Activation of the extrinsic apoptotic pathway by TNF-α in Human Salivary Gland (HSG) cells in vitro, suggests a role for the TNF receptor (TNF-R) and Intercellular Adhesion Molecule-1 (ICAM-1) in Sjögren's Syndrome-associated autoimmune sialadenitis

Abstract

Studies suggest that apoptosis plays a major role in destruction of salivary glands in Sjögren's Syndrome. We hypothesise that apoptosis results in the exposure of cryptic T cell epitopes and an autoimmune response which is pathway-specific.

Objective

To activate the extrinsic or intrinsic apoptotic pathway to examine morphology, adhesion molecules, markers of antigen processing, and autoantigens on apoptotic bodies in vitro.

Methods

Tumor necrosis factor-α (TNF-α) or staurosporine, a protein kinase inhibitor, was used to trigger the extrinsic or intrinsic apoptotic pathway in a Human Salivary Gland cell line (HSG), in vitro. Activated genes were profiled by cDNA array. Apoptotic bodies were visualised using light and SEM. Proteins were evaluated by immunofluorescence and confirmed by functional binding assays with Jurkat lymphocytes.

Results

TNF-α-triggered extrinsic apoptosis resulted in “sticky” aggregated, apoptotic bodies which displayed cleaved α-fodrin autoantigen. In contrast, intrinsic apoptosis induced by staurosporine, resulted in dispersed cell blebs which were α-fodrin-negative. cDNA arrays revealed that TNF-α, but not staurosporine, upregulated transcriptional expression of Intercellular Adhesion Molecule-1 (ICAM-1) and Macrophage Inflammatory Protein-3 (CCL20).

Conclusion

The apoptotic pathway controls morphological, structural and functional properties of apoptotic bodies. Collectively, TNF-α-dependent activation of the extrinsic apoptotic pathway leads to upregulation of ICAM-1 and CCL20 in HSG in vitro. This suggests that pathogenesis in Sjögren's Syndrome may involve a TNF-controlled cross-talk between apoptotic ductal and CCL20-attracted dendritic cells via the CD137/CD137L signalling pathway.

Introduction

It is increasingly recognised that apoptosis occurs in minor salivary glands in patients with Sjögren's Syndrome causing tissue destruction although the precise mechanism of autoimmune response remains unclear.1, 2 It is proposed that specific antigens revealed may depend upon the particular enzymatic modification of proteins which occurs during apoptosis. Currently, this proposition remains speculative and the multiple steps leading to autoimmune pathogenesis are unclear. These observations have led us to propose that production of novel immunogenic antigens by salivary gland cells, antigen processing/presentation and activation of autoreactive lymphocytes occurs in an apoptotic pathway-specific manner. The rationale is that proteolytic activities occurring during apoptosis may reveal previously cryptic “self protein” determinants which elicit an autoimmune response.

There are two main apoptotic pathways which have been widely studied, namely extrinsic and intrinsic. The first is the death receptor pathway which among other triggers can be activated by TNF-α, a proinflammatory cytokine produced in response to tissue damage, infection, and other environmental challenges. Binding of TNF to 55-kDa TNF receptor (TNF-R1) results in initiation of multiple signalling pathways, one of which leads to programmed cell death or apoptosis.3, 4, 5, 6 TNF-induced activation of apoptosis is mediated by the recruitment of the adaptor protein FADD (FAS-associated death domain) to the intracellular of TNF receptor domain known as TRADD (TNF-R associated death domain protein). These initial events lead to the assembly of the death-inducing signalling complex, or DISC, which activates the downstream effector caspase 8 and 10 and the executioner caspase 3.

A second pathway, referred to as the “mitochondrial” pathway, is on the other hand activated by a number of non-specific inhibitors which increase the permeability of the mitochondrial outer membrane. Staurosporine, a protein kinase inhibitor, has been characterised as a strong inducer of apoptosis in many different cell systems in vitro and is generally believed to induce apoptosis by the mitochondrial pathway causing the release of cytochrome c from mitochondria and the loss of mitochondrial membrane potential.⁷ However, overexpression of anti-apoptotic BCL2 that prevents the efflux of cytochrome c and the initiation of apoptosis does not protect from staurosporine-induced apoptosis suggesting that the exact mechanism of its actions is not completely understood.

To investigate the pathogenic role of apoptosis in salivary gland disease, we compared the morphology of, and display of cleaved α-fodrin autoantigen on, apoptotic cells generated from HSG ductal cells in response to challenge with TNF-α, or staurosporine. Next, to study gene expression associated with activation of each apoptotic pathway, we used cDNA arrays. Finally, to determine whether TNF-α- or staurosporine-generated apoptotic HSG cells can support an interaction with T lymphocytes, we employed a binding assay using human Jurkat T lymphocytes.

Our data show that TNF-α, but not staurosporine, leads to expression of the cleaved α-fodrin autoantigen on apoptotic HSG cells which are also capable of T cell binding, similar to binding/activation of T cells observed in Sjögren's Syndrome. Since it is known that stimulated HSG cells express HLA antigens and have other properties of professional antigen presenting cells (APCs),⁸ we speculate that TNF-induced apoptotic ductal cells may serve as surrogate APCs and trigger an autoimmune response by displaying the processed autoantigens within class II MHC expressed on their surface for their recognition by autoreactive T cells.