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. 2002 Jul;129(1):19–26. doi: 10.1046/j.1365-2249.2002.01899.x

The genotype of mice influences the autoimmune response to spliceosome proteins induced by cytomegalovirus gB immunization

J LIPES 1, E SKAMENE 1, M M NEWKIRK 1
PMCID: PMC1906413  PMID: 12100018

Abstract

In previous studies we have established a link between cytomegalovirus (CMV) infection and an autoimmune response to the U1–70 k protein of the spliceosome in man. This autoimmune response, generally referred to as the anti-RNP (ribonucleoprotein) antibodies, is observed in about 30% of patients with systemic lupus erythematosus (SLE). We have also found that the CMV glycoprotein B (CMV gB) when expressed in a adenovirus vector (Ad) could induce a significant anti-U1–70 k antibody response in several strains of mice, such as C3H, MRL and BALB/c. In the present study we examined the autoimmune response induced by immunization with Ad-gB in A/J and C57BL/6 (B6) mice and determined whether there was any autoimmune phenotype similar to that observed in patients with SLE. Thus groups of A/J and B6 mice were immunized with Ad/gB or with Ad alone and then observed for possible skin or kidney disease. In addition the autoantibody response to the spliceosome was measured, and the target antigens identified by immunoblot techniques. All of the A/J mice mounted a very high IgG response primarily to the U1–70 k protein of the spliceosome, with evidence of a rapid spreading of the autoantibody response to other components of the complex. In contrast, B6 mice mounted only a very low titre autoantibody response and failed to show signs or symptoms of autoimmunity. The A/J but not the B6 mice were found to have deposits of IgG in their kidneys, which were consistent with abnormal levels of blood urea nitrogen in the A/J but not B6 mice. This study demonstrates the importance of the genetic background in the susceptibility to autoimmunity.

Keywords: autoantibodies, cytomegalovirus, spliceosome, systemic lupus erythematosus

INTRODUCTION

Systemic lupus erythematosus (SLE) is a multi-systemic disease where cells and tissues in the body are damaged by autoantibodies and immune complexes, directed most often at DNA or nuclear proteins (snRNPs) [1]. Although the cause of SLE is still largely unknown, it probably develops due to interactions between the environment and several susceptibility genes [2]. Studies of the monozygotic twin concordance rate for the clinical expression of the disease has been found to be 25–69%, whereas that of the presence of serum autoantibodies can be as high as 92% (for review see [3]), suggesting a strong genetic component. Ninety per cent of SLE patients are women, often of childbearing age, and the disease is more common in certain population groups such as African-Americans [4]. The disease can cause diffuse damage and predominantly affects the skin, musculoskeletal, renal, cardiopulmonary and nervous systems.

Viral infections such as cytomegalovirus (CMV) have often been implicated as possible aetiological agents of autoimmune diseases, including SLE [5–7]. Epidemiological studies have shown that there is a significant association between human cytomegalovirus infection and clinical SLE [7]. Recent studies have shown an association between antibodies to the spliceosome and CMV infection in normal (latently infected) individuals as well as in patients with SLE [8]. CMV is a member of the herpes family of viruses and causes long-lasting latent infections with the potential for reactivation [9–11]. Infection in the immunocompromised host results in significant mortality and maternal transmission of CMV to the neonate is responsible for severe congenital birth defects, including mental retardation [12]. Because the morbidity of CMV neonatal infection is so severe, but coupled with the fact that SLE predominantly affects women of childbearing age, the development of a safe and successful vaccine is of great importance.

The major envelope glycoprotein of CMV, gB (also known as gpUL55), which is conserved throughout the human herpes viruses, with highest homology to human herpes virus 6 and 7, has been the focus of subunit vaccine research because protective CMV specific immunity can be induced [13,14]. In previous studies it was shown that an adenovirus-gB construct could elicit an autoimmune response to the U1–70 kDa spliceosome protein in a number of mouse strains [15]. Autoantibodies to the U1-70 kDa protein comprise the predominant response to the spliceosome and are seen typically in mixed connective tissue disease (MCTD) (RNP response) and SLE (Sm/RNP response) [1]. One possible mechanism, which could account for this breakdown in self-tolerance, is via molecular mimicry, whereby an exogenous immunogen (Ad-gB) induces autoantibodies due to similarities between the antigen and the host's native proteins [16]. However, it is likely that clinical disease will only occur in genetically susceptible hosts. It is now recognized that as autoimmunity progresses, there is a recognition of multiple different epitopes by autoreactive B and T cells in the process known as epitope spreading [16–18].

In the present study two mouse strains, namely A/J and C57BL/6, were compared for their ability to mount an autoimmune response induced by the CMV vaccine Ad-gB, as well as to spread their autoantibodies to different proteins in the spliceosome. In addition, the autoimmune phenotype and kidney-associated pathology induced by Ad-gB was characterized. All A/J mice immunized with Ad-gB mounted an autoimmune response to the spliceosome components, while C57BL/6 mice failed to show signs or symptoms of autoimmunity, which illustrates the importance of the genetic background in the susceptibility to autoimmunity.

MATERIALS AND METHODS

Mice and immunizations

The 23 female C57BL/6 (B) (Charles River, Montreal, QC, Canada) and 26 A/J (A) (Jackson Laboratories, Bar Harbor, MA, USA) mice, 4–6 weeks old, were housed in a conventional mouse colony at the Montreal General Hospital Research Institute. These strains were selected for study due to the availability of ~40 recombinant congenic A×B and B×A strains which have been generated from these founder strains, and can be used in future experiments for genetic linkage analyses. As a first step it was important to determine differences in the immune response and phenotype in the parental strains. The McGill University animal care committee approved these studies. Immunizations with Ad-gB, Ad alone or a truncated construct of gB containing amino acids 1–700 Ad-gB (1–700)] were performed as described previously [15]. The truncated gB lacks the cytoplasmic domain, which contains a large number of charged amino acids, a region which could potentially contribute to the molecular mimicry of U1–70 k. A third injection of the immunogen was given 3 weeks after the second injection. Mice were euthanized at 14–16 weeks of age. One group of six female mice of each strain was not immunized and served as a control for the renal function assays.

Blood and urine collection

Blood was collected from the saphenous vein before the start of the experiment and then at 3-week intervals until sacrifice. Sixteen-hour urine samples were collected by housing mice in individual metabolic chambers. Because A/J mice produce very little if any urine during an overnight period, it was necessary to inject mice intraperitonealy with 0·5 ml of sterile PBS to hydrate them just prior to the urine collection. Urine was collected 2 weeks prior to sacrifice and then just prior to sacrifice.

Autoantibody detection by immunoblot analysis

The autoimmune responses to U1–70 k and other spliceosome proteins were detected by immunoblot techniques. Bovine calf thymus (BCT, Pel-Freeze, Rogers AK, USA) and purified bovine Sm (ImmunoVision, Springdale, AZ, USA) were used as a substrate and resolved on 10% SDS polyacrylamide gels using 300 μg per gel (approximately 14 lanes) as described previously [15]. The proteins were transferred to PVDF strips (Immobilon P, Millipore, Bedford, MA, USA) as described previously [8,15]. Following transfer, the PVDF strips were blocked with PBS 0·1% Tween 20 for 1h at 37°C and then incubated with selected sera diluted 1:1000 in PBS-Tween for 2h at 37°C. After 3 15-min washes with PBS-Tween, the strips were incubated with peroxidase conjugated goat antimouse IgG diluted 1:15000 (Jackson ImmunoResearch, BioCan, Mississauga, ON, Canada) for 1h at 37°C. A second wash was repeated as above, and bound antibodies were visualized using a chemiluminescence substrate according to the manufacturer's directions (Western Blot Chemiluminescence Reagent, NEN Boston, MA, USA). The strips were exposed to Biomax MR Film (Kodak, Rochester, NY, USA) and the bands quantified by laser densitometry (SciScan 5000, US Bio-chemical). Immunoblots also contained biotinylated molecular markers (Bio-Rad, Richmond, CA, USA). The procedure was the same as for the sera, except HRP-avidin (Vector 1/10000 dilution) was used with a 20-min incubation at room temperature, with washes in PBS/Tween as above, prior to the chemiluminescence step.

Immunohistochemistry

To investigate kidney pathology, IgG deposits were detected in the glomeruli by immunohistochemistry analysis on selected A/J (n = 8) and B6 (n = 5) mice. Snap-frozen kidney specimens were maintained at −70°C prior to embedding in OCT (Tissue-Tek, Torrance, CA, USA) and cryostat sectioning at 10 μm on poly l-lysine coated slides. Following sectioning the tissue sections were fixed with 4% paraformaldehyde and then washed twice in 0·1m Tris saline, pH 7·4 for 30 min. Endogenous peroxidase was quenched with 0·6% hydrogen peroxide in Tris saline for a 30-min incubation. Non-specific background staining by the primary antibody (raised in goats) was blocked with 10% goat serum for 30 min. To detect the antibody containing complexes, the kidney sections were incubated with HRP conjugated F(ab′)2 fragments of goat antimouse IgG (Fc-specific) at 1:500 in Tris saline at 4°C in a humid chamber for 16–18 h. Following two washes, the sections were incubated in the substrate 0·1m Tris saline containing 0·5 mg/ml of DAB (Sigma, Oakville, ON, Canada) and 1 ml of 8% NiCl for 15 min]. Two additions of 30 μl 30% H2O2 per 200 ml Tris saline/DAB followed, with 1 min separation between additions, and the reaction was terminated after 5 min. The sections were then washed for 30 min in tap water, counterstained for 30 s in 0·1% toluidine blue, cleaned and mounted using Permount. IgG deposits in the glomeruli were evaluated using a semiquantitative scale of 1–4, where 1 = no deposits as determined by a scorer blinded to strain and treatment. Inflammatory infiltrates were noted.

Detection of antibodies to Sm/RNP by ELISA

IgG antibodies to calf thymus RNP and Sm were detected by ELISA as described previously [15].

Urine analysis

The amount of protein in the 16-h urine specimen was measured using a sensitive microplate method. A standard curve was generated using bovine serum albumin (BSA). To 5 μl of urine, 300 μl of Coomasie Plus Protein Assay reagent (Pierce, Rockville, IL, USA) was added and the samples were incubated for 30 s. Absorbance was measured using an automatic plate reader (ALT Labinstruments, Grodig, Austria) at a wavelength of 550 nm. Protein concentrations were extrapolated from the standard curve, and the total protein in the 16-h collection calculated. Urine creatinine levels of mice were detected using the Creatinine kit 555 A (Sigma) following the manufacturer's instructions. Blood urea nitrogen (BUN) was measured in the serum of the mice at sacrifice, using the Infinity BUN assay (Sigma) according to the manufacturer's instructions.

Statistics

The statistical analyses (i.e. Student's t-test with Yates’ correction, linear correlation, Tukey–Kramer multiple comparisons test, used as appropriate) were calculated with InStat3 software (Graphpad, San Diego, CA, USA).

RESULTS

Physical signs and symptoms of disease

Throughout the study there were no anatomical signs indicative of an autoimmune disease such as hair or weight loss in either the A/J or B6 mice.

Kidney function

Total protein in the urine, as determined at sacrifice (Table 1) showed a trend in both strains of mice examined to be higher in the Ad-gB immunized than in the Ad control, although the results were not significantly different, which may reflect in part the small sample size. The Ad-gB (1–700) immunized mice had proteinuria levels that were lower than in the mice immunized with the full-length gB, but the differences were not significant. BUN measurements indicated that there was a lower basal level in A/J mice when compared to C57Bl/6 (Table 1). These data were consistent with other studies in the laboratory where unimmunized female A/J mice have a significantly lower BUN of 9·66 ± 2·10 in contrast to C57Bl/6, which have a BUN of 13·37 ± 2·76, p = 0·036. Twofold higher levels of BUN were detected in the Ad-gB immunized A/J mice (either full-length or truncated construct), whereas there was no difference in the BUN levels in any of the groups of C57BL/6 mice. The mean BUN values observed in the Ad-gB (either full-length or truncated construct) immunized A/J mice were significantly higher than the unimmunized A/J of the same age (P < 0·01 for Ad-gB (1–700); P < 0·05 for Ad-gB). There was no significant correlation between BUN and creatinine levels. The abnormalities in BUN suggest early kidney disease.

Table 1.

Proteinuria and creatinine levels (mean ± s.d.) at sacrifice in A/J and B6 mice after im-munization with Ad-gB or control constructs

Strain Immunogen Total urine protein (mg) BUN (mg/dL) Urine creatinine(mg/16h) Urine creatinine1(mg/16h)
A/J Ad 0·011 7·9 ± 0·282 0·039 0·039
n = 1 n = 2 n = 1 n = 1
Ad-gB (1–700) 0·044 ± 0·042 21·5 ± 9·0 0·058 ± 0·056 0·062 ± 0·059
n = 7 n = 7 n = 4 n = 4
Ad-gB 0·087 ± 0·100 19·2 ± 3·8 0·070 ± 0·081 0·079 ± 0·084
n = 10 n = 9 n = 9 n = 9
B6 Ad 0·015 16·9 ± 7·42 0·518 0·401
n = 1 n = 2 n = 1 n = 1
Ad-gB (1–700) 0·032 ± 0·009 18·9 ± 4·4 0·196 ± 0·107 0·149 ± 0·114
n = 3 n = 3 n = 3 n = 2
Ad-gB 0·099 ± 0·087 13·6 ± 6·3 0·112 ± 0·235 0·101 ± 0·213
n = 12 n = 12 n = 12 n = 12
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1

Corrected for weight of animal to 25·0 g

2

unimmunized control female A/J 9·66 ± 2·10 (n = 6), C57Bl/6 13·37 ± 2·76 (n = 6).

Of the A/J mice immunized with Ad-gB, 7/10 had elevated proteinuria levels compared with Ad treatment and those immunized with Ad-gB (1–700), 5/7 had higher levels. Almost all the B6 mice had slightly raised proteinuria following immunization with the full (11/12) or truncated construct (3/3).

Kidney immunohistochemistry

From immunohistochemistry studies of A/J and B6 kidneys, IgG containing immune deposits were investigated. Results showed deposits in the mesangium of the kidney glomerulus of A/J mice following Ad-gB treatment (Fig. 1), with 12% of 65 glomeruli evaluated showing a score of 4 for IgG deposits, and 62% with a score of 3, when the semiquantitative scale was used. For A/J mice immunized with Ad-gB (1–700) none of 58 glomeruli had a score of 4, with 22% having a score of 3. For A/J mice immunized with Ad alone, as shown in Fig. 1 the deposits scored either 1 (64% of 45 glomeruli) or 2 (36%)and stained with much less intensity. As can be seen in Fig. 1e, not all of the glomeruli had deposits (note the negative glomerulus at top). The B6 mice tested did not reveal significant IgG glomerular deposits following either treatment (for Ad-gB immunized mice, 2% of 58 glomeruli scored 3, with 73 and 24% scoring 2 and 1, respectively). Figure 1 is representative of the renal IgG deposits and the degree of lymphocyte infiltration, which were detected in the A/J and B6 kidney sections. Inflammatory infiltrates were observed predominantly in the A/J glomeruli that scored either 3 or 4 for IgG deposits.

Fig. 1.

Fig. 1

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Immunohistochemical analysis of kidney sections from A/J (a–g) and B6 (H) mice, immunized with either Ad alone (a.b), or Ad-gB (c–h). Sections in (c), (d) and (e–g) are from two separate mice. Sections in panels (b, d, g) were not stained with the primary antibody, and thus serve as control for the substrate. Note (e) and (f) are of the same section, of different magnification. Panel (e) contains several glomeruli with marked deposits except one, which is negative for IgG (top).

Circulating antibodies

IgG antibodies directed towards the spliceosome were measured prior to vaccination and at sacrifice in both A/J and B6 mice (Fig. 2). The A/J mice mounted a highly significant high titre autoantibody response when immunized with the full-length Ad-gB at sacrifice when compared to preimmune levels (P < 0·001). Ad immunization alone did not differ significantly from the preimmune group (P > 0·05). The anti-Sm/RNP levels in B6 mice vaccinated with Ad-gB did not show a significant difference between the preimmune levels and those at sacrifice nor compared to the Ad control (P > 0·05). Moreover, when measured at 1/500 dilution there was a highly significant difference between the autoimmune response to Ad-gB in the A/J versus the B6 at sacrifice (P < 0·001). When the cut-off value for the preimmune autoantibody O.D. (mean plus 2 s.d.) was established at 0·159, all 10 A/J mice treated with Ad-gB were considered positive and all had a high titre response greater than 1:2000. In addition, the A/J mice receiving Ad-gB (1–700) were also positive, all having titres greater than 1:500. Only seven of 12 B6 mice treated with Ad-gB had values above the cut-off at 1/200. Mean O.D.s were calculated at 1/2000 in A/Js mice and at sacrifice the mean IgG titres for A/J mice inoculated with Ad-gB was 6800 ± 4131 (s.d.) and 10 188 ± 8022 with Ad-gB (1–700).

Fig. 2.

Fig. 2

Fig. 2

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Detection of IgG anti-Sm/RNP antibodies in preimmune sera and at sacrifice in (a) A/J and (b) B6 mice by ELISA at 1/2000 and 1/200 dilution, respectively. Groups of 2–12 mice were vaccinated with Ad, Ad-gB (1–700) or Ad-gB. *Pre-immune levels were calculated in 12 B6 mice at 1/200 dilution. **A/J versus B6 at sacrifice at 1/500 dilution P < 0·001. (a) P > 0·05 (compared to preimmune levels). (b–c) P < 0·001 (compared to preimmune levels). (d–f) P > 0·05 (compared to preimmune levels). Error bars represent s.e.m.

Immunoblot analysis

Prior to immunization (Fig. 3) neither the A/J nor the B6 mice mounted an immune response to the spliceosome proteins. Following three immunizations with the full-length Ad-gB, the autoantibody responses in all the A/J mice were directed predominantly toward the U1-70 kDa protein (Fig. 3,Table 2), which increased in intensity with time. B6 mice did not mount an immune response to either the U1-70 kDa or any other component of the spliceosome. In contrast the A/J mice showed evidence of epitope spreading as antibodies directed towards the B/B′ component of the spliceosome were detected in 60% of the mice, and against the D protein in 40%, both at 4 weeks and at sacrifice. The intensities of these responses were maximal at 4 weeks and appeared to diminish with time. When the bovine calf thymus proteins were used as a target preparation, all A/J mice at sacrifice had circulating antibodies reactive with a 55-kDa protein (identity unknown). This reactivity was not observed when the purified Sm proteins were used in the immunoblots (data not shown). A/J mice immunized with Ad-gB (1–700) also produced autoantibodies directed towards spliceosome proteins, however, in lower amounts (Table 2).

Fig. 3.

Fig. 3

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Immunoblot analysis of mouse sera demonstrating IgG binding to spliceosome proteins from bovine calf thymus. Lanes 1 and 8, molecular weight markers; lane 2, MRL response following one Ad-gB immunization (negative control); lanes 3 and 4, A/J immunized with Ad; lanes 5–7, A/J immunized with the Ad-gB (1–700) construct; lanes 9–11, A/J immunized with the full length Ad-gB; lanes 12–14, preimmune sera from A/J mice.

Table 2.

IgG response to spliceosome proteins (U1-70, 55 kDa, B′/B, D) as detected by density measurements (mean ± s.d.) of immunoblot analysis prior to immunization, at 4 weeks and at sacrifice in A/J and B6 mice

Strain Immunization Protein Preimmune Week 4 #+/# tested Density (kIOD) Sacrifice #+/# tested Density (kIOD)
A/J Ad U1 – 2/2 708·5 ± 398·1 1/1 552·0
Ad-gB (1–700) – 8/8 392·8 ± 120·3 8/8 410·3 ± 263·0
Ad-gB – 8/8 672·8 ± 227·3 10/10 1078·0 ± 865·6
Ad 55 – – – 1/1 25·0
Ad-gB (1–700) – 4/8 33·5 ± 10·6 8/8 48·25 ± 61·9
Ad-gB – 7/8 82 ± 39·8 10/10 39·7 ± 35·2
Ad B′/B – 2/2 13·5 ± 6·4 1/1 24·0
Ad-gB (1–700) – 5/8 15·0 ± 1·79 5/8 38·2 ± 59·4
Ad-gB – 5/8 107·8 ± 49·2 6/10 20·2 ± 13·6
Ad D – 2/2 3·5 ± 2·1 1/1 2·0
Ad-gB (1–700) – 3/8 5·68 ± 0·67 1/8 4·1 ± 5·5
Ad-gB – 3/8 37·7 ± 17·1 4/10 9·3 ± 6·1
B6 Ad ALL1 – – – – –
Ad-gB (1–700) – – – – –
Ad-gB – – – – –
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1

No Ab recognition of any of the proteins (U1, 55, B′/B and D).

Two A/J mice immunized with Ad alone also showed evidence of antibodies directed towards the various spliceosome proteins, suggesting that the inflammation caused by the adenovirus alone was sufficient to induce this response in this strain of mice, although the Ad-gB induced a much higher response (Table 2).

DISCUSSION

This study illustrates the importance of the genetic susceptibility in the induction of an autoimmune response to a vaccine against the ubiquitous cytomegalovirus in that A/J but not B6 mice mounted an autoantibody response to specific spliceosome proteins. B6 mice appear to be the most resistant strain in mounting this antibody response when compared to this and previous studies, showing the induction of the autoimmune response following Ad-gB vaccination [15,19] in several other strains of mice. A number of previous studies has examined the ability of these two strains to mount an antibody response to different antigens and whereas A/J are generally the better antibody producers [20–22], this is not always the case [23] and is dependent on the antigen in question. Studies with H2 congenic mice would facilitate a better understanding of the role of MHC in this immune response.

James et al.[24,25] has demonstrated previously that the induction of an autoimmune response in mice can be strain-specific. Following immunization with the PPPGMRPP peptide derived from Sm B/B′, A/J mice in their study were shown to spread their response from the peptide of immunization and developed antibodies towards the entire nRNP protein. Conversely, the C57BL/6 mice did not show any epitope spreading and thus did not develop antibodies against any other proteins apart from the peptide of immunization. Our results are analogous but with a different immunogen, which in this case is foreign, where A/J but not B6 were capable of spreading their autoimmune response. It is of interest that Mason et al.[26] repeated the PPPGMRPP immunization experiment performed by James and were not able to confirm the results with A/J mice.

What initiates the breakdown in tolerance is currently unknown, but because the mouse U1-70 kDa protein and CMV gB C-terminal regions share some structural similarity, having commonly charged amino acids [27,28], molecular mimicry is a plausible model of autoimmune induction. The lower titre autoantibody response and renal immunopathology seen subsequent to Ad-gB (1–700) inoculation, a construct of gB which lacks the C-terminal region, further supports this model. Following loss of self-tolerance to U1–70 kDa, additional spliceosome proteins can be targeted by B-cells through intermolecular epitope spreading [16–18]. Immunoblot results at 4 weeks following the initial vaccination already revealed autoantibodies directed at spliceosome proteins other than U1-70 kDa and therefore the progress of epitope spreading appeared to be rapid in the A/J mice. In previous studies [15] C3H and MRL/mpj mice showed a response directed only against the U1-70 kDa protein after the same time period. This may outline further the importance of genetic susceptibility in the rate of progression and severity of the autoimmune response.

In mixed connective tissue disease (MCTD), the detection of anti-RNP antibodies almost always in the absence of the anti-Sm response is a diagnostic criteria. In contrast in patients with SLE, antibodies directed to U1snRNP are detected in 30–40% of patients [29], and the anti-Sm response (B/B′ and D) is found in 20–30%. Furthermore antibodies directed towards U1 without the anti-Sm response occurs in less than 10% of SLE patients [29]. In the A/J strain, antibodies were detected to both the U1 snRNP and to Sm in the majority of the mice after immunization, which is more SLE-like. The high level responsiveness reflects the inbred nature of the mouse strain (in contrast to humans with SLE) as well as to the timing of analysis with respect to the exposure to the antigen.

ELISA results used to detect the anti-Sm/RNP response demonstrate high levels of IgG in the A/J mice compared with the B6. The A/J strain had almost twice the frequency of developing antibodies towards Sm/RNP compared with the B6 mice. The role of circulating autoantibodies in disease pathogenesis is still largely unknown, yet evidence shows that the presence of autoantibodies to Sm/RNP are often found in SLE patients that exhibit a higher frequency of such disease manifestations as arthritis, Raynaud's phenomenon, mean number of nail fold capillary loops [30] and late onset renal disease [31]. One possibility is that autoreactive B-cells could be underlying the process of epitope spreading which could lead to further disease progression in the genetically susceptible host.

In order to establish a clinical phenotype, our studies focused on renal disease as a possible outcome. There was a trend towards increased proteinuria in the A/J mice in those that mounted the high autoimmune response in response to the full-length Ad-gB when compared to truncated construct Ad-gB (1–700)] and Ad alone. A similar pattern was observed in the B6 mice following immunization, although these mice did not mount the high-titre anti-RNP/Sm response. The BUN levels were significantly elevated in the Ad-gB immunized A/J but not B6 mice when compared to unimmunized female mice of the same age. However, the levels observed were indicative of early kidney disease only, as BUN values of >50 mg/dL are more indicative of severe kidney disease. In addition the creatinine values observed were also consistent with the concept of early kidney disease. Increased levels of urine and serum creatinine were detected in A/J but not B6 following CMV Ad-gB or Ad-gB (1–700) vaccinations, although the values were not statistically different from the control group, reflecting in part the small sample size. The degree of proteinuria and raised plasma BUN and creatinine levels suggest an early stage of kidney damage which was not as yet clinically significant (i.e. there was no sign of weight loss). Importantly, immunochemical analysis of the kidneys revealed the presence of glomerular IgG containing immune complexes in A/J but not B6 mice following immunization with CMV gB. The deposition of autoantibodies in the glomeruli is implicated in the development of lupus nephritis [32,33]. Previous studies have shown that A/J develop renal disease in response to daunomycin at an accelerated rate compared to B6 [34]. Similarly in response to mercury, A/J but not B6 mount an antibody response to chromatin and histone [35], again a response seen in SLE.

Establishment of a phenotype at this time-point, rather than following the mice for a much longer period of time, i.e. to end-stage disease, will facilitate future studies where the genetic basis of the autoantibody titre and epitope spreading characteristics, as well as renal disease, can be examined in recombinant congenic strains established from crosses of B6 and A/J. Such studies will help identify genes that accelerate or prevent either phenotype and determine whether the responses are linked. Previous genetic studies have in some cases linked the ability to generate autoantibodies, and thus immune complexes and the nephritis that occurs as a model system of SLE [36,37], whereas in another system the two phenotypes were influenced by separate genes [38,39]. Further studies will allow us to determine which of these scenarios are influencing the autoimmune response and kidney disease in this model of a subset of patients with SLE.

Acknowledgments

The authors would like to thank Drs Eva Gonczol and Klara Berencsi (Wistar Institute, Philadelphia) for the Ad-gB, Ad and Ad-gB (1–700) and the technical assistance of Ewurabena Simpson. The study was supported in part by a grant to E. Skamene from the Canadian Institutes for Health Research. Jed Lipes was the recipient of an award from the Canadian Genetic Diseases Network.

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