Pasquale Scagliusi

Tumor Necrosis Factor Inhibitors Block Apoptosis of Human Epithelial Cells of the Salivary Glands

Inhibition of tumor necrosis factor‐α (TNF‐α) in organ‐specific autoimmune disease is proving efficacious for a large number of patients. A wide array of biological agents has been designed to inhibit TNF‐α, such as adalimumab (fully humanized) and etanercept (soluble TNF‐α receptor fusion constructs p75 subunit). Recently, we suggested that anti‐Ro and anti‐La autoantibodies (Abs) isolated from patients with Sjögrens syndrome, an autoimmune rheumatic disease, are able to trigger cell death through extrinsic apoptotic mechanisms in human salivary gland epithelial cells (SGEC). We analyzed if primary human SGEC cultures, established from biopsy of labial minor salivary glands, are able to produce TNF‐α, an inductor of the extrinsic apoptotic pathway, when treated with anti‐Ro autoantibodies. A comparative study was performed to test the efficacy of adalimumab and etanercept to block TNF‐α‐mediated apoptosis. ELISA assay and RT‐PCR were employed to visualize TNF‐α production, and apoptosis was evaluated by DNA ladder and flow cytometry. We found that cell treatment with anti‐Ro autoantibodies determines TNF‐α production that reaches a maximum at 16 h and is decreased (P < 0.05) at 24 and 48 h. Adalimumab seems to be more efficacious than etanercept in blocking TNF‐α‐mediated apoptosis. The YOPRO‐1 (+) and propidium iodide (−) method revealed 60% of apoptotic cells after 24 h of incubation with anti‐Ro compared with 15% of apoptotic cells treated with anti‐Ro plus adalimumab and 25% of apoptotic cells treated with anti‐Ro plus etanercept. The antiapoptotic effect of adalimumab and etanercept was supported by inhibition of DNA laddering induced by anti‐Ro Abs. These data validate the therapeutic efficacy of the anti‐TNF reagents in the treatment of autoimmune disorders.

Autoantibodies from Sjögren's syndrome trigger apoptosis in salivary gland cell line.

Abstract:  The presence of serum autoantibodies has been associated with Sjögrens syndrome (SS), an autoimmune rheumatic disease that targets salivary and lachrymal glands. The association of apoptosis with autoantibodies production seems to play a role in the pathogenesis of glandular damage. The best‐defined antibodies in SS are those reacting with the ribonucleoprotein antigens SS‐A (Ro) and SS‐B (La). Anti‐Ro antibodies are found in about 70–90%, and anti‐La in approximately the same frequency, of patients with primary SS. The objective of this work was to explore whether anti‐Ro and anti‐La autoantibodies purified from Sjögren IgG fractions are able to trigger apoptotic process in the human salivary gland cell line A‐253. Anti‐Ro and anti‐La autoantibodies were purified on protein G Sepharose affinity column and used for the A‐253 cell treatment. Apoptosis induced by autoantibodies was revealed by FACS analysis, and the active caspase‐3 and the cleaved caspase‐3 substrate poly (ADP‐ribose) polymerase (PARP) was demonstrated by colorimetric assay and Western blot. This report shows that anti‐Ro and anti‐La autoantibodies, but not healthy IgG, activate the caspase‐3 and determine the cleavage of PARP in A‐253 cells. Apoptosis triggered by Sjögren autoantibodies could be responsible for the impairment of the secretory function in the salivary glands.

Selective TNF-alpha gene silencing attenuates apoptosis in human salivary gland epithelial cells.

RNA interference (RNAi) was used in this study for selective knockdown of TNF-alpha gene expression in anti-Ro/SSA autoantibodies (Abs)-treated human salivary gland epithelial cells. Our findings reveal that selective TNF-alpha gene silencing resulted in the subsequent attenuation of the pro-apoptotic effects of anti-Ro/SSA Abs; this could have therapeutic effects in autoimmune diseases.

Modulation of the Fcγ receptors induced by anti-Ro and anti-La autoantibodies: observations in salivary gland cells

Only few reports have shown protein expression of the Fcγ receptors (FcγRs) molecules on human salivary gland cells. In this study we investigate a possible upregulation of FcγRs following anti-Ro and anti-La autoantibodies treatment. Anti-Ro and anti-La autoantibodies were purified from IgG fractions obtained from 14 patients with primary Sjögren’s syndrome (SS), using Sepharose 4B-Ro and Sepharose 4B-La affinity columns. Flow cytometry and RT-PCR were used to study the FCγRI, FCγRII and FCγRIII receptors expression and upregulation by anti-Ro and anti-La on a salivary gland cell line. The present data document that the anti-Ro and anti-La autoantibodies determine an increase of the FcγRs expression on salivary gland cells, and provide evidence that both the high affinity FcγRI and the low affinity FcγRII and FcγRIII are overexpressed. Treatment with IgG isolated from healthy donors had no effect on the basal FCγRs expression.

Anti‐Ro/SSA autoantibody‐mediated regulation of extracellular matrix fibulins in human epithelial cells of the salivary gland

Objectives: The fibulins are a family of extracellular matrix (ECM) molecules that regulate the organ shape along with other growth factors and stromal cells and have recently been shown to be involved in a variety of cellular functions including proliferation, migration, differentiation, and survival. Important changes in acinar and ductal morphology and function, together with pronounced ECM remodelling, are detectable in the labial salivary glands (LSGs) of patients with Sjögrens syndrome (SS). Here we report the in vitro expression of the recently identified ECM proteins fibulin‐6 and fibulin‐7 by human salivary gland epithelial cells (SGECs). The ability of anti‐Ro/SSA autoantibodies (Abs) to modulate fibulin‐6 and fibulin‐7 expression was investigated. Methods: Semiquantitative reverse transcription polymerase chain reaction (RT‐PCR) and real‐time PCR were used to analyse fibulin‐6 and fibulin‐7 mRNA expression. Confocal microscopy and fluorescence‐activated cell sorting (FACS) were used to study expression of the proteins in primary human SGEC cultures, established from biopsies of minor LSGs, in both untreated control cells and anti‐Ro/SSA Abs‐treated cells. Results: The methods used show the expression of fibulin‐6 and fibulin‐7 in SGECs. Treatment of cells with anti‐Ro/SSA Abs results in a down‐regulation of fibulin‐6 mRNA expression whereas no significant differences were observed in fibulin‐7 expression between untreated and treated cells. Conclusion: Dysregulation of fibulin expression in SGECs by anti‐Ro/SSA Abs may contribute to disorganization of the ECM environment and thus cause injury to the salivary gland architecture and functionality observed in SS.

Fibulin-6 expression and anoikis in human salivary gland epithelial cells: implications in Sjogren's syndrome.

Important changes in acinar and ductal morphology and function, together with pronounced extracellular matrix (ECM) remodelling, are detectable in the labial salivary glands of Sjögrens syndrome (SS) patients. The objective of this work was to determine the effect of treatment with the anti-Ro/SSA auto-antibodies, characterizing SS, on the expression of fibulin-6, a member of the fibulins family of the ECM, in primary human salivary gland epithelial cell (SGEC) cultures established from biopsies of labial minor salivary glands obtained from healthy donors. The induction of cell detachment and anoikis in SGECs treated with anti-Ro/SSA auto-antibodies were also investigated. Changes in fibulin-6 mRNA expression were measured by semi-quantitative reverse transcriptase-PCR and real-time PCR. Fibulin-6 expression in cells treated with anti-Ro/SSA auto-antibodies was evaluated by flow cytometric analysis and confocal laser scanning microscopy. SGECs undergoing death by anoikis were identified and quantified using Calcein blue/YOPRO-1 dyes. Herein, we present the first evidence of fibulin-6 expression in SGEC that results distributed in the cytoplasm surrounding the inner side of the plasma membrane. Fibulin-6 was down-regulated in SGECs treated with anti-Ro/SSA auto-antibodies in which a marked cell detachment and a reduction of cell viability were observed. Notably, a reduction of fibulin-6 expression in SGECs treated with anti-Ro/SSA auto-antibodies corresponds to an increase of anoikis cell death. Our observations demonstrate a dysregulation of fibulin-6 in the pathological processes observed in SS and provide new evidence that disorganization of the ECM environment could damage the architecture and function of the salivary glands.