NF-κB is a key regulator of synovial inflammation. In the initiation phase of rheumatoid arthritis (RA) dendritic cells and T cells are likely to be important, whereas in established arthritis other cells play a key role as well. We investigated the relative effect of upstream NF-κB inhibition specifically in T cells versus non-cell type specific NF-κB inhibition in different stages of rat adjuvant arthritis (AA), using either the T-cell specific PKC-theta inhibitory factor (PIF) peptides or non-cell type specific IKK-beta blocking NEMO binding domain (NBD) peptides. The effects of the NBD peptide on human RA synovial tissue in culture were also evaluated.
AA was induced in Lewis rats by intradermal injection of heat-killed mycobacteria. Rats develop clinical signs of arthritis 10–12 days after immunization. PIF or NBD peptides (150 μg) were administered either intraperitoneally or intra-articularly into the ankle joint at different time-points. The course and severity of arthritis was monitored using water-displacement plethysmometry. On day 21 rats were sacrificed and tissue specimens were collected for routine histology and X-rays of the ankle joints. Human RA synovial tissue was collected by arthroscopy and cultured ex vivo ± NBD (100 μM). Tumor necrosis factor alpha induced IL-6 production was measured in the supernatant after 7 days by ELISA.
Intra-articular injection of the T-cell specific PKC-theta inhibitor PIF on days 10 and 12 did not result in amelioration of arthritis nor reduced bone erosion. However, intraperitoneal injection of PIF on days -1, 1 and 3 resulted in reduced T-cell proliferation on day 5 in response to TCR triggering (50%; P = 0.03) or cognate antigen (PPD) (30%; P = 0.05). Furthermore, this resulted in a slightly reduced severity of arthritis (area under the curve 18.47 versus 22.86; P = not significant) and radiological damage (erosion score 4 versus 6; P = not significant). Non-cell type specific NF-κB blockade by the NBD peptide resulted, however, in significantly reduced severity of arthritis (P < 0.0001) and radiological damage (P = 0.04) when injected intra-articularly on days 10 and 12. In addition, proinflammatory cytokine expression was significantly lower in synovial tissue of NBD-treated rats. Incubation of human RA synovial tissue with NBD peptides resulted in 33% inhibition of tumor necrosis factor alpha induced IL-6 production in the supernatant (P < 0.01).
Specific NF-κB blockade using a small molecule inhibitor of IKK beta has anti-inflammatory effects in AA and human RA synovial tissue. Inhibition of PKC-theta has clear effects on T-cell proliferation when administered around the induction of arthritis, but very limited effects on the clinical course of arthritis, suggesting that NF-κB activity in other cell types contributes significantly to the inflammatory response. These results indicate that IKK-beta-targeted NF-κB blockade is superior to T-cell specific PKC-theta inhibition in established arthritis.
This study was supported by a EULAR Young Investigator Award to SWT.