**2.1. Antinuclear antibodies**

**1.1. Triggering factors**

120 Autoantibodies and Cytokines

**1.2. Outline of pathogenesis**

In the pathogenesis of primary Sjögren's syndrome, certain genetic factors play a significant role, such as the presence of HLA-B8, HLA-DW3, HLA-DR3, and DRw52 genes or interferon regulatory factor 5 (IRF 5) gene polymorphism [1, 2]. Not only viral infections have been recognized as the pSS triggering factors, mainly Epstein-Barr virus (EBV) [3], but also human T-cell lymphotropic virus type-1 (HTLV-1), cytomegalovirus (CMV), and hepatitis C virus (HCV) [4, 5]. Bacterial infections *Staphylococcus aureus*, *Chlamydia pneumoniae*, *Chlamydia trachomatis*, *M. hominis*, *U. urealyticum*, and *H. pylori* may also play part in the development of pSS [6–8]. In many of the autoimmune rheumatic diseases, ultraviolet (UV) radiation is a recognized factor influencing the activity of the autoimmune process. The UV radiation causes movement of antigen molecules, bound with/to ribonucleoprotein, between cytoplasm and cell membrane (SS-A/Ro) and cell nucleus and cytoplasm (SS-B/La). UVB radiation affects epithelial damage, activates plasma dendritic cells, and increases the risk of their apoptosis and (risk?) of the IFN signaling pathway activation. Different observations concerning UVA

The hormonal state of the individual may also play a role in the pSS development. The imbalance of sex hormones and its receptors, dependent on hypothalamic-pituitary-adrenal axis (HPA or HTPA axis), interferes with the ratio of estrogens to androgens, especially in genetically predisposed patients [11]. This influences the stimulation of the autoimmune process and may explain the more frequent occurrence of pSS in women, especially in middle-aged. In recent years, attention has been paid to the role of the deficiency of dehydroepiandrosterone (DHEA) and of DHEA-S, its metabolite, in pSS and other autoimmune diseases [12].

As the epithelial cell damage and apoptosis provide basis for the pSS pathogenesis, the first step of the whole process is the activation of innate immunity, as the virus or bacteria antigen activates pattern recognition receptors (PRR) via Toll-like receptors (especially TLR 3, 7, 9). Activation of innate immunity leads, in turn, to the damage of epithelial cells, their apoptosis and the release of antigens and RNA complexes that strongly stimulate plasmacytoid dendritic cells (pDCs). These produce interferon alpha (IFN-α)—a factor strongly stimulating epithelial cells, dendritic cells and neutrophils to produce B-cell activating factor (BAFF) [13–16]. All processes initiating epithelial damage lead to the apoptosis of cells, activation of congenital and acquired immune systems and the cascade effect of pathophysiological phenomena, resulting primarily in the overproduction of BAFF and other B-cell stimulating cytokines including APRIL (proliferation inducing ligand), similar in its actions to BAFF. Both BAFF and APRIL belong to the tumor necrosis factor superfamily (TNF) [17, 18]. The antigens released from damaged cells, primarily SS-A and SS-B ribonucleoproteins are targets for B cells and cause the production of specific anti-SS-A/Ro and anti-SS-B/La autoantibodies. Plasmocytoid dendritic cells also stimulate T lymphocytes, particularly the CD4+ subtype, which is later the main component of infiltrates in the endocrine glands. The Th1-type immune response is predominant, with activation of Th17 cells secreting interleukin 17 (IL-17). Th1 cells produce IFNγ, which, in addition to the increase of BAFF secretion, induces the production of plasminogen

radiation suggest that it inhibits the production of autoantibodies [9, 10].

A primary test for autoantibodies, finding the use in the diagnostics of pSS and other systemic autoimmune rheumatic diseases (SARD), is the determination of anti-nuclear antibodies (ANAs) [20, 21]. ANA are found in 80–90% of patients with pSS. These antibodies react with the components of the cell nucleus and are most often tested with indirect immunofluorescence (IF) on HEp-2 (human epithelial cell) cell line. In pSS, ANA often occur in higher titers (above 1:320), but may be also detected in lower titers (1:160) and in the concurrent presence of other autoantibodies. In **Table 1**, the prevalence of ANA antibodies in different autoimmune diseases was presented.

In recent years attention has been paid to the frequent occurrence of dense fine speckled pattern (DFS70) on HEp-2 in both healthy people and patients with ANA associated autoimmune rheumatic diseases (AARD). DFS70 antibodies bind a ubiquitinated protein called lens epithelium derived growth factor (LEDGF), which occurrence was associated in first observations [22] with asthma and atopic dermatitis. However, the high prevalence of DFS70 autoantibodies in normal population, without any symptoms of any AARD, was observed. Therefore, in the case of positive result of the screening for ANA antibodies in individuals without symptoms suggestive of a systemic autoimmune rheumatic disease (SARD/AARD), it is advisable to detect DFS70 antibodies using specific tests (e.g., ELISA/EIA; CLIA/CIA) [23, 24]. Even up to 1/3 of positive cases for ANA are also positive for DFS70 antibodies [23, 24].

### **2.2. Extractable nuclear antigens**

In 1959, Holman et al. recorded a reaction of sera from SLE patients with extractable nuclear antigens (ENA) isolated from a crushed calf thymus. This observation confirmed the reaction of autoantibodies in the SLE sera with soluble nuclear antigens. The nomenclature of ENA autoantibodies derived from the group, in which they were first described, and corresponds to the nuclear function of the antigen (RNP) or the name of the patient providing the prototype serum (Ro, La, Sm, Jo, Mi), as well as the disease from which the patient suffered (SSA,


SLE—systemic lupus erythematosus; SS, SSc—systemic sclerosis, pSS—primary Sjögren's syndrome, MCTD—mixed connective tissue disease.

**Table 1.** Prevalence of ANA antibodies in different autoimmune diseases [20, 21].

SSB, SSC, Scl-70, PM-1, PM-Scl) [23]. The set of all ENA includes more than 100 soluble and cytoplasmic antigens. In clinical practice, until present day, only few of the them are finding use as a immunological hallmarks of certain autoimmune diseases or being used as immunological prognostic factors. Among them the most prominent are as follows: anti-RNP (antiribonucleoprotein anti-U(1)RNP), anti-Sm RNP, anti-SSA/Ro, anti-SSB/La, anti-Sm (Smith) antibody, anti-Scl-70 (anti-topoisomerase antibodies), anti-Jo-1 (anti-histidyl-transfer RNA synthase antibodies). The pattern of positive and negative results obtained with an ENA panel should be evaluated in conjunction with all clinical findings. Main autoantibodies and disease which they are typical to are presented in **Table 2**. Selected autoimmune diseases along with their predominant autoantibodies are presented in **Table 2**. **Figure 1** shows a simplified diagnostic algorithm of immunological diagnosis.
