TLR-2 activation induces EC invasion and migration and MMP-2 and 9 expression in RA synovial explants.

<p>(<b>A</b>) Representative photomicrograph shows HMVEC invasion following Pam3CSK4 (1 µg/ml), stimulation. At 24 hours invading cells attached to lower membrane were fixed (1% glutaraldehyde) and stained (0.1% crystal violet) (Mag×40). (<b>B</b>) Representative bar graph quantifying HMVEC invasion. (n = 4). *<i>p</i><0.05 significantly different to control. (C) Representative photomicrograph showing cells repopulating the wound in response to Pam3CSK4 (1 ug/ml). (D) Representative gel of MMP-2 and 9 activity by gelatine zymography in response to Pam3CSK4 in RA synovial explants (n = 3).</p

TLR2 activation induces ICAM-1 cell surface expression HMVEC.

<p>Human dermal microvascular endothelial cells were stimulated with Pam3CSK4 (1 ug/ml) and expression of ICAM-1 was detected by flow cytometric analysis. (A) Representative flow cytometry histogram demonstrating induced ICAM-1 expression on HMVEC following stimulation with Pam3CSK4 (black line) compared to basal (grey line). <b>B</b>. Quantification of ICAM-1 expression following incubation with Pam3CSK4. Data represented as mean fluorescent intensity (mean±sem, n = 4). * p<0.05 significantly different from unstimulated.</p

TLR2 activation induces EC tube formation and Ang2 expression.

<p>Human dermal microvascular endothelial cell tubule formation on matrigel matrix following stimulation with Pam3CSK4 (1 ug/ml). (A) Representative image of baseline tube formation (left panel) and tube formation following stimulation with Pam3CSK4 (right panel). (B) Quantitative analysis of the number of connecting branches at baseline and in response to Pam3CSK4. The tube analysis was determined from 5 sequential fields (Magnification×40) focussing on the surface of the matrigel (n = 4). (C–D) The effect of Pam3CSK4 on angiopoietin-2 expression in HMVEC (n = 4) and RA synovial explants (n = 6). Data represented as the mean+/−sem. *p<0.05 significantly different from baseline. (E) Ang2, Tie2 and TLR2 expression in RA synovial tissue sections.</p

TLR2 induced EC invasion is inhibited by anti-Tie2.

<p>(A) Representative photomicrographs showing anti-Tie2 blocks Pam3CSK4 induced HMVEC invasion, with no effect observed for IgG control mAb. At 24 hours invading cells attached to lower membrane were fixed (1% glutaraldehyde) and stained (0.1% crystal violet) (Mag×40). (B) Representative bar graph quantifying HMVEC invasion. (n = 4). *<i>p</i><0.05 significantly different.</p

Interleukin-6 does not upregulate pro-inflammatory cytokine expression in an ex vivo model of giant cell arteritis

Objective: The aim of this study was to examine the pro-inflammatory effects of IL-6 in ex vivo temporal artery explant cultures. Methods: Patients meeting 1990 ACR classification criteria for GCA were prospectively recruited. Temporal artery biopsies were obtained and temporal artery explants cultured ex vivo with IL-6 (10-40 ng/ml) in the presence or absence of its soluble receptor (sIL-6R; 20 ng/ml) for 24 h. Explant supernatants were harvested after 24 h and assayed for IFN-γ, TNF-α, Serum amyloid A, IL-1β, IL-17, IL-8, angiotensin II and VEGF by ELISA. Myofibroblast outgrowths, cytoskeletal rearrangement and wound repair assays were performed. Results: IL-6 augmented production of VEGF, but not of any of the other pro-inflammatory mediators assayed. No differences were observed in the explants cultured in the presence or absence of the sIL-6R or between those with a positive (n = 11) or negative (n = 17) temporal artery biopsy. IL-6 did not enhance myofibroblast proliferation or migration. Western blot analysis confirmed signalling activation, with increased expression of pSTAT3 in response to IL-6+sIL-6R. Conclusion: IL-6 stimulation of temporal artery explants from patients with GCA neither increased expression of key pro-inflammatory mediators nor influenced myofibroblast proliferation or migration.</p

IL-17A expression is localized to the inflamed joint.

<p>IL-17A protein levels were measured by MSD Assay in paired serum/synovial fluid samples (n = 20) (<b>A</b>). Synovial fluid levels were significantly higher than serum levels. Values expressed as median ± range, *p<0.01, significance level. Immunohistochemistry was performed in synovial tissue sections from patients with inflammatory arthritis (n = 19). (<b>B</b>).The number of mononuclear cells (white bars) staining for IL-17A was higher than the number of IL-17A positive polymorphonuclear cells (grey bars). Results are expressed as the number of IL-17A positive cells per high powered field (HPF). (<b>C</b>) Representative images of IL-17A expression in RA (i) vs. PsA (ii) and mononuclear IL-17A expression (iii) and polymorphonuclear IL-17A expression (iv).</p

Localisation of IL-17A to neutrophils and mast cells within the inflamed synovium.

<p>Representative images of RA synovial tissue section stained with antibodies against tryptase, CD15 and IL-17A. Merged images indicating co-localisation – yellow.</p

Results are expressed as mean (standard deviation) percentage.

<p>Results are expressed as mean (standard deviation) percentage.</p

Increased systemic expression of IL-17A at low pO2.

<p>(A) Patient synovial fluid samples (n = 22) were assessed by MSD assay for the expression of IL-17A. Cytokine levels were then grouped according to patient tpO₂ levels or >20mmHg. No significant difference in synovial fluid levels was observed between the two groups. (B) Synovial tissue pO₂ levels (n = 18) were also examined in relation to the expression of IL-17A positive mononuclear (white bars) and polymorphonuclear cells (grey bars). Patients with tpO₂ levels <20mmHg (n = 9) had significantly more IL-17A positive mononuclear cells than those with tpO₂ levels >20mmHg (n = 9) (p<0.05). Patients with tpO₂ levels <20mmHg (n = 9) also had a higher number of IL-17A positive polymorphonuclear cells than those with tpO₂ levels >20mmHg (n = 9). This difference was not statistically different. (C) Representative images of IL-17A expression on mononuclear cells from a patient with high tpO2 levels vs a patient with low tpO2 levels are shown.</p

The effect of 3% hypoxia on IL-17A and IL-6 <i>in vitro</i>.

<p>Peripheral blood mononuclear cells (n = 12) and neutrophils (n = 6) were cultured for 24hrs under 3% hypoxic conditions and normoxia. Hypoxia had no effect on IL-17A expression (A), but significantly induced IL-6 expression in mononuclear cells (B). Hypoxia had no effect on IL-17A(C) or IL-6 (D) in neutrophils. Data expressed as the mean <u>±</u> SEM. P<0.05 significantly different from normoxic conditions.</p