Clinical Immunology Unit and
1 Department of Medicine, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, Hong Kong
Correspondence to: P.-L. Lim
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Abstract |
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Keywords: idiotype, epitope, peptide, autoimmunity, mimicry
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Introduction |
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A clue to understanding the pathogenesis of SLE may come from unraveling the mechanisms involved in the generation of anti-DNA and other autoantibodies. There has been intense investigation in this area but, so far, there are no clear answers. It is not certain, for example, what the real immunogen is that initiates an anti-DNA response. A possible candidate is bacterial DNAs (3), which, with their high unmethylated CpG content, can stimulate the immune system like any foreign antigen. Mammalian DNAs, on the one hand, have more conserved sequences and are consequently less immunogenic, but which, on the other hand, are more appropriately targeted by the autoimmune response (4). It is possible, however, that these and other nuclear antigens can become more immunogenic in the lupus environment through abnormal presentation to the immune apparatus (5). Substances which mimic DNA provide an attractive alternative, but no naturally occurring surrogate has been identified; a synthetic peptide surrogate, however, was recently described (6). For other autoantigens, such as Sm (7) and smooth muscle (8), viral homologues are known which can induce production of the respective autoantibody following infection by the virus. Autoantibodies may also be generated by epitope spreading (9) in which, for example, immunization with a Sm peptide induces antibodies not only to Sm, but also to other antigens that are normally associated with Sm. Finally, a mechanism based on the idiotype (id)anti-id network theory of Jerne (10) has been advanced to explain how anti-DNA antibodies may be generated. This assumes that the original stimulus is still DNA, but during the course of the immune response and following the induction of the primary anti-DNA antibodies (Ab1), successive generations of antibodies are produced which are directed at the specificity determinants or id of the antibody molecule and one of these anti-id antibodies (Ab3) is specific for DNA, i.e. an id is present in Ab2 which is a DNA mimotope. Thus, immunization of normal or lupus-prone mice with some types of anti-DNA antibodies, or with anti-id antibodies to these proteins, were found to result in the production of anti-DNA antibodies (reviewed in 11). Both human and mouse antibodies, and more recently peptides of these antibodies including those based on the VHCDR3 segment (third complementarity-determining region in heavy chain), were found to be effective (12,13). The nature of the DNA mimotope in these cases has not been identified.
Little is known, however, about the prevalence or clinical importance of anti-id antibodies, particularly those that may be induced by the anti-DNA antibodies. These appear to have both positive and negative immunoregulatory roles, as well as beneficial and deleterious effector functions (1416). We had previously argued that because half of our SLE patients developed anti-id antibodies to anti-DNA antibodies which bear the T14 id, these anti-id antibodies (which are directed to the DXP'1-encoded VHCDR3 segment) may be pathogenic by forming inflammatory immune complexes with the primary antibody (14,17). The prototypic T14 antibody was described by van Es et al. (18). We attempt to extend these observations to animals in the present study using mice which produce a transgene-encoded anti-phosphorylcholine (PC) antibody (19). This murine antibody, which bears the T15 id, does not bind to DNA and is not related to T14 except that it has a VHCDR3 segment encoded largely by the DFL16.1 gene, the mouse equivalent of DXP'1. The conserved motif shared by these D genes is YYGS(G)S (see Table 1). We investigate whether anti-id antibodies to the transgene-encoded protein could be induced by immunizing the animals with a peptide based on the VHCDR3 of the transgene, so that the effect of having both sets of id+ and anti-id antibodies present continually in the animal could be studied. We find that anti-id antibodies could indeed be elicited as easily as in animals without the transgene, but importantly, anti-DNA antibodies were also induced. This raises the intriguing possibility that anti-DNA antibodies may arise easily in nature as a result of the inadvertent induction by antibodies that are not necessarily DNA specific, but which are teleologically important or which are specifically induced in the course of an infection. T15+ antibodies are a notable example of these (20). We show, further, that this potential extends to human DXP'1-encoded antibodies. The present report focuses on the nature of the various anti-peptide antibodies generated and not their biologic functions.
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Methods |
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Animals and immunization
Six-month-old CBA/N xid/xid mice which harbored the T15+ transgene were obtained as described previously (19). Transgene-negative littermates of these mice were used as controls.
The animals were injected with alum-precipitated peptideHSA or HSA (50 µg) in complete Freund's adjuvant i.p. A booster dose (10 µg) was given i.p. 2 weeks later in incomplete Freund's adjuvant, and blood was obtained from the retro-orbital plexus on days 0, 7, 14 and 21 following the primary dose. All procedures were approved by the Ethics Committee of the University.
Comparison of data between groups of animals was performed using the MannWhitney non-parametric test (Prism 3.0; GraphPad Software, San Diego, CA).
B cell hybridomas
Mice immunized with two i.p. doses of P9HSA were given another dose of the peptide (10 µg, in saline) i.v. before spleen cells were obtained and fused with Sp2/0 myeloma cells according to conventional procedures (14), using polyethylene glycol (mol. wt 1450; Sigma). Clones were screened for reactivity against P9toxoid and selected hybridomas were grown as ascites. The IgM antibodies were purified from the ascites fluid by precipitation with 50% cold saturated ammonium sulfate followed by cryoprecipitation.
Antipeptide and anti-DNA ELISA
Antipeptide antibodies were detected essentially as described (14). Briefly, P9toxoid (2 µg/ml) was coated in bicarbonate buffer, pH 9.6, on Immulon-2 microtiter plates (Dynex, Chantilly, VA) at 4°C overnight. The plates were blocked with 2.5% (w/v) BSA (Sigma) in PBS (pH 7.6) for 30 min and then incubated with the test serum (diluted at 1:500 in blocking buffer, in duplicates) at 37°C for 2 h. Following washing, the assay was developed with goat anti-mouse IgG (whole molecule) conjugated with horseradish peroxidase (Sigma; 1:1000 dilution) for 2 h at 37°C. Substrate (o-phenylenediamine; Sigma) was then added and incubated for 30 min at 37°C and the results were read in an ELISA reader (Dynex). [Preliminary studies had indicated that the results were similar whether the serum was titrated or assayed at a single dilution (at non-saturating antibody concentrations).]
A similar protocol was used to detect anti-DNA antibodies from mouse sera (1:100 dilution) using goat anti-mouse Ig (whole molecule) antibody (Sigma). The antigens used were purified salmon sperm dsDNA obtained from Pharmacia Biotech (Uppsala, Sweden) (20 µg/ml) or single-stranded (ss) DNA, prepared by boiling the dsDNA for 10 min and quickly chilling it on ice for 10 min.
Anti-id ELISA
Purified TEPC15 (IgA,) was used as the detecting antigen instead of the virtually identical S107 protein for convenience. This was coated overnight at 0.5 µg/ml on microtiter plates. The test mouse serum (diluted 1:100 in 2.5% BSA/PBS) was incubated for 2 h at 37°C. Following washing, biotinylated goat anti-mouse IgG or mouse IgM (both Fc specific) (Sigma; 1:4000 dilution) was added and incubated for 2 h. ExtrAvidin peroxidase (Sigma; 1:2500 dilution) was then added and incubated for 45 min, and the assay developed as described above. Responsiveness was determined using, as cut-off, the mean response of the HSA-immunized mice + 3 SE.
Isotype and inhibition ELISA
Isotypic analysis was performed as described, but using biotinylated rabbit anti-mouse Ig subclass-specific antibodies (IgM, IgG1, IgG2a, IgG2b, IgG3) (PharMingen, San Diego, CA; 1:2000 dilution) and ExtrAvidin peroxidase (Sigma; 1:2000 dilution).
In the inhibition ELISA, the basic ELISA protocol described above was followed except that the test serum was pre-incubated with the free peptide used as inhibitor (60 µl; starting, 80 µg/ml) for 2 h at 37°C; 100 µl of the mixture was then used in the assay.
Anti-DNA immunofluorescence assay
Fixed smears of Crithidia lucilliae obtained from our routine laboratory were pre-blocked with 2% normal goat serum for 30 min and then incubated with the selected human serum for 2 h at 37°C. The smear was washed twice with PBS and incubated with fluorescein-labeled goat anti-human Ig (whole molecule) (Sanofi Diagnostics Pasteur, Chaska, MN). After 1 h, the smear was washed and examined by fluorescence microscopy. In the inhibition assay, the selected human serum was pre-incubated with the unconjugated test peptide for 2 h at 37°C before use.
mRNA sequencing
Total RNA was isolated from hybridoma cells using the RNeasy kit (Qiagen, Valencia, CA) and amplified by RT-PCR using Superscript II (Gibco/BRL, Bethesda, MD) and oligo-d(T)16 (Perkin-Elmer, Foster City, CA). Specific VH and VL fragments were obtained using the following PCR conditions: 35 cycles of 1 min each at 95, 55 and 72°C, and final elongation at 72°C for 10 min. The primers used were:
5' VH primers:
5'-TGC GGC CCA GCC GGC CCR SGT CAA RCT GCA-3' (clone 2H4)
(SfiI)
5'-AGG TCA ADC TGC AGC AGT CAG G-3' (clone 11G2)
5'-CAG GTC AAG CTG CAG CAG TCT GG-3' (clone 6F4)
3' Cµ primers:
5'-ACC TGC GGC CGC GAA GGA CTG ACT CTC-3' (clone 2H4)
(Not1)
5'-GCA GGA GAC GAG GGG GA-3' (clone 6F4)
5'-GGC CAC CAG ATT CTT ATC A-3' (clone 11G2)
5' V primer:
5'-CCA GAT GTG AGC TCG TGA TGA CCC AGA CTC CA-3'
3' C primer:
5'-AAG ACC TTA GAA GGG AAG ATA GG-3'
The PCR products were purified using QIAquick gel extraction kit (Qiagen) and sequenced directly using ABI Prism dRhodamine terminator cycle sequencing kit (Perkin-Elmer) in an ABI Prism 310 genetic analyzer (Perkin-Elmer). The sequences were analyzed according to GenBank (National Center for Biotechnology Information, Bethesda, MD).
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Results |
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To determine whether the antipeptide antibodies recognize native Id determinants in T15+ antibodies, an assay was established which exploits the isotypic difference between the detecting antigen (intact IgA protein of TEPC15) and the anti-id antibodies (IgG or IgM). High levels of IgG, but very little IgM, anti-id antibodies were found in the majority (90.4%) of the animals (Fig. 1), suggesting a thymus-dependent response. Again, the results were similar whether transgenic or non-transgenic mice were used (Fig. 1
). Hence, for simplicity, subsequent analyses utilize pooled data from both groups of animals.
The fine specificities of the antipeptide and anti-id antibodies are different from each other. Thus, using smaller (6mer) segments of the P9 peptide (Table 1) as probes in inhibition assays, it was found that in all mice examined, the whole peptide, as well as the P9b subpeptide which covers the C-terminal half of P9, were inhibitory in the anti-whole peptide assay; the other subpeptides were not (Fig. 2A
). This implies that the antibodies are directed largely to the FDV region in P9. In contrast, in the anti-id assay, while the whole peptide was inhibitory, the P9b subpeptide was not (Fig. 2B
). In addition, in some of these animals, the N-terminal (P9a) and central (P9c) subpeptides were also inhibitory, albeit weakly, suggesting that the (Y)GSS residues are important for antigenicity. Other anti-id antibodies presumably recognize a less defined or a conformational epitope. The fact that in all cases the P9a and P9c subpeptides were not inhibitory in the anti-whole peptide assay suggests that the anti-id antibodies form only a very small fraction of the antipeptide antibodies.
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Both the murine and human T15+ peptides induce anti-DNA antibodies
The antibodies produced in the P9-immunized mice were examined further by constructing hybridomas from the animals. Three IgM clones (2H4, 6F4 and 11G2) were obtained which showed reactivity to the P9 peptide (Fig. 3). Clone 2H4 was found to produce the transgene-encoded antibody as well, demonstrated by the dual presence of IgM antipeptide and IgG2b anti-PC activities in antigen-specific ELISAs (data not shown). In all clones, the antibody also bound very well to the human peptides, P6 and P7 (Fig. 3
). Whereas 2H4 and 11G2 showed no reactivity to irrelevant non-Ig peptides (including those derived from EpsteinBarr virus), 6F4 was polyreactive.
Sequence analysis of the transcripts from the clones revealed that different VH and VL (variable region of heavy and light chain respectively) genes were used in the antibodies (Fig. 3). Interestingly, in the double-producer clone, 2H4, only one VL transcript was detected which belonged to the transgene. Interestingly, too, the VH segment of the 2H4 antipeptide antibody, encoded by the VHJ558, DSP2 and JH4 genes, is used commonly by various DNA-binding antibodies (GenBank, NCBI, Bethesda, MD). The CDR3 region, YSNYGAIDY, in fact, is very similar to that (QAYSNYGAMDY) of a Z-DNA-binding antibody (23). The possibility that 2H4 could be DNA specific was consequently examined. The antibody was indeed found to bind very well to both dsDNA and ssDNA. The 6F4 antibody, but not 11G2, was also reactive with the DNA substrates. It is not clear why 6F4 is polyreactive, although the VH (VH11) and VL (V
8) genes used have been described in polyreactive antibodies (24). The VHCDR3 of 6F4 is unusually `bulky' as it comprises the residues H (N region), NW (DQ52) and AWFAY (JH3). Whether this contributes to the polyreactivity of the antibody remains an interesting possibility particularly since this region of the molecule has been implicated in such a role previously (24,25), but not always (26). For comparison, the VHCDR3 of 11G2 is quite different, YYSNYVRVLLTGAKGL.
Based on the above finding, the P9-immunized mice were examined to see if anti-DNA antibodies had been induced by the peptide in these animals. Indeed, 21% of the animals showed elevated levels of antibodies to dsDNA, while a higher number (67%) produced anti-ssDNA antibodies that was highly significant (Fig. 4A). The number of responders to dsDNA may be underestimated due to the high background of the assay.
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SLE patients produce peptide-specific anti-DNA antibodies
Direct demonstration proving that P9 and P6 (P7, not done) are DNA surrogates is shown in Fig. 6. Anti-dsDNA antibodies present in SLE sera normally stain the kinetoplast and nucleus of C. lucilliae, as revealed by fluorescence microscopy. Addition of the P9 or P6 peptide to the serum specifically abolished this reactivity in four out of 10 patients examined. It is not clear, at this stage, why only some sera are inhibitable by the peptides. In all cases, the P9 subpeptides and the control peptide (P10) were not inhibitory.
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Discussion |
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Based on the specificity tests described in Fig. 2(B and C), it appears that the single most critical amino acid residue defining the cross-reactive T15+ id is glycine. This is supported by an examination of the crystal structure of the VHCDR3 segment of a T15-related Ig (McPC603). As apparent (Fig. 7
), glycine is located at the apex of the CDR3 loop and is prominently exposed to the outside. It should be noted the T15+ id has been variously defined by previous investigators depending on the reagent antibody used, all based on the prototypic T15+ Ig, TEPC15. Of particular note is the mAb, NL16, used by Nishinarita et al. (27), which recognized both the D segment and the V
22 light chain. This antibody failed to detect an Ig (140.7C6) which was very similar to TEPC15 except for a mutated D segment (YYDGS), supporting the importance of the germline-encoded glycine residue in idiotypy. The serine residue adjacent to glycine in the germline sequence does not appear to be important since a TEPC15-like Ig (H8) which has asparagine in this position was also recognized by NL16 (27). This also appears to be the case in our system since alanine found in this position in the 18/2 sequence (P6) as a result of somatic mutation had no effect on the cross-reactivity of this peptide with P9 or P7. The importance of glycine in the YYGSS motif in idiotypy was also described for an antibody which was specific for the 4-hydroxy-3-nitro-5-iodophenylacetyl hapten, but which also utilized DFL16.1 (28). The id of this Ig, as determined by several mAb, was lost in a mutant which had a glycine to arginine substitution, although antigen binding was retained.
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It is tempting to speculate that the DNA mimotope exists in all Ig which utilize the DFL16.1 or DXP'1 gene. The mimotope is thus potentially widespread, because DXP'1, for example, is very commonly utilized in anti-DNA antibodies (32). Identification of such a mimotope is important because this provides a chemical basis for the numerous observations made previously that immunization of mice with anti-DNA (or anti-id) antibodies can result in the production of anti-DNA antibodies (11). The idanti-id network theory has often been used to explain this phenomenon and anti-id (Ab2) antibodies are presumed to bear the DNA mimotopes. We have not only identified such a mimotope, but have also shown that this is not necessarily confined to Ab2 but can exist in the primary antibody (Ab1), i.e. DFL16.1- or DXP'1-encoded anti-DNA antibodies can directly induce other anti-DNA antibodies to be produced. More importantly, the present finding shows that anti-DNA antibodies can be induced by antibodies of any specificity, not necessarily those that are DNA-specific, so long as these are encoded by DXP'1 or DFL16.1, i.e. they can be produced as bystander proteins in a normal anti-idiotypic response. A possible scenario is that, during an infection by a PC-containing microorganism such as Trichinella spiralis, T15+ anti-PC antibodies are generated. Anti-id antibodies are consequently produced and some of these can be DNA specific. Although there is no actual documentation of this, Shefner et al. (33) noted a transient increase in the level of anti-DNA antibodies in BALB/c mice when these animals were immunized with a PCprotein conjugate. Similar views have been expressed previously that autoimmunity may follow a common infection (11) or result from a normal functioning immune system (34). Viruses thus play a role in autoimmunity (35) not only because they have shared antigens with the host (8), but also, as we contend, because of the type of antibodies they elicit.
It remains presumptuous, however, that anti-DNA antibodies are normally induced by T15+ antibodies in a healthy person or animal. This is slightly different from the experimental situation described herein because the immunogen used was a synthetic peptide conjugated to a foreign carrier (HSA) and this was artificially administered in Freund's adjuvant. Evidently, in this case, HSA provided the T cell epitope(s) necessary for the memory response and, in the transgenic animals, the abrogation of peripheral tolerance (36) to the transgene-encoded `self' protein (data not shown). This means that B cells specific for the T15 id or for DNA are present normally in both the transgenic and non-transgenic animals. In the natural state, T cell epitopes are presumed to be continually generated in the Ig molecule through somatic hypermutation or creation at the VDJ junctions. Such epitopes are known to exist in VH segments (12,3739). Thus, Ig fragments bearing linked B and T cell epitopes when presented to appropriate B cells can stimulate the cell, become endocytosed and processed in the same way as normal antigens for MHC class II presentation to T cells. In this way, `natural' antibodies (40) specific for DNA can be formed in a normal person or animal. T cells may be primed in the first place by dendritic cells and macrophages in the spleen and other organs following normal Ig catabolism. Ig fragments thus generated can also be presented to B cells directly by these cells in apoptotic blebs (5) or indirectly by follicular dendritic cells. Obviously, responsiveness will depend on whether adequate quantities of the relevant fragmentall bearing the same linked B and T cell epitopes and, hence, monoclonally derivedare available. What distinguishes the normal state from the autoimmune condition is perhaps the up-regulation of antibody production in the latter due to genetic factors (41) or the more appropriate presentation of the antigen (5), or alternatively that regulatory mechanisms exist normally to control the production. It is not clear, for example, why only a proportion of the peptide-immunized animals in our study were able to produce antibodies to dsDNA when virtually all of them could produce anti-id or anti-peptide antibodies.
From a teleological standpoint, T15+ antibodies are both important and interesting. The present study adds more intrigue to these. They are known to play a very important role in natural immunity (42,43) and, more recently, were found to be associated with murine atherosclerosis as well as the normal physiology of apoptotic cell clearance (44). More unusually, these proteins, including the human counterpart, can bind to their own selves (20). The id responsible for this unusual property is located in the CDR2/framework region 3 location of the murine T15 antibody and, hence, is different from that which induces the anti-DNA or anti-id antibodies described herein. However, there is a common feature among these various ids in that they all involve conserved structures present in both mouse and human. Similar interspecies, cross-reactive ids have been found in other Ig (45), indicating that, in general, such ids may serve an evolutionary function (20).
Another important revelation of the study is that the bulk of antibodies generated to a peptide may be biologically irrelevant, because they do not bind to the native antigen. In our case, these antibodies are directed to the FDV residues of the peptide, a region which is buried inside the native molecule (Fig. 7). Thus, studies which rely on measuring antibodies to short peptides as an alternative to determining the antibody response to a native antigen can be quite misleading.
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Acknowledgments |
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Abbreviations |
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CDR complementarity-determining region in Ig |
ds double stranded |
HSA human serum albumin |
id idiotype (idiotope) |
LPS lipopolysaccharide |
PC phosphorylcholine |
SLE systemic lupus erythematosus |
ss single stranded |
T15+ Ig of the T15 idiotype |
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Notes |
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Received 8 January 2001, accepted 23 May 2001.
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References |
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