Ectopic germinal center formation in Sjögren’s syndrome: Significance of lymphoid organization

This thesis is based on the following papers, which will be referred to in the text by their Roman numerals: I. Salomonsson S, Jonsson MV, Skarstein K, Hjälmström P, Wahren-Herlenius M, Jonsson R. Cellular basis of ectopic germinal center formation and autoantibody production in the target organ of patients with Sjögren’s syndrome. Arthritis & Rheumatism 2003;48(11):3187-201. II. Jonsson MV, Szodoray P, Jellestad S, Jonsson R, Skarstein K. Association between circulating levels of the novel TNF family members APRIL and BAFF and lymphoid organization in primary Sjögren’s syndrome. Journal of Clinical Immunology, 2005;25(3):189-201. III. Jonsson MV, Skarstein K, Jonsson R, Brun JG. Germinal centers in primary Sjögren’s syndrome indicate a certain clinical immunological phenotype. Submitted. IV. Jonsson MV, Salomonsson S, Gunnvor Øijordsbakken, Skarstein K. Elevated serum levels of soluble E-cadherin in patients with primary Sjögren’s syndrome. Scandinavian Journal of Immunology, 2005;62(6):552-9. V. Jonsson MV, Delaleu N, Brokstad KA, Berggreen E, Skarstein K. Impaired salivary gland function in NOD mice – association with changes in cytokine profile but not with salivary gland histopathology. Arthritis & Rheumatism, in press.Sjögren’s syndrom (SS) is an autoimmune, chronic inflammatory disorder predominantly affecting the salivary and lacrimal glands. The overall aim of this study was to determine clinicopathological features in human and murine disease with regard to organization of ectopic lymphoid tissue, and to explore possible strategies for detection of patients at increased risk for extra-glandular manifestations. Mononuclear cell infiltrates in the shape of germinal centers (GC) were observed in the salivary glands of approximately 1/4th of patients. Phenotypic markers for GC components such as T and B cells, proliferating cells, follicular dendritic cells and plasma cells confirmed the ectopic GC formation. The pattern and distribution of homing and retentive chemokines CXCL12, CXCL13 and CCL21, and adhesion molecules/integrin pairs ICAM/LFA and VCAM/VLA, was described in various lymphoid organizations in minor salivary glands. In addition, local autoantibody production was detected and correlated with serum levels. Focal infiltrates and GC could be observed within the same gland, and were separated by altered B and T cell ratios, higher degree of proliferation and the localization of plasma cells in the periphery of infiltrates. Serum levels of BAFF and APRIL were elevated in pSS, and were in part linked to focus score, elevated serum IgG and autoantibody levels. In a large cohort of pSS, ectopic GC were also associated with higher focus scores, lower mean un-stimulated salivary secretion, Ro/SSA and La/SSB autoantibodies, elevated RF-titres and increased serum IgG. Not all morphological GC could be confirmed by CD21/CD23/CD35 labeling, but clinical features remained comparable. E-cadherin, an adhesion molecule important for epithelial tissue integrity, was investigated in minor salivary glands. E-cadherin is the ligand of integrin αEβ7/CD103 and lymphocytes expressing this integrin were increased in SS compared to non-SS. E-cadherin+ infiltrating cells were identified as CD68+ macrophages. Serum levels of sE-cadherin were increased in pSS compared to healthy blood donors and most likely mirror the chronic inflammatory state. The non-obese diabetic (NOD) mouse is an animal model of SS. We observed significant changes in inflammation between 8 and 17 weeks of age, while hyposalivation was first observed between 17 and 24 weeks. In 1/3rd of mice older than 17 weeks, proliferating cells were observed in the focal infiltrates. Significant differences were detected in serum cytokine levels of IL-2, IL-5 and GM-CSF, and IL-4 and TNF-α in saliva. Salivary secretion correlated with IL-4, IFN-γ and TNF-α levels in saliva of NOD mice, but not with inflammatory changes in the salivary glands. Focal sialadenitis preceded hyposalivation, which occurred without a significant change in inflammation in NOD mice. Proliferating inflammatory cells indicate contribution of local factors in progression of SS-like disease. In conclusion, ectopic germinal centers occur in a sub-group of patients with SS and are characterized by autoantibody production, progressive disease and increased serum IgG. It remains unclear whether the observed GC are a result of long-standing inflammation or indeed have functional properties and thus play an active role in the pathogenesis. A future challenge will be to identify and target trafficking molecules which drive the chronic inflammation in SS, without affecting migration and function of leukocytes required for protective immunity

Understanding fatigue in Sjögren’s Syndrome: Outcome measures, biomarkers and possible interventions

Sjögren’s syndrome (SS) is an autoimmune disease affecting the salivary and lacrimal glands. Symptoms range from dryness to severe extra-glandular disease involving manifestations in the skin, lungs, nervous system, and kidney. Fatigue occurs in 70% of patients, characterizing primary SS (pSS) and significantly impacting the patient’s quality of life. There are some generic and specific instruments used to measure fatigue in SS. The mechanisms involved with fatigue in SS are still poorly understood, but it appears fatigue signaling pathways are more associated with cell protection and defense than with pro-inflammatory pathways. There are no established pharmacological treatment options for fatigue in pSS. So far, exercise and neuromodulation techniques have shown positive effects on fatigue in pSS. This study briefly reviews fatigue in pSS, with special attention to outcome measures, biomarkers, and possible treatment options.publishedVersio

Impaired vascular responses to parasympathetic nerve stimulation and muscarinic receptor activation in the submandibular gland in non-obese diabetic mice

Introduction Decreased vascular responses to salivary gland stimulation are observed in Sjögren's syndrome patients. We investigate whether impaired vascular responses to parasympathetic stimulation and muscarinic receptor activation in salivary glands parallels hyposalivation in an experimental model for Sjögren's syndrome. Methods Blood flow responses in the salivary glands were measured by laser Doppler flowmeter. Muscarinic receptor activation was followed by saliva secretion measurements. Nitric oxide synthesis-mediated blood flow responses were studied after administration of a nitric oxide synthase inhibitor. Glandular autonomic nerves and muscarinic 3 receptor distributions were also investigated. Results Maximal blood flow responses to parasympathetic stimulation and muscarinic receptor activation were significantly lower in nonobese diabetic (NOD) mice compared with BALB/ c mice, coinciding with impaired saliva secretion in nonobese diabetic mice (P < 0.005). Nitric oxide synthase inhibitor had less effect on blood flow responses after parasympathetic nerve stimulation in nonobese diabetic mice compared with BALB/c mice (P < 0.02). In nonobese diabetic mice, salivary gland parasympathetic nerve fibres were absent in areas of focal infiltrates. Muscarinic 3 receptor might be localized in the blood vessel walls of salivary glands. Conclusions Impaired vasodilatation in response to parasympathetic nerve stimulation and muscarinic receptor activation may contribute to hyposalivation observed in nonobese diabetic mice. Reduced nitric oxide signalling after parasympathetic nerve stimulation may contribute in part to the impaired blood flow responses. The possibility of muscarinic 3 receptor in the vasculature supports the notion that muscarinic 3 receptor autoantibodies present in nonobese diabetic mice might impair the fluid transport required for salivation. Parasympathetic nerves were absent in areas of focal infiltrates, whereas a normal distribution was found within glandular epithelium.publishedVersio

Complement C4 Copy Number Variation is Linked to SSA/Ro and SSB/La Autoantibodies in Systemic Inflammatory Autoimmune Diseases

Objective Copy number variation of the C4 complement components, C4A and C4B, has been associated with systemic inflammatory autoimmune diseases. This study was undertaken to investigate whether C4 copy number variation is connected to the autoimmune repertoire in systemic lupus erythematosus (SLE), primary Sjögren's syndrome (SS), or myositis. Methods Using targeted DNA sequencing, we determined the copy number and genetic variants of C4 in 2,290 well-characterized Scandinavian patients with SLE, primary SS, or myositis and 1,251 healthy controls. Results A prominent relationship was observed between C4A copy number and the presence of SSA/SSB autoantibodies, which was shared between the 3 diseases. The strongest association was detected in patients with autoantibodies against both SSA and SSB and 0 C4A copies when compared to healthy controls (odds ratio [OR] 18.0 [95% confidence interval (95% CI) 10.2–33.3]), whereas a weaker association was seen in patients without SSA/SSB autoantibodies (OR 3.1 [95% CI 1.7–5.5]). The copy number of C4 correlated positively with C4 plasma levels. Further, a common loss-of-function variant in C4A leading to reduced plasma C4 was more prevalent in SLE patients with a low copy number of C4A. Functionally, we showed that absence of C4A reduced the individuals’ capacity to deposit C4b on immune complexes. Conclusion We show that a low C4A copy number is more strongly associated with the autoantibody repertoire than with the clinically defined disease entities. These findings may have implications for understanding the etiopathogenetic mechanisms of systemic inflammatory autoimmune diseases and for patient stratification when taking the genetic profile into account.publishedVersio

Salivary glands of primary Sjögren’s syndrome patients express factors vital for plasma cell survival

Introduction: The presence of circulating Ro/SSA and La/SSB autoantibodies has become an important marker in the classification criteria for primary Sjögren’s syndrome (pSS). Plasma cells producing these autoantibodies are mainly high affinity plasma cells originating from germinal centre reactions. When exposed to the right microenvironment these autoimmune plasma cells become long-lived and resistant to immunosuppressive treatment. Since autoimmune plasma cells have been detected in the salivary glands of SS patients, we wanted to investigate if the glandular microenvironment is suitable for plasma cell survival and if glandular residing plasma cells are the long-lived plasma cell subset. Methods: Single, double and triple immunohistochemistry as well as immunofluorescence staining was performed on minor salivary gland tissue retrieved from pSS, chronically inflamed and normal subjects. Results: We detected significant numbers of CD138+, non-proliferating, Bcl-2 expressing plasma cells in the salivary glands of pSS patients with high focus score (FS). Furthermore, we demonstrated that CXCL12 and interleukin (IL)-6 survival factors were highly expressed in pSS salivary gland epithelium and by focal mononuclear infiltrating cells. Notably, adipocytes when present in the salivary gland tissue were an important source of CXCL12. We clearly demonstrate that plasma cells are localised in close proximity to CXCL12 and IL-6 expressing cells and thus that the environment of salivary glands with high FS provide factors vital for plasma cell survival. Conclusions: Plasma cells residing in the salivary glands of pSS patients with high FS showed phenotypic characteristics of the long-lived plasma cell subtype. Furthermore, the pSS salivary gland microenvironment provided niches rich in factors vital for plasma cell survival

Impaired vascular responses to parasympathetic nerve stimulation and muscarinic receptor activation in the submandibular gland in non-obese diabetic mice

Introduction Decreased vascular responses to salivary gland stimulation are observed in Sjögren's syndrome patients. We investigate whether impaired vascular responses to parasympathetic stimulation and muscarinic receptor activation in salivary glands parallels hyposalivation in an experimental model for Sjögren's syndrome. Methods Blood flow responses in the salivary glands were measured by laser Doppler flowmeter. Muscarinic receptor activation was followed by saliva secretion measurements. Nitric oxide synthesis-mediated blood flow responses were studied after administration of a nitric oxide synthase inhibitor. Glandular autonomic nerves and muscarinic 3 receptor distributions were also investigated. Results Maximal blood flow responses to parasympathetic stimulation and muscarinic receptor activation were significantly lower in nonobese diabetic (NOD) mice compared with BALB/ c mice, coinciding with impaired saliva secretion in nonobese diabetic mice (P < 0.005). Nitric oxide synthase inhibitor had less effect on blood flow responses after parasympathetic nerve stimulation in nonobese diabetic mice compared with BALB/c mice (P < 0.02). In nonobese diabetic mice, salivary gland parasympathetic nerve fibres were absent in areas of focal infiltrates. Muscarinic 3 receptor might be localized in the blood vessel walls of salivary glands. Conclusions Impaired vasodilatation in response to parasympathetic nerve stimulation and muscarinic receptor activation may contribute to hyposalivation observed in nonobese diabetic mice. Reduced nitric oxide signalling after parasympathetic nerve stimulation may contribute in part to the impaired blood flow responses. The possibility of muscarinic 3 receptor in the vasculature supports the notion that muscarinic 3 receptor autoantibodies present in nonobese diabetic mice might impair the fluid transport required for salivation. Parasympathetic nerves were absent in areas of focal infiltrates, whereas a normal distribution was found within glandular epithelium

Understanding fatigue in Sjögren’s Syndrome: Outcome measures, biomarkers and possible interventions

Sjögren’s syndrome (SS) is an autoimmune disease affecting the salivary and lacrimal glands. Symptoms range from dryness to severe extra-glandular disease involving manifestations in the skin, lungs, nervous system, and kidney. Fatigue occurs in 70% of patients, characterizing primary SS (pSS) and significantly impacting the patient’s quality of life. There are some generic and specific instruments used to measure fatigue in SS. The mechanisms involved with fatigue in SS are still poorly understood, but it appears fatigue signaling pathways are more associated with cell protection and defense than with pro-inflammatory pathways. There are no established pharmacological treatment options for fatigue in pSS. So far, exercise and neuromodulation techniques have shown positive effects on fatigue in pSS. This study briefly reviews fatigue in pSS, with special attention to outcome measures, biomarkers, and possible treatment options

Radiographic features in 2D imaging as predictors for justified CBCT examinations of canine-induced root resorption

Objectives: This retrospective observational study aimed to evaluate the diagnostic accuracy of two-dimensional radiographs on canine-induced root resorption (CIRR) in lateral incisors and identify predictors of CIRR in patients with impacted maxillary canines (IMC). Methods: Ninety-nine patients aged 9–17 years, with 156 IMCs, were included in the study. All had CBCT-volumes and two-dimensional radiographs consisting of at least one panoramic radiograph. Two radiologists jointly viewed all cases twice. First, radiographic features related to the IMC and possible CIRR were recorded from two-dimensional radiographs. Then, CIRR was determined from CBCT and according to position and extension classified as mild, moderate and severe. Results: CIRRs was detected in 80% of lateral incisors (mild: 45%; moderate: 44%; severe: 11%). The sensitivity was generally low at mild and moderate cut-offs (29 and 29%), and somewhat higher for severe (50%). Corresponding specificities were 48%, 63% and 68%. Canine cusp-tip superimposing the lateral incisor’s middle third and root/crown ratio >1 was positively associated with mild CIRR, with an odds ratio (OR) of 3.8 and 6.7, respectively. In addition, the root development stage was positively associated with moderate/severe CIRR when the canine root was nearly or fully developed (OR = 3.1). Conclusions: The diagnostic accuracy of two-dimensional radiographs was inadequate for detecting CIRR amongst patients referred for CBCT examinations. Based on our results, none of the suggested two-dimensional radiographic features could predict moderate/severe CIRR except for root development stage. IMC in a later stage of root development seems to be associated with a higher risk of moderate/severe CIRR