The results of this study indicate that supernatants of SCL derived from RA synovial tissue can attract more monocytes from peripheral blood than OA SCL and skin fibroblasts. MCP-1 and, to a lesser degree, IL-8 played the major roles in SCL-induced trafficking of monocytes. These results begin to provide an explanation for the extensive accumulation of myeloid cells in rheumatoid synovium. Importantly, RA SCL induced more monocyte migration after stimulation with TNF-α, one of the major inflammatory cytokines produced in the rheumatoid synovium.
Peripheral blood monocytes express many chemokine receptors [9,10], including CCR1, CCR2, CCR5, CCR8, and CXCR4. The current analysis indicates that, along with these receptors, CXCR1, CXCR2, and CXCR3 are also expressed by monocytes. CCR1, CCR2, CCR5, CXCR1, CXCR2, CXCR3, and CXCR4 notably appear to be expressed by monocytes obtained from most donors.
Ligands of these chemokine receptors are candidates to be involved in monocyte trafficking. These ligands would include RANTES, MIP-1α and MCP-3 (CCR1), MCP-1, MCP-2, MCP-3, MCP-4, MCP-5 (CCR2), RANTES, MIP-1α, and MIP-1β (CCR5), IL-8 and CGP-2 (CXCR1), IL-8, GROα, and epithelial-derived neutrophil attractant 78 (CXCR2), IP-10 and MIG (CXCR3), and stromal cell derived factor-1 (CXCR4) . Migration assays using supernatants of RA SCLs were employed to determine which of these chemokine and receptor interactions might be involved in monocyte migration in the RA synovium.
Stromal cells are one of the important cell populations in RA synovium. SCL can produce many cytokines, chemokines, and can promote viability and functional activation of T cells and B cells [14–16]. The current findings also indicate that supernatants of RA SCLs attract more monocytes than the supernatants of OA SCL and skin fibroblasts. This result suggests that the supernatants from RA SCLs may contain more chemokines than or different chemokines to those of OA SCL or fibroblast lines and, therefore, are able to attract additional monocytes. This is likely to contribute to the more marked accumulation of monocytes in RA compared with OA synovium.
An analysis of chemokines produced by RA SCL indicated that these cells expressed MCP-1 and IL-8 mRNAs, and also secreted MCP-1 and IL-8. It is noteworthy that there were some discrepancies between mRNA expression and protein production. From the mRNA expression, we expected RA SCLs to produce reasonable amounts of IP-10, RANTES, MIP-1α, and MIP-1β. RA SCLs, however, secreted only small amounts of RANTES, and IP-10 and MIP-1β secretion was not detected. According to previous reports, mRNAs of MCP-1, IL-8, and RANTES were found in RA SCL or fibroblasts, but only MCP-1 was reported to be secreted by RA SCL or fibroblasts without cytokine stimulation [4,5,17,18]. The current study indicates a wider profile of chemokine expression and secretion by RA SCL, and also a greater production of IL-8 and MCP-1 by RA SCL compared with OA SCL, implying a greater proinflammatory potential.
Experiments with blocking mAbs were carried out to examine the chemokines in the supernatants of SCL that accounted for monocyte migration. The data were consistent with the conclusion that MCP-1 and, to a lesser extent, IL-8 accounted for the capacity of RA SCL supernatants to stimulate monocyte migration. Whether additional cytokines produced by RA SCL also contributed to monocyte migration is currently not known, but the combination of the chemokine data and the mAb blocking results suggests that MCP-1 and IL-8 play a dominant role in monocyte migration measured by SCL.
TNF-α is one of the major cytokines produced in inflammatory sites such as RA synovium, and it is thought to play a central proinflammatory role . We examined the influence of TNF-α on monocyte migration induced by RA SCL. RA SCLs stimulated by TNF-α secreted more MCP-1, IL-8, and RANTES than those that were unstimulated, and began to produce IP-10. Moreover, supernatants of TNF-α-stimulated RA SCLs attracted more monocytes in migration assay. MCP-1 and IL-8 played the main roles in monocyte migration induced by TNF-α-stimulated RA-SCL, as documented by blocking experiments.
It has recently been reported that T cells expressing CCR5 gather at inflammatory sites such as RA or multiple sclerosis, and that interactions between CCR5 and its ligands (RANTES, MIP-1α, MIP-1β) are thought to be important in the accumulation of inflammatory cells at these sites [19–21]. CCR5 expressed by monocytes, however, was not apparently active in transmitting transmigratory signals. RANTES is also the ligand of CCR1, but the major receptor for RANTES is thought to be CCR5. In this regard, expression of CCR1 by monocytes was lower than that of CCR5. Moreover, antibody to MCP-1 and IL-8 blocked 75% and 20% of migration of monocytes, respectively. According to this information, RANTES is unlikely to play a major role in monocyte migration by RA SCL supernatants.
MCP-1 has been shown to play an important role in the development of arthritis in MRL-lpr mice , whereas the migration of monocytes into inflammatory sites was reduced in the CCR2-deficient mouse . These results are consistent with the current findings, demonstrating the important role of MCP-1 in monocyte migration. Blocking the effect of RANTES has, however, also been shown to be effective in ameliorating collagen-induced arthritis in DAB/1 mice and adjuvant-induced arthritis in rats [24,25]. RANTES might, therefore, play a different role in the initiation of inflammatory arthritis in experimental animals compared with propagating chronic inflammation in RA.
The overlapping roles of MCP-1 and IL-8 in the migration of inflammatory cells have recently been highlighted in studies of neutrophil trafficking. It was reported that MCP-1 plays a role in neutrophil trafficking in inflammation, although neutrophil trafficking was previously believed to be regulated only by IL-8, and not by MCP-1 . These previous results, together with the current data, indicate that both MCP-1 and IL-8 play major roles in regulating the trafficking of myeloid cells into inflammatory sites.
The role of specific chemokines in arthritis is still controversial, and further investigation is necessary to delineate to specific roles of these effector molecules. Moreover, the role of tissue cells in regulating the migration of inflammatory cells into the synovium is also not fully established. The current data, however, strongly imply that monocyte accumulation in rheumatoid synovium is regulated by SCL via production of the chemokines MCP-1 and IL-8.