Rheumatology Advance Access originally published online on August 25, 2006
Rheumatology 2007 46(3):479-483; doi:10.1093/rheumatology/kel270
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Clinical and immunological characteristics of patients with Sjögren's syndrome in relation to 
-fodrin antibodies
Department of Medicine B, 1Institute of Clinical Chemistry and Laboratory Medicine, Muenster University Hospital, D-48129 Muenster, Germany.
Correspondence to: P. Willeke, MD, Department of Medicine B, Muenster University Hospital, Albert Schweitzer Str. 33, D-48129 Muenster, Germany. E-mail: willeke{at}mednet.uni-muenster.de
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Abstract |
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Objectives. To analyse the prevalence of
-fodrin antibodies in patients with primary (pSS) and secondary Sjögren's syndrome (sSS) and the relation to clinical, serological and immunological features.
Methods. Serum IgA and IgG antibodies to the 120 kDa -fodrin were determined by ELISA technique in 62 pSS patients and 28 sSS patients. Results were correlated with clinical symptoms and laboratory findings as well as with the HLA-DR genotype. Additionally, antibody concentrations were correlated with the numbers of peripheral blood mononuclear cells (PBMCs) secreting interleukin (IL)-6, IL-10, interferon-
(INF)-, and tumour necrosis factor- determined by ELISPOT analysis. Lymphocytes and monocytes were examined flow-cytometrically for the expression of activation markers. Healthy age- and sex-matched volunteers served as controls.
Results. The sensitivity of IgA and IgG -fodrin antibodies was 35 and 31%, respectively, in pSS patients. In sSS patients, the sensitivity was 29 and 21%, respectively. In pSS patients with IgG antibodies, recurrent parotid swelling was significantly more prevalent. Also the number of INF- secreting PBMCs and the percentage of CD4/CD71+ lymphocytes as well as CD14/HLA-DR+ monocytes were significantly increased in this group compared with -fodrin-negative patients or with controls. Interestingly, these patients also had a shorter disease duration. No association of -fodrin antibodies with the HLA-DR genotype was found.
Conclusion. Due to the low prevalence, serum antibodies to -fodrin turned out to be of limited diagnostic value in our study. However, our data suggest that IgG antibodies to -fodrin are indicative of clinical and immunological activity in pSS especially in patients with shorter disease duration and may thus serve as a marker of disease activity.
KEY WORDS: -Fodrin, Sjögren's syndrome, Interferon-, ELISPOT, CD71, HLA-DR, Disease duration
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Introduction |
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Sjögren's syndrome (SS) is an autoimmune disorder characterized by chronic inflammation of the exocrine glands leading to keratoconjunctivitis sicca and xerostomia and other systemic manifestations. It can occur as primary SS (pSS) or in combination with other autoimmune diseases as secondary SS (sSS). Focal lymphocytic gland infiltration with up-regulated T-helper type I (Th1) cytokine expression, hypergammaglobulinaemia and the production of different autoantibodies are hallmarks of the disease.
In 1997, Haneji et al. [1] identified autoantibodies in a 120 kDa cleavage product of -fodrin as a specific and sensitive serological marker in patients with pSS. This cleavage product is formed during apoptosis in the inflamed salivary gland tissue and represents an organ-specific autoantigen that may be responsible for the development of autoimmune lesions and the perpetuation of tissue destruction [1, 2]. Moreover, a specific T-cell response against 120 kDa -fodrin with up-regulation of Th1 cytokines has been reported in animal models and pSS patients [1, 3].
Recent studies have raised doubts about the diagnostic value of these antibodies due to a lack of sensitivity and specificity [4–6]. However, with respect to several findings suggesting a pathogenic role of -fodrin in the development of SS [7, 8], we wondered whether there might be distinctive clinical, serological and immunological features in antibody-positive vs antibody-negative SS patients.
As it is well known that the production of autoantibodies is partly determined by an immunogenetic predisposition, we further analysed a potential association of anti--fodrin autoantibodies with distinct HLA-DR genotypes.
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Patients and methods |
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Patients and healthy controls
Sixty-two patients with pSS and 28 patients with sSS were included in this study. Patient characteristics and laboratory findings are given in Table 1. The diagnosis of pSS was based on the American–European consensus criteria [9]. The diagnosis of rheumatoid arthritis, systemic lupus erythematosus, systemic sclerosis and Sharp-syndrome was based on the classification criteria proposed by the American College of Rheumatology and Sharp et al. [10–13], respectively. None of the participating pSS patients received immunosuppressants while sSS patients received 2.5–7.5 mg prednisolone per day as well as different DMARDs (hydroxychloroquine n = 9, azathioprine n = 3 methotrexate n = 10 and leflunomide n = 3).
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Twenty-two age- and sex-matched healthy volunteers served as controls for ELISPOT and flow-cytometric assay. For the analysis of HLA-DR association, 158 healthy sex-matched Caucasians served as controls. The study protocol was approved by the local independent ethics committee. Patients and controls gave informed consent.
Determination of -fodrin antibodies and other laboratory parameters
IgA and IgG antibodies of the 120 kDa -fodrin molecule were determined by ELISA technique (Orgentec, Germany) according to the manufacturer's instruction.
Routine laboratory parameters (i.e. erthrocyte sedimentation rate, C-reactive protein, white and red blood cell count, serum creatinine and serum-electrophoresis) were determined simultaneously. RF-isotypes as well as the levels of IgG antibodies to Ro and La were analysed by ELISA (Pharmacia-Upjohn, Germany). Further, we performed the Waaler–Rose test and the latex–fixation test for RF (Dade-Behring, Germany). Serum concentrations of IgG, IgA, IgM as well as complement levels (C3c and C4) were measured by nephelometry (Dade-Behring, Germany).
ELISPOT analysis and flow cytometry
Cell isolation and culture as well as ELISPOT analysis of interleukin (IL)-6, IL-10, tumour necrosis factor- (TNF-) and interferon- (INF-) (Endogen, Boston, MA, USA) were performed as described earlier [14].
Flow-cytometric determination of activated CD4+ T-cells (CD4/CD25, CD4/CD45RO, CD4/CD69 and CD4/CD71) and activated CD14+ monocytes (CD14/HLA-DR) was performed as described earlier [15].
HLA-DR typing
Generic HLA-DR typing was performed by using an enzyme-linked probe hybridization assay (ELPHA; Biotest, Germany) according to the manufacturer's instruction.
Statistical analysis
Data were analysed using the statistic software package SPSS 12.0. Non-parametric tests were used for statistical analysis since a normal distribution of values could not be assumed. Spearman correlation test was used to correlate laboratory results and clinical data. Chi-square and Fisher's exact tests were applied to analyse qualitative variables. The P-values obtained in multiple comparisons were adjusted using Bonferroni correction. A P-value of <0.05 was considered as significant.
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Results |
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TopAbstractIntroductionPatients and methodsResultsDiscussionReferences |
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Prevalence of
-fodrin antibodiesIn pSS as well as in sSS patients, the prevalence of anti-
-fodrin antibodies was relatively low (Table 1). The sensitivity of IgA and IgG antibodies to -fodrin was 35 and 31%, respectively, in pSS patients. In sSS patients the sensitivity for IgA and IgG antibodies was 29 and 21%, respectively. Any one of the antibody isotypes was detected in 30 pSS patients (48%) and 12 sSS patients (43%).
Association of -fodrin antibodies in pSS and sSS patients with clinical features and laboratory findings
Out of 62 pSS patients, 23 had recurrent parotid swelling. In these patients, we found an increased prevalence of IgG antibodies to -fodrin (n = 13) compared with antibody-negative patients (P < 0.05, data not shown). In addition, pSS patients with IgG antibodies to -fodrin had a shorter disease duration. The disease duration inversely correlated with -fodrin antibody titers in this group (R = –0.384; P < 0.01).
For other glandular or extraglandular manifestations, no differences between the groups were observed. There were no associations of anti-Ro, anti-La or RF isotypes with -fodrin autoantibodies.
In patients with sSS, no association of -fodrin antibodies with clinical or laboratory features could be observed.
Frequency of cytokine secreting PBMCs
In the subgroup of pSS patients with IgG antibodies to -fodrin, the number of INF--secreting peripheral blood mononuclear cells (PBMCs) was significantly increased compared with -fodrin negative patients or with controls (P < 0.01; Fig. 1). The number of INF- secreting cells correlated with IgG antibody titres (R = 0.46; P < 0.01). In contrast, there were no significant differences in IL-6-, IL-10- and TNF--secreting PBMCs between the different subgroups.
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There were no significant differences in the frequency of cytokine-secreting PBMCs between pSS and sSS patients or between patients and controls (data not shown).
Expression of activation markers on lymphocyte subsets and monocytes
The percentage of CD4/CD71+ T-cells was significantly increased in pSS patients with IgG antibodies to -fodrin compared with pSS patients without these antibodies (P < 0.05) and compared with controls (P < 0.01, data not shown). In the antibody positive group, even the percentage of CD14/HLA-DR+ monocytes was increased compared with controls (P < 0.05, data not shown).
There were no significant differences in the expression of other activation markers (CD4/CD25, CD4/CD45RO and CD4/CD69) in pSS or sSS patients with and without antibodies of either isotype against -fodrin.
HLA-DR associations
The prevalence of HLA-DR3 was increased in pSS patients compared with healthy controls (P < 0.01) while in sSS patients, no differences in the HLA-DR distributions were found compared with controls.
Due to the low prevalence of Ro-antibody-negative patients in our cohort, it was not possible to test for an association of anti-Ro antibodies and the HLA-DR alleles. The presence of -fodrin autoantibodies in either patient group was not associated with HLA-DR alleles.
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Discussion |
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TopAbstractIntroductionPatients and methodsResultsDiscussionReferences |
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Our data clearly demonstrate differential clinical and immunological characteristics in primary SS patients with and without antibodies to
-fodrin.
However, the diagnostic sensitivity of -fodrin antibodies in pSS and sSS was rather low in our study which confirms recent data [4–6]. Earlier studies have reported higher antibody prevalences [2, 16]. These differences might be explained, at least in part, by differences in the test methods applied [6]. In addition, genetic and environmental factors might contribute to the observed discrepancies. In this regard, a lower prevalence of -fodrin antibodies has been reported in American pSS patients compared with Japanese pSS patients [7]. It has been suggested that immunosuppressive therapy can down-regulate the production of -fodrin antibodies [2]. In our study, none of the pSS patients received immunosuppressants. Thus, the low sensitivity in pSS patients cannot be explained by treatment effects.
In pSS patients with IgG-antibodies to -fodrin, an increased prevalence of recurrent parotid swelling was observed. As previously reported, recurrent parotid swelling is associated with a high focus score of lymphocytic infiltration in labial salivary glands of pSS patients [17]. Witte et al. [18] reported a positive correlation of -fodrin antibody serum concentrations and the degree of lymphocytic infiltration in salivary glands. Thus, -fodrin antibodies are associated with clinical characteristics of the disease in pSS patients.
-Fodrin has been shown to be directly involved in secretory events of the parotid acinar cells [19]. The cleavage of -fodrin induces a strong antibody response to the 120 kDa -fodrin cleavage product [7]. These antibodies do not react with the intact molecule, indicating that the epitope recognized is exposed after -fodrin cleavage [7].
Currently, no available data demonstrate a direct pathogenic role of autoantibodies against -fodrin. However, in animal models as well as in SS patients, a clear correlation between the autoantibody production and inflammatory infiltration in the salivary glands has been reported [3, 20]. Thus, it may well be that the presence of -fodrin antibodies reflects crucial underlying immunopathological processes initiated by apoptosis-driven exposition of -fodrin to antigen-specific CD4+ T-lymphocytes.
The notion that -fodrin antibodies may participate in early pathogenic processes is supported by our finding that pSS patients with IgG-antibodies to -fodrin had a shorter disease duration. In addition, antibodies in the sera of children with pSS or sSS were observed even before anti-Ro or anti-La antibodies became positive [21]. Further, it has been shown that -fodrin can induce significantly higher proliferative responses of PBMCs in patients with short disease duration compared with those with longer disease duration [8].
We found an increased number of INF--secreting PBMCs in pSS patients with IgG antibodies to -fodrin. The number of INF--secreting PBMCs even correlated with the antibody concentrations. INF- is a major cytokine that is present in the salivary glands of pSS patients [22]. Interestingly, the recombinant 120 kDa -fodrin has been shown to induce the production of INF- in PBMCs from pSS patients [1, 8]. Experimental data suggest that INF- is an essential element in initiating a cascade of events that lead to lymphocytic infiltration and acinar cell loss in SS [23, 24]. INF- knockout mice do not exhibit increased acinar cell apoptosis or leucocytic salivary gland infiltration [23]. INF- has been shown to induce two chemokines (IP-10 and Mig) in the salivary glands of patients with pSS, which play an important role in the accumulation of activated T-cells in the affected organ [24]. Recently, a novel IP-10 antagonist has been shown to ameliorate the progression of autoimmune sialadenitis in MRL/lpr mice [25].
Another important finding of our study was an increased percentage of CD71 positive T-helper cells and HLA-DR positive monocytes in pSS patients with IgG antibodies to -fodrin. This again demonstrated that the presence of -fodrin antibodies in the serum of pSS patients reflects the systemic activation of major immune effector cells. The transferrin receptor CD71 is important for the iron uptake of proliferating lymphocytes and monocytes. Its expression is low on resting lymphocytes and greatly up-regulated in activated T-cells [26]. An increased subset of CD71 positive T-cells in the periductal foci of salivary glands and increased CD71-positive PBMCs have been reported in pSS patients [27, 28]. Monocytes expressing surface HLA-DR are crucial for effective antigen presentation to immune effector cells. INF- has been shown to induce the expression of HLA-DR on monocytes as well as salivary epithelial cells of pSS patients [29, 30]. Thus, the increased percentage of HLA-DR-positive monocytes is most probably a consequence of increased INF- secretion in this group.
We found an increased prevalence of HLA-DR3 in pSS patients in our cohort confirming previous findings [31]. Antibodies against Ro and La have been shown to be associated with HLA-DR3, possibly due to HLA haplotype-dependent differences in presentation of autoantigens and subsequent stimulation of the immune response [31]. For -fodrin antibodies, no such associations have been found in our study suggesting that HLA-DR polymorphism does not play a major role in the generation of anti--fodrin autoantibodies.
We conclude that -fodrin antibodies, due to their relatively low prevalence, do not aid much in the diagnosis of SS. However, pSS patients with IgG antibodies to -fodrin exhibit characteristic clinical and immunological features indicative of local and systemic inflammation. Thus, these antibodies may preferentially serve as markers of disease activity. In addition, our finding of an increased prevalence of anti--fodrin antibodies of the IgG-isotype in pSS patients with shorter disease duration suggests that these antibodies may participate in early pathogenic processes. Since these findings are restricted to patients with primary SS, it can be hypothesized that there are different immunological pathways involved in primary and secondary SS. This difference should deserve further elucidation.
The authors have declared no conflict of interest.
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