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TNF blocker drugs modulate human TNF-α-converting enzyme pro-domain shedding induced by autoantibodies
Immunobiology 215 (2010) 874–883
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Immunobiology journal homepage: www.elsevier.de/imbio
TNF blocker drugs modulate human TNF-a-converting enzyme pro-domain shedding induced by autoantibodies$ Margherita Sisto a,n,1, Sabrina Lisi 1,a, Dario D. Lofrumento b, Simone Caprio c, Vincenzo Mitolo a, Massimo D’Amore d a
Department of Human Anatomy and Histology, section of Cell Biology, University of Bari Medical School, Bari, Italy Department of Biological and Environmental Sciences and Technologies, University of Salento, Lecce, Italy c Department of Odontostomatology and Surgery, University of Bari Medical school, Bari, Italy d Department of Internal Medicine and Public Medicine, Section of Rheumatology, University of Bari Medical School, Bari, Italy b
a r t i c l e in f o
a b s t r a c t
Article history: Received 6 October 2009 Received in revised form 27 November 2009 Accepted 28 November 2009
Novel biologic therapies targeted against specific components of the immune system, including blockade of TNF-a have revolutionized therapeutic approaches to inflammatory conditions and systemic inhibitors of TNF-a have been approved for the treatment of a wide variety of autoimmune diseases. No studies aimed to elucidate the effects of anti-TNF-a blockers on tumour necrosis factor-a convertase (TACE) expression and activation have yet been published. TACE is the principal protease involved in the activation of pro-TNF-a and is a target for anti-TNF-a therapy. Here we focused on regulation of TACE expression in human salivary gland epithelial cells (SGEC) treated by anti-Ro/SSA ¨ autoantibodies (autoAbs), characterizing primary Sjogren’s syndrome and on the effect of anti-Ro/SSA autoAbs on TACE pro-domain shedding and activation. To test the hypothesis that anti-TNF-a blocker drugs affect TACE expression, we used Adalimumab and Etanercept to block TNF-a and evaluate the effects of these biological agents on post-translational regulation of TACE. Anti-Ro/SSA autoAbs determines TACE pro-domain shedding suggesting that TACE activity is necessary for the release of TNF-a observed in anti-Ro/SSA autoAbs-stimulated cells. The comparative efficacy analysis of the regulation of TACE activity by Adalimumab and Etanercept revealed that Adalimumab appear to be significantly more efficacious than Etanercept in preventing TACE activation caused by anti-Ro/SSA autoAbs. It is intriguing to consider that regulation of TACE may participate in the pathogenic role of autoantibodies and the modulation of TACE expression by TNF-a antagonists might contribute to the beneficial effect of these drugs in inflammatory and autoimmune diseases. & 2009 Elsevier GmbH. All rights reserved.
Keywords: Adalimumab Autoantibodies Autoimmune disease Etanercept Shedding TACE
Introduction TNF-a is a crucial pro-inflammatory and immunoregulatory cytokine that is central to the pathogenesis of various inflammatory and autoimmune conditions. A number of controlled trials
Abbreviations: Anti-Ro/SSA AutoAbs, antinuclear autoantibodies in the blood directed against Ro/SSA; autoAbs, autoantibodies; ADAM, a disintegrin and metalloproteinase; FACS, Fluorescence-Activated Cell Sorting; Healthy IgG, H IgG, IgG obtained from the healthy volunteers; LSG, LSGs, labial salivary glands; M.F.I., mean fluorescence intensities; PBS, phosphate buffer saline; PCR, polymerase chain reaction; PMA, phorbol ester phorbol 12-myristate 13-acetate; pSS, primary ¨ ¨ Sjogren’s syndrome; Ro/SSA, Ro/Sjogren’s syndrome ribonucleoprotein A antigen; ¨ RT, reverse transcriptase; SGEC, salivary gland epithelial cell; SS, Sjogren’s syndrome; TNF-a, tumour necrosis factor-a, a proinflammatory cytokine; TACE, TNF-a converting proteolytic enzyme. $ This work was supported by grant (No.: 20216000056) from the Italian Ministry for Universities and Research. n Corresponding author. Tel.: + 39 080 547 8315; fax: +39 080 547 8327. E-mail address: [email protected] (M. Sisto). 1 Authors have equal contribution in this work and both are equally considered as ‘‘ first author’’. 0171-2985/$ - see front matter & 2009 Elsevier GmbH. All rights reserved. doi:10.1016/j.imbio.2009.11.005
have shown effectiveness for TNF-a inhibition and the strategies to block TNF-a have revolutionized the therapeutic approaches (Ackermann and Kavanaugh, 2007). Among the biological agents that neutralise the activity of TNF-a that have entered the clinic, Etanercept (Enbrel, Amgen and Wyeth Pharmaceutical) is a soluble TNF-a antagonist that competitively inhibits the interaction of TNF-a with cell-surface receptors and Adalimumab (Humira; Abbott Laboratories), is the first fully human (100% human peptide sequences) therapeutic monoclonal antibody that blocks TNF-a and is currently being evaluated in clinical trials (Furst et al., 2003; Ackermann and Kavanaugh, 2007; Ramos-Casals et al., 2008). The impaired interaction with receptors prevents TNF-a-mediated cellular responses and modulates the activity of other TNF-a-regulated pro-inflammatory cytokines (Ackermann and Kavanaugh, 2007). TACE (tumour necrosis factor-a convertase, ADAM-17), is the principal protease involved in the activation of pro-TNF-a (Black et al., 1997) and is a target for anti-TNF-a therapy because it processes membranebound precursor TNF to generate a soluble form of the cytokine that can act both in an autocrine manner and distally from the
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producing cells. The discovery of TACE permitted pharmaceutical companies to convert nonselective metalloproteinase inhibitors into selective compounds with drug-like properties (Moss et al., 2008). However, no studies aimed to elucidate the effects of antiTNF-a blockers on TACE expression and activation have yet been published. TACE, originally discovered as the processing protease of precursor TNF-a, is a type I transmembrane metalloproteinase involved in the shedding of the extracellular domain of several transmembrane proteins such as cytokines, growth factors, receptors or adhesion molecules imposing this enzyme as a centerpiece in mammalian cell biology (Black et al., 1997; Moss et al., 1997). It is synthesized as zymogen and contains a prodomain, catalytic domain, a disintegrin and cysteine-rich region, a transmembrane segment and a cytoplasmic tail (Moss et al., 1997; Killar et al., 1998) As for other proteolytically active metalloprotease/disintegrins, two forms of TACE are found in cells; a full-length precursor and a mature form lacking the pro¨ domain (Schlondorff et al., 2002). Pro-domain removal occurs in a late Golgi compartment, consistent with the proposed role of a furin-type proprotein convertase in this process (Peiretti et al., ¨ 2003). Mature TACE was detected on the cell surface (Schlondorff et al., 2002), but the mechanism by which TACE pro-domain shedding is regulated is poorly understood. Due to the firm evidence that TACE is the major TNF-a convertase, this enzyme has attracted considerable interest as a specific therapeutic target in diseases known to benefit from anti-TNF-a treatment including Crohn’s disease and ulcerative colitis collectively termed inflammatory bowel diseases (Brynskov et al., 2002). Healthy human salivary gland epithelial cells (SGEC), established from biopsies of labial minor salivary glands of healthy donors, produce the cell death-inducing cytokine TNF-a when treated with the antiRo/SSA autoantibodies (Sisto et al., 2008;Sisto et al., 2009). TNF-a stimulates TACE expression in human cells (Bzowska et al., 2004) and an increased knowledge of the physiological mechanisms regulating TACE is of particular interest to help understand their roles in disease conditions. Here we focused on regulation of TACE expression in SGEC treated by anti-Ro/SSA autoAbs, purified from IgG fractions of patients with primary ¨ Sjogren’s syndrome (pSS), and on the effect of anti-Ro/SSA autoAbs on TACE pro-domain shedding and activation. To test the hypothesis that anti-TNF-a blockers affect TACE expression, we used Adalimumab and Etanercept to block TNF-a and evaluate the effects of these biological agents on post-translational regulation of TACE.
Materials and methods Labial minor salivary glands cells culture and experimental design Labial minor salivary gland biopsies were obtained from ten healthy individuals awaiting removal of salivary mucoceles from the lower lip. Informed consent from the patients and approval by the local ethics committee were obtained. The healthy subjects had no complaints of oral dryness, no autoimmune disease and normal salivary function. Labial minor glands were harvested from the lower lip under local anaesthesia through normal mucosa. The explant outgrowth technique (Sens et al., 1985) was applied to establish non-neoplastic salivary gland epithelial cells cultures (SGEC) from limited amounts of glandular tissue. The cells were isolated from the labial glands by microdissection and collagenase (Worthington Diagnostic Division, Millipore, freehold, NJ, USA) digestion in physiological saline containing 1 mM Ca2 + . Following dispersal, cells were resuspended in McCoy’s 5a modified medium supplemented with 10% (v/v) fetal
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bovine serum, 1% (v/v) antibiotic solution, 2 mM L-Glutamine, 20 ng/ml (w/v) epidermal growth factor (EGF, Promega, Madison, WI), 0.5 mg/ml (w/v) insulin (Novo, Bagsvaerd, Denmark) and incubated at 37 1C, 5% CO2 in air for two weeks. Contaminating fibroblasts were selectively removed by treatment of the cultures with 0.02% EDTA. The epithelial origin of cultured cells was routinely confirmed by staining with monoclonal antibodies against epithelial-specific markers, including the various cytokeratins and epithelial membrane antigens and the absence of myoepithelial, fibroblastoid and lymphoid markers, using immunocytochemistry as previously described (Kapsogeorgou et al., 2001a; Kapsogeorgou et al., 2001b). For treatments SGEC were collected from culture media by centrifugation at 250g and resuspended at 1 106 cells/ml in McCoy’s 5a medium modified as indicated above. Cell suspension (105 cells/well) was added to each well of a 6-well plate (Thomas Scientific, Swedesboro, NJ, USA) and allowed to incubate for 24 h at 37 1C under 5% CO2. After incubation, cells were washed and treated as follows: (1) anti-Ro/SSA autoAbs (50 mg/ml) dissolved in McCoy’s 5a for 24 h; (2) IgG fractions extracted from sera of healthy donors (H IgG, 50 mg/ml) for 24 h; (3) Etanercept (50 mg/ml) for 24 h; (4) anti-Ro/ SSA autoAbs (50 mg/ml) plus 50 mg/ml of Etanercept for 24 h; (5) Adalimumab (50 mg/ml) for 24 h; (6) anti-Ro/SSA autoAbs (50 mg/ml) plus Adalimumab (50 mg/ml) for 24 h. Control was represented by untreated cells. Anti-Ro/SSA autoantibodies purification Anti-Ro/SSA autoAbs were purified from IgG fractions of patients with pSS, all fulfilling the American–European Consensus Group Classification criteria for SS (Vitali et al., 2002), using human Ro/SSA antigen-Sepharose 4B affinity columns as described (Lisi et al., 2007). The autoAbs obtained were concentrated to 200 mg/ml by ultrafiltration method (Amicon Ultra-4; Millipore Corporation, Bedford, MA, USA) and used at different concentrations in the experimental procedures. AutoAbs preparations resulted free of endotoxin contamination, as assessed by a Limulus amebocyte assay (Sigma, St. Louis, MO, USA). Assay for TACE expression RNA isolation, cDNA synthesis and PCR As first step, reverse transcription polymerase chain reaction (RT-PCR) was used to determine the optimal concentration of anti-Ro/SSA autoAbs to employ in the experimental procedure. SGEC were cultured in complete McCoy’s 5a modified medium at 37 1C in the presence of various concentrations of anti-Ro/SSA autoAbs (10, 25, 50 mg/ml). After 24 h of incubation, cells were collected, washed twice and RT-PCR for TACE mRNA expression was performed. Total RNA was extracted from SGEC using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions. DNase-treated RNA (2 mg) was reverse transcribed to cDNA using the Superscript First Strand cDNA Synthesis kit (Invitrogen), according to the manufacturer’s instructions. PCR was performed in a 50 ml reaction mixture composed of 2 mM of each sense and antisense primer, 1 PCR buffer, 2.4 mM MgCl2, 0.2 mM each dNTP, 10 ml of transcribed cDNA, and 0.04 U/ml Taq DNA polymerase. After the initial denaturation at 94 1C for 5 min, 35 cycles were used (denaturation at 94 1C for 30 s, annealing at 58 1C for 30 s, and extension at 72 1C for 1 min) followed by 10 min at 72 1C. Equal amounts of PCR products were run on a 1.5% agarose gel containing ethidium bromide. The expected size of PCR products is 495 bp for GADPH, 380 bp for TACE. The primers were designed according to published sequences (ref. source: www.ncbi.nlm.nih.gov). TACE
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amplification primers were sense 50 -ACTGGACCACCAGAGAATGG30 and antisense, 50 -GGCCAAACCACACAAGAACT-30 ; GADPH amplification primers were sense 50 -TTCACCACCATGGAGAAGGC-30 and antisense 50 -GGCATGGACTGTGGTCATGA-30 . The bands obtained were submitted to densitometric analysis using 1D Image Analyses Software (Kodak Digital Science, Rochester, NY). The results are expressed as arbitrary units. Once determined the optimal concentration of anti-Ro/SSA autoAbs to employ in the experimental procedure , the second step was determine the TACE mRNA expression after the various treatments performed on SGEC. RT-PCR was performed as above described. All RT-PCR products visualized by agarose gel electrophoresis in Tris-acetate-EDTA buffer (40 mM Tris, pH 8.0, 40 mM acetic acid, 2 mM EDTA) followed by ethidium bromide staining, were densitometrically analyzed, using gel image software (Bio-Profil Bio-1D; ltf Labortechnik GmbH, Wasserburg, Germany) after standardizing the amount of amplification product according to the expression of the housekeeping gene GADPH. Results were averaged from twelve sets of independent experiments and expressed as arbitrary units. The identities of all the amplified fragments were confirmed by sequencing. Real time PCR assay Total RNA from cultured cells were prepared and reverse transcription was conducted as described above. TaqMan expression assays, including fluorescent probes, forward and reverse primers for human TACE (Assays-On-Demand, Applied Biosystems, Foster City, CA, USA), and the internal control gene b-2 microglobulin (part no. 4326319E; b2M) were purchased from Applied Biosystems. All other reagents used in real-time PCR assays were purchased from the same manufacturer. Real-time quantitative PCR was performed in a 96-well microtiter plate with an ABI PRISM 7700 (Applied Biosystems). Each reaction contained 5 ml of cDNA template, 2.5 ml of 20 probe and primers mixture, 12.5 ml of TaqMan Universal PCR Master Mix, No AmpErase UNG (Applied Biosystems), in a total volume of 25 ml. Reactions were amplified for 40 cycles (65 1C for 5 min and 95 1C for 10 min, followed by 40 denaturation cycles at 95 1C for 15 s and annealing/extension at 58 1C for 1 min). The threshold was determined as 10 times the SD of the baseline fluorescence signal. The cycle number at the threshold was used as the threshold cycle (Ct). The different expression of TACE in untreated and treated samples was deducted from 2 DDCt where DDCt= DCt variously treated cells –DCt untreated cells. TACE protein expression TACE protein levels were determined in treated and untreated control cells. We adopted different approaches to investigate TACE protein expression in SGEC, in absence or presence of anti-Ro/SSA autoAbs stimulation and/or Adalimumab/Etanercept: immunofluorescence, Western blot and flow cytometry. In the immunofluorescence assay, variously treated SGEC were fixed for 10 min in 3.7% (v/v) paraformaldehyde diluted in PBS plus 0.1% (v/v) Triton X-100. After treatment with 0.2% (w/v) BSA (bovine serum albumin) in PBS for 10 min, to minimize non-specific autoAbsorption of antibodies, the coverslips were incubated, for 45 min at room temperature, with the goat anti-human TACE polyclonal Ab (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and for 30 min with the donkey anti-goat IgG-FITC (Santa Cruz Biotechnology). Finally, the slides were mounted and examined using a confocal laser scanning microscopy system (Leica, TCS-SP2, Germany) using a l = 488 nm Argon-Crypton laser for FITC. For Western blot, SGEC were washed twice, detached, collected and centrifuged at 600g for 10 min. The supernatant was removed and the pellet was incubated with lysis buffer [1% (v/v)
Triton X-100, 20 mM Tris–HCl, 137 mM NaCl, 10% (v/v) glycerol, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF), 20 mM leupeptin hemisulfate salt, 0.2 U/ml aprotinin] for 30 min on ice. After incubation, the obtained lysate was vortexed and then centrifuged at 12 800g for 10 min; the protein concentration in the supernatant was spectrophotometrically determined by Bradford’s protein assay, and the lysate was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins (25 mg/lane) and prestained standards (BioRad Laboratories, Hercules, CA, USA) were loaded on 10% SDSpolyacrylamide precast gels. After electrophoresis, the resolved proteins were transferred from gel to nitrocellulose membranes (Invitrogen). A blot buffer [20 mM Tris/150 mM glycine, pH 8, 20% (v/v) methanol] was used for gel and membrane saturation and blot. The blot conditions were the following: 200 mA (constant amperage), 200 V for 110 min. Blots were then blocked by PBS pH 7.2 with 0.1% (v/v) Tween 20, 5% w/v non-fat dried milk for 1 h and washed three times with 0.1% (v/v) Tween 20-PBS 1 (T-PBS). Membranes were then incubated for 90 min with goat anti-human TACE polyclonal Ab (Santa Cruz Biotechnology), and for 30 min with the donkey anti-goat IgG-HRP (Santa Cruz Biotechnology). Proteins recognized by the antibody were revealed using chemoluminescence luminal reagent (Santa Cruz Biotechnology) according to the protocol. As control for TACE protein expression, lysates from monocytes, obtained from healthy donors, treated or not with phorbol ester phorbol 12myristate 13-acetate (PMA) for 12 h, were submitted to the same blot condition of SGEC. Monocytes are used widely as a positive control for TACE expression and function (Doedens and Black, 2000; Edwards et al., 2008). The beta(s)-actin protein level was determined by Western blot and used as a protein loading control. Surface TACE staining was performed in SGEC using flow cytometric analysis. SGEC were incubated with monoclonal antihuman TACE-phycoerythrin Ab (R&D Systems, Minneapolis, MN USA). Intracellular TACE staining was performed in permeabilized cells using the Fluorescence-Activated Cell Sorting (FACS) permeabilizing solution and technique developed by BD Bioscences (BD Bioscences, San Jose, CA, USA). Surface and intracellular expression of TACE was analyzed by a Becton Dickinson (BD, Becton Dickinson, Germany) FACSCantotm II flow cytometer and BD FACS Diva software. Values are given as percentages of positive cells and M.F.I. Enzyme-linked immunosorbent assay The culture supernatants from cells cultured for 24 h in the absence or presence of anti-Ro/SSA autoAbs (50 mg/ml) and/or Adalimumab/Etanercept (50 mg/ml) were tested for TACE contents using commercial enzyme-linked immunosorbent assay (ELISA) kit (R&D, Abingdon, UK). A monoclonal antibody specific for TACE has been pre-coated onto a microplate. Standards and samples were pipetted into the wells and any TACE present in the supernatants was bound by the immobilized antibody. After washing away any unbound substances, an enzyme-linked monoclonal antibody specific for TACE was added to the wells. Following a wash to remove any unbound antibody–enzyme reagent, a substrate solution was added to the wells and color develops in proportion to the amount of TACE bound in the initial step. The color development was stopped and the intensity of the color was measured. Detection level for TACE was 10 pg/ml. Statistics The data were analyzed for normality using the Wilks Shapiro Test. Differences in means for paired observations were analyzed
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by Student’s t-test. In all instances values of p o0.05 were considered statistically significant.
demonstrated in our previous published works (Sisto et al., 2006; Lisi et al., 2007).
Results
Anti-Ro/SSA autoAbs determine TACE activation
Dose-dependent effects of anti-Ro/SSA AutoAbs on TACE mRNA
To investigate the potential effect of anti-Ro/SSA autoAbs treatment on TACE expression in SGEC several methods were employed. The flow cytometric analysis investigated whether anti-Ro/SSA autoAbs treatment could modulate the cellular localization of TACE protein. SGEC were cultured in presence of anti-Ro/SSA autoAbs for 24 h and membrane TACE (surface TACE) and intracellular TACE expression were assessed by using antihuman TACE monoclonal Ab. Fig. 2, examples of flow cytometric images from one representative experiment, demonstrated that control SGEC predominantly express TACE on the cell surface (95%71.3, mean fluorescence intensity (M.F.I.)= 70087150 for surface TACE versus 71% 71.7, M.F.I.=59807127 for intracellular
The studies performed indicated that the regulation of TACE mRNA expression in response to anti-Ro/SSA autoAbs is dosedependent, maximal effects of autoAbs being obtained for doses at 50 mg/ml (Fig. 1 A,B). The optimal concentration of 50 mg/ml for anti-Ro/SSA autoAbs was employed in all experimental procedures. When the SGEC were treated with IgG fractions extracted from sera of healthy donors (H IgG, 50 mg/ml), no changes in TACE mRNA expression was observed in comparison with untreated SGEC (Fig. 1 C,D) confirming the inability of H IgG to stimulate human salivary gland epithelial cells, as
Fig. 1. Dose-dependent effect of anti-Ro/SSA autoAbs treatment on TACE mRNA expression. A: RT-PCR analysis of TACE mRNA extracted from SGEC treated with growing concentrations of anti-Ro/SSA autoAbs. M, marker; Control = untreated SGEC. RT-PCR of GADPH was used as control. B: densitometric analysis (data represent the mean 7 SE of twelve independent experiments). C: RT-PCR analysis of TACE mRNA extracted from untreated control SGEC and SGEC treated with IgG fractions extracted from sera of healthy donors (H IgG). M, markers; Control = untreated SGEC. D: densitometric analysis (data represent the mean 7 SE of twelve independent experiments).
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Fig. 2. Flow cytometric analysis of membrane-bound and intracellular TACE expression in SGEC following anti-Ro/SSA autoAbs treatment. Examples of flow cytometric images from one representative experiment. Results obtained by flow cytometry were expressed as the relative mean fluorescence intensity (M.F.I.). Data represent the mean 7 SE of M.F.I. from ten independent experiments.
TACE, p o0.01). Surface TACE expression in untreated SGEC remained unchanged in the 24 h incubation period (M.F.I. =7008 7150). After 24 h following anti-Ro/SSA autoAbs treatment, a decrease of surface TACE (67%71.32, M.F.I.= 44107133) and an increase of intracellular TACE (98% 71.78, M.F.I.=158437156) were observed (p o0.01). Furthermore, 24 h following anti-Ro/SSA autoAbs treatment, an increase of TACE protein synthesis was observed as demonstrated by the mean of the M.F.I. (Fig. 2). To confirm the flow cytometric observations regarding TACE expression after 24 h of incubation with anti-Ro/SSA autoAbs, we investigated TACE intracellular localization in permeabilized cells by confocal microscopy (Fig. 3A) demonstrating that anti-Ro/SSA autoAbs treatment determines an increase of the diffuse cytoplasmic immunostaining of TACE in comparison with untreated control cells (Fig. 3A,a,b). In order to investigate the post-translational regulation of TACE following anti-Ro/SSA autoAbs treatment, the presence of TACE protein was analyzed by Western blotting (Fig. 3B). A band corresponding to a fulllength molecule containing the pro-domain was identified in untreated SGEC (110 kDa). Anti-Ro/SSA autoAbs treatment induces the appearance of TACE processed form devoid of pro-domain (85 kDa). Whether modulation of TACE protein expression, regulated by anti-Ro/SSA autoAbs treatment, was accompanied by the concomitant release of soluble TACE was assessed by ELISA in the conditioned media of anti-Ro/SSA autoAbs-treated SGEC. However, no detectable levels of soluble TACE were found (Fig. 3C), suggesting TACE internalization.
TACE is a target for Adalimumab and Etanercept When SGEC were treated with anti-Ro/SSA autoAbs plus Adalimumab/Etanercept a decreased intracellular TACE expression was observed and the immunofluorescence signal was reported to that observed in untreated SGEC (Fig. 3 A,e,g). In addition western blot revealed that, when treated with anti-Ro/ SSA autoAbs plus Adalimumab and anti-Ro/SSA autoAbs plus Etanercept, SGEC predominantly express the inactive proform of TACE (Fig. 3B). Flow cytometric analysis was employed to verify whether there is a difference in the regulation of TACE expression following Adalimumab or Etanercept treatment. After Adalimumab treatment, membrane-bound TACE levels in anti-Ro/ SSA autoAbs-treated SGEC were restored (94%71.53, M.F.I.=85407144 for anti-Ro/SSA autoAbs plus Adalimumabtreated SGEC versus 67% 71.32, M.F.I.=4410 7133 anti-Ro/SSA autoAbs-treated SGEC, p o0.01). Adalimumab, moreover, determined a remarkable significant (p o0.01) decrease of the mean fluorescence intensities (M.F.I. = 158437156 for anti-Ro/ SSA autoAbs-treated SGEC versus M.F.I.= 74077163 for anti-Ro/ SSA autoAbs plus Adalimumab-treated SGEC). Notably, treatment of SGEC with Adalimumab alone did not affect the surface and intracellular TACE levels in comparison with untreated control cells (93%71.27, M.F.I.= 73817139 for surface TACE in Adalimumab-treated SGEC versus 95% 71.3, M.F.I.=7008 7150 for surface TACE in untreated cells; 75%71.89, M.F.I.= 56917127 for intracellular TACE in
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Fig. 3. Expression and localization of TACE in SGEC before and after anti-Ro/SSA autoAbs and/or Adalimumab/Etanercept treatment. A: Confocal microscopy of TACE protein expression. Scale bar = 20 lm. B: Western blot analysis of TACE protein expression in SGEC before and after anti-Ro/SSA autoAbs and/or Adalimumab/Etanercept treatment. Monocytes treated or not with PMA were used as control cells. Visualized bands correspond to inactive and active TACE protein. The b-actin was used as a protein loading control. C: ELISA for soluble TACE. Standard: 100 pg/ml of recombinant human TACE 1: untreated control cells; 2: anti-Ro/SSA autoAbs-treated SGEC; 3: anti-Ro/SSA autoAbs plus Adalimumab-treated SGEC; 4: Adalimumab-treated SGEC; 5: anti-Ro/SSA autoAbs plus Etanercept-treated SGEC; 6: Etanercept-treated SGEC; (data represent the mean7 SE of five independent experiments)(reads were performed at k = 450 nm).
Adalimumab-treated SGEC versus 71%71.7, M.F.I.=5980 7127 in untreated cells) (Fig. 4). Flow cytometric analysis performed after Etanercept treatment revealed an increase of membranebound TACE levels and a decrease of intracellular TACE levels in anti-Ro/SSA autoAbs-treated SGEC (80%71.64, M.F.I.= 73457123 for surface TACE in anti-Ro/SSA autoAbs plus Etanercept-treated SGEC versus 67%71.32, M.F.I.= 44107133 anti-Ro/SSA autoAbs-treated SGEC, po0.01; 90%7155, M.F.I.= 72807152 for intracellular TACE in Etanercept-treated SGEC versus 98% 71.78, M.F.I.= 158437156 in untreated cells). As observed with Adalimumab, the treatment of SGEC with Etanercept alone did not affect the surface and intracellular TACE levels in comparison with untreated control cells (94% 71.37, M.F.I.=7290 7125 for surface TACE in Etanercept-treated SGEC versus 95%71.3, M.F.I.= 70087150 for surface TACE in untreated cells; 70%71.93, M.F.I.= 58247133 for intracellular TACE in Etanercept-treated SGEC versus 71% 71.7, M.F.I.= 59807127 in untreated cells) (Fig. 5).
Effect of Adalimumab and Etanercept on TACE mRNA For semiquantitative RT-PCR analysis, primary human SGEC cultures were treated with human anti-Ro/SSA autoAbs (50 mg/ml) and/or Adalimumab/Etanercept (50 mg/ml) for 24 h. As shown in Fig. 6(A,B) as compared to untreated cells, expression of TACE gene was increased (more than two fold) in anti-Ro/SSA autoAbs-treated SGEC. These changes are in agreement with results obtained by real time PCR (Fig. 6C) that showed that the TACE mRNA copy number in untreated control cells was lower than in anti-Ro/SSA autoAbs-treated cells. RT-PCR and real-time PCR analyses demonstrated that following Adalimumab and Etanercept treatment a significant decrease of TACE mRNA transcription was observed in comparison with mRNA levels in anti-Ro/SSA autoAbs-treated SGEC (p o0.01). In anti-Ro/SSA autoAbs-treated SGEC, the addition of Adalimumab determined a decrease in TACE mRNA copy number significantly higher than the decrease observed following the addition of Etanercept (p o0.01) (Fig. 6A–C).
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Fig. 4. Flow cytometric analysis of membrane-bound and intracellular TACE expression in SGEC following Anti-Ro/SSA autoAbs plus Adalimumab treatment. Examples of flow cytometric images from one representative experiment. Results obtained by flow cytometry were expressed as the relative mean fluorescence intensity (M.F.I.). Data represent the mean7 SE of M.F.I. from ten independent experiments.
Fig. 5. Flow cytometric analysis of membrane-bound and intracellular TACE expression in SGEC following anti-Ro/SSA autoAbs plus Etanercept treatment. Examples of flow cytometric images from one representative experiment. Results obtained by flow cytometry were expressed as the relative mean fluorescence intensity (M.F.I.). Data represent the mean7 SE of M.F.I. from ten independent experiments.
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Fig. 6. Effect of anti-Ro/SSA autoAbs and/or Adalimumab/Etanercept treatment on TACE mRNA expression. A: RT-PCR analysis of TACE mRNA expression. M, marker. RTPCR of GADPH was used as control. B: densitometric analysis of TACE mRNA expression. C: real-time PCR quantification of TACE mRNA expression. Representative histograms of mRNA levels of TACE in SGEC, untreated and treated with anti-Ro/SSA autoAbs and anti-Ro/SSA autoAbs plus Adalimumab/Etanercept. The mRNA levels of the housekeeping gene b-2 microglobulin were quantified between untreated control cells and the variously treated cells (mean 7 SE of five independent experiments).
Discussion Cytokines play important roles in inflammatory processes. ADAMs-mediated shedding of cytokines has been reported, and much attention has been given to the possible use of ADAMs inhibitors as therapeutics for various diseases. The most wellstudied member of the ADAMs family is TNF-a converting enzyme (TACE), since it is involved in the regulation of several key physiological and pathological processes (Black et al., 1997). The experiments described herein were undertaken in an attempt to demonstrate TACE expression on human SGEC and to elucidate the mechanisms by which the anti-Ro/SSA autoantibodies activate TACE pro-domain shedding. Anti-Ro/SSA autoAbs stimulation of human SGEC appeared to have an effect on TACE localization and expression, suggesting that increased TACE activity is necessary for the release of TNF-a observed in antiRo/SSA autoAbs-stimulated cells. Furthermore, herein, a regulation of TACE expression by the TNF-a antagonists Adalimumab and Etanercept in human SGEC was demonstrated. The compara-
tive efficacy analysis of the two TNF-a antagonists revealed that Adalimumab appear to be significantly more efficacious than Etanercept in preventing TACE activation caused by anti-Ro/SSA autoAbs. The presence of the TACE protein in human SGEC has not been previously described but this is not surprising in light of the ubiquitous expression of this sheddase in a variety of nonimmune human cells, including endothelial cells, small muscle cells, chondrocytes and human colonic epithelial cells (Black et al., 1997; Moss et al., 1997; Killar et al., 1998; Edwards et al., 2008). We used flow cytometric analysis and immunohistochemistry to access localization of TACE protein in human SGEC. A strong intensity of immune staining for TACE protein was observed, as expected, in these cells. These data were confirmed by Western blotting of cellular protein lysate showing that untreated SGEC express the inactive form of TACE protein. The fundamental nature of the biological processes controlled by TACE proteinase means that dysregulation of this enzyme expression will contribute to pathology. TACE have been
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implicated in cancer (Mochizuki et al., 2004), neurological (Tachida et al., 2008 and cardiovascular diseases (Morange et al., 2008), asthma (Trifilieff et al., 2002), and infection (Haga et al., 2008). In autoimmune diseases, up-regulation of TACE was recently demonstrated in inflammatory synovial tissue from patients with rheumatoid arthritis, in peripheral blood mononuclear cells of patients with multiple sclerosis (Patel et al., 1996; Ohta et al., 2001; Seifert et al., 2002) and an altered TACE activity in Crohn’s disease mucosa has been suggested (Brynskov et al., 2002). However, a correlation between autoantibodies production characterizing autoimmune diseases and TACE expression has not previously been investigated. To our knowledge, our report provides the first observation of a relation between TACE and pathogenic autoantibodies. Growing evidence indicates that expression of the TACE gene can be regulated. Increased levels of TACE mRNA were observed in HL-60 cells stimulated with lipopolysaccharide (Ding et al., 2001), in murine retinal endothelial cells exposed to vascular endothelial growth factor (Majka et al., 2002), and in osteoarthritis-and rheumatoid arthritis-affected cartilage (Patel et al., 1996). Our study demonstrated a dose-dependent increase of TACE mRNA expression in anti-Ro/SSA autoAbs-treated SGEC (Figs. 1,6). Since autoantibodies were shown to induce the increase of TACE mRNA synthesis, we could expect an increase in TACE protein concentration after treatment with anti-Ro/SSA autoAbs. This hypothesis was confirmed by the increase of the mean of the M.F.I. observed through flow cytometric analysis (Fig. 2). We next tested the possibility that anti-Ro/SSA autoAbs treatment of SGEC could determine TACE activation through pro-domain shedding. Western blotting revealed the mature form of enzyme in anti-Ro/SSA autoAbs-treated SGEC (Fig. 3). The mechanism by which TACE shedding was activated, following anti-Ro/SSA autoAbs treatment of SGEC, was investigated by analysing whether the treatment could modulate the cellular localization of TACE protein. Treatment of the cells with anti-Ro/ SSA autoAbs affected the cellular amount and distribution of TACE. In fact, as shown in Fig. 3, anti-Ro/SSA autoAbs treatment determines an increase of the diffuse cytoplasmic immunostaining of TACE, in comparison with untreated control cells. The different localization of TACE in response to autoantibodies suggests a TACE pro-domain removal stimulated by anti-Ro/SSA autoantibodies. If TACE pro-domain removal occurs in our experiments, we would expect to detect a cell-associated remaining transmembrane fragment after proteolysis. We compared the levels of TACE in lysates of unstimulated and anti-Ro/SSA autoAbs-stimulated cells by Western blot analysis performed with anti-human TACE antibody raised against the catalytic domain of TACE. A band of 110 kDa corresponding to the inactive TACE protein is clearly visible only in the lysates from untreated control SGEC. Instead, a band of 85 kDa, probably corresponding to the mature form of the protein, is only detectable in anti-Ro/SSA autoAbs–stimulated cells (Fig. 3). To detect the presence of any soluble fragment of TACE, an ELISA was performed on the conditioned media of anti-Ro/SSA autoAbstreated SGEC. However, no soluble forms of TACE were found, leading us to hypothesize that TACE might be internalized in response to autoantibodies. Based on these observations we speculate that the induction of TACE pro-domain shedding by anti-Ro/SSA autoAbs treatment might be due to a stimulated movement of inactive TACE from the membrane to the intracellular compartment, where pro-domain shedding occurs, followed by trafficking of active TACE from intracellular stores to the plasma membrane. Our current findings support the assertion of several authors that have suggested TACE internalization in response to cellular stimulation (Doedens and Black, 2000; Soond et al., 2005; Obeid et al., 2007. To further
address the question whether there is a correlation between pro-domain shedding, activation, TACE internalization and release of TNF-a protein observed in SGEC upon anti-Ro/SSA autoAbs treatment (Sisto et al., 2009), we used Adalimumab and Etanercept to block TNF-a and evaluated the effects of these biological agents on TACE mRNA and protein expression. In the present work we found that in anti-Ro/SSA autoAbs-treated SGEC, Adalimumab and Etanercept have an effect both on TACE gene and protein expression. These biological agents brought TACE mRNA and surface TACE expression to marginally higher levels than those observed in untreated SGEC, as shown by RT-PCR, real time PCR and flow cytometry (Figs. 4–6). These data were confirmed by immunohistochemistry and Western blot, demonstrating that, anti-Ro/SSA autoAbs plus Adalimumab/or Etanercept-treated SGEC express the inactive proform of TACE predominantly on the plasma membrane (Fig. 3). Results obtained revealed that Adalimumab appear to be significantly more efficacious than Etanercept in preventing TACE activation (Figs. 4–6). Taken together, the results obtained suggested the hypothesis that the effect of anti-Ro autoAbs on TACE expression and intracellular distribution in SGEC is exerted by TNF-a production. Thus, it is intriguing to consider that regulation of TACE expression may contribute to the pathogenic role of autoantibodies. Besides, we have shown that Adalimumab and Etanercept have an effect on TACE regulation in human SGEC. Regulation exerted by TNF-a antagonists on TACE, might contribute to their beneficial effect in inflammatory and autoimmune diseases. Further studies are needed to prove the clinical efficacy of Adalimumab, Etanercept and of other anti-TNF-a reagents in the relevant pathology.
Acknowledgements We are grateful to M.V.C. Pragnell, B.A., for critical reading of the manuscript.
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Contents lists available at ScienceDirect
Immunobiology journal homepage: www.elsevier.de/imbio
TNF blocker drugs modulate human TNF-a-converting enzyme pro-domain shedding induced by autoantibodies$ Margherita Sisto a,n,1, Sabrina Lisi 1,a, Dario D. Lofrumento b, Simone Caprio c, Vincenzo Mitolo a, Massimo D’Amore d a
Department of Human Anatomy and Histology, section of Cell Biology, University of Bari Medical School, Bari, Italy Department of Biological and Environmental Sciences and Technologies, University of Salento, Lecce, Italy c Department of Odontostomatology and Surgery, University of Bari Medical school, Bari, Italy d Department of Internal Medicine and Public Medicine, Section of Rheumatology, University of Bari Medical School, Bari, Italy b
a r t i c l e in f o
a b s t r a c t
Article history: Received 6 October 2009 Received in revised form 27 November 2009 Accepted 28 November 2009
Novel biologic therapies targeted against specific components of the immune system, including blockade of TNF-a have revolutionized therapeutic approaches to inflammatory conditions and systemic inhibitors of TNF-a have been approved for the treatment of a wide variety of autoimmune diseases. No studies aimed to elucidate the effects of anti-TNF-a blockers on tumour necrosis factor-a convertase (TACE) expression and activation have yet been published. TACE is the principal protease involved in the activation of pro-TNF-a and is a target for anti-TNF-a therapy. Here we focused on regulation of TACE expression in human salivary gland epithelial cells (SGEC) treated by anti-Ro/SSA ¨ autoantibodies (autoAbs), characterizing primary Sjogren’s syndrome and on the effect of anti-Ro/SSA autoAbs on TACE pro-domain shedding and activation. To test the hypothesis that anti-TNF-a blocker drugs affect TACE expression, we used Adalimumab and Etanercept to block TNF-a and evaluate the effects of these biological agents on post-translational regulation of TACE. Anti-Ro/SSA autoAbs determines TACE pro-domain shedding suggesting that TACE activity is necessary for the release of TNF-a observed in anti-Ro/SSA autoAbs-stimulated cells. The comparative efficacy analysis of the regulation of TACE activity by Adalimumab and Etanercept revealed that Adalimumab appear to be significantly more efficacious than Etanercept in preventing TACE activation caused by anti-Ro/SSA autoAbs. It is intriguing to consider that regulation of TACE may participate in the pathogenic role of autoantibodies and the modulation of TACE expression by TNF-a antagonists might contribute to the beneficial effect of these drugs in inflammatory and autoimmune diseases. & 2009 Elsevier GmbH. All rights reserved.
Keywords: Adalimumab Autoantibodies Autoimmune disease Etanercept Shedding TACE
Introduction TNF-a is a crucial pro-inflammatory and immunoregulatory cytokine that is central to the pathogenesis of various inflammatory and autoimmune conditions. A number of controlled trials
Abbreviations: Anti-Ro/SSA AutoAbs, antinuclear autoantibodies in the blood directed against Ro/SSA; autoAbs, autoantibodies; ADAM, a disintegrin and metalloproteinase; FACS, Fluorescence-Activated Cell Sorting; Healthy IgG, H IgG, IgG obtained from the healthy volunteers; LSG, LSGs, labial salivary glands; M.F.I., mean fluorescence intensities; PBS, phosphate buffer saline; PCR, polymerase chain reaction; PMA, phorbol ester phorbol 12-myristate 13-acetate; pSS, primary ¨ ¨ Sjogren’s syndrome; Ro/SSA, Ro/Sjogren’s syndrome ribonucleoprotein A antigen; ¨ RT, reverse transcriptase; SGEC, salivary gland epithelial cell; SS, Sjogren’s syndrome; TNF-a, tumour necrosis factor-a, a proinflammatory cytokine; TACE, TNF-a converting proteolytic enzyme. $ This work was supported by grant (No.: 20216000056) from the Italian Ministry for Universities and Research. n Corresponding author. Tel.: + 39 080 547 8315; fax: +39 080 547 8327. E-mail address: [email protected] (M. Sisto). 1 Authors have equal contribution in this work and both are equally considered as ‘‘ first author’’. 0171-2985/$ - see front matter & 2009 Elsevier GmbH. All rights reserved. doi:10.1016/j.imbio.2009.11.005
have shown effectiveness for TNF-a inhibition and the strategies to block TNF-a have revolutionized the therapeutic approaches (Ackermann and Kavanaugh, 2007). Among the biological agents that neutralise the activity of TNF-a that have entered the clinic, Etanercept (Enbrel, Amgen and Wyeth Pharmaceutical) is a soluble TNF-a antagonist that competitively inhibits the interaction of TNF-a with cell-surface receptors and Adalimumab (Humira; Abbott Laboratories), is the first fully human (100% human peptide sequences) therapeutic monoclonal antibody that blocks TNF-a and is currently being evaluated in clinical trials (Furst et al., 2003; Ackermann and Kavanaugh, 2007; Ramos-Casals et al., 2008). The impaired interaction with receptors prevents TNF-a-mediated cellular responses and modulates the activity of other TNF-a-regulated pro-inflammatory cytokines (Ackermann and Kavanaugh, 2007). TACE (tumour necrosis factor-a convertase, ADAM-17), is the principal protease involved in the activation of pro-TNF-a (Black et al., 1997) and is a target for anti-TNF-a therapy because it processes membranebound precursor TNF to generate a soluble form of the cytokine that can act both in an autocrine manner and distally from the
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producing cells. The discovery of TACE permitted pharmaceutical companies to convert nonselective metalloproteinase inhibitors into selective compounds with drug-like properties (Moss et al., 2008). However, no studies aimed to elucidate the effects of antiTNF-a blockers on TACE expression and activation have yet been published. TACE, originally discovered as the processing protease of precursor TNF-a, is a type I transmembrane metalloproteinase involved in the shedding of the extracellular domain of several transmembrane proteins such as cytokines, growth factors, receptors or adhesion molecules imposing this enzyme as a centerpiece in mammalian cell biology (Black et al., 1997; Moss et al., 1997). It is synthesized as zymogen and contains a prodomain, catalytic domain, a disintegrin and cysteine-rich region, a transmembrane segment and a cytoplasmic tail (Moss et al., 1997; Killar et al., 1998) As for other proteolytically active metalloprotease/disintegrins, two forms of TACE are found in cells; a full-length precursor and a mature form lacking the pro¨ domain (Schlondorff et al., 2002). Pro-domain removal occurs in a late Golgi compartment, consistent with the proposed role of a furin-type proprotein convertase in this process (Peiretti et al., ¨ 2003). Mature TACE was detected on the cell surface (Schlondorff et al., 2002), but the mechanism by which TACE pro-domain shedding is regulated is poorly understood. Due to the firm evidence that TACE is the major TNF-a convertase, this enzyme has attracted considerable interest as a specific therapeutic target in diseases known to benefit from anti-TNF-a treatment including Crohn’s disease and ulcerative colitis collectively termed inflammatory bowel diseases (Brynskov et al., 2002). Healthy human salivary gland epithelial cells (SGEC), established from biopsies of labial minor salivary glands of healthy donors, produce the cell death-inducing cytokine TNF-a when treated with the antiRo/SSA autoantibodies (Sisto et al., 2008;Sisto et al., 2009). TNF-a stimulates TACE expression in human cells (Bzowska et al., 2004) and an increased knowledge of the physiological mechanisms regulating TACE is of particular interest to help understand their roles in disease conditions. Here we focused on regulation of TACE expression in SGEC treated by anti-Ro/SSA autoAbs, purified from IgG fractions of patients with primary ¨ Sjogren’s syndrome (pSS), and on the effect of anti-Ro/SSA autoAbs on TACE pro-domain shedding and activation. To test the hypothesis that anti-TNF-a blockers affect TACE expression, we used Adalimumab and Etanercept to block TNF-a and evaluate the effects of these biological agents on post-translational regulation of TACE.
Materials and methods Labial minor salivary glands cells culture and experimental design Labial minor salivary gland biopsies were obtained from ten healthy individuals awaiting removal of salivary mucoceles from the lower lip. Informed consent from the patients and approval by the local ethics committee were obtained. The healthy subjects had no complaints of oral dryness, no autoimmune disease and normal salivary function. Labial minor glands were harvested from the lower lip under local anaesthesia through normal mucosa. The explant outgrowth technique (Sens et al., 1985) was applied to establish non-neoplastic salivary gland epithelial cells cultures (SGEC) from limited amounts of glandular tissue. The cells were isolated from the labial glands by microdissection and collagenase (Worthington Diagnostic Division, Millipore, freehold, NJ, USA) digestion in physiological saline containing 1 mM Ca2 + . Following dispersal, cells were resuspended in McCoy’s 5a modified medium supplemented with 10% (v/v) fetal
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bovine serum, 1% (v/v) antibiotic solution, 2 mM L-Glutamine, 20 ng/ml (w/v) epidermal growth factor (EGF, Promega, Madison, WI), 0.5 mg/ml (w/v) insulin (Novo, Bagsvaerd, Denmark) and incubated at 37 1C, 5% CO2 in air for two weeks. Contaminating fibroblasts were selectively removed by treatment of the cultures with 0.02% EDTA. The epithelial origin of cultured cells was routinely confirmed by staining with monoclonal antibodies against epithelial-specific markers, including the various cytokeratins and epithelial membrane antigens and the absence of myoepithelial, fibroblastoid and lymphoid markers, using immunocytochemistry as previously described (Kapsogeorgou et al., 2001a; Kapsogeorgou et al., 2001b). For treatments SGEC were collected from culture media by centrifugation at 250g and resuspended at 1 106 cells/ml in McCoy’s 5a medium modified as indicated above. Cell suspension (105 cells/well) was added to each well of a 6-well plate (Thomas Scientific, Swedesboro, NJ, USA) and allowed to incubate for 24 h at 37 1C under 5% CO2. After incubation, cells were washed and treated as follows: (1) anti-Ro/SSA autoAbs (50 mg/ml) dissolved in McCoy’s 5a for 24 h; (2) IgG fractions extracted from sera of healthy donors (H IgG, 50 mg/ml) for 24 h; (3) Etanercept (50 mg/ml) for 24 h; (4) anti-Ro/ SSA autoAbs (50 mg/ml) plus 50 mg/ml of Etanercept for 24 h; (5) Adalimumab (50 mg/ml) for 24 h; (6) anti-Ro/SSA autoAbs (50 mg/ml) plus Adalimumab (50 mg/ml) for 24 h. Control was represented by untreated cells. Anti-Ro/SSA autoantibodies purification Anti-Ro/SSA autoAbs were purified from IgG fractions of patients with pSS, all fulfilling the American–European Consensus Group Classification criteria for SS (Vitali et al., 2002), using human Ro/SSA antigen-Sepharose 4B affinity columns as described (Lisi et al., 2007). The autoAbs obtained were concentrated to 200 mg/ml by ultrafiltration method (Amicon Ultra-4; Millipore Corporation, Bedford, MA, USA) and used at different concentrations in the experimental procedures. AutoAbs preparations resulted free of endotoxin contamination, as assessed by a Limulus amebocyte assay (Sigma, St. Louis, MO, USA). Assay for TACE expression RNA isolation, cDNA synthesis and PCR As first step, reverse transcription polymerase chain reaction (RT-PCR) was used to determine the optimal concentration of anti-Ro/SSA autoAbs to employ in the experimental procedure. SGEC were cultured in complete McCoy’s 5a modified medium at 37 1C in the presence of various concentrations of anti-Ro/SSA autoAbs (10, 25, 50 mg/ml). After 24 h of incubation, cells were collected, washed twice and RT-PCR for TACE mRNA expression was performed. Total RNA was extracted from SGEC using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions. DNase-treated RNA (2 mg) was reverse transcribed to cDNA using the Superscript First Strand cDNA Synthesis kit (Invitrogen), according to the manufacturer’s instructions. PCR was performed in a 50 ml reaction mixture composed of 2 mM of each sense and antisense primer, 1 PCR buffer, 2.4 mM MgCl2, 0.2 mM each dNTP, 10 ml of transcribed cDNA, and 0.04 U/ml Taq DNA polymerase. After the initial denaturation at 94 1C for 5 min, 35 cycles were used (denaturation at 94 1C for 30 s, annealing at 58 1C for 30 s, and extension at 72 1C for 1 min) followed by 10 min at 72 1C. Equal amounts of PCR products were run on a 1.5% agarose gel containing ethidium bromide. The expected size of PCR products is 495 bp for GADPH, 380 bp for TACE. The primers were designed according to published sequences (ref. source: www.ncbi.nlm.nih.gov). TACE
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amplification primers were sense 50 -ACTGGACCACCAGAGAATGG30 and antisense, 50 -GGCCAAACCACACAAGAACT-30 ; GADPH amplification primers were sense 50 -TTCACCACCATGGAGAAGGC-30 and antisense 50 -GGCATGGACTGTGGTCATGA-30 . The bands obtained were submitted to densitometric analysis using 1D Image Analyses Software (Kodak Digital Science, Rochester, NY). The results are expressed as arbitrary units. Once determined the optimal concentration of anti-Ro/SSA autoAbs to employ in the experimental procedure , the second step was determine the TACE mRNA expression after the various treatments performed on SGEC. RT-PCR was performed as above described. All RT-PCR products visualized by agarose gel electrophoresis in Tris-acetate-EDTA buffer (40 mM Tris, pH 8.0, 40 mM acetic acid, 2 mM EDTA) followed by ethidium bromide staining, were densitometrically analyzed, using gel image software (Bio-Profil Bio-1D; ltf Labortechnik GmbH, Wasserburg, Germany) after standardizing the amount of amplification product according to the expression of the housekeeping gene GADPH. Results were averaged from twelve sets of independent experiments and expressed as arbitrary units. The identities of all the amplified fragments were confirmed by sequencing. Real time PCR assay Total RNA from cultured cells were prepared and reverse transcription was conducted as described above. TaqMan expression assays, including fluorescent probes, forward and reverse primers for human TACE (Assays-On-Demand, Applied Biosystems, Foster City, CA, USA), and the internal control gene b-2 microglobulin (part no. 4326319E; b2M) were purchased from Applied Biosystems. All other reagents used in real-time PCR assays were purchased from the same manufacturer. Real-time quantitative PCR was performed in a 96-well microtiter plate with an ABI PRISM 7700 (Applied Biosystems). Each reaction contained 5 ml of cDNA template, 2.5 ml of 20 probe and primers mixture, 12.5 ml of TaqMan Universal PCR Master Mix, No AmpErase UNG (Applied Biosystems), in a total volume of 25 ml. Reactions were amplified for 40 cycles (65 1C for 5 min and 95 1C for 10 min, followed by 40 denaturation cycles at 95 1C for 15 s and annealing/extension at 58 1C for 1 min). The threshold was determined as 10 times the SD of the baseline fluorescence signal. The cycle number at the threshold was used as the threshold cycle (Ct). The different expression of TACE in untreated and treated samples was deducted from 2 DDCt where DDCt= DCt variously treated cells –DCt untreated cells. TACE protein expression TACE protein levels were determined in treated and untreated control cells. We adopted different approaches to investigate TACE protein expression in SGEC, in absence or presence of anti-Ro/SSA autoAbs stimulation and/or Adalimumab/Etanercept: immunofluorescence, Western blot and flow cytometry. In the immunofluorescence assay, variously treated SGEC were fixed for 10 min in 3.7% (v/v) paraformaldehyde diluted in PBS plus 0.1% (v/v) Triton X-100. After treatment with 0.2% (w/v) BSA (bovine serum albumin) in PBS for 10 min, to minimize non-specific autoAbsorption of antibodies, the coverslips were incubated, for 45 min at room temperature, with the goat anti-human TACE polyclonal Ab (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and for 30 min with the donkey anti-goat IgG-FITC (Santa Cruz Biotechnology). Finally, the slides were mounted and examined using a confocal laser scanning microscopy system (Leica, TCS-SP2, Germany) using a l = 488 nm Argon-Crypton laser for FITC. For Western blot, SGEC were washed twice, detached, collected and centrifuged at 600g for 10 min. The supernatant was removed and the pellet was incubated with lysis buffer [1% (v/v)
Triton X-100, 20 mM Tris–HCl, 137 mM NaCl, 10% (v/v) glycerol, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF), 20 mM leupeptin hemisulfate salt, 0.2 U/ml aprotinin] for 30 min on ice. After incubation, the obtained lysate was vortexed and then centrifuged at 12 800g for 10 min; the protein concentration in the supernatant was spectrophotometrically determined by Bradford’s protein assay, and the lysate was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins (25 mg/lane) and prestained standards (BioRad Laboratories, Hercules, CA, USA) were loaded on 10% SDSpolyacrylamide precast gels. After electrophoresis, the resolved proteins were transferred from gel to nitrocellulose membranes (Invitrogen). A blot buffer [20 mM Tris/150 mM glycine, pH 8, 20% (v/v) methanol] was used for gel and membrane saturation and blot. The blot conditions were the following: 200 mA (constant amperage), 200 V for 110 min. Blots were then blocked by PBS pH 7.2 with 0.1% (v/v) Tween 20, 5% w/v non-fat dried milk for 1 h and washed three times with 0.1% (v/v) Tween 20-PBS 1 (T-PBS). Membranes were then incubated for 90 min with goat anti-human TACE polyclonal Ab (Santa Cruz Biotechnology), and for 30 min with the donkey anti-goat IgG-HRP (Santa Cruz Biotechnology). Proteins recognized by the antibody were revealed using chemoluminescence luminal reagent (Santa Cruz Biotechnology) according to the protocol. As control for TACE protein expression, lysates from monocytes, obtained from healthy donors, treated or not with phorbol ester phorbol 12myristate 13-acetate (PMA) for 12 h, were submitted to the same blot condition of SGEC. Monocytes are used widely as a positive control for TACE expression and function (Doedens and Black, 2000; Edwards et al., 2008). The beta(s)-actin protein level was determined by Western blot and used as a protein loading control. Surface TACE staining was performed in SGEC using flow cytometric analysis. SGEC were incubated with monoclonal antihuman TACE-phycoerythrin Ab (R&D Systems, Minneapolis, MN USA). Intracellular TACE staining was performed in permeabilized cells using the Fluorescence-Activated Cell Sorting (FACS) permeabilizing solution and technique developed by BD Bioscences (BD Bioscences, San Jose, CA, USA). Surface and intracellular expression of TACE was analyzed by a Becton Dickinson (BD, Becton Dickinson, Germany) FACSCantotm II flow cytometer and BD FACS Diva software. Values are given as percentages of positive cells and M.F.I. Enzyme-linked immunosorbent assay The culture supernatants from cells cultured for 24 h in the absence or presence of anti-Ro/SSA autoAbs (50 mg/ml) and/or Adalimumab/Etanercept (50 mg/ml) were tested for TACE contents using commercial enzyme-linked immunosorbent assay (ELISA) kit (R&D, Abingdon, UK). A monoclonal antibody specific for TACE has been pre-coated onto a microplate. Standards and samples were pipetted into the wells and any TACE present in the supernatants was bound by the immobilized antibody. After washing away any unbound substances, an enzyme-linked monoclonal antibody specific for TACE was added to the wells. Following a wash to remove any unbound antibody–enzyme reagent, a substrate solution was added to the wells and color develops in proportion to the amount of TACE bound in the initial step. The color development was stopped and the intensity of the color was measured. Detection level for TACE was 10 pg/ml. Statistics The data were analyzed for normality using the Wilks Shapiro Test. Differences in means for paired observations were analyzed
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by Student’s t-test. In all instances values of p o0.05 were considered statistically significant.
demonstrated in our previous published works (Sisto et al., 2006; Lisi et al., 2007).
Results
Anti-Ro/SSA autoAbs determine TACE activation
Dose-dependent effects of anti-Ro/SSA AutoAbs on TACE mRNA
To investigate the potential effect of anti-Ro/SSA autoAbs treatment on TACE expression in SGEC several methods were employed. The flow cytometric analysis investigated whether anti-Ro/SSA autoAbs treatment could modulate the cellular localization of TACE protein. SGEC were cultured in presence of anti-Ro/SSA autoAbs for 24 h and membrane TACE (surface TACE) and intracellular TACE expression were assessed by using antihuman TACE monoclonal Ab. Fig. 2, examples of flow cytometric images from one representative experiment, demonstrated that control SGEC predominantly express TACE on the cell surface (95%71.3, mean fluorescence intensity (M.F.I.)= 70087150 for surface TACE versus 71% 71.7, M.F.I.=59807127 for intracellular
The studies performed indicated that the regulation of TACE mRNA expression in response to anti-Ro/SSA autoAbs is dosedependent, maximal effects of autoAbs being obtained for doses at 50 mg/ml (Fig. 1 A,B). The optimal concentration of 50 mg/ml for anti-Ro/SSA autoAbs was employed in all experimental procedures. When the SGEC were treated with IgG fractions extracted from sera of healthy donors (H IgG, 50 mg/ml), no changes in TACE mRNA expression was observed in comparison with untreated SGEC (Fig. 1 C,D) confirming the inability of H IgG to stimulate human salivary gland epithelial cells, as
Fig. 1. Dose-dependent effect of anti-Ro/SSA autoAbs treatment on TACE mRNA expression. A: RT-PCR analysis of TACE mRNA extracted from SGEC treated with growing concentrations of anti-Ro/SSA autoAbs. M, marker; Control = untreated SGEC. RT-PCR of GADPH was used as control. B: densitometric analysis (data represent the mean 7 SE of twelve independent experiments). C: RT-PCR analysis of TACE mRNA extracted from untreated control SGEC and SGEC treated with IgG fractions extracted from sera of healthy donors (H IgG). M, markers; Control = untreated SGEC. D: densitometric analysis (data represent the mean 7 SE of twelve independent experiments).
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Fig. 2. Flow cytometric analysis of membrane-bound and intracellular TACE expression in SGEC following anti-Ro/SSA autoAbs treatment. Examples of flow cytometric images from one representative experiment. Results obtained by flow cytometry were expressed as the relative mean fluorescence intensity (M.F.I.). Data represent the mean 7 SE of M.F.I. from ten independent experiments.
TACE, p o0.01). Surface TACE expression in untreated SGEC remained unchanged in the 24 h incubation period (M.F.I. =7008 7150). After 24 h following anti-Ro/SSA autoAbs treatment, a decrease of surface TACE (67%71.32, M.F.I.= 44107133) and an increase of intracellular TACE (98% 71.78, M.F.I.=158437156) were observed (p o0.01). Furthermore, 24 h following anti-Ro/SSA autoAbs treatment, an increase of TACE protein synthesis was observed as demonstrated by the mean of the M.F.I. (Fig. 2). To confirm the flow cytometric observations regarding TACE expression after 24 h of incubation with anti-Ro/SSA autoAbs, we investigated TACE intracellular localization in permeabilized cells by confocal microscopy (Fig. 3A) demonstrating that anti-Ro/SSA autoAbs treatment determines an increase of the diffuse cytoplasmic immunostaining of TACE in comparison with untreated control cells (Fig. 3A,a,b). In order to investigate the post-translational regulation of TACE following anti-Ro/SSA autoAbs treatment, the presence of TACE protein was analyzed by Western blotting (Fig. 3B). A band corresponding to a fulllength molecule containing the pro-domain was identified in untreated SGEC (110 kDa). Anti-Ro/SSA autoAbs treatment induces the appearance of TACE processed form devoid of pro-domain (85 kDa). Whether modulation of TACE protein expression, regulated by anti-Ro/SSA autoAbs treatment, was accompanied by the concomitant release of soluble TACE was assessed by ELISA in the conditioned media of anti-Ro/SSA autoAbs-treated SGEC. However, no detectable levels of soluble TACE were found (Fig. 3C), suggesting TACE internalization.
TACE is a target for Adalimumab and Etanercept When SGEC were treated with anti-Ro/SSA autoAbs plus Adalimumab/Etanercept a decreased intracellular TACE expression was observed and the immunofluorescence signal was reported to that observed in untreated SGEC (Fig. 3 A,e,g). In addition western blot revealed that, when treated with anti-Ro/ SSA autoAbs plus Adalimumab and anti-Ro/SSA autoAbs plus Etanercept, SGEC predominantly express the inactive proform of TACE (Fig. 3B). Flow cytometric analysis was employed to verify whether there is a difference in the regulation of TACE expression following Adalimumab or Etanercept treatment. After Adalimumab treatment, membrane-bound TACE levels in anti-Ro/ SSA autoAbs-treated SGEC were restored (94%71.53, M.F.I.=85407144 for anti-Ro/SSA autoAbs plus Adalimumabtreated SGEC versus 67% 71.32, M.F.I.=4410 7133 anti-Ro/SSA autoAbs-treated SGEC, p o0.01). Adalimumab, moreover, determined a remarkable significant (p o0.01) decrease of the mean fluorescence intensities (M.F.I. = 158437156 for anti-Ro/ SSA autoAbs-treated SGEC versus M.F.I.= 74077163 for anti-Ro/ SSA autoAbs plus Adalimumab-treated SGEC). Notably, treatment of SGEC with Adalimumab alone did not affect the surface and intracellular TACE levels in comparison with untreated control cells (93%71.27, M.F.I.= 73817139 for surface TACE in Adalimumab-treated SGEC versus 95% 71.3, M.F.I.=7008 7150 for surface TACE in untreated cells; 75%71.89, M.F.I.= 56917127 for intracellular TACE in
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Fig. 3. Expression and localization of TACE in SGEC before and after anti-Ro/SSA autoAbs and/or Adalimumab/Etanercept treatment. A: Confocal microscopy of TACE protein expression. Scale bar = 20 lm. B: Western blot analysis of TACE protein expression in SGEC before and after anti-Ro/SSA autoAbs and/or Adalimumab/Etanercept treatment. Monocytes treated or not with PMA were used as control cells. Visualized bands correspond to inactive and active TACE protein. The b-actin was used as a protein loading control. C: ELISA for soluble TACE. Standard: 100 pg/ml of recombinant human TACE 1: untreated control cells; 2: anti-Ro/SSA autoAbs-treated SGEC; 3: anti-Ro/SSA autoAbs plus Adalimumab-treated SGEC; 4: Adalimumab-treated SGEC; 5: anti-Ro/SSA autoAbs plus Etanercept-treated SGEC; 6: Etanercept-treated SGEC; (data represent the mean7 SE of five independent experiments)(reads were performed at k = 450 nm).
Adalimumab-treated SGEC versus 71%71.7, M.F.I.=5980 7127 in untreated cells) (Fig. 4). Flow cytometric analysis performed after Etanercept treatment revealed an increase of membranebound TACE levels and a decrease of intracellular TACE levels in anti-Ro/SSA autoAbs-treated SGEC (80%71.64, M.F.I.= 73457123 for surface TACE in anti-Ro/SSA autoAbs plus Etanercept-treated SGEC versus 67%71.32, M.F.I.= 44107133 anti-Ro/SSA autoAbs-treated SGEC, po0.01; 90%7155, M.F.I.= 72807152 for intracellular TACE in Etanercept-treated SGEC versus 98% 71.78, M.F.I.= 158437156 in untreated cells). As observed with Adalimumab, the treatment of SGEC with Etanercept alone did not affect the surface and intracellular TACE levels in comparison with untreated control cells (94% 71.37, M.F.I.=7290 7125 for surface TACE in Etanercept-treated SGEC versus 95%71.3, M.F.I.= 70087150 for surface TACE in untreated cells; 70%71.93, M.F.I.= 58247133 for intracellular TACE in Etanercept-treated SGEC versus 71% 71.7, M.F.I.= 59807127 in untreated cells) (Fig. 5).
Effect of Adalimumab and Etanercept on TACE mRNA For semiquantitative RT-PCR analysis, primary human SGEC cultures were treated with human anti-Ro/SSA autoAbs (50 mg/ml) and/or Adalimumab/Etanercept (50 mg/ml) for 24 h. As shown in Fig. 6(A,B) as compared to untreated cells, expression of TACE gene was increased (more than two fold) in anti-Ro/SSA autoAbs-treated SGEC. These changes are in agreement with results obtained by real time PCR (Fig. 6C) that showed that the TACE mRNA copy number in untreated control cells was lower than in anti-Ro/SSA autoAbs-treated cells. RT-PCR and real-time PCR analyses demonstrated that following Adalimumab and Etanercept treatment a significant decrease of TACE mRNA transcription was observed in comparison with mRNA levels in anti-Ro/SSA autoAbs-treated SGEC (p o0.01). In anti-Ro/SSA autoAbs-treated SGEC, the addition of Adalimumab determined a decrease in TACE mRNA copy number significantly higher than the decrease observed following the addition of Etanercept (p o0.01) (Fig. 6A–C).
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Fig. 4. Flow cytometric analysis of membrane-bound and intracellular TACE expression in SGEC following Anti-Ro/SSA autoAbs plus Adalimumab treatment. Examples of flow cytometric images from one representative experiment. Results obtained by flow cytometry were expressed as the relative mean fluorescence intensity (M.F.I.). Data represent the mean7 SE of M.F.I. from ten independent experiments.
Fig. 5. Flow cytometric analysis of membrane-bound and intracellular TACE expression in SGEC following anti-Ro/SSA autoAbs plus Etanercept treatment. Examples of flow cytometric images from one representative experiment. Results obtained by flow cytometry were expressed as the relative mean fluorescence intensity (M.F.I.). Data represent the mean7 SE of M.F.I. from ten independent experiments.
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Fig. 6. Effect of anti-Ro/SSA autoAbs and/or Adalimumab/Etanercept treatment on TACE mRNA expression. A: RT-PCR analysis of TACE mRNA expression. M, marker. RTPCR of GADPH was used as control. B: densitometric analysis of TACE mRNA expression. C: real-time PCR quantification of TACE mRNA expression. Representative histograms of mRNA levels of TACE in SGEC, untreated and treated with anti-Ro/SSA autoAbs and anti-Ro/SSA autoAbs plus Adalimumab/Etanercept. The mRNA levels of the housekeeping gene b-2 microglobulin were quantified between untreated control cells and the variously treated cells (mean 7 SE of five independent experiments).
Discussion Cytokines play important roles in inflammatory processes. ADAMs-mediated shedding of cytokines has been reported, and much attention has been given to the possible use of ADAMs inhibitors as therapeutics for various diseases. The most wellstudied member of the ADAMs family is TNF-a converting enzyme (TACE), since it is involved in the regulation of several key physiological and pathological processes (Black et al., 1997). The experiments described herein were undertaken in an attempt to demonstrate TACE expression on human SGEC and to elucidate the mechanisms by which the anti-Ro/SSA autoantibodies activate TACE pro-domain shedding. Anti-Ro/SSA autoAbs stimulation of human SGEC appeared to have an effect on TACE localization and expression, suggesting that increased TACE activity is necessary for the release of TNF-a observed in antiRo/SSA autoAbs-stimulated cells. Furthermore, herein, a regulation of TACE expression by the TNF-a antagonists Adalimumab and Etanercept in human SGEC was demonstrated. The compara-
tive efficacy analysis of the two TNF-a antagonists revealed that Adalimumab appear to be significantly more efficacious than Etanercept in preventing TACE activation caused by anti-Ro/SSA autoAbs. The presence of the TACE protein in human SGEC has not been previously described but this is not surprising in light of the ubiquitous expression of this sheddase in a variety of nonimmune human cells, including endothelial cells, small muscle cells, chondrocytes and human colonic epithelial cells (Black et al., 1997; Moss et al., 1997; Killar et al., 1998; Edwards et al., 2008). We used flow cytometric analysis and immunohistochemistry to access localization of TACE protein in human SGEC. A strong intensity of immune staining for TACE protein was observed, as expected, in these cells. These data were confirmed by Western blotting of cellular protein lysate showing that untreated SGEC express the inactive form of TACE protein. The fundamental nature of the biological processes controlled by TACE proteinase means that dysregulation of this enzyme expression will contribute to pathology. TACE have been
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implicated in cancer (Mochizuki et al., 2004), neurological (Tachida et al., 2008 and cardiovascular diseases (Morange et al., 2008), asthma (Trifilieff et al., 2002), and infection (Haga et al., 2008). In autoimmune diseases, up-regulation of TACE was recently demonstrated in inflammatory synovial tissue from patients with rheumatoid arthritis, in peripheral blood mononuclear cells of patients with multiple sclerosis (Patel et al., 1996; Ohta et al., 2001; Seifert et al., 2002) and an altered TACE activity in Crohn’s disease mucosa has been suggested (Brynskov et al., 2002). However, a correlation between autoantibodies production characterizing autoimmune diseases and TACE expression has not previously been investigated. To our knowledge, our report provides the first observation of a relation between TACE and pathogenic autoantibodies. Growing evidence indicates that expression of the TACE gene can be regulated. Increased levels of TACE mRNA were observed in HL-60 cells stimulated with lipopolysaccharide (Ding et al., 2001), in murine retinal endothelial cells exposed to vascular endothelial growth factor (Majka et al., 2002), and in osteoarthritis-and rheumatoid arthritis-affected cartilage (Patel et al., 1996). Our study demonstrated a dose-dependent increase of TACE mRNA expression in anti-Ro/SSA autoAbs-treated SGEC (Figs. 1,6). Since autoantibodies were shown to induce the increase of TACE mRNA synthesis, we could expect an increase in TACE protein concentration after treatment with anti-Ro/SSA autoAbs. This hypothesis was confirmed by the increase of the mean of the M.F.I. observed through flow cytometric analysis (Fig. 2). We next tested the possibility that anti-Ro/SSA autoAbs treatment of SGEC could determine TACE activation through pro-domain shedding. Western blotting revealed the mature form of enzyme in anti-Ro/SSA autoAbs-treated SGEC (Fig. 3). The mechanism by which TACE shedding was activated, following anti-Ro/SSA autoAbs treatment of SGEC, was investigated by analysing whether the treatment could modulate the cellular localization of TACE protein. Treatment of the cells with anti-Ro/ SSA autoAbs affected the cellular amount and distribution of TACE. In fact, as shown in Fig. 3, anti-Ro/SSA autoAbs treatment determines an increase of the diffuse cytoplasmic immunostaining of TACE, in comparison with untreated control cells. The different localization of TACE in response to autoantibodies suggests a TACE pro-domain removal stimulated by anti-Ro/SSA autoantibodies. If TACE pro-domain removal occurs in our experiments, we would expect to detect a cell-associated remaining transmembrane fragment after proteolysis. We compared the levels of TACE in lysates of unstimulated and anti-Ro/SSA autoAbs-stimulated cells by Western blot analysis performed with anti-human TACE antibody raised against the catalytic domain of TACE. A band of 110 kDa corresponding to the inactive TACE protein is clearly visible only in the lysates from untreated control SGEC. Instead, a band of 85 kDa, probably corresponding to the mature form of the protein, is only detectable in anti-Ro/SSA autoAbs–stimulated cells (Fig. 3). To detect the presence of any soluble fragment of TACE, an ELISA was performed on the conditioned media of anti-Ro/SSA autoAbstreated SGEC. However, no soluble forms of TACE were found, leading us to hypothesize that TACE might be internalized in response to autoantibodies. Based on these observations we speculate that the induction of TACE pro-domain shedding by anti-Ro/SSA autoAbs treatment might be due to a stimulated movement of inactive TACE from the membrane to the intracellular compartment, where pro-domain shedding occurs, followed by trafficking of active TACE from intracellular stores to the plasma membrane. Our current findings support the assertion of several authors that have suggested TACE internalization in response to cellular stimulation (Doedens and Black, 2000; Soond et al., 2005; Obeid et al., 2007. To further
address the question whether there is a correlation between pro-domain shedding, activation, TACE internalization and release of TNF-a protein observed in SGEC upon anti-Ro/SSA autoAbs treatment (Sisto et al., 2009), we used Adalimumab and Etanercept to block TNF-a and evaluated the effects of these biological agents on TACE mRNA and protein expression. In the present work we found that in anti-Ro/SSA autoAbs-treated SGEC, Adalimumab and Etanercept have an effect both on TACE gene and protein expression. These biological agents brought TACE mRNA and surface TACE expression to marginally higher levels than those observed in untreated SGEC, as shown by RT-PCR, real time PCR and flow cytometry (Figs. 4–6). These data were confirmed by immunohistochemistry and Western blot, demonstrating that, anti-Ro/SSA autoAbs plus Adalimumab/or Etanercept-treated SGEC express the inactive proform of TACE predominantly on the plasma membrane (Fig. 3). Results obtained revealed that Adalimumab appear to be significantly more efficacious than Etanercept in preventing TACE activation (Figs. 4–6). Taken together, the results obtained suggested the hypothesis that the effect of anti-Ro autoAbs on TACE expression and intracellular distribution in SGEC is exerted by TNF-a production. Thus, it is intriguing to consider that regulation of TACE expression may contribute to the pathogenic role of autoantibodies. Besides, we have shown that Adalimumab and Etanercept have an effect on TACE regulation in human SGEC. Regulation exerted by TNF-a antagonists on TACE, might contribute to their beneficial effect in inflammatory and autoimmune diseases. Further studies are needed to prove the clinical efficacy of Adalimumab, Etanercept and of other anti-TNF-a reagents in the relevant pathology.
Acknowledgements We are grateful to M.V.C. Pragnell, B.A., for critical reading of the manuscript.
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