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Toll-like receptor 2 (TLR2) induces migration and invasive mechanisms in rheumatoid arthritis.

McGarry T, Veale DJ, Gao W, Orr C, Fearon U, Connolly M - Arthritis Res. Ther. (2015)

Bottom Line: Pam3CSK4 specifically induced β1-integrin binding in RASFC (p <0.05), with no effect observed for β2-4, β6, αvβ5 or α5β1.Finally, blockade of TLR2 with OPN301 significantly decreased spontaneous release of MMP-3, MMP-2 and MMP-9 and increased TIMP-3 secretion from RA synovial explant cultures (p <0.05).TLR2 activation induces migrational and invasive mechanisms, which are critically involved in the pathogenesis of RA, suggesting TLR2 as a potential therapeutic target for the treatment of RA.

View Article: PubMed Central - PubMed

Affiliation: Department of Rheumatology, St. Vincent's University Hospital, Elm Park, Dublin Academic Health Care and The Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Belfield, Dublin 4, Ireland. trudy.mc-garry@ucdconnect.ie.

ABSTRACT

Introduction: This study investigates the role of Toll-like receptor 2 (TLR2) in the regulation of migratory and invasive mechanisms in rheumatoid arthritis (RA).

Methods: Invasion, migration, matrix metalloproteinase (MMP)-1, -3 and tissue inhibitor of matrix metalloproteinase-3 (TIMP-3) expression, β-integrin binding, cytoskeletal rearrangement and Ras-related C3 botulinum toxin substrate 1 (Rac1) activation in response to a TLR2-ligand, Pam3CSK4 (1 μg/ml), in ex vivo RA synovial tissue explants, primary RA synovial fibroblasts (RASFC) and microvascular endothelial cells (HMVEC) were assessed by Transwell Matrigel™ invasion chambers, enzyme-linked immunosorbent assay (ELISA), multiplex adhesion binding assay, reverse transcription polymerase chain reaction (RT-PCR), F-actin immunofluorescent staining, matrigel synovial outgrowths, Rac1 pull-down assays/Western blot and zymography. β1-integrin expression in RA/control synovial tissue was assessed by immunohistology. The effect of Pam3CSK4 on cell migration, invasion, MMP-3 and Rac1 activation was examined in the presence or absence of anti-β1-integrin (10 μg/ml) or anti-IgG control (10 μg/ml). The effect of an anti-TLR-2 mAb (OPN301)(1 μg/ml) or immunoglobulin G (IgG) control (1 μg/ml) on RASFC migration and RA synovial tissue MMP activity was assessed by wound assays, ELISA and zymography.

Results: Pam3CSK4 significantly induced cell migration, invasion, MMP-1, MMP-3, MMP-2 and MMP-9 expression and induced the MMP-1/TIMP-3 and MMP-3/TIMP-3 ratio in RASFC and explants (p <0.05). β1-integrin expression was significantly higher in RA synovial tissue compared to controls (p <0.05). Pam3CSK4 specifically induced β1-integrin binding in RASFC (p <0.05), with no effect observed for β2-4, β6, αvβ5 or α5β1. Pam3CSK4 increased β1-integrin mRNA expression, Rac1 activation, RASFC outgrowths and altered cytoskeletal dynamic through induction of filopodia formation. Pam3CSK4-regulated cell migration and invasion processes, but not MMP-3, were inhibited in the presence of anti-β1-integrin (p <0.05), with no effect observed for anti-IgG control. Furthermore, anti-β1-integrin inhibited Pam3CSK4-induced Rac1 activation. Finally, blockade of TLR2 with OPN301 significantly decreased spontaneous release of MMP-3, MMP-2 and MMP-9 and increased TIMP-3 secretion from RA synovial explant cultures (p <0.05). Incubation of RASFC with OPN301 RA ex vivo conditioned media inhibited migration and invasion compared to IgG control.

Conclusions: TLR2 activation induces migrational and invasive mechanisms, which are critically involved in the pathogenesis of RA, suggesting TLR2 as a potential therapeutic target for the treatment of RA.

No MeSH data available.


Related in: MedlinePlus

Representative photomicrograph demonstrating cells repopulating the wound in response to Pam3CSK4 (1 μg/ml) in RASFC and HMVEC (i). Bar graph quantifying RASFC and HMVEC (both n = 4) 24 h migration in response to Pam3CSK4 (ii). Data is represented as mean ± SEM, *p <0.05, significantly different to control. HMVEC human microvascular endothelial cells, RASFC rheumatoid arthritis synovial fibroblast cells
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Fig1: Representative photomicrograph demonstrating cells repopulating the wound in response to Pam3CSK4 (1 μg/ml) in RASFC and HMVEC (i). Bar graph quantifying RASFC and HMVEC (both n = 4) 24 h migration in response to Pam3CSK4 (ii). Data is represented as mean ± SEM, *p <0.05, significantly different to control. HMVEC human microvascular endothelial cells, RASFC rheumatoid arthritis synovial fibroblast cells

Mentions: RASFC and HMVEC monolayers were wounded and stimulated in the presence or absence of Pam3CSK4 (1 μg/ml). Figure 1i shows representative images of wound repair in response to Pam3CSK4 for RASFC and HMVEC, where repopulation of wound margins was observed. Semi-quantitative analysis demonstrated a significant increase in cell migration in response to Pam3CSK4 (Fig. 1ii).Fig. 1


Toll-like receptor 2 (TLR2) induces migration and invasive mechanisms in rheumatoid arthritis.

McGarry T, Veale DJ, Gao W, Orr C, Fearon U, Connolly M - Arthritis Res. Ther. (2015)

Representative photomicrograph demonstrating cells repopulating the wound in response to Pam3CSK4 (1 μg/ml) in RASFC and HMVEC (i). Bar graph quantifying RASFC and HMVEC (both n = 4) 24 h migration in response to Pam3CSK4 (ii). Data is represented as mean ± SEM, *p <0.05, significantly different to control. HMVEC human microvascular endothelial cells, RASFC rheumatoid arthritis synovial fibroblast cells
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4495696&req=5

Fig1: Representative photomicrograph demonstrating cells repopulating the wound in response to Pam3CSK4 (1 μg/ml) in RASFC and HMVEC (i). Bar graph quantifying RASFC and HMVEC (both n = 4) 24 h migration in response to Pam3CSK4 (ii). Data is represented as mean ± SEM, *p <0.05, significantly different to control. HMVEC human microvascular endothelial cells, RASFC rheumatoid arthritis synovial fibroblast cells
Mentions: RASFC and HMVEC monolayers were wounded and stimulated in the presence or absence of Pam3CSK4 (1 μg/ml). Figure 1i shows representative images of wound repair in response to Pam3CSK4 for RASFC and HMVEC, where repopulation of wound margins was observed. Semi-quantitative analysis demonstrated a significant increase in cell migration in response to Pam3CSK4 (Fig. 1ii).Fig. 1

Bottom Line: Pam3CSK4 specifically induced β1-integrin binding in RASFC (p <0.05), with no effect observed for β2-4, β6, αvβ5 or α5β1.Finally, blockade of TLR2 with OPN301 significantly decreased spontaneous release of MMP-3, MMP-2 and MMP-9 and increased TIMP-3 secretion from RA synovial explant cultures (p <0.05).TLR2 activation induces migrational and invasive mechanisms, which are critically involved in the pathogenesis of RA, suggesting TLR2 as a potential therapeutic target for the treatment of RA.

View Article: PubMed Central - PubMed

Affiliation: Department of Rheumatology, St. Vincent's University Hospital, Elm Park, Dublin Academic Health Care and The Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Belfield, Dublin 4, Ireland. trudy.mc-garry@ucdconnect.ie.

ABSTRACT

Introduction: This study investigates the role of Toll-like receptor 2 (TLR2) in the regulation of migratory and invasive mechanisms in rheumatoid arthritis (RA).

Methods: Invasion, migration, matrix metalloproteinase (MMP)-1, -3 and tissue inhibitor of matrix metalloproteinase-3 (TIMP-3) expression, β-integrin binding, cytoskeletal rearrangement and Ras-related C3 botulinum toxin substrate 1 (Rac1) activation in response to a TLR2-ligand, Pam3CSK4 (1 μg/ml), in ex vivo RA synovial tissue explants, primary RA synovial fibroblasts (RASFC) and microvascular endothelial cells (HMVEC) were assessed by Transwell Matrigel™ invasion chambers, enzyme-linked immunosorbent assay (ELISA), multiplex adhesion binding assay, reverse transcription polymerase chain reaction (RT-PCR), F-actin immunofluorescent staining, matrigel synovial outgrowths, Rac1 pull-down assays/Western blot and zymography. β1-integrin expression in RA/control synovial tissue was assessed by immunohistology. The effect of Pam3CSK4 on cell migration, invasion, MMP-3 and Rac1 activation was examined in the presence or absence of anti-β1-integrin (10 μg/ml) or anti-IgG control (10 μg/ml). The effect of an anti-TLR-2 mAb (OPN301)(1 μg/ml) or immunoglobulin G (IgG) control (1 μg/ml) on RASFC migration and RA synovial tissue MMP activity was assessed by wound assays, ELISA and zymography.

Results: Pam3CSK4 significantly induced cell migration, invasion, MMP-1, MMP-3, MMP-2 and MMP-9 expression and induced the MMP-1/TIMP-3 and MMP-3/TIMP-3 ratio in RASFC and explants (p <0.05). β1-integrin expression was significantly higher in RA synovial tissue compared to controls (p <0.05). Pam3CSK4 specifically induced β1-integrin binding in RASFC (p <0.05), with no effect observed for β2-4, β6, αvβ5 or α5β1. Pam3CSK4 increased β1-integrin mRNA expression, Rac1 activation, RASFC outgrowths and altered cytoskeletal dynamic through induction of filopodia formation. Pam3CSK4-regulated cell migration and invasion processes, but not MMP-3, were inhibited in the presence of anti-β1-integrin (p <0.05), with no effect observed for anti-IgG control. Furthermore, anti-β1-integrin inhibited Pam3CSK4-induced Rac1 activation. Finally, blockade of TLR2 with OPN301 significantly decreased spontaneous release of MMP-3, MMP-2 and MMP-9 and increased TIMP-3 secretion from RA synovial explant cultures (p <0.05). Incubation of RASFC with OPN301 RA ex vivo conditioned media inhibited migration and invasion compared to IgG control.

Conclusions: TLR2 activation induces migrational and invasive mechanisms, which are critically involved in the pathogenesis of RA, suggesting TLR2 as a potential therapeutic target for the treatment of RA.

No MeSH data available.


Related in: MedlinePlus