Massimo D’Amore

Pro-inflammatory role of Anti-Ro/SSA autoantibodies through the activation of Furin-TACE-amphiregulin axis.

Prolonged inflammation can be detrimental because it may cause host toxicity and tissue damage. Indeed, excessive production of inflammatory cytokines is often associated with many autoimmune diseases. In this study we demonstrate that the anti-Ro/SSA autoantibodies (Abs) stimulate the production of pro-inflammatory cytokines IL-6 and IL-8 by human healthy salivary gland epithelial cells (healthy SGEC). The secretion of these cytokines is due to amphiregulin (AREG) that is overexpressed in healthy SGEC treated with anti-Ro/SSA Abs and in Sjögrens syndrome. We have discovered that the up-regulation of AREG occurs through TNF-alpha produced following anti-Ro/SSA Abs treatment. The gene silencing technique was used to study the AREG-TNF-alpha-IL-6/IL-8 secretion pathway, demonstrating that: (i) TNF-alpha gene silencing provokes a significant decrease of proinflammatory cytokines production and AREG expression in anti-Ro/SSA Abs-treated healthy SGEC; (ii) AREG gene silencing has a potent inhibitory effect on TNF-alpha-induced IL-6 and IL-8 secretion in healthy SGEC treated with anti-Ro/SSA Abs. These findings indicate that TACE-mediated AREG shedding plays a critical role in TNF-alpha-induced IL-6 and IL-8 secretion by the human healthy salivary gland epithelial cells, suggesting that this may be one of the possible intracellular mechanisms involved in the salivary glands inflammatory response in Sjögrens syndrome.

Expression of pro-inflammatory TACE-TNF-α-amphiregulin axis in Sjögren’s syndrome salivary glands

The tumor-necrosis-factor-converting-enzyme (TACE)-TNF-α-Amphiregulin (AREG) axis plays an important pathogenic role in inflammatory and autoimmune disorders. However, the pathological roles of these proteins in the chronic autoimmune disease Sjögren’s syndrome (SS) remain to be elucidated. It is known that the TACE–AREG axis is clearly part of a larger cascade of signals that starts with the activation of Furin, responsible for maturation of TACE that, in turn, determines the production of active TNF-α, directly involved in the up-regulation of AREG expression. This study showed that Furin, TACE, TNF-α, and AREG proteins, detected in acinar and ductal cells of human salivary glands from SS patients, increased remarkably in comparison with biopsies of labial salivary glands from healthy controls. The changes in Furin, TACE, TNF- α, and AREG proteins’ level detected in salivary glands biopsies of SS patients could be responsible for pro-inflammatory cytokines overexpression characterizing Sjögren’s syndrome.

Neuropilin-1 is upregulated in Sjögren's syndrome and contributes to pathological neovascularization.

Neuropilin-1 (NRP1) is a transmembrane co-receptor for members of the vascular endothelial growth factor family. Recent studies revealed an important role of NRP1 in angiogenesis and progression of many diseases. The role of NRP1 in the development of Sjögren’s syndrome (SS), one of the most common rheumatic diseases, has not yet been investigated. Molecular studies and protein expression techniques were performed to elucidate the gene and protein expression profile of NRP1 in human salivary gland epithelial cells (SGEC) from primary SS. We used human microarrays and transient transfection with a mutant form of the negative inhibitory κBα proteins (IκBαDN) to investigate whether selective inhibition of nuclear Factor-κB (NF-κB) improves NRP1-mediated pro-angiogenic factors release from SS SGEC. The selective NRP1 function inhibition with an antibody to human NRP1, was employed to evaluate the therapeutic potential of targeting NRP1. We demonstrate that NRP1 is expressed in SGEC of both human healthy biopsies and in SS samples, and increased NRP1 expression in SS SGEC is significantly associated with pro-angiogenic factors release. Neutralizing anti-NRP1 antibody decreased pro-angiogenic factor production from SS SGEC and blocking NF-κB activation could be a way to inhibit NRP1-mediated angiogenesis in Sjögren’s syndrome.

Advances in the understanding of the Fc gamma receptors-mediated autoantibodies uptake

Receptors for the Fc fragment of immunoglobulin G (FcγRs) are important molecules not only to mediate and control the effectors’ functions of IgG antibodies, but they also control the autoimmunity-tolerance balance in the periphery. In humans, three different types of FcγRs, belonging to the Ig gene superfamily, have been identified; FcγRI (cluster of differentiation (CD64), FcγRII (CD32) and FcγRIII (CD16). A wide range of inflammatory and autoimmune diseases, such as vasculitis, glomerulonephritis, and autoimmune hemolytic anemia, seems to be mediated, in part, by FcγRs. Recent findings supposed that, under certain conditions, FcγRs are involved in the penetration of antibodies into cells and FcγRs constitute one of the main effector mechanisms through which autoantibodies exert their action. In this review, we concentrate on the role of human FcγRs in autoantibodies penetration and summarize the current knowledge on the structure, ligand binding capacity and their role in autoimmunity and pathogenic effect of autoantibodies.

Regulation of mRNA caspase-8 levels by anti-nuclear autoantibodies

Apoptosis of the acinar and ductal epithelial cells of the salivary glands has been proposed as a mechanism possibly responsible for the impairment of the secretory function in Sjögren’s syndrome, an organ-specific autoimmune disorder characterized by destruction of these glandular structures. The presence of serum autoantibodies (Abs) directed against the ribonucleoproteic antigens Ro and La is one of the classification criteria used to identify Sjögren patients, and there is increasing evidence of the direct involvement of Abs in tissue pathogenesis. Our recent report demonstrated that anti-Ro and anti-La Abs are able to trigger the extrinsic pathway of apoptosis in the human salivary gland cells. To better understand how the anti-Ro and anti-La Abs exert their apoptotic effect, human caspase-8 gene expression was examined in primary human salivary gland epithelial cell (SGEC) cultures established from biopsies of labial minor salivary glands. To measure mRNA expression changes of initiating caspase-8, the real-time polymerase chain reaction was employed. This was combined with western blot to study the activation of caspase-8 detecting the cleaved form of caspase-8 and the cleaved poly (ADP-ribose) polymerase, downstream consequences of caspases activation. Data obtained suggest that the anti-Ro and anti-La Abs determine a transcriptional up-regulation and activation of caspase-8. Study of the mRNA in SGEC experimental model may provide insight into the signal transduction pathway stimulated by anti-nuclear autoantibodies.

Altered IkBα expression promotes NF-kB activation in monocytes from primary Sjögren’s syndrome patients

Aims: To study the importance of I&kgr;B&agr; in NF-&kgr;B signal transduction, we analysed the I&kgr;B&agr; expression in monocytes from Sjögrens syndrome (SS) patients versus healthy controls. Methods: Monocytes were obtained from the peripheral blood of 30 SS patients and 23 healthy subjects. I&kgr;B&agr; expression was studied by semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR), real-time PCR, immunoblotting, flow cytometry and enzyme linked immunosorbent assay (ELISA). Results: Analysis of the gene and protein expression profiles of SS monocytes revealed a down-regulation of I&kgr;B&agr;, and in all the Sjögrens syndrome cases examined, serum I&kgr;B&agr; levels were significantly decreased in comparison with controls. Conclusions: Our findings clearly demonstrate changes in the levels of I&kgr;B&agr; in SS monocytes, suggesting that the attenuated expression of I&kgr;B&agr; could contribute to the deregulation of NF-&kgr;B pathways in the SS pathogenesis. Decreased expression of I&kgr;B&agr; may specifically amplify cytokines production and inflammatory response linked to Sjögrens syndrome.

Saponins from Tribulus terrestris L. protect human keratinocytes from UVB-induced damage.

Chronic exposure to solar UVB radiation damages skin, increasing the risk to develop cancer. Hence the identification of compounds with a photoprotective efficacy is essential. This study examined the role of saponins derived from Tribulus terrestris L. (TT) on the modulation of apoptosis in normal human keratinocytes (NHEK) exposed to physiological doses of UVB and to evaluate their antitumoral properties. In NHEK, TT saponins attenuate UVB-induced programmed cell death through inhibition of intrinsic apoptotic pathway. In squamous cell carcinomas (SCC) TT saponins do not make the malignant keratinocytes more resistant to UVB and determine an enhanced apoptotic response. The photoprotective effect of TT saponins is tightly correlated to the enhancement of NER genes expression and the block of UVB-mediated NF-κB activation. Collectively, our study shows experimental evidence that TT has a preventive efficacy against UVB-induced carcinogenesis and the molecular knowledge on the mechanisms through which TT saponins regulate cell death suggests great potential for TT to be developed into a new medicine for cancer patients.

The metalloproteinase ADAM17 and the epidermal growth factor receptor (EGFR) signaling drive the inflammatory epithelial response in Sjögren’s syndrome

Primary Sjögren’s syndrome (pSS) is a chronic autoimmune disorder that particularly compromises the function of exocrine glands. The pathogenetic mechanisms of this autoimmune exocrinopathy have not been fully elucidated. Since increasing evidence actually suggests that the epidermal growth factor receptor (EGFR) pathway has a major impact on the inflammatory/immune reactions of the epithelial cells, in the apparent effort of enhancing innate immune defense while opposing overactivation of pro-inflammatory functions, the focus of the work presented here is clarify whether the EGFR–extracellular-signal-regulated kinase (ERK) pathway plays a role in the pro-inflammatory responses mounted by pSS salivary gland epithelial cells (SGEC). Investigations revealed that the EGFR-mediated activation of the downstream effectors ERK1/2 in pSS SGEC appeared to require ADAM17-dependent release of the endogenous EGFR ligand amphiregulin and transactivation of the EGFR. Moreover, blockade of amphiregulin bioactivity using a neutralizing Ab significantly reduced EGFR transactivation and ERK1/2 phosphorylation. In addition, pSS SGEC treated with the specific ADAM17 inhibitor TAPI-1 and with the EGFR inhibitor AG1478 exhibited deactivated AREG/EGFR/ERK signaling pathway and reduced pro-inflammatory cytokines released.

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.

A potential role of the GRO-α/CXCR2 system in Sjögren’s syndrome: regulatory effects of pro-inflammatory cytokines

Chemokines, small pro-inflammatory cytokines, are involved in migration of inflammatory cells in inflamed tissues and recent studies established their role in angiogenesis, hematopoiesis, cancer and autoimmune conditions. Growth related oncogene-alpha (GRO-α), a member of the CXC chemokine family, and its receptor CXCR2 are involved in the inflammatory processes. Since there is no previous report that supports a possible role of GRO-α/CXCR2 receptor complex during inflammation and neovascularization existing in the autoimmune disease Sjögren’s syndrome (SS), in this study, we examined CXCR2 and its ligand GRO-α expression in SS tissues. Immunohistochemistry revealed that GRO-α and its receptor CXCR2 were expressed at high levels in diseased tissues compared to healthy controls. In addition, human salivary gland epithelial cells (SGEC) cultures were submitted to a pro-inflammatory microenvironment using cytokines IL-6 and TNF-α in order to demonstrate that CXCR2 may change its initial expression pattern to another under inflammatory condition. The data show an increased expression of CXCR2 depending on the inflammatory cytokine used in culture in a time-dependent manner. Furthermore, silencing of the pro-angiogenic chemokine GRO-α is proportionally correlated with decreased expression of CXCR2 in pro-inflammatory cytokine-stimulated SGEC indicating the GRO-α/CXCR2 complex as a novel therapeutic target for the chronic inflammatory disease Sjögren’s syndrome.