Seronegative Autoimmune Diseases

CONTEMPORARY CHALLENGES IN AUTOIMMUNITY Seronegative Autoimmune Diseases Cristiano Alessandri, Fabrizio Conti, Paola Conigliaro, Riccardo Mancini, Laura Massaro, and Guido Valesini Dipartimento di Clinica e Terapia Medica, Sapienza Università di Roma, Rome, Italy A close relationship exists between autoimmunity and autoantibodies; despite this, some patients are persistently negative for disease-specific autoantibodies. These conditions have been defined as seronegative autoimmune diseases. Although the prevalence of seronegative autoimmune diseases is low, they may represent a practical problem because they are often difficult cases. There are also situations in which autoantibodies are positive in healthy subjects. In particular, three different conditions can be described: latent autoimmunity, preclinical autoimmunity, and postclinical autoimmunity. Here, we analyze briefly the meaning of autoantibody negativity in the seronegative autoimmune diseases, focusing in particular on the specificities associated with systemic lupus erythematosus, antiphospholipid syndrome, and rheumatoid arthritis. Key words: autoimmune diseases; seronegative AD; SLE; APS; RA Introduction It is well known that there is a close relationship between autoimmunity and autoantibodies targeting intracellular or extracellular antigens. Indeed, an alteration of the immune homeostasis leads to the production of both autoantibodies and autoreactive cells that may play a pathogenic role in autoimmune diseases (AD). It is still a matter of debate whether autoantibodies result from a specific immune response to an immunogenic antigen or whether they are produced because of a defect in immune regulation. Moreover, even though circulating autoantibodies are frequently observed in linkage with infections, drugs, or aging, most of them are markers of AD and may be involved in their pathophysiology. Autoantibodies can further inform us about basic mechanisms of loss of tolerance, inflammation, and tissue damage; in these cases a positive test for specific autoantibodies, especially with high titer, strongly supports the diagnosis of AD. It is noteworthy, for Address for correspondence: Professor Guido Valesini, Dipartimento di Clinica e Terapia Medica, Reumatologia, Sapienza Università di Roma, V.le del Policlinico 155, 00161 Rome, Italy. Voice: +39 0649974631; fax: +39 0649974642. [email protected] example, that anti-double-stranded (ds)DNA antibodies are associated with disease flare in systemic lupus erythematosus (SLE), anticyclic citrullinated peptide (anti-CCP) antibodies are a specific marker of rheumatoid arthritis (RA), and antiphospholipid antibodies (aPL) correlate significantly with clinical manifestation of antiphospholipid syndrome (APS). Nevertheless, despite the tightened association between autoantibodies and AD, some patients affected by these types of disease are persistently negative for disease-specific autoantibodies. The lack of evidence of circulating autoantibodies in these cases may be a consequence of real absence or just difficulty in detecting some peculiar specificities. In the first case, it can be assumed the autoantibodies are not the (unique) pathogenic factor; in the second case, it could be the method of detection or immunological alterations (Table 1). Although the prevalence of seronegative AD is low, they may represent a practical problem because they are often difficult cases. There are situations in which autoantibodies are positive in healthy subjects. In particular, three different conditions can be described: latent autoimmunity, preclinical autoimmunity, and postclinical autoimmunity (Table 2). In the Contemporary Challenges in Autoimmunity: Ann. N.Y. Acad. Sci. 1173: 52–59 (2009). c 2009 New York Academy of Sciences. doi: 10.1111/j.1749-6632.2009.04806.x  52 Alessandri et al.: Seronegative AD TABLE 1. Lack of Evidence of Circulating Autoantibodies 1. Real absence (?) 2. Difficult to detect some specificities – Serum dilution – Immunofluorescence substrate – Prozone phenomenon – Low Ig levels – Hidden autoantibodies – Autoantibodies complexed and deposited in tissues first case, autoantibodies usually are IgM natural antibodies or low-affinity IgG.1 Otherwise, autoantibodies can be detected in the serum of healthy first-degree relatives of patients with autoimmune diseases, such as SLE, APS, and RA.2–6 In the second case, autoantibodies can predict the future development of AD. For example, it has been demonstrated that several antibodies can be detected many years before the diagnosis or the first clinical manifestation.7–9 Finally, some antibodies, such as antithyroid microsomial and anti-thyroglobulin, can be detected in patients with a previous thyroiditis, representative of a postclinical autoimmunity.10 Seronegative SLE and APS SLE is a multisystemic autoimmune disease that involves almost all the organs in the human body with a broad spectrum of clinical manifestations. It has been suggested that several autoantibodies play a role in the pathogenesis of SLE. Noteworthy, more than 100 different specificities are detectable in the SLE sera (reviewed in Ref. 11). Whereas anti-nuclear antibodies (ANA) are found in nearly all SLE patients, increased anti-dsDNA antibody titers are characteristic of the acute phase of the disease and predict clinical exacerbations,12 and anti-Ro and anti-La antibodies correlate with neonatal lupus and lupus dermatitis.13 Nevertheless, conflicting results have been reported about the association between other autoantibodies and their clinical association. 53 Interestingly enough, some patients fulfill the clinical criteria for the diagnosis of SLE but result repeatedly as ANA negative by the screening test used to diagnose SLE [i.e., indirect immunofluorescence technique (IIF)]. This subset is called ANA-negative SLE, and it is characterized by cutaneous features, particularly photosensitivity.14–21 It has been suggested that the lack of ANA could be due to serum dilution, IIF substrate choice, prozone phenomenon (excess of circulating antigens), or low levels of serum immunoglobulins associated with massive proteinuria.18 Rarely, serological markers may also be initially absent but they later become positive.21 Interestingly, ANA-negative SLE patients may show positivity against dsDNA, U1-ribonucleoprotein (U1RNP), SSA, and SSB.14,15,19 In general, positive anti-Sm and anti-dsDNA antibody tests are not consistent with negative tests for ANA. However, Morris et al. have reported a positive test for anti-dsDNA antibody in ANA-negative SLE.15 Moreover, Sugisaki et al. observed antiribosomal P antibodies in ANA-negative SLE.20 Recently two cases of ANA-negative SLE have been reported showing increased proportion of B cells lacking RP105, a member of the toll-like receptor family.17 The authors also described an association of this subset of cells with disease activity in ANA-negative SLE. APS is a disease characterized by arterial and venous thrombosis, recurrent miscarriages, or fetal loss associated with circulating aPL. Diagnosis of APS requires the combination of at least one clinical and one laboratory criterion. Anti-cardiolipin antibodies (aCL) and anti-β2glycoprotein 1 antibodies (aβ2-GP1), measured by standardized ELISA, and the lupus anticoagulant (LA), detected by clotting assays according to the guidelines of the International Society on Thrombosis and Haemostasis, are the recommended tests for the detection of aPL.22 As persistent positivity is required for the diagnosis, these tests should be present in two or more occasions at least 12 weeks apart. 54 Annals of the New York Academy of Sciences TABLE 2. Autoantibodies in Healthy People Latent Autoimmunity Ref. Natural Ab FDR SLE - ANA/aENA FDR SjS - aENA FDR APS - aCL FDR PBS - AMA FDR RA - RF 1 2, 3 3 4 5 6 Preclinical Autoimmunity Ref. ANA in SLE Anti-dsDNA in SLE Anti-ENA in SLE Anti-CCP in RA IgM-RF in RA 9 9 9 7, 8 7, 8 Postclinical Autoimmunity Ref. Anti-HTG antimicrosomial 10 10 Ab, antibody; SLE, systemic lupus erythematosus; ANA, antinuclear antibodies, AENA, anti-ENA; SjS, Sjogren syndrome; APS, antiphospholipid syndrome; aCL, anticardiolipin; PBC, primary biliary cirrhosis; AMA, antimitrochrondrial antibodies; RA, rheumatoid arthritis; RF, rheumatoid factor; anti-dsDNA, anti-double-stranded DNA; anti-CCP, anticyclic citrullinated peptide; anti-HTG, antihuman thyroglobulin; FDR, first-degree relatives. APS could be identified in the setting of other AD, mainly SLE (secondary APS). In addition, patients with APS may have other clinical noncriteria features, including livedo reticularis, thrombocytopenia, cognitive dysfunction, seizures, migraine, valvular heart disease, and nephropathy. In daily clinical practice it is not unusual to find patients with clinical signs suggestive of APS who are persistently negative for the routinely used assays to detect aCL, aβ2GP1, and LA. The classic profile of such patients is represented by a young female with a thrombotic event showing a normal screening for other causes of thrombophilia (i.e., antithrombin III, protein C and protein S deficiency, hyperhomocysteinemia, Factor V Leiden, and prothrombin mutations) and with other clinical features suggestive of APS, such as fetal losses, livedo reticularis, and thrombocytopenia, but without evidence of circulating aPL. Therefore, a seronegative APS (SN-APS) has been recently hypothesized for these patients with clinical features suggestive of APS who are persistently negative for aCL, aβ2GP1, and LA.23 In this situation, the intriguing possibility of undiscovered autoantibodies directed against phospholipids or protein cofactors, which are not detected by conventional assays (hidden aPL), has been suggested.24–26 A number of reports have described the heterogeneity of aPL, including antibodies directed to cofactor proteins (prothrom- bin, protein S, protein C, annexin V, annexin II, oxidized low-density lipoprotein), phospholipid–protein complexes, and anionic phospholipids.22,27 New antigenic targets for aPL in APS have been proposed. In particular, it has been described that antibodies directed to lyso(bis)phosphatidic acid (aLBPA) may represent a marker of APS showing similar sensitivity and specificity compared to anti-β2GP1 antibodies.28 In addition, aLBPA are strongly associated with the presence of LA.28,29 Moreover, antiprothrombin (aPT) have been reported as the only antibodies detected in few patients with SLE and history of thrombosis but persistently negative for aCL or LA (at least three times, 6 weeks apart).30 Therefore, testing for aPT may be helpful in some selected cases. In a multicenter retrospective study antiphosphatidylethanolamine antibodies (aPE) were detected in 15% of 270 patients with thrombosis and mainly found in the absence of the other laboratory criteria of APS, even though the retrospective design of the study did not permit the evaluation of persistence of aPE positivity.31 Recently, new diagnostic tools, such as TLC immunostaining, has been proposed to evaluate aPL reactivity in APS and other AD.32–34 Nevertheless, this technique is still not suitable for screening purposes in SN-APS, and larger prospective studies need to be conducted to assess the clinical relevance 55 Alessandri et al.: Seronegative AD Figure 1. Seronegative spondyloarthropathies and seronegative rheumatoid arthritis (RA).41 as a reference test in patients with suspected APS but persistently negative for conventional aPL. Seronegative RA Classically, RA has been considered an AD since the production of rheumatoid factor (RF) was first observed.35 RF is an autoantibody directed against determinants on the Fc fragment of IgG molecules. It can belong to different isotypes (IgM as well as IgG, IgA). About 80% of patients affected by RA are positive for RF and high serum levels are associated with an aggressive disease, extra-articular manifestations, and a worse outcome.36 In particular, the various isotypes seem to correlate differently with disease progression. The presence of IgA RF has been associated with increased joint damage and disability.37 However, because of the moderate specificity of RF (around 66%), the search for other autoantibodies more specific for the diagnosis of RA has been stimulated.38 Antibodies in RA can be classified as those associated with RA and those specific for RA. The most relevant specific autoantibodies appear to be the anti-CCP. They were first described in 1979 as antikeratin antibodies.39 These autoantibodies show high specificity (96%) and sensitivity comparable with RF. Of interest, they correlate with disease severity and with radiological progression of the disease.40 Moreover, recent studies have demonstrated the presence of both anti-CCP and RF up to 10 years before the onset of synovitis.7,8 The concept of seronegative arthritis arose in 1978 from the observation of a number of diseases that were negative for the RF. They included seronegative spondyloarthropathies and seronegative RA41 (Fig. 1). The first classification of seronegative spondyloarthropathies comprised a group of disorders that have in common the negativity of RF, clinical manifestation, and familial aggregation. This classification originally included ankylosing spondylitis, psoriatic arthritis, reactive arthritis (former Reiter’s syndrome), 56 enteropathic arthropathies, and Behçet’s syndrome.42,43 Seronegative RA may evolve into a classic RA or remain seronegative. In the group of seronegative RA, three main disorders need to be considered for differential diagnosis: elderly onset RA (EORA), polymyalgia rheumatica (PMR), and remitting seronegative symmetrical synovitis with pitting edema (RS3PE). EORA affects male and female humans equally beginning after 60 years of age. The onset is often acute and large proximal joints, such as shoulders, are involved. Systemic manifestations, such as fatigue, weight loss, depression, flu-like syndrome, and high erythrocyte sedimentation rate (ESR), appear more frequently than in RA. RF can be negative in 1–48% of patients, depending on the studies.44–46 This could be attributed to age-related changes in the immune system. However, it has to be remembered that the prevalence of RF in elderly healthy people is increased 10–15% after age 60–65 and 30% by age 90.47 The outcome of EORA is often worse than the younger-onset RA, possibly because of the co-morbidities associated with age. RF is associated with an adverse prognosis, and EORA patients with positive RF display more disease activity, radiological damage, and mortality than seronegative patients.44,48 The differential diagnosis of EORA includes seronegative spondyloarthropathies, crystal-induced arthritis, osteoarthritis, malignancies, and paraneoplastic arthritis. EORA may be difficult to distinguish with PMR and RS3PE because they can share some clinical and serological features. The correct diagnosis is important because of the different therapeutic strategy and prognosis. A useful serological biomarker of disease may be represented by the presence of antiCCP antibodies. In fact, the prevalence of antiCCP antibodies in EORA is 65%, and they are not detected in PMR. They have a sensitivity of 56% and specificity of 92% with a negative and positive predictive value of 63% and 90%, respectively. Anti-CCP could allow the identification of EORA with a polymyalgic on- Annals of the New York Academy of Sciences set. Moreover, two studies, although in a small number of patients, revealed that some EORA patients (30–40%) were negative for RF and positive for anti-CCP antibodies, confirming the higher specificity of anti-CCP compared with RF.49,50 However, PMR and EORA may overlap. RS3PE syndrome was described in 1985 as a seronegative RA.51 It occurs especially in men older than 60 and it is characterized by symmetrical synovitis usually localized in the peripheral joints, tenosynovitits, and pitting edema of upper or lower limbs. Constitutional symptoms are not frequent. Laboratory findings demonstrate high ESR and negative RF. Patients usually respond to steroid treatment, and the prognosis is good. The remission usually occurs within 18 months, although the disease can evolve into RA, spondyloarthropathy, connective tissue disease, or vasculitis.52 Seronegative arthritis at the onset may also represent an undifferentiated arthritis. Synovitis of recent onset represents a challenge for an early diagnosis, estimating a prognosis, and commencing a treatment in the so-called window of opportunity. Some patients meet the American College of Rheumatology (ACR) criteria at onset of the disease for the diagnosis of RA. However, about 25% of patients do not fulfill the criteria and they are identified as undifferentiated arthritis. This subset of patients may evolve toward a defined diagnosis, remain undifferentiated or go into spontaneous remission. The pathogenic mechanisms that underlie the development of the synovitis may differ according to the genetic background and the environmental factors that may precipitate the disease. In this context the presence of autoantibodies, such as RF and anti-CCP, together with the immune reactions of synovitis may drive the fate of disease progression.53 In fact, recent studies aimed to establish a scoring system to assess the outcome of early undifferentiated arthritis. The presence of RF or anti-CCP antibodies appears to be one of the criteria to predict the outcome.54,55 Alessandri et al.: Seronegative AD In conclusion, the diagnosis of seronegative AD is a difficult task because of their unclear clinical presentation and atypical features. Moreover, autoantibodies may be initially absent but later become positive, and we need to keep in mind the intriguing possibility of autoantibodies directed against unknown antigens, which are not detected by conventional assays. Nevertheless, in daily clinical practice it is not unusual to find patients with clinical signs suggestive of AD who are persistently negative for autoantibodies. The diagnosis and management of seronegative AD may represent a practical problem because they are often difficult cases. Finally, because of the length of this chapter, we are unable to summarize all of the cuttingedge issues that surround this research. For this reason, we refer to the recent literature on this subject.56–60 Acknowledgments This work was supported by Fondazione Umberto Di Mario ONLUS per il Progresso Delle Scienze Biomediche. Conflicts of Interest The authors declare no conflicts of interest. References 1. Tomer, Y. & Y. Shoenfeld. 1988. The significance of natural autoantibodies. Immunol. Invest. 17: 389–424. 2. Miles, S. & D. Isenberg. 1993. A review of serological abnormalities in relatives of SLE patients. Lupus 2: 145–150. 3. Arnett, F.C., R.G. Hamilton, J.D. Reveille, et al. 1989. 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