A model of potential contribution of different factors in SS pathogenesis
Association of non-MHC class genes with SS susceptibility according to distinct pathogenetic pathways
Type I and II IFN pathways
LTA/LTB/TNF gene clusters
The observation that lymphocytes infiltrating exocrine glands and parenchymal organs surround epithelia suggests a central role of the epithelial cell in the formation and further organization of characteristic immunopathological lesions in SS . Especially, salivary gland epithelial cells have been investigated and found to undergo increased apoptosis , leading to release of autoantigens (such as Ro/SSA and La/SSB) which in turn drive the production of disease-specific autoantibodies. The immunocomplexes that are generated through this process result in type I interferon (IFN) production by plasmacytoid dendritic cells (PDCs) in individuals with genetic predisposition. Subsequently, type I IFN can reinforce epithelial activation and BAFF overexpression, as well as autoantibody production. The complex role of activated epithelia as antigen-presenting cells and cells secreting chemokines and cytokines or expressing chemotactic molecules places them at the center of the immunological process. Epithelial cells ultimately contribute to further aggregation of inflammatory cells, activation of lymphocytes (both T and B), and autoantibody production, thus closing the vicious circle of autoimmunity. This series of events can possibly culminate in extensive tissue damage and even B-cell monoclonal expansion [4, 31–35]. Therefore, the term “autoimmune epithelitis,” stressing the key role and active involvement of epithelial cells, has been fairly proposed to describe SS .
Diagnosis and Differential Diagnosis
2016 American College of Rheumatology/European League Against Rheumatism classification criteria for primary SS. The classification of pSS applies to any individual who meets the inclusion criteria (a), does not have any of the conditions listed as exclusion criteria (b), and has a score of at least 4 when the weights from the five selected criteria items are summed (c). Several changes from 2002 AECG classification criteria were made. Subjective ocular or oral symptoms are now considered a prerequisite, rather than criteria contributing to the total score as they were before. Sialography and scintigraphy have been omitted, and a higher threshold for the ocular staining score has been implemented. The list of exclusion criteria was revisited, as the newly identified IgG4-related disease was added and lymphoma was removed, while more accurate techniques are required to rule out known, confounding entities (PCR confirmation of active hepatitis C). Finally, anti-SSB/La autoantibodies positivity was concluded to have no diagnostic value in the absence of anti-SSA/Ro and was therefore withdrawn. To be noted that patients on anticholinergic drugs should be objectively evaluated for their sicca symptoms after a sufficient time of these medications has elapsed
(a) Inclusion criteria
Positive response to at least 1 of the following questions:
1. Have you had daily, persistent, troublesome dry eyes for more than 3 months?
2. Do you have a recurrent sensation of sand or gravel in the eyes?
3. Do you use tear substitutes more than 3 times a day?
4. Have you had a daily feeling of dry mouth for more than 3 months?
5. Do you frequently drink liquids to aid in swallowing dry food?
Suspicion of Sjögren’s syndrome from the ESSDAI questionnaire (at least 1 domain with a positive item)
(b) Exclusion criteria
1. History of head and neck radiation treatment
2. Active hepatitis C infection
6. Graft-versus-host disease
7. IgG4-related disease
Labial salivary gland with focal lymphocytic sialadenitis and focus score ≥ 1 foci/4 mm2
Score ≥ 4
Ocular staining score ≥ 5 (or van Bijsterveld score ≥ 4) in at least one eye
Positive Schirmer’s test (≤5 mm/5 min in at least one eye)
Unstimulated whole saliva flow rate ≤ 0.1 ml/min
SS differential diagnosis. Other causes of sicca symptoms and processes infiltrating the exocrine glands need to be ruled out in order to reach the correct diagnosis. IgG4-related disease is a newly identified entity, added in the exclusion criteria list of the recently reviewed American/European Consensus SS classification criteria. It is a multi-organ, immune-mediated condition that has unified several diseases once considered to be individual. It includes Mikulicz disease (sialo/dacryo-adenitis and salivary/lacrimal gland enlargement), Küttner tumor, Riedel’s thyroiditis, orbital inflammatory pseudotumor, pituitary hypophysitis, and hypertrophic pachymeningitis in the head and neck region, as well as autoimmune pancreatitis, interstitial pneumonitis, interstitial nephritis, prostatitis, retroperitoneal fibrosis, and inflammatory aortic aneurysm. Elevated serum IgG4 levels (≥135 mg/dl) and infiltration of abundant IgG4-positive plasma cells into affected organs help with differential diagnosis
Sicca symptoms (xerophthalmia and/or xerostomia)
Lymphocytic infiltration of exocrine glands
Non-lymphocytic infiltration of exocrine glands
Use of medications
Antihypertensives, antihistamines, antidepressants, isotretinoin, etc.
Previous head and neck radiation
Vitamin A deficiency
Any functional or anatomical defect of the eyelid
Chronic blepharitis, chronic conjunctivitis, or another chronic eye inflammation
Chronic viral infections
Hepatitis C, AIDS, HTLV-1
Chronic graft-versus-host disease
Sarcoidosis, tuberculosis, leprosy, syphilis
Hyperlipoproteinemia, diabetes mellitus, amyloidosis, hemochromatosis
Prevalence of specific autoantibodies in SS patients and correlation with clinical manifestations, other serological features, and disease outcomes. This table includes traditional autoantibodies for disease classification, autoantibodies identified from murine models, and autoantibodies typically associated with other autoimmune diseases. The hoped-for result of as early as possible diagnosis has recently led to the marketing of a new diagnostic kit (Sjö® test). The cumulative sensitivity of traditional (ANA, RF, anti-SSA/Ro, anti-SSB/La) and novel (anti-CA VI, anti-SP-1, anti-PSP) antibodies of Sjö® panel has been estimated to be 91.8%, whereas the sensitivities for anti-SSA/SSB alone and for the novel biomarkers alone were found to be 74.9% and 49.8%, respectively. Additionally, the cumulative specificity for the complete Sjö® panel was estimated at 79.8%. Further potential biomarkers currently under investigation are anti-kallikrein antibodies, antibodies against carbamylated proteins, and antibodies against TRIM38 proteins, among others (not shown in the table)
Type of autoantibodies
Prevalence of autoantibody positivity in pSS patients
Usually associated with female sex, younger age at diagnosis, more prominent lymphocytic infiltrate of the exocrine glands, and potentially a higher prevalence of extraglandular manifestations
Attention needed in case of pregnancy, due to potential congenital heart block of the baby (complete heart block occurring in approximately 2% of cases)
Anti-nuclear antibodies (ANA)
Associated with female gender, younger age at diagnosis, parotid gland enlargement, extraglandular manifestations, cytopenia, hypergammaglobulinemia, as well as increased frequency of RF, anti-Ro/SSA, anti-La/SSB, and antiphospholipid antibodies positivity
Rheumatoid factor (RF)
Linked to earlier disease onset, female predominance, positive salivary gland biopsy, more frequent extraglandular features, and higher use of corticosteroids, among others
Also, increased frequency of anti-La/SSB, anti-Ro/SSA, cryoglobulins, and ANA positivity, as well as low C3/C4 and hypergammaglobulinemia
One of the indisputable risk factors for non-Hodgkin’s lymphoma development and SS-related death. Also, linked to earlier disease onset, higher frequency of extraglandular features (vasculitis, renal involvement, peripheral neuropathy, Raynaud’s phenomenon) and cytopenia, as well as higher prevalence of parotid gland enlargement
Anti-thyroperoxidase antibodies (anti-TPO)
Anti-thyroglobulin antibodies (anti-TG)
Autoimmune thyroid disease prevalence in SS seems to be 10–30%
Furthermore, SS prevalence in already diagnosed autoimmune thyroid disease has been reported to be 3–32% (10 times higher probability of SS in autoimmune thyroid disease than in the general population)
Sicca symptoms are even more frequent in the context of autoimmune thyroid disease (37% of patients develop xerostomia and 23% isolated keratoconjunctivitis sicca)
Autoimmune thyroid disease-associated SS is linked to milder SS phenotype, but also to greater risk of developing further autoimmune diseases (such as autoimmune liver and inflammatory bowel diseases), requiring closer follow-up
Antibodies against cyclic citrullinated peptides (anti-CCP)
Anti-CCP-positive pSS patients do not seem to have major clinical differences from anti-CCP-negative individuals, but there is a possible association with nonerosive arthritis
Anti-mitochondrial antibodies (AMA)
Depending on laboratory technique for detection; indirect immunofluorescence, Western blot, or ELISA
Specific for primary biliary cirrhosis (PBC)
Also, higher prevalence of Raynaud’s phenomenon, peripheral neuropathy, hypergammaglobulinemia, and high ESR
Valuable for separating patients with autoimmune liver involvement from those with chronic viral liver disease
Anti-smooth muscle antibodies (ASMA)
Autoimmune hepatitis (only 1.7–4% in pSS patients)
Anti-centromere antibodies (ACA, comprising of CENP-A, CENP-B, and CENP-C)
Overlapping features between SS and systemic sclerosis (SSc)
Up to 40% of the ACA-positive pSS patients can progress to systemic sclerosis
Associated with delayed disease onset, but increased frequency of keratoconjunctivitis sicca, Raynaud’s phenomenon, peripheral neuropathy, and lymphoma
Lower frequency of anti-Ro/La antibodies and higher prevalence of other coexisting autoimmune disorders, such as PBC
SS patients usually recognize CENP-C alone, whereas recognition of both CENP-B and CENP-C is more frequent in SSc
Antibodies against carbonic anhydrase (anti-CA; 13 known isoenzymes)
Anti-CA II antibodies linked to renal involvement and particularly distal renal tubular acidosis
Anti-CA VI and anti-CA XIII antibodies have also been shown to correlate with urine pH and inversely with serum sodium levels (cross-reactivity between anti-CA VI and anti-CA XIII is a possible scenario, as CA VI is the only isoenzyme secreted in saliva and expressed in the parotid and submandibular glands, but not in the kidney)
Anti-CA VI is considered a novel, early SS biomarker, included in commercially available Sjö® diagnostic panel. It is the most prevalent novel autoantibody of the kit among both SS and non-SS dry eye patients (52% and 43%, respectively). Anti-CA VI positivity has been linked to more severe xerophthalmia, presence of xerostomia, younger age, and negative MSGB
Antibodies to 21-hydroxylase (anti-21[OH])
Anti-21[OH] positivity was not linked to overt adrenal insufficiency, but it was associated with adrenal hyporesponsiveness and evidence of more prominent B-cell activation in MSG tissue samples
Decreased prevalence of subjective xerophthalmia and increased frequency of leukopenia were also noted for anti-21[OH]-positive SS individuals
Anti-muscarinic receptor antibodies
Associated with cytopenia and higher ESSDAI scores
Could partially account for the salivary gland hypofunction, the gastroesophageal symptoms, and the bladder smooth muscle hyperresponsiveness, observed in pSS patients
Antibodies against citrullinated alpha-enolase peptides (anti- CEP-1)
60% of anti-citrullinated protein antibodies (ACPA)-positive pSS patients
Less than 10% of unselected pSS patients
Associated with higher urine pH levels at first evaluation
(linked to distal renal tubular acidosis, nephrocalcinosis, and impaired bone health)
Antibodies against salivary protein 1 (anti-SP-1)
52% of SS patients
19% of SS patients with negative anti-Ro/anti-La
Antibodies against murine SP-1 (no known human protein analogue) seem to identify targets in human parotid glands
Anti-SP-1 antibodies are considered novel, early SS biomarkers, included in commercially available Sjö® diagnostic panel
Patients with lower focus scores in MSGB tend to be tested positive for anti-SP-1 more often than those with higher focus scores (who generally test positive for anti-Ro/anti-La). Especially patients expressing only anti-SP-1 antibodies (no anti-Ro/anti-La) have low or negative MSGB focus score
Anti-SP-1 can also be used as a marker to separate SS-associated RA from RA not complicated with SS
Antibodies against parotid secretory protein (anti-PSP)
PSP is a protein involved in the binding and clearance of infectious agents
Anti-PSP is considered a novel, early SS biomarker, included in commercially available Sjö® diagnostic panel
Can also be positive in non-SS dry eye disease and rarely in RA and healthy controls
29% of pSS patients, but 47% of SLE patients
Almost 2 times more prevalent in non-SS sicca than SS patients
Anti-α-fodrin antibodies serum concentrations have been associated with the degree of lymphocytic infiltration in salivary glands
Usually found in early disease stages
Most probably not useful for SS diagnostic purposes
Salivary Gland Biopsy
The advantages of MSGB include easy accessibility, avoidance of skin incisions, and local anesthesia. Parotid biopsy is reserved only to rule out lymphoma in case of persistent parotid enlargement [74, 75]. MSGB sensitivity and specificity are considered to be higher than 75% and 90%, respectively [64, 76, 77].
Oral Involvement Assessment Tests
The objective evaluation of xerostomia and oral involvement in SS, except for the generally accepted MSGB, remains a challenge [75, 78]. The easiest, most common, and affordable way to assess major and minor salivary gland secretory capacity is the measurement of salivary flow or sialometry. Unstimulated whole saliva flow rate equal to or below 0.1 ml/min is considered abnormal (0.3–0.4 ml/min are expected for healthy individuals) .
Sialography may be used to demonstrate the morphology of the ducts, while scintigraphy can assess the salivary gland functionality. The nonspecific results of both techniques and the involvement of radiation for the latter have led to their removal from the most recent SS classification criteria [75, 79].
Ultrasound (US), being noninvasive, inexpensive, and radiation-free, is drawing a lot of attention for the purposes of major salivary gland imaging. Multiple studies have shown good agreement and comparable results between salivary gland ultrasound and sialography and even diagnostic superiority compared to scintigraphy .
In addition to its value as a diagnostic tool, the prognostic value of ultrasound has also been explored. Increased parenchymal dyshomogeneity scores of major salivary glands have been found to correlate with SSA, SSB, ANA, RF, higher levels of IgG, salivary gland enlargement, cutaneous vasculitis and/or purpura, GC-like structures in salivary gland biopsy, CD4 T-cell lymphopenia, Raynaud’s phenomenon, and disease activity scores. Last but not least, there is some evidence that ultrasonographic images of salivary glands change in response to treatment (rituximab versus placebo) for SS .
Elastography is an added feature to the classic US modality, which can increase sensitivity compared to B-mode US alone and differentiate between SS patients and sicca controls .
Mainly aqueous deficiency but also meibomian gland dysfunction and neuropathic pain contribute to increased tear evaporation rate, reduced tear film stability, and ocular discomfort in SS patients .
Disease Activity Indexes
There are two indexes, introduced by European League Against Rheumatism (EULAR), which are used to assess SS activity from the patient’s and the clinician’s point of view. The first one is the EULAR Sjögren’s Syndrome Patient Report Index (ESSPRI), which consists of three visual-analogue scales measuring severity of sicca symptoms, fatigue, and pain . The second one is the EULAR Sjögren’s Syndrome Disease Activity Index (ESSDAI), which assesses the activity in 12 different domains representing organ systems. Since its introduction, ESSDAI has become the gold standard in terms of disease activity assessment because it serves as point of reference among physicians; it is widely used in randomized controlled trials as outcome measure and has been correlated with biomarkers and lymphoma development risk [42, 43].
Clinical Manifestations and Disease Management
Glandular manifestations and their treatment in SS patients
Symptoms and signs
Irregularity of the corneal image, irritation, redness, photosensitivity
No MG damage (aqueous deficiency)
Stop offending drugs, environmental changes, artificial tears, gels, ointments
Ω3 suppl., CIS collyrium (0.05%) pulse steroids, punctal plugs, secretagogues, moisture chamber spectacles
Topical autologous serum, contact lenses, permanent punctal occlusion
Systemic anti-inflammatory medication, eyelid surgery
MG damage (evaporative)
Stop offending drugs, environment modification, lipid-rich tear substitutes, warm compress, massage
CIS: 2–2.5 mg/kg/d, topical steroids, AZI, DXC, secretagogues, punctal plugs, moisture chamber spectacles
Topical autologous serum, contact lenses
Dryness, caries, angular cheilitis
Salivary gland enlargement
Dental fluorination, masticatory stimulation, chlorhexidine
Pilocarpine hydrochloride: max 20 mg/d in divided doses
Cevimeline hydrochloride: max 30 mg × 3
CS, 0.25–0.5 mg/kg/d for 10–15 d
Recurrent autoimmune pancreatitis (5%)
AZA 2–3 mg/kg/d, RTX, pancreatic enzymes
Extraglandular manifestations and their treatment in SS patients
Symptoms and signs
Myalgias, arthralgias, Jaccoud arthropathy, rare arthritis
HCQ, MTX, or combination of both, small dose CS < 15mg qd
Cold-related color skin changes
Vasodilators, especially calcium channel blockers
Increased need for resting hours, disruption of sleep patterns
Nordic (active) walking, HCQ in some cases
Small airway disease (13%)
Mild to moderate dyspnea and dry cough, xerotrachea
LFTs abnormalities, jaundice
Interstitial cystitis (0.3%)
Hypokalemic hyperchloremic distal renal tubular acidosis/nephrocalcinosis
Pollakiuria, nycturia, urinary urgency, pelvic or suprapubic pain
Urine alkalization (bicarbonate, electrolyte supplements)
CS, CIS, surgical intervention
Immune complex-mediated disease
Vasculitis in 5%
Purpura – palpable in 5%
RTX for necrotizing vasculitis (cycles of 2gr q15 days interval/6 months)
Membranous or membranoproliferative
CS, RTX, CYC
Peripheral sensorimotor neuropathy or pure sensory neuropathy
Small fiber neuropathy
CS, 0.5–1 mg/kg, and IVIGs, RTX
CS, 0.5–1 mg/kg, and CYC/AZA, 2–3 mg/kg/d
Anticholinergics, antidepressants, gabapentinoids
CS, PE, RTX
Wait and watch policy
R-CHOP = if diffuse large B cell
Lymphomagenesis and Lymphoproliferation
SS is unique among autoimmune diseases as for malignant transformation risk. Those patients seem to have a 10–44-fold greater risk of developing lymphoma compared to general population, whereas systemic lupus erythematosus patients and rheumatoid arthritis patients only have a seven-fold and four-fold greater risk, respectively [67, 121]. In other words, 2.7–9.8% of SS patients are diagnosed with non-Hodgkin lymphoma (NHL) and that risk increases by 2.2% per year of age [78, 122].
Mucosa-associated lymphoid tissue (MALT) lymphomas represent 60% of cases . Most common sites are the salivary glands, especially the parotid and submandibular glands, but other mucosal sites of MALT lymphoma development include the orbits, nasopharynx, stomach, thyroid, and lung . Other subtypes of lymphoma found in these patients are the diffuse large B-cell lymphoma (DLBCL) and the nodal marginal zone lymphoma (NMZL), which – together with MALT – account for more than 90% of total SS-associated lymphoma cases .
Current belief on etiopathogenesis of lymphoma focuses on four interrelated axes: chronic inflammation, B-cell activation, defective immunosurveillance, and epigenetic alterations [4, 104]. Focus score of at least 3 in MSGBs of SS patients has been identified as an independent and important predicting factor for NHL development, and the use of this threshold has a positive predictive value of 16% and a negative predictive value of 98% for that kind of malignant transformation . Furthermore, the activation of P2X7 receptor-NLRP3 inflammasome complex (with subsequent increase of pro-inflammatory cytokines IL-18 and IL-1β serum levels, among others) correlates with lymphocytic infiltration severity, ESSDAI scores, and lymphoproliferation risk. The hypothesis of inflammasome activation secondary to increased accumulation of proinflammatory nucleic acid shreds, ineffectively degraded and cleared, has recently been supported [127, 128].
Another factor promoting chronic inflammation seems to be IFNγ, as the mRNA levels in MSGBs have been associated with a higher degree of lymphocytic infiltration, shown to be a predisposing factor for lymphomagenesis . Moreover, a functional variant of TNFAIP3 gene (rs2230926), encoding the A20 protein, leads to unopposed NF-κB pathway activation and is therefore involved in inflammatory process perpetuation, B-cell survival, and more aggressive disease phenotype with earlier disease onset and increased lymphoproliferation risk .
B-cell activating factor (BAFF), produced by various immune cells but also by the salivary epithelial cells and regulated by both type I and II IFNs, is of utmost importance for the maturation, proliferation, and survival of B cells. BAFF levels are found increased in SS patients’ serum with a history of lymphoma, and high levels persist for years after treatment and remission [4, 131, 132]. Another equally important observation is the association of specific BAFF polymorphisms with SS-related lymphomagenesis and in particular the significantly different prevalence of the protective AA genotype of the rs12583006 polymorphism, as well as of the protective haplotypes TACAC and TACC and of the risk haplotype TTTC in SS patients prone to NHL development (high risk determined by the presence of adverse predictors), compared to low-risk individuals . Additionally, a specific mutation (His159Tyr) of BAFF receptor (BAFF-R) is of interest, as it has been identified in more than two thirds of SS-associated MALT lymphoma. This mutation, leading to NF-kB pathway activation, was linked to earlier development of lymphoma or adverse immunological features (hypergammaglobulinemia and positive RF) [78, 134].
Finally, Fms-like tyrosine kinase 3 ligand (Flt-3 l) is a protein that acts as a cytokine and a growth factor, activating Flt-3 (or CD135) on the surface of hematopoietic progenitor cells, and therefore considered to be a mediator of B-cell survival. Higher levels of Flt-3l are strongly associated with history of lymphoma, detectable years before malignant transformation, and not affected by treatment .
The vicious circle of autoreactive B-cell chronic stimulation and immunocomplexes formation might finally lead to the favorable, monoclonal expansion of rheumatoid factor (RF)-reactive B cells and to the lymphomatous transformation, under defective immunosurveillance . As a matter of fact, IFNα mRNA levels in MSGBs seem to strongly correlate with the expression of pro-apoptotic molecules (tumor suppressor gene p53 and auto-antigen Ro52, with the latter negatively regulating the anti-apoptotic B-cell lymphoma 2 (Bcl-2) gene) . Moreover, decreased prevalence of a specific TREX1 variant (rs11797 AA genotype) in SS-related non-MALT cases was observed. This variant was shown to associate with higher mRNA IFNα levels in SS salivary gland tissues .
Further oncogenic mechanisms, likely to contribute to malignant turn in SS, are the over-expression of Bcl-2 due to a translocation involving chromosomes 14 and 18, leading to inhibition of apoptosis and increased B-cell survival. Apart from IFNα effect on p53 levels, specific mutations of this tumor suppressor gene were described 20 years ago in MSGBs from SS-associated NHL cases [4, 132, 137].
Epigenetic changes, involving methylating enzymes and transcription of noncoding micro-RNAs, are also implicated in lymphomagenesis . MiR200b miRNAs, which regulate the expression of oncogenes and tumor suppressor genes, have been found to be downregulated in MSGBs with advanced lymphocytic infiltration and MALT lymphoma . Especially miR200b-5p strongly discriminates SS patients who already have or will develop NHL from the rest of them or from sicca controls . As for methylating enzymes, DNA methyltransferase (DNMT)3B and methyl-CpG-binding protein 2 (MeCP2) have been found decreased in SS-lymphoma patients , while methylene-tetrahydrofolate reductase (MTHFR) gene variants, leading to defective methylation and impaired stability of DNA, have lately been suggested as susceptibility factors for non-MALT lymphoma .
It becomes clear that the multifactorial process of lymphomagenesis is rather complicated, and the finely tuned balance between opposing forces can become deranged and lead to adverse outcomes. An example of that is the IFNγ/IFNα mRNA ratio in MSGBs, which has emerged as a histopathological biomarker for the prediction of in situ lymphoma development .
SS-related hematological malignancies are correlated with an eightfold higher mortality risk compared to general population , and one in five deaths of SS is attributable to lymphoma . Follow-up every 6 months is recommended for high-risk patients. Overall, NHL 5-year survival is estimated at approximately 92%, but higher disease activity is linked to higher possibility of relapse and death [143–145]. Wait and watch strategy is suitable for MALT lymphomas localized in the salivary glands, while rituximab and chemotherapy are employed in case of disseminated or aggressive disease [106, 146, 147, 149].
Which of the following clinical features would set Sjögren’s syndrome (SS) on top of your differential diagnosis list?