Structural, Molecular and Immunological Properties of Linear B-Cell Epitopes of Ro60KD Autoantigen

Antibodies to Ro60KD protein are found with high frequency in sera from patients with systemic lupus erythematosus (SLE) and primary Sjögren's syndrome (pSS). Two major epitopes of the Ro60KD antigen, the TKYKQRNGWSHKDLLRSHLKP (169–190) and the ELYKEKALSVETEKLLKYLEAV (211–232), were synthesized and their antigenic and structural properties were studied. Using a large panel of SLE and pSS patients' sera, it was found that the anti-Ro60KD reactivity of both Ro60KD epitopes is rather limited (≈45%), although they retain their original disease specificity. The epitope p.169–190 possessed sequence similarity with the peptide RPDAEYWNSQKDLLEQKRGR, shared in the β-chain of different HLA-DR molecules, among them the HLA-DR3 (which is associated with anti-Ro/Sjögren's syndrome A (SSA) response in patients with SLE). The antigenicity of the HLA-DR3 RPDAEYWNSQKDLLEQKRGR peptide was found to be similar to the 169–190 homologous Ro60KD epitope, recognized mainly by SLE sera. Structural studies showed that the 211–232 Ro60KD epitope exhibits pronounced helical characteristics, while the 169–190 epitope and the HLA-DR3 homologous peptide possess a somewhat lower percentage of α-helix. A β-folded structure was identified in the latter two peptides. Although the diagnostic value of the reported Ro60KD epitopes seems to be rather limited, correlations with other ribonucleoprotein epitopes (La/Sjögren's syndrome B, Ro52KD) may prove complementary to each other and valuable in clinical use. The ordered structure of the HLA-DR3 homologous peptide, exposed to the autoantibody binding, may offer an initiative in further investigation of the role of the HLA haplotypes, associated with the anti-Ro/SSA response, in the autoimmune stimulus.     

Hypotheses on basis mechanisms arising from recent research

Clinical Rheumatology -     

Fine antigenic specificities of antibodies in sera from patients with D-penicillamine-induced myasthenia gravis.

A small fraction of patients with rheumatoid arthritis and other diseases on D-penicillamine treatment may develop antibodies against the acetylcholine receptor (AChR) and symptoms of myasthenia gravis (MG). The mechanism leading to this phenomenon is not known. We have studied the fine antigenic specificities of the anti-AChR antibodies in 19 D-penicillamine-induced MG (pen-MG) patients and compared them with those of antibodies from 204 idiopathic MG patients (the data for 122 obtained from earlier experiments). Antigenic specificities of the circulating antibodies were determined by the capacity of monoclonal antibodies (MoAbs), against certain determinants on the AChR, to inhibit binding of the serum antibodies to the AChR. Monoclonal antibodies against alpha, beta and gamma subunits were used. The anti-AChR antibody patterns of pen-MG patients were very similar to those of idiopathic MG patients. Antibodies to the main immunogenic region, which is located on the extracellular surface of the alpha-subunit, were the predominant group. The variations of antibody specificities in serial sera collected from individual patients at different times were usually small, as were those of idiopathic MG. These results strongly suggest that the antibody repertoire in the sera of idiopathic and pen-MG patients is very similar.     

Conjunctival epithelial cells from patients with Sjögren's syndrome inappropriately express major histocompatibility complex molecules,La(SSB) antigen,and heat-shock proteins

The La(SSB) antigen has been detected within the cytoplasm and on the membrane of conjunctival cells (CC) from patients with Sjögren's syndrome, whereas it was weakly expressed in the nucleus of normal cells. The diseased CC were shown to overproduce major histocompatibility complex (MHC) class I antigens and express MHC class II antigens. Anti-heat-shock protein monoclonal antibody bound to the cell membrane in patients but not in normal controls.On sabbatical leave from Department of Internal Medicine, Medical School, Ioannina, Greece.     

Induction of salivary gland epithelial cell injury in Sjogren's syndrome: in vitro assessment of T cell-derived cytokines and Fas protein expression

Sjogren's syndrome (SS) is an exocrinopathy characterized by T cell infiltrates, salivary gland epithelial cell (SGEC) apoptosis and high Fas and FasL expression. To address the participation of T cell-derived cytokines and of Fas apoptotic pathway in SS glandular lesions, we utilized non-neoplastic SGEC lines established from SS patients and controls. Possibly attesting to their intrinsic activation, cell lines derived from SS patients displayed significantly higher constitutive Fas and FasL than controls. Surface co-expression of Fas and FasL was not associated with spontaneous fratricide apoptosis. SGEC were resistant to anti-Fas-mediated apoptosis (possibly owing to the constitutive expression of anti-apoptotic proteins cFLIP and Bcl-2), but became sensitive after protein or RNA synthesis inhibition. IFN-gamma and TNF-alpha were able to upregulate surface Fas and FasL, whereas IL-1beta downregulated surface FasL. IFN-gamma (but not several other cytokines) reduced the survival of SGEC in a dose- and time-dependent manner and induced Fas/FasL-mediated apoptosis, directly and via anoikia. Dexamethasone inhibited the upregulation of Fas and FasL by IFN-gamma and the induction of SGEC apoptosis and detachment by anti-Fas mAb or IFN-gamma. Our findings indicate the injurious role of IFN-gamma for the salivary epithelia of SS patients through the induction of Fas-mediated apoptosis and anoikia.     

Clinically significant and biopsy-documented renal involvement in primary Sjögren syndrome

Clinically significant renal involvement in patients with primary Sj?gren syndrome (pSS) has been described previously only in isolated case reports. The prevalence and significance of the 2 described syndromes, interstitial nephritis (IN) and glomerulonephritis (GMN), are not well known. In a cohort of 471 patients with pSS who were followed for a mean of 10 years, 20 patients (4.2%) developed overt renal disease. Eighteen patients underwent a percutaneous renal biopsy; 2 patients declined. Ten patients had IN, 8 patients had GMN, and 2 patients presented with both entities. Glomerular histology disclosed changes compatible with membranoproliferative GMN in 5 patients and mesangial proliferative GMN in 4 patients. Patients with IN had a younger disease onset compared with patients with GMN (mean, 36.8 compared with 46.0 yr, p 5 0.063). Patients with GMN had longer disease duration compared with patients with IN (mean, 2.2 compared with 8.0 yr, p 5 0.001). The majority of patients with GMN (80%) had mixed monoclonal cryoglobulinemia IgMk (type II) and lower complement C4 levels. Two patients (both with GMN) developed chronic renal failure requiring hemodialysis. Overall, clinically significant renal involvement is infrequent in pSS. IN occurs early in the disease process, while GMN is a late sequela and may have a less favorable prognosis.     

Alveolitis correlates with clinical pulmonary involvement in primary Sj?gren's syndrome

Bronchoalveolar lavage (BAL) was performed in 23 patients with primary Sj?gren's syndrome (1Ss) and ten healthy controls to evaluate alveolitis and correlate it with pulmonary and systemic manifestations. Patients with 1Ss had higher BAL total cell count (9.2 +/- 6.7 millions/ml vs 6.1 +/- 2.9 millions/ml) and higher percentage of lymphocytes 23.3 +/- 15.6 percent vs 6.5 +/- 2.9 percent, p less than 0.001) than controls. Twelve patients (group A) constituted the "high alveolitis" group (lymphocytes greater than 15.2 percent) and ten (group B) constituted the "low alveolitis" group (lymphocytes less than 15.2 percent). Group A had more frequent cough (6/12 vs 2/10, p = 0.07), dyspnea (4/12 vs 1/10), and roentgenologic evidence of interstitial lung disease (5/12 vs 0/10, p less than 0.05). They also had lower total lung capacity (85.6 +/- 14.2 percent pred vs 105.8 +/- 23.3 percent pred, p less than 0.05) and Dco (87.7 +/- 20.6 percent pred vs 103.6 +/- 21.0 percent pred). All patients with +3 or +4 grading or lymphocytic infiltrates in lip biopsy specimen belonged in group A (5/12). Finally, T-helper/T-suppressor ratio was lower in group A than in group B. The intensity of alveolitis was not correlated with clinical or serologic manifestations of systemic disease.     

Serum isoamylases in patients with autoimmune rheumatic diseases

We studied sera of 107 patients with autoimmune rheumatic diseases (46 with classical rheumatoid arthritis (RA), 36 with systemic lupus erythematosus (SLE) and 25 with primary Sj?gren's syndrome (SS). None of these patients had abdominal pain or gastrointestinal symptoms at the time of blood collection. We used as controls 81 normal age and sex matched volunteers. The presence of hyperamylasemia i) of P-type in 6 of 46 patients (13%) with RA and ii) of P-type and S-type in 11 of 36 patients (30.5%) with SLE and 6 of the 25 patients (24%) with primary SS suggests that asymptomatic pancreatic damage in autoimmune rheumatic diseases may occur frequently especially in patients with SLE. We conclude that the hyperamylasemia in these patients probably reflects a slow, subclinical, inflammatory process of the exocrine glands.     

Sj?gren's syndrome in progressive systemic sclerosis

Forty-four sequential, unselected patients with progressive systemic sclerosis (PSS) were prospectively evaluated for evidence of coexistent Sj?gren's syndrome (SS). This diagnosis was established when a patient with focal lymphocytic infiltration in the labial salivary gland (LSG) biopsy, scoring greater than or equal to 2+ in Tarpley's scale, had keratoconjunctivitis sicca (KCS) (positive rose bengal test) and/or xerostomia (subjective xerostomia and decreased parotid flow rate). Ten patients had an LSG biopsy score of greater than or equal to 2+, 3 a 1+ score, 17 had mild to moderate fibrosis only and 14 had normal tissue. Nine of the 10 patients with a greater than or equal to 2+ score had SS, according to applied criteria, suggesting a 20.5% prevalence of SS in our population with PSS. On the other hand, pure fibrosis in the biopsy was felt to be secondary to PSS. Parotid gland enlargement was present in 44.4% of the patients with SS, but was extremely uncommon in the fibrosis and normal tissue groups. Subjective xerophthalmia and xerostomia, although elicited by specific questionnaire in the majority of the patients with SS, did not constitute major complaints. Serious internal manifestations, with the exception of esophageal and pulmonary involvement, were unusual in all groups. Anti-Ro (SSA) antibodies were detected in 33.3% of the patients with SS and 11.8% of those with fibrosis. Our study suggests that SS in scleroderma is relatively common and, although lacking prominent exocrine gland symptomatology, resembles primary SS in some clinical and serologic respects.     

Secondary Sj?gren's syndrome in rheumatoid arthritis

One hundred and forty-three sequential, unselected patients with rheumatoid arthritis (RA) were prospectively evaluated for evidence of secondary Sj?gren's syndrome (sSS). All of them had a labial salivary gland (LSG) biopsy, since a greater than or equal to 2+ score (Tarpley's classification) was considered mandatory for the diagnosis of sSS. One hundred eleven were completely investigated. The diagnosis of sSS was established when a patient with a positive LSG biopsy (greater than or equal to 2+ score) had keratoconjunctivitis sicca (KCS) (positive rose bengal test and/or the combination of subjective xerophthalmia and positive Schirmer's I) and/or xerostomia (subjective xerostomia and decreased parotid flow rate = [PFR]). Forty-four patients had an LSG biopsy score of greater than or equal to 2+, 28 a 1+ score and 39 a negative biopsy. Thirty-four of the 44 patients with positive biopsy had sSS, suggesting a 31% prevalence of sSS in RA. The 1+ score group represents a nonspecific category, resembling the negative biopsy group. Rose bengal test was very sensitive for sSS, whereas Schirmer's I and PFR were not. Parotid gland enlargement was unusual, and extraglandular manifestations, with the exception of diffuse interstitial lung disease, were relatively uncommon in all biopsy groups. No patient complained of xerophthalmia or xerostomia on his/her own. Anti-Ro (SSA) antibodies, detected in 23.5% of the patients with sSS, correlated well with positive LSG biopsy. Our study suggests that sSS in RA is common, benign and subclinical, requiring a positive lip biopsy and specific tests for its diagnosis.