Is NF-κB a useful therapeutic target in rheumatoid arthritis?

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Figure 1 .

Schematic diagram of NF-κB activation.

Figure 2 .

Requirements for a good therapeutic target.

Figure 3.

TNFα production in rheumatoid synovial cell cultures is NF-κB dependent. (A) More than 90% of rheumatoid synovial cells can be infected with replication deficient adenoviruses at an m.o.i. of 40 as shown by measuring ß-galactosidase activity by FACS. Rheumatoid T cells, macrophage-like and fibroblast-like cells are all efficiently infected. (B) Rheumatoid synovial cells were infected with an adenovirus overexpressing IκBα (AdvIκBα) or a control adenovirus without insert (Adv0) at an m.o.i. of 40. Supernatants were collected after 48–72 hours and examined for TNFα production by ELISA.

Figure 4 .

IκBα overexpression selectively inhibits cytokine expression in RA joint cultures. It blocks proinflammatory but not anti-inflammatory cytokines and mediators (IL10, IL1RA, sTNFR, IL11).

Figure 5 .

IκBα down regulates the production of MMP-1 and MMP-3 but not TIMP-1. Rheumatoid synovial cells were infected with an adenovirus overexpressing IκBα (AdvIκBα) or a control adenovirus without insert (Adv0) at an m.o.i. of 40. Supernatants were collected after 48–72 hours and examined by ELISA for the presence of MMPs and TIMP-1.

Figure 6 .

Dendritic cells take up adenovirus effectively. (A) Mature dendritic cells were generated by five days' culture in 50 ng/ml GM-CSF and 10 ng/ml IL4 and a further three days in monocyte conditioned medium. Then they were plated on a 96 well, flat bottom plate at a density of 2x10⁵ cells/well and left either uninfected with an m.o.i. ranging from 40 to 500 of an adenovirus without insert (Adv0) or an adenovirus overexpressing ß-Gal expression. Fluorescein-di-(ß-D)-galactopyranoside (Sigma) was used as a substrate of ß-galactosidase and cell fluorescence was analysed by FACS analysis. (B) and (C) 10x10⁶ cells for each condition were left either uninfected or infected with Adv0 or AdvIκBα at an m.o.i. of 300. Two days after infection, cells were left unstimulated or stimulated with LPS (50 ng/ml) for 60 minutes. Cytosolic and nuclear extracts were then prepared and examined for IκBα or p42/44MAPK (loading control) expression by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (B), or for NF-κB DNA binding activity by electrophoretic mobility shift assay (C).

Figure 7 .

AdvIκBα inhibits the mixed lymphocyte reaction. Mature DC were left uninfected, infected with Adv0, or infected with AdvIκBα at an m.o.i. of 300. DC were then plated in graded doses for 10⁵ purified, allogeneic T cells in triplicate in a 96 cell, round bottom microtitre plate on day 1 after adenovirus infection. Proliferation was determined on day six by a [³H]thymidine ([³H]TdR) uptake assay. Each point represents the mean (SEM) of three separate experiments.

Figure 8 .

AdvIκBα inhibits three critical components of antigen presentation. The effect of AdvIκBα infection on dendritic cell antigen presenting and costimulatory molecules as well as the costimulatory cytokine IL12 was studied. IκBα was overexpressed in mature DC for two days and then cells were collected and stained by FACS for HLA (A) or CD80/86 (B), or stimulated by soluble CD40L for 24 hours, and then IL12 production was assayed by ELISA (C).

Figure 9 .

PSI pretreatment inhibits DC antigen presentation. DC were pretreated with vehicle or PSI (0.1 µM, 0.5 µM, 1 µM) for four hours. After washing, graded numbers from these four batches of DC were plated with 105 allo-lymphocytes on a 96 well plate. Proliferation was determined on day 6 using the [³H]TdR uptake assay. Each point represents the mean (SEM) of five separate experiments.

Figure 10 .

Potential pitfalls of NF-κB inhibition.