Rheumatoid arthritis (RA) is an autoimmune inflammatory disease characterized by the presence of rheumatoid factor (RF) and destruction of bone and cartilage in multiple joints. Pathologic features of the affected joints include hyperplasia of synovial tissue, with increased angiogenesis necessary to oxygenate the tissue (1, 2).
Angiogenesis consists of multiple processes such as degradation of vascular basement membranes and surrounding extracellular matrix, and migration and proliferation of endothelial cells (3). Several mediators have been reported as angiogenic factors that are important for these processes. Vascular endothelial growth factor (VEGF) is a potent angiogenic factor which promotes migration and proliferation of endothelial cells (4, 5). VEGF also induces vascular permeability (4, 5) and mediates inflammation (6).
There is a great deal of evidence suggesting that VEGF plays an important role in the pathogenesis of RA. VEGF is produced by macrophages, fibroblasts surrounding microvessels, vascular smooth muscle cells, synovial lining cells in synovium (7), neutrophils in synovial fluid (8), and peripheral blood mononuclear cells (PBMCs) (9) from patients with RA. VEGF levels are significantly higher in synovial fluids from patients with RA than in those from patients with osteoarthritis (OA) or other arthritides (6, 10, 11). In addition, serum VEGF levels correlate with disease activity scores and radiologic progression in patients with RA (12). However, the mechanism of VEGF production in RA is not fully understood. Hypoxia and interleukin-1 (IL-1) have been reported to induce VEGF production in synovial fibroblasts (13, 14). Tumor necrosis factor α (TNFα) up-regulates VEGF production in PBMCs in RA patients (9). Indeed, Paleolog et al reported that therapy with the anti-TNFα monoclonal antibody (mAb) cA2 (infliximab) not only improved clinical symptoms of RA, but also reduced serum VEGF levels in RA patients (15). They also showed that the combination of anti-TNFα mAb and IL-1 receptor antagonist (IL-1Ra) suppressed VEGF production from RA synovial membrane cells in vitro (15). Therefore, TNFα and IL-1 are believed to be capable of inducing VEGF production in RA.
IL-6 is a pleiotropic cytokine with various biologic activities, such as induction of acute-phase reaction, regulation of immune response, and promotion of hematopoiesis (16). Furthermore, IL-6 activates osteoclasts in the presence of soluble IL-6 receptor (sIL-6R) (17). Therefore, overproduction of IL-6 may be involved in the appearance of RF, elevation of acute-phase proteins, hypergammaglobulinemia, thrombocytosis, and destruction of the joints in patients with RA (18). Indeed, IL-6 levels both in synovial fluid and in serum from RA patients have been reported to be higher than those in patients with other arthritides (19, 20). We have reported that treatment of refractory RA with MRA, a humanized anti–IL-6R mAb, resulted in improvement of both clinical symptoms and laboratory findings (18). Thus, IL-6 plays an important role in the pathogenesis of RA. Moreover, it has been reported that IL-6 induces VEGF production in human myeloma cells (21), epidermoid carcinoma cells, rat skeletal muscle myoblasts, and rat glioma cells (22) in vitro. We have also demonstrated that IL-6 blockade reduced hyaline vascular vessels in the affected lymph node in a patient with Castleman's disease (23), in which VEGF is produced by infiltrating plasma cells (24). This evidence suggests that IL-6 may regulate VEGF production in RA.
To investigate whether IL-6 is indeed a regulator of VEGF production in RA, we analyzed serum VEGF levels before and after anti–IL-6R mAb therapy in RA patients. We also examined the ability of IL-6, acting synergistically with IL-1β and/or TNFα, to induce VEGF production in RA synovial fibroblasts in vitro.
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The fact that anti–IL-6R mAb therapy reduced serum VEGF levels in the RA patients indicates that IL-6 is involved in VEGF production in RA. Anti–IL-6R mAb therapy also improved disease activity in these patients, and the reduction in serum VEGF may be the result of disease amelioration. Another possible mechanism for the reduction in serum VEGF levels is that the blockade of IL-6 action directly inhibits VEGF production in RA. Because IL-6 reportedly induces VEGF production in some cells in vitro (21, 22), we analyzed VEGF production in RA synovial fibroblasts stimulated with IL-6. As we expected, IL-6 in the presence of sIL-6R induced VEGF production, but the activity was weak. Since IL-6 has been shown to suppress the growth of synovial fibroblasts (28), this increase in VEGF was not the result of an increase in the number of cells. Quantitative RT-PCR analysis confirmed that VEGF induction occurred through the augmentation of transcription.
IL-1β alone also significantly induced VEGF production in RA synovial fibroblasts, while TNFα did not. However, TNFα might have introduced the signal into the cells, since IL-6–induced VEGF production was augmented by TNFα. In addition, we have reported that synovial fibroblasts proliferate and produce IL-6 in response to TNFα (28). These results, taken together with the finding that the combination of IL-1β and TNFα did not have a synergistic or additive effect on VEGF production, indicate that the stimulatory signal of TNFα on VEGF expression may be weaker than that of IL-1β.
Although the VEGF-inducing activity of each cytokine was not very strong, IL-6, in synergy with IL-1β and TNFα, induced VEGF production in RA synovial fibroblasts. Of these cytokines, IL-6 is pivotal in VEGF induction in synovial fibroblasts, because a synergistic effect was observed between IL-6 and IL-1β and between IL-6 and TNFα, but not between IL-1β and TNFα. This in vitro finding has important implications. Since these cytokines all exist together in vivo, especially at high levels in affected joints, it is necessary to consider their interactions in order to understand their in vivo pathologic as well as physiologic roles in RA.
In this study, cells were stimulated with IL-6 in the presence of sIL-6R because they constitutively express gp130 signal transducer, but little IL-6R, on their cell surface. In addition, IL-1β did not induce IL-6R expression. Therefore, the synergistic effect of IL-6 and IL-1β may be mediated through second messengers. It has been reported that several signal-transducing molecules, such as signal transducer and activator of transcription 3, p42/p44 mitogen-activated protein kinase (MAP kinase), nuclear factor κB, and p38 MAP kinase, are involved in VEGF production (34–37). IL-6 reportedly activates the former two, while IL-1 and TNFα activate the latter two, and therefore the IL-6–mediated signal for VEGF production may be different from IL-1– and TNFα-mediated signals. The mutual action of these signaling molecules may exert a synergistic effect between IL-6 and IL-1 or between IL-6 and TNFα. IL-1 and TNFα may compensate for one another for VEGF-inducing activity because they share the same signaling pathways. Therefore, IL-6 blockade seems the most favorable for inhibition of VEGF production in the presence of the triple combination of IL-6, IL-1, and TNFα, as in the affected joints of patients with RA.
Anti–IL-6R mAb eliminated the synergistic effect of IL-6, IL-1β, and TNFα on VEGF production in RA synovial fibroblasts, while IL-1Ra and anti-TNFα mAb did not, even though we used high concentrations of IL-1Ra and anti-TNFα mAb to block the activity. Anti-TNFα mAb did not inhibit VEGF production. The combination of IL-6 and IL-1β induced the maximal response, similar to that obtained with the triple combination of IL-6, IL-1β, and TNFα. Since IL-1Ra up to 2,000-fold in excess of the amount of IL-1β present did not suppress the synergistic effect, IL-1Ra may not be strong enough to block the signal of IL-1β in its synergistic action with IL-6 on VEGF production. In fact, IL-1Ra at 20,000-fold in excess of the amount of IL-1β partially inhibited VEGF production. These findings, taken together with the short half-life of IL-1Ra in vivo, indicate that IL-1Ra does not seem favorable for suppression of VEGF production.
Our data suggest that IL-6 blockade is the most effective of the 3 inhibitors in suppressing VEGF production by synovial fibroblasts. This is supported by the evidence that serum VEGF levels in our patients treated with anti–IL-6R mAb were normalized. Treatment with anti-TNFα mAb cA2 has also been reported to significantly reduce serum VEGF levels in RA patients (15), although the VEGF-inducing activity of TNFα was weak and another anti-TNFα mAb did not inhibit VEGF production in the presence of IL-6 and IL-1β in our in vitro experiment. Since cA2 suppresses IL-6 and IL-1 production in vivo in RA patients (38), the decrease in serum VEGF may be mediated through the suppression of IL-6 and IL-1 production by cA2 therapy. However, the suppression of IL-6 by cA2 is transient (38), and this may be the reason that the reduction in serum VEGF in the patients treated with anti-TNFα mAb was only partial. Similar mechanisms may explain the partial reduction in CRP and serum amyloid A protein levels in such patients (38).
The findings of this study indicate that IL-6 blockade directly suppresses VEGF production in synovial fibroblasts and may consequently reduce serum VEGF levels in patients with RA. The inhibition of VEGF production could be one of the mechanisms by which anti–IL-6R mAb therapy is effective in this disease.
Induction of VEGF by proinflammatory cytokines has been observed not only in synovial fibroblasts from RA patients, but also in other cell types including malignant cells (21). Moreover, since VEGF is involved in the progression of malignant neoplasms, anti–IL-6R mAb therapy may be effective in the treatment of the neoplasms. Further studies will be needed to elucidate the molecular mechanism of VEGF production by proinflammatory cytokines.