We show that CCR7 and CCL21 are coexpressed on synovial tissue endothelial cells and that CCR7 ligands CCL19 and CCL21 are capable of inducing macrophages and RA fibroblasts to produce potent proangiogenic factors (6
). Therefore, studies were performed to determine whether CCL19 or CCL21 ligation to CCR7 may directly contribute to angiogenesis in RA. Our data demonstrate that while CCL21 induces HMVEC chemotaxis, CCL19 was not effective in this process. We further demonstrate that CCL21-mediated HMVEC chemotaxis and/or tube formation are mediated through ligation to CCR7 on HMVECs and activation of the PI3K pathway. Lastly, we document that CCL21 can form blood vessels at concentrations detected in the RA joint. Hence, these results support a novel role for CCL21 in RA angiogenesis.
CCL21 and CCR7 colocolize on RA endothelium where their expression is correlated. Conversely, results from previous studies show that CCL21 was only weakly expressed on RA blood vessels while demonstrating that 100% of CCL21 producing vessels were LYVE-1+ in the RA synovium (24
). Our data demonstrate that close to 50% of LYVE-1+ fields positively stained for CCL21 while CCR7 immunostaining was present on all LYVE-1+ fields. The variability of our results may be due in part to heterogeneity of RA disease or patient treatment. Hence RA synovium utilized by Manzo et al. (24
) may have been obtained from patients with less disease activity since only 12 of 27 patients were on substantial treatment and as a result inflammatory factors such as IL-17 that elevate expression of CCL21 may not have been present or may have been expressed in lower concentrations. Thus, CCL21 immunostaining on blood vessels demonstrated a weak intensity. Unlike Manzo et al. (24
), our lower percentage of CCL21 staining in LYVE-1+ cells may be due to selection of RA synovial tissues. While these investigators selected 9 RA tissues that were rich in lymphoid aggregates in our study CCL21 and LYVE-1 immunostaining were performed in random RA synovium.
We show that in macrophages, stimulation with RA synovial fluid modulates the expression of CCR7 and CCL21 (6
). Further, activation with IL-17 and RA synovial fluid significantly increases the expression levels of CCR7 in RA fibroblasts and endothelium and concentrations of CCL21 in endothelial cells. While in HMVECs, TNF-α treatment had no effect on CCL21 expression (data not shown), levels of this chemokine were greatly elevated in human dermal lymphatic endothelial cells stimulated with TNF-α (25
) suggesting that expression of CCL21 is differentially regulated in lymphatic compared to blood vessels. HIF-1α is a hypoxic transcription factor that has been shown to modulate CCR7 expression (8
) and similar to its downstream target is expressed in RA synovial tissue macrophages and endothelial cells (26
). Like CCR7, expression levels of HIF-1α are modulated by TLR4 ligation in RA synovial fluid macrophages (26
) suggesting that TLR4 endogenous ligands in RA synovial fluid (27
) may be important for regulation of HIF-1α and CCR7 pathway.
Consistent with our findings in RA synovial tissue, skin HMVECs express elevated levels of CCR7 compared to controls, therefore these cells were employed as surrogates for RA endothelial cells to examine the direct effect of CCL19 and CCL21 on angiogenesis. We demonstrate that CCL21 induces HMVEC chemotaxis at concentrations available in the human RA joint, which is due to its ligation to CCR7. Unlike CCL21, CCL19 had no effect on HMVEC migration. Although CCL19 and CCL21 have similar affinity to CCR7, ligation of these chemokines mediates different signaling effects. CCL19, but not CCL21, activates CCR7 phosphorylation and internalization, resulting in receptor desensitization (28
). This suggests that CCR7-induced cell responses to CCL19 may have a shorter time-span compared to those of CCL21. Consistently, previous studies have shown that while CCL19 was chemotactic for RA synovial tissue fibroblasts, CCL21 was unable to attract these cells (7
). Although CCL19-activated RA synovial tissue fibroblasts produce VEGF, this effect was not noted with CCL21 stimulation (7
). These results suggest that CCL19 or CCL21 ligation of CCR7 can differentially modulate angiogenesis through induction of different signaling pathways.
Next, experiments were performed to investigate signaling pathways that were associated with CCL21-induced HMVEC chemotaxis and tube formation. Inhibition of the CCL21-activated pathways in HMVECs demonstrated that only activation of PI3K significantly reduces CCL21-mediated chemotaxis and tube formation, and suppression of ERK and JNK pathways had no effect on this process. Consistently, B cell chemotaxis mediated by CCL21 was markedly reduced by inhibitors to PI3K while suppression of ERK and JNK pathways were ineffective (30
). In contrast, monocyte derived dendritic cell migration to CCL21 was dependent on phospholipase C but not PI3K signaling pathway (31
). Further, others have shown that PI3K signaling plays an important role in VEGF and FGF mediated endothelial migration (32
), suggesting that PI3K is involved in the mediation of angiogenesis by various inflammatory factors.
Since CCL19 and CCL21 are both highly elevated in RA synovial fluid (6
) the contribution of these two chemokines was examined in RA synovial fluid-mediated HMVEC chemotaxis. Neutralization of CCL21 and not CCL19 (data not shown) in RA synovial fluid partially reduced RA synovial fluid-mediated HMVEC chemotaxis. RA synovial fluid-mediated HMVEC chemotaxis was also mediated through CCR7, confirming the importance of this receptor in CCL21-mediated angiogenesis. Like CCL21 (6
) other proangiogenic factors present in human RA synovial fluid are mostly produced by RA synovial tissue fibroblasts (VEGF, bFGF, VCAM1, IL-6 and ELR+ CXC chemokines) or macrophages (TNF-α, IL-8 and IL-1β) (35
). Previous reports demonstrate that although both CCR7 ligands CCL19 and CCL21 play a key role in the migration of T cells (36
), neutrophils (37
) and dendritic cells (25
) expression of CCL21 in CCL19-/- mice was sufficient for dendritic cell trafficking, maturation and function suggesting that CCL21 may be the more critical CCR7 ligand in the inflammatory process (38
We next demonstrate that CCL21 can directly contribute to blood vessel formation at concentrations detected in RA synovial fluid and tissue through extravasation of endothelial cells as well as EPCs as the matrigel plugs are acellular and other proangiogenic factors can not be produced.
In conclusion, endothelial migration and tube formation induced by CCL21 were mediated through activation of the PI3K pathway and ligation to CCR7. Neutralization of CCL21 or CCR7 significantly downregulated RA synovial fluid mediated endothelial migration, suggesting that CCL21 plays an important role in RA angiogenesis.