The Toll-Like Receptor Adaptor Proteins MyD88 and Mal/TIRAP Contribute to the Inflammatory and Destructive Processes in a Human Model of Rheumatoid Arthritis
2007; Elsevier BV; Volume: 170; Issue: 2 Linguagem: Inglês
10.2353/ajpath.2007.060657
ISSN1525-2191
AutoresSandra Sacre, Evangelos Andreakos, Serafim Kiriakidis, Parisa Amjadi, Anna M. Lundberg, Grey Giddins, Marc Feldmann, Fionula M. Brennan, Brian M. J. Foxwell,
Tópico(s)Immunotherapy and Immune Responses
ResumoThe widespread distribution of Toll-like receptors (TLRs) and their ligands raises the question whether they contribute to the production of inflammatory and tissue destructive molecules in rheumatoid arthritis (RA). We examined the expression and function of TLR2 and TLR4 and their downstream signaling adaptors MyD88 and Mal/TIRAP in synovial membrane cultures from RA tissue. Both TLR2 and TLR4 were detected by flow cytometry, and stimulation with TLR2 and TLR4 ligands augmented the spontaneous production of tumor necrosis factor-α, interleukin (IL)-6, and IL-8, indicating that TLR2 and TLR4 are functional in these cultures. In addition, overexpression of dominant-negative forms of MyD88 and Mal/TIRAP significantly down-regulated the spontaneous production of cytokines tumor necrosis factor-α, IL-6, and vascular endothelial growth factor, and enzymes MMP-1, MMP-2, MMP-3, and MMP-13 in RA synovial membrane cell cultures. Because TLR2 and TLR4 require both MyD88 and Mal/TIRAP for signaling, this study suggests that TLR function may regulate the expression of these factors in the RA synovium. Conditioned media from synovial membrane cell cultures stimulated human macrophages in a MyD88- and Mal-dependent manner, suggesting the release of a TLR ligand(s) from these cells. Thus, TLRs not only protect against infection but may also promote the inflammatory and destructive process in RA. The widespread distribution of Toll-like receptors (TLRs) and their ligands raises the question whether they contribute to the production of inflammatory and tissue destructive molecules in rheumatoid arthritis (RA). We examined the expression and function of TLR2 and TLR4 and their downstream signaling adaptors MyD88 and Mal/TIRAP in synovial membrane cultures from RA tissue. Both TLR2 and TLR4 were detected by flow cytometry, and stimulation with TLR2 and TLR4 ligands augmented the spontaneous production of tumor necrosis factor-α, interleukin (IL)-6, and IL-8, indicating that TLR2 and TLR4 are functional in these cultures. In addition, overexpression of dominant-negative forms of MyD88 and Mal/TIRAP significantly down-regulated the spontaneous production of cytokines tumor necrosis factor-α, IL-6, and vascular endothelial growth factor, and enzymes MMP-1, MMP-2, MMP-3, and MMP-13 in RA synovial membrane cell cultures. Because TLR2 and TLR4 require both MyD88 and Mal/TIRAP for signaling, this study suggests that TLR function may regulate the expression of these factors in the RA synovium. Conditioned media from synovial membrane cell cultures stimulated human macrophages in a MyD88- and Mal-dependent manner, suggesting the release of a TLR ligand(s) from these cells. Thus, TLRs not only protect against infection but may also promote the inflammatory and destructive process in RA. Rheumatoid arthritis (RA) is an autoimmune disease primarily characterized by synovial inflammation and destruction of cartilage and bone. Cytokines and matrix metalloproteinases (MMPs) play important roles in these processes, a fact highlighted by the clinical effectiveness of anti-cytokine biologicals (antibodies or soluble receptors) targeting tumor necrosis factor (TNF)-α, interleukin (IL)-1, and IL-6 receptor.1Feldmann M Brennan FM Maini RN Role of cytokines in rheumatoid arthritis.Annu Rev Immunol. 1996; 14: 397-440Crossref PubMed Scopus (2301) Google Scholar, 2Feldmann M Maini RN Anti-TNF alpha therapy of rheumatoid arthritis: what have we learned.Annu Rev Immunol. 2001; 19: 163-196Crossref PubMed Scopus (1154) Google Scholar However, it is still unclear what regulates cytokine production or triggers and prolongs the expression of inflammatory and tissue-destructive mediators in RA. Toll-like receptors (TLRs) recognize microbial products termed pathogen-associated molecular patterns in the response to infection. In humans, there are at least 10 TLRs that have different pathogen-associated molecular pattern specificities, eg, TLR4 for lipopolysaccharide (LPS), TLR2 for lipoproteins and TLR3, -7, and -8 for single- or double-stranded RNA. These ligands are potent inducers of inflammatory cytokines. The TLR signal transduction pathway that activates nuclear factor (NF)-κB shares many components with IL-1R signaling mechanisms, due to the common use of the signaling adaptor molecule MyD88 that binds to both TLRs and IL-1R. However, unlike the IL-1R family, some TLRs also require other TIR adaptors such as MAL/TIRAP (TLR2 and 4), TRIF (TLR3 and 4), and TRAM (TLR4) to function.3Kawai T Akira S Pathogen recognition with Toll-like receptors.Curr Opin Immunol. 2005; 17: 338-344Crossref PubMed Scopus (470) Google Scholar TLRs have also been reported to recognize a number of endogenous ligands, (eg, fibronectin fragments,4Okamura Y Watari M Jerud ES Young DW Ishizaka ST Rose J Chow JC Strauss JF The extra domain A of fibronectin activates Toll-like receptor 4.J Biol Chem. 2001; 276: 10229-10233Crossref PubMed Scopus (978) Google Scholar hyaluronan fragments,5Termeer C Benedix F Sleeman J Fieber C Voith U Ahrens T Miyake K Freudenberg M Galanos C Simon JC Oligosaccharides of hyaluronan activate dendritic cells via Toll-like receptor 4.J Exp Med. 2002; 195: 99-111Crossref PubMed Scopus (1160) Google Scholar self-mRNA,6Karikó K Ni H Capodici J Lamphier M Weissman D mRNA is an endogenous ligand for Toll-like receptor 3.J Biol Chem. 2004; 279: 12542-12550Crossref PubMed Scopus (828) Google Scholar HMGB17Park JS Gamboni-Robertson F He Q Svetkauskaite D Kim JY Strassheim D Sohn JW Yamada S Maruyama I Banerjee A Ishizaka A Abraham E High mobility group box 1 protein interacts with multiple Toll-like receptors.Am J Physiol. 2006; 290: C917-C924Crossref Scopus (785) Google Scholar). These potential danger signals would indicate tissue damage, are likely to be abundant in chronically inflamed tissue,8Brentano F Schorr O Gay RE Gay S Kyburz D RNA released from necrotic synovial fluid cells activates rheumatoid arthritis synovial fibroblasts via Toll-like receptor 3.Arthritis Rheum. 2005; 52: 2656-2665Crossref PubMed Scopus (308) Google Scholar, 9Roelofs MF Joosten LA Abdollahi-Roodsaz S van Lieshout AW Sprong T van den Hoogen FH van den Berg WB Radstake TR The expression of Toll-like receptors 3 and 7 in rheumatoid arthritis synovium is increased and costimulation of Toll-like receptors 3, 4, and 7/8 results in synergistic cytokine production by dendritic cells.Arthritis Rheum. 2005; 52: 2313-2322Crossref PubMed Scopus (272) Google Scholar and could potentially initiate or sustain an inflammatory response. There is considerable evidence from rodent models that activation of the TLRs can induce or exacerbate inflammatory arthritis.10Joosten LA Koenders MI Smeets RL Heuvelmans-Jacobs M Helsen MM Takeda K Akira S Lubberts E van de Loo FA van den Berg WB Toll-like receptor 2 pathway drives streptococcal cell wall-induced joint inflammation: critical role of myeloid differentiation factor 88.J Immunol. 2003; 171: 6145-6153PubMed Google Scholar However, its relevance to human disease is limited because all of these studies used microbial products such as LPS and mycobacterial DNA to induce arthritis. So far, data on any role for TLRs in RA have been circumstantial. In humans, infection of the joints induces strong immune responses that often lead to a destructive septic arthritis. In addition, activation of fibroblast-like synoviocytes with TLR ligands results in NF-κB activation and increased expression of inflammatory cytokines, chemokines, adhesion molecules, and MMPs.11Kyburz D Rethage J Seibl R Lauener R Gay RE Carson DA Gay S Bacterial peptidoglycans but not CpG oligodeoxynucleotides activate synovial fibroblasts by Toll-like receptor signaling.Arthritis Rheum. 2003; 48: 642-650Crossref PubMed Scopus (171) Google Scholar, 12Andreakos E Sacre SM Smith C Lundberg A Kiriakidis S Stonehouse T Monaco C Feldmann M Foxwell BM Distinct pathways of LPS-induced NF-{kappa}B activation and cytokine production in human myeloid and nonmyeloid cells defined by selective utilization of MyD88 and Mal/TIRAP.Blood. 2004; 103: 2229-2237Crossref PubMed Scopus (170) Google Scholar Interestingly, peptidoglycans and bacterial DNA derived from gut-colonizing bacteria have been detected in RA joints, but the relevance is unclear because they are also found in osteoarthritic joints.13van der Heijden IM Wilbrink B Tchetverikov I Schrijver IA Schouls LM Hazenberg MP Breedveld FC Tak PP Presence of bacterial DNA and bacterial peptidoglycans in joints of patients with rheumatoid arthritis and other arthritides.Arthritis Rheum. 2000; 43: 593-598Crossref PubMed Scopus (251) Google Scholar Immunohistological staining has detected TLR2 and TLR4 in the RA joint synovial tissue although, curiously, the Asp299Gly polymorphism that inactivates TLR4 function has been associated with RA susceptibility but not severity.14Radstake TR Franke B Hanssen S Netea MG Welsing P Barrera P Joosten LA van Riel PL van den Berg WB The Toll-like receptor 4 Asp299Gly functional variant is associated with decreased rheumatoid arthritis disease susceptibility but does not influence disease severity and/or outcome.Arthritis Rheum. 2004; 50: 999-1001Crossref PubMed Scopus (132) Google Scholar This study investigates whether there is a role for the TLRs in chronic inflammatory processes of RA. Using a human disease model of RA, total synovial tissue cultures,15Brennan FM Chantry D Jackson A Maini R Feldmann M Inhibitory effect of TNF alpha antibodies on synovial cell interleukin-1 production in rheumatoid arthritis.Lancet. 1989; 2: 244-247Abstract PubMed Scopus (826) Google Scholar, 16Brennan FM Chantry D Jackson AM Maini RN Feldmann M Cytokine production in culture by cells isolated from the synovial membrane.J Autoimmun. 1989; 2: S177-S186Crossref PubMed Scopus (100) Google Scholar we show that TLR2 and TLR4 are present and responsive to exogenous ligands. More importantly, we show that signaling mediated by the pan-TLR adaptor MyD88 and by Mal/TIRAP, which is used by TLR2 and TLR4, is involved in the spontaneous production of cytokines and MMPs in RA synovial membranes and that the RA membrane cell cultures release a factor(s) that can stimulate macrophages in a MyD88- and Mal-dependent manner. These data provide evidence, for the first time to our knowledge, that the TLR signaling system is involved in the pathogenesis of a human chronic inflammatory disease. Phenol-chloroform-purified Escherichia coli LPS and Pam3Cys-Ser-Lys4 (Pam3C) were purchased from Alexis (Nottingham, UK), and lipoteichoic acid (LTA) and peptidoglycan (PGN) were from Invivogen (San Diego, CA). The directly conjugated fluorescein isothiocyanate-labeled TLR2 and TLR4 antibodies used for fluorescence-activated cell sorting (FACS) analysis were purchased from Imgenex (San Diego, CA). Anti-CD3-PE and anti-CD68-PE and their isotype controls were purchased from Becton Dickinson (Oxford, UK), and IgG2a-fluorescein isothiocyanate was purchased from Abcam (Cambridge, UK). Recombinant, replication-deficient adenoviral vectors encoding β-galactosidase (Adβ-gal) or IκBα were kind gifts of Quantum Biotech (Canada) and Dr R. de Martin (University of Vienna, Vienna, Austria). Adenoviruses encoding dominant-negative forms of MyD88 (AdMyD88dn) and Mal/TIRAP (AdMal/TIRAPdn) and the GFP control (AdGFP) were constructed in-house.12Andreakos E Sacre SM Smith C Lundberg A Kiriakidis S Stonehouse T Monaco C Feldmann M Foxwell BM Distinct pathways of LPS-induced NF-{kappa}B activation and cytokine production in human myeloid and nonmyeloid cells defined by selective utilization of MyD88 and Mal/TIRAP.Blood. 2004; 103: 2229-2237Crossref PubMed Scopus (170) Google Scholar All viruses used in this study are E1/E3 deleted, belong to the Ad5 serotype, and have been previously used in other studies.12Andreakos E Sacre SM Smith C Lundberg A Kiriakidis S Stonehouse T Monaco C Feldmann M Foxwell BM Distinct pathways of LPS-induced NF-{kappa}B activation and cytokine production in human myeloid and nonmyeloid cells defined by selective utilization of MyD88 and Mal/TIRAP.Blood. 2004; 103: 2229-2237Crossref PubMed Scopus (170) Google Scholar, 17Senftleben U Cao Y Xiao G Greten FR Krahn G Bonizzi G Chen Y Hu Y Fong A Sun SC Karin M Activation by IKKalpha of a second, evolutionary conserved, NF-kappa B signaling pathway.Science. 2001; 293: 1495-1499Crossref PubMed Scopus (1137) Google Scholar, 18Wrighton CJ Hofer-Warbinek R Moll T Eytner R Bach FH de Martin R Inhibition of endothelial cell activation by adenovirus-mediated expression of I kappa B alpha, an inhibitor of the transcription factor NF-kappa B.J Exp Med. 1996; 183: 1013-1022Crossref PubMed Scopus (230) Google Scholar, 19Oitzinger W Hofer-Warbinek R Schmid JA Koshelnick Y Binder BR de Martin R Adenovirus-mediated expression of a mutant IkappaB kinase 2 inhibits the response of endothelial cells to inflammatory stimuli.Blood. 2001; 97: 1611-1617Crossref PubMed Scopus (72) Google Scholar, 20Smith C Andreakos E Crawley JB Brennan FM Feldmann M Foxwell BM NF-kappaB-inducing kinase is dispensable for activation of NF-kappaB in inflammatory settings but essential for lymphotoxin beta receptor activation of NF-kappaB in primary human fibroblasts.J Immunol. 2001; 167: 5895-5903Crossref PubMed Scopus (73) Google Scholar, 21Andreakos E Smith C Monaco C Brennan FM Foxwell BM Feldmann M Ikappa B kinase 2 but not NF-kappa B-inducing kinase is essential for effective DC antigen presentation in the allogeneic mixed lymphocyte reaction.Blood. 2003; 101: 983-991Crossref PubMed Scopus (66) Google Scholar Viruses were propagated in 293 human embryonic kidney cells (American Type Culture Collection, Rockville, MD), purified by ultracentrifugation through two cesium chloride gradients, and viral titers determined by plaque assay as previously described.22Graham FL Prevec L Methods for construction of adenovirus vectors.Mol Biotechnol. 1995; 3: 207-220Crossref PubMed Scopus (437) Google Scholar Human macrophages were derived from monocytes after differentiation for 4 days with 100 ng/ml M-CSF as previously described.23Foxwell B Browne K Bondeson J Clarke C de Martin R Brennan F Feldmann M Efficient adenoviral infection with IkappaB alpha reveals that macrophage tumor necrosis factor alpha production in rheumatoid arthritis is NF-kappaB dependent.Proc Natl Acad Sci USA. 1998; 95: 8211-8215Crossref PubMed Scopus (240) Google Scholar RA synovial membrane cells were isolated from patients undergoing joint replacement surgery as previously described.15Brennan FM Chantry D Jackson A Maini R Feldmann M Inhibitory effect of TNF alpha antibodies on synovial cell interleukin-1 production in rheumatoid arthritis.Lancet. 1989; 2: 244-247Abstract PubMed Scopus (826) Google Scholar, 16Brennan FM Chantry D Jackson AM Maini RN Feldmann M Cytokine production in culture by cells isolated from the synovial membrane.J Autoimmun. 1989; 2: S177-S186Crossref PubMed Scopus (100) Google Scholar The study was approved by the Riverside Research ethics committee, and waste tissue (synovium after joint replacement surgery) was obtained only after receiving signed informed consent from the patient and anonymyzing the tissue to protect patient identity. Immediately after isolation, cells were used for mRNA analysis, stained by FACS, or cultured at 1 × 105 cells/well in 96-well tissue culture plates (Falcon, Becton Dickinson) in RPMI 1640 containing 10% (v/v) fetal bovine serum and 100 U/ml penicillin/streptomycin. For exogenous stimulation experiments, RA membrane cell cultures were incubated with 20 μg/ml LTA, 10 μg/ml PGN, 10 ng/ml Pam3Cys-Ser-Lys4, or 10 ng/ml LPS and supernatants collected after 24 hours. For adenoviral gene transfer experiments, cells were incubated with adenoviral vectors at a multiplicity of infection of 100, washed after 2 hours, and cultured in complete medium for 48 hours, at which time supernatants were collected. In all cases, viability of the cells was not significantly affected throughout this time period when examined by the MTT cell viability assay (Sigma, Poole, UK).24Mosmann T Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays.J Immunol Methods. 1983; 65: 55-63Crossref PubMed Scopus (46517) Google Scholar Supernatants were subsequently examined for the presence of cytokines and MMPs by enzyme-linked immunosorbent assay (ELISA). Total RA synovial membrane cells were plated at 2.5 × 106 cells/well in six-well tissue culture plates (Falcon) and were either uninfected or infected with adenovirus. Cytosolic proteins were obtained and subsequently separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on a 10% (w/v) polyacrylamide gel and transferred onto polyvinylidene difluoride membrane for Western blotting. Antibodies for MyD88 and IκBα were purchased from QED Bioscience Inc. (San Diego, CA) and Santa Cruz Biotechnology (Santa Cruz, CA), respectively. Rabbit polyclonal antibody directed against the N-terminal peptide MASSTSLPAPGSRPK of human Mal/TIRAP was designed in our laboratory.12Andreakos E Sacre SM Smith C Lundberg A Kiriakidis S Stonehouse T Monaco C Feldmann M Foxwell BM Distinct pathways of LPS-induced NF-{kappa}B activation and cytokine production in human myeloid and nonmyeloid cells defined by selective utilization of MyD88 and Mal/TIRAP.Blood. 2004; 103: 2229-2237Crossref PubMed Scopus (170) Google Scholar Supernatants were analyzed for cytokine levels by ELISA according to the manufacturer's instructions. TNF-α, IL-1β, IL-6, IL-8, and vascular endothelial growth factor (VEGF) ELISAs were purchased from Pharmingen (Becton Dickinson). MMP-1, MMP-2, MMP-3, and MMP-13 ELISAs were purchased from Amersham (Buckinghamshire, UK). Absorbance was read on a spectrophotometric ELISA plate reader (Labsystems Multiscan Biochromic) and analyzed using the Ascent software program (Thermo Labsystems, Altrincham, UK). RNA was isolated using a RNA blood isolation kit (Qiagen, Valencia, CA) according to the manufacturer's instructions and then treated with turbo DNase (Ambion, Austin, TX) according to manufacturer's instructions. Total RNA was reverse-transcribed with Superscript II RNase H− reverse transcriptase and oligo(dT) primer (Life Technologies, Inc., Grand Island, NY). For human TLR2 amplification, the primers 5′-GCCAAAGTCTTGATTGATTGG-3′ and 5′-TTGAAGTTCTCCAGCTCCTG-3′ were used. For human TLR4, the primers 5′-TGGATACGTTTCCTTATAAG-3′ and 5′-GAAATGGAGGCACCCCTTC-3′ were used. Subsequent PCR amplification consisted of 35 cycles with an annealing temperature of 62°C for TLR2 and 58°C for TLR4 and performed in a Dyad PCR machine (MJ Instruments, Waltham, MA). Macrophages cultured in a 96-well plate were infected with recombinant adenovirus containing a NF-κB luciferase reporter gene (kindly provided by Dr. B. Davidson, University of Iowa, Ames, IA) at a multiplicity of infection of 50:1. The cells were rested at least 4 hours before an additional infection with AdGFP, AdMyD88dn, or AdMaldn at a multiplicity of infection of 100:1. After 24 hours, cells were stimulated for 6 hours with filtered RA supernatants. The cells were washed once in phosphate-buffered saline (PBS) and lysed with 100 μl of CAT lysis buffer [0.65% (v/v) of Nonidet P-40, 10 mmol/L Tris-HCl, pH 8, 0.1 mmol/L ethylenediaminetetraacetic acid, pH 8, and 150 mmol/L NaCl]. Fifty μl of cell lysate were mixed with 120 μl of luciferase assay buffer [25 mmol/L Tris-phosphate, pH 7.8, 8 mmol/L MgCl2, 1 mmol/L ethylenediaminetetraacetic acid, 1% (v/v) Triton X-100, 1% (v/v) glycerol, 1 mmol/L dithiothreitol, and 0.5 mmol/L ATP] in the well of a luminometer cuvette strip. Luciferase activity was measured with a luminometer (Thermo Labsystems) by adding 30 μl of luciferin (Bright-Glo luciferase assay system; Promega, Madison, WI) per assay point. Cells were washed, fixed in 2% paraformaldehyde, and then blocked with 10% human serum (PAA, Pasching, Austria) in PBS containing 0.01% azide for 30 minutes at 4°C with or without 0.1% saponin (Sigma, St. Louis, MO) for intracellular or cell surface staining, respectively. Cells were then incubated with α-TLR2, α-TLR4, α-CD3, α-CD68, or isotype control antibodies for 1 hour at 4°C and then washed before analysis on a Becton-Dickinson LSR flow cytometer. Mean, SD, SEM, and statistical tests were calculated using GraphPad version 3 (GraphPad Software Inc., San Diego, CA). For statistical analysis of parametric data, a one-tailed Student's t-test for normally distributed data were used. For nonparametric data, a one-tailed Wilcoxon signed rank test was applied. Because most of the endogenous TLR ligands so far described have been for TLR2 and TLR4, the expression of these receptors in RA synovial membrane cells was examined. TLR2 and TLR4 mRNA was detected by RT-PCR in all four RA patients examined (Figure 1A). The presence of TLR2 and TLR4 was also detected by FACS. In addition to the cell surface expression, considerable staining was also found intracellularly, particularly for TLR2 (Figure 1B), in contrast to the belief that these TLRs are mostly cell surface localized. An analysis of the major cell populations showed that macrophages (CD68+) almost universally expressed both TLRs (Figure 1C), whereas a considerable proportion of CD68− cells (mainly fibroblasts and T cells) did not express TLR2 and/or TLR4 (Figure 1C). Further analysis showed that the TLR2/4-negative population was mostly confined to CD3+ T cells (data not shown). The addition of the TLR2 ligands, Pam3Cys-Ser-Lys4 (PAM3), PGN, or LTA, and the TLR4 ligand LPS consistently yielded a significant twofold to fourfold increase in the production of TNF-α and IL-8, above that spontaneously produced by these cultures (Figure 2, A and B). For IL-6, the effect of exogenous TLRs was less pronounced, with only PGN (TLR2) and LPS (TLR4) recording a significant increase (Figure 2C). Because the data above showed that functional TLR2 and TLR4 are expressed in RA synovial membranes, the contribution of signaling by these receptors to the endogenous production of inflammatory cytokines and MMPs was examined. This study was performed using adenoviral gene transfer of dominant-negative inhibitory forms of MyD88 and Mal/TIRAP. These constructs have been previously used to examine TLR4 signaling in primary human cells,12Andreakos E Sacre SM Smith C Lundberg A Kiriakidis S Stonehouse T Monaco C Feldmann M Foxwell BM Distinct pathways of LPS-induced NF-{kappa}B activation and cytokine production in human myeloid and nonmyeloid cells defined by selective utilization of MyD88 and Mal/TIRAP.Blood. 2004; 103: 2229-2237Crossref PubMed Scopus (170) Google Scholar and this combination of adaptors are used by TLR2 and TLR4.25Horng T Barton GM Flavell RA Medzhitov R The adaptor molecule TIRAP provides signalling specificity for Toll-like receptors.Nature. 2002; 420: 329-333Crossref PubMed Scopus (685) Google Scholar, 26Yamamoto M Sato S Hemmi H Sanjo H Uematsu S Kaisho T Hoshino K Takeuchi O Kobayashi M Fujita T Takeda K Akira S Essential role for TIRAP in activation of the signalling cascade shared by TLR2 and TLR4.Nature. 2002; 420: 324-329Crossref PubMed Scopus (817) Google Scholar An adenoviral construct for IκBα previously used in RA tissue27Andreakos E Smith C Kiriakidis S Monaco C de Martin R Brennan FM Paleolog E Feldmann M Foxwell BM Heterogeneous requirement of IkappaB kinase 2 for inflammatory cytokine and matrix metalloproteinase production in rheumatoid arthritis: implications for therapy.Arthritis Rheum. 2003; 48: 1901-1912Crossref PubMed Scopus (69) Google Scholar was the positive control because all of the parameters examined have been shown to be NF-κB-dependent.27Andreakos E Smith C Kiriakidis S Monaco C de Martin R Brennan FM Paleolog E Feldmann M Foxwell BM Heterogeneous requirement of IkappaB kinase 2 for inflammatory cytokine and matrix metalloproteinase production in rheumatoid arthritis: implications for therapy.Arthritis Rheum. 2003; 48: 1901-1912Crossref PubMed Scopus (69) Google Scholar, 28Bondeson J Foxwell B Brennan F Feldmann M Defining therapeutic targets by using adenovirus: blocking NF-kappaB inhibits both inflammatory and destructive mechanisms in rheumatoid synovium but spares anti-inflammatory mediators.Proc Natl Acad Sci USA. 1999; 96: 5668-5673Crossref PubMed Scopus (200) Google Scholar A viral construct expressing β-galactosidase was used as the negative control. Adenoviral gene transfer into RA synovial membrane cultures led to several fold higher levels of expression of MyD88dn and Maldn compared with the endogenous levels (Figure 3A), as previously described for IκBα.28Bondeson J Foxwell B Brennan F Feldmann M Defining therapeutic targets by using adenovirus: blocking NF-kappaB inhibits both inflammatory and destructive mechanisms in rheumatoid synovium but spares anti-inflammatory mediators.Proc Natl Acad Sci USA. 1999; 96: 5668-5673Crossref PubMed Scopus (200) Google Scholar The expression of these constructs had a profound effect on the production of most inflammatory cytokines measured. Thus, MyD88dn resulted in a statistically significant but variable decrease in the spontaneous production of TNF-α (28 ± 15% inhibition, P < 0.05), IL-6 (73 ± 13% inhibition, P < 0.05), IL-8 (67 ± 14% inhibition, P < 0.01), and VEGF (44 ± 16% inhibition, P < 0.01) but not IL-1β (28 ± 22% inhibition, P > 0.05), probably attributable to the wider scatter of IL-1 production (Figure 3, B–F). Expression of Maldn also resulted in a similar inhibition of the spontaneous TNF-α (33 ± 16% inhibition, P < 0.05), IL-6 production (70 ± 14% inhibition, P < 0.05), IL-8 (48 ± 23% inhibition, P < 0.01), and VEGF (40 ± 21% inhibition, P < 0.05) but again not IL-1β (20 ± 35% inhibition, P > 0.05). IκBα was used as a positive control and resulted in a statistically significant inhibition in TNF-α (48 ± 14% inhibition, P < 0.05), IL-1β (47 ± 10% inhibition, P < 0.05), IL-6 (94 ± 1% inhibition, P < 0.05), IL-8 (72 ± 16% inhibition, P < 0.05), and VEGF (59 ± 9% inhibition, P < 0.01) (Figure 3, B–F), confirming previous studies.27Andreakos E Smith C Kiriakidis S Monaco C de Martin R Brennan FM Paleolog E Feldmann M Foxwell BM Heterogeneous requirement of IkappaB kinase 2 for inflammatory cytokine and matrix metalloproteinase production in rheumatoid arthritis: implications for therapy.Arthritis Rheum. 2003; 48: 1901-1912Crossref PubMed Scopus (69) Google Scholar, 28Bondeson J Foxwell B Brennan F Feldmann M Defining therapeutic targets by using adenovirus: blocking NF-kappaB inhibits both inflammatory and destructive mechanisms in rheumatoid synovium but spares anti-inflammatory mediators.Proc Natl Acad Sci USA. 1999; 96: 5668-5673Crossref PubMed Scopus (200) Google Scholar Given the evidence above that MyD88 and Mal were required for part of the inflammatory cytokine production from enzymatically dispersed synovial membrane cultures, the effect of the same constructs was assessed on the spontaneous expression of MMP-1 (collagenase-1), MMP-2 (gelatinase A), MMP-3 (stromelysin-1), and MMP-13 (collagenase-3), four important enzymes considered to be involved in the tissue destruction and remodeling in RA. We found that MyD88dn significantly inhibited MMP-1 (51 ± 13% inhibition, P < 0.05), MMP-2 (72 ± 8% inhibition, P < 0.05), MMP-3 (54 ± 20% inhibition, P < 0.01), and MMP-13 (67 ± 8% inhibition, P < 0.01) (Figure 4, A–D). Likewise, Maldn significantly inhibited MMP-1 (48 ± 18% inhibition, P < 0.05), MMP-2 (61 ± 14% inhibition, P < 0.05), MMP-3 (52 ± 20% inhibition, P < 0.01), and MMP-13 (68 ± 10% inhibition, P < 0.01) (Figure 4, A–D). The inhibitory effect on MMPs of MyD88dn and Maldn parallels that seen by IκBα overexpression (Figure 4, A–D). The fact that Mal/TIRAP is a specific adaptor for TLR2 and TLR4 signaling and is not involved in IL-1R signaling29Fitzgerald KA Palsson-McDermott EM Bowie AG Jefferies CA Mansell AS Brady G Brint E Dunne A Gray P Harte MT McMurray D Smith DE Sims JE Bird TA O'Neill LA Mal (MyD88-adapter-like) is required for Toll-like receptor-4 signal transduction.Nature. 2001; 413: 78-83Crossref PubMed Scopus (1001) Google Scholar, 30Horng T Barton GM Medzhitov R TIRAP: an adapter molecule in the Toll signaling pathway.Nat Immunol. 2001; 2: 835-841Crossref PubMed Scopus (829) Google Scholar suggests that TLR signaling contributes to MMP expression in the RA synovium. To determine whether a potential TLR ligand(s) was released from the synovial cell cultures, supernatants were collected from cultures after 24 hours and filtered to remove any cell debris. These supernatants were tested for LPS and found to be free from contamination. Supernatants were used to stimulate M-CSF-derived macrophages expressing a consensus sequence NF-κB reporter gene and either a control GFP construct or one containing the MyD88dn or the Maldn. NF-κB activity was measured because the supernatants used to stimulate the macrophages would contain cytokines. The supernatants induced activation of NF-κB that was inhibited by MyD88dn (80 ± 10%, P < 0.01) and by Maldn (57 ± 17%, P < 0.05), suggesting the presence of a TLR ligand in the conditioned media (Figure 5). Stimulation of macrophages with conditioned media harvested from M-CSF-derived macrophages after 24 hours was unable to stimulate NF-κB activation in unrelated macrophages (data not shown). TNF-α, IL-1, and more recently, IL-6, are recognized as effective targets for the treatment of RA. However, the stimuli that drive the production of these cytokines in the disease, specifically within the synovium are still unclear. This study is the first to provide evidence that TLRs may be involved in regulating inflammatory cytokine production in human RA disease tissue. In addition, it is the first to indicate that the production of MMPs involved in destructive processes could also be dependent o
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