表达人自身抗原SSA/Ro60重组体的构建及鉴定

目的：构建表达人自身抗原ＳＳＡ／Ｒｏ６０的重组体,为研究自身免疫病中的抗原体作用提供物质基础。方法：采用逆转录－ＰＣＲ技术克隆自身抗原ＳＳＡ／Ｒｏ６０ ｃＤＮＡ,定向插入表达载体ＰＧＥＸ－２Ｔ,并经酶切电泳,ＤＮＡ测序鉴定分析。结果：经鉴定,证明成功构建了表达人自身抗原ＳＳＡ／Ｒｏ６０的重组体。结论：ＳＳＡ／Ｒｏ６０表达型重组体可用于自身抗原ＳＳＡ／Ｒｏ６０的大量生产并用于诊断。     

Epitope mapping of the 52-kD Ro/SSA autoantigen.

Autoantibodies to Ro/SSA are commonly found in sera of patients with systemic lupus erythematosus (SLE) and primary Sjögren's syndrome. The presence of these antibodies is related to lymphopenia, photosensitive dermatitis, and pulmonary and renal disease, suggesting that they have an immunopathologic role [1-6]. We previously isolated a cDNA clone which encodes the 52-kD human Ro/SSA protein. In this study we have determined the number and location of epitopes recognized by SLE sera using recombinant proteins encoded by the full-length or overlapping subclones of this cDNA. An immunodominant epitope was detected using Western blots and ELISA on the NH2-terminal side of this protein's putative leucine zipper. The data suggest that 11 amino acids are critical for the recognition of this molecule by these autoantibodies. Although the titres of anti-52-kD Ro/SSA antibodies vary between different patient sera, no heterogeneity in the location of antigenic epitopes to which their autoantibodies bound was detected. This homogeneous pattern of reactivity to a single rather than multiple regions of this protein is unusual for lupus autoantigens which have been identified, and suggests that these antibodies may have arisen as by a cross-reaction to an epitope on another molecule.     

Ro60/SSA基因转染HEp-2细胞作为间接免疫荧光检测底物及其应用

本研究拟改造间接免疫荧光技术（IIF）抗核抗体（ANA）检测法的底物HEp-2细胞，建立抗60kDRo60／SSA抗体免疫荧光检测法。采用PCR扩增人源Ro60 cDNA，克隆入真核表达载体pEGFP-C1，并转染HEp-2细胞。通过荧光显微镜、免疫印迹法（IBT）及IIF鉴定转染细胞（HEp-Ro60）中Ro60-绿色荧光蛋白（GFP）融合蛋白的表达和抗原性。分别以HEp-Ro60以及HEp-2为底物的IIF检测10份抗Ro／SSA对流免疫电泳（CIE）检测阳性、其他抗体阴性血清以及对照血清。获得的转染细胞传十几代后仍具有较强的Ro60-GFP表达，融合蛋白保持Ro60的抗原性。IIF检测中HEp-Ro60的滴度比HEp-2增加了6．7倍（P〈0．01），而且2例HEp-2细胞上IIF检测为阴性的血清在HEI-Ro60上为阳性。10例阳性血清中有8例出现了特征性荧光模式。结论是HEp-Ro60可用于IIF检测抗Ro抗体，并增加了ANA检出的敏感性。     

A calreticulin-like protein co-purifies with a '60 kD' component of Ro/SSA, but is not recognized by antibodies in Sjögren's syndrome sera.

In this study, we used human tonsils for the isolation of the 60 kD component of the Ro/SSA autoantigen, following the method described by Wu et al. (J Immunol Methods 1989; 121:219-24). Western blot analyses were carried out using Ro/SSA-reactive human Sjögren's syndrome sera, to follow the autoantigen through the purification procedure. A 60 kD Ro/SSA component was eluted as a broad peak from a Mono Q column. Within this peak, a much more abundant protein, co-migrating with the Ro/SSA component on SDS-PAGE, was also eluted. The more abundant protein was further purified on a Superose 12 column and its N-terminal sequence was shown to be identical to that of human calreticulin. The 60 kD Ro/SSA autoantigen was also further purified on the Superose 12 column and was eluted as an asymmetric peak, with the majority being eluted at a position corresponding to 60 kD, whereas the calreticulin-like protein was eluted from the same column as an apparent dimer of approximately 120 kD. A panel of five Ro/SSA-reactive human sera reacted with the purified Ro/SSA antigen, but not with the calreticulin-like protein. Therefore, it is clear that the calreticulin-like protein is not a Ro autoantigen and is distinct from the 60 kD Ro/SSA antigen. As the calreticulin-like protein is a much more abundant protein than the 60 kD Ro/SSA component, its co-purification with the autoantigen on ion-exchange and its close migration with the autoantigen on SDS-PAGE may explain why peptide sequences for human calreticulin were derived from apparent 60 kD Ro/SSA antigen preparations.     

Epitope mapping with synthetic peptides of 52-kD SSA/Ro protein reveals heterogeneous antibody profiles in human autoimmune sera.

The reactivity of autoantibodies present in the sera of 489 patients with Sjögren's syndrome (SS), systemic lupus erythematosus (SLE) and other autoimmune diseases was investigated by ELISA using recombinant 52-kD SSA/Ro protein (rRo52) and 39 overlapping synthetic peptides representing the entire sequence of Ro52. We report that IgG antibodies reacting with rRo52 were present in the sera of a large number of patients with SS (67% of patients with primary SS and 46% of patients with SS associated with SLE), whereas they were less frequent (10-25%) in SLE, rheumatoid arthritis (RA), juvenile chronic arthritis (JCA) and mixed connective tissue disease (MCTD), and absent in scleroderma. Among the 39 peptides tested, five were recognized by sera from 30-65% of patients with SS, namely peptides representing residues 2-11, 107-122, 107-126, 277-292 and 365-382. Patients with JCA had raised levels of IgG antibodies reacting with peptides 2-11 and 365-382, and 51% of patients with MCTD had raised levels of IgG antibodies reacting with peptide 365-382. None of the five peptides was recognized by more than 20% of sera from patients with SLE and RA. Interestingly, and of importance in the field of diagnostic tests based on peptides, the reactivity of antibodies to the Ro52 synthetic peptides varied greatly according to the origin of sera. Inhibition experiments using either patients' sera or antibodies induced in rabbits against Ro52 peptides showed that the four domains 2-11, 107-122, 277-292 and 365-382 are accessible on the surface of the Ro52 protein. These regions may thus be involved in the induction of specific antibodies in autoimmune patients.     

Suppressor cells induced in the human autologous mixed lymphocyte reaction

Theophylline, a phosphodiesterase inhibitor, also reversibly inhibits the SRBC receptor of a subpopulation of human T lymphocytes. We have previously reported that the precursor for the Concanavalin A (Con A) inducible suppressor cell (SC) is found predominantly in the theophylline-sensitive (Ts) population; whereas, the precursor of the mitomycin resistant suppressor cell induced in the allogeneic mixed lymphocyte reaction (MLR) is predominantly in the theophylline-resistant (TR) subset. This study examines the theophylline sensitivity and functional characteristics of suppressor cells induced in autologous MLR (AMLR). Both TS and TR proliferate moderately in AMLR but differ in their ability to form suppressors. AMLR activated Ts and TR cells demonstrate early blastogenesis and weak suppression in the Con A assay system, but suppression in an MLR assay system appeared to be confined largely to the TR subset. AMLR suppressors were mitomycin sensitive. These data illustrate that suppressors generated in allogeneic and autologous MLR have significant functional and phenotypic similarities, and strengthen the association between these two immunologic phenomena.     

Ro60/SSA基因转染HEp-2细胞作为间接免疫荧光检测底物及其应用

本研究拟改造间接免疫荧光技术(IIF)抗核抗体(ANA)检测法的底物HEp-2细胞,建立抗60kDRo60/SSA抗体免疫荧光检测法。采用PCR扩增人源Ro60 cDNA,克隆入真核表达载体pEGFP-C1,并转染HEp-2细胞。通过荧光显微镜、免疫印迹法(IBT)及IIF鉴定转染细胞(HEp-Ro60)中Ro60-绿色荧光蛋白(GFP)融合蛋白的表达和抗原性。分别以HEp-Ro60以及HEp-2为底物的IIF检测10份抗Ro/SSA对流免疫电泳(CIE)检测阳性、其他抗体阴性血清以及对照血清。获得的转染细胞传十几代后仍具有较强的Ro60-GFP表达,融合蛋白保持Ro60的抗原性。IIF检测中HEp-Ro60的滴度比HEp-2增加了6.7倍(P<0.01),而且2例HEp-2细胞上IIF检测为阴性的血清在HEp-Ro60上为阳性。10例阳性血清中有8例出现了特征性荧光模式。结论是HEp-Ro60可用于IIF检测抗Ro抗体,并增加了ANA检出的敏感性。     

自身抗原52 kD-Ro/SSA的序列分析及抗原性预测

目的：分析自身抗原５２ｋＤ－Ｒｏ／ＳＳＡ的序列结构，预测其抗原位点。方法：将自身抗原５２ｋＤ－Ｒｏ／ＳＳＡ的氨基酸序列输入“蛋白质结构预测”软件包，分析蛋白质分子生、表面可及性、柔性，并模拟其二级结构，预测其主要抗原位点。结果：自身抗原５２ｋＤ－Ｒｏ／ＳＳＡ是多抗原位点，其氨基酸序列中１２０￣１３０、３００￣３２０、３６０￣３８０位间抗原性较强。结论：确定５２ｋＤ－Ｒｏ／ＳＳＡ的抗原优势表位，为研     

CD8+/DR+ T gamma cells inhibit the autologous mixed lymphocyte reaction.

The proliferative response of T cells during autologous mixed lymphocyte reactions (AMLR) was affected by depletion of IgG Fc receptor+ T lymphocytes (Tg). Removal of Tg cells resulted in enhanced proliferation, and EA-rosette isolated Tg cells, when added to AMLR cultures as irradiated third components, reduced the uptake of 3H-thymidine by 63-87% in a dose-dependent manner. Negative selection using an avidin-biotin affinity chromatography technique demonstrated that the suppression was mediated by DR+ Tg cells; the major proportion of which also expressed the CD8 antigen. By comparing AMLR supernatants collected from control (lacking Tg) and suppressed (containing Tg) cultures on days 2, 3, and 4, it was established that supernatants from suppressed cultures had significantly reduced levels of cytokine activity. These data indicate that the CD8+/DR+ Tg cells function as suppressor cells during an AMLR and reduce the proliferative response by inhibiting AMLR responder T cells from producing the cytokines necessary for in vitro growth.     

IgG antibodies from patients with primary Sjögren''s syndrome and systemic lupus erythematosus recognize different epitopes in 60-kD SSA/Ro protein.

Five synthetic peptides corresponding to the N-, the C- and a central domain in 60-kD SSA/Ro protein were prepared and tested with sera from 112 patients with systemic lupus erythematosus (SLE), 55 with primary Sjögren's syndrome (pSS) and 29 with rheumatoid arthritis. Among these five fragments, one representing residues 21-41, was recognized by antibodies in 57% of pSS patients. Interestingly, this peptide was recognized by only a few (≤7%) of SLE sera, while 63% of pSS sera and 46% of SLE sera tested in parallel possessed antibodies reacting in ELISA with purified 60-kD SSA protein. The ELISA results were compared with the pattern of reactivity obtained in immunodiffusion and immunoblotting. The results indicate that the sensitivity of ELISA using peptide 21-41 and pSS sera was in the same range as immunoblotting and higher than immunodiffusion. Thus the peptide 21-41 proved useful for the detection of anti-SSA antibodies in the sera of patients with pSS. Furthermore, a positive ELISA using peptide 21–41 could be of potential use to discriminate pSS with systemic features from SLE. The fact that peptide 21–41 is recognized by antibodies in pSS but only by very few SLE sera implies that different mechanisms are involved in the anti-SSA immune response in these two autoimmune diseases.     

Heterogeneity of the Ro/SSA antigen and autoanti-Ro/SSA response: evidence of the four antigenically distinct forms.

Precipitating antibodies to the Ro/SSA antigen occur in the sera of 40% of patients with sytemic lupus erythematosus (SLE) and in 40–70% of the sera of patients with primary Sjögren's syndrome. Previous work has shown that lymphocyte extracts contain two Ro/SSA antigens with protein moieties of 60 kD and 52 kD and that erythrocyte haemolysate contain two analogous but antigenically distinct Ro/SSA molecules of 60 kD and 54 kD. Frequency analysis of the various specificities in 43 sera with precipitating anti-Ro/SSA and studies with affinity-eluted antibodies suggest that the lymphocyte 60 kD and erythrocyte 60 kD Ro/SSA molecules are related as are the lymphocyte 52 kD and erythrocyte 54 kD Ro/SSA proteins. Anti-Ro/SSA sera when accompanied by other precipitins (anti-La/SSB and anti-U1RNP) react preferentially with certain Ro/SSA isoforms. Evidence is also presented for a 45 kD form of Ro/SSA. These data suggest that the antigenic heterogeneity of the Ro/SSA antigen is immunologically relevant and that there are two families of Ro/SSA antigens: one comprising of the two 60-kD proteins in the erythrocyte and lymphocyte and the other the erythrocyte 54 kD and lymphocyte 52 kD Ro/SSA proteins, respectively.     

Nuclear-Targeting Autoantibodies Induced Nuclear PARP Cleavage Accompanied by More Pronounced Decrease of Peripheral White Blood Cells Than Ro/SSA and La/SSB Antigen-Targeting Autoantibodies

Autoantibody production and leukocytopenia may be linked in patients with lupus erythematosus (LE). Unclear is the ability of different autoantibody species to induce apoptosis and cell loss. Laboratory routine analyses (white blood cell counts, autoantibody detection), and flow cytometry (annexin V, CD3, CD4, CD8) have been performed in 126 consecutive LE-patients. Nuclei of PBMC were investigated flow cytometrically for the presence of the 85 kDa poly-(ADP-ribose)-polymerase (PARP) fragment. Peripheral total white blood cells (WBC), lymphocytes, T-cells, CD3+ CD4+, and CD3+ CD8+ cells were significantly decreased in patients with LE (P from 1.2 × 10^–14 to P < .0008). In the presence of either antinuclear (P from 1.2 × 10^–14 to P < .0008) or anti-dsDNA antibodies (P from 2.9 × 10^–12 to P < .007) were significantly diminished. Differences in cell numbers in LE patients with versus without anti-Ro/SSA were less pronounced: significant differences could be only obtained in lymphocytes and T-cells (P < .02). Anti-La/SSB antibodies were accompanied by significant increased leukocytes (P < .02). PARP cleavage (85 kDa) in nuclei was preferentially observed in cases with nuclear targeting autoantibodies. These results indicate that nuclear targeting autoantibodies are associated to lower peripheral blood cells counts than Ro/SSA, and La/SSB cytoplasmic targeting autoantibodies. This provides an explanation for the pathogenesis of cytopenias associated with SLE.     

Different temporal expression of immunodominant Ro60/60 kDa-SSA and La/SSB apotopes

Opsonization of apoptotic cardiocytes by maternal anti-Ro/SSA and anti-La/SSB antibodies contributes to tissue injury in the neonatal lupus syndrome. The objective of the current study was to quantify the surface membrane expression of Ro/La components during different phases of apoptosis and map the Ro/La apotopes (epitopes expressed on apoptotic cells) bound by cognate antibodies. Multi-parameter flow cytometry was used to define early and late apoptotic populations and their respective binding by monospecific anti-Ro and anti-La IgGs. Anti-Ro60 bound specifically to early apoptotic Jurkat cells and remained accessible on the cell surface throughout early and late apoptosis. In contrast, anti-La bound exclusively to late apoptotic cells in experiments controlled for non-specific membrane leakage of IgG. Ro52 was not accessible for antibody binding on either apoptotic population. The immunodominant NH₂-terminal and RNA recognition motif (RRM) epitopes of La were expressed as apotopes on late apoptotic cells, confirming recent in vivo findings. An immunodominant internal epitope of Ro60 that contains the RRM, and is recognized by a majority of sera from mothers of children with congenital heart block (CHB) and patients with primary Sjögren's syndrome, was also accessible as an apotope on early apoptotic cells. The distinct temporal expression of the immunodominant Ro60 and La apotopes indicates that these intracellular autoantigens translocate independently to the cell surface, and supports a model in which maternal antibody populations against both Ro60 and La apotopes act in an additive fashion to increase the risk of tissue damage in CHB.     

Severe T lymphocyte immunodeficiency associated with hypogammaglobulinemia: Defective lymphokine secretion but enhanced autologous mixed lymphocyte reaction

The multiparameter immunologic study of T cells of a patient with acquired hypogammaglobulinemia was investigated, since he had a normal B-cell number and function. His peripheral blood lymphocytes were found to contain predominant CD4+ CD45R+ T cells with a clear deficiency of CD4+ CDw29+ as well as CD8+ T cells. His T cells proliferated in response to phytohemagglutinin (PHA) and pokeweed mitogen (PWM), but no immunoglobulin was secreted in PWM-induced patient's T-cell and normal B-cell differentiations. His T cells were also found to posses concanavalin A (Con A)-induced suppressor function when cocultured with normal T cells, as well as IgG-, IgA-, and IgM-specific suppressor function on PWM-induced normal T- and B-cell differentiations. The patient's T cells were found to secrete elevated amounts of interleukin-2 but failed to secrete two important B-cell stimulating factors, B-cell growth factor and B-cell differentiation factor, in response to PHA. An investigation of immunoregulatory T-cell function in the autologous mixed lymphocyte reaction (AMLR) and allogeneic mixed lymphocyte reaction (MLR) indicated that the patient's T cells produced an enhanced AMLR but were deficient in MLR. These results suggest that the abnormalities we have identified in this patient with hypogammaglobulinemia reflect an intrinsic defect of T cells in the humoral immune response to produce three major immunoglobulins.     

Anti-Ro/SSA and La/SSB antibodies

The Ro/La system is considered as an heterogeneous antigenic complex, constituted by three different proteins (52?kDa Ro, 60?kDa Ro and La) and four small RNAs particles. Anti-Ro/SSA are the most prevalent specificity among many autoimmune diseases, such as systemic lupus erythematosus (SLE), SS/SLE overlap syndrome, subacute cutaneous LE (SCLE), neonatal lupus and primary biliary cirrhosis. In contrast, anti-La/SSB is more associated with Sjögren's syndrome (SS). The differences between 52?kDa, 60?kDa Ro and La could explain why different assays did not show equivalent performance in anti-Ro and anti-La autoantibodies detection. The RNA precipitation assay had the highest sensitivity and specificity, usually considered as the reference methods. CIE is considered the most reliable to detect anti-Ro/SSA antibodies in routine practice, performing better than immunoblotting (IB) and some ELISAs. It shows a high sensitivity (89%) and specificity (100%). ELISA is generally considered a safe, rapid, sensitive and specific tecnique. Therefore, its high sensitivity often corresponds to a very low clinical specificity and the assay can give false positive results. Therefore, it is very important to search anti-Ro and anti-La only in selected patients, using the assay with high specificity and good predictive value, in order to have clinically significant and true positive results.     

Endoplasmic reticulum stress causes autophagy and apoptosis leading to cellular redistribution of the autoantigens Ro/Sjögren's syndrome‐related antigen A (SSA) and La/SSB in salivary gland epithelial cells

The aim of this study was to examine the levels of endoplasmic reticulum (ER) stress in minor salivary glands, to investigate the interplay between ER stress-induced autophagy and apoptosis in human salivary gland (HSG) cells and to test the effect of ER stress-induced apoptosis on the cellular redistribution of the two major Sjögren’s syndrome (SS) autoantigens Ro/Sjögren’s syndrome-related antigen A (SSA) and La/Sjögren’s syndrome-related antigen B (SSB). Minor salivary gland biopsies from SS patients and sicca controls were examined by immunohistochemistry for the expression of 78 kDa glucose-regulated protein/binding immunoglobulin protein (GRP78/BiP) as an indicator of unfolded protein response (UPR). HSG cells were treated with thapsigargin (TG) and cell viability, autophagy and apoptosis were assessed. Immunoblot was applied to detect the conversion of LC3I to LC3II and the protein levels of GRP78/BiP and X-box binding protein-1 (XBP-1). Apoptosis was evaluated by a single-stranded DNA enzyme-linked immunosorbent assay (ELISA). Ro/SSA and La/SSB localization was visualized using immunofluorescence. GRP78/BiP was expressed by acinar and ductal epithelial cells in salivary glands of patients and sicca controls. TG treatment induced autophagy, as indicated by enhanced protein expression of LC3II. The protein levels of UPR marker XBP-1 were increased after TG treatment, while GRP78/BiP levels were decreased. TG treatment resulted in induction of HSG apoptosis. Ro/SSA and La/SSB autoantigens were localized predominantly to the cytoplasm in resting cells, while they were redistributed to cell membrane and blebs in the apoptotic cells. In conclusion, ER stress is activated in minor salivary gland epithelial cells from SS patients and controls. ER stress-induced apoptosis in HSG cells leads to cell surface and apoptotic blebs relocalization of Ro/SSA and La/SSB autoantigens.     

Autologous mixed lymphocyte reaction in man. XIII. Characterization of the T-T autologous mixed lymphocyte reaction

In this study we have demonstrated that in the T-TA autologous mixed lymphocyte reaction (AMLR), OKT4⁺ T cells are the major responders; however, in the presence of additional interleukin-2 (IL-2), OKT8⁺ T cells also respond by proliferation. Both OKT4⁺ and OKT8⁺ T cells, activated in the T-non-T AMLR, act as stimulators in the T-TA AMLR. OKT4⁺ T cells activated in the T-TA AMLR suppress the proliferative response of the fresh T-non-T AMLR; control OKT4⁺ cells show no immunoregulatory activity in this system. In contrast, control OKT8⁺ T cells spontaneously suppress the proliferation of the T-non-T AMLR, but activation of OKT8⁺ T cells in the T-TA AMLR does not result in a further increase in the suppressor activity of OKT8⁺ T cells. In summary, in the T-non-T and T-TA AMLR phenotypically similar T-cell subpopulations proliferate but express distinct immunoregulatory functions and perhaps regulate the tempo of the AMLR.     

The evolution of red blood cell and lymphocyte Ro/SSA.

Recent studies have demonstrated that the Ro/SSA autoantigen is heterogeneous as is the corresponding autoimmune response. In addition the autoimmune responses is highly species specific and preferentially reactive with the human antigen. Quantitative ELISA study shows that red blood cell Ro/SSA evolves much more rapidly than lymphocyte Ro/SSA and Western Blot analysis shows that the quantitative ELISA results are mirrored by changes in the 60 kD Ro/SSA molecules but not the 52 kD and 54 kD Ro/SSA molecules. The 52 kD and 54 kD Ro/SSA molecules seem to be relatively conserved as indicated by the Western immunoblotting experiments. These studies add weight to the concept that the antigenic epitopes of these related proteins are under the control of separate genes which have undergone different rates of evolution.     

T cell interactions in active rheumatoid arthritis: insights from the human autologous mixed lymphocyte reaction as a model of T cell activation cascade.

The autologous mixed lymphocyte reaction (AMLR) represents the activation, proliferation and differentiation of T cells in response to signals from autologous non-T cells. Using monoclonal anti-Leu8 antibody to isolate subpopulations of human CD4+ and CD8+ T cells, we have investigated the role of these subpopulations in the T cell activation cascade during the course of AMLR. In normal subjects, CD4+Leu8+ cells are necessary for the initiation of the AMLR response, and sequentially lead to activation and proliferation of both CD4+Leu8- cells and CD8+Leu8+ cells. The activated CD8+Leu8+ cells, in turn, induce CD8+Leu8- cells to generate proliferation of the latter cells. Soluble mediators could be involved in the T cell activation cascade induced by the AMLR. Patients with active rheumatoid arthritis have a profound defect in the AMLR. Further analysis indicates that rheumatoid arthritis CD8+ T cells are markedly defective as responding cells in the AMLR. The impaired AMLR response by CD8+ cells cannot be reconstituted with AMLR-derived supernatants from normal T cells. The data suggest that the defective CD8+ T cell function may contribute to the pathogenesis of the disease.