Massimo D'Amore

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.

Sjögren's syndrome autoantibodies provoke changes in gene expression profiles of inflammatory cytokines triggering a pathway involving TACE/NF-κB.

We explore the association of the inflammatory gene expression profile observed in the chronic inflammatory autoimmune disorder Sjögrens syndrome (SS) with changes in TNF-α converting enzyme (TACE), tumor necrosis factor (TNF)-α and nuclear factor (NF)-κB levels showing that pathways that include TNF-α signaling converge on NF-κB contributing to exacerbate the diseases. The treatment of human salivary gland epithelial cells (SGECs) with SS anti-Ro/SSA autoantibodies (Abs) result in a progressive increase in NF-κB–DNA binding, that includes a marked enhancement in NF-κB subunit p65 protein–DNA binding. A human cytokine multi-analyte array demonstrated that the NF-κB proinflammatory target genes, increased by anti-Ro/SSA Abs treatment, includes CXC chemokines (CXCL1, CXCL6 and CXCL9), CC chemokines (CCL2, CCL13 and CCL20), interleukins (IL-1α, IL-1β, IL-1F8, IL-6, IL-8, IL-9, IL-13, IL-17 and IL-22) and their receptors (IL-1RN, IL-10Rα, IL-13Rα, CCR1, CCR2, CCR3, CCR4 and CXCR1). Blockade of TACE through the use of the specific inhibitor TAPI-1 regulates proinflammatory cytokines production in SGEC treated with anti-Ro/SSA Abs inhibiting NF-κB nuclear translocation and activation. To further investigate the role of NF-κB on anti-Ro/SSA Abs-determined proinflammatory gene expression, we used the inhibitory protein IκB-α dominant negative super-repressor as inhibitor of NF-κB–DNA binding, demonstrating that transfection with dominant-negative IκB-α in anti-Ro/SSA-treated SGEC determined a marked reduction of proinflammatory cytokines gene expression. Although further studies are needed to clarify the mechanisms underlying SS, our results demonstrate that SS Abs exert their pathogenic effects via triggering the TACE/TNF-α/NF-κB axis.

Sjögren's syndrome pathological neovascularization is regulated by VEGF-A-stimulated TACE-dependent crosstalk between VEGFR2 and NF-κB.

We explore the involvement of tumor necrosis factor α (TNF-α)-converting enzyme (TACE) in vascular endothelial growth factor (VEGF) and its receptor 2 (VEGFR2) (VEGF-A/VEGFR2)-mediated angiogenesis in Sjögren’s syndrome (SS), one of the most common autoimmune rheumatic diseases. To test the hypothesis that SS autoantibodies (Abs) regulate VEGF-A/VEGFR2 expression by a TACE-dependent nuclear factor-κB (NF-κB) activation pathway, their effects on the expression and activation of the VEGF-A/TACE/VEGFR2/NF-κB pathway were determined in human salivary gland epithelial cells (SGEC). An enhanced angiogenesis in SS salivary gland biopsies was observed, associated with an increased VEGF-A expression and activation of VEGF-A/VEGFR2 signaling. Human cytokine array analysis of the pro-inflammatory and pro-angiogenic protein response in SGEC treated with SS Abs revealed an overexpression of multiple pro-angiogenic factors. TACE RNA knockdown, the use of anti-VEGF-A monoclonal antibody and the inhibition of NF-κB activity significantly abrogated the release of pro-angiogenic factors, demonstrating that VEGF-A/TACE/VEGFR2/NF-κB axis dysfunction may be contributory to pathogenesis and exacerbation of this autoimmune condition.

Molecular identification of Mycobacterium avium subspecies paratuberculosis in an Italian patient with Hashimoto's thyroiditis and Melkersson–Rosenthal syndrome

Mycobacterium avium subspecies paratuberculosis (MAP) causes Johne’s disease, a chronic granulomatous intestinal condition that affects ruminants, including cattle, sheep, goats and farmed deer (Naser et al., 2004). Water and milk supplies have both been suggested as vehicles of MAP transmission between cattle and humans (Pickup et al., 2006; Groenendaal & Zagmutt, 2008). Milk may be contaminated directly within the udder or indirectly as a result of faecal contamination (Giese & Ahrens, 2000). This bacterium has attracted considerable interest owing to the rapidly growing body of scientific evidence suggesting similarities between Johne’s disease in ruminants and Crohn’s disease in humans, which has led to speculation that MAP might be one of the causative agents in Crohn’s disease (Naser et al., 2004). However, because of the complex nature of human Crohn’s disease, as well as the conflicting experimental evidence, a definitive link between MAP and Crohn’s disease can neither be confirmed nor excluded at present. Some authors believe there is no evidence that a single pathogen, including MAP, is involved in the pathogenesis of inflammatory bowel diseases (Packey & Sartor, 2009), while others have reported that MAP may asymptomatically colonize apparently healthy individuals (Juste et al., 2008). Therefore, no causal link has been scientifically established between MAP and Crohn’s disease, although it is important to note that much evidence supports the possibility of such a relationship. The MAP debate has gathered momentum in recent years because of the more frequent detection of MAP in the blood and tissues of Crohn’s disease patients as compared with controls. The over-representation of MAP DNA in Crohn’s disease patients suggests a probable role for MAP in the aetiology of this pathological condition (Bentley et al., 2008).

ADAM17 at the interface between inflammation and autoimmunity

The discovery of the disintegrin and metalloproteinase 17 (ADAM17), originally identified as tumor necrosis factor-a converting enzyme (TACE) for its ability as sheddase of TNF-α inspired scientists to attempt to elucidate the molecular mechanisms underlying ADAM17 implication in diseased conditions. In recent years, it has become evident that this protease can modify many non matrix substrates, such as cytokines (e.g. TNF-α), cytokine receptors (e.g. IL-6R and TNF-R), ligands of ErbB (e.g. TGF-α and amphiregulin) and adhesion proteins (e.g. Lselectin and ICAM-1). Several recent studies have described experimental model system to better understand the role of specific signaling molecules, the interplay of different signals and tissue interactions in regulating ADAM17-dependent cleavage of most relevant substrates in inflammatory diseases. The central question is whether ADAM17 can influence the outcome of inflammation and if so, how it performs this regulation in autoimmunity, since inflammatory autoimmune diseases are often characterized by deregulated metalloproteinase activities. This review will explore the latest research on the influence of ADAM17 on the progression of inflammatory processes linked to autoimmunity and its role as modulator of inflammation.

Proposing a relationship between Mycobacterium avium subspecies paratuberculosis infection and Hashimoto's thyroiditis

Abstract Humans are widely exposed to Mycobacterium avium subspecies paratuberculosis (MAP), a proven multi-host chronic enteric pathogen that has recently been linked to autoimmune diabetes. In the present study we used a MAP species-specific polymerase chain reaction with the insertion element IS900-specific probe to detect MAP infection in members of the same family suffering from Hashimotos thyroiditis.

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.

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 Sjögrens 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.

Induction of TNF-alpha-converting enzyme-ectodomain shedding by pathogenic autoantibodies

The release of the soluble form of tumor necrosis factor (TNF)-alpha from the plasma membrane occurs through the activation of the secretase tumor necrosis factor-alpha-converting enzyme (TACE). The current study was designed to examine whether the anti-Ro/SSA autoantibodies (Abs) are capable to regulate TACE expression in non-neoplastic human salivary gland epithelial cells (SGEC) cultures. We investigated the effect of anti-Ro/SSA Abs on the localization and abundance of cell-surface TACE and on TACE pro-domain-shedding and activation. In addition, the potential physiological consequences of TNF-alpha blockage by the biological agent Adalimumab on post-translational regulation of TACE are discussed. Anti-Ro/SSA Abs were purified from IgG fractions of patients with primary Sjögrens syndrome, using Sepharose 4B-Ro/SSA affinity columns. Flow cytometry, reverse transcription-PCR, western blot and immunohistochemistry were used to study TACE expression on SGEC and TACE regulation by Abs. Our study demonstrated a dose-dependent increase of TACE messenger RNA (mRNA) expression in anti-Ro/SSA Abs-treated SGEC, followed by internalization, pro-domain shedding and activation of TACE protein, suggesting that increased TACE activity is necessary for the release of TNF-alpha observed in anti-Ro/SSA Abs-stimulated SGEC. Adalimumab treatment brought TACE mRNA and surface TACE expression to levels than those observed in untreated SGEC. These data suggest that the effect of anti-Ro/SSA Abs on TACE expression and intracellular distribution is exerted by TNF-alpha production.

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.