The expression of VEGF and RANKL in the synovial fluid, serum, and synovial tissues of RA patients.

<p><b>(A)</b> The synovial fluid samples of 32 RA patients were collected and their VEGF and soluble RANKL concentrations were determined by sandwich ELISA. <b>(B)</b> The serum samples of 32 RA patients were collected and their VEGF and soluble RANKL concentrations were determined by sandwich ELISA. Each dot expresses the results from an individual patient. <b>(C)</b> The synovial tissues of patients with RA and osteoarthritis (OA) were simultaneously labeled with anti-VEGF (green), anti-RANKL (red), and CD55 (white) antibodies and then photographed under appropriate filters. The merged image shows co-localization of the three markers (yellow). Sections were counterstained with DAPI staining. The figures are representative of three independent experiments (original magnification 400×).</p

VEGF-induced RANKL expression in RA synovial fibroblasts.

<p><b>(A)</b> After RA synovial fibroblasts were cultured with 0–50 ng/ml of VEGF for 72 h, the RANKL mRNA expression determined by RT-PCR. Data were normalized to beta-actin and reported in relative expression units. The figure is representative of three experiments. <b>(B)</b> RA synovial fibroblasts were cultured with VEGF for 72 h, and RANKL concentration in the cultured media was measured by sandwich ELISA. <b>(C)</b> RA synovial fibroblasts were cultured with VEGF for 72 h and then stained with anti-RANKL antibodies (red) (original magnification 400×). The figures are representative of three independent experiments. <b>(D)</b> Triplicate wells of RA synovial fibroblasts were transfected with 1 μg of pGL3-RANKL reporter plasmids and 1 μg of pRLTk control plasmid. Both firefly and renilla luminescence were measured after 24 h incubation with 20ng/ml of VEGF. <b>(E)</b> After RA synovial fibroblasts were cultured with VEGF for 72 h, the concentrations of IL-1β, TNF-α, and IL-6 in the cultured media was determined by sandwich ELISA. The data represent the mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01.</p

Induction of osteoclastogenesis by VEGF-pretreated RA synovial fibroblasts.

<p><b>(A)</b> RA synovial fibroblasts were cultured with 20 ng/ml of VEGF for 72 h and RANKL production was quantified using ELISA in the cultured media. <b>(B)</b> RA synovial fibroblasts were preincubated with 20 ng/ml of VEGF for 72 h and Src inhibitor (10 nM) or PKC inhibitor (5 nM) and then cocultured with CD14+ monocytes from the peripheral blood in the presence of M-CSF. After 21 days of culturing, TRAP-positive multinucleated cells were counted. The figure represents one of three independent experiments. <b>(B)</b> The gene expressions of TRAP, RANK, CTR, cathepsin K, and MMP-9 from differentiated osteoclasts measured by real-time PCR. Data were normalized to beta-actin and reported in relative expression units. The data represent the mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01.</p

VEGF-induced osteoclast differentiation from CD14+ monocytes isolated from peripheral blood.

<p><b>(A)</b> CD14+ monocytes isolated from peripheral blood of RA patients were cultured with 25 ng/ml M-CSF and 0–50 ng/ml VEGF or 30 ng/ml RANKL. After maximal 21 days of culturing, TRAP-positive multinucleated cells were counted. The figures represent one of three independent experiments. <b>(B)</b> The gene expression of osteoclast markers such as TRAP, RANK, CTR, cathepsin K, and MMP-9 from differentiated osteoclasts measured by real-time PCR. Data were normalized to beta-actin and reported in relative expression units. The data represent the mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01.</p

The signaling pathways for VEGF-induced osteoclast differentiation from peripheral blood monocytes.

<p><b>(A)</b> CD14+ monocytes were cultured with M-CSF and 20 ng/ml of VEGF in the presence of 20ng/ml of anti-VEGFR1, 20ng/ml of anti-VEGFR2, or 10nM of p38 MAPK inhibitor. After 21 days of culturing, TRAP+ multinucleated cells were counted. The figure represents one of three independent experiments. <b>(B)</b> CD14+ monocytes were cultured with M-CSF and 20 ng/ml of VEGF in the presence of Src inhibitor (10 nM), or PKC inhibitor (5 nM). After 21 days of culturing, TRAP+ multinucleated cells were counted. The figure represents one of three independent experiments. <b>(C)</b> The gene expression of TRAP, RANK, CTR, cathepsin K, and MMP-9 from differentiated osteoclasts was measured by real-time PCR. Data were normalized to beta-actin and reported in relative expression units. The data represent the mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01.</p

The signaling pathways involved in the VEGF-induced RANKL expression in RA synovial fibroblasts.

<p><b>(A)</b> RA synovial fibroblasts were pretreated with anti-VEGFR1 (20 ng/ml), anti-VEGFR2 (20 ng/ml), SB203580, a p38 MAPK inhibitor (10 nM), Src inhibitor (10 nM), or PKC inhibitor (5 nM) for 1 h, and then cultured with 20 ng/ml VEGF for 72 h. The expression of RANKL mRNA was determined by real time-PCR. Data were normalized to beta-actin and reported in relative expression units. <b>(B)</b> RA synovial fibroblasts were stimulated with 20 ng/ml VEGF, the phosphorylated forms of Src, PKC, and ERK were detected by western blotting. The figures are representative of three independent experiments. <b>(C)</b> Stimulation of RA synovial fibroblasts with VEGF activated the phosphorylation of p-Src, Src, p-PKC, PKC, p-ERK and ERK as detected by Western blotting and shown by the ratio of phosphorylated to total proteins. Data were normalized to beta-actin and reported in relative expression units. The figure represents one of three independent experiments. The data represent the mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01.</p

Protective effect of 1α,25-dihydroxyvitamin D3 on effector CD4⁺ T cell induced injury in human renal proximal tubular epithelial cells

<div><p>Background</p><p>The aim of this study was to investigate the protective effect of 1α,25-dihydroxyvitamin D3 [1,25(OH)₂D3] on effector CD4⁺ T cells or on inflammatory cytokine-induced injury in human renal proximal tubular epithelial cells (HRPTEpiC).</p><p>Methods</p><p>First, we investigated the effect of 1,25(OH)₂D3 on CD4⁺ T cell proliferation. Second, we examined the effect of 1,25(OH)₂D3 on inflammatory cytokine secretion or fibrosis in HRPTEpiC induced by inflammatory cytokines or activated CD4⁺ T cells using ELISA and real-time PCR. Lastly, we compared urine inflammatory-cytokine (IL-6, IL-8) or KIM-1 levels in kidney transplant recipients low serum 25-hydroxyvitamin D (25(OH)D) group (< 20 ng/mL) (n = 40) and normal 25(OH)D group (n = 50).</p><p>Results</p><p>Pre-incubation with 1,25(OH)₂D3 significantly reduced the percentages of Th1 and Th17 cells compared to that of Th0 condition (P < 0.05 for each). In contrast, 1,25(OH)₂D3 increased the proportion of Th2 and Treg cells in a dose-dependent manner (P < 0.05 for each). Treatment of HRPTEpiC with inflammatory cytokines (TNF-α, IL-17, and TGF-β) or effector CD4⁺ T cells resulted in increased production of IL-6, IL-8, or KIM-1 from HRPTEpiC in a dose-dependent manner. However, treatment with 1,25(OH)₂D3 significantly reduced the level of these cytokines (P < 0.05 for all). Western blot analysis demonstrated that the mTOR/STAT3/ERK pathway was downregulated by 1,25(OH)₂D3 in HRPTEpiC. Furthermore, the concentrations of urine IL-6/creatinine (P < 0.05) and Kim-1/creatinine (P < 0.05) were higher in the low 25(OH)D group than in the normal 25(OH)D group in kidney transplant recipients.</p><p>Conclusion</p><p>The results of this study suggests that vitamin D may have a significant role in the regulation of inflammation in allograft tissue in kidney transplant recipients.</p><p>Trial registration</p><p>All participants provided written informed consent in accordance with the Declaration of Helsinki. This study was approved by the Institutional Review Board of Seoul St. Mary’s Hospital (<a href="https://clinicaltrials.gov/ct2/show/KC13TNMI0701" target="_blank">KC13TNMI0701</a>).</p></div

Distribution of chemokine receptor CCR4⁺CCR6^–, CCR4^–CCR6⁺ and CCR4⁺CCR6⁺ subpopulations of CD4⁺ T lymphocytes.

<p><b>(A)</b> PBMCs were stained with anti-CD4 PE-cy7, anti-CCR4 PE, anti-CCR6 APC and anti-IL-17 FITC. CD4+ T cells were gated for further analysis. <b>(B)</b> The proportion (%) of CCR4⁺CCR6^–/CD4⁺ T cells <b>(C)</b> CCR4^–CCR6⁺/CD4⁺ T cells <b>(D)</b> CCR4⁺CCR6⁺/CD4⁺ T cells in each patient group. <b>(E)</b> After surface staining with anti-CD4, CCR4 and CCR6 mAbs, analysis of IL-17 in CD4⁺ T cell subsets by intracellular flow cytometry was done. <b>(F)</b> The proportion (%) of IL-17⁺/CCR4⁺CCR6⁺CD4⁺ T cells in each patient group. * <i>P</i><0.05 for each comparison. LTS, long term stable; CAD, chronic allograft dysfunction; ES, early stable; ESRD, end stage renal disease; HC, healthy control.</p

Effect of 1,25(OH)₂D3 on the production of KIM-1 and fibronectin 1 from HRPTEpiC induced by recombinant human IL-17 (rhIL-17), human TNF- α or TGF-beta.

<p><b>(A)</b> The expression of KIM-1 by HRPTEpiC was pretreated with 1,25(OH)₂D3 (10 nM) as indicated, and then cultured for 24 hours with TNF-α (50 ng/mL) and/or IL-17 (50ng/mL) (n = 3). The expression of KIM-1 was measured by real-time PCR. Note that addition of 1,25(OH)₂D3 significantly decrease KIM-1 expression which was increased by TNF-α and IL-17. *P<0.05, **P<0.01 vs. Nill and ^†P<0.05 vs. TNF- α 50 and ^‡P<0.05 vs. TNF-α+IL-17. <b>(B)</b> The expression of Fibronectin-1 by HRPTEpiC was pretreated with 1,25(OH)₂D3 (10 nM) as indicated, and then cultured for 24 hours with TGF-β (10 ng/mL) and/or IL-17 (50ng/mL) (n = 3). The expression of fibronectin-1 was measured by real-time PCR. 1,25(OH)₂D3 significantly decreased fibronectin-1 expression, which was increased by TGF-β (10 ng/mL) and IL-17. **P<0.01 vs. Nill and ^#P<0.05 vs. TGF- β 10 and ^##P<0.05 vs. TGF- β+IL-17.</p

Distribution of T_naïve, T_CM, T_EM subpopulations of CD4⁺T lymphocytes and IL-17⁺/T_EM subpopulations of CD4⁺ T lymphocytes.

<p><b>(A)</b> PBMCs were stained with anti-CD4 PE-cy7, anti-CD45RA–FITC, anti-CCR7 APC and anti-IL-17 PE. CD4+ T cells were gated for further analysis. <b>(B)</b> The proportion (%) of T_naïve/CD4⁺ T (CD45RA^<i>+</i>CCR7⁺/CD4⁺ Tcells) <b>(C)</b> T_CM/CD4⁺ T (CD45RA^–CCR7⁺/CD4⁺Tcells) <b>(D)</b> T_EM/CD4⁺ T (CD45RA^–CCR7^–/CD4⁺ Tcells) <b>(E)</b> After surface staining with CD45 and CCR7 mAbs, analysis of IL-17 in CD4⁺ T cell subsets by intracellular flow cytometry was done. <b>(F)</b> The proportion (%) of IL-17⁺/T_EM. in each patient group. * <i>P</i><0.05 for each comparison. LTS, long term stable; CAD, chronic allograft dysfunction; ES, early stable; ESRD, end stage renal disease; HC, healthy control.</p