Fig 1.
Osteoblasts expressed CXCR1 and CCR6 receptors.
Flow cytometric analysis of CXCR1, CXCR3 and CCR6 receptor expression by primary bone cells. Open histogram: isotype control; shaded histogram: chemokine receptor antibody.
Table 1.
Functional chemokine receptor expression in human primary bone cells after 48 h of culture with or without CXCL8 (200 pg/ml) or CCL20 (500 pg/ml).
Fig 2.
CXCL8 (2, 20, and 200 pg/ml) and CCL20 (5, 50, and 500 pg/ml) enhanced Ki-67 gene expression, but only 200 pg/ml of CXCL8 and 500 pg/ml of CCL20 enhanced early osteogenic marker gene expression in osteoblasts.
(A) CXCL8 (200 pg/ml) enhanced Ki-67 expression (day 8,14). (B) CCL20 (500 pg/ml) enhanced ki-67 expression (day 6,8). (C) CXCL8+CCL20 (20 pg/ml+50 pg/ml) enhanced ki-67 expression (day 8). (D) TNF-α (100 ng/ml) enhanced Ki-67 expression (day 6,8,14). (E) CXCL8 (200 pg/ml) enhanced ALP and COL1 expression (day 14). (F) CCL20 (500 pg/ml) enhanced ALP expression (day 14). Values are median with 5–95 percentile range of treatment-over-control ratios from 3 experiments, n = 9. Significant effect of chemokines or TNF-α, *p<0.05, **p<0.01.
Fig 3.
CXCL8 (200 pg/ml) and CCL20 (500 pg/ml) affected osteoblast-to-osteoclast communication at day 14.
(A) CXCL8 (200 pg/ml) enhanced MCSF and IL-6 gene expression. (B) CCL20 (500 pg/ml) enhanced IL-6 gene expression. (C) CXCL8+CCL20 (20 pg/ml+50 pg/ml) enhanced IL-6, OPG, and CYR61 gene expression. (D) TNF-α (100 ng/ml) enhanced MCSF, IL-6, and OPG gene expression. (E) CXCL8 (200 pg/ml), CCL20 (500 pg/ml), CXCL8+CCL20 (20 pg/ml+50 pg/ml), and TNF-α (100 ng/ml) enhanced IL-6 production by osteoblasts. Values are median with 5–95 percentile range of treatment-over-control ratios from 3 experiments, n = 9. Significant effect of chemokines and TNF-α, *p<0.05, **p<0.01.
Fig 4.
Effect of CXCL8 (200 pg/ml), CCL20 (500 pg/ml), CXCL8-CM, and CCL20-CM on osteoclast formation.
(A) CXCL8 (200 pg/ml) and CCL20 (500 pg/ml) did not affect TRACP-positive multinucleated cell (TRACP+ MNC) number. (B) CM from osteoblasts cultured without chemokines enhanced TRACP+ MNC number. (C) CXCL8-CM enhanced osteoclastogenesis. IL-6 inhibition reduced this effect. (D) CCL20-CM enhanced osteoclastogenesis. IL-6 inhibition reduced this effect. (E) CM from osteoblasts cultured with CXCL8+CCL20 (20 pg/ml+50 pg/ml) enhanced osteoclastogenesis. IL-6 inhibition reduced this effect. (F) TNF-α-CM enhanced osteoclastogenesis (osteoclasts with >5 nuclei). IL-6 inhibition reduced this effect. Values are mean±SEM from 3 experiments, n = 9. Significant effect of chemokine, control-CM, and chemokine-CM, *p<0.05, **p<0.01, ***p<0.001. Significant effect of IL-6 inhibitor, #p<0.05, ###p<0.001.
Fig 5.
Effect of CM from osteoblasts cultured with CXCL8 (200 pg/ml) or CCL20 (500 pg/ml) on osteoclastic bone resorption.
(A) Resorption pits (blue area; arrows) on a bone slice after 21 days of culture of PBMCs in the presence of control-CM. CXCL8-CM, CXCL8+CCL20-CM, and TNF-α-CM resulted in resorption pit formation, similar to those formed in the presence of control-CM (data not shown). (B) Resorption pits (blue area; arrows) on a bone slice after 21 days of culture of PBMCs in the presence of CCL20-CM. (C) CCL20 (500 pg/ml)-CM enhanced osteoclastic bone resorption compared to control-CM. CXCL8 (200 pg/ml)-CM, CXCL8 (20 pg/ml) +CCL20 (50 pg/ml)-CM, and TNF-α (100 ng/ml)-CM did not affect osteoclastic bone resorption. Values are mean±SEM from 3 experiments, n = 9. Significant effect of CM, *p<0.05.
Fig 6.
Illustration of how CXCL8 and CCL20 might influence bone remodeling during systemic inflammation such as RA and might contribute to osteoporosis.