Pedigreed Rabbits Peptide Immunizations of Noninbred

Models of Systemic Lupus Erythematosus: Development of Autoimmunity Following Peptide Immunizations of Noninbred Pedigreed Rabbits This information is current as of September 29, 2021. Geeta Rai, Satyajit Ray, Robyn E. Shaw, Paula F. DeGrange, Rose G. Mage and Barbara A. Newman J Immunol 2006; 176:660-667; ; doi: 10.4049/jimmunol.176.1.660 http://www.jimmunol.org/content/176/1/660 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2006 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Models of Systemic Lupus Erythematosus: Development of Autoimmunity Following Peptide Immunizations of Noninbred Pedigreed Rabbits1

Geeta Rai,* Satyajit Ray,* Robyn E. Shaw,† Paula F. DeGrange,† Rose G. Mage,2* and Barbara A. Newman2*

Reported in this study are the initial results from studies to develop rabbit models of systemic lupus erythematosus (SLE) by immunizations using two distinct peptides on branched polylysine backbones (multiple Ag peptide)-peptides. Eleven rabbits received a peptide from the Sm B/B؅ spliceosomal complex previously shown to be immunogenic in rabbits, and 13 rabbits received a peptide from the rabbit N-methyl-D-aspartate receptor NR2b. All 24 animals in different generations of pedigreed, noninbred rabbits produced peptide-speciﬁc responses. Anti-nuclear autoantibody responses, including anti-dsDNA, were seen in 17 of 24 Downloaded from rabbits. To date, two rabbits have been observed to have seizure-like events and a third nystagmus. A model for eliciting development of SLE in genetically related yet heterogeneous rabbits may more closely resemble development of human SLE than do some models in inbred mice. Through selective breeding, it may also ultimately provide additional information about the genetics and etiology of SLE and serve as a model for assessing new treatment options. The Journal of Immunology, 2006, 176: 660–667.

ystemic lupus erythematosus (SLE) is a complex, chronic from other species. Our current understanding of rabbit Ig genes, http://www.jimmunol.org/ autoimmune disorder that is etiologically multifactorial, their allelic forms (allotypes) and the mechanisms of diversifica- S with both genetic and environmental factors contributing tion of rearranged Ab H and L chain sequences during develop- to disease initiation and progression (1). Clinical presentation, ment of the primary preimmune repertoire by somatic hypermu- which may manifest as rash, inflammatory arthritis, glomerulone- tation and gene conversion as well as during specific responses phritis, and generalized cognitive decline, is accompanied by the after immunization (8), has laid the groundwork that now allows presence of autoantibodies to various nuclear Ags including better understanding of rabbit models of autoimmune diseases, in- dsDNA. A subset of patients diagnosed with neuropsychiatric lu- cluding those having neurological components (9–14). Data from pus (NL) exhibit severe and sudden onsets of psychosis or other rabbit models will complement knowledge derived from the more acute neurological impairment, including seizure disorders, and frequently used mouse models of autoimmune diseases that have by guest on September 29, 2021 carry a poorer prognosis. Autoantibody-Ag immune complex for- revealed roles for B- and T cell-mediated immunity (15–17), cy- mation accompanied by complement-mediated inflammatory pro- tokines (18, 19), dendritic cells (20, 21), apoptosis (22, 23), and, in cesses is believed to contribute to the pathological kidney damage particular, begin to decipher the genetic basis for susceptibility to (2, 3); however, the basis for autoimmune damage in NL is largely development of SLE (24, 25). Studies of spontaneously SLE-prone unexplored (4–6). inbred mouse strains as well as mutant, transgenic, and knockout Rabbit polyclonal Abs produced after immunization are popular murine models (26–31) have revealed that a variety of genetic contemporary research reagents. Recent studies (7, 8) have defined defects can contribute to development of SLE in general but have mechanisms that result in the rabbit’s unique ability to produce not pinpointed those that lead to NL. Human genome-wide studies some highly specific high-affinity Abs that are difficult to obtain to date have linked multiple genetic loci to SLE (32–35); the extent to which the findings using inbred or homogeneous artificially mu- tated strains will apply to heterogeneous outbred human popula- *Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, tions remains to be determined. It is our hope that in the future, National Institutes of Health, Bethesda, MD 20892; and †Spring Valley Laboratories, Woodbine, MD 21797 treatment targets may be identified through genetic studies. A rab- Received for publication September 2, 2005. Accepted for publication October bit model may contribute and complement the information ob- 21, 2005. tained in other species and serve as a useful tool in designing The costs of publication of this article were defrayed in part by the payment of page therapeutic interventions. charges. This article must therefore be hereby marked advertisement in accordance The National Institute of Allergy and Infectious Diseases with 18 U.S.C. Section 1734 solely to indicate this fact. (NIAID)-pedigreed rabbits are Ig allotype-defined noninbred ani- 1 This work was supported by the Intramural Research Program of the National In- stitutes of Health, National Institute of Allergy and Infectious Diseases. mals. The genetic heterogeneity of these animals is closer to the human situation. Therefore, they constitute a valuable resource for 2 Address correspondence and reprint requests to Dr. Rose G. Mage, Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Insti- breeding, selection, and development of models to study Ag- tutes of Health, Building 10, Room 11N311, 10 Center Drive-MSC1892, Bethesda, driven production of autoantibodies similar to those seen in SLE, MD 20892-1892; E-mail address: [email protected] or Dr. Barbara A. Newman, Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, and especially to aid in identification of those that may be relevant National Institutes of Health, Building 10, Room 11N311, 10 Center Drive- to NL pathology. We chose rabbits from a pedigreed group for MSC1892, Bethesda, MD 20892-1892; E-mail address: [email protected] immunization with two different peptides on multiple Ag-branched 3 Abbreviations used in this paper: SLE, systemic lupus erythematosus; NL, neuro- polylysine backbones (BB) (multiple Ag peptide (MAP)-peptides). psychiatric lupus; MAP, multiple Ag peptide; BB, backbone; NMDA, N-methyl-D- aspartate; GR, glutamate receptor; TMB, 3,3Ј,5,5Ј-tetramethylbenzidine; ANA, anti- One was a peptide derived from the spliceosomal Smith (Sm) Ag nuclear Ab; RT, room temperature; ENA, extractable nuclear Ag. studied previously (11) (called in this study peptide SM), and the

other was a peptide sequence from the neuronal postsynaptic glu- Table I. tamate receptor N-methyl-D-aspartate (NMDA) NR2b chain (pep- Immunized rabbits, sexes, and Ig allotypes tide GR), based on previous reports that a pathogenic mouse anti- dsDNA Ab that bound kidney tissue recognized a consensus Groupa Rabbit Codeb Rabbit no. Sex Allotypec peptide sequence DWEYS found within NMDA NR2b and that 1 SM-1 XX129–3 M a1/1 b4/5 some murine and human anti-dsDNA Abs bound the peptide or the SM-2 2XX127–4 F a1/1 b5/5 receptor itself (36–38). BALB/c mice immunized with MAP- SM-3 2XX288–2 M a1/1 b9/9 DWEYS developed anti-dsDNA, anti-cardiolipin, and anti-histone SM-4 1XX288–4 F a1/1 b5/9 autoantibodies, with glomerular deposition of immune complexes SM-5 2XX127–2 M a1/1 b9/9 SM-6 2XX92–6 F a1/1 b9/9 (39). Recently, anti-NR2 Abs have also been suggested to cause GR-7 XX129–5 M a1/1 b4/5 brain pathology and cognitive dysfunction (40). Finally, in some GR-8 2XX127–5 F a1/1 b9/9 SLE patients showing signs of NL, serum Abs that may bind NR2a GR-9 1XX288–3 M a1/1 b5/5 and NR2b receptors were found (41, 42). GR-10 2XX288–6 F a1/1 b5/9 BB-11 2XX127–1 M a1/1 b5/5 The results reported in this study show that immunization of BB-12 1XX78–8 F a1/1 b9/9 rabbits with the SM- or GR-MAP peptide led to development of 2 SM-13 LL191–1 M a1/1 b4/5 anti-nuclear autoantibodies, anti-dsDNA, as well as neurological SM-14 LL191–2 F a1/1 b5/5 symptoms in some rabbits. It is notable that with both SM- and SM-15 2LL179–1 M a1/1 b9/9 GR-peptide immunizations, autoantibodies speciﬁc for Ags very SM-16 1LL163–3 F a1/1 b9/9 SM-17 LL164–4 F a1/1 b9/9 different from the sequence of the immunizing peptide were pro- GR-18 1LL178–2 M a1/1 b9/9 Downloaded from duced, providing the basis for exploring a potential model of SLE GR-19 1LL178–3 F a1/1 b4/4 with Ag-driven production of autoantibodies and subsequently GR-20 1LL178–4 F a1/1 b9/9 arising clinical symptoms associated with NL. GR-21 1LL178–5 M a1/1 b4/9 GR-22 1LL178–6 F a1/1 b4/9 GR-23 1LL178–8 F a1/1 b4/4 Materials and Methods BB-24 LL164–1 M a1/1 b9/9

Animals BB-25 LL164–3 F a1/1 b9/9 http://www.jimmunol.org/ BB-26 2LL179–3 M a1/1 b5/9 The animal studies described in this study were reviewed and approved by BB-27 1LL163–4 F a1/1 b5/9 the animal care and use committees of NIAID/National Institutes of Health 3 GR-28 LL108–1 M a1/1 b5/9 (Bethesda, MD; animal study protocol LI6) and of the Spring Valley Lab- GR-29 LL108–3 F a1/1 b5/5 oratories (Woodbine, MD), where the NIAID allotype-defined rabbit col- GR-30 LL108–4 F a1/1 b9/9 ony is housed. The three immunization groups studied comprised a total of BB-31 2LL179–2 M a1/1 b9/9 31 1- to 2-year-old rabbits maintained in a room separate from the main a colony. Rabbits of Groups 2 or 3 were descendants of Groups 1 or 2 rabbits Rabbits of Groups 1 and 3 received peptides (SM or GR) synthesized on MAP-8 and/or their siblings. The animals’ designations, sexes, and allotypes at the BB, and rabbits of Group 2 received the peptides on MAP-4. BB indicates control ␬ rabbits that received BB alone. M, Male; F, female. VHa H chain and C␬b L chain loci are summarized in Table I. There were b SM animals received MAP-peptide derived from the Sm Ag spliceosomal B/BЈ by guest on September 29, 2021 10 males and 14 females among the immunized rabbits. All rabbits were complex. GR animals received MAP-peptide derived from the NMDA glutamate ␬ homozygous for the Ig H chain VHa1 allotype but varied in their L chain receptor. c ␬ b allotypes. Ig heavy chain VHa allotypes and L chain C␬b allotypes. Antigens laboratory (Antech Diagnostics) for complete blood counts and chemistry Two peptide immunogens were each synthesized on branched lysine panels. MAP-8 and MAP-4 BB (AnaSpec) (43). The “SM” peptide sequence PPPGMRPP corresponds to major antigenic regions at positions 191–198, Assay for anti-peptide Abs 216–223, and 231–238 of the nuclear protein Sm B/BЈ, as reported previously (11). The “GR” peptide sequence DEWDYGLP corresponds to a Serum Abs to MAP-peptide immunogens were assayed by standard solid- known rabbit sequence of an extracellular epitope of the 2b subunit of phase ELISA. Polystyrene 96-well plates (Corning; catalog no. 3590) were neuronal postsynaptic NMDA receptors. It is related but not identical with coated with 50 ␮l/well of either SM, GR, or BB (MAP-8 or MAP-4) at 10 the peptide DWEYS reported earlier (36–42). ␮g/ml in bicarbonate buffer (pH 9.6) and incubated overnight at 4°C. Plates MAP-8 and MAP-4 BB without peptide were synthesized to serve as were washed three times with PBS (pH 7.2) containing 0.1% Tween 20 and control Ags, and all synthesized structures were analyzed by HPLC and blocked with 100 ␮l of blocking solution for1hat37°C (Quality Bio- MALDI-TOF mass spectrometry (National Institutes of Health Peptide Syn- logicals). Wells were then incubated1hat37°C with 50 ␮l/well sera thesis and Analysis Unit, Research Technologies Branch, Rockville, MD). titrated by 4-fold dilutions in blocking solution, washed five times, incubated for1hat37°C with 50 ␮l of a 1/2000 dilution (0.4 ng/␮l) of Immunization affinity-purified HRP-conjugated goat anti-rabbit IgG (H ϩ L) secondary Ab (Jackson ImmunoResearch Laboratories), developed with 3,3Ј,5,5Ј- Each rabbit received initial s.c. injections of either MAP-8-peptide (Groups tetramethylbenzidine (TMB) (INOVA Diagnostics), and the resulting OD 1 and 3), MAP-4-peptide (Group 2), or control BB (in each group) (0.5 read at 450 nm. mg/0.5 ml; borate-buffered saline (pH 8.0)) emulsified with 0.5 ml of CFA. Boosts were given s.c. at 3-wk intervals with the same Ag concentration Detection of anti-nuclear Abs (ANA) by indirect emulsified with IFA. Sera were collected immediately before immunization immunofluorescence (preimmune) and 1 wk after each boost (postboost) and were aliquoted and stored at Ϫ20°C for assays. Group 1 received a total of nine boosts before Commercially available slides coated with fixed Hep-2 cells (Antibodies, sacrifice. Groups 2 and 3 received 10 boosts before two rabbits were Inc.) were incubated with rabbit antisera diluted 1/10 in 5% goat serum sacrificed, and the remainder kept for observation without additional (Jackson ImmunoResearch Laboratories) for 30 min. at room temperature immunization. (RT). ANA binding was detected by fluorescence microscopy following 30-min incubation at RT with 12.5 ng/␮l FITC-goat anti-rabbit IgG Fc Clinical assessment (Southern Biotechnology Associates). Fluorescent binding patterns were compared with reference pictures provided by Antibodies, Inc. Rabbits were observed continually by video surveillance cameras equipped for night vision (Bullet Multicam color IR; Central Alarm Systems) and Assays for serum autoantibodies recorded onto DVD so that abnormal behavior, particularly seizure activity or other evidence of neurological dysfunction, could be detected. Whole Serum autoantibodies to total extractable nuclear Ags (ENA) and to com- blood and sera collected after boosts were sent to a veterinary diagnostic ponent Ags Sm, Rnp, SS-A (Ro 60 and 52 kDa), SS-B (La), Scl-70, and 662 MODELS OF SLE IN PEDIGREED RABBITS

FIGURE 1. Results of ELISA of anti-peptide responses of (A) SM-MAP-8- and (B) GR-MAP-8-immunized rabbits showing sera of pre- and postimmune 2nd, 3rd, 5th, 7th, and 9th boosts at a dilution of 1/1024.

Jo-1 were assayed by ELISA using adaptations of commercially available immunizing peptide in ELISA at titers exceeding 105 by the 5th human diagnostic kits (INOVA Diagnostics). Assays for autoantibodies to boost (Fig. 1). Ab response patterns among all SM peptide-immu- Downloaded from calf thymus dsDNA were adapted similarly using two different commer- nized rabbits showed only slight variations, with the response cially available kits (Vidia, Vestec (Kit A); Zeus Scientiﬁc (Kit B)). Man- ufacturers’ instructions were followed. Brieﬂy, 100 ␮l of rabbit sera diluted peaking as early as 49 days after the primary immunization (post- 1/100 in the proprietary sample diluents were added to Ag-coated wells and 2nd boost) and persisting through the 9th boost (240 days) (Fig. incubated for 60 min at 37°C (Kit A) or 30 min at RT (Kit B). Wells were 1A); responses of GR peptide-immunized rabbits were more vari- then washed, incubated for 60 min at 37°C (Kit A) or 30 min at RT (Kit able (Fig. 1B). Antisera from neither peptide group ever exhibited B) with secondary Ab HRP-goat anti-rabbit IgG Fc (Jackson ImmunoRe- http://www.jimmunol.org/ search Laboratories), and developed with TMB for reading OD at 450 nm. cross-reactivity with the alternate peptide; similarly, there was no All results are presented as absorbance of the postboost sera OD minus the detectable reactivity with the MAP-4 or MAP-8 BB. Control rab- preimmune sera OD at 450 nm. An OD difference Ͼ0.1 above preimmune bits immunized with only MAP-4 or MAP-8 BB showed negligi- OD was considered positive. ble or no Ab response to the BB, and no reactivity in ELISA with either of the MAP-peptides. Results Serum response to MAP-peptide immunization Rabbit pedigree All 24 rabbits that were immunized with SM or GR peptides, on The pedigree of the rabbits used in this study is shown in Fig. either the MAP-8 or MAP-4 BB, produced antisera that bound the 2. Within each symbol of the peptide-immunized rabbits are by guest on September 29, 2021

FIGURE 2. Pedigree of 24 peptide- and six control BB-immunized rabbits comprising immunization Groups 1, 2, and 3 and their relatives with an overview of lupus-like responses. Rabbits in Groups 2 and 3 were either the direct descendants or the offspring of relatives of Group 1 rabbits. Squares indicate males and circles females; red, blue, and black symbolize SM-, GR-, and MAP-4/-8 BB-immunized rabbits, respectively. Within each symbol of the peptide-immunized rabbits are representations of anti-dsDNA, anti-Sm, anti-Rnp, and other autoantibody (anti-ENA, anti-SS-A, and anti-SS-B) responses that may be traced through descendants. The Journal of Immunology 663

FIGURE 3. ANA immunofluorescence. A, Preimmune serum of SM-13 at a dilution 1/10 shows negligible immunofluorescence on Hep-2 cells as compared with 3rd postboost (B), 5th postboost (C), and 7th postboost sera (D). The speckled pattern sug- gests the presence of Abs against Sm and/or Rnp. Maximum fluorescence was seen 3rd postboost. Both preimmune (E) and postboost (F–H) sera of control BB-26 exhibited very faint immunofluorescence.

representations of anti-dsDNA, anti-Sm, anti-Rnp, and other au- no preference in reactivity of females over males and also no ob- toantibody responses (anti-ENA, anti-SS-A, and anti-SS-B) that vious correlation with the L chain allotype. Downloaded from may be traced through descendants. Lupus-like serology was elicited among Group 1 rabbits and among their relatives and descen- Autoantibody responses measured by ANA immunoﬂuorescence dants in Groups 2 and 3 (Fig. 2 and Table I). Autoantibody re- Among the 24 immunized rabbits, six developed anti-nuclear Abs sponses in the members of the pedigree immunized thus far reveal detected by immunoﬂuorescence assays on Hep-2 cells using http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 4. Anti-nuclear Ag autoantibody responses and their persistence following peptide immunization. Solid lines indicate SM, and dashed lines indicate GR rabbits. Group 1 rabbits are denoted by unshaded symbols, Group 2 by shaded symbols, and Group 3 by symbols containing crossed lines. Results of analyses of sera diluted 1/100 are presented as absorbance of the postboost sera OD minus the preimmune sera OD (450 nm) as measured by ELISA for anti-Sm (A), anti-Rnp (B), anti-SS-A (C), anti-SS-B (D), and anti-ENA (E). 664 MODELS OF SLE IN PEDIGREED RABBITS

ing HRP-conjugated goat anti-rabbit IgG (Fc) as the secondary Ab. Fig. 4 shows results of ELISA for autoantibody responses to ENA and its components Sm, Rnp, SS-A (Ro), and SS-B (La), and Fig. 5 shows results of assays for Abs to dsDNA. In Figs. 4 and 5, ELISA results presented as postboost OD minus preimmune OD are shown at four time points: preimmune (day 0); postboost 3 (day 70); postboost 5 (day 112); and postboost 7 (day 154). Some rabbits exhibited high preimmune values. In Fig. 6, where results of all assays are summarized, the maximum differences found between the OD at 450 nm in preimmune and postboost sera for each test are given, and the actual preimmune OD is shown in parentheses. A difference of 0.1 OD over the preimmune OD was selected as a cut off. Although only 3 of 24 rabbits tested positive with the commercial ELISA for Ab to total ENA (containing lower concentrations of each component), Abs were detected in more of the rabbits when higher concentrations of the ENA components were assayed individually: 7 of 24 (3 GR and 4 SM) were positive for Sm (Fig. 4A); 7 of 24 (3 GR and 4 SM) were positive for Rnp FIGURE 5. Autoantibodies to dsDNA following peptide immunization. Downloaded from Sera diluted 1/100 were assayed by ELISA. Results are presented as post- (Fig. 4B); 3 of 24 (1 GR and 2 SM) were positive for SS-A (Ro 60 boost sera OD minus preimmune sera OD. and 52 kDa) (Fig. 4 C); and 3 of 24 (all GR) were positive for SS-B (La) (Fig. 4D). The variation in Ab response patterns over time FITC-labeled secondary Ab speciﬁc for Fc of rabbit IgG (SM-1, after the 3rd boost (day 70) did not correlate with the proﬁles of the GR-9, SM-13, SM-15, GR-19, GR-20, GR-30). The Abs’ staining Ab responses to the peptide immunogen itself (Fig. 1). The results patterns appeared nuclear and speckled, suggesting the presence of show clearly that rabbits can develop autoantibody responses to http://www.jimmunol.org/ either anti-Sm or anti-RNP. Fig. 3 shows an example of staining by ENA components when immunized with either of the two unre- serum from rabbit SM-13 compared with a control (BB-26). Pre- lated peptides (conjugated to either MAP-4 or MAP-8 BB), immune sera from these six rabbits and postboost sera from rabbits whereas no rabbit immunized with either BB alone showed such immunized with control MAP-4/8 BB were all negative for ANA. response. Overall, 42% (10 of 24) of all the immunized rabbits (7 of 13 of Autoantibody responses to ENA, its component Ags, and to the GR; and 3 of 11 of the SM) developed anti-dsDNA responses dsDNA (Figs. 5 and 6). All sera from rabbits immunized with the control Assays using commercially available kits designed for human MAP-4 or MAP-8 BB were negative for anti-dsDNA autoantibod- samples were successfully adapted to rabbit serum samples by us- ies, as they were for the other Abs to nuclear Ags assayed with the by guest on September 29, 2021

FIGURE 6. Summary of autoantibody responses following MAP-peptide immunization. Preimmune and postboost sera, 3rd, 5th, and 7th, were tested. Shaded boxes represent responsive rabbits; the numbers in the boxes indicate the maximum difference between postboost and preimmune sera OD; the absolute preimmune OD value at 450 nm is given in the parenthesis. In the ANA column, results have been presented as a semi- quantitative measure of the fluorescence on a scale of 1 to 3: 3ϩ, optimum fluorescence; 2ϩ, dim fluorescence; 1ϩ, very subdued fluorescence as compared with the preimmune sera. The three values represent the fluorescence after 3rd, 5th, and 7th boosts. The Journal of Immunology 665 exception of one control rabbit BB-31. This rabbit had a high netic uniformity of inbred mouse strains and begins to approach preimmune anti-dsDNA value (0.8) that increased over time (0.9, the heterogeneity among humans more closely. 1.2, 1.2 at 3rd, 5th, and 7th postboost, respectively) for reasons not To obtain a more in-depth understanding of the etiology and yet understood. Two rabbits (SM-1 and GR-9) that have thus far pathogenesis of SLE, several recent investigations in animal mod- had documented seizure-like episodes, described further below, els have focused on understanding Ag-driven mechanisms. Putter- and captured by video4 were among those with the highest anti- man and Diamond (39) identified a peptide mimetope that was dsDNA levels. bound by a monoclonal anti-dsDNA Ab, R4, as well as by other anti-dsDNA Abs. A related sequence was later shown to occur in Comparison of Ab responses to MAP-4 vs MAP-8 peptide the NMDA (NR2b) receptor (37). Our immunization study in rab- conjugates bits was initiated using the actual peptide sequence from the rabbit MAP-8 peptide conjugates were used for immunization of pedi- NR2b. We found that this MAP-peptide could not only elicit anti- gree Groups 1 and 3; MAP-4 conjugates were used for Group 2 dsDNA responses but also other autoantibodies found in lupus (Table I and Fig. 2). The decision to use MAP-4 was based on the patients. The generation of Abs binding to Ags other than the possibility that these could be synthesized more reliably, but the immunizing peptide, may result from somatic mutation or, in rab- response pattern observed led to a return to MAP-8. Among rabbits bits, gene conversion of rearranged H and L chain sequences in immunized with the MAP-4 peptides, 7 of 11 developed autoan- peptide-specific B cells (48). It is known, for example, that anti- tibody responses to one or more nuclear autoantigens (ENA, SM, dsDNA-producing cells can be generated by somatic mutation of B Rnp, SS-A (Ro), or SS-B (La) (Figs. 4 and 6), but only 3 of 11 cells producing anti-phosphorylcholine Abs during in vivo re- developed detectable anti-dsDNA (Figs. 5 and 6). In contrast, 7 of sponses (49, 50). Epitope spreading can also be used to explain the Downloaded from 13 of the MAP-8 peptide-immunized rabbits had anti-dsDNA Abs. generation of novel autoantibody specificities. The spreading can In Group 3, all three (GR-28, -29, -30) MAP-8-immunized prog- occur between proteins that are known to be physically associated eny of responders from Group 1 produced detectable autoantibod- in vivo, such as the spreading from Sm B/BЈ immunization to ies to one or more nuclear Ags and two (GR-28, -29) to dsDNA. include other regions of the small nuclear ribonucleoprotein par- Clinical observations and hematological studies ticle (11, 51, 52), or it may involve recognition of self Ags that appear to differ in molecular structure. For example, it has been http://www.jimmunol.org/ The video recordings have been reviewed thus far for Group 1 shown that immunization with short peptides from the 60-kDa Ro only. The extensive monitoring of the rabbits by animal care staff particles can lead to noncross-reactive intra- and intermolecular and by video surveillance led to observations of three rabbits in this group that exhibited neurologically related symptoms. GR-8 epitope spreading (53–56). was diagnosed with nystagmus (involuntary oscillatory move- In an earlier study that used nonpedigreed rabbits, James et al. ments of the eye) that is found associated with clinical SLE (44– (11) provided support for the molecular mimicry theory of auto- 46). GR-9 and SM-1 showed seizure-like episodes on day 141 (29 immune disease. They were able to initiate autoimmune responses Ј days after the 5th boost) and day 59 (17 days after the 2nd boost), in some normal rabbits immunized with an Sm B/B -derived respectively. Rabbit SM-1 manifested episodic, possibly seizure- epitope PPPGMRPP, which is very similar to PPPGRRP from the by guest on September 29, 2021 like behavior at least five times in 1 day. The first event occurred EBV-encoded nuclear Ag (EBNA 1). The fact that this work was at 10:33 a.m. and was characterized by a short period of extreme not reproduced by another group (57) is possibly explained by agitation followed by 4 min of abrupt unconsciousness and then structural variation among MAP-8-peptide Ag synthesized in dif- sudden awakening.4 The nature of this episode resembles “atonic ferent batches, undetected infections of some rabbits, and other seizures” in man, which are characterized by sudden loss of pos- unknown environmental and genetic factors. Given that only a few tural muscle tone and brief impairment of consciousness (47). The of the 15 rabbits studied by the James group (11) exhibited lupus- other four behaviors of SM-1 were similar with extreme agitation like responses to immunization, an important difference may sim- followed by a brief period of abrupt unconsciousness and took ply have been the small sample size of six rabbits in the second place at 1:09 p.m., 2:19 p.m., 4:09 p.m., and 5:19 p.m. study. The present study included 24 allotype-defined, pedigreed, Complete blood counts and serum chemistry panels for Ͼ47 noninbred rabbits maintained in a controlled but not specific patho- parameters including serum creatinine and total protein was done gen-free environment. Seventeen of these 24 rabbits developed for all rabbits after each boost, but no other significant change or some lupus-like autoantibody responses, and at least three rabbits variation was noticed in any of them. Although some fluctuation manifested neurological symptoms in the form of seizure-like ep- was seen in the absolute numbers of lymphocytes, no definite pat- isodes or nystagmus; mild lymphopenia may also have occurred in tern was observed. some rabbits. In this and the previous report (11), not all rabbits developed lupus-like symptoms, which is similar to the human Discussion scenario. There is no general unifying theory to explain initiating events that Reviewing the Ab responses we observed, sera from all rabbits lead to SLE in patients. The fact that we do not know the specific immunized with SM or GR peptide bound to the immunizing MAP pathways by which genetic and immune-modulating environmen- peptide on the ELISA plates, and no response to the MAP BB was tal factors interact, such that one individual develops SLE whereas detected at the limit of sensitivity of MAP-peptide ELISA. Al- others do not, is evidence that additional new approaches are though SM peptide is derived from a sequence within the Sm Ag, needed. The need for additional specific prognostic indicators and only 5 of 11 SM peptide-immunized rabbits’ sera bound when treatment modalities also remains. Rabbits of the NIAID colony assayed against the commercially available Sm ELISA kit. Perhaps have been bred to maintain Ig allotypes, and pedigrees of multiple the Sm Ag coated on the plate did not expose the immunizing breeding generations permit us to trace both their genetics and peptide epitope sufficiently to bind all anti-peptide Abs. It is also immune responsiveness to chosen Ags. This genetically related yet possible that rabbit sera that were positive in the Sm ELISA rec- more heterogeneous background differs substantially from the ge- ognized other epitopes; epitope spreading reported in both earlier studies (11, 57) may have occurred to different extents in different 4 The online version of this article contains supplemental material. rabbits. Similar technical issues may also account for our finding 666 MODELS OF SLE IN PEDIGREED RABBITS that although all rabbit sera that were positive for ENA were pos- unpublished observations), where analyses in progress show that itive for at least one of the component Ags, ENA-negative sera MAP-4- and MAP-8-immunized rabbits have some distinct pat- from eight rabbits including both SM and GR peptide-immunized terns of clustering. rabbits (SM-6, GR-9, SM-14, SM-15, GR-20, GR-28, GR-29, GR- The rabbit model introduced in this study presents numerous 30) bound to one or more of the component Ags of the ENA in opportunities for increasing the understanding of the autoimmune ELISA for Sm, Rnp, Ro, and La. In addition to epitope availability responses associated with human SLE. Genetic and immune re- discussed above, the likelihood that the epitopes necessary for sponse heterogeneity clearly exists within patient populations, and binding were present at sufficient density is greater with wells the appearance of autoantibodies well before the onset of clinical coated with a single Ag. Furthermore, the assay kits were origi- symptoms and pathology has been documented (71–74). The pres- nally intended and optimized for use with human samples and ence of autoantibodies in the absence of abnormal laboratory val- were adapted for use with our rabbit samples. It is noteworthy that ues in the rabbits documented in this study could reflect an early although the GR peptide is unrelated to Sm Ag, three rabbits stage of development of lupus-like disease; hence, some members (GR-9, GR-29, and GR-30) exhibited Ab response to Sm, three of Groups 2 and 3 are being monitored as they age, before being (GR-20, GR-28, and GR-30) responded to Rnp, three (GR-19, GR- euthanized for immunohistological studies. 28, GR-30) responded to SS-B (La), and one (GR-30) responded to Although no animal model can exactly replicate the complexity SS-A (Ro) (Figs. 4 and 6). of human disease, great potential exists for a noninbred rabbit It has recently been documented that NMDA receptors, known model contributing significantly to information gathered previ- to be essential for multiple brain functions and classically consid- ously from other species. Ongoing protein microarray analyses are ered to be localized within the CNS, are also present on tissues of further characterizing the full repertoire of autoantibodies pro- Downloaded from peripheral organs, including bone osteocytes, osteoblasts and os- duced by the rabbits described here. Furthermore, Western blotting teoclasts (58, 59), testis (60), pancreas (61), skin keratinocytes, and immunohistochemical studies in progress will further elucidate (62), heart (63), and adrenal glands (64). Specific anti-GR peptide reactivity with potential neuroimmune targets. Finally, ongoing anal- or anti-NMDA receptor Abs may have bound peripheral NMDA yses of gene expression in peripheral blood leukocytes following pep- receptors, caused excessive cell death, resultant release of nuclear tide immunization will hopefully serve to enhance understanding of

Ags, including spliceosomal Ags and dsDNA, and hence triggered NL and provide avenues for therapeutic intervention. http://www.jimmunol.org/ Ab responses to these autoantigens. In all autoantibody assays, initial screenings done using Abs Acknowledgments ϩ specific for rabbit H L chains gave higher background values in We are grateful to Dr. Hai Qi for his help with formatting the seizure video. some preimmune sera. For this reason, IgG Fc-specific secondary We appreciate the valuable suggestions about the manuscript from Ab was used in ELISA for autoantibodies. High-affinity anti- Drs. Mike Mage, Richard Siegel, and Ronald Wilder, and all the members dsDNA IgG Ab and the resulting immune complex formation is of the Molecular Immunogenetics Section, Laboratory of Immunology, considered a key component in the development of pathology seen National Institute of Allergy and Infectious Diseases, and thank Shirley clinically in SLE (65–68). Assays for anti-dsDNA are known to Starnes for outstanding editorial assistance. We dedicate this paper to the have substantial variability (69, 70). Configuration and density of memory of the late Dr. Timothy O’ Neill, who provided enthusiastic vet- by guest on September 29, 2021 dsDNA on coated wells, the presence of ssDNA, and details of erinary support and advice during the initial phase of this study. assay protocols may influence charge-sensitive anti-dsDNA Ab binding. We used two different assay kits with standard calf thy- Disclosures mus DNA-coated wells, both developed for human diagnosis, to The authors have no financial conflict of interest. increase the probability of detecting anti-dsDNA responses. Inter- estingly, most rabbits that made higher anti-dsDNA responses had References lower or no response to autoantigens Sm, Rnp, Ro, or La (Sm-1, 1. Manson, J. J., and D. A. Isenberg. 2003. 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