Systemic lupus erythematosus (SLE) is characterized by the presence of a vast array of autoantibodies directed against organ and non-organ specific auto-antigens; such as anti-dsDNA antibody, anti-Smith antibodies, anti-SSA and anti-SSB antibodies. Among these autoantibodies, anti-dsDNA antibody is the serological marker of systemic lupus erythematosus and has proven to be involved in the pathogeneses of LE glomorulonephritis.1,2 The level of anti-dsDNA antibody is often associated with SLE activity. Autoantibody to La/SSB is commonly found in the sera of patients with primary Sjögren's syndrome (pSS) and SLE, especially in some LE subsets such as neonatal lupus erythematosus (NLE) and subacute cutaneous lupus erythematosus (SCLE). Clinically, patients with anti-La/SSB antibody often have mild disease and a better prognosis compared with anti-La/SSB negative lupus patients.3-5 In this study, we immunized rabbits with synthetic SSB peptide and DNA to investigate if there is any interaction between the production of these two antibodies.
SSB peptide synthesis
The amino-acid sequence of SSB was analyzed for selection of epitope-containing fragment. Amino acid 214-225 of the SSB antigen (KQKLEEDAEMKS) was selected as the target fragment. The peptide was synthesized by the Fmoc strategy based on the organic chemistry solid-phase peptide synthesis from carboxyl to N-terminal with an automatic peptide synthesizer (ABI433, USA). Peptide resin was collected and the peptides were cleaved from the resin and extracted. Purification was done with high performance liquid chromatograph (Water600E, USA) with C18 reversed phase high performance liquid chromatography. This yielded a purity of above 95%. The product was dried with a freezing drier machine (Virtis, USA). This peptide was then conjugated to keyhole limpet hemocyanin (KLH) to yield the SSB peptide-KLH conjugate that was used for animal immunization.
Ten healthy New Zealand white rabbits (provided by Experimental Animal Center, Peking University People's Hospital, China) were divided into 5 groups. Group 1 were immunized with SSB peptide-KLH conjugate (KQKLEEDAEMKS-Y-KLH), marked as SSB1, SSB2; Group 2 were immunized with calf thymus DNA (Dingguo corporation, Beijing), marked as DNA1, DNA2; Group 3 were immunized with SSB peptide-KLH and DNA, marked as SSB+DNA1, SSB+DNA2; Group 4 were immunized with KLH alone and marked as KLH1, KLH2; Group 5 were pseudo-immunized with phosphate buffered saline (PBS), marked as PBS1, PBS2. The antigens were emulsified with complete Freud's adjuvant in the first immunization and were emulsified with incomplete Freud's adjuvant in the subsequent 3 booster immunizations. The concentration of the immunization antigens was 1 mg/ml. One ml of each immunogen was injected subcutaneously into the back of the rabbits. Each immunization was performed at intervals of 20 days. Successive bleedings of the immunized animals were performed after 3 booster immunizations (days 34, 51 and 72). The animals were sacrificed on day 72 and the blood and tissue samples were obtained.
Sera from rabbits immunized with different antigens were collected before and after each immunization and were tested for their reactivity against the immunogens. Specific ELISA assays were performed to detect the antibodies in rabbit sera after immunizations. 96-well polystyrene plates (Corning) were coated with a solution of the coating SSB peptide, calf dsDNA or KLH (pH 9.6) and were left overnight at 4°C. The concentration of the coating antigens were 20 μg/ml for dsDNA, 2 μg/ml for SSB peptide and 20 μg/ml for KLH. The plates were washed with PBS-T (0.01 mol/L PBS pH 7.4 plus 0.05 %Tween-20) four times. Non-specific binding was blocked using blocking buffers containing bovine serum albumin (BSA), 1% w/v in phosphate buffered saline (PBS) pH 9.6. After 1 hour incubation at 37°C, the plates were washed with PBS-T four times. Serial diluted sera from rabbits were added. Normal rabbit serum, at a dilution of 1:1000, was used as the negative control. After 30 minutes' incubation at 37°C the plates were washed with PBS four times. Alkaline phosphatase-conjugated goat anti-rabbit IgG (Sigma, USA) was subsequently added at 0.1 ml/well and left for 40 minutes at 37°C. Tetramethyl benzidine (TMB) was used as a chromogenic substrate. 2N H2SO4 was used as a stop solution. The absorbance was measured at 450 nm by an ELISA reader. A positive reaction was definded as 2.0 times the OD value of samples of normal control.
Histopathology and immunofluorescence assay
Histolological examination of the heart, liver, kidney, spleen and skin tissues were performed by routine methods. A direct immunofluorescence assay was also performed by using FITC-conjugated anti-rabbit IgG.
Anti-SSB and anti-dsDNA antibody production
Immunization of the rabbits with synthetic SSB peptides and calf thymus DNA induced anti-SSB and anti-dsDNA antibody production. Anti-SSB and anti-dsDNA antibodies were detected after the second immunization. The antibody level was increased with each immunization and the booster immunizations were performed 3 times. Both anti-SSB and anti-dsDNA antibodies veached the highest level after the second booster immunization and remained at that level despite subsequent immunizations. The level of SSB antibody in the co-immunization group was higher than that of the SSB peptide immunization group (Figure 1). However, the level of anti-dsDNA antibody in the co-immunization group was lower than that of the DNA immunization group (Figure 2) and the level of anti-SSB antibody was higher than anti-dsDNA antibody in the co-immunization group (Figure 3). Neither anti-SSB nor anti-dsDNA antibodies were found in the sera of rabbits immunized with KLH or in those pseudo-immunized with PBS.
Histology and direct immunofluorescence assay
Histological examination of the heart, liver, kidney, spleen and skin tissues of the rabbits showed no morphological changes. Direct immunofluorescence assay also revealed no immune complex deposition in these tissues.
The clinical relevance of anti-dsDNA antibody and anti-SSB antibody had been widely studied. Anti-dsDNA antibody is found in the sera of patients with SLE and often associate with disease activity, especially in LE nephritis. It is becoming increasingly clear that antinucleosome (DNA-histones) and possibly anti-dsDNA antibodies are the major players in the pathogenesis of SLE. DNA and its immune complex may contribute to the pathogenesis of SLE.6-8 On the other hand, antibodies to Ro/SSA and La/SSB are found mostly in patients with Sjögren's syndrome (SS) as well as in some patients with SLE. Studies have shown that photosensitivity in LE were associated with the expression of Ro/SSA and La/SSB antigens in the keratinocytes of the skin.9-11 Wasicek et al3 found that a lower frequency of anti-dsDNA antibody and lower prevalence of renal disease were seen in patients having both anti-SSA and anti-SSB antibodies than in those having anti-SSA antibody alone. Zhang et al reported that patients with SCLE of the annular erythema type had a higher frequency of anti-SSB antibody than patients with papulosquamous type. Moreover, these patients usually had mild disease and less had developed SLE than those with papulosquamous type, suggesting that anti-SSB antibody might be a marker of mild disease and predict a better prognosis for patients with LE.
Usually patients with SS or SLE had more than one type of autoantibodies. Anti-Sm antibody was often associated with anti-U1RNP antibody and anti-SSB was often associated with anti-SSA antibody. Anti-dsDNA antibody was also found to co-exist with other autoantibodies. However, it is not clear if there is any interaction between these antibodies. Zhang et al12-14 had studied the relationship of anti-Ro/SSA and anti-La/SSB antibodies to anti-dsDNA antibody in sera from patients with SLE. They found that some anti-Ro/SSA and anti-La/SSB antibodies were anti-idiotypes to the idiotopes of anti-dsDNA antibodies and that they could both mask and down-regulate these anti-dsDNA antibodies; indicating that such anti-idiotypic antibodies might play some roles in the regulation of anti-dsDNA antibody levels in SLE.
SSB is an autoantigen which, along with two other proteins (Ro52 and Ro60), constitutes a complex with hYRNAs and forms ribonucleoprotein particles (hYRNPs). Autoimmune responses against the hYRNPs are characterized by a remarkable diversity, illustrated by autoantibodies directed towards different proteins of the complex (Ro52, Ro60 and SSB), recognizing several conformational or linear epitopes.1-3 Human La/SSB is a phosphoprotein consisting of 408 amino-acids with a molecular weight ranging from 48 to 51 kD. In recent years, investigations have been done on SSB antigen structure and epitopes and several epitopes have been found. The 1-107 aa, 111-242 aa and 346-408 aa fragments of SSB antigen were proven to contain immunodominant epitopes.15-18 Chang et al19 found that the 192-223 aa oligopeptide from the SSB antigen had the same amino acid sequence of "EAKLRA" as laminin and was located in an immunodominant epitope. Kohsaka et al20 described substructure of SSB and divided functional domains of SSB into SSB-A, SSB-B and SSB-C. The main epitope of SSB-A was located at 88-101 aa, SSB-B at 283-338 aa and SSB-C at 179-220 aa. Apostolos21 found that animals immunized with the epitope of SSB protein developed an early immune response against the epitope, as well as against the other B-cell epitopes of SSB, even the recombinant human La/SSB. We selected the 214-225 aa oligo from La/SSB for our synthetic peptide. It is located in a domain of reported dominant epitopes.
In this study, both anti-SSB and anti-DNA antibodies were produced following immunization. The level of anti-SSB antibodies were higher than that of anti-dsDNA antibodies in the co-immunization group, suggesting there might be some "competition" in the production of these two antibodies. And in this competition, production of anti-SSB might overwhelm anti-dsDNA antibody production. Furthermore, by comparing the antibody level in the co-immunization group with those immunized with SSB peptide or DNA, it was demonstrated that anti-dsDNA antibody production was inhibited in the co-immunization group. Although the mechanism of this phenomenon is unknown, the inhibition of anti-dsDNA antibody might lead to less anti-dsDNA antibody-related inflammation and tissue damage. Therefore, the inhibition of anti-dsDNA antibody in this experiment is of clinical significance. It might reduce anti-dsDNA antibodymediated immunoreactions and therefore reduce the severity of the disease as was seen in LE patients who had anti-SSB antibody.
Although production of antibodies after immunization with SSB and DNA was seen, no histological abnormality and immunological deposition was seen in the heart, liver, kidney, spleen and skin tissues in these animals, suggesting that, in addition to autoantibody production, other factors are required to induce inflammation and tissue damage in SLE or other autoimmune diseases.
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