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Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES

Objective

To characterize the expression of interleukin-7 (IL-7) and IL-7 receptor (IL-7R) in rheumatoid arthritis (RA) synovial tissue and to examine their regulation and pathogenic role in macrophages, endothelial cells, and synovial tissue fibroblasts in RA.

Methods

Expression of IL-7 and IL-7R in RA and normal synovial tissue was demonstrated by immunohistochemistry. Expression and regulation of IL-7 and IL-7R in RA peripheral blood in vitro–differentiated macrophages, RA synovial tissue fibroblasts, and human microvascular endothelial cells (HMVECs) were determined by real-time reverse transcription–polymerase chain reaction and/or flow cytometry. Enzyme-linked immunosorbent assay was used to examine production of proangiogenic factors by IL-7–activated macrophages, RA fibroblasts, and endothelial cells.

Results

IL-7 and IL-7R were coexpressed on RA synovial tissue lining and sublining macrophages and endothelial cells. Expression of IL-7 and its receptor was significantly elevated in RA synovial fluid and peripheral blood macrophages as well as RA fibroblasts, compared to normal cells. Toll-like receptor 4 ligation (with lipopolysaccharide) and tumor necrosis factor α (TNFα) stimulation modulated expression of IL-7 and IL-7R on RA macrophages and HMVECs. However, in RA fibroblasts, lipopolysaccharide and TNFα activation increased expression of IL-7R only. IL-7 also mediated RA pathogenesis by inducing production of potent proangiogenic factors from macrophages and endothelial cells.

Conclusion

We have identified, for the first time, regulators of IL-7 and IL-7R expression in RA fibroblasts, RA peripheral blood in vitro–differentiated macrophages, and endothelial cells. Our results document a novel role of IL-7 in RA angiogenesis.

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.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES

Antibodies and immunohistochemistry.

The studies were approved by the institutional review board, and all donors provided written informed consent. The RA synovial tissue samples were obtained from the practices of orthopedic surgeons and were deidentified; therefore, information on disease severity and treatment was unavailable. RA and normal synovial tissue was formalin fixed, paraffin embedded, and sectioned at the pathology core facility (Northwestern University). Synovial tissue was immunoperoxidase stained using Vector Elite ABC Kits, with diaminobenzidine (Vector) as a chromogen. Briefly, slides were deparaffinized in xylene for 15 minutes at room temperature, followed by rehydration by transfer through graded alcohol. Antigens were unmasked by incubating slides in proteinase K digestion buffer (Dako) for 10 minutes at room temperature. Endogenous peroxidase activity was blocked by incubation for 5 minutes with 3% H2O2. Nonspecific binding of avidin and biotin was blocked using an avidin–biotin blocking kit (Dako). Tissue samples were incubated with antibodies to human IL-7 (1:80; R&D Systems) or IL-7R (1:100; Santa Cruz Biotechnology) or IgG control antibody (Beckman Coulter). Slides were counterstained with Harris' hematoxylin and treated with lithium carbonate for bluing. Each slide was evaluated by 2 observers (MVV and AMM) under blinded conditions (20–23). Tissue sections were scored for lining and sublining macrophages and endothelial cell staining on a 0–5 scale, where 0 = no staining, 1 = few cells stained positively, 2 = some cells stained positively (fewer than half), 3 = approximately half of the cells stained positively, 4 = majority of the cells stained positively, and 5= all cells stained positively. Score data were pooled, and the mean ± SEM was calculated for each data group.

To localize IL-7 or IL-7R to macrophages or endothelial cells in RA synovial tissue, slides were deparaffinized as mentioned above and the antigen was unmasked by incubating slides in proteinase K digestion buffer for 10 minutes at room temperature. Using an Invision G2 kit (Dako), RA synovial tissue specimens were stained with IL-7 (1:25) or IL-7R (1:100) (both from Santa Cruz Biotechnology), with diaminobenzidine (brown staining) used as a chromogen. Subsequently, tissue samples were blocked (double staining blocker included in the Invision G2 kit) and stained with von Willebrand factor (vWF; 1:1,000) or CD68 (1:100) (both from Dako), with fast red (red staining) used as a chromogen according to the instructions of the manufacturer (Dako).

Flow cytometry.

In order to identify IL-7R+ cells, normal and RA monocytes and macrophages were washed with fluorescence-activated cell sorting buffer (5% fetal bovine serum [FBS] in phosphate buffered saline [PBS]). Thereafter, cells were blocked with 50% human serum and 0.5% bovine serum albumin in PBS for 30 minutes at room temperature. Cells were then stained with phycoerythrin-conjugated anti-CD127 (IL-7R; BD PharMingen) and fluorescein isothiocyanate–conjugated anti-CD14 (Becton Dickinson Immunocytometry Systems) or isotype control antibodies (BD PharMingen). IL-7R+ cells were identified as those that were CD14+CD127+.

Cell isolation and culture.

Normal and RA peripheral blood and RA synovial fluid mononuclear cells were isolated by Histopaque gradient centrifugation (Sigma-Aldrich) as previously described (24, 25). Monocyte/macrophages were isolated from normal and RA peripheral blood or RA synovial fluid using a negative selection kit according to the instructions of the manufacturer (StemCell Technologies) (26). Monocytes were subsequently differentiated to macrophages by culturing for 7 days in RPMI containing 20% FBS.

Quantification of chemokines and cytokines.

Human IL-8, Ang-1, Ang-2, vascular endothelia growth factor, CXCL16, IL-1β, TNFα, and IL-6 were quantified by enzyme-linked immunosorbent assay (ELISA). All ELISA procedures were performed according to the instructions of the manufacturer (R&D Systems).

Isolation of RA synovial tissue fibroblasts.

Synovial tissue fibroblasts were isolated from fresh RA synovial tissue by mincing and digestion in a solution of Dispase, collagenase, and DNase (25). Cells were used between passages 3 and 9 and were cultured in Dulbecco's modified Eagle's medium containing 10% FBS.

Cell treatment.

RA peripheral blood in vitro–differentiated macrophages, RA synovial tissue fibroblasts, and HMVECs were treated with LPS (10 ng/ml; Sigma-Aldrich), IL-1β (10 ng/ml; R&D Systems), TNFα (10 ng/ml; R&D Systems), IL-17 (50 ng/ml; R&D Systems), or RA synovial fluid (1:4 dilution). After 6 hours cells were harvested, and IL-7R and IL-7 messenger RNA (mRNA) levels were quantified by real-time reverse transcription–polymerase chain reaction (RT-PCR). RA synovial tissue fibroblasts, normal peripheral blood in vitro–differentiated macrophages, or HMVECs (Lonza) were treated with IL-7 (10 ng/ml; R&D Systems), and cell-conditioned medium was harvested after 24 or 48 hours of treatment. Alternatively, normal peripheral blood differentiated macrophages were treated with IL-1β (10 ng/ml) for 24 hours, after which cells were washed and were either left untreated or treated with IL-7 (10 ng/ml); the cell-conditioned medium was harvested after a further 24 or 48 hours, and IL-8 and Ang-1 were quantified.

Real-time RT-PCR.

Total cellular RNA from cells of the different types was extracted using TRIzol (Invitrogen). Subsequently, reverse transcription and real-time RT-PCR were performed to determine IL-7 and IL-7R expression levels as previously described (24, 25, 27). Relative gene expression was determined by the ΔΔCt method, and results were expressed as fold increases.

Statistical analysis.

Data were analyzed using Student's 2-tailed t-test for paired and unpaired samples. P values less than 0.05 were considered significant.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES

Elevated levels of IL-7 and IL-7R in RA synovial tissue.

To characterize the expression pattern of IL-7 and IL-7R in RA patients compared to healthy individuals, RA and normal synovial tissue specimens were stained with antibodies against IL-7 and IL-7R. We found that IL-7 immunostaining was markedly higher on RA synovial tissue lining and sublining macrophages and endothelial cells, compared to normal synovial tissue (Figure 1). Further, RA synovial tissue lining and sublining macrophages and endothelial cells exhibited significantly higher levels of IL-7R compared to normal synovial tissue (Figure 2). We also found that IL-7 and IL-7R colocalized on vWF+ and CD68+ cells (Figures 1D and E and Figures 2D–F), indicating that IL-7 and its receptor are expressed in RA synovial tissue macrophages (in lining and sublining) as well as in endothelial cells in the sublining. Since IL-7R and its ligand are coexpressed on the same cell types, this suggests that cells producing IL-7 may also be responsive to its activation. Further, up-regulation of IL-7 on the surface of endothelial cells may play an important role in mediating transendothelial migration.

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Figure 1. Interleukin-7 (IL-7) levels are increased in rheumatoid arthritis (RA) synovial tissue (ST) compared to normal (NL) synovial tissue, and macrophages (mac) from RA peripheral blood (PB) and synovial fluid (SF) are an important source of IL-7 expression. A–C, Normal synovial tissue (A) and RA synovial tissue (B) were stained with anti-human IL-7, and positive immunostaining was scored on a scale of 0–5 (C). D and E, RA synovial tissue was stained for IL-7 (brown) and von Willebrand factor (red) (D) or for IL-7 (brown) and CD68 (red) (E), in order to distinguish endothelial cells (Endo) or macrophages that expressed IL-7. F, IL-7 mRNA levels were determined in normal and RA peripheral blood monocytes (mono) and macrophages as well as in RA synovial fluid macrophages, by real-time reverse transcription–polymerase chain reaction. Data are shown as the fold increase above levels in normal peripheral blood monocytes, normalized to GAPDH. Values in C and F are the mean ± SEM (n = 8–15 experiments in C; n = 8–22 experiments in F). ∗ = P < 0.05. Original magnification × 200 in A and B; × 400 in C and D.

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Figure 2. Higher levels of IL-7 receptor (IL-7R) in RA synovial tissue than in normal synovial tissue. A–C, Normal synovial tissue (A) and RA synovial tissue (B) were stained with anti-human IL-7R, and positive immunostaining was scored on a scale of 0–5 (C). Values in C are the mean ± SEM (n = 8–15 experiments). ∗ = P < 0.05. D–F, RA synovial tissue was stained for IL-7R (brown) and von Willebrand factor (red) (D) or for IL-7R (brown) and CD68 (red) (E and F), in order to distinguish endothelial cells or macrophages that expressed IL-7R. Original magnification × 200 in A, B, and E; × 400 in D and F. See Figure 1 for other definitions.

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High levels of IL-7 and IL-7R expression on RA synovial fluid macrophages.

Since levels of IL-7 and IL-7R were elevated in RA synovial tissue lining and sublining macrophages, we investigated whether expression of these factors was increased in RA peripheral blood and synovial fluid macrophages compared to normal peripheral blood monocytes and macrophages. We found that expression of mRNA for IL-7 was elevated by 17-fold in RA synovial fluid macrophages and 7-fold in RA peripheral blood macrophages compared to their counterpart normal cells, as assessed by real-time RT-PCR (Figure 1F). Levels of IL-7 mRNA expression were 4-fold greater in RA peripheral blood monocytes compared to normal monocytes (Figure 1F).

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Figure 3. Up-regulation of IL-7 receptor (IL-7R) in RA synovial fluid and peripheral blood macrophages compared to normal peripheral blood macrophages. A, IL-7R mRNA levels in normal and RA peripheral blood monocytes and macrophages and in RA synovial fluid macrophages were determined by real-time reverse transcriptase–polymerase chain reaction. Data are shown as the fold increase above levels in normal peripheral blood monocytes, normalized to GAPDH. B, Normal and RA peripheral blood monocytes and macrophages were immunostained with CD14 and CD127 in order to determine the percentage of IL-7R+ cells. Values in A and B are the mean ± SEM (n = 8–22 experiments in A; n = 4–5 experiments in B). ∗ = P < 0.05. C, The percentage of CD14+CD127+ cells among normal and RA monocytes and macrophages was assessed by flow cytometry. Representative results are shown. PE = phycoerythrin; FITC = fluorescein isothiocyanate (see Figure 1 for other definitions).

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By microarray analysis, IL-7R was identified as one of the most highly altered genes in RA synovial fluid macrophages (5-fold increase compared to normal macrophages; P = 1.10 × 10−8) (data not shown). Furthermore, concentrations of IL-7R were significantly higher in RA synovial fluid macrophages (45-fold) and RA peripheral blood macrophages (10-fold) compared to normal macrophages, as determined using real-time RT-PCR (Figure 3A). Also, RA peripheral blood monocytes expressed 8-fold greater levels of IL-7R compared to normal monocytes (Figure 3A). Consistent with the mRNA results, flow cytometry experiments demonstrated that IL-7R was significantly elevated in RA monocytes and macrophages compared to counterpart normal cells (Figures 3B and C). These results suggest that RA synovial fluid and peripheral blood macrophages may be an important source of IL-7 production and response.

Regulation of IL-7 and IL-7 expression in RA macrophages by proinflammatory factors.

To determine which factors modulate expression of IL-7 and IL-7R in RA peripheral blood in vitro–differentiated macrophages, untreated cells and cells treated with LPS, IL-1β, TNFα, IL-17, or RA synovial fluid were analyzed. The results demonstrated that both IL-7 expression and IL-7R expression were significantly induced by LPS, TNFα, and IL-1β activation of RA peripheral blood macrophages compared to untreated cells (Figures 4A and B). However, only expression levels of IL-7 were up-regulated (3.5-fold) by IL-17 stimulation, while RA synovial fluid activation in macrophages resulted in increased IL-7R expression compared to untreated cells (Figures 4A and B). These findings suggest that expression of IL-7 and expression of IL-7R are, to some extent, similarly modulated in RA peripheral blood in vitro–differentiated macrophages.

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Figure 4. Proinflammatory factors induce the expression of IL-7 and IL-7 receptor (IL-7R) in RA peripheral blood in vitro–differentiated macrophages. RA peripheral blood in vitro differentiated–macrophages were left untreated or were treated with lipopolysaccharide (LPS; 10 ng/ml), IL-1β (10 ng/ml), tumor necrosis factor α (TNFα; 10 ng/ml), IL-17 (50 ng/ml), or RA synovial fluid (1:4 dilution), and expression of IL-7 (A) and IL-7R (B) was determined by real-time reverse transcriptase–polymerase chain reaction. Data are shown as the fold increase above levels in untreated RA peripheral blood macrophages, normalized to GAPDH. Values are the mean ± SEM (n = 4–9 experiments). ∗ = P < 0.05. See Figure 1 for other definitions.

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Elevated IL-7 and IL-7R expression levels in RA synovial tissue fibroblasts and induction of IL-7R expression by proinflammatory factors.

Based on the histologic results, we investigated whether expression of IL-7 and IL-7R was elevated in RA synovial tissue fibroblasts compared to normal synovial tissue fibroblasts. Results obtained by real-time RT-PCR demonstrated that IL-7 and IL-7R expression was greater in RA compared to normal fibroblasts (7-fold and 15-fold, respectively) (Figures 5A and B). Further, we demonstrated that, whereas LPS and TNFα up-regulated the expression of IL-7R on RA fibroblasts (Figure 5C), expression of IL-7 was unaffected by LPS, IL-1β, TNFα, IL-17, and RA synovial fluid activation of RA fibroblasts (data not shown). Since expression of IL-7R on RA fibroblasts was greater and more responsive to activation compared to expression of IL-7, RA fibroblasts may be activated by IL-7R ligation, but are not the main production source for IL-7.

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Figure 5. Elevated levels of IL-7 and IL-7 receptor (IL-7R) on RA synovial tissue fibroblasts, and modulation of IL-7 and IL-7R expression levels by Toll-like receptor 4 ligation with lipopolysaccharide (LPS) and tumor necrosis factor α (TNFα) activation in human microvascular endothelial cells (HMVECs). A and B, Levels of mRNA for IL-7 (A) and IL-7R (B) in normal and RA synovial tissue fibroblasts were determined by real- time reverse transcriptase–polymerase chain reaction (RT-PCR). C, RA synovial tissue fibroblasts were left untreated or were treated with LPS; 10 ng/ml), IL-1β (10 ng/ml), TNFα (10 ng/ml), IL-17 (50 ng/ml), or RA synovial fluid (1:4 dilution), and expression of IL-7R was measured by RT-PCR. (Since IL-7 expression levels were unaffected in stimulated RA fibroblasts, these data are not shown.) D and E, HMVECs were treated as described for RA fibroblasts in C, and expression of IL-7 (D) and IL-7R (E) was determined by RT-PCR. Data are shown as the fold increase (above levels in normal synovial tissue fibroblasts in A and B, above levels in untreated RA synovial tissue fibroblasts in C, and above levels in untreated HMVECs in D and E), normalized to GAPDH. Values are the mean ± SEM (n = 7–15 experiments in A and B; n = 4–9 experiments in C; n = 4–6 experiments in D and E). ∗ = P < 0.05. See Figure 1 for other definitions.

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Regulation of IL-7 and IL-7R expression on endothelial cells by proinflammatory factors.

Since IL-7 and IL-7R were expressed by RA synovial tissue vascular endothelial cells, we examined factors that may modulate their expression on HMVECs. We found that increases in both IL-7 (Figure 5D) and IL-7R (Figure 5E) expression levels were induced by LPS and TNFα stimulation of HMVECs, while IL-1β activation affected only IL-7R expression. Expression of IL-7 on endothelial cells may thus play an important role in facilitating transendothelial migration of IL-7R+ cells.

IL-7 induces production of proangiogenic factors from macrophages and HMVECs.

To examine the mechanism by which IL-7 mediates pathogenesis in RA, IL-7–activated macrophages, RA synovial tissue fibroblasts, and HMVECs were screened for various proinflammatory factors. IL-7 induced the production of IL-8 from macrophages (Figure 6A), as well as Ang-1 from macrophages (Figure 6C) and HMVECs (Figure 6E), after 48 hours. However, IL-7 did not induce production of IL-6, CCL2/monocyte chemotactic protein 1, CCL5/RANTES, CCL19, or CCL21 from RA synovial tissue fibroblasts, macrophages, or HMVECs (data not shown). Further, production of TNFα and IL-1β by macrophages or HMVECs was not mediated by IL-7 stimulation (data not shown).

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Figure 6. Interleukin-7 (IL-7) activates production of proangiogenic factors from macrophages and human microvascular endothelial cells (HMVECs). Macrophages (A–C) or HMVECs (E) from patients were treated for 24–48 hours with phosphate buffered saline (PBS) or with IL-7 (10 ng/ml), and levels of IL-8 (A) and angiopoietin 1 (Ang-1) (C and E) in conditioned medium were determined by enzyme-linked immunosorbent assay (ELISA). To determine whether elevated levels of IL-7R receptor (IL-7R) on macrophages would result in earlier detection or increased levels of proangiogenic factors, cells were pretreated with IL-1β (10 ng/ml) for 24 hours prior to IL-7 treatment, and levels of IL-8 (B) and Ang-1 (D) in conditioned medium were determined by ELISA. Proangiogenic factors were not detected in IL-7–activated rheumatoid arthritis fibroblasts (data not shown). Values are the mean ± SEM (n = 5 experiments). ∗ = P < 0.05.

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Since IL-7 was capable of inducing the production of IL-8 and Ang-1 after 48 hours, we investigated whether increased levels of IL-7R in macrophages may affect the production level or the response time of these factors. To increase IL-7R levels in macrophages, cells were pretreated with IL-1β prior to IL-7 stimulation. We found that pretreatment of macrophages with IL-1β resulted in earlier detection (at 24 hours) of IL-7–induced increases in IL-8 levels, but levels of IL-8 were not significantly higher than those observed at 48 hours with IL-7 treatment but no IL-1β pretreatment (Figures 6A and B). In contrast, the levels of Ang-1 at 24 hours in IL-1β–pretreated macrophages were significantly greater than those observed in non–IL-1β–pretreated cells following 48 hours of IL-7 activation (Figures 6C and D). These results indicate that the pathogenic role of IL-7 in RA is mediated through production of proangiogenic factors and that expression of IL-7R on macrophages has a role in the IL-7 response.

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES

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.

AUTHOR CONTRIBUTIONS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES

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.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES
  • 1
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