Dharmesh D. Desai1 and
Tony N. Marion
Department of Microbiology and Immunology, University of Tennessee Health Science Center, 858 Madison Avenue, Memphis, TN 38163, USA
1 Present address: Department of Medicine, Division of Rheumatology, Columbia University, New York, NY 10032-3702, USA
Correspondence to:
T. N. Marion
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Abstract
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Spontaneous anti-DNA antibodies in autoimmune mice have the characteristics of antibodies produced by antigen-specific, clonally selective B cell stimulation. The nature of the somatically derived antibody variable region structures recurrent among spontaneous anti-DNA antibodies suggests that DNA or DNAprotein complexes may provide the antigenic stimulus for autoimmune anti-DNA antibody. Previously we have demonstrated that native mammalian DNA in complexes with an immunogenic DNA-binding peptide Fus1 from Trypanosoma cruzi can induce anti-DNA antibody in mice not genetically prone to autoimmune disease. The induced anti-DNA has similar specificity, structure and immunopathological function as autoimmune anti-DNA. The present experiments were designed to further characterize the immune response to DNApeptide complexes. There was considerable variation in the antibody responses of mice from different strains to DNAFus1 immunizations. The range was from virtually no response in C57BL/6 mice to most robust responses in NZW mice. The full-length 52 amino acid carboxy-extension protein of ubiquitin (CEP) in T. cruzi (TCEP) protein from which Fus1 was derived functions equally well as an immunogenic carrier for DNA. Anti-DNA responses were generally weak even though anti-Fus1 and anti-TCEP responses were very strong. The results are discussed with respect to the contrasting roles of T cell help and peripheral B cell tolerance in controlling immune and autoimmune antibody responses to DNA.
Keywords: anti-DNA antibodies, autoimmunity, immunization, mice, tolerance
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Introduction
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Antibodies against mammalian, B-form DNA (nDNA) are diagnostic markers for systemic lupus erythematosus, and contribute to the pathogenesis of lupus nephritis in mice and men (1,2). Spontaneous antibodies to native DNA in autoimmune mice appear to be produced in response to clonally selective, antigen-specific, B cell stimulation (36); nevertheless, the nature and form of the immunogen(s) that induce autoimmune anti-DNA have not been conclusively determined. The progressive selection for nDNA specificity as anti-DNA antibody responses mature in autoimmune (NZBxNZW)F1 mice has implicated nDNA or complexes containing nDNA as the selective stimuli for autoimmune anti-DNA antibody production (7). We have hypothesized that the immunogenic DNA complexes in autoimmune mice may be complexes of DNA with DNA-binding proteins, either autologous or heterologous, in which case and under the appropriate conditions such complexes should be immunogenic in normal mice. In this case, according to Landsteiner's definition (8), nDNA could be considered to be a hapten. Attempts to induce anti-nDNA antibody by immunization with nDNA, either covalently or non-covalently bound to various protein carriers such as histones, poly-L-lysine, bovine
-globulin and mBSA, have generally been unsuccessful (9). As an alternative carrier for DNA we have used the highly immunogenic, 27 amino acid nucleic acid-binding Fus1 peptide. Normal, non-autoimmune-prone mice immunized with complexes of native, mammalian DNA and Fus1 peptide produced anti-DNA antibody that was serologically, structurally and functionally similar to autoimmune anti-DNA antibody (16,17). Fus1 peptide is a synthetic peptide derived from the 52 amino acid carboxy-extension protein of ubiquitin (CEP) in Trypanosoma cruzi (TCEP). TCEP and Fus1 are highly basic and their structures include one C2C2 zinc-finger DNA-binding motif. CEP is associated with the 60S ribosomal subunit (1315) and has been proposed to play a role in ribosome biogenesis (14). CEP have been found in all eukaryotes examined to date and share a high degree of structural homology (1013).
The present experiments were undertaken to provide a better understanding of the parameters that control the immunogenicity of DNAFus1 in mice. Specifically the experiments were designed (i) to determine whether Fus1 binding to DNA is dependent upon the zinc-finger DNA-binding motif, (ii) to determine how mice from different inbred strains would respond to DNAFus1 immunizations, (iii) to determine whether TCEP could also function as an immunogenic carrier for DNA in mice and (iv) to determine whether autologous mouse Fus1 (mFus1) could be an immunogenic carrier for DNA. Although DNATCEP was at least as immunogenic as DNAFus1, the antibody response to DNATCEP was dominated by antibodies specific for TCEP rather than DNA. Autologous mFus1 was a very poor immunogen with or without DNA. Even though Fus1 and TCEP can induce strong T cell help, judging from induced anti-Fus1 or anti-TCEP titers, anti-DNA titers induced with DNAFus1 and DNATCEP were relatively weak. The results suggest that peripheral B cell tolerance to DNA effectively limits induced anti-DNA antibody responses even in the presence of effective T cell help.
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Methods
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Nucleotide sequence for mouse CEP
Poly(A) RNA was isolated from the BALB/c mouse-derived myeloma cell line P3-X63-Ag8.653 (18) using an oligo(dT)cellulose column. cDNA was synthesized by MMLV reverse transcriptase (Life Sciences, St Petersburg, FL) using 2.5 µg poly(A) RNA and the oligonucleotide primer A17, 5'-GCTGCAGCATGCGTCGACTTTTTTTTTTTTTTTTT-3'. The cDNA was PCR-amplified by Taq polymerase using the sense primer Mou1, 5'-GCTGATCATTGAGCCATCCCTTCGTC-3', and the antisense primer A1, 5'-GCTGCAGCATGCGTCGAC-3'. The PCR amplification consisted of 30 cycles with each cycle containing a denaturation step at 95°C for 2 min, annealing step at 55°C for 2 min and an extension step at 72°C for 2 min. The cDNA and the oligonucleotide primers, A1 and Mou1, were generous gifts from Dr John T. Swindle (The University of Tennessee, Memphis, TN). The Mou1 primer was designed based upon a previously published partial sequence of the mouse ubiquitin-carboxyl extension protein (19). The amplified DNA was gel purified (Geneclean kit; Bio101, Vista, CA) and sequenced with the Mou1 primer using a double-stranded DNA cycle sequencing kit (BRL, Grand Island, NY).
Assays for DNA binding to TCEP or Fus1
For the electrophoretic mobility shift assay, 50 pmol of Fus1 (3080 Da), mFus1 (3202 Da), or TCEP (6167 Da) were titrated in 10 µl binding reactions that contained 10.5 ng (0.15 pmol) of 3' 32P-labeled 123 bp DNA, 10% glycerol and 500 µg/ml BSA in TBE (89 mM Tris/89 mM boric acid/2 mM EDTA, pH 8.0). Bound versus free DNA was visualized by 5% non-denaturing PAGE in TBE. Gels were dried, and the DNA, visualized by autoradiography. The 123 bp DNA fragments were generated by complete digestion of a 123 bp ladder DNA (Gibco/BRL, Grand Island, NY) with the restriction enzyme AvaI (New England BioLabs, Beverly, MA) (20). The restriction digest was resolved on a 1% agarose gel. The 123 bp DNA fragments were excised and purified using a Qiaex kit (Qiagen, Valencia, CA). The 123 bp DNA was 3' end-labeled with [
-32P]dCTP using Klenow fragment (Life Sciences).
For the affinity chromatography assay, varying amounts of Fus1 were added to 20 µg of biotin-labeled 200 bp DNA in PEN (10 mM phosphate/1 mM EDTA/150 mM NaCl, pH 7.4) in a final volume of 400 µl. Each mixture was transferred to a 5 ml snap-cap tube containing 1.75 ml streptavidinagarose beads (Sigma, St Louis, MO) packed by centrifugation for 10 min at 1100 g. The contents of the snap-cap tubes were incubated at 37°C for 2 h on an inversion shaker and then centrifuged as above. Supernatants were transferred to new tubes. A competitive inhibition ELISA using Fus1-coated ELISA plates and the mAb Fus1.5.1 was used to measure free Fus1 in each supernatant. As a control, 1.5 µg Fus1 alone were incubated with the streptavidinagarose beads. After centrifugation 1.44 µg remained in the supernatant as measured by the competitive inhibition ELISA. This indicated no significant interaction between Fus1 and the streptavidinagarose beads. The 200 bp DNA was generated by sonicating calf thymus DNA (Sigma) solubilized in TE (21). Sonicated DNA was biotinylated using photobiotin acetate (Pierce, Rockford, IL) according to the manufacturer's directions. No DNA was detected in supernatants after incubation with the streptavidinagarose beads and centrifugation as above.
The zinc-dependence of Fus1 binding to DNA was tested by pre-incubating 0.3 µg Fus1 peptide with 3.75x10 7 mol of the chelators EDTA or 1,10-phenanthroline for 2 h at room temperature in 75 µl borate saline buffer. The chelated Fus1 peptide was then transferred to pre-blocked DNA-coated ELISA plates. Bound Fus1 was detected by Fus1.5.1 mAb. The non-chelating structural analog of 1,10-phenanthroline, 1,7-phenanthroline, was used as a negative control. Both 1,10-phenanthroline and 1,7-phenanthroline were generous gifts from Dr William Taylor (University of Tennessee, Memphis). Stoichiometric calculations were based upon the following molecular masses: Fus1, 3080.7 Da; mFus1, 3201.9 Da; TCEP, 6167.5 Da; and 1 kb DNA, 6.6x105 Da.
Antigens, mice and immunizations
Fus1, mFus1 and TCEP were synthesized by the Molecular Resource Center (University of Tennessee, Memphis) based upon the amino acid sequences indicated in Fig. 1
. The molecular masses of the synthetic peptides were confirmed by mass spectrometry (Quality Controlled Biochemicals, Hopkinton, MA).

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Fig. 1. Comparison of the amino acid sequence for T. cruzi and mouse CEP. The amino acid sequence of the T. cruzi CEP (11) is indicated above the amino acid sequence of the mouse CEP. Solid lines and colons indicate amino acid identity and similarity, respectively. T. cruzi and mouse CEP are 62% identical and 75% similar. The amino acid sequences for Fus1 and mFus1 are indicated within the box. The conserved cysteine residues of the conserved C2C2 zinc-finger DNA binding motif are indicated by an asterisk.
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Female BALB/cAnNHsd, C3H/HeNHsd, C57BL/6NHsd, NZB/Hsd, NZW/Hsd and (NZBxNZW)F1 mice were obtained from Harlan Sprague-Dawley (Indianapolis, IN) at 68 weeks of age. SWR/J and B6.C-H2bm12/KhEg mice were obtained from Jackson Laboratories (Bar Harbor, ME). Mice were housed in a pathogen-free environment at the animal facilities of the University of Tennessee, Memphis and had no detectable serum IgG antibodies to the relevant antigens prior to immunization.
For all immunizations, DNA refers to calf thymus DNA sheared to 13 kbp average size. Complexes of DNA and TCEP or mFus1 were generated similarly to DNAFus1 complexes (16). Antigen was emulsified in complete Freund's adjuvant (Difco, Detroit, MI) for the primary immunization and incomplete Freund's adjuvant (Difco) for the second immunization. Antigens were injected in PEN for subsequent immunizations. Sera were collected 10 days after each immunization. Immunizations were always spaced at least 3 weeks apart. All mice were immunized at least 4 times, i.e. primary plus three boosting immunizations, and there were always at least five mice per experimental group.
Assay for serum antibody
For all assays, DNA refers to calf thymus DNA sheared to 13 kbp average size. Serum antibody binding to DNA was measured in a direct-binding, solid-phase ELISA as described previously (4). Serum antibody binding to Fus1, TCEP or mFus1 was detected by using ELISA plates coated with 5 µg/ml of Fus1, TCEP or mFus1 respectively in borate saline buffer, pH 8.5. Mouse sera were titrated in 3-fold serial dilutions. Bound antibodies were detected with biotinylated goat anti-mouse IgG (Southern Biotechnology, Birmingham, AL) followed by streptavidinalkaline phosphatase. Percent binding was calculated based on the maximum absorbency of an anti-Fus1 mAb, Fus1.5.1. Serum titers were calculated as the reciprocal dilution that gave 50% maximal binding.
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Results
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Nucleotide sequences of mouse and T. cruzi CEP
Mouse and T. cruzi (11) CEP are 65% identical and 62% similar (Fig. 1
). The mouse CEP cDNA sequence is 90% identical with human (22), 92% identical with chicken (23) and 97% identical with rat (24). Mouse, rat, chicken and human CEP have identical amino acid sequences. All of the cDNA nucleotide differences are in wobble positions of the respective codons (data not shown). The 27 amino acid synthetic peptides, Fus1 and mFus1, were derived from amino acids 1844 within TCEP and mouse CEP respectively (Fig. 1
). Fus1 and mFus1 are 67% identical and 74% similar. Both Fus1 and mFus1 peptides are highly basic with a theoretical pI of 10.1 and 10.2 respectively. Fus1 and mFus1 each contain a single C2C2 zinc-finger DNA-binding motif which is conserved among all known CEP proteins (22).
Fus1 binding to DNA
The results of our initial experiments suggested that the anti-DNA response was optimal in mice immunized with DNAFus1 complexes formed by mixing 100 µg DNA with 10 µg Fus1 per immunization per mouse (16). To determine how much peptide can be bound to a given amount of DNA, increasing amounts of Fus1 were added to biotinylated 200 bp DNA. No unbound Fus1 was detected at a weight:weight ratio of DNA:Fus1 as low as 2.5:1 (Table 1
). This would be equivalent to 100 µg DNA:40 µg Fus1. The results also indicate that Fus1 may bind to DNA at a relatively high density. At a weight:weight ratio of 2.5:1, DNA:Fus1, on average 17 molecules of Fus1 peptide would be bound per molecule of 200 bp DNA. This would be ~1 molecule of Fus1 peptide for every 12 bp of DNA. These results indicate that no free or unbound Fus1 was present in the DNAFus1 immunogens used in previous immunizations.
Sequence-specific binding of zinc-finger DNA-binding proteins to DNA generally requires Zn2+ (25). Even though binding of Fus1 to DNA appeared to be independent of nucleotide sequence, there was still a possibility that Fus1 binding would be dependent upon Zn2+. As indicated in Fig. 2
, a 300-fold molar excess of either EDTA or 1,10-phenanthroline did not affect the ability of Fus1 to bind DNA. These results indicate that free Zn2+ was not required for Fus1 binding to DNA and that the C2C2 zinc-finger motif was not required for Fus1 to form immunogenic complexes with DNA. Together all of the above results suggest that Fus1 binding to DNA was independent of nucleotide sequence.

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Fig. 2. Zinc-chelators do not inhibit Fus1 binding to DNA. Fus1 peptide was pre-incubated in 300-fold molar excess of the indicated chelators EDTA and 1,10 phenanthroline and the non-chelating control 1,7-phenanthroline. Binding to DNA was measured in a solid-phase ELISA with a monoclonal anti-Fus1 antibody (Fus1.5). Results are presented as percent of maximum binding to of the peptide to DNA in the absence of chelators. Results are means of triplicate determinations ± SEM.
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An electrophoretic mobility shift assay was performed to quantitatively assess whether TCEP and mFus1 bind DNA similarly to Fus1. The mol. wt of TCEP (6167 Da) is twice that of Fus1 (3080 Da). The theoretical pIs of TCEP and Fus1 are roughly the same, 10.3 and 10.1 respectively. As indicated in Fig. 3
, Fus1, mFus1, and TCEP each had a similar effect on the electrophoretic mobility of a 123 bp fragment of DNA at approximately the same molar ratio of peptide to DNA.

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Fig. 3. DNA binding by Fus1, mFus1 and TCEP. The DNA-binding ability of Fus1 (A), mFus1 (B) and TCEP (C) was compared by mobility shift PAGE. Lanes 28 represent increasing 2-fold dilutions of peptide, as indicated above the lanes, in the presence of 0.15 pmol of 32P-labeled 123 bp DNA fragment. Lane 1 indicates the mobility of the 123 DNA fragment in the absence of peptide.
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Variations in the response to DNAFus1 immunization in mice from different strains
In previous experiments, BALB/c (H-2d) and C3H (H-2k) mice produced both anti-DNA and anti-Fus1 antibodies in response to DNAFus1 immunizations (16). In the present experiments, mice from other strains, each with a different H-2 haplotype, were tested for their immune responsiveness to DNAFus1. C57BL/6 (H-2b) was chosen as another mouse strain with no known genetic predisposition for autoimmunity. NZB (H-2d), NZW (H-2z), SWR (H-2q) and (NZBxNZW)F1 (H-2d/z) were all chosen because of the known genetic association of these strains or their F1 hybrids with lupus-like autoimmunity (2,26). B6.C-H2bm12 (H-2bm12) was chosen because of the known effect of H-2bm12 on anti-DNA antibody production in NZB mice (27).
C57BL/6 mice responded poorly to immunization with Fus1 and DNAFus1 (Table 2
). The H-2bm12 yielded only minor improvement in the C57BL/6 response to DNAFus1 and then only for the production of anti-Fus1 antibody. SWR mice developed serum anti-DNA titers comparable to those induced in BALB/c and C3H mice (16). Because of the high mouse to mouse variation, however, there was no significant difference in serum anti-DNA titers between SWR mice immunized with DNAFus1 and Fus1 (Table 2
). This result may simply indicate the autoimmune tendency of SWR mice. Only SWR mice immunized with DNAFus1 or Fus1 had detectable anti-Fus1 antibody. As expected, (NZBxNZW)F1 mice produced anti-DNA antibodies in response to immunization with DNAFus1 (Table 2
). Once initiated, the anti-DNA response in these mice was sustained (data not shown). This no doubt simply reflects the autoimmune predisposition of (NZBxNZW)F1 mice. Surprisingly, the anti-Fus1 titers for the (NZBxNZW)F1 mice immunized in the above experiment were higher than their anti-DNA titers.
Both NZB and NZW mice produced anti-DNA antibody in response to DNAFus1 immunization (Fig. 4
); however, the response of NZW mice was much more robust than that of NZB. The results in Fig. 4
represent the anti-DNA response of NZB and NZW mice after two immunizations with DNAFus1. After five immunizations with DNAFus1, there was only minimal increase in anti-DNA titers over those measured after two immunizations in both NZB and NZW mice. After two immunizations with Fus1 in adjuvant, NZW mice had serum anti-DNA titers of
200 (data not shown). After two immunizations with DNAFus1, NZB mice produced only barely detectable anti-Fus1 antibody, serum titers <30 (data not shown). Four of the five NZW mice, on the other hand, produced serum anti-Fus1 titers
3000. After four to five immunizations with DNAFus1 or two immunizations with 100 µg Fus1, NZB mice did produce high serum anti-Fus1 antibody titers. In summary, the results indicate that anti-DNA antibody production in response to DNAFus1 immunization varies from strain to strain. NZW mice were the strongest responders to DNAFus1.

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Fig. 4. IgG anti-DNA in NZB and NZW mice immunized with DNAFus1. NZB (open symbols) and NZW (filled symbols) mice received two immunizations each with DNAFus1, 100 µg DNA:10 µg Fus1 per mouse per immunization. Sera were collected 10 days after the second immunization. Titers are the reciprocal serum dilutions that gave the indicated percent of maximum binding to calf thymus DNA in a direct ELISA.
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TCEPDNA complexes induce anti-DNA similarly to Fus1-DNA
Even though the Fus1 peptide was immunogenic and could induce T cell help for anti-DNA antibody production, this same function could not be guaranteed for TCEP. BALB/c, C3H and NZW mice were immunized 5 times each with 20 µg of TCEP alone or in complexes with 100 µg of DNA per mouse per immunization. TCEP at 20 µg per mouse was used to prepare the immunogens since that amount is the molar equivalent of 10 µg of Fus1 peptide, which induces an optimum anti-DNA response when mixed with 100 µg of DNA (16). Mice from all three strains whether immunized with TCEP or DNATCEP developed high titers of IgG anti-TCEP antibody (Table 3
). Except for one C3H mouse, mice from all three strains immunized with DNATCEP made IgG anti-DNA antibody (Fig. 5
). All five of the BALB/c mice and four of five NZW mice immunized with TCEP alone also produced IgG anti-DNA. Only one of five C3H mice immunized with TCEP also made anti-DNA antibody. Whether this derives from the ability of TCEP to bind DNA in situ after immunization is at present unknown. These results indicate that DNATCEP is at least as effective as DNAFus1 for inducing anti-DNA antibodies in mice.

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Fig. 5. IgG anti-DNA induced by immunization with TCEP or DNATCEP. The data represent the maximum IgG anti-DNA titer achieved by (A) BALB/c, (B) C3H or (C) NZW mice after five immunizations with either 20 µg TCEP alone or 100 µg DNA: 20 µg TCEP, as indicated. The number of mice per group is indicated.
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Immunogenicity of DNAmFus1
Since CEP is a highly conserved protein apparently common to all eukaryotes, mice also express a homologue of the CEP protein and the Fus1 peptide. These experiments were designed to determine if the immune response to Fus1 would break tolerance to mFus1. The experiments also tested the potential for mFus1 to be immunogenic in mice either alone or in complexes with DNA. Sera from BALB/c, C3H and NZW mice that had been immunized 4 times each with either Fus1 or DNAFus1 were tested for antibodies reactive to mFus1. All of the mice developed high serum titers of IgG anti-Fus1 antibody. Titers were generally between 5000 and 10,000 after four or five immunizations (Fig. 6
). None of the antisera from BALB/c mice were cross-reactive with mFus1. Roughly one-half of the C3H antisera were cross-reactive with mFus1, and one of four Fus1 immune and three of six DNAFus1 immune antisera from NZW mice were cross-reactive with mFus1. The Fus1-specific antisera from both C3H and NZW mice were generally much less reactive with mFus1 than with Fus1.

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Fig. 6. Cross-reactivity of serum IgG anti-Fus1 with mFus1. Sera from three different strains of mice immunized with (A) 10 µg Fus1 or (B) 100 µg DNA:10 µg Fus1 were assayed for binding to Fus1 and mFus1 by direct ELISA on Fus1 and mFus1-coated ELISA plates respectively. The titer of IgG serum antibody binding to mFus1 (open symbols) and Fus1 (closed symbols) is indicated on the ordinate.
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The anti-DNA response initiated by DNAFus1 does not become an autoimmune response driven by autologous DNApeptide antigens (16). To determine if the self-peptide mFus1 can be immunogenic, BALB/c, C3H and NZW mice were immunized with mFus1 alone or DNAmFus1 complexes. MFus1 and DNAmFus1 were poor immunogens for inducing anti-DNA or anti-peptide antibodies in C3H mice, even though C3H anti-Fus1 antibodies were the most cross-reactive with mFus1 (Fig. 6
and Table 4
). BALB/c mice responded poorly to mFus1 alone; however, most of the BALB/c mice immunized with DNAmFus1 produced anti-mFus1 antibodies (Table 4
), but little to no anti-DNA. The anti-DNA response after DNAmFus1 immunization was no better than that after mFus1 in BALB/c mice. Interestingly the anti-mFus1 antibody cross-reacted with Fus1 in only eight of the 16 BALB/c mice that produced anti-mFus1 antibody. All of the NZW mice produced IgG anti-DNA after four immunizations with either mFus1 or DNAmFus1. In contrast to immunization with DNAFus1, there was no increase in serum anti-DNA after only two immunizations with DNAmFus1 in NZW mice (data not shown). By the fourth immunization, the majority of NZW were producing anti-DNA; however, the average serum titer was similar to that for NZW immunized with mFus1. Even after five immunizations with 100 µg mFus1 peptide per immunization, only very low titer IgG anti-mFus1 antibodies were elicited and then in only half of the NZW mice immunized. In summary, mFus1 when combined with DNA was not as effective as Fus1 for inducing anti-DNA. BALB/c were the only mice to make significant anti-mFus1 antibody, mostly in response to DNAmFus1 immunization.
Lin et al. (28) demonstrated that co-immunization with the self protein mouse cytochrome c and the related protein human cytochrome c can break tolerance for the self cytochrome c. Mamula et al. (29) were able to achieve a similar result with the nuclear autoantigen snRNP. BALB/c and NZW mice were immunized with complexes formed with DNA and both Fus1 and mFus1 (Table 4
). After five immunizations, all of the BALB/c mice produced high titers of antibody that bound Fus1. The antibodies in two of the five BALB/c reacted equally well with mFus1. None of the five BALB/c mice produced anti-DNA. Four of five of the NZW mice produced high titers of antibody to Fus1 that reacted with mFus1 relatively weakly. The NZW mice all produced relatively low titers of anti-DNA in response to the mFus1-DNAFus1 immunizations and the anti-DNA was present only after the fourth immunization (data not shown). These results indicate that DNAFus1 was a better immunogen for inducing anti-DNA in NZW mice. The results also indicate that coincident immunization with Fus1 was not generally effective in breaking tolerance to mFus1.
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Discussion
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Immunization of normal, non-autoimmune-prone mice with DNAFus1 has been used to induce anti-DNA antibody with similar structure, specificity and pathogenetic potential as autoimmune anti-DNA (16,17). These results have been important because they indicate that even non autoimmune-prone mice have the immune potential to produce disease-inducing anti-DNA antibody. The amino acid sequence of the immunogenic, DNA-binding, 27 amino acid Fus1 peptide was derived from the CEP protein from T. cruzi (TCEP). The 52 amino acid CEP protein is a carboxy extension protein of ubiquitin and is highly conserved among all eukaryotes (11). CEP and Fus1 are highly basic and contain a single C2C2 zinc-finger nucleic acid binding domain (22). The present results indicate that Fus1 binding to DNA is independent of the zinc-dependent conformation of the zinc-finger motif and most likely dependent upon the highly basic charge of the peptide. TCEP functioned at least as well as Fus1 as an immunogenic carrier for DNA, and DNATCEP complexes induced antibody to both DNA and TCEP. mFus1, the mouse homologue of Fus1, on the other hand, was generally a poor immunogen and likewise was not an effective carrier for DNA. Even though Fus1 and mFus1 are structurally very similar, antibodies to Fus1 generally do not react with mFus1 or do so only weakly. Both Fus1 (16) and TCEP have been more immunogenic when injected as complexes with DNA than when injected as isolated peptides.
There were differences in the immune responses of mice with different H-2 haplotypes to immunizations with either Fus1 or DNAFus1. NZW, H-2z mice produced the highest titers of anti-DNA and anti-Fus1 antibodies in response to Fus1 and DNAFus1 immunizations, even higher than those produced by autoimmune-prone H-2d NZB mice. This result is extremely interesting in light of the known H-2z linkage for autoimmune anti-DNA antibody production in (NZBxNZW)F1 backcross mice (3032). Also consistent with this result, the majority of Fus1-specific, CD4+ T cell lines and hybrids generated from DNAFus1 immunized (NZBxNZW)F1 mice were H-2z restricted (M. Wang et al., in preparation). The bm12 mutation in Aßb enhances anti-DNA autoantibody production in NZB.H-2bm12 mice (27); however, anti-DNA and anti-Fus1 responses of B6.C-H2bm12 mice to DNAFus1 and Fus1 were only slightly better than the very weak responses of normal B6 mice. Similarly, H-2q SWR mice responded poorly to Fus1 and DNAFus1 immunizations although they did produce anti-DNA antibody. In previously published results, antigen-presenting cells (APC) from SWR mice were also unable to stimulate T cells from lupus-prone (SWRxNZB)F1 that were specific for histone peptides derived from nucleosomes (33,34). H-2d/q APC and I-Ad-transfected L cells were the most effective APC for nucleosome-derived histone peptides. The pI and amino acid sequence of Fus1 are similar to those of the reactive histone peptides. The results indicate that the potential for anti-DNA antibody production in response to DNAFus1 immunization can be correlated with autoimmune anti-DNA antibody production in NZB and NZW mice, but not necessarily with other autoimmune strains.
The anti-DNA antibody responses induced by both DNAFus1 and DNATCEP have been relatively poor compared to anti-hapten responses induced by haptenated proteins and are weaker than autoimmune anti-DNA responses. Serum levels of anti-DNA induced with DNAFus1 (16) and DNATCEP have generally been between 5 and 10 µg/ml but may be as high as 200 µg/ml. Serum anti-DNA titers in autoimmune (NZBxNZW)F1 mice are generally
2400, which is equivalent to
400 µg/ml of mAb DNA6 (35). After secondary DNAFus1 or DNATCEP immunizations, subsequent immunizations did not induce increased anti-DNA titers. Anti-Fus1 and anti-TCEP titers were much higher than anti-DNA titers after multiple immunizations with DNATCEP and DNAFus1, and anti-TCEP and anti-Fus1 titers did continue to increase after tertiary and quaternary immunizations, and reached titers comparable to those induced by most heterologous protein antigens. There is no obvious reason to suspect that Fus1- or TCEP-induced T cell help would not be as effective for DNA-specific B cells as Fus1- or TCEP-specific B cells. Most likely the relatively poor DNA-specific antibody response induced by immunization with DNAFus1 or DNATCEP was due to an inhibition of DNA-specific B cells that could not affect Fus1- or TCEP-specific B cells. The majority of DNA-specific B cells in DNAFus1 or DNATCEP immunized mice would most likely have bound to internally derived nucleosomal DNA that would neither be bound to follicular dendritic cells nor contain Fus1 or TCEP. Consequently those B cells would have died by apoptosis because they had bound free nucleosomes or nucleosomal DNA and would not have interacted with follicular dendritic cells or received cognate T cell help (3638). In mice transgenic for production of an anti-double-stranded DNA antibody, B cells are excluded from lymphoid follicles and rapidly turn over at the TB interface (39). This was an extremely important result because it suggested that soluble nucleosomal antigens including DNA must exist within and can be bound by B cells in the follicular and interfollicular environment. TCEP- and Fus1-specific B cells would not have been subject to peripheral tolerance induction.
The majority of pre-autoimmune (SWRxNZB)F1 mice immunized with nucleosomes also produced <10 µg/ml of serum anti-DNA or anti-histone-DNA (33). Serum levels of anti-DNA induced by inoculation of mice with polyomavirus BK (40) or immunization with a recombinant expression vector for the SV40 large T antigen (41) were also generally low. In both of these experimental systems, Th cells were specific for peptides derived from proteins that are bound to nucleosomal DNA. In both experimental systems T cell help was not limiting. Every DNA-specific B cell that presents relevant histone or large T antigen peptides respectively should receive cognate T cell help. The nucleosomes bound by such B cells, however, would most likely be soluble nucleosomes encountered within the extracellular milieu rather than bound to the surface of follicular dendritic cells in immune complexes. Soluble nucleosomes may induce B cell tolerance even in the presence of T cell help (42). The secondary immune characteristics of the autoimmune anti-DNA antibody response in mice may depend upon more than just the provision of T cell help to DNA-specific B cells. Autoimmune B cells must also be able to thwart peripheral tolerance mechanisms that apparently block clonal expansion of DNA-specific B cells in mice not genetically prone to autoimmune disease. The results from recent studies indicate that the systemic lupus erythematosus-associated genetic locus Sle-1 functions to reduce B cell tolerance to nucleosomal antigens (43,44).
Even though induced anti-DNA titers were generally low in non-autoimmune-prone C3H and BALB/c mice immunized with DNAFus1, mice from both strains developed proteinuria and had glomerular IgG deposits after three to four immunizations (16,33,40). The same was true for polyomavirus BK inoculated (16,33,40) mice and (SWRxNZB)F1 mice (16,33,40). These results suggest that continuous or repeated production of relatively low levels of anti-DNA antibody over a period of weeks or months may be sufficient to initiate lupus nephritis.
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Acknowledgments
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The authors wish to acknowledge Dr Meera Krishnan for helpful discussion throughout the tenure of this study, and Dr Ole Petter Rekvig for helpful discussion and critical review of the manuscript. This research was supported by grants AI26833 and AR45219 from the National Institute of Allergy and Infectious Diseases and the National Institute for Arthritis and Musculoskeletal and Skin Diseases respectively (NIH).
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Abbreviations
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Fus1 peptide derived from an internal amino acid sequence of CEP |
mFus1 mouse homologue of Fus1 |
nDNA native DNA |
TCEP ubiquitin carboxy extension protein from T. cruzi |
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Notes
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Transmitting editor: J. F. Kearney
Received 5 June 2000,
accepted 28 July 2000.
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