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The Journal of Immunology, 2005, 175: 8409-8414.
Copyright © 2005 by The American Association of Immunologists

Immunization with Short Peptides from the 60-kDa Ro Antigen Recapitulates the Serological and Pathological Findings as well as the Salivary Gland Dysfunction of Sjögren’s Syndrome

R. Hal Scofield1,*,{dagger},{ddagger}, Sima Asfa*, David Obeso*, Roland Jonsson§ and Biji T. Kurien*

* Arthritis and Immunology Program, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104; {dagger} Department of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73109; {ddagger} Medical Service, Department of Veterans Affairs Medical Center, Oklahoma City, OK 73104; and § Broegelmann Research Laboratory, University of Bergen, Bergen, Norway


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Sjögren’s syndrome is a poorly understood autoimmune inflammatory illness that affects the salivary and lacrimal glands as well as other organ systems. We undertook the present study to determine whether mice immunized with short peptides from the 60-kDa Ro (or SSA) Ag, which is a common target of the autoimmunity of Sjögren’s syndrome, develop an illness similar to Sjögren’s syndrome. BALB/c mice were immunized with one of two short peptides from 60-kDa Ro that are know to induce epitope spreading. The animals were analyzed for the presence of anti-Ro and anti-La (or SSB) in the sera by immunoblot and ELISA. Salivary glands were collected and examined by histology after H&E staining. Salivary lymphocytes were purified and studied for cell surface makers by fluorescence-activated cell sorting. Timed stimulated salivary flow was measured. As reported previously, BALB/c mice immunized with 60-kDa Ro peptides developed an immune response directed against the entire Ro/La ribonucleoprotein particle that was similar to that found in humans with lupus or Sjögren’s syndrome. Functional studies showed a statistical decrease in salivary flow in immunized mice compared with controls. Furthermore, there were lymphocytic infiltrates in the salivary glands of immunized animals that were not present in controls. The infiltrates consisted of both CD4– and CD8+ T lymphocytes as well as B lymphocytes. BALB/c mice immunized with 60-kDa Ro peptides develop anti-Ro, salivary gland lymphocyte infiltrates, and salivary dysfunction that is highly reminiscent of human Sjögren’s syndrome.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Sjögren’s syndrome is a chronic, progressive autoimmune illness characterized by involvement of the salivary and lacrimal glands. Patients usually present with keratoconjunctivitis sicca and xerostomia but may develop, or present initially with, systemic manifestations involving the lungs, kidneys, skin, muscles, bone marrow, joints, and/or vasculature. The disease is usually classified as primary or secondary in that secondary Sjögren’s syndrome occurs along with another inflammatory autoimmune illness such as rheumatoid arthritis, systemic lupus erythematosus, primary biliary cirrhosis, polymyositis, or scleroderma (1). In either form, the sera of patients with the disease contain autoantibodies. As in other systemic autoimmune diseases, the commonly recognized autoantigens in Sjögren’s syndrome are found in every cell type, not just in the affected organs. When identified with sensitive techniques, >75% of Sjögren’s syndrome patients have Abs with specificity for the Ro/La (or SSA/SSB) ribonucleoprotein particle (2). A number of other autoantibodies are also found in the sera of patients with Sjögren’s syndrome, some of which, such as anti-muscarinic receptor, may be involved in the pathogenesis of glandular dysfunction and are tissue specific (3). The etiology, either environmental or genetic, of Sjögren’s syndrome is not known.

There are several animals models of Sjögren’s syndrome, but none of these completely reproduces all aspects of the disease. Lymphocytic infiltration of the salivary glands and reduced salivary and lacrimal gland function have been noted in NOD mice (4). Strikingly, the NOD mouse is the only previously reported model of Sjögren’s syndrome that shows exocrine glandular dysfunction. NOD mice have autoantibodies binding {alpha}-fodrin and muscarinic receptors, but there is no evidence of anti-Ro or anti-La, which are so commonly found in the sera of humans with Sjögren’s syndrome. The salivary glands of MRL-lpr/lpr mice, a model of lupus in which a defect in the fas gene is found, are infiltrated with activated T lymphocytes. After transfer of these cells into SCID mice, salivary and lacrimal lesions develop. However, there is no salivary gland dysfunction in MRL-lpr/lpr mice or their derivatives (5). C57BL/6 mice carrying the lpr/lpr gene develop infiltrates of the salivary gland and high-titer anti-Ro and anti-La, but salivary or lacrimal gland function has not been studied (6).

In a number of experimental systems, including experimental autoimmune encephalitis, autoimmune ovarian disease, and systemic lupus erythematosus, immunization with components of autoantigens leads to epitope spreading and disease in immunized animals (reviewed in Ref.7). We have immunized animals with short peptides from lupus-related autoantigens such as small nuclear ribonucleoprotein and found initiation of an immune response toward the peptide of immunization, quickly followed by a full B cell response to the array of small nuclear ribonucleoprotein and Sm protein that form the slpiceosome and are targets of the immune response in systemic lupus erythematosus. Furthermore, these animals develop a clinical syndrome similar to systemic lupus erythematosus (8). We have also conducted similar experiments using monomer peptides from the 60-kDa Ro Ag (9, 10) and found epitope spreading with an autoimmne B cell response quantitatively and qualitatively similar to the mature anti-Ro/anti-La response found in humans with systemic lupus erythematosus or Sjögren’s syndrome. Also, we and others have used fragments or whole polypeptides of La or 52-kDa Ro (11, 12, 13, 14) to produce similar findings of epitope spreading. However, animals immunized with components of the Ro/La ribonucleoprotein complex have not developed a lupus-like illness, although pups born of 52 Ro-immunized mothers have congenital heart block that is similar to that seen in human infants born to mothers with anti-Ro and anti-La (15). We undertook the present study to determine whether mice immunized with short peptides from the 60-kDa Ro autoantigen develop an illness similar to Sjögren’s syndrome.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Animals

BALB/c mice were purchased from The Jackson Laboratory or raised in our local colony, the founders of which were purchased from The Jackson Laboratory.

Immunization

The animals were immunized with peptide Ro480–494 (amino acid sequence AIALREYRKKMDIPA) or Ro274–290 (amino acid sequence QEMPLTALLRNLGKMT) from 60-kDa Ro or immunized as controls. Immunization was conducted as reported previously (10, 16). Briefly, 100 µg of monomer peptide in PBS were emulsified 1:1 in CFA for the initial immunization with subsequent immunization in IFA on days 14, 35, 63, and 51. Animals were bled via the tail vein on days 21, 42, 56, 70, 84 105, 126, 147, 168, and 262. Surviving animals were killed by exsanguination on day 263 of the protocol. Control animals followed an immunization schedule identical to the Ro peptide-immunized animals, including both CFA and IFA, but were immunized with Freund’s adjuvant alone, saline emulsified in Freund’s adjuvant, or control peptides emulsified in Freund’s adjuvant. The control peptide consisted of a reversed sequence of Ro274–290 (i.e., TMKGLNRNLLATLPMEQ), a peptide consisting of these same amino acids in a random order (RTLMLKAGTLNLPMEQL), or Ro413–425 amino acid sequence (VAFACDMVPFPVTTDM), a peptide previously shown by us to not induce epitope spreading (10). Another set of animals followed an identical immunization protocol but was killed on day 126.

Peptides

The peptides were synthesized at the University of Oklahoma Health Sciences Center Molecular Biology Facility using standard Fmoc chemistry. Peptides were purified to apparent homogeneity by HPLC. Purity was analyzed by mass spectroscopy. Multiple antigenic peptides (MAPs)2 representing the previously described B cell epitopes of 60-kDa Ro (17, 18) were synthesized by the same facility and used in ELISA.

Enzyme-linked immunosorbent assay

ELISAs were conducted using purified 60-kDa Ro (9, 10) or the Ro-MAPs (10), as described previously, with sera diluted at 1/100. Results of the MAPs ELISA were considered positive when the OD >3 SDs above control and preimmune sera developed. In addition, positive values had to have an OD >0.40. For ELISA to determine whether saliva had anti-Ro, the Ro protein was coated onto plates as described previously (9, 10), then saliva was used at a dilution of 1/10 with PBS. The remainder of the assay was conducted using the identical protocol as for sera.

Immunoblot

These assays were conducted as described previously, using recombinant mouse 60-kDa Ro expressed as a fusion protein with maltose-binding protein. A lacz-MBP fusion protein served as a control (16). Ro-MAPs were used in immunoblot as described previously (19). Mouse sera were used individually, not pooled, and studied at a 1/100 dilution.

Salivary lymphocyte purification and analysis

Single-cell suspensions of salivary gland lymphocytes were prepared according to a published protocol as follows. Salivary glands were removed by dissection immediately after the mice were killed and placed in a 1.5-ml conical tube containing RPMI 1640 medium supplemented with glutamine, penicillin, streptomycin, and FBS. The salivary gland was then placed inside a sterile petri plate. The tissue was minced with scissors and then with a razor blade, transferred to conical tube containing 0.75 ml of RPMI 1640 that was supplemented additionally with collagenase (Sigma-Aldrich) at 1.5 mg/ml, and mixed end-over-end for 30 min at 37°C using a Shaker rotisserie (Thermolyne Labquake). The contents were then filtered through a Falcon cell strainer (70-µm nylon; BD Biosciences), and the filtrate was saved. The retentate was retransferred to another conical tube containing 0.75 ml of RPMI 1640 and collagenase as before, and the mixing was repeated. The sample was filtered as before, and the filtrate was combined with the earlier filtrate.

The filtrate, in aliquots containing 50,000 cells, was used in a FACS (Beckman Coulter) to analyze the number of CD3+ and CD19+ cells to determine the number of T and B cells. The cells were incubated separately with mAbs to these markers (Serotec). The cells were then stained with PE-labeled secondary Abs and analyzed on a FACS.

Pathology

Salivary glands were collected from mice on day 263 (or 126) of the immunization protocol. One gland from each animal was fixed immediately in 10% Formalin and embedded in paraffin. Thin sections were cut and stained with H&E. Lymphocytic infiltrates (>50 cells each) were scored by an observer who was blinded to the immunization group of an individual mouse, and who examined 10 sections from each mouse.

Saliva

Mouse saliva was collected according to a previous published protocol (20), with modifications. Briefly, animals were fasted for 16–18 h before the procedure. An i.p. of injection of 2.5% 2,2,2-tribromoethanol at 0.01 ml/g body weight was given to each animal as anesthesia. Saliva secretion was then stimulated with an i.p. injection of 0.020 mg of isoproterenol/100 g body weight and 0.05 mg of pilocarpine/100 g body weight in the same syringe. Total saliva was then obtained from the oral cavity over a 10-min period using capillary tubes. The saliva was placed in sterile plastic containers and stored at –70°C until further use.

Statistics

Categorical variables such as the presence or absence of infiltrates were subjected to a {chi}2 analysis. The timed saliva flow did not fit a Gaussian distribution and therefore was analyzed using nonparametric rank order testing.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Ten animals were immunized with Ro480 and 10 animals were immunized with Ro274 according to the protocol outlined in Materials and Methods. Control animals were immunized with Freund’s adjuvant alone, saline alone, or with the control peptides. As we reported previously and present only briefly in this report, such immunization rapidly leads to anti-peptide Ab, and this was followed quickly (within 2–3 wk) by Abs binding other peptide epitopes of 60-kDa Ro as assessed by ELISAs using the 21 Ro-MAPs, which represent the previously identified B cell epitopes of 60-kDa Ro (data not shown). Most of the Ro480 and Ro274 animals also bound to whole mouse 60-kDa Ro in immunoblot using sera obtained at day 147 (Fig. 1). There was no binding by any mouse to a lacz-MBP fusion protein used as a control (Fig. 1). Animals immunized with control peptides, either a reversed or a scrambled sequence of Ro274, had no binding to Ro274 (Fig. 2). Nor was there binding to any epitope of 60-kDa Ro or whole 60-kDa Ro (data not shown). Likewise, Freund’s adjuvant alone or saline in Freund’s adjuvant immunization produced no Ro peptide or whole Ro Abs. Animals immunized with Ro413–425 develop anti-413–425 but have no Ab binding whole 60-kDa Ro or other epitopes of 60-kDa Ro (10).



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  		FIGURE 1. Mouse Ro60-MBP fusion protein was purified from inclusion bodies as a soluble protein and subjected to SDS-PAGE, followed by transfer to nitrocellulose. Blots were probed with 1/100 dilutions of mouse sera. Animals were immunized with either Ro peptide 274 or Ro peptide 480, or as controls with saline alone, with Freund’s adjuvant alone, or with an unrelated peptide.

 


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  		FIGURE 2. Ro274-immunized animals 2–5 (as shown in Fig. 1) were studied by ELISA with the Ro274 peptide, a scrambled sequence of Ro274, a reversed sequence of Ro274, or no Ag coated on the well.

 
Intramolecular spreading was assessed in several ways. A direct Ag ELISA in which La was coated on the surface of microtiter plate wells demonstrated that several of the BALB/c mice immunized with either Ro480 or Ro274 had Abs in their sera binding this autoantigen (Fig. 3). Inhibition studies confirmed the specificity of these Abs. Exposure of the sera to soluble La inhibited binding to the solid-phase La Ag (Fig. 3). Meanwhile, exposure to solid-phase Ro60 did not inhibit binding to La (data not shown). Thus, as we have shown previously (9, 16, 21), intermolecular spreading in the B cell compartment is not due to cross-reacting Abs in the experiments undertaken. Only one animal bound 52- kDa Ro on immunoblot using a mouse cell lysate as the electrophoresed Ag source (data not shown). So, intermolecular spreading was similar to that we have reported previously in that even within a cohort of genetically identical mice, there was heterogeneity.



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  		FIGURE 3. OD developed in ELISA is given on the y-axis for studies in which La was coated on the solid phase. Sera were used at a 1/100 dilution. Individual mice are a point along the x-axis with immunogen given for each mouse group. •, Studies with inhibition by soluble-phase Ag; {blacksquare}, studies using La as a soluble inhibitor.

 
We next wanted to test the central hypothesis of this report, namely, that immunization with peptides from 60-kDa Ro would lead to a Sjögren’s-like disease in the BALB/c mice. This question was assessed in two ways. First, we determined whether there was an infiltrate of lymphocytes in the salivary glands of immunized animals. Second, we determined whether altered function of the salivary glands was present. Concerning the former, salivary glands were collected on day 263 of the protocol and examined for the presence of groups of infiltrating lymphocytes. We found that animals immunized with the Ro peptides did, in fact, have lymphocytic infiltration similar to that seen in human Sjögren’s syndrome (Fig. 4). Similar to intermolecular epitope spreading in these mice, the presence and degree of pathology in the form of lymphocytic infiltrates varied among the individual mice, but immunized mice clearly had infiltrates, whereas control mice had little or no infiltration of lymphocytes in the salivary glands (Table I).



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  		FIGURE 4. Pathology of the salivary glands from animals immunized with Ro274 or Ro480 or control is given at low-power (top row) and high-power (bottom row) magnification. Lymphocytic infiltrations are clearly found in the Ro peptide-immunized animals but not in the control animals.

 

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  		Table I. Lymphocytic infiltration in the salivary glands of BALB/c mice immunized with Ro480 or Ro274 as well as control mice was assesseda

 
We have preliminarily examined the cellular make-up of these infiltrates. One parotid gland from each animal was used for study of resident lymphocytes by fluorescence-activated cell sorting. From 4 to 6 million lymphocytes were present in the parotid gland of Ro peptide-immunized animals, whereas from 1 to 2 million lymphocytes were found in the glands of control animals. Thus, the percentage of cells that were of immune origin was much higher in the Ro peptide-immunized animals than in the control animals. Single-cell suspensions were produced, and fluorescence-activated cell sorting was performed with anti-CD3 and anti-CD19 on cells from each animal. Both B and T lymphocyte subtypes were found in the salivary glands of Ro peptide-immunized animals with 45% CD4+ T cells, 18% CD8+ T cells, and 35% CD19+ B cells, similar to findings in humans with Sjögren’s syndrome (22).

In some other animal models with salivary gland infiltrates, there is no glandular dysfunction, whereas in the NOD mouse, there is both pathology and dysfunction. Thus, we wanted to determine whether our Ro peptide-immunized animals had clinical, physiological sequelae of the pathological process that gave lymphocytic infiltrates in the form of reduced saliva production. To that end, we measured timed, stimulated salivary flow in the BALB/c mice immunized with the Ro peptides as well as in the controls (Fig. 5). Similar to both intermolecular spreading and salivary infiltration in which there was a discordance among animals, there were Ro peptide-immunized animals with normal saliva flow and animals with severely impaired saliva flow. Thus, there was not a normal distribution of salivary flow in any group of animals. Median flow in the controls was 47 µl, whereas the median flow was 23 and 28 in the Ro274- and Ro480-immunized animals, respectively (Fig. 5). So, saliva production was statistically significantly impaired in Ro peptide-immunized animals compared with controls (p < 0.05, Mann-Whitney U test). We assayed saliva for the presence of anti-Ro, also. Ro peptide-immunized animals had IgA anti-Ro in their saliva samples, but control animals did not (data not shown).



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  		FIGURE 5. The median, maximum, minimum, 75th percentile, and 25th percentile are given for stimulated salivary flow in control (n = 10) as well as Ro peptide-immunized animals (n = 10 in each group). When examined as a single group, or separately, compared with the control animals, there was a statistically decreased flow in the Ro274 and Ro480 peptide-immunized animals (p < 0.05, Mann-Whitney U test) but not among the Ro413 peptide-immunized animals.

 
To demonstrate reproducibility of the model and a shorter time course to disease development, a second set of mice were immunized with the Ro274 peptide, and controls were immunized with adjuvant only. Saliva flow studies were performed on day 120, and pathology studies were performed on day 126. Similar to the initial group studied on day 263, there was a statistically significant decrease in salivary flow as well as salivary gland lymphocytic infiltrates in the Ro peptide-immunized animals compared with controls (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Sjögren’s syndrome is a relatively common disease. Among the autoimmune rheumatic diseases, the disease is possibly the second most common, only surpassed by rheumatoid arthritis (23). The disease is most commonly diagnosed in middle-aged women but can occur at any age and in men. Sjögren’s syndrome is likely underdiagnosed such that the true prevalence and incidence in the population are underappreciated. A population-based study in Greece found at least 5% of women >65 years of age had the disease (24). Many persons have dry eyes and mouth for which medical care is not sought, and these persons tend to have higher levels of anti-Ro and anti-La than those without sicca (25). Several classification criteria have been proposed, including those of Fox and Saito (26) and a European study group (27). Recently, a new set of criteria has been proposed by a combined European and North American group (28). There are little data concerning the heritability of Sjögren’s syndrome. Only two sets of twins with definite Sjögren’s syndrome have been reported, to our knowledge (29, 30). Thus, the concordance of disease among twin pairs is unknown, and the genetic contribution to the risk of disease is not known.

There is a relative paucity of research into this disease compared with other autoimmune diseases, many of which are less frequent than or have about the same frequency in the human population as Sjögren’s syndrome. The reasons underlying this lack of investigative effort are likely multiple and complex. Sjögren’s syndrome may not be considered a life-threatening, serious illness. Of course, Sjögren’s syndrome can have serious effects on the lungs, kidney, and nervous system in addition to its oral and ocular effects as well as life-threatening lymphoproliferative disease (31). Another factor that may have impacted research into the condition is the relative lack of animal models compared with diseases such as lupus, multiple sclerosis, type 1 diabetes mellitus, and rheumatoid arthritis. Each of these diseases has a long history of a well-described animal model in which pathological and immunological studies as well as preclinical trials of therapy can be conducted.

In contrast, animal models of Sjögren’s syndrome have been much less satisfactory and have a shorter history. The NOD mouse is an inbred strain that has been used for many years as a model of type 1 diabetes. More recently, lymphocytic infiltrates of the salivary glands and reduced salivary and lacrimal gland function have been noted in these animals (reviewed in Ref.4). Strikingly, the NOD mouse is the only previously reported model of Sjögen’s syndrome that shows exocrine glandular dysfunction. There are circulating Abs that bind the surface as well as the nucleus of salivary gland cells. NOD mice make Abs against {alpha}-fodrin (32), but there is little evidence that anti-Ro or anti-La Abs are present in these mice. There is, however, evidence that Abs are critical to disease in this model. NOD mice without functional B cells (NOD IgM null) have salivary infiltrates consisting of T cells but have normal salivary gland function. Infusion of IgG from NOD mice or from humans with Sjögren’s syndrome induces a decrement of salivary gland function in the B cell-deficient NOD mouse, though (33). Abs against muscarinic receptors may be important in this regard. Like other reported animals with Sjögren’s syndrome, the NOD might be considered a model of secondary Sjögren’s syndrome because another autoimmune disease is present, namely type 1 diabetes. The diabetes in these animals is dependent on the MHC haplotype. Studies with NOD congenic for H-2b demonstrate that although diabetes does not develop, salivary gland infiltrates do develop (20). Thus, these congenic animals are a definite model of primary Sjögren’s syndrome. Because the cellular infiltrate, cell surface binding Abs and glandular dysfunction are present, the NOD mouse and its variants have and will no doubt continue to prove useful in deciphering the pathogenesis of Sjögren’s syndrome. Nonetheless, while replicating the disease in several ways, this model does not have appreciable anti-Ro and anti-La, a near-universal finding in human disease.

The MRL-lpr/lpr mouse has a genetic defect in the fas gene and therefore a defect in the apoptotic death of lymphocytes. There is massive accumulation of lymphocytes and development of a disease similar to human systemic lupus erythematosus. This animal has been used extensively to study lupus autoimmunity (5). The salivary glands of MRL-lpr/lpr mice are infiltrated with activated T lymphocytes. After transfer of these cells into SCID mice, salivary and lacrimal lesions develop (34). Anti-CD4 or anti-V{beta}8 mAbs prevent development of lesions in the SCID recipients (20). However, there are several inherent problems with the MRL-lpr/lpr mouse as a model of Sjögren’s syndrome. Clearly, this is a model of secondary, not primary, disease because these mice also have a lupus-like illness. Second, there is no decreased function of the salivary glands (35). Third, these animals do not develop anti-Ro or anti-La in a manner similar to human patients. In one study, 6 of 17 mice had Abs to 52-kDa Ro, but the titer was high in only 3 mice. Meanwhile, 1 of 17 mice was reported to have anti-60-kDa Ro, and no titer or inhibition data were given (36). Finally, the infiltrate in the salivary glands of these mice consists of T cells alone, in contrast to that found in human disease, which is a mix of CD4+ and CD8+ T cells along with B lymphocytes.

Several other animal models of Sjögren’s syndrome have been reported, but many of these have been the subject of only a few reports, or even only a single report. An acute sialoadenitis occurs in a graft-vs-host transplant model (37). Other than a pathological description, no other data have been reported. Mice transgenic for the envelope protein of the hepatitis C virus develop salivary lesions resembling Sjögren’s syndrome (38), and humans with hepatitis C can develop a sicca illness (39). The NFS/sld mouse bears a mutation resulting in sublingual gland differentiation arrest. After neonatal thymectomy, these mice develop pathology in the other salivary and lacrimal glands that resembles that found in human Sjögren’s syndrome, with females affected more often than males (40). The infiltrate was predominantly composed of CD8+ lymphocytes, and although there were anti-salivary Abs in the sera, no anti-Ro or anti-La was detected (40). Mice homozygous for the almphoplasia (aly) mutation lack lymph nodes and Peyer’s patches. These animals develop inflammatory infiltrates (CD4 lymphocyte rich) of the salivary glands, lungs, pancreas, and lacrimal glands. The mechanism by which this defect gives the absent lymph node and absent Peyer’s patches phenotype is not known (41).

Fleck et al. (6) reported infection of C57BL/6-lpr/lpr mice with murine cytomegalovirus with resultant acute and chronic sialoadenitis. This inflammation persisted after clearance of the virus. High levels of anti-Ro, anti-La, rheumatoid factor, and anti-dsDNA were produced. Thus, this is the only previously reported mouse model of Sjögren’s syndrome with convincingly high levels of anti-Ro and anti-La, although whether the anti-Ro is anti-60-kDa Ro or anti-52-kDa Ro, or both, is not clear. Pathology of other organs is not given, but the presence of anti-dsDNA, which is usually considered specific to lupus, implies that this might be a model of secondary Sjögren’s syndrome. Furthermore, salivary function has not been studied (6). C57BL/6 mice with mutant fas-ligand (B6-gld/gld) also develop salivary lymphocytic infiltrates after infection with mouse CMV (42). Local salivary delivery of wild-type fas ligand via an adenoviral vector resulted in a marked reduction in the salivary infiltrates, however. Production of autoantibodies or saliva is not given for the B6-gld/gld mice (42).

The present work has developed a new model of Sjögren’s syndrome in which pathology within the salivary gland, salivary gland dysfunction, and production of high titer anti-Ro and anti-La are all found. This work is an extension of our previous work with immunizing animals with short peptide from the 60-kDa Ro protein Ag with resultant T cell and B cell epitope spreading. Immunization as a model of autoimmune rheumatic illnesses and their associated autoimmunity has been reviewed recently (43). We found that immunization of some but not all mouse strains (16) with the Ro-derived peptides results in an Ab response that targets the entire 60-kDa Ro molecule. This response is highly similar to that found in humans with anti-Ro (9, 10, 16, 21) and includes intermolecular spreading such that the La protein and 52-kDa Ro are also targeted by the immune response.

BALB/c mice immunized with either the Ro274 or Ro480 peptide recapitulate the findings in human Sjögren’s syndrome quite well. The pathology found in the salivary glands of these animals is highly similar to that found in the human minor salivary gland, parotid gland, or lacrimal gland among patients with Sjögren’s syndrome. In particular, it is striking in both humans and our mice that the abnormality of salivary flow is not fully explained by the degree of destruction of the gland. This finding suggests another component of the disease, which interferes with saliva and tear production. In humans, this may prove to be Abs blocking the muscarinic receptors that are vital to salivary and lacrimal gland function. Perhaps the presently reported mice, similar to the NOD, will prove to have anti-muscarinic autoantibodies. In contrast to other models of Sjögren’s syndrome, only the NOD mouse and the immunization model described in this study have reduced saliva production, a key feature of disease in humans with Sjögren’s syndrome. We submit that this feature is critical to any model of Sjögren’s syndrome.

Finally, the model reported has high-titer anti-Ro and anti-La that is similar to that found in humans with Sjögren’s syndrome. The importance of these Abs has recently been re-emphasized by the new combined European-American classification criteria (28) in which patients must have either a diagnostic biopsy or anti-Ro. So, a model that incorporates the presence of these characteristic Abs is much more similar to human disease than one that does not. The relationship of these Abs to the pathogenesis of the disease is unknown. An animal model in which these autoantibodies can be studied in relation to salivary gland dysfunction and pathology will likely help elucidate the role of anti-Ro and anti-La in the etiology and pathogenesis of Sjögren’s syndrome.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
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.

1 Address correspondence and reprint requests to Dr. R. Hal Scofield, 825 Northeast 13th Street, Oklahoma City, OK 73104. E-mail address: hal-scofield{at}omrf.ouhsc.edu Back

2 Abbreviation used in this paper: MAP, multiple antigenic peptide. Back

Received for publication February 19, 2004. Accepted for publication October 13, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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