pSS Minor Salivary Glands Research Articles

Different operators and histologic techniques in the assessment of germinal center-like structures in primary Sjögren's syndrome minor salivary glands.

ObjectiveA standardization of minor salivary gland (MSG) histopathology in primary Sjögren’s syndrome (pSS) has been recently proposed. Although there is strong agreement that germinal center (GC)-like structures should be routinely identified, due to their prognostic value, a consensus regarding the best protocol is still lacking. Aim of this study was to compare the performance of different histological techniques and operators to identify GC-like structures in pSS MSGs. MSG biopsies from 50 pSS patients were studied.MethodsThree blinded operators (one pathologist and two rheumatologists with different years of experience in pSS MSG assessment) assessed 50 MSGs of which one slide was stained with haematoxylin and eosin (H&E) and consecutive slides were processed to investigate CD3/CD20, CD21 and Bcl-6 expression.ResultsBy assessing 225 foci, the best agreement was between H&E-stained sections evaluated by the rheumatologist with more years of experience in pSS MSG assessment and CD3/CD20 segregation. In the foci with CD21 positivity, the agreement further increased. Bcl-6- foci could display a GC, detected with other staining, but not vice versa.ConclusionGC assessment on H&E-stained sections should be performed with caution, being operator-dependent. The combination of H&E with CD3/CD20 and CD21 staining should be recommended as it is reliable, feasible, able to overcome the bias of operator experience and easily transferrable into routine practice.

Interleukin-15 as a potential new target in Sjögren's syndrome-associated inflammation

IL-15 is a key regulatory cytokine that shares many biological properties with IL-2. Recently, it has been shown that IL-15 could be up-regulated in T cell-mediated inflammatory disorders, such as rheumatoid arthritis and inflammatory bowel diseases. However, the role and expression of IL-15 in the inflammatory autoimmune disease Sjögren's syndrome (SS) has not been investigated. In the present study we evaluated the expression of IL-15 mRNA and protein in minor salivary gland (MSG) biopsy specimens and in human salivary gland epithelial cell (SGEC) cultures obtained from patients with primary SS (pSS) and compared their expression with that seen in normal healthy control subjects. IL-15 gene and protein analysis revealed that SGEC are able to produce IL-15. Results obtained demonstrated that the number of IL-15(+) cultured SGEC was significantly higher in cells derived from patients with pSS in comparison with SGEC from healthy subjects; similar results were obtained for IL-15 immunoreactivity by using immunohistochemistry that revealed a strong expression both in acinar and in ductal cells from pSS MSG. These studies could provide a rational basis to determine whether IL-15 could be a good candidate for anti-cytokine therapy in chronic inflammatory pSS diseases.

TLR2 signals via NF-κB to drive IL-15 production in salivary gland epithelial cells derived from patients with primary Sjögren's syndrome.

Toll-like receptors (TLRs) are pattern recognition receptors linking innate and adaptive immune responses, which resulted overexpressed in primary Sjögren's syndrome (pSS). Interleukin-15 (IL-15) is a pro-inflammatory cytokine which was recently demonstrated to be involved in pSS pathogenesis. The study was undertaken to clarify whether TLR2 is involved in the production of IL-15 in human salivary gland epithelial cells (SGEC) from pSS patients. SGEC primary cell cultures were established from pSS minor salivary gland tissues explanted from patients with a sure diagnosis of SS. After neutralization of TLR2 with a blocking monoclonal antibody, IL-15 production was assayed by immunoblotting and flow cytometry, IL-15 in the culture supernatants was measured by ELISA, and mRNA levels were assessed by RT-PCR and real-time PCR. The production of IL-15 by pSS SGEC decreased in culture supernatants and in protein lysates (p<0.01) when TLR2 signaling was inhibited in pSS SGEC. In addition, a control at the transcriptional level was also detected; in fact, inhibition of nuclear factor (NF)-κB through the transfection of pSS SGEC with the dominant-negative inhibitory κBα proteins (IκBα) vector (IκBαDN) abrogated the stimulatory effect of TLR2 on IL-15 production. These data suggest that TLR2 activation is involved in the induction of IL-15 production by pSS SGEC and promotes inflammation through NF-κB activation. Therefore, therapeutic strategies that target TLR2/IL-15 pathway might be strong candidates for preventing or treating pSS.

Mobilization of lymphatic endothelial precursor cells and lymphatic neovascularization in primary Sjögren's syndrome.

Although lymphatic neovascularization may be a key feature of chronic inflammation, it is almost unexplored in primary Sjögren's syndrome (pSS). A recent study revealed a pro‐lymphangiogenic function of interleukin (IL)‐17, a leading player in pSS pathogenesis. The aims of the study were to investigate lymphangiogenic mediators and lymphatic vasculature in pSS, as well as their possible association with IL‐17. Circulating lymphatic endothelial precursor cells (LEPCs) and Th17 cells were enumerated in pSS patients and healthy donors. VEGF‐C and IL‐17 levels were assessed in paired serum samples. Lymphatic vasculature, VEGF‐C/VEGF receptor (VEGFR)‐3 and IL‐17 were evaluated in pSS minor salivary glands (MSGs) and compared with normal and non‐specific chronic sialadenitis (NSCS) MSGs. Circulating LEPCs were expanded in pSS and correlated with circulating Th17 cells, IL‐17 and VEGF‐C. In pSS MSGs, a newly formed lymphatic capillary network was found within periductal inflammatory infiltrates and the number of interlobular lymphatic vessels was significantly increased compared with normal and NSCS MSGs. Strong VEGF‐C expression was detected in pSS ductal epithelial cells and periductal inflammatory cells. Numerous VEGFR‐3+ infiltrating mononuclear cells were exclusively observed in pSS MSGs. VEGFR‐3 expression was strongly increased in lymphatic capillaries of pSS MSGs. IL‐17+ inflammatory cells were preferentially observed around lymphatic vessels in pSS MSGs. This study supports the notion that lymphvasculogenesis and lymphangiogenesis are active in pSS, thereby unmasking a novel aspect of disease pathogenesis. In addition, our results suggest another possible pathogenic role of IL‐17 in pSS, further supporting its therapeutic targeting in this disease.

Interferon gamma-inducible protein 16 in primary Sjögren's syndrome: a novel player in disease pathogenesis?

IntroductionThere is evidence that interferon is involved in the pathogenesis of primary Sjögren’s syndrome (pSS). The interferon-inducible IFI16 protein, normally expressed in cell nuclei, may be overexpressed, mislocalized in the cytoplasm and secreted in the extracellular milieu in several autoimmune disorders. This leads to tolerance breaking to this self-protein with consequent development of anti-IFI16 antibodies. The aim of this study was to identify the pathogenic and clinical significance of IFI16 and anti-IFI16 in pSS.MethodsIFI16 and anti-IFI16 were assessed in the serum of 67 pSS patients and over 100 healthy donors by enzyme-linked immunosorbent assay. IFI16 was also evaluated by immunohistochemistry in minor salivary glands of 15 pSS patients and 10 subjects with sicca symptoms but without any clinical, serological or histological features of pSS.ResultspSS patients display higher serum levels of both IFI16 and anti-IFI16 compared to healthy donors. IFI16 concentration was directly correlated with disease duration and focus score and inversely correlated with age at diagnosis. Moreover, IFI16 positivity was associated with concurrent positivity for rheumatoid factor. Interestingly, the direct correlation between IFI16 positivity and focus score was independent of disease duration and age at diagnosis. pSS minor salivary glands display marked expression and cytoplasmic mislocalization of IFI16 by acinar and ductal epithelial cells as well as infiltrating lymphocytes and peri/intralesional endothelium compared to minor salivary glands with normal architecture or nonspecific chronic sialadenitis. Within the mononuclear cell infiltrate, IFI16 expression appears to parallel the distribution of T lymphocytes.ConclusionOur data suggest that the IFI16 protein may be involved in the pathogenesis of glandular inflammation occurring in pSS.

Telocytes in minor salivary glands of primary Sjögren's syndrome: association with the extent of inflammation and ectopic lymphoid neogenesis.

It has been recently reported that telocytes, a stromal (interstitial) cell subset involved in the control of local tissue homeostasis, are hampered in the target organs of inflammatory/autoimmune disorders. Since no data concerning telocytes in minor salivary glands (MSGs) are currently available, aim of the study was to evaluate telocyte distribution in MSGs with normal architecture, non-specific chronic sialadenitis (NSCS) and primary Sjögren’s syndrome (pSS)-focal lymphocytic sialadenitis. Twelve patients with pSS and 16 sicca non-pSS subjects were enrolled in the study. MSGs were evaluated by haematoxylin and eosin staining and immunofluorescence for CD3/CD20 and CD21 to assess focus score, Tarpley biopsy score, T/B cell segregation and germinal center (GC)-like structures. Telocytes were identified by immunoperoxidase-based immunohistochemistry for CD34 and CD34/platelet-derived growth factor receptor α double immunofluorescence. Telocytes were numerous in the stromal compartment of normal MSGs, where their long cytoplasmic processes surrounded vessels and encircled both the excretory ducts and the secretory units. In NSCS, despite the presence of a certain degree of inflammation, telocytes were normally represented. Conversely, telocytes were markedly reduced in MSGs from pSS patients compared to normal and NSCS MSGs. Such a decrease was associated with both worsening of glandular inflammation and progression of ectopic lymphoid neogenesis, periductal telocytes being reduced in the presence of smaller inflammatory foci and completely absent in the presence of GC-like structures. Our findings suggest that a loss of MSG telocytes might have important pathophysiological implications in pSS. The specific pro-inflammatory cytokine milieu of pSS MSGs might be one of the causes of telocyte loss.

Mobilization of lymphatic endothelial progenitor cells and lymphatic neovascularization in primary Sjögren’s syndrome

Primary Sjogren’s syndrome (pSS) is a systemic autoimmune disorder charac- terized by chronic lymphocytic infiltration of exocrine glands leading to progressive functional impairment. Lympangiogenesis is a common finding in chronic inflamma- tory diseases; however, its role in pSS remains to be elucidated. Inflammation induces the production of growth factors for lymphatic vessels, such as vascular endothelial growth factor-C (VEGF-C). Recent growing evidence indicates that bone marrowderived lymphatic endothelial progenitor cells (LEPCs) may differentiate into lymphatic endothelial cells via VEGF-C/VEGFR-3 signaling contributing to lymphangiogenesis. This study was designed to investigate the circulating levels of LEPCs and the occurrence of lymphangiogenesis in pSS. Pheripheral blood mononuclear cells were collected from 10 female pSS patients and 11 healthy females. LEPCs, defined as CD34+CD133+VEGFR-3+ cells, were identified by FACS using CD34-FITC, CD133-APC and VEGFR-3-PE antibodies. Results were expressed as percentage of CD133+VEGFR-3+ cells among CD34+ cells. Labial minor salivary gland (MSG) biop- sies were obtained from 12 female pSS patients and 16 sicca non-pSS control females. MSGs were evaluated by haematoxylin-eosin and immunofluorescence for CD3/CD20 and CD21 to assess focus score, Tarpley biopsy score, T/B cell segregation and germi- nal center-like structures. Lymphatic vessels were identified by immunohistochemistry for podoplanin (D2-40), a mucin-type transmembrane protein expressed by lymphatic endothelial cells but not by blood vessels. VEGF-C/VEGFR-3 expression in MSGs was investigated by immunofluorescence. An average ten-fold increase in circulating levels of LEPCs was found in pSS (35.2±2.7%) compared with controls (3.4±0.8%) (p=0.0003). In control MSGs, lymphatic vessels were only detected around excretory ducts in the interlobular connective tissue. In pSS MSGs, the number of lymphatic vessels was increased around interlobular excretory ducts and a newly formed lymphatic capil lary network was found within inflammatory foci. A strong expression of VEGF-C was detected in ductal cells, vessels and inflammatory cells in pSS MSGs. VEGFR-3 expres- sion was observed in a subset of vessels and infiltrating mononuclear cells. Our find ings suggest that LEPC mobilization and MSG lymphatic vessel reorganization may take center stage in the chronic inflammatory process of pSS.

Chronic inflammation enhances NGF-β/TrkA system expression via EGFR/MEK/ERK pathway activation in Sjögren’s syndrome

Primary Sjögren's syndrome (pSS) is a chronic autoimmune exocrine disease associated with variable lymphocytic infiltration of the affected organs (primarily salivary and lachrymal glands). To investigate the potential implication of nerve growth factor-β (NGF-β) and its high affinity receptor tyrosine kinase receptor A (TrkA) in the regulation of pSS inflammatory responses, we studied their expression in the human salivary gland epithelial cells (SGEC) cultures from pSS minor salivary glands (MSG) biopsies and their relationship with histopathological disease parameters. Here, we demonstrated an increased expression of the NGF-β/TrkA system in pSS SGEC, correlated with the MSG inflammation grade. The results demonstrate that the pro-inflammatory cytokines TNF-α and IL-6 enhance NGF-β production; on the contrary, NGF-β production was reduced in the presence of both Raf-1 kinase and MEK inhibitors. Furthermore, TNF-α/IL-6 treatment increased ERK1/2 phosphorylation. Inhibition of the EGF/EGFR system also decreased NGF-β release by pSS SGEC, indicating that the chronic inflammatory condition characteristic of pSS enhances NGF-β production via EGFR/Raf-1/MEK/ERK pathway activation. NGF-β and TrkA expression is elevated in salivary gland epithelial cells of primary Sjögren's syndrome (pSS). Overexpression of NGF-β/TrkA system in pSS occurs via EGFR/Raf-1/MEK/ERK pathway. In pSS, NGF-β overexpression was prevented by EGFR/Raf-1/MEK/ERK pathway inhibition.

Neovascularization is prominent in the chronic inflammatory lesions of Sjögren's syndrome.

Angiogenesis is a common finding in chronic inflammatory diseases; however, its role in Sjögren's syndrome (SS) remains to be elucidated. Previous SS studies have demonstrated an increase in VEGF-A/VEGFR-2 system expression in minor salivary gland (MSG) biopsies from patients with SS, but differences in the new blood vessel formation between the different grades of disease severity have not been reported. Therefore, experiments were performed to demonstrate angiogenesis during different phases of primary SS (pSS) and to define the relationship between the microvessel density (MVD), macrophage infiltration and histiocyte distribution in SS MSG inflammatory lesions. In this series of experiments, immunohistochemistry was used to examine angiogenesis in serial sections of pSS MSG. Patients with pSS were classified accordingly with the grade of inflammatory lesions as I = low-grade (low focus score of 1 or 2), II = intermediate-grade (focus score of 3–6) and III = extensive inflammation in the MSG (high focus score of 12). Histological examination demonstrated that the MVD increased with the severity of the inflammatory lesions, and in addition, we found an increased infiltration of inflammatory and pro-angiogenic cells.These findings reveal that angiogenesis is intimately involved in the progression of pSS, may be central to the propagation of the chronic immune response observed in pSS and could represent a novel potential biomarker of pSS disease activity.

IL-17-producing double-negative T cells are expanded in the peripheral blood, infiltrate the salivary gland and are partially resistant to corticosteroid therapy in patients with Sjögren’s syndrome

A small CD3+ T-cell population, that lacks both CD4 and CD8 molecules, defined as double negative (DN), is expanded in the peripheral blood of patients with systemic lupus erythematosus, produces IL-17 and accumulates in the kidney during lupus nephritis. Since IL-17 production is enhanced in salivary gland infiltrates of patients with primary Sjögren's syndrome (pSS), we aimed to investigate whether DN T cells may be involved in the pathogenesis of salivary gland damage. Fifteen patients with SS and 15 normal controls (NC) were enrolled. Peripheral blood mononuclear cells were stimulated with anti-CD3 antibody and cultured in presence or absence of dexamethasone (Dex). Phenotypic characterization was performed by flow cytometry in freshly isolated cells and after culture. Minor salivary glands (MSG) from pSS were processed for immunofluorescence staining. Total circulating DN T cells were increased in pSS compared to NC (4.7±0.4% vs 2.6±0.4%). NC and pSS freshly isolated DN T cells produce consistent amounts of IL-17 (67.7±5.6 in NC vs 69.2±3.3 in pSS). Notably, DN T cells were found in the pSS-MSG infiltrate. Dex was able to down-regulate IL-17 in vitro production in NC (29±2.6% vs 15.2±1.9% vs 13±1.6%) and pSS (49±4.8% vs 16±3.8% vs 10.2±0.8%) conventional Th17 cells and in DN T cells of NC (80±2.8% vs 3.8±2.1% vs 4.2±1.8%), but not of pSS (81±1.5% vs 85.4±0.8% vs 86.2±1.7%). DN T cells are expanded in pSS PB, produce IL-17 and infiltrate pSS MSG. In pSS, conventional Th17 cells are inhibited by Dex, but DN T cells appear to be resistant to this effect. Taken together, these data suggest a key role of this T-cell subset in the perpetuation of chronic sialoadenitis and eventually in pSS prognosis.

Expression of SSA antigen epitopes in minor salivary glands from patients with primary Sjögren's syndrome

To investigate the correlation between the differential expression of 60 000 SSA epitopes in minor salivary glands (MSG) from patients with primary Sjögren's syndrome (pSS) and glandular inflammation. The screening and soluble expression of single-chain fragment V monoclonal antibodies (Scfv McAb) against SSA antigen epitopes (P1: 480 - 494, P2: 310 - 323 and P3: 230 - 241) were performed by phagemid antibody expression system. The expression of epitopes was detected by immunohistochemical assay (IH) in minor salivary glands from these patients. The correlation between epitopes expression and glandular inflammation was analyzed quantitatively. Immunohistochemical assay of MSG with McAb against P1-P3 epitopes showed that the epithelial cells of glandular tubes and striated duct were stained in membrane and cytoplasm. The expression of P1 epitope was higher than the other two in grading score (chi(2) = 12.94, P < 0.01). And a positive correlation was found between the extent of glandular infiltration and the grade of P1 epitope expression (t = 2.27, P < 0.05) but not with P2 or P3 epitope. Aberrant redistribution of intracellular SSA antigen epitopes onto the cell membrane of involved cells may break the immune tolerance and thus induce the production of pathogenic autoantibodies involved in triggering and maintaining the tissue-specific autoimmune response in pSS MSG. A significantly high membranous expression of P1 and a positive correlation between P1 and the inflammation of MSG suggest that P1 may be the dominant determinant of the antigen-driven immune response in pSS.

Systemic and Local Interleukin-17 and Linked Cytokines Associated with Sjögren’s Syndrome Immunopathogenesis

Recently recognized as a distinct CD4(+) T helper (Th) lineage, Th17 cells have been implicated in host responses to infections and in pathogenesis associated with autoimmune diseases. This cytokine is implicated in primary Sjögren's syndrome (pSS) immunopathology because of the increased levels of circulating interleukin (IL)-17 in pSS. Plasma and minor salivary glands (MSGs) from patients with pSS were therefore evaluated for CD4(+) T cells, T regulatory cells, IL-17, and supporting cytokines by immunohistochemistry, RT-PCR, and microbead assays. MSGs from pSS patients contain IL-17-expressing cells as a dominant population within inflammatory lesions. IL-17 protein expression progressively increased with higher biopsy focus scores (P < 0.0001), in parallel with detection by RT-PCR. Transforming growth factor-beta, IL-6 and IL-23, which are requisite promoters of Th17 differentiation, were found in abundance compared with the amounts in control tissues. Although transforming growth factor-beta is also a pivotal differentiation factor for immunosuppressive Foxp3(+) T regulatory cells (Tregs), an increase in Foxp3(+) Tregs was evident in biopsy specimens with mild and moderate inflammation but this increase was disproportionate to escalating pro-inflammatory Th17 populations in advanced disease. Furthermore, the Th17-centric cytokines IL-17, IL-6, IL-23, and IL-12 were significantly elevated in pSS plasma. These data identify a profusion of IL-17-generating cells and supporting cytokines within diseased pSS MSGs without a compensatory increase in immunomodulatory Tregs; this imbalance seems to foster a pathogenic milieu that may be causative and predictive of infiltrative injury and amenable to therapeutic intervention.

Nuclear BCL10 in primary Sjögren’s syndrome

The events following triggering of antigen receptors and subsequent activation of the transcription factor nuclear factor kappa B (NFkappaB) need to be carefully controlled to prevent abnormal immune responses. BCL10 links the antigen receptor to NFkappaB. The aim of this study was to determine the expression pattern of BCL10 and NFkappaB in minor salivary gland infiltrates of patients with primary Sjögren's syndrome (pSS). Minor salivary glands from patients with primary SS (n = 17) and sicca controls (n = 4) were evaluated by single and double immunohistochemistry and immunofluorescence for confocal microscopy. BCL10 and NFkappaB-p65 expression were evaluated in the infiltrating lymphocytes. Ectopic germinal centers (GCs) were investigated by CD21. Tonsil, lymph node and lymphoma tissue were used as positive controls. BCL10 nuclear positive cells were observed in focal lymphocytic infiltrates in the investigated minor salivary glands and were not restricted to patients with ectopic GCs. By double-staining, some of the BCL10 nuclear positive cells were identified as B cells. There was, however, no constitutive activation of NFkappaB as depicted by the exclusive cytoplasmic expression of p65 in the infiltrating lymphocytes in the pSS. Nuclear expression of BCL10 in infiltrating lymphocytes was a common occurrence in pSS minor salivary glands indicating it as a possible marker of autoimmune induced chronic inflammation. There was, however, no constitutive activation of NFkappaB.