Immunofluorescence

The existence of autoantibodies is characteristic of several rheumatic diseases and indicates impaired self-tolerance. The analysis and detection of these autoantibodies represent an important factor in the diagnosis of rheumatic diseases. Immunofluorescence is an important technique in basic science and also one of the most commonly used techniques for the characterization of autoantibodies in clinical medicine thus facilitating the diagnosis and evaluation of rheumatic diseases. Addition of serum samples from individual patients to an antigen substrate (present in fixed cells or tissue) results in a specific reaction between the autoantibodies and the respective antigens. Incubation with a second, fluorescence-linked, anti-human antibody which binds to the primary antigen-bound autoantibodies from patient serum results in fluorescence patterns which are characteristic of certain rheumatic diseases.     

Autoantibodies in connective tissue disease

Autoimmune connective tissue diseases are heterogeneous rheumatic diseases with the potential to affect multiple body systems. Autoantibodies are a characteristic feature of these diseases and are typically highly disease specific. In addition to aiding diagnosis, many autoantibodies have established associations with clinically important disease complications including internal organ involvement. In this chapter, we review the autoantibodies relevant to autoimmune connective tissue diseases, excluding systemic lupus erythematosus, with particular reference to the associated clinical features and how identification of such an autoantibody may inform prognosis and clinical management. We also discuss the practicalities of testing for autoantibodies along with potential difficulties and pitfalls.     

Serologic laboratory findings in malignancy

Autoantibodies are extremely promising diagnostic and prognostic biomarkers of cancer, and have the potential to promote early diagnosis and to make a large impact by improving patient outcome and decreasing mortality. Moreover, autoantibodies may be useful reagents in the identification of subjects at risk for cancer, bearing premalignant tissue changes. Great efforts are being made in many laboratories to validate diagnostic panels of autoantibodies with high sensitivity and specificity that could be useful in a clinical setting. It is likely that prospective studies of sufficiently large cohorts of patients and controls using high-throughput technology may allow the identification of biomarkers with diagnostic significance, and perhaps of discrete antigen phenotypes with clinical significance. The identification of TAAs may also be essential for the development of anticancer vaccines, because autoantibodies found in cancer sera target molecules involved in signal transduction, cell-cycle regulation, cell proliferation, and apoptosis, playing important roles in carcinogenesis. On this basis, molecular studies of antigenantibody systems in cancer promise to yield valuable information on the carcinogenic process. TAAs identified by serum antibodies in cancer sera can be natural immunogenic molecules, useful as targets for cancer immunotherapy. An important problem encountered in the practice of medicine is the identification of healthy individuals in the general population who unknowingly are at high risk of developing cancer. For the rheumatologist, a related problem is the identification of those patients with rheumatic diseases who are at high risk for developing a malignant process. These problems encountered in the fields of cancer and the rheumatic diseases can in the future be helped by new diagnostic instruments based on antibodies. The need for promoting the early diagnosis of cancer is a recognized major public health problem in need of significant research support for the validation of multiple promising but inconclusive studies, with the intention of producing diagnostic panels of autoantibodies in various types of cancers. Cancer developing in patients with rheumatic diseases is also an important problem requiring prospective longterm follow-up studies of patients with rheumatic diseases, particularly because some of the new biologic therapies seem to increase the cancer risk. It is possible that a panel of autoantibodies common to patients with cancer and the rheumatic diseases may prove to be of value in the identification of those patients with ADs at high risk for neoplasms.     

Anti-interleukin-6 autoantibodies in rheumatic diseases. Increased frequency in the sera of patients with systemic sclerosis.

OBJECTIVE. To investigate the presence and the roles of anti-interleukin-6 (anti-IL-6) autoantibodies in rheumatic diseases, and to further elucidate clinical and pathophysiologic significance of anticytokine autoantibodies. METHODS. Anti-IL-6 IgG autoantibodies were measured by the 125I-IL-6 binding activity of IgG, which was isolated from serum by protein A-Sepharose. RESULTS. Nine of 52 sera (17.3%) from patients with systemic sclerosis (SSc) contained anti-IL-6 antibodies, whereas only 1.9% of sera from normal subjects and 0-5% of sera from patients with other rheumatic diseases were positive for the antibodies. Moreover, anti-IL-6 autoantibodies were found predominantly among patients with the limited form of SSc (42.9%), compared with those with the diffuse form (7.9%). CONCLUSION. Anti-IL-6 IgG autoantibodies were detected in patients with SSc, particularly those with the limited form of the disease, at a significantly increased frequency compared with normal subjects and patients with other rheumatic diseases. These results suggest that the development of anti-IL-6 autoantibodies and IL-6 may have a role in the pathophysiology of SSc.     

Anti–interleukin-6 autoantibodies in rheumatic diseases

Objective. To investigate the presence and the roles of anti–interleukin-6 (anti–IL-6) autoantibodies in rheumatic diseases, and to further elucidate clinical and pathophysiologic significance of anticytokine autoantibodies. Methods. Anti–IL-6 IgG autoantibodies were measured by the ¹²⁵I–IL-6 binding activity of IgG, which was isolated from serum by protein A–Sepharose. Results. Nine of 52 sera (17.3%) from patients with systemic sclerosis (SSc) contained anti–IL-6 antibodies, whereas only 1.9% of sera from normal subjects and 0–5% of sera from patients with other rheumatic diseases were positive for the antibodies. Moreover, anti–IL-6 autoantibodies were found predominantly among patients with the limited form of SSc (42.9%), compared with those with the diffuse form (7.9%). Conclusion. Anti–IL-6 IgG autoantibodies were detected in patients with SSc, particularly those with the limited form of the disease, at a significantly increased frequency compared with normal subjects and patients with other rheumatic diseases. These results suggest that the development of anti–IL-6 autoantibodies and IL-6 may have a role in the pathophysiology of SSc.     

Laboratory testing in autoimmune rheumatic diseases

There are a number of pathological conditions in which tissue damage occurs in association with immune activation directed against components of normal tissue. The initial damaging events usually involve cells of the immune system, the T-cells, but the cell damage releases antigens that become targets for an antibody response. The detection and quantification of autoantibodies has become an important component in the diagnosis and management of autoimmune rheumatic diseases such as rheumatoid arthritis, systemic lupus erythematosus, the systemic vasculitides and systemic sclerosis. Each of these diseases is associated with a particular autoantibody or group of autoantibodies. They are usually detected by their reaction against tissue components using subjective methods such as indirect immunofluorescence. Any positive samples are further analysed using more specific and quantitative methods for the 'quantification' of the specific autoantibody concentration. It is important that these autoantibodies are not considered to be 'gold standard' tests: they are no more than markers of the disease with significant limitations. They are best used as part of a diagnostic panel rather than as a marker indicating one particular disease. Techniques are gradually improving, giving numerical results rather than titres, but a lack of standardization makes these results extremely variable. Many of the markers show no correlation with disease activity. Their use should be restricted to the initial investigation and not repeated every time the patient is followed up. Other markers do, however, correlate with disease activity and can be used to monitor disease. When investigating patients who have symptoms associated with autoimmune rheumatic diseases, analytes such as immunoglobulins, complement components and C-reactive protein may all be measured.     

Autoantibodies against C-reactive protein (CRP) in sera of patients with systemic rheumatic diseases

An assessment of the frequency of serum autoantibodies against modified C-reactive protein (mCRP) in systemic rheumatic diseases and the association of these autoantibodies with clinical and laboratory findings in patients with systemic lupus erythematosus (SLE). Serum levels of autoantibodies against mCRP were measured by an enzyme-linked immunosorbent assay in 125 patients with SLE and in 213 patients with other systemic rheumatic diseases. The frequency of patients with high antimodified CRP antibody levels was 32% in SLE, 22% in systemic sclerosis (SSc), 19% in polymyositis/dermatomyositis (PM/DM), 43% in primary Sjögren's syndrome (pSS), 29% in rheumatoid arthritis (RA), 33% in mixed connective tissue disease (MCTD), and 43% in overlap syndrome. Serum levels of anti-mCRP antibody were significantly lower in SLE patients with persistent proteinuria (P < 0.001), cellular casts (P < 0.01), and hypoalbuminemia (P < 0.05). Serum anti-mCRP antibody levels in SLE showed a direct correlation with serum IgG levels (P < 0.001), serum anti-SS-A antibody levels (P < 0.01), serum anti-SS-B antibody levels (P < 0.01), and serum anti-U1-RNP antibody levels (P < 0.05). Inhibition experiments revealed that nonnative epitopes on the CRP molecule, termed mCRP, were the main target of the anti-mCRP antibodies detected. Autoantibodies against mCRP were frequently found in sera from patients with systemic rheumatic diseases, and may have a role in the immunopathogenesis of systemic rheumatic diseases, which are characterized by persistent inflammation.     

Autoantibodies against C-reactive protein (CRP) in sera of patients with systemic rheumatic diseases

Abstract

An assessment of the frequency of serum autoantibodies against modified C-reactive protein (mCRP) in systemic rheumatic diseases and the association of these autoantibodies with clinical and laboratory findings in patients with systemic lupus erythematosus (SLE). Serum levels of autoantibodies against mCRP were measured by an enzyme-linked immunosorbent assay in 125 patients with SLE and in 213 patients with other systemic rheumatic diseases. The frequency of patients with high antimodified CRP antibody levels was 32% in SLE, 22% in systemic sclerosis (SSc), 19% in polymyositis/dermatomyositis (PM/DM), 43% in primary Sjögren's syndrome (pSS), 29% in rheumatoid arthritis (RA), 33% in mixed connective tissue disease (MCTD), and 43% in overlap syndrome. Serum levels of anti-mCRP antibody were significantly lower in SLE patients with persistent proteinuria (P < 0.001), cellular casts (P < 0.01), and hypoalbuminemia (P < 0.05). Serum anti-mCRP antibody levels in SLE showed a direct correlation with serum IgG levels (P < 0.001), serum anti-SS-A antibody levels (P < 0.01), serum anti-SS-B antibody levels (P < 0.01), and serum anti-U1-RNP antibody levels (P < 0.05). Inhibition experiments revealed that nonnative epitopes on the CRP molecule, termed mCRP, were the main target of the anti-mCRP antibodies detected. Autoantibodies against mCRP were frequently found in sera from patients with systemic rheumatic diseases, and may have a role in the immunopathogenesis of systemic rheumatic diseases, which are characterized by persistent inflammation.     

Autoantibodies to tissue transglutaminase in Sjögren's syndrome and related rheumatic diseases

OBJECTIVE: Sj?gren's syndrome (SS) has been reported in up to 15% of patients with biopsy proven celiac disease (CD). The diagnosis of CD in the setting of SS and other systemic rheumatic diseases can be difficult because they are often associated with a number of gastrointestinal symptoms and diseases. Although the diagnosis of CD is often confirmed by a small bowel biopsy, marker autoantibodies directed against the endomysium of transitional epithelium (EMA) and tissue transglutaminase (tTG) are highly correlated with biopsy-proven disease and serve as a valuable screening test. We used an IgA-anti-tissue transglutaminase antibody (anti-tTG) ELISA to assess the prevalence of anti-tTG in an unselected cohort of patients with SS and other systemic rheumatic diseases. METHODS: Sera from 50 patients with SS, 50 with systemic lupus erythematosus (SLE), 50 with rheumatoid arthritis (RA), 30 with systemic sclerosis (SSc), and 50 healthy controls were tested for autoantibodies to tTG. A comparison group of 40 sera from patients with biopsy-confirmed CD was also included. IgA anti-tTG was measured by a commercially available ELISA kit (Inova, San Diego, CA) that employs purified tTG. RESULTS: Six of the 50 (12%) IgA sufficient SS patients had anti-tTG compared to 2 (4%) normal sera, 3 (6%) SLE, 2 (7%) SSc, and 1 (2%) RA. By comparison, in the CD cohort, 33 (83%) had anti-tTG. Five of 6 SS patients with anti-tTG had symptoms, signs, or small bowel biopsy findings consistent with a diagnosis of CD. IgA anti-tTG and EMA were accompanied by other IgA autoantibodies in SS sera. CONCLUSION: Anti-tTG ELISA is a reliable method to indicate a coexisting diagnosis of CD in patients with SS. Interestingly, the frequency of false positive tTG tests in any of the systemic rheumatic diseases is not significantly greater than in controls. Further, our study shows that anti-tTG is more prevalent in SS than in other systemic rheumatic diseases. The tTG ELISA may be used as a screening test to identify patients with SS who are at risk and require further evaluation for the presence of CD.     

ASE-1: an autoantigen in systemic lupus erythematosus

ASE-1 is a 55 kDa nucleolar autoantigen. We show that autoantibodies to this antigen occur at a higher frequency in the sera of patients with SLE than in other systemic rheumatic diseases and that the specificity of ASE-1 as a serum marker of SLE increases as the number of epitopes recognized by the sera increases. Autoantibodies to ASE-1 were temporally associated with autoantibodies to HsEg5 but were not found in conjunction with other known serum markers of SLE. The frequency of antibodies to ASE-1 epitopes in a SLE cohort was approximately the same as anti-dsDNA. However, anti-dsDNA is associated with renal involvement, whereas ASE-1 reactivity shows an association with a history of serositis. We conclude that ASE-1 is correlated with serositis and that ASE-1 should be added to a list of autoantigens that are considered important serological features of SLE.     

Hypertrophic osteoarthropathy, cutaneous vasculitis, and mixed-type cryoglobulinemia in a patient with nasopharyngeal carcinoma

We describe a 27-year-old woman with a primary nasopharyngeal carcinoma who developed hypertrophic osteoarthropathy and cutaneous vasculitis. Her serum contained antibodies to Epstein-Barr virus and U1 RNP antigens. Cryoproteins isolated from her serum contained antibodies to U1 RNP and a protein with a molecular weight of 32 kd which reacted specifically with antibodies to U1 RNP. HLA typing revealed HLA-B7 and DR1; these have been reported to be increased in Japanese patients with rheumatic diseases who have autoantibodies to U1 RNP. These findings indicate that some features of the paraneoplastic syndrome in this patient might have been caused by immune complexes, part of which were formed by specific autoantibodies produced under genetically controlled conditions of immune responsiveness.     

Role of autoantibody testing

Autoantibodies are the serological hallmark of autoimmune disease. Though their pathogenic role is debatable, they play an important role in the management of a patient with rheumatic disease. However, due to their presence in the general population as well as in multiple autoimmune diseases, the presence of an autoantibody alone does not make a diagnosis; the result has to be interpreted along with clinical findings. Similarly, the absence of autoantibody does not exclude a disease.The common autoantibodies used in clinical practice include rheumatoid factor, anti-CCP antibodies, antinuclear antibodies (ANAs), anti-neutrophil cytoplasmic antibodies (ANCA) and anti-phospholipid antibodies. Once an autoantibody to a broad antigen is detected in a patient, sub-specificity analysis can improve the utility of the antibody. Autoantibodies are detected in the serum using different assays and results of which can vary; thus, it is important for a clinician to know the method used, its sensitivity and specificity to help in the proper interpretation of the laboratory results. This review will address these issues.     

Synovial membrane histopathology in the differential diagnosis of rheumatoid arthritis, gout, pseudogout, systemic lupus erythematosus, infectious arthritis and degenerative joint disease

The synovial membrane histologic sections from patients with six common rheumatic diseases were reviewed without knowledge of the clinical diagnosis. After histopathologic evaluation, the synovial membrane characteristics were grouped according to the patient's clinical diagnosis, and included 29 patients with rheumatoid arthritis, 13 with systemic lupus erythematosus, 17 with degenerative joint disease, 10 with acute bacterial arthritis, 8 with gout, and 13 with pseudogout. The only specific characteristics identified were bacteria (infectious arthritis), crystals (gout, pseudogout), and lymphoid follicles (rheumatoid arthritis). Nevertheless, other characteristic features of differential diagnostic utility were recognized, including the intensity and nature of synovial lining cell hyperplasia and of leukocyte infiltration. Light microscopic histopathologic changes in the common rheumatic diseases are not specific, but are of diagnostic utility. Complete and exhaustive review of each pathologic synovial membrane characteristic provides more justification for the routine use of synovial membrane biopsy as an adjunct to arthrocentesis in the evaluation of common rheumatic diseases.     

Simultaneous identification of various antinuclear antibodies using an automated multiparameter line immunoassay system

The objective was to determine the sensitivity and specificity of an automated multiparameter line immunoassay system compared with other techniques for the identification of autoantibodies in rheumatic diseases. We studied sera from 90 patients. Anti-U1RNP, anti-Sm, anti-Ro/SS-A, anti-La/SS-B, anti-Jo 1 and anti-Scl 70 antibodies were identified by counterimmunoelectrophoresis (CIE) techniques, enzyme-linked immunosorbent assay (ELISA), immunoblotting (IB) using extracts of rabbit thymus and human placenta, and an automated multiparameter line immunoassay system (INNO-LIA ANA UPDATE K-1090) that detects nine different antibodies simultaneously (anti-U1RNP, anti-Sm, anti-Ro/SS-A, anti-La/SS-B, anti-Scl 70, anti-Jo 1, anticentromere, antihistone, and antiribosomal P protein). The line immunoassay system equaled or surpassed the other techniques in the identification of anti-Sm, anti-La/SS-B, anti-Jo 1 and anti-Scl 70 antibodies (sensitivity 100%, specificity 94-100%) and was similarly effective in the case of anti-U1RNP (sensitivity 87.5%, specificity 93.9%) and anti-Ro/SS-A (sensitivity 91.4%, specificity 87.2%) antibodies. In addition, this technique detected more 52 and 60 kD anti-Ro/SS-A sera than IB. Nine antibodies can be detected with this method at a cost of 25.38 Euros per serum sample. In five hours, 19 sera can be studied. The approximate cost of detecting these nine antibodies with an automated ELISA system would be 28.93 Euros, which allows 10 sera to be studied in four hours. In conclusion, the automated multiparameter line immunoassay system is a valid method for the detection of autoantibodies in rheumatic diseases. Its most notable advantages are automated simultaneous detection of several autoantibodies in the same serum and its lower cost compared with ELISA techniques.     

Analysis of autoantibodies to cell cycle-associated antigens

To determine the specificities of autoantibodies targeting cell cycle-associated antigens in rheumatic diseases, we studied 30 sera which were obtained from patients visiting our hospital and which exhibited a variegated speckled pattern in indirect immunofluorescence (IF). The immunoreactivities of the sera were analyzed by Western blotting (WB) and IF. Various reactivities to cellular components were observed in IF. The sera reacted with proteins of various molecular weights in WB. Serum OH from a patient with rheumatoid arthritis showed fine speckled nucleoplasmic and nucleolar staining at interphase, discrete dot staining associated with chromosomes and the midbody at mitosis, and reacted with a 34-kD polypeptide in WB. The target antigen was different from proliferating cell nuclear antigen (PCNA). The 34-kD antigen was characterized by IF using double staining procedures and cell synchronization. The results showed that the expression of the antigen was mainly observed between the late G1 and M-phases. This study indicated that various cell-cycle-associated antigens were recognized in sera from patients with rheumatic diseases, and suggested that a 34-kD antigen recognized by serum OH was a novel cell cycle-associated autoantigen. Received: October 26, 2000 / Accepted: February 26, 2001     

Spectrum of autoantibodies against a dynamin-related protein, dymple

OBJECTIVE: To characterize the clinical features of patients with autoantibodies against dymple, a dynamin-related protein. METHODS: Serum samples from 281 patients with rheumatic diseases were examined. The characteristics of antidymple and antibody-reactive determinants were investigated by immunoblotting with the recombinant dymple antigen, including its deletion mutants, and by immunofluorescence studies with affinity-purified serum. RESULTS: Five serum samples (2%) were found to have antidymple. All of these patients were male, and 4 of them had interstitial pneumonitis. Their sera were considered to mainly recognize the N-terminus of dymple, which contains GTP-binding motifs. CONCLUSION: Dymple, which joins the cytoplasmic autoantigens, could be a marker for a newly recognized subset of connective tissue diseases.     

Clinical use of serological tests for antineutrophil cytoplasmic antibodies. What do the studies say?

Trustworthy and clinically useful laboratory test results depend on high quality laboratory knowledge and performance and on close collaboration with experienced clinicians. Just like a diagnosis can only be made by contrasting it to a number of differential diagnoses, a prototype autoantibody profile can only be recognized if autoantibodies that partially mimic the former are just as well-known. In antineutrophil cytoplasmic antibody (ANCA)-associated small vessel vasculitides the characteristic autoantibody profile can only be demonstrated by use of at least two different methodologies and an assay-specific cut-off selection that clearly distinguishes between strongly positive and disease control low positive to negative values. Local serum banks containing samples from patients with well-characterized chronic immunoinflammatory diseases and ANCA-associated vasculitides are needed for such important studies whether in-house or commercial assays are used.     

Cancer and autoimmunity: autoimmune and rheumatic features in patients with malignancies

OBJECTIVES: To review the autoimmune and rheumatic manifestations of patients with malignancy. METHODS: A Medline search of all published papers using keywords related to malignancies, autoimmunity, rheumatic diseases, and paraneoplastic syndromes. RESULTS: Patients with malignant diseases may develop autoimmune phenomena and rheumatic diseases as a result of (a) generation of autoantibodies against various autoantigens, including oncoproteins (P185, 1-myc, c-myc, c-myb), tumour suppression genes (P53), proliferation associated antigens (cyclin A, B1, D1, E; CENP-F; CDK, U3-RNP), onconeural antigens (Hu, Yo, Ri, Tr), cancer/testis antigens (MAGE, GAGE, BAGE, SSX, ESO, SCP, CT7), and rheumatic disease associated antigens (RNP, Sm). The clinical significance of the various autoantibodies is not clear. Anti-oncoprotein and anti-tumour suppression gene antigens are detected before the diagnosis of the cancer or in the early stages of the malignant disease, suggesting a potential diagnostic or prognostic role. Anti-onconeural antibodies are pathogenic and are associated with specific clinical neurological syndromes (anti-Hu syndrome and others). (b) Paraneoplastic syndromes, a wide range of clinical syndromes, including classic autoimmune rheumatic diseases that develop among patients with cancer. (c) Rheumatism after chemotherapy, a clinical entity characterised by the development of musculoskeletal symptoms after combination chemotherapy for malignancy. CONCLUSION: Autoimmune and rheumatic features are not rare among patients with malignancies. They are the result of various diverse mechanisms and occasionally they may be associated with serious clinical entities.     

Antibodies to amyloid A protein in rheumatic diseases

Summary Circulating autoantibodies against amyloid A protein (AA) were demonstrated by enzyme immunoassay in 18/62 patients with rheumatoid arthritis (RA) and in 9/27 patients with systemic lupus erythematosus (SLE). In the subset of RA patients who had developed amyloid, the frequency of antibodies to AA was lower than in those without amyloid (P<0.05). The antibody levels showed some variation in serial serum samples during follow-up (1–4 years) of patients with amyloidosis or SLE, but did not correlate with disease activity. In contrast to the patients with rheumatic diseases, patients with inflammatory bowel disease and acute bacterial peritonitis had antibody levels within the range of the healthy control subjects. The results show that autoantibodies to protein AA may occur in rheumatic diseases; their occurrence does not, however, identify subjects with tissue amyloid deposits. Absorption experiments suggest that the antibodies may be directed to circulating amyloid A protein.