Fig. 15.1
Illustration demonstrating principle of autoantibody identification by indirect immunofluorescence
Fig. 15.2
Examples of IIF staining patterns that can be found in patients with RP secondary to autoimmune rheumatic diseases.(a) Speckled–nucleolar sparing is the most common and least specific pattern for rheumatic disease, associated with SS-A/Ro, SS-B/La, Sm, and U1RNP and thus occurs in MCTD, PSS, and SLE. (b) Centromere (ACA) pattern is identified by a characteristic speckled pattern with each speckle representing an antibody targeting centromere proteins. The pattern reflects the phase of cell division; for example central to the photo is a linear pattern in the center of the cell as chromosomes align during metaphase. (c) Homogeneous describes an even distribution of fluorescence throughout the cell nucleus. It is associated with SLE and drug-induced lupus-specific autoantibodies including dsDNA, anti-nucleosome, and anti-histone antibodies. (d) Nucleolar is most often seen in association with systemic sclerosis reflecting the presence of PM-Scl, U3RNP, Th/To, RNA polymerase I, Ku, Nor 90, or anti-Scl 70. (e) Fine cytoplasmic speckle may be missed on conventional ANA testing and occurs in the presence of Jo-1, ribosomal RNP, and anti-mitochondrial antibodies. (f) Anti–neutrophil cytoplasmic antibodies (ANCA) are autoantibodies that target antigens within neutrophils and as such human neutrophils are the substrate used for autoantibody detection rather than HEp2 cells. ANCA staining can be reported as cytoplasmic (demonstrated above) or perinuclear associated with PR3 or MPO, respectively. A perinuclear pattern may be hidden by strongly positive ANA staining. In the presence of the latter, ELISA may be required to further characterize potential pANCA
A positive ANA can be found in as many as 25–30 % of the healthy population, but often at a low titer of 1:40 [6, 7] and as a nonspecific ANA. The prevalence increases with age and in the presence of intercurrent infection or malignancy. The cutoff titer for reporting positive ANA should therefore ideally be chosen on a local level according to the population and it is the responsibility of the requesting clinician to interpret low-titer, nonspecific results within the correct clinical context.
Many autoantibodies associated with RP typically exist at high titer; for example the median titer of anticentromere antibody (ACA) in systemic sclerosis (SSc) in one study was 1:5,120 [8]. However, ACA can exist at lower titers, and therefore IIF staining patterns at lower dilutions need adequate reporting. It should be noted that other specific autoantibodies may occur in lower titer; for example anti-scleroderma 70 (anti-Scl 70) autoantibodies in SSc and a low-titer ANA should not therefore be automatically dismissed. It is the presence of specific autoantibodies rather than the absolute titer that is important in disease classification although we shall later discuss possible associations between autoantibody titer and disease activity in RP-associated diseases.
IIF cannot confirm the antigenic target of autoantibodies to extractable nuclear antigens (ENA, e.g., Ku, Jo-1, SS-A/Ro60, Ro52, SS-B/La, RNA polymerases, and PM/Scl,) although certain IIF patterns such as anticentromere antibody (ACA) can be regarded as diagnostic.
Enzyme-Linked Immunosorbent Assay
ELISA is used to confirm the antigen target and allows quantification of autoantibody levels (e.g., double-stranded DNA [dsDNA] titer) once they have been identified, or can be used for specific antibody detection (e.g., anti-β2 glycoprotein-I [β2-GP-I] and anti-cardiolipin [aCL]). Its method is not dissimilar to IIF but in the place of FITC, the secondary antibody is conjugated with an enzyme. An enzyme-specific substrate is then added which changes color on binding [9]. Spectroscopy is used to quantify the strength of the color change allowing estimation of quantification of autoantibody concentration from a standard curve generated with known concentrations of antigen. Up to 35 % of clinically relevant ANA results may be reported as falsely negative [10–13] and disease-specific autoantibodies may also be missed due to failure of the relevant autoantigen to be included in the assay system. Therefore a negative autoantibody by ELISA may be unduly reassuring.
Immunoblotting
This is a semiquantitative technique where antigens are separated by electrophoresis on a polyacrylamide gel according to their molecular weight and transferred to a nitrocellulose strip. An adaption more widely used is a line blot where commercially derived autoantigens are individually placed at specific positions on a membrane strip. Human serum to be tested is applied to the strip and any autoantibodies present in the sera bind to the autoantigens at their set positions. Any bound autoantibodies are then detected by a secondary anti-human antibody conjugate [14].
Immunoprecipitation
Immunoprecipitation (IP) is a sensitive method of identifying autoantibodies and has the advantage of identifying autoantibodies that are otherwise difficult to detect using routine methods. It requires use of radioactive material, is more costly, and thus is more commonly used for research purposes [15]. IP uses radiolabelled autoantigens mixed with human sera, separation by polyacrylamide gel electrophoresis according to molecular weight, and detection of autoantigen by autoradiography (Fig. 15.3).
Fig. 15.3
An autoradiograph illustrating a selection of autoantibodies, including the main SSc-specific autoantibodies (anti-Scl 70, RNA polymerases, Th/To, PM/Scl, U3RNP, Ku) detected by immunoprecipitation. These may be found where RP is associated with autoimmune rheumatic disease. Arrows identify the position of the bands corresponding to the respective radiolabelled autoantigen-autoantibody complex that have separated according to their molecular weight (electrophoresis)
Autoantibodies and Raynaud’s Phenomenon Associated with Autoimmune Rheumatic Disease
RP is common in ARD and these diseases are often associated with specific autoantibodies (Table 15.1) [16–19]. Autoantibody testing can aid the early identification of those patients with or at risk of developing future ARD. In the absence of other clinical features of ARD, the presence of RP in conjunction with autoantibodies is sometimes termed autoimmune RP. The presence of autoantibodies increases the likelihood of developing ARD. Patients with RP referred to one secondary unit had an overall risk of 14 % of developing an associated ARD [20]. The risk increased to 30 % if the ANA was positive (i.e., autoimmune RP) [20] and fell to 7 % if the ANA was negative [21]. A meta-analysis of ten articles reviewing a total of 639 patients demonstrated that 1/3 of patients initially classified as autoimmune RP differentiate to SSc within 5 years of presentation [22].
Table 15.1
The prevalence of Raynaud’s phenomenon and autoantibody positivity in autoimmune rheumatic disease
Prevalence of RP (%) | Prevalence of autoantibodies (%) | |
|---|---|---|
Systemic sclerosis | >95 | >90 |
SLE | 10–44 | >95 |
Polymyositis/dermatomyositis | 25–29 | 80–90 |
Primary Sjogren’s syndrome | 13–33 | 50–80 |
Mixed connective tissue disease | 85–94.5 | 99 |
Rheumatoid arthritis | 10–15 | 30–50 |
Systemic Sclerosis
RP occurs in more than 95 % of patients with SSc and is often the earliest manifestation of the disease [23]. The initial observation of antibodies directed to the nucleus and nucleolus in the sera of patients with SSc provided both evidence of autoimmunity and an early diagnostic marker in SSc [24, 25]. In their landmark paper of 1968, Rothfield and Rodnan identified the presence of ANA in 60 % of patients with SSc [26]. By the late 1980s, novel cellular substrates and refined serological techniques had increased the prevalence of ANAs in the Pittsburgh cohort to 76 % [27]. More recent work has reported the prevalence of ANAs in SSc in more than 90 % of cases [28, 29]. There remain a small proportion of patients with SSc (~5–10 %) who are negative for ANAs using standard techniques of identification such as IIF, immunoblotting, and ELISA. Novel mutually exclusive SSc-specific autoantibodies continue to be identified in SSc and there are likely to be other, as yet, unidentified autoantibodies in the small percentage of otherwise ANA-negative SSc [30].
The major SSc-specific autoantibodies are ACA, anti-Scl70, anti-Th/To, anti-RNA polymerase (anti-RNA Pol), and anti-fibrillarin (U3RNP). Additional rarer SSc-specific autoantibodies such as anti-U11/U12-RNP and anti-Nor-90 have also been identified [31, 32]. The major SSc-specific autoantibodies are typically mutually exclusive and each antibody predicts a well-characterized clinical phenotype [28].
The presence of SSc-specific autoantibodies (and/or abnormal nailfold capillaries) provides important prognostic information for the progression to definite SSc. Indeed, such is the importance of autoantibodies that they receive weighting in the 2013 ACR/EULAR classification criteria for SSc [33] and have been included for previous classification criteria for “early” and “very early” SSc in the absence of skin or internal organ manifestations of SSc [34–36]. Patients with RP in conjunction with positive anti-Scl 70 or ACA have a 63-fold increased risk of progression to ARD including SSc compared to those who are ANA negative (p = 0.000009) [37]. In this small prospective study of patients with RP, ten developed features of an evolving ARD over a mean follow-up of 4 years [37]. All ten patients had either ACA or anti-Scl 70-positive sera at baseline (100 %) compared to 13 (25 %) of the remaining 53 clinically stable patients [37]. In a large secondary care cohort, ANA positivity gave a hazard ratio of 9.70 (2.11, 44.48) (p = 0.003) for progression to SSc compared to ANA-negative RP [38]. The presence of ACA further increased the risk by a hazard ratio of 3.94 (1.74, 8.94) (p = 0.001) compared to ANA positivity alone [38]. In a 20-year prospective study [39] of a secondary care cohort of patients with isolated RP and no clinical features of SSc, 11.5 % carried SSc-specific autoantibodies in the presence of normal nailfold capillaries. Of these, 35.4 % progressed to develop definite SSc compared to only 3.4 % of those with RP and a positive (but nonspecific) ANA [39]. None of the primary RP group (i.e., negative ANA and normal nailfold capillaries) progressed to develop SSc (p < 0.0001) [39]. The presence of both abnormal nailfold capillaries in addition to SSc-specific autoantibodies improved the predictive value of progression to definite SSc and collectively provided an odds ratio of 50 (p < 0.0001), a positive predictive value of 79 %, and negative predictive value of 93 %. In this study, the presence of SSc-specific autoantibodies provided an eightfold increased risk of developing SSc and the median time for progression was 4.6 years from RP onset [39].
Although the reported risk of progression seems to vary across the literature, it is clear that the presence of specific autoantibodies significantly increases the risk of progression to SSc. Conversely, less than 0.2 % of primary RP (negative ANA and normal nailfold capillaries at baseline) develop an ARD [40]. Only 0.5 % of patients with primary RP develop new autoantibodies during follow-up [40]. Thus if both autoantibodies and nailfold capillaries are persistently normal on two occasions the likelihood of RP progressing to an SSc is minimal [40].
RP is a clinical feature across all of the SSc antibody specificities although certain antibodies are associated with a more pronounced peripheral micro-vasculopathy and the formation of digital ischaemic lesions such as digital ulcers (DU) [7, 41–46]. Anti-Scl 70, anti-RNA polymerase III, ACA, U1RNP, and U3RNP are all independently associated with a higher risk of severe RP with DU and digital necrosis in SSc [41]. Anti-Scl 70 is associated with a higher frequency of severe RP (57 % of anti-Scl 70-positive SSc develop digital ulceration compared to 32.2 % of anti-Scl 70 negative) [41]. Anti-RNA polymerase III and anti-Scl 70 are associated with younger age at onset of RP and a shorter duration from RP onset to the first episode of DU than ACA. DU occurs up to 5 years earlier from the time of RP onset in anti-Scl 70-positive compared with ACA-positive SSc [41, 47]. Despite this, patients with ACA are more likely to require surgical digital amputation (14.6 % ACA positive compared to 7.9 % anti-Scl 70) [41]. The titer of ACA may also be relevant and has a positive association with severe RP [8].
The frequency of antiphospholipid antibodies (aCL and β2-GP-I) in SSc has been reported to be as high as 20–41 % [48]. The presence of antiphospholipid antibodies is associated with more pronounced peripheral microvascular dysfunction in SSc, reflected as both abnormal nailfold capillaries and the presence of digital pitting [48].
Autoantibodies associated with severe peripheral vascular complications in SSc (e.g., ACA, U3RNP, APLA particularly β2-GP-I) are also associated with a higher prevalence of other vascular complications in SSc including pulmonary arterial hypertension (PAH) [49–52].
PMScl and U1RNP can be found in SSc or as part of an overlap syndrome (with myositis and/or SLE). Both are typically associated with symptoms of RP, particularly when the clinical phenotype closely resembles SSc (discussed later).
Mixed Connective Tissue Disease
Anti-RNP autoantibodies (in particular U1RNP) are the hallmark of MCTD and historical diagnostic criteria included their presence as a major criterion [53]. Patients often display characteristics of several ARDs that feature in this overlap syndrome (SSc, SLE, myositis, and RA). In a review of 91 patients with MCTD, RP was the most common feature occurring in 94.5 % of patients [54].
Myositis
RP is present in about 25 % of patients with idiopathic inflammatory myositis, which is less common than in either SSc or MCTD [19] (Table 15.1). The frequency of RP increases to 38 % in association with Jo-1 [55] or the anti-synthetase syndromes. In contrast, patients with myositis with anti-signal recognition particle autoantibodies (SRP) tend to have fewer extra-muscular features including RP [56]. In this small study of 12 patients only 1 had RP in association with SRP [56].
Systemic Lupus Erythematosus
RP occurs in approximately half (40–49 %) of SLE patients [57, 58]. Whilst SLE-specific autoantibodies are associated with different systemic manifestations (e.g., the relationship between anti-dsDNA and renal disease) there does not seem to be an association of SLE-specific autoantibodies with either the frequency or severity of RP. Approximately 5.6 % of SLE patients carry ACA, and these patients have a high prevalence of RP symptoms (75 % ACA-positive patients versus 33 % ACA negative) [59].
Primary Sjogren’s Syndrome
RP occurs in 13–30 % of primary Sjogren’s syndrome (PSS). In a study of 320 patients with PSS, RP preceded the onset of sicca symptoms in 45 % of cases [62]. The identification of ANA, anti-/Ro, and anti-La is more common when RP was present [62]. Similar to in SLE, the presence of ACA in PSS is associated with a higher prevalence of RP symptoms (75 versus 18 %) [63] and RP typically represents a more prominent symptom [63–66]. In prospective studies, a quarter (23 %) of ACA-positive patients initially classified as PSS progress to SSc [63–65, 67, 68]. ANCA are not commonly identified in PSS; however, one cross-sectional study identified RP symptoms in 44 % of ANCA-positive PSS compared to only 8 % of ANCA-negative PSS (p = 0.01) [69].
Vasculitis
There are no reported associations between specific autoantibodies and the prevalence or severity of RP occurring in vasculitis.
Autoantibodies in the Pathogenesis of Raynaud’s
There is some evidence that autoantibodies may not just be markers of rheumatic disease but may contribute to pathogenesis. Autoantibodies may contribute to tissue damage in systemic autoimmune rheumatic disease through complement activation [70], inducing opsonization and stimulation or inhibition of cellular receptors [71]. Endothelial injury and dysfunction are thought to contribute to the pathogenesis of RP, particularly in SSc [72]. Autoantibodies such as ACA and anti-Scl 70 are highly specific markers in SSc but there is no compelling evidence to suggest that they have a role in pathogenesis [44].
The identification of anti-endothelial cell antibodies (AECA) provides possible evidence for a direct pathogenic role of antibodies in vascular dysfunction in SSc. AECA have been identified in up to 86 % of patients with SSc and are associated with increased frequency of RP with digital ulceration [73–75]. The titer of AECA is also positively associated with an increased incidence of severe RP (p < 0.01) [74], which suggests a pathogenic relationship. AECA titer is similarly associated with occurrence of other vascular complications including PAH (p < 0.001) [74] and vascular damage on nailfold capillaroscopy [73, 76]. Endothelial dysfunction with resultant increase in endothelial cell markers and adhesion molecules is considered a core pathogenic phenomenon in SSc [77–82]. A dose-dependent increase in cell adhesion molecules is seen in vitro when healthy human endothelial cells are exposed to AECA in SSc serum [83] suggesting that AECA are functional antibodies. AECA have been shown to activate antibody-dependent cell-mediated cytotoxicity and endothelial cell apoptosis, which is thought to encourage fibroblast differentiation and thus collagen deposition [84, 85]. AECA are also thought to further activate coagulation pathways exacerbating the vascular insult in SSc [86]. The identification of AECA is limited as they are not readily detectable using routine methods of autoantibody detection.
AECA have been identified in other ARDs including MCTD and are associated with prominent vascular symptoms including RP [87]. Studies have reported a higher prevalence of AECA in MCTD than in SSc (77 % MCTD versus 36 % SSc) [88, 89]. Increased levels of AECA have been found in patients with MCTD deemed to have “active” versus “inactive” disease [88]. Sera from patients with higher AECA levels induce over-expression of endothelial E-selectin [88].
Higher levels of AECA have been detected in PSS with RP compared to without RP (p < 0.01) [90]. Similarly in a study of AECA in SLE, higher AECA levels were detected in those with RP than those without; however, results did not reach significance [91].
The identification of antibodies directed towards platelet-derived growth factor receptor (anti-PDGFR) in SSc has generated significant interest and may be relevant to RP pathogenesis because of the potential ability of anti-PDGFR to generate reactive oxygen species [92]. The high reported prevalence of anti-PDGFR antibodies has been limited to a few studies and has yet to be widely replicated [92–94]. Nonetheless, such studies raise the possibility of the presence of additional autoantibodies in ARD, which may contribute to RP but which existing cellular substrates and methods of antibody detection have yet to identify.
The presence of ACL antibodies in SSc may indirectly contribute to the pathogenesis of RP by inducing platelet activation, stimulation of endothelial cells, and inhibition of protein C activation resulting in micro-thrombosis [95].
The identification and confirmation of pathogenic autoantibodies in RP would be of significant clinical value with potential for targeted therapies that may reduce the morbidity of both sequelae of severe RP such as digital ulceration and other vascular complications of ARD such as PAH. However, the timeline for such work to migrate from bench side to bedside is lengthy and unlikely to be of clinical value in the near future.
Conclusion
Autoantibodies are important tools in the classification of RP, and help predict the risk of progression to a defined ARD. Autoantibodies such as ACA are associated with RP irrespective of the major clinical phenotype (e.g., SLE or PSS) and future work elucidating the exact mechanisms leading to expression of such autoantibodies may provide novel treatment strategies for vascular dysfunction in ARD. The identification of autoantibodies with a potential direct pathogenic role is an exciting development and offers fascinating insight into the possible associations between autoimmunity and vascular disease in RP.
Expert Opinion
Autoantibody investigations should be used as part of an initial clinical assessment of RP where there are additional clinical features that raise suspicion of an associated ARD (Fig. 15.4). Where RP occurs in conjunction with high-titer ANA or disease-specific autoantibodies at any titer, but without a definite diagnosis of ARD, patients should be monitored for disease progression and to identify systemic ARD involvement at an early stage. Screening for subclinical systemic involvement of ARD can be tailored depending on autoantibody specificity and their associated clinical manifestations. Monitoring should include a clinical assessment, urine dipstick testing, blood pressure, and consideration of echocardiography and pulmonary function tests where indicated. If baseline autoantibody investigations are negative but clinical suspicion of ARD remains high then repeat autoantibody testing may be warranted especially if new or worsening clinical features occur.
