Autoimmune Disease Not Results In

Immunization with a Peptide of Sm B/B′ Results in Limited Epitope Spreading But Not Autoimmune Disease

This information is current as Lesley J. Mason, Linda M. Timothy, David A. Isenberg and of September 29, 2021. Jatinderpal K. Kalsi J Immunol 1999; 162:5099-5105; ; http://www.jimmunol.org/content/162/9/5099 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 © 1999 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunization with a Peptide of Sm B/B؅ Results in Limited Epitope Spreading But Not Autoimmune Disease1

Lesley J. Mason, Linda M. Timothy, David A. Isenberg, and Jatinderpal K. Kalsi2

An experimental model of systemic lupus erythematosus has recently been described in normal animals. We sought to conﬁrm and extend this model, which involved immunization of normal rabbits and mice with a peptide of Sm B/B؅, PPPGMRPP. This peptide is an early target of the immune response in anti-Sm-positive patients with lupus. The peptide was used in a multiple Ag peptide format, with multiple copies of PPPGMRPP bound to an inert lysine backbone. New Zealand White rabbits and A/J and C57BL/ 10ScSn mouse strains were immunized with PPPGMRPP-MAP. Pepscan assays were used to determine the epitope spreading of -the anti-PPPGMRPP-MAP response to other octamers of SmB/B؅ following immunization. We obtained high titer anti PPPGMRPP-MAP IgG responses in the New Zealand White rabbits and A/J mice. The rabbits immunized with PPPGMRPP-

MAP showed varying degrees of epitope spreading, while the A/J mice showed no spreading. We observed no autoantibodies to Downloaded from dsDNA or other anti-nuclear autoantibodies in our animals by ELISA or immunoﬂuorescence, although anti-nuclear autoantibodies were found by Western blotting in some of the rabbits. No evidence of clinical disease was seen in our normal animals. These data underline the difﬁculties often associated with the reproduction of animal models in different laboratories. The Journal of Immunology, 1999, 162: 5099–5105.

ystemic lupus erythematosus (SLE) is a multisystem au- is a possible mechanism by which the generation of multiple au- http://www.jimmunol.org/ toimmune disease characterized by the presence of auto- toantibodies could arise, the theory being that once immunological S antibodies to a variety of nuclear Ags (1). The principal tolerance to one component of a particle is broken, the immune anti-nuclear autoantibodies (ANA) found in the sera of patients are response can diversify, allowing the recognition of new epitopes against DNA, histones, small nuclear ribonucleoproteins (snRNP), within the complex (9, 10). Experimental proof that this phenom- and Ro/La particles. Abs to the spliceosomal Ag Sm are found in enon occurs in lupus is limited, although there is recent evidence approximately one-third of patients with lupus and are virtually from our laboratory4 and others (11, 12) that epitope spreading disease specific. These Abs may constitute up to 20% of the Ig may be occurring in patients with SLE. repertoire of these patients (2, 3). The spliceosome is a large mac- Recent experiments by James et al. (13, 14) showed that romolecular complex of uridine-rich snRNP particles that is re- epitope spreading occurred in normal NZW rabbits and certain by guest on September 29, 2021 sponsible for the splicing of pre-mRNA. It has been shown that the inbred mouse strains (markedly A/J and AKR/J) that had main antigenic targets of anti-Sm Abs are the common Sm been immunized with a MAP peptide derived from Sm B/BЈ, polypeptides B, BЈ, and D, which are present in all Sm snRNP PPPGMRPP-MAP. This work was particularly notable because particles. These polypeptides appear to have been highly con- the epitope spreading within the structurally associated snRNP served during evolution; B and D proteins are present in most proteins, Sm D, and U1 snRNP polypeptides 70K, A and C, eukaryotes (4). The presence of Sm BЈ is species and tissue spe- subsequently extended to include production of Abs to dsDNA cific, with both B and BЈ present in rabbits but only the B protein and was also associated with the development of symptoms found in mice (5). reminiscent of SLE. The proline-rich sequence PPPGMRPP, The immunopathogenesis of SLE, including the origin of the which occurs three times toward the carboxyl-terminal end of wide spectrum of autoantibodies that are found in patients, remains Sm B/BЈ, has been demonstrated to be the predominant anti- elusive (6). However, studies have revealed that autoantibodies to genic epitope in anti-Sm-positive patient sera (15). different components of the same nuclear particle often occur in To our knowledge, there is no published study to date indepen- linked sets and in an ordered hierarchy of expansion with time in dently analyzing this model of lupus autoimmunity. Our group has the sera of individual patients with SLE (7, 8). Epitope spreading attempted to generate the model of lupus described by James et al. (13, 14). Our findings confirm the ability of PPPGMRPP-MAP to trigger spreading of the autoimmune response in NZW rabbits, but Center for Rheumatology/Bloomsbury Rheumatology Unit, Department of Medicine, contrary to the findings of James et al. (13, 14), our results do not University College London, London, United Kingdom support the generation of lupus-like autoimmunity as a result of Received for publication October 6, 1998. Accepted for publication February 5, 1999. immunization with PPPGMRPP-MAP. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Materials and Methods 1 This work was supported by a project grant (KO522) from the Arthritis Research Animals Campaign. NZW female rabbits, weighing approximately 2.5 kg, were supplied by 2 Address correspondence and reprint requests to Dr. J. K. Kalsi, Center for Rheu- Froxfield Farms (Petersfield, U.K.). Accurate identification of the individ- matology/Bloomsbury Rheumatology Unit, Arthur Stanley House, 40-50 Tottenham ual NZW rabbits was ensured throughout the experimental procedures by St., London, United Kingdom W1P 9PG. 3 Abbreviations used in this paper: SLE, systemic lupus erythematosus; ANA, anti- nuclear autoantibody; snRNP, small nuclear ribonucleoprotein; NZW, New Zealand 4 J. K. Kalsi, W. Ng, S. Muller, and D. A. Isenberg. Expansion of the autoantibody White; MAP, multiple Ag peptide; ENA, extractable nuclear Ag. response in patients with systemic lupus erythematosus. Submitted for publication.

Copyright © 1999 by The American Association of Immunologists 0022-1767/99/$02.00 5100 Sm B/BЈ PEPTIDE (PPPGMRPP-MAP) IMMUNIZATION OF NORMAL ANIMALS the s.c. injection of microchip code numbers, which could be read with a with 50 ␮g/ml poly-L-lysine (Sigma) in distilled water. After washing, hand-held scanner. Female A/J mice (H-2a, 5–6 wk old) and C57BL/10/ plates were divided into three parts coated with 5 ␮g/ml dsDNA or 2.5 ScSn mice (H-2b, 3–4 wk old) were obtained from Harlan U.K. (Bicester, ␮g/ml ssDNA or control wells with distilled water for1hat37°C. Plates U.K.). Mice were identified by tail marking or ear punching. All the ani- were washed twice and blocked with 2% casein (BDH, Poole, U.K.) in mals were supplied barrier reared and were acclimatized for 2 wk in the PBSfor1hat37°C. Sera at varying dilutions in PBS/Tween-20 were animal facility before immunization. All procedures were conducted under incubated for1hat37°C, and bound Abs were detected as described license from the Home Office in accordance with the Animals (Scientific above. Procedures) Act 1986. Western blotting Immunogens Autoantibodies against nuclear Ags were detected using an ANA-Bioblot The peptide used for immunization was in a multiple Ag peptide (MAP) kit (Biocode Biotechnology, Sclessin, Belgium). Nuclear Ags extracted format, in this case consisting of eight copies of a synthetic peptide at- from human HeLa cells were separated by SDS-PAGE and blotted onto tached to a immunogenically inert core matrix of lysine residues (MAP, nitrocellulose together with precolored m.w. markers facilitating accurate Applied Biosystems, Foster City, CA). The MAP format peptide used in interpretation of the results. These ready-to-use strips were incubated for this study was PPPGMRPP-MAP, a major antigenic region of Sm B/BЈ 15 min with a 1/100 dilution of rabbit or mouse serum in Tris-buffered that occurs three times at positions 191–198, 216–223, and 231–238. saline containing blocking proteins and Tween-20. Bound Abs were de- (PPPGMRPP-MAP was supplied by J. A. James and J. B. Harley, Okla- tected using affinity-purified goat anti-rabbit or anti-mouse ␬-chain-specific homa Medical Research Foundation, Oklahoma City, OK). Control groups IgG conjugated to alkaline phosphatase diluted 1/1000 and then developed were included that received the MAP lysine backbone with adjuvant or with the substrates, 5-bromo-1-chloro-3-indolyl phosphate and nitroblue sterile saline with adjuvant and also a naive unimmunized group of tetrazolium (Promega, Madison, WI). Results were assessed with reference animals. to the control strips provided, showing the positions of Sm, RNP, Ro, La, Jo-1, Scl-70, and Ribosomal Po Ag bands. Immunizations Downloaded from Indirect immunofluorescence for detection of ANA The protocol for immunization adhered closely to that used by James et al. (13); we used the same peptide doses, adjuvants, and injection routes. The Serum samples were screened for ANA using standard indirect immuno- rabbits were immunized on day 0 with peptide diluted in sterile saline fluorescence techniques. Prefixed Hep-2 cells (INOVA Diagnostics, San emulsified 1/1 (v/v) with CFA (Difco, Detroit, MI) and were boosted on Diego, CA) and rat liver and kidney cryosections were used as substrates. days 26, 53, and 99 with peptide emulsified in IFA. At each immunization After incubation with varying dilutions of serum, ANA binding was de- the rabbits received 500 ␮g of peptide; half was injected s.c., and half was tected using goat anti-rabbit IgG FITC (Sigma) or goat anti-mouse IgG injected i.p. A final boost containing 500 ␮g of peptide in saline without FITC (Harlan Sera-lab, Loughborough, U.K.). http://www.jimmunol.org/ adjuvant was given i.v. on day 152. Immunization groups consisted of six rabbits immunized with PPPGMRPP-MAP, three rabbits received MAP- Solid phase peptide synthesis and pepscan assay lysine backbone and three rabbits were given saline and adjuvant alone. Pepscan assays were used to investigate the extent of any epitope spreading The A/J and C57BL/10/ScSn mice were divided into the following im- of the immune response within Sm B/BЈ. The 233 octamer peptides, over- munization groups: PPPGMRPP-MAP (n ϭ 10), MAP-lysine backbone lapping by seven amino acids, encompassing the entire length of Sm B/BЈ (n ϭ 7), saline (n ϭ 7), and naive unimmunized mice (n ϭ 7). All mice were simultaneously synthesized from F-moc-protected amino acids were immunized with 100 ␮g of peptide emulsified with CFA on day 0, (Calbiochem-Novabiochem) using the solid phase multipin peptide syn- half administered s.c. and half given i.p., and were boosted with 100 ␮gof thesis system (16) developed by Chiron Mimotopes (Victoria, Australia). peptide, half administered s.c. and half given i.p., in IFA on days 14, 28, Incubation steps of the pepscan assays were performed by lowering the and 64 (A/J) and on days 14, 28, and 63 (C57BL/10/ScSn). solid-phase peptides, bound to the pins, into solutions contained in micro- by guest on September 29, 2021 Clinical assessment titer plates, and washing steps were conducted by agitation in PBS/0.05% Tween-20 in plastic boxes. Pins were incubated for1hat37°C in blocking Throughout the experiment, the animals were under constant observation buffer consisting of 1% OVA (Sigma), 1% BSA, 1% casein, and 0.1% for the development of any clinical disease symptoms. Before the initial Tween-20 in PBS. Sera was diluted to 1/500 in blocking buffer and incu- immunization, following each boost, and at frequent intervals, the animals bated with pins overnight at 4°C. After washing in three changes of PBS, were weighed, urine was taken to assess proteinuria using Albustix (Bayer pins were immersed for1hat37°C in affinity-purified goat anti-mouse or Diagnostics, Basingstoke, U.K.), animals were bled, and serum was stored anti-rabbit ␬-chain-specific IgG conjugated to alkaline phosphatase diluted for analysis. 1/10,000 or 1/2,000, respectively, in blocking buffer and developed with the substrate p-nitrophenol phosphate. OD was measured at 410 nm. Fol- Anti-peptide Ig ELISAs lowing the assay, peptides were regenerated by sonication of the pins for 15 min in disruption buffer (0.13 M sodium dihydrogen phosphate with 1% Standard solid phase assays were used to assess the IgG Ab response to the SDS (BDH) and 0.1% 2-ME (Sigma), pH 7.2) at 60°C to ensure removal immunizing peptide. Polystyrene 96-well plates (Maxisorp, Nunc, Rosk- of the reaction products. Pins were then washed twice for 2 min in distilled ilde, Denmark) were half-coated with either PPPGMRPP-MAP or MAP ␮ water at 60°C and boiled for 3 min in methanol before being air-dried and lysine backbone at 8 g/ml in bicarbonate buffer, pH 9.6; the other half of stored at 4°C with silica desiccant. Successful regeneration of the unbound each plate was coated with buffer alone. After incubation overnight at 4°C, peptides was verified throughout these experiments by performing the as- plates were washed three times with PBS (pH 7.2) and blocked with 1% say with the omission of the serum incubation stage. (w/v) BSA (Sigma, Poole, U.K.) in PBS. Serum samples, at varying dilutions in PBS/1% BSA/0.05% Tween-20 (Sigma), were applied to coated Histology and uncoated wells and incubated for1hat37°C. Following three washes, bound Abs were detected by incubation for1hat37°C with a 1/10,000 Formalin-fixed, paraffin-embedded, kidney sections from the rabbits and dilution of affinity-purified goat anti-mouse or anti-rabbit ␬-chain-specific mice were stained with hematoxylin and eosin. These were then examined IgG conjugated to alkaline phosphatase conjugate (Sigma) and, following by a histopathologist for morphological evidence of kidney disease. Ace- washing, developed with the substrate p-nitrophenol phosphate (Sigma). tone fixed, frozen sections of the rabbits’ kidneys were stained for Ig dep- OD was measured at 410 nm. osition using horseradish peroxidase-conjugated monoclonal anti-rabbit Igs (clone RG16, Sigma), and bound Abs were detected with 3,3Ј-diamino- Autoantibody ELISAs benzidine. Similarly, paraffin wax kidney sections from the A/J mice were stained with rabbit anti-mouse Ig-horseradish peroxidase (Dako, Denmark) Autoantibodies to Sm, Sm/RNP, and Ro (60 and 52 kDa) were measured and developed with 3,3Ј-diaminobenzidine. using standard solid-phase ELISAs. Ag-precoated wells were obtained from Shield Diagnostics (Dundee, U.K.) along with uncoated control wells, Results sample diluent, and borate washing buffer. Bound Abs were detected as described for the anti-peptide IgG ELISA. Ab response to PPPGMRPP-MAP Autoantibodies to denatured ssDNA and to native dsDNA were detected Both the PPPGMRPP-MAP-immunized NZW rabbits and A/J mice using a solid phase ELISA. Pure DNA was prepared from salmon sperm DNA (Calbiochem-Novabiochem, Nottingham, U.K.) by phenol/chloro- demonstrated high titer anti-PPPGMRPP-MAP IgG Ab responses, form extraction, and ssDNA was prepared by boiling for 10 min and cool- 1/25,000 and 1/10,000, respectively. The anti-PPPGMRPP-MAP re- ing on ice for 15 min. Microtiter plates (Nunc) were coated for1hat37°C sponse was seen after the primary immunization and persisted for at The Journal of Immunology 5101

FIGURE 2. Western blot of rabbit and mouse sera for ANAs. Strips 1 and 18 are reference strips showing the positions of the labeled ENA bands. The ANA binding of sera from NZW rabbits immunized with PPPGMRPP-MAP are shown: rabbit 1 on days 26 (strip 2), 76 (3), and 230 (4); rabbit 6 on days 26 (strip 5), 76 (6), and 230 (7); and rabbit 5 on days 26 (strip 8), 76 (9), and 230 (10). Strip 11 shows the lack of binding by sera from a rabbit immunized with saline as a control, and strip 12 was incubated with anti-rabbit ␬-chain-specific IgG alkaline phosphatase conjugate, Downloaded from before substrate development. The complex binding pattern of sera from a FIGURE 1. Anti-PPPGMRPP IgG Ab response with time. A, A 1/1000 pool of two unimmunized MRL/Mp-lpr/lpr mice is shown for comparison dilution of sera from NZW rabbits immunized with PPPGMRPP-MAP, in strip 13. Strips 14, 15, and 16 show the binding (pools of four mice) of MAP lysine backbone, or saline. B, A 1/200 dilution of sera from A/J mice sera from A/J mice immunized with PPPGMRPP-MAP and lysine MAP immunized with PPPGMRPP-MAP, lysine MAP backbone, or saline or backbone and from unimmunized mice, respectively. Strip 17 was incu- from naive unimmunized mice. The days when animals were immunized bated with anti-mouse ␬-chain-specific IgG alkaline phosphatase conjugate are indicated (1). OD values were measured at 410 nm, with a reference http://www.jimmunol.org/ before substrate development. The precolored m.w. markers can be seen filter of 490 nm. running across all the strips at 22, 43, and 84 kDa. All sera were used at a 1/100 dilution. least 327 days in the rabbits (Fig. 1A) and 217 days in the mice (Fig. 1B). Sera from the control groups of animals showed no anti- immunized with PPPGMRPP-MAP did not bind to Sm B/BЈ at any PPPGMRPP-MAP response. No Ab response to the MAP lysine time point tested, but the bands suggested Ab binding to RNP A on backbone was observed in sera from the NZW rabbits and A/J mice. days 26 and 76 and to Ro (SS-A) on day 230. The other three The C57BL/10 mice were nonresponders to PPPGMRPP-MAP in our rabbits immunized with PPPGMRPP-MAP (rabbits 2, 3, and 4) hands, having no anti-PPPGMRPP-MAP IgG in their sera following showed no binding to Sm B/BЈ. It is possible that Abs might arise by guest on September 29, 2021 the full immunization schedule. These mice were therefore sacrificed spontaneously during the lifetime of a rabbit or as a result of the on day 125. immunization process, which could cross-react with nuclear Ags in the Western blot. We tested control sera from MAP backbone and Autoantibody response saline-immunized rabbits to exclude the possibility that bands seen The A/J mice from all immunization groups had no detectable on the Western blots arose from such cross-reacting Abs. No ANA anti-Sm IgG Abs when tested by ELISA on days 13, 27, 41, 63, 84, binding was observed by Western blotting of sera from these con- 106, 273, and 429. The A/J sera were also tested up until day 429 trol rabbits. The A/J mice immunized with PPPGMRPP-MAP for anti-Sm/RNP, anti-Ro, anti-ssDNA, and anti-dsDNA IgG Abs showed no binding to Sm B/BЈ or the other ENAs by Western by ELISA and were negative for all these autoantibodies. The blotting (data shown for day 84; Fig. 2). Furthermore, these ANAs NZW rabbit sera were negative in all immunization groups when were undetectable when the sera were incubated at dilutions rang- tested by ELISA for anti-Sm and anti-Sm/RNP on days 26, 76, ing from 1/10 to 1/80 on slides containing Hep2 cells or rat liver 327, and 365 and were also negative for anti-Ro on days 327 and and kidney sections. 365 and for anti-ssDNA and anti-dsDNA IgG Abs on days 26, 76, 327, and 365. Sera from MRL/Mp-lpr/lpr mice, a spontaneous Epitope spreading model of SLE, were used as a positive control in these assays, with Pepscan assays were used to investigate B cell epitope spread- the expected 20–30% of animals giving high titers of anti-Sm and ing of the immune response away from the original epitope, virtually all having anti-DNA Abs (data not shown). However, PPPGMRPP. The 233 octamer peptides, overlapping by seven over time, the development of ANAs in the sera from three of the amino acid residues, of Sm B/BЈ were synthesized according to rabbits immunized with PPPGMRPP-MAP was revealed by West- the sequence of 240 amino acids published by Rokeach et al. ern blotting of nuclear Ags extracted from HeLa cells (Fig. 2). (17). Each peptide is referred to by the position of the first After immunization with PPPGMRPP-MAP, serum from rabbit 1 amino acid residue of the octamer in the sequence of Sm B/BЈ. showed a faint Sm B/BЈ band by Western blotting on day 26; this A pool of sera from three unimmunized MRL/Mp-lpr/lpr mice band was stronger by day 76 and still persisted on day 230. We was used as a positive control. As expected, autoantibodies in also observed in sera from rabbit 1 faint bands of binding to other this serum pool bound to many of the peptides along the whole extractable nuclear Ags (ENA). Serum from rabbit 6, also immu- sequence of Sm B/BЈ (Fig. 3A). The highest peaks of binding nized with PPPGMRPP-MAP, showed no binding to Sm B/BЈ in were found at the sites of the octapeptides corresponding to and the Western blot. However, bands suggesting Ab binding to RNP adjacent to PPPGMRPP itself. Sera taken from nine A/J mice, C and ribosomal Po were observed for rabbit 6 on day 26. By day immunized with PPPGMRPP-MAP, at four time points (days 76 serum from rabbit 6 gave strong bands of binding to Sm B/BЈ, 27, 84, 161, and 300) during the experiment showed strong which still persisted on day 230. Sera from a third rabbit (rabbit 5) peptide binding to PPPGMRPP and adjacent epitopes, but no 5102 Sm B/BЈ PEPTIDE (PPPGMRPP-MAP) IMMUNIZATION OF NORMAL ANIMALS

MAP. The peptide pins incubated with anti-rabbit ␬-chain-speciﬁc IgG conjugate and substrate showed no reactivity. All six rabbits immunized with PPPGMRPP-MAP showed strong binding to octamers starting at positions 190–192, 215–217, and 230–232, which correspond to the three occurrences of PPPGMRPP in the sequence of Sm B/BЈ (191, 216, and 231) and to their adjacent epitopes. Rabbit 1 showed the greatest degree of spreading to epitopes earlier in the sequence of Sm B/BЈ, such as epitopes starting at positions 5 and 8 (KSSKMLQH and KMLQHIDY). Of particular note was an epitope starting at position 28 (IGTFKAFD) that gave a persistently high peak in the pepscans of sera from rabbits 1 and 6. The adjacent epitope 29 (GTFKAFDK) was also present for approximately 100 days in rabbit 5, and epitopes at nearby positions 32 and 33 also featured in rabbit 1 and brieﬂy in rabbit 3 on day 76. In rabbit 1, two other epitopes also gave high peaks in the pepscans; an epitope at position 42 (LCDCDEFR) was present by day 230 onward, and epitope 54 (KNAKQPER) was present on day 402. The epitopes at positions 75 (ENLVSMTV) and 77 (LVSMTVEG) were markedly Downloaded from persistent in rabbit 1 from days 76–402, and an epitope at 79 (SMTVEGPP) appeared on day 76 in rabbit 6. In the same region, epitope 85 (PPPKDTGI) appeared in rabbits 6 and 5. Indeed, as one might expect, such epitopes containing two or more prolines, as in the immunizing PPPGMRPP-MAP peptide, were common.

For example, epitopes commencing from positions 183–186 http://www.jimmunol.org/ (TPPPGIMA-PGIMAPPP) occurred in four of the six rabbits (no. 2, 4, 5, and 6) immunized with PPPGMRPP-MAP. Also occurring in the same four rabbits were epitopes from positions 222–223 (PPPPGIRG-PPPGIRGP). Other proline-rich epitopes giving high peaks were in rabbit 1 at position 203 (IGLPPARG) and in rabbit 6 at position 199 (MGPPIGLP).

FIGURE 3. Pepscans of autoantibodies in mouse sera binding to oc- Clinical observations Ј by guest on September 29, 2021 tapeptides of Sm B/B . The bars are numbered according to the position of Despite extensive monitoring, no evidence of clinical disease, in- the first amino acid residue of each octamer within the sequence of Sm cluding proteinuria, alopecia, or loss of weight was observed in B/BЈ. The occurrence of PPPGMRPP at positions 191, 216, and 231 is any of the normal animals. The formalin-fixed, paraffin-embedded indicated (2). A, A 1/500 dilution of sera from a pool of three naive unimmunized (aged 18 wk) MRL/Mp-lpr/lpr mice; B–D, a 1/500 dilution kidney sections stained with hematoxylin and eosin showed no of sera from a pool of 4 A/J mice immunized with PPPGMRPP-MAP on evidence of disease in the A/J mice. Of the six rabbits immunized day 27 (B), day 84 (C), and day 300 (D). OD values were measured at 410 with PPPGMRPP-MAP, only rabbit 1 showed evidence of any nm, with a reference filter of 490 nm. histological abnormality. This presented as a mild focal interstitial lymphocytic nephritis, which in the absence of proteinuria in this rabbit was unlikely to represent a pathological result of the immunization. Rabbits 5 and 6, which also showed epitope spreading, spreading to other epitopes. The pepscans obtained using a pool showed no such histological abnormalities. Examination of the of sera from four of the A/J mice immunized with PPPGMRPP- slides stained by immunohistochemistry revealed no evidence of MAP are shown for days 27, 84, and 300 (Fig. 3, B–D). Sera Ig deposition in any of the NZW rabbits or A/J mice immunized from the A/J mice immunized with MAP lysine backbone, sa- with PPPGMRPP-MAP. line alone, and the unimmunized mice showed no reactivity with the octapeptides of Sm B/BЈ. Pepscans that omitted the serum incubation step and where the peptide pins were im- Discussion mersed in the anti-mouse ␬-chain-specific IgG conjugate and The source and nature of the Ags in SLE that initiate the events then developed with the substrate showed no reactivity. that result in autoimmune disease are a subject of constant debate. Following the analysis of all six NZW rabbits immunized with It is still uncertain, for example, whether the eliciting agent is a PPPGMRPP-MAP, we found that two rabbits (rabbits 1 and 6) cryptic self Ag, revealed by processes such as inflammation or showed substantial epitope spreading to other epitopes of Sm B/BЈ, apoptosis, or indeed a foreign molecular mimic. In the model we which persisted for at least 376 days (Fig. 4, A–D). Another rabbit have studied a possible candidate would be the PPPGRRP region (rabbit 5) showed moderate epitope spreading for approximately of the Epstein-Barr nuclear Ag-1. A third possibility, is simply that 200 days (Fig. 4, E and F), and the remaining three rabbits (rabbits the mechanisms controlling tolerance have broken down in SLE, 2–4) showed very limited epitope spreading (representative rabbit allowing the emergence of autoimmunity. The diversity of auto- 4 is shown in Fig. 4G). Sera from the control rabbits immunized antibodies and the expression of different clinical profiles in SLE with MAP lysine backbone or saline alone showed negligible re- suggest that different triggers could be important in individual activity with the octapeptides of Sm B/BЈ (a representative rabbit patients. immunized with lysine MAP backbone is shown in Fig. 4H), as did It has been suggested that the phenomenon of epitope spreading the sera from a rabbit bled before immunization with PPPGMRPP- may be involved in the development of the autoimmune response The Journal of Immunology 5103

FIGURE 4. Pepscans of autoantibodies in NZW rabbit sera binding to octapeptides of Sm B/BЈ. The bars are numbered according to the position of the first amino acid residue of each octamer within the sequence of Sm B/BЈ. The occurrence of PP- PGMRPP at positions 191, 216, and 231 is indicated (2). A 1/500 dilution of individual sera from rabbits immunized with PP- PGMRPP-MAP was used: rabbit 1 on day 76 (A) and day 402 (B), rabbit 6 on day 76 Downloaded from (C) and day 402 (D), rabbit 5 on day 76 (E) and day 327 (F), and rabbit 4 on day 76 (G). Background binding in a rabbit immunized with the lysine MAP backbone is shown on day 76 (H). OD values were measured at 410 nm, with a reference filter of 490 nm. http://www.jimmunol.org/ by guest on September 29, 2021 in lupus. Epitope spreading can occur at both the B and T cell 216), 217, and 231 by day 26. By day 76 after the primary immu- levels, as demonstrated in models of other autoimmune diseases, nization, autoantibodies from the sera of all six of these rabbits did for example experimental autoimmune encephalomyelitis (18, 19) show strong binding in the pepscan assays to PPPGMRPP and its and the insulin-dependent NOD mouse (20). The expansion of the adjacent epitopes. immune response is thought to be facilitated by the close proximity Not unexpectedly, the observed epitope spreading was often or association of Ags (21). In SLE this could explain the occur- directed toward other proline-rich epitopes in the Sm B/BЈ se- rence of linked sets of autoantibodies (7, 8). Murine experiments quence, such as 183–186 (TPPPGIMA-PGIMAPPP) and 222–223 demonstrate that immunization with snRNP A protein also results (PPPPGIRG-PPPGIRGP), which both occurred in four of the six in the production of Abs to Sm (22), and similarly, immunization rabbits immunized with PPPGMRPP-MAP. PPPGIRGP (223) was with La Ag leads to the generation of anti-Ro Abs (23). The pres- indeed one of the MAP peptides used by James et al. in their ence of whole intact Ag has been shown to be important in the rabbit model (13), as it has been shown to be another early ordered expression of the autoantibody response (24). In a recent epitope mapped in anti-Sm/RNP-positive patients with SLE model James et al. (13, 14) suggest that an epitope, PPPGMRPP, (15). The two rabbits that showed the greatest and most persis- derived from Sm B/BЈ Ag may initiate the autoimmune response in tent epitope spreading, rabbits 1 and 6, did exhibit other normal animals that ultimately leads to the expression of clinical epitopes that had fewer amino acids in common with the im- features of lupus. munizing peptide. These included epitopes 28 (IGTFKAFD), 29 We have investigated this model and present our findings herein. (GTFKAFDK), 42 (LCDCDEFR), 75 (ENLVSMTV), and 77 We immunized genetically identical NZW rabbits and A/J and (LVSMTVEG). Some of these epitopes, 28/29 and 42, have C57BL/10 mice with PPPGMRPP-MAP peptide obtained from been shown to be antigenic regions in anti-Sm and anti-nRNP- James and colleagues. We have shown evidence of epitope spread- positive patients (15). Sera from our MRL/Mp-lpr/lpr mice also ing in three of the six NZW rabbits that we immunized with gave a peak in the region of epitope 42, and the data presented PPPGMRPP-MAP, confirming in part the data reported by James by James et al. (14) show that their A/J mice exhibit a peak in et al. (13). Intriguingly, although we observed an Ab response to this region only 28 days after immunization with PPPGMRPP- the immunogen, PPPGMRPP, from the beginning of the experi- MAP. Peaks in the region of epitopes 75–77 also appear in ment (as shown by ELISA in Fig. 1A), detailed examination of the pepscans from our MRL/Mp-lpr/lpr mice and in pepscan data anti-peptide response in the pepscans (on day 26; data not shown) from a NZW rabbit after immunization with PPPGMRPP-MAP revealed that in two of the three rabbits (rabbits 1 and 6) in which reported by James et al. (13). James et al. have shown an epitope spreading was observed, the highest responses were ini- epitope in region 168–170 (YPPGRGTP-PGRGTPPP) to be im- tially to octamers other than PPPGMRPP. Rabbit 5 was the ex- portant in patients (15), rabbits (13), and A/J mice (14). We ception, with predominant epitopes at positions 191, 215, (not found no peaks corresponding to this region in pepscans of our 5104 Sm B/BЈ PEPTIDE (PPPGMRPP-MAP) IMMUNIZATION OF NORMAL ANIMALS

NZW rabbits. However, sera from rabbit 5 did bind strongly to has a long history of interference with the results of animal ex- a proline-rich epitope at position 174 (TPPPPVGR). Many of periments, especially those involving the immune system (25). It is the epitopes bound by our NZW rabbits contain proline-rich interesting to note that although our rabbits were all female and sequences, but others often feature arginine (R) and lysine (K). housed in the same environment, only half of them exhibited sig- This adds weight to the hypothesis put forward by James et al. nificant epitope spreading, and this occurred to varying degrees (15) that the presence of such positively charged amino acids and to different epitopes in the individual rabbits. Also, of a total seems to be an important factor determining the antigenicity of of nine rabbits (five males and four females) immunized by James a peptide sequence. Three of our six NZW rabbits immunized et al. (13) with PPPGMRPP-MAP using the same immunization with PPPGMRPP-MAP have shown epitope spreading within schedule that we employed, only one developed Abs to dsDNA Ј Sm B/B . However, although the Western blotting data for these and clinical symptoms reminiscent of SLE. However, sera from all three rabbits suggests epitope spreading to Ags beyond Sm nine of these rabbits did bind to octapeptides of spliceosomal pro- Ј B/B , we have been unable to confirm this by ELISA or immu- teins beyond Sm B/BЈ. Perhaps if we had immunized larger num- nofluorescence techniques. In addition, we have found no evi- bers of rabbits, some of these may have developed further auto- dence of diversification of this response to include Abs to immunity. It is notable in MRL/Mp-lpr/lpr mice, a spontaneous dsDNA or the development of clinical disease in any of our model of SLE, only approximately 25% of these mice develop rabbits. anti-Sm Abs despite being a genetically inbred strain (30). The data published by James et al. (14) indicate that of all the Although James et al. (13, 14) have successfully used the MAP strains of mice they tested, the A/J strain exhibited the greatest

system to generate autoimmunity in normal animals, there are sev- Downloaded from degree of epitope spreading to other regions of Sm B/BЈ and Sm D. eral authors who report the limited value of MAPs for raising This spreading was already marked only 28 days after immuniza- anti-peptide Abs that are cross-reactive with the cognate protein. tion with PPPGMRPP-MAP. Sera from their mice also showed high autoantibody binding to Sm and U1 snRNP, and 80% of the These limitations are reviewed by Briand et al. (31). These authors mice had Abs to dsDNA. Some of these mice also showed clinical reported that this lack of cross-reactivity occurred especially when symptoms reminiscent of lupus (14). Intriguingly, despite sustain- the MAP was constructed from C-terminal peptides. PPPGMRPP Ј ing high titers of anti-PPPGMRPP Abs we saw no epitope spread- is found at three positions within Sm B/B , all of these are toward http://www.jimmunol.org/ ing within Sm B/BЈ and no autoantibody response to Sm, U1 the C-terminal end, with the 231 position being at the very termi- snRNP, or dsDNA. We also observed no evidence of clinical dis- nus. McLean et al. (32) report that none of their animals immu- ease in any of the A/J mice we immunized. The C57BL/10 mice nized with different MAPs produced sera that recognized the par- that we immunized with PPPGMRPP-MAP did not show any anti- ent protein in Western blots. It is thought likely that the peptides PPPGMRPP IgG response by ELISA, whereas James et al. (14) in the MAP construction assume an unusual conformation that found that C57BL/10 mice gave a high titer antipeptide response, does not mimic the structure of either the monomeric peptide or although this strain did not subsequently exhibit epitope spreading the parent protein. This may explain why we were able to dem- or snRNP binding. onstrate ANAs by Western blotting but not by ELISA in the sera The discrepancies between our findings and those of James et al. from our PPPGMRPP-immunized rabbits. by guest on September 29, 2021 (13, 14) could be due to several factors, including batch-to-batch vari- In a situation where comparable numbers and as identical as ation between different preparations of PPPGMRPP-MAP peptide, possible conditions have been employed to reproduce a model of environmental factors, and any differences in immunization proce- autoimmune disease but with differing results, it is important to dures. We suspect that the exact reproducibility of the MAP peptide isolate and define those factors that might allow the expression of synthesis, even within the same facility, may be the most important of autoimmunity. The immunological mechanism should have been these factors. It is possible that any structural variation in the Ag may the same in these two experimental sets; however, it is possible affect the presentation of Ag to the immune system. The peptides we that a slight alteration to the level of signaling or in the presenta- used were obtained from the same biochemical facility to try and tion of the Ag resulted in autoimmune disease in one set of animals avoid this source of variation; however, it was not possible to use but not in the other. Dissection of any subtle environmental or exactly the same batch of PPPGMRPP-MAP as that employed by genetic variables might provide clues to the pathogenesis of auto- James et al. (13, 14) due to the limited quantity of peptide produced immunity in lupus. at each synthesis. The environmental conditions under which the an- In conclusion, we observed limited epitope spreading in some imals were housed appear to have been comparable, although we NZW rabbits but not in any A/J mice, as a result of immuni- cannot fully exclude the possibility of an unknown infectious agent zation with PPPGMRPP-MAP. Immunization with this Sm having affected the development of autoimmunity (25). The immuni- B/BЈ peptide did not lead to the development of autoantibodies zation protocol reported in the original paper by James et al. (13) was to dsDNA or to the development of autoimmune disease in our adhered to very closely; we used the same peptide doses, adjuvants, normal animals. We were thus unable to confirm the generation and injection routes. We allowed more than adequate time for the of autoimmune disease in normal animals following immuni- development of autoimmunity, since we followed the NZW rabbits zation with PPPGMRPP-MAP, as reported by James et al. (13, and A/J mice for 402 and 429 days, respectively, after immunization with PPPGMRPP-MAP. 14). This once again underlines the difficulties often experi- There are many examples of animal models of autoimmunity enced in reproducing animal models in different laboratories. that have proven difficult to reproduce; some of these are discussed by Isenberg et al. (26) when describing their attempt to reproduce the 16/6 Id lupus model (27). Leiter et al. (28) report variability in Acknowledgments the frequency of development of diabetes in NOD mice, probably We thank Selena Blades for carrying out the immunohistochemistry, due to variable diet or microbial exposure. Goverman et al. (29) Marlene Swana for assistance with ANA immunofluorescence, and Dr. report how spontaneous EAE can develop in transgenic mice Meryl Griffiths for examining the histological sections. We also thank Dr. housed in a nonsterile facility, but not in those maintained in a Judith James, Dr. John Harley, and Tim Gross for useful discussions during specific pathogen-free facility. Mouse hepatitis virus, in particular, the course of our experiments. The Journal of Immunology 5105

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