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*
Division of Rheumatology, Department of Medicine, and
Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY 10461
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The anti-dsDNA Abs found in patients with SLE and in the murine models of the disease display characteristics of Abs arising in an Ag-driven response (9, 10, 11, 12). However, the antigenic trigger for the production of anti-dsDNA Abs has yet to be conclusively identified. Bacterial (but not mammalian) DNA, complexes of DNA and DNA binding proteins, and bacterial polysaccharides can induce anti-dsDNA Abs in various experimental models (13, 14, 15, 16, 17).
We previously screened a phage peptide display library with the murine IgG2b Ab R4A to identify a peptide mimotope for autoantigen. R4A binds to dsDNA and fibronectin and deposits in glomeruli of nonautoimmune mice. The 5-mer peptide DWEYS in- hibited binding of R4A to dsDNA as well as binding of R4A to renal tissue (18). We have recently demonstrated that nonautoimmune BALB/c mice develop lupus-like autoimmunity when immunized with the peptide DWEYSVWLSN (19) (containing the DWEYS sequence) attached to a polylysine backbone. Peptide-immunized mice develop anti-dsDNA Abs as well as other autoantibodies characteristic of lupus, including anti-histone, anti-cardiolipin, and anti-Sm/ribonucleoprotein (RNP) Abs. At 3 mo of age, immunohistochemical studies demonstrate the presence of IgM and IgG deposits in renal glomeruli of immunized mice.
To further understand the molecular basis for the generation of autoreactivity in peptide-immunized BALB/c mice, we analyzed 24 monoclonal IgM Abs derived from BALB/c mice immunized with multimeric DWEYSVWLSN, and reactive with peptide and/or autoantigen. We focussed on IgM Abs initially to try to identify Abs that may be in their germline configuration as well as somatically mutated Abs. Two important observations can be made from an analysis of these hybridomas. Using peptide libraries, it is possible to derive true peptide molecular mimics for dsDNA. Furthermore, because VH and VL genes and VH-VL combinations used in the autoantibody response in peptide-immunized BALB/c mice were similar to those used in the anti-dsDNA response in (NZB x NZW)F1 mice, peptide-induced autoimmunity may be a useful model to study the autoimmunity that arises in response to foreign Ag.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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R4A is a murine IgG2b anti-dsDNA Ab that deposits in glomeruli of nonautoimmune BALB/c mice. This Ab is encoded by S107 VH11-unknown D-JH4 and VK1-JK1 gene segments (20). The immunizing peptide DWEYSVWLSN was derived as previously described (18) by screening a phage peptide-display library with R4A. DWEYSVWLSN inhibits the binding of R4A to dsDNA (18) and induces anti-dsDNA and other autoantibodies by immunization (19).
Peptides and immunization
BALB/cJ mice were obtained from The Jackson Laboratory (Bar Harbor, ME). The immunizations were performed as described previously (19). Briefly, DWEYSVWLSN was prepared for immunization on an eight-branched polylysine backbone (multiple antigenic peptide (MAP) from ANA SPEC, San Jose, CA; peptide on MAP from Research Genetics, Huntsville, AL). Six-week-old female mice were immunized s.c. with 100 µg of MAP-peptide in CFA H37 Ra (Difco, Detroit, MI), followed by a boost of 100 µg of MAP-peptide in IFA s.c. 7 and 14 days later. Mice were housed in the Albert Einstein College of Medicine specific-pathogen free animal facility.
As described previously (19), only mice immunized with MAP-peptide in adjuvant developed significant autoantibody titers. Mice immunized with 1) MAP backbone alone in CFA and boosted twice with IFA (19), or 2) MAP conjugated to an irrelevant peptide using the same adjuvant schedule (data not shown) failed to generate significant increases in serum autoantibody levels.
Spleen cell fusions
One week (fusion 1) and 2 mo (fusion 2) after the final boost, mice were sacrificed, and the spleen cells were fused to the NSO myeloma cell line at a splenocyte to myeloma cell ratio of 1:1 using standard hybridoma technology (21). Hybrids were screened for binding to peptide, dsDNA, Sm/RNP, and cardiolipin by ELISA (see below). Positive wells were cloned by limiting dilution.
ELISAs
ELISAs for peptide and autoantigen reactivity were performed as previously described (19). Salmon sperm dsDNA (Calbiochem Novabiochem, La Jolla, CA) was purified by filtration with a 0.45-µm pore size filter (Millipore, Bedford, MA), adsorbed to Immulon II 96-well microtiter plates (Dynatech, Chantilly, VA) at a concentration of 100 µg/ml in PBS, and dried overnight at 37°C. Cardiolipin (Fluka, Ronkonkoma, NY) at a concentration of 75 µg/ml in ethanol was adsorbed to Immulon II 96-well microtiter plates at room temperature overnight. MAP-DWEYSVWLSN (Research Genetics, Huntsville, AL) at 20 µg/ml in PBS and Sm/RNP (Immunovision, Springdale, AR) at 10 µg/ml in PBS were adsorbed to Immulon II 96-well microtiter plates at 4°C overnight. Plates (for all Ags) were then blocked with 3% FCS (HyClone, Logan, UT) in PBS for 1 h at 37°C, and incubated with hybridoma supernatant for 2 h at room temperature. Plates were washed five times with PBS/0.05% Tween-20, and alkaline phosphatase-conjugated goat anti-mouse IgM and IgG (Southern Biotechnology Associates, Birmingham, AL) diluted 1/1000 in 3% FCS/PBS was added for 1 h at 37°C. The ELISAs were developed by adding the alkaline phosphatase substrate p-nitrophenyl phosphate (Sigma, St. Louis, MO), and the OD was monitored at 405 nm using a ELISA Titertek Reader (Titertek, Huntsville, AL).
For reactivity with BSA (Roche, Indianapolis, IN), cytochrome c, lysozyme, fibronectin (Sigma) and phosphorylcholine linked to keyhole limpet hemocyanin, plates were coated with Ag at 10 µg/ml in PBS or carbonate coating buffer (for fibronectin) overnight. The ELISA was then performed as described above. Cells from positive wells for peptide and/or autoantigen (dsDNA, Sm/RNP, cardiolipin) were cloned, and the culture supernatants were isotyped and quantitated by ELISA using purified mouse Ig standards from Sigma. Normalized supernatants were assayed on the peptide and autoantigen panel, and positive clones were selected for sequencing.
RNA isolation and PCR
Total RNA was extracted from 2 x 107
cells using the Ultraspec RNA isolation system and the manufacturer’s
protocol (Biotecx, Houston, TX) or the cesium chloride extraction
method. Two micrograms of total RNA was reversed transcribed using the
Superscript Pre-Amplification system (Life Technologies, Gaithersburg,
MD), and an oligo(dT)- or a 3' gene-specific oligonucleotide primer
(
constant region TGGATGGTGGGAAGATG and IgM constant region
GCAGGAGACGAGGGGGA). This was followed by PCR amplification using the
constant region primer and a set of seven 5' degenerate primers for
the light chain, and the IgM heavy chain primer and a set of 10
degenerate 5' primers for the IgM heavy chain (22). The
PCR reactions were performed in a Perkin-Elmer 9700 thermal cycler
(Perkin-Elmer, Palo Alto, CA) for 35 cycles, with denaturation at
94°C for 50 s, annealing at 50°C for 50 s, and extension
at 72°C for 40 s. The PCR products were purified using the
QIAquick PCR purification kit (Qiagen, Valencia, CA).
Sequencing and data analysis
Automated sequencing of the PCR products was performed at the DNA sequencing facility of the Albert Einstein College of Medicine using the ABI 377 sequencer (Applied Biosystems, Foster City, CA). Analysis was performed comparing the obtained sequences to the GenBank database using the Advanced Blast search program from the National Center for Biotechnology Information, National Institutes of Health.
SDS-PAGE and Western blotting
Protein separation and Western blotting were performed as
previously described (23). Briefly, 
250 ng of Ig from
the hybridoma supernatants was boiled for 5 min in sample buffer with
and without the presence of DTT (Calbiochem, La Jolla, CA). The samples
were loaded in a 4–15% Tris-HCl Precast SDS-PAGE minigel (Bio-Rad,
Hercules, CA) and run at 100 V for 10 min for the stacking gel and 150
V for 40 min for the running gel. Polyvinylidene difluoride membranes
(NEN Life Science Products, Boston, MA) were presoaked in methanol for
15 s, washed in water for 2 min, and soaked in transfer buffer (25
mM Tris, 192 mM glycine, and 10% methanol) for 5 min at room
temperature. The proteins were transferred to the polyvinylidene
difluoride membrane at 200 mA for 1 h at room temperature using a
minigel transfer apparatus from Bio-Rad. The membrane was blocked with
5% nonfat milk in PBS for 1 h at room temperature and washed
twice in PBS/0.05% Tween 20. The membrane was incubated with
HRP-labeled goat anti-mouse IgM (Southern Biotechnology Associates,
Birmingham, AL) at a dilution of 1/5000 in 5% milk/PBS for 30 min at
room temperature and washed twice with PBS/0.05% Tween-20. The
chemiluminescence assay was developed using the
ECL+ Western blotting detection system (Amersham,
Aylesbury, U.K.) and was photographed with ECL Hyperfilm (Amersham)
after about 60 s.
Inhibition ELISAs
Salmon sperm dsDNA (Calbiochem Novabiochem, La Jolla, CA) was adsorbed to Immulon II 96-well microtiter plates (Dynatech) at a concentration of 100 µg/ml in PBS and dried overnight at 37.C. The plates were blocked with 3% FCS/PBS for 1 h at 37.C. Abs at 20 µg/ml in PBS were incubated with serial dilutions of the immunizing peptide MAP-DWEYSVWLSN for 1 h at room temperature and transferred to the dsDNA-coated plates for 2 h at 37.C (final Ab concentration, 10 µg/ml). The ELISA was then continued as described above.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To understand the generation of autoimmunity following immunization of BALB/c mice with the dsDNA surrogate DWEYSVWLSN, we analyzed IgM mAbs derived from these mice. Two fusions were performed, and IgM-producing B cells were isolated. Abs 23-7, 36-3, 20-1, and 12-16 were isolated from fusion 1, while the rest of the Abs were isolated from fusion 2. As there was no major differences in the gene usage and somatic mutation in Abs from each of these fusions (see below), the Abs from both fusions have been considered together as a group.
Three groups of Abs are discernible based on their antigenic
specificity (Table I
): 1) Abs reactive
with peptide alone (26-20, 23-7, 32-13, 8-3, 39-9), 2) anti-peptide
Abs cross-reactive with one or more autoantigens (dsDNA, Sm/RNP,
cardiolipin, fibronectin; 15-16, 18-1, 5-1, 25-30, 29-3, 5-16, 2-4,
16-19, 18-18, 16-9, 19-43, 31-26, 29-5, 37g), and 3) autoantibodies
(almost all reactive with DNA alone) that do not bind to peptide
(19-19, 36-3, 38-11, 20-1, 12-16).
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To study the nature of the polyreactivity of some of the elicited
autoantibodies, we studied whether polyreactive Abs (defined
arbitrarily as binding to three or more of the peptide and autoantigen
panels) would also bind to irrelevant Ags (Table II). Of the 10 tested autoantibodies
meeting the definition for polyreactivity, only 16-9 and 19-43 reacted
strongly with all of the tested Ags; 29-3 bound weakly to BSA,
cytochrome c, and lysozyme, and 18-18 bound weakly to
phosphorylcholine-keyhole limpet hemocyanin; other Abs in this panel
(5-1, 29-5, 15-16, 5-16, 32-13, 37g) did not bind to any of the
irrelevant self or foreign Ags. Thus, the polyreactivity detected is
not pan-reactivity but, rather, probably reflects the presence of a
common antigenic epitope among a variety of autoantigens.
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, the immunizing peptide
inhibited 90% of the binding of 16-9 and 19-43 to dsDNA, supporting a
specific binding of these Abs to the tested Ag mediated through an
identical or contiguous Ag binding site. Furthermore, Sm/RNP and
cardiolipin showed a similarly high degree of inhibition of the 16-9
and 19-43 Abs binding to dsDNA (data not shown).
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VH and VL gene usage
The nucleotide sequences of the heavy and light chain variable
regions are available through GenBank (accession nos. AF178584–178607
for the heavy chains and AF178608–178631 for the light chains). The
VH and VL gene usage of the
monoclonal IgM Abs is given in Table III.
The VH and VL gene families
used in the autoantibody response in immunized mice are similar to the
repertoire present in the anti-dsDNA response in spontaneous as
well as induced lupus, with representation of multiple heavy and light
chain gene families. Eight Abs used a J558 heavy chain, with two Abs
(38-11, 18-18) exhibiting the highest degree of homology to the J558
BW-16 VH gene closely associated with the
anti-DNA response in (NZB x NZW)F1 mice
(24). While 16-19 is more closely homologous to a
different J558 family member, this Ab appears to be clonally related to
18-18 (see below), and therefore the 16-19 heavy chain may also be
encoded by VHBW-16. Four Abs used a
VH derived from the VH606
family. The presumed VH606 germline encoding the
heavy chain of these Abs has been reported in the IgM anti-DNA
response in (NZB x NZW)F1 mice
(25). A VH gene from the
VH36–60 family used to encode one Ab was
homologous to the germline gene used to encode an anti-arsonate Ab,
but showed poor homology to a VH36–60 gene
encoding for an anti-DNA Ab in an (NZB x
NZW)F1 mouse (12). The
VH11 S107 gene used in the 20-1 Ab was 96%
homologous to an anti-DNA Ab derived from an (NZB x
NZW)F1 mouse immunized with bacterial DNA
(26). Two Abs used a 7183-derived heavy chain that was
98% homologous to the VH238 V segment used in
the anti-dsDNA response in a MRL/lpr mouse
(9), and two Abs used the Vgam3.2-derived heavy chain used
to encode for anti-insulin Abs (27). Three Abs used a
VHQ52 heavy chain that was 100% homologous to
the germline gene used in an IgM anti-DNA Ab derived from a BALB/c
mouse (28).
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Somatic mutation
Somatic mutation was not essential to encode an autoantibody response. The 25-30 Ab, which bound peptide and Sm/RNP, had both heavy and light chains that were germline encoded. Yet, as reported previously for IgM anti-DNA Abs isolated from (NZB x NZW)F1 lupus-prone mice (29), most of the IgM Abs isolated displayed somatic mutations throughout the heavy and light chain variable regions. In the heavy chain, the number of mutated bases ranged from 0–20, with most Abs having <10 mutations. Similarly, most of the light chains differed from the putative germline gene by <10 bases.
Arginines in VH CDR3
Anti-DNA Abs in spontaneous murine lupus are characterized by an
increased number of arginine residues in the
VHCDR3 (11). This observation had
suggested that the acquisition of an arginine through N-terminal
additions, D region reading frame shifts, or other mechanisms in
spontaneous murine lupus substantially contributes to binding to dsDNA,
and that this residue is selected for during affinity maturation of the
anti-DNA response. It is thought that arginine contributes to DNA
binding through formation of ionic bonds with the negatively charged
phosphodiester backbone of DNA. An arginine in
VHCDR3 is present in 11 of the 14 mAbs that binds
with moderate to high affinity to dsDNA (
0.5 OD units, Table I
),
compared with only 3 of 10 Abs that show weak or no dsDNA binding
(Table IV). One Ab that binds to dsDNA,
38-11, has two N-nucleotide-encoded arginines in
VHCDR3 at positions 95 and 97.
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The 20-1 Ab is encoded by the S107
VH11-DHQ52-JH2
and VK1-JK1 segments
(20). The R4A Ab used to isolate the DWEYS peptide has a
very similar structure, encoded by S107
VH11-unknown D-JH4 and
VK1-JK1 segments. The heavy
chain variable regions of R4A and 20-1 differ only by a conserved
glycine to alanine change in FR3 (Fig. 2A). Both D regions have an
arginine at position 95, while the JH regions
differ by two amino acids (Fig. 2A). The light chains of
20-1 and R4A also have small differences, a single amino acid in FR1
and two amino acid differences in light chain CDR1 (Fig. 2B). Although we cannot directly compare the antigenic
specificities of R4A (IgG2b) to those of 20-1 (IgM) because of the
differences in isotype, we assume that the loss of binding to peptide
by 20-1 is due to the differences from R4A in the light chain.
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There appear to be four pairs of clonally related Abs, sharing
identical VH, DH,
JH, VL, and
JL gene segments. 16-19 and 18-18 differ by five
nucleotide substitutions in the heavy chain, leading to three amino
acid substitutions in FR1 and a serine to glycine change in CDR2. In
the light chain there are eight nucleotide differences, leading to two
amino acid substitutions in FR1 and four amino acid substitutions in
FR2. Abs 5-1 and 5-16 have five identical substitutions throughout the
heavy chain variable region, have an identical
VHCDR3, and share the same truncated
JH4. Ab 5-1 and 5-16 have a
VK2-encoded light chain with nine nucleotide
differences, leading to one substitution each in FR1, CDR2, and FR3,
and three replacements in FR3. These light chain differences probably
account for the minor differences in binding of 15-16 compared with 5-1
(Table I). Ab 19-19 has a heavy chain VDJ identical to those of 5-1 and
5-16, but the light chain is encoded by a VK21E
light chain. As 19-19 binds DNA with less affinity than Abs 5-1 and
5-16, it is tempting to postulate that this Ab has a receptor-edited
light chain in a partially successful attempt to shift away from
autospecificity. Abs 16-9 and 19-43 have six nucleotide differences in
the heavy chain, leading to a single amino acid difference in FR2 and
two amino acid differences in CDR2. An identical
VK21E, presumably germline-encoded, gene encodes
the light chains of 16-9 and 19-43. Both 16-9 and 19-43 are broadly
cross-reactive with all the lupus-specific and nonspecific Ags
examined, with the CDR2 differences probably accounting for the small
differences in binding affinities (Tables I and II). Abs 8-3 and 39-9,
which bind peptide alone, have a single nucleotide difference leading
to an amino acid change in the heavy chain FR1, and six nucleotide
differences in the light chain leading to four amino acid differences
in FR1.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In spontaneous murine SLE, pathogenic autoantibodies are primarily of the IgM and IgG isotypes, and the IgG Abs are clonally related to the IgM Abs. The multiple features of structural similarity of anti-DNA Abs isolated from peptide-immunized mice and described above to spontaneous anti-DNA Abs from lupus mice strongly support the relevance of this model to the study of systemic autoimmunity. We are now isolating cross-reactive IgG Abs from peptide-immunized BALB/c mice with similar specificities to the IgM Abs described in this paper. Analysis of these IgG autoantibodies is in progress.
(NZB x NZW)F1 mice undergo a spontaneous isotype switch from IgM to IgG in the anti-DNA response at about 6 mo of age. Tillman et al. (12) studied many IgM anti-DNA Abs from (NZB x NZW)F1 mice and found that IgM anti-DNA Abs showed evidence of somatic mutation with selection for changes in the Ag binding pocket, suggesting an Ag-driven response. Furthermore, in individual mice, IgM and IgG anti-DNA Abs may be clonally related, suggesting that high affinity IgG anti-DNA Abs arise by clonal expansion and differentiation of IgM anti-DNA Abs present earlier in autoimmune mice. Similar results have been reported by Hirose et al., who found that at least some IgG anti-DNA-producing B cells originate from clonally related IgM anti-DNA Abs (29). The model they suggest for the progression of the anti-DNA response in the (NZB x NZW)F1 lupus mouse is clonal expansion and selection of nonmutated IgM anti-DNA Abs, followed by somatic mutation beginning before isotype switching, isotype switching to IgG, and further accumulation of mutations increasing the affinity for DNA. We isolated here from mice immunized with a peptide mimotope for dsDNA clonally expanded Abs of the IgM isotype that displayed somatic mutations. Because IgM and IgG deposition is present in renal glomeruli of these mice, we believe that the Abs described in this report are themselves potentially pathogenic and are precursors of the pathogenic IgG anti-DNA Abs that are present in peptide-immunized BALB/c mice.
It has been postulated that in lupus, peptides processed from
anti-DNA Abs may activate autoreactive T cells, leading to
auto-up-regulation and T cell help for anti-DNA Ab production. Hahn
and coworkers (31) reported spontaneous T cell
autoreactivity to idiopeptides derived from certain J558 anti-DNA
Abs before the onset of disease in autoimmune mice. Tolerizing young
(NZB x NZW)F1 mice to these peptides had a
significant effect on disease, with decreased titers of anti-DNA
Abs, improved renal function, and prolonged survival (32).
The heavy chains of four of the eight J558 Abs described in this paper
have a 80% amino acid homology to the heavy chain of the A6.1
anti-DNA Ab from which these immunogenic (and tolerogenic)
idiopeptides were derived. Interestingly, five of the Abs we describe
in this report had a >80% amino acid homology (four Abs had >90%)
in the CDR2-FR3 region (residues 58–69) from which the p58 peptide was
derived. This leads us to hypothesize that one possible mechanism for
the autoimmunity seen in peptide-immunized mice is through
up-regulation of autoreactive T cells against self-peptide determinants
derived from Abs arising in the anti-peptide response. While Hahn
and coworkers (31) demonstrated that BALB/c mice had no
spontaneous immunity to VH-derived peptides, the
activation of B cells expressing Abs with a p58-like peptide in their
sequence may have converted the mice to responders.
How do autoantibodies that no longer bind peptide arise in response to peptide immunization? As the portion of the DWEYSVWLSN peptide that mimics DNA is DWEYS (18), we have begun to analyze epitope recognition in DWEYSVWLSN, comparing the binding of our mAb panel to MAP-DWEYSVWLSN and MAP-DWEYSAAAAA. While for many Abs the binding was comparable (data not shown), several Abs (18-1, 29-3, 32-13) displayed a much higher binding to MAP-DWEYSAAAAA than to MAP-DWEYSVWLSN. Furthermore, the 12-16 Ab, which bound dsDNA but not peptide (MAP-DWEYSVWLSN), bound well to MAP-DWEYSAAAAA. This suggests that some anti-DNA Abs from MAP-DWEYSVWLSN-immunized mice that do not recognize the complete immunizing peptide may still recognize the DWEYS peptide motif that mimics DNA.
What are the implications of the use of peptide on a polylysine backbone to induce an autoimmune response to understanding a possible role of foreign mimotopes in breaking tolerance? Although Ags with highly repetitive epitopes are usually T independent, peptide-induced autoimmunity is a T cell-dependent response, as indicated by the presence of somatic mutations in the elicited autoantibodies and data not shown (M. Khalil and B. Diamond, manuscript in preparation). The nature of the T cell epitope in peptide-induced autoimmunity is of obvious interest and is being actively pursued at this time. Preliminary data suggest that the MAP backbone may be part of the T cell epitope, or alternatively, may play a role in targeting the Ag to a specific intracellular compartment for processing (M. Khalil and B. Diamond, manuscript in preparation).
Although the anti-dsDNA specificity is closely associated with
nephritis, lupus nephritis can occur in patients with SLE and in mouse
models for the disease without high titers of anti-dsDNA Abs. The
etiology of glomerulonephritis in these instances is not clear
(2). We isolated anti-peptide Abs that did not bind or
bound weakly to DNA. Several of these Abs bound fibronectin (Table I).
Although we do not yet have evidence that such Abs are pathogenic, we
demonstrate here that Abs arising in an anti-foreign response
(peptide) can acquire a pathogenic, potentially nephritogenic
specificity (fibronectin) without binding to dsDNA. We postulate that a
similar scenario may contribute to lupus nephritis presenting without
the serological hallmark of anti-dsDNA Abs.
Using a peptide bound by the anti-DNA Ab R4A, we elicited the 20-1 Ab, which shows close structural homology to R4A and also binds DNA. Thus, the DWEYSVWLSN peptide was also an immunogenic mimic of DNA and elicits an Ab structurally homologous to the R4A Ab used to initially select the peptide. Other laboratories have produced similar results. Demangel et al. recently reported that a phage clone bound by an anti-malaria Ab elicited an anti-peptide response that also bound malaria (33). Comparing two malaria-binding mAbs from the immunized mice to the parent Ab initially used to derive the peptide immunogen, similarities were found in the heavy and light chain variable region sequences. Similarly, Valadon et al. found that a peptide that in- hibits binding of capsular polysaccharide of Cryptococcus neoformans to a protective anti-polysaccharide Ab elicits anti-peptide mAbs (albeit that do not bind polysaccharide) with a high VK-JK homology to the parent mAb (34). The similarity in DNA binding and structure between R4A and 20-1 described here confirms the utility of the phage peptide display technology in defining peptide antigenic mimics for nonprotein Ags. Nevertheless, while 20-1 is similar to R4A, by peptide immunization we elicited cross-reactive Abs encoded by several different heavy and light chain gene families, indicating that a structurally similar binding site can be encoded for by more than one heavy and light chain combination.
The VHBW-16 heavy chain variable region gene is closely associated with the anti-dsDNA response in lupus mice in both the (NZB x NZW)F1 and MRL/lpr models (24). Moreover, Abs with a heavy chain encoded by this J558 family member have been demonstrated to be pathogenic. Interestingly, VHBW-16 encoded heavy chains have also been recently identified in several models of experimental anti-dsDNA responses in nonautoimmune mice (35, 36). Eilat et al. studied the characteristics of Abs using VHBW-16-encoded heavy chains in nonautoimmune mice with an induced anti-dsDNA response and in diseased (NZB x NZW)F1 mice (24). In comparison to mice with spontaneous lupus, VHBW-16-positive Abs from mice with induced lupus were clonally restricted, had a lower frequency of class switching from IgM to IgG, and had decreased affinity for DNA. Although we did not measure the affinity of the Abs encoded by the VHBW-16 heavy chain to DNA, these Abs were IgM, and two of the three were clonally related. Our data are consistent with the observations by Eilat et al. and confirm that anti-dsDNA Ab responses in nonautoimmune mice induced by a variety of stimuli may be genetically similar to the anti-dsDNA Ab response in spontaneous lupus.
It is of interest to compare the autoantibodies derived from peptide-immunized mice to those reported in other well-described models of induced autoimmunity. Gilkeson et al. (37) analyzed a panel of 10 anti-DNA Abs derived from three BALB/c mice immunized with bacterial DNA. There was a predominant utilization of J558 family genes (9 of 10) and the JH4 gene segment (6 of 10), with a variety of D region genes. In the light chain VK21 (5 of 10) and VK23 (2 of 10) predominated, with a variety of JL gene segments. In the analysis of Ab VHCDR3, arginines were present in four Abs, but no induced anti-DNA Abs contained more than one arginine in CDR3 or an arginine at position 100 or 100a. These anti-DNA Abs from normal mice displayed a broad range of reactivity with polynucleotides, cardiolipin, and lupus-associated autoantigens (Sm, Ro, and La) (38). Marion et al. (35) described a panel of six anti-DNA Abs (four IgG and two IgM) derived from a single BALB/c mouse following immunization with a DNA binding peptide (Fus 1) in complex with native mammalian DNA. V region structures and DNA specificities of induced anti-DNA Abs were similar to those of spontaneous anti-DNA Abs from (NZB x NZW)F1 mice. Many of the induced anti-DNA Abs described by Gilkeson et al. (37) and Krishnam and Marion (35), like the induced Abs we now describe, are highly homologous to Abs arising spontaneously in murine lupus. These results in the peptide-induced autoimmunity model suggest that nucleic acid is not essential to trigger the production of anti-DNA Abs similar in structure and antigenic specificity to those produced spontaneously in autoimmune lupus mice or following exposure to an immunogenic form of DNA. Induction of anti-DNA Abs by peptide immunization that are structurally homologous to anti-DNA Abs in spontaneous lupus would support the idea that protein Ags may be responsible for loss of tolerance to dsDNA in systemic lupus.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Chaim Putterman, Division of Rheumatology, Department of Medicine, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461. E-mail address:
3 Abbreviations used in this paper: SLE, systemic lupus erythematosus; RNP, ribonucleoprotein; CDR, complementarity-determining region; MAP, multiple antigenic peptide; FR, framework region.
Received for publication September 27, 1999. Accepted for publication December 15, 1999.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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R. Pal, U. S. Deshmukh, Y. Ohyama, Q. Fang, C. C. Kannapell, F. Gaskin, and S. M. Fu Evidence for Multiple Shared Antigenic Determinants within Ro60 and Other Lupus-Related Ribonucleoprotein Autoantigens in Human Autoimmune Responses J. Immunol., December 1, 2005; 175(11): 7669 - 7677. [Abstract] [Full Text] [PDF]
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E N Voynova, A I Tchorbanov, T A Todorov, and T L Vassilev Breaking of tolerance to native DNA in nonautoimmune mice by immunization with natural protein/DNA complexes Lupus, July 1, 2005; 14(7): 543 - 550. [Abstract] [PDF]
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C. M. Grimaldi, R. Hicks, and B. Diamond B Cell Selection and Susceptibility to Autoimmunity J. Immunol., February 15, 2005; 174(4): 1775 - 1781. [Abstract] [Full Text] [PDF]
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 L. D. Erickson, L.-L. Lin, B. Duan, L. Morel, and R. J. Noelle A genetic lesion that arrests plasma cell homing to the bone marrow PNAS, October 28, 2003; 100(22): 12905 - 12910. [Abstract] [Full Text] [PDF]
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Z. Liang, C. Chen, and C. Mohan Molecular Signatures of Anti-nuclear Antibodies: Contributions of Specific Light Chain Residues and a Novel New Zealand Black V{kappa}1 Germline Gene J. Immunol., October 1, 2003; 171(7): 3886 - 3894. [Abstract] [Full Text] [PDF]
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 J. J. Marchalonis, G. K. Whitfield, and S. F. Schluter Rapid Evolutionary Emergence of the Combinatorial Recognition Repertoire Integr. Comp. Biol., April 1, 2003; 43(2): 347 - 359. [Abstract] [Full Text] [PDF]
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 J. A. D. Navoa, S. Laal, L.-A. Pirofski, G. R. McLean, Z. Dai, J. B. Robbins, R. Schneerson, A. Casadevall, and A. Glatman-Freedman Specificity and Diversity of Antibodies to Mycobacterium tuberculosis Arabinomannan Clin. Vaccine Immunol., January 1, 2003; 10(1): 88 - 94. [Abstract] [Full Text] [PDF]
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B Deocharan, X Qing, E Beger, and C Putterman Antigenic triggers and molecular targets for anti-double-stranded DNA antibodies Lupus, December 1, 2002; 11(12): 865 - 871. [Abstract] [PDF]
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 J. J. MARCHALONIS, S. KAVERI, S. LACROIX-DESMAZES, and M. D. KAZATCHKINE Natural recognition repertoire and the evolutionary emergence of the combinatorial immune system FASEB J, June 1, 2002; 16(8): 842 - 848. [Abstract] [Full Text] [PDF]
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E. Beger, B. Deocharan, M. Edelman, B. Erblich, Y. Gu, and C. Putterman A Peptide DNA Surrogate Accelerates Autoimmune Manifestations and Nephritis in Lupus-Prone Mice J. Immunol., April 1, 2002; 168(7): 3617 - 3626. [Abstract] [Full Text] [PDF]
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M. Khalil, K. Inaba, R. Steinman, J. Ravetch, and B. Diamond T Cell Studies in a Peptide-Induced Model of Systemic Lupus Erythematosus J. Immunol., February 1, 2001; 166(3): 1667 - 1674. [Abstract] [Full Text] [PDF]
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Y Shoenfeld Eppur si muove (Galileo Galilei 1564-1642): the idiotypic dysregulation of autoantibodies as part of the etiology of SLE Lupus, September 1, 2000; 9(7): 481 - 483. [PDF]
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