Session Title: Vasculitis
Session Type: Abstract Submissions (ACR)
Activation of dendritic cells (DCs) is one of the earliest inciting events in Giant Cell Arteritis (GCA). TLR 2 is expressed on DCs in normal temporal arteries and is also found ubiquitously throughout the macro vasculature. 1 Stimulating temporal arteries implanted into SCID mice with TLR 2 activates dendritic cells. 2 Therefore TLR 2 is likely to play a major role in the pathogenesis of GCA while the exact mechanisms involved are yet to be fully elucidated.
This study examines the functional effects of TLR 2 on induction of pro-inflammatory cytokines, angiogenesis and cell migration in GCA.
15 patients with biopsy proven GCA and meeting 1990 ACR classification criteria for GCA were prospectively recruited. To directly examine the effects of TLR 2 on pro-inflammatory cytokines, growth factors and gelatinase expression in GCA, ex-vivo temporal artery (TA) whole tissue explant models were established. PBMCs and TA explants were cultured in the presence of Pam3CSK4 (a TLR 2 agonist) (1μg/ml) for 24 hours. Supernatants were harvested and assayed for IL-6, IL-8 Ang2, and VEGF by ELISA and MMP-2 and MMP-9 by gelatin zymography. Endothelial cell tube formation was assessed following culture with TLR 2-induced TA explant conditioned media. To examine the effect of TLR 2 on migration/invasion in GCA, TA explants were embedded in Matrigel, stimulated with Pam3CSK4 and myofibroblast outgrowths observed. Myofibroblasts were also isolated from TA explants, cultured and wound repair assays performed. To examine the effects of TLR 2 on cytoskeletal architecture, cultured myofibroblasts were treated with Pam3CSK4 and stained for F-actin.
In PBMC cultures, Pam3CSK4 induced a 7.7 and 3.6 fold increase in expression of IL6 and IL8 respectively, from a basal IL-6 level of 34.58 ± 6.77 pg/ml to 266.1 ± 117.6 pg /ml and basal IL-8 level of 1769 ± 731.7 pg/ml to 6388 ± 1632 pg/ml.
In temporal artery explant cultures stimulation with Pam3CSK4 increased expression of IL- 6 from 26,800 ± 9209 pg/ml to 47494 ± 10,946pg/ml (p=0.01). Expression of IL-8 was also increased from basal levels of 20,593 ± 8224 pg/ml to 40,793 ± 16, 670 pg/ml (p=0.058).
Stimulation with Pam3CSK4 significantly increased Ang 2 expression from basal levels of 727.9 ± 254.5 pg/ml to 1415 ± 459.6 pg/ml (p=0.011). There was a trend towards increased expression of VEGF, from basal levels of 107.5 ± 33.38 pg/ml to 200 ± 69.95pg/ml but this was not statistically significant. (p=0.19) Differential effects for MMP2/9 expression were observed on zymography. Pam3CSK4 induced endothelial cell tube formation. Pam3CSK4 also promoted myofibroblast outgrowths from the TA explants and cytoskeletal disassembly in the cultured myofibroblasts.
In an ex vivo temporal artery culture model, Pam3CSK4, a TLR 2 agonist, enhances production of pro inflammatory mediators, promotes angiogenesis, myofibroblast migration and cytoskeletal rearrangement. TLR2 signalling may therefore play a role in driving vascular inflammation and remodelling in GCA, and may represent a potential therapeutic target in GCA.
1 Pryshchep et al, Circulation 2008
2 Makrupa et al, Journal of Exp Med 2006
G. M. McCarthy,
D. J. Veale,
E. S. Molloy,
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ACR Meeting Abstracts - https://acrabstracts.org/abstract/toll-like-receptor-2-agonism-induces-inflammation-angiogenesis-and-cell-migration-in-giant-cell-arteritis/