Interleukin-7 (IL-7) is a member of the IL-2/IL-15 family of cytokines, which can be secreted or presented on stromal cells, epithelial cells, endothelial cells, fibroblasts, and smooth muscle cells (1–4). Levels of circulating IL-7 have been shown to be elevated in rheumatoid arthritis (RA) patients compared to healthy individuals (5, 6). Additionally, increased concentrations of IL-7 have been detected in RA synovial fluid compared to osteoarthritis synovial fluid (7, 8). However, the cells responsible for producing IL-7 in the circulation as well as in RA synovial fluid are unknown.
In concert with other growth factors, IL-7 can contribute to the expansion of T cell precursors (9). Mature T cells are also modulated by IL-7, first by costimulation of T cells through cytokine production, second by promoting Th1 differentiation, and last by inhibiting programmed cell death through proteins of the Bcl-2 family, thereby maintaining T cell homeostasis (10–13). Further, dendritic cell development and maturation and antigen presentation are partially controlled by IL-7 (14).
It has been shown that IL-7–activated RA synovial fluid macrophages differentiate into osteoclasts (15). IL-7 was also demonstrated to be the most potent factor in the induction of bone-resorbing cell differentiation, among a panel of 16 cytokines (tumor necrosis factor α [TNFα], IL-1, IL-6, IL-8, and others) and growth factors (granulocyte–macrophage colony-stimulating factor, macrophage colony-stimulating factor, and others) (15, 16). Consistent with this, IL-7−/− mice have exhibited a significant increase in bone mass due to reduced RANKL concentration (17). These results suggest that IL-7 plays an important role in bone resorption by inducing osteoclast differentiation as well as RANKL production.
Coculture of T cells and monocytes stimulated with IL-7 is associated with TNFα production by monocytes (5, 6). However, IL-7–activated T cells or monocytes cultured separately fail to produce TNFα. Interestingly, the investigators who reported these findings have also shown that RA patients whose disease is responsive to anti-TNFα therapy have significantly lower circulating IL-7 levels compared to anti-TNFα nonresponders (6).
Expression levels of IL-7 receptor (IL-7R) have been demonstrated to correlate with numbers of T cells and IL-7 expression levels (18). Further, blockade of IL-7R in RA peripheral blood and synovial fluid significantly reduced endogenous IL-7–induced interferon-γ production (18), suggesting that IL-7 and IL-7R play an important role in RA pathology by activating T cells. Consistent with this, blockade of IL-7R ameliorates joint inflammation in collagen-induced arthritis by reducing T cell trafficking and production of proinflammatory factors, such as TNFα, IL-1β, IL-6, and matrix metalloproteinases, by macrophages (19).
In the present study, we demonstrated that IL-7 and IL-7R are coexpressed on RA synovial tissue lining and sublining macrophages and endothelial cells, thereby identifying novel IL-7 activation–responsive target cells. Further, expression of IL-7 and IL-7R is greatly increased in RA synovial fluid and peripheral blood macrophages as well as in RA peripheral blood monocytes, compared to normal cells. We also showed that activation with Toll-like receptor 4 ligand lipopolysaccharide (LPS), IL-1β, and TNFα can modulate expression of IL-7 and IL-7R in RA peripheral blood in vitro–differentiated macrophages. Expression of both IL-7 and IL-7R were markedly higher in RA compared to normal fibroblasts; however, only IL-7R expression was affected by LPS and TNFα stimulation in RA fibroblasts. In contrast, Toll-like receptor 4 ligation and TNFα stimulation of human microvascular endothelial cells (HMVECs) significantly induced expression of both IL-7 and its receptor. Finally, we demonstrated that the pathologic role of IL-7 is exerted via activation of macrophages and endothelial cells to produce proangiogenic factors such as IL-8 and angiopoietin 1 (Ang-1). Hence, therapy directed against IL-7R ligation may reduce leukocyte migration by inhibiting angiogenesis in RA.
- Top of page
- MATERIALS AND METHODS
- AUTHOR CONTRIBUTIONS
In this study, we showed that RA synovial tissue lining and sublining macrophages and endothelial cells express higher levels of IL-7 and IL-7R compared to tissue from normal controls. Our data demonstrate that macrophages are an important source of IL-7 production in RA, and expression levels of this cytokine and its receptor are greatly up-regulated in RA synovial fluid and peripheral blood macrophages compared to their normal counterparts. LPS, IL-1β, and TNFα activation modulates expression of IL-7 and IL-7R in RA peripheral blood macrophages. Consistent with the histologic results, RA synovial tissue fibroblasts expressed elevated levels of IL-7 and IL-7R compared to normal fibroblasts. In RA fibroblasts, while IL-7R expression was regulated by LPS and TNFα activation, IL-7 levels were unaffected by these stimuli. In contrast, expression of both IL-7 and IL-7R was greatly increased in HMVECs treated with LPS and TNFα versus untreated cells. Finally, we documented a novel role of IL-7 in the induction of key proangiogenic factors from macrophages and HMVECs. Our results suggest that macrophages in RA synovial tissue and synovial fluid are an important source of IL-7 production, and elevated levels of IL-7R on these cells allow them to respond to IL-7 stimulation by producing proangiogenic factors in RA.
Previous studies have shown that IL-7R is expressed on CD4+ and natural killer T cells (28). Others have demonstrated that IL-7 is expressed by lymphoid follicles (29). In contrast, in the present study, IL-7 and IL-7R were coexpressed on RA sublining macrophages and endothelial cells as well as on RA synovial tissue lining, where macrophages and RA fibroblasts are in close proximity and interact with one another. Further, it was shown by others that the number of macrophages in the synovial tissue lining and sublining correlates with IL-7+ cells in RA synovial tissue, indicating that macrophages may be major producers of IL-7 (5). Since IL-7 and IL-7R are expressed by RA fibroblasts, macrophages, and endothelial cells, this may suggest that these cells can be directly activated through IL-7 ligation to produce proinflammatory factors in the synovium.
Elevation of IL-7 levels in peripheral blood prior to the onset of RA suggests that IL-7 may play an important role in the initiation of disease (30). Additionally, plasma IL-7 levels correlate with C-reactive protein levels (31), suggesting that IL-7 may also have an essential role in established disease. Similar to our findings in RA synovial tissue, we demonstrated that expression of IL-7 and IL-7R is elevated in RA synovial fluid and peripheral blood macrophages compared to normal cells. Earlier studies have shown that RA synovial fluid macrophages activated with IL-7 differentiate to osteoclasts, suggesting that ligation of IL-7 to IL-7R expressed on RA synovial fluid macrophages has important involvement in osteoclastogenesis (15). Studies performed on human monocytes and on mice have demonstrated that IL-7–induced osteoclast formation is dependent on RANKL production (32–34).
Interestingly, expression of both IL-7 and IL-7R on RA peripheral blood macrophages was elevated by treatment with LPS, IL-1β, and TNFα, while IL-17 and RA synovial fluid stimulation of these cells modulated expression only of IL-7 and only of IL-7R, respectively. In accordance with the present results, others have shown that elevated levels of IL-1β and TNFα in RA synovial fluid lead to increased IL-7 production in stromal cells (35). Levels of TNFα and IL-7 in RA synovial fluid and tissue also have been found to correlate with one another, suggesting that TNFα can contribute to IL-7 production (28). Additionally, IL-7 can stimulate monocyte-dependent TNFα production in monocyte and T cell cocultures (5, 6). The feedback regulation between IL-7 and TNFα was documented in a recent study in which it was demonstrated that TNFα blockade treatment reduced circulating levels of IL-7 in RA patients who were anti-TNFα responders (6). This is consistent with our findings, as TNFα in RA macrophages can potentiate IL-7 function by up-regulating IL-7R on these cells. Taken together, these results suggest that macrophages are important cell types that can express and respond to IL-7, and proinflammatory factors such as LPS, IL-1β, and TNFα are in part responsible for this process.
IL-7 protein was previously detected in synovial tissue fibroblasts from patients with RA, but not from patients with osteoarthritis (36). In contrast to our findings, those investigators found IL-7 production in RA fibroblasts to be greatly increased by TNFα activation. The conflicting results may be due to differences in passage number, growth conditions, or methods used for quantifying IL-7. Since RA fibroblasts were extracted from deidentified tissue samples in the present study, the treatment information was unknown, and there is no patient information provided in the report by Harada et al (36). However, it may be speculated that the differences in results may be due to differences in treatment strategies. While our data show that both IL-7 and its receptor were elevated in RA compared to normal fibroblasts, only IL-7R expression levels were modulated by LPS and TNFα stimulation in RA fibroblasts, suggesting that, although these cells are responsive to IL-7 stimulation, they may not be the main source of its production.
Expression of IL-7 and IL-7R by endothelial cells is consistent with their pathologic role in RA angiogenesis. Similar to RA macrophages, expression of IL-7 and IL-7R is regulated by LPS and TNFα activation in HMVECs. IL-7R is also modulated by IL-1β in HMVECs, whereas IL-7 is not. When IL-7–activated RA fibroblasts, macrophages, and HMVECs were screened for a variety of inflammatory factors, we found that IL-7 was able to induce production of IL-8 and Ang-1 from macrophages and secretion of Ang-1 from HMVECs. Production of potent proangiogenic factors in IL-7–activated macrophages and endothelial cells suggests that IL-7 is important in angiogenesis in RA and is consistent with a previous report of reduced joint vascularization and fibroblast growth factor levels following IL-7R blockade in a collagen-induced arthritis model (19). In contrast to our data, others have found that IL-7 is capable of inducing production of TNFα, IL-1, and IL-6 from monocyte/macrophages (37). The inconsistency is probably due to the lower dose of IL-7 (10 ng/ml) used in our studies: Alderson et al (37) noted that a higher IL-7 concentration (100 ng/ml) was needed for detection of the aforementioned monokines.
In conclusion, fibroblasts and endothelial cells from RA synovial tissue and macrophages from RA synovial fluid, synovial tissue, and peripheral blood express higher levels of IL-7 and IL-7R compared to control cells. We have demonstrated for the first time that LPS and TNFα can regulate expression of IL-7 and IL-7R in RA macrophages and HMVECs, as well as IL-7R in RA fibroblasts. Finally, potent proangiogenic factors are secreted from IL-7–activated macrophages and endothelial cells, highlighting a novel role of IL-7 in RA angiogenesis.
- Top of page
- MATERIALS AND METHODS
- AUTHOR CONTRIBUTIONS
All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Shahrara had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Study conception and design. Pickens, Shahrara.
Acquisition of data. Pickens, Chamberlain, Volin, Pope, Talarico, Mandelin.
Analysis and interpretation of data. Pickens, Chamberlain, Volin, Shahrara.