Defining therapeutic targets by using adenovirus: blocking NF-kappaB inhibits both inflammatory and destructive mechanisms in rheumatoid synovium but spares anti-inflammatory mediators

Abstract

The role of the transcription factor NF-kappaB in the pathogenesis of rheumatoid arthritis has long been a subject of controversy. We used an adenoviral technique of blocking NF-kappaB through overexpression of the inhibitory subunit IkappaBalpha, which has the advantage that it can be used in the diseased tissue itself, with >90% of the synovial macrophages, fibroblasts, and T cells infected. We found that the spontaneous production of tumor necrosis factor alpha and other pro-inflammatory cytokines is NF-kappaB-dependent in rheumatoid synovial tissue, in contrast to the main anti-inflammatory mediators, like IL-10 and -11, and the IL-1 receptor antagonist. Of even more interest, IkappaBalpha overexpression inhibited the production of matrix metalloproteinases 1 and 3 while not affecting their tissue inhibitor. Blocking NF-kappaB in the rheumatoid joint thus has a very beneficial profile, reducing both the inflammatory response and the tissue destruction. The adenoviral technique described here has widespread applicability, allowing rapid testing of the effects of blocking a potential therapeutic target in either cultures of normal cells or in the diseased tissue itself.

Figure 1

In excess of 90% of rheumatoid synovial cells can be infected with adenovirus. β-galactosidase activity in total rheumatoid synovial membrane cell cultures (A), T lymphocytes (B), macrophages (C), or synoviocytes (D) were detected by double gating for size/granularity and cell surface markers, in cultures infected with 40:1 of either Adv0 (broken line) or Advβgal (solid line). Shown is a representative of five experiments. In E, the means of β-galactosidase-positive cells are plotted ± SEM (n = 5). In F, rheumatoid synovial cells were infected with 40:1 of either Adv0 or AdvIκBα, and cytosolic extracts were prepared. IκBα expression was determined by Western blotting, and the expression of the p42–44 mitogen-activated protein kinases was used as a control.

Figure 2

AdvIκBα infection inhibits the spontaneous production of pro-inflammatory cytokines from rheumatoid synovial cells. Shown is the effect of infection of rheumatoid synovial cells with 40:1 of either Adv0 or AdvIκBα on the spontaneous production of IL-1β (A), IL-8 (B), and IL-6 (C). Error bars indicate SEM (n = 6).

Figure 3

IκBα over-expression permanently inhibits TNFα and IL-6 production. Shown is the effect of infection of rheumatoid synovial cells with 40:1 of either Adv0 or AdvIκBα on the spontaneous production of TNFα (A) or IL-6 (B) over time. Symbols denote uninfected (■), Adv0-infected (♦), or AdvIκBα-infected (●) cells. Shown is a representative of three independent experiments.

Figure 4

Inhibition of NF-κB through AdvIκBα infection has only marginal effects on the spontaneous production of anti-inflammatory mediators. Shown is the effect of infection of rheumatoid synovial cells with 40:1 of either Adv0 or AdvIκBα on the spontaneous production of IL-10 (A), the IL-1 receptor antagonist (B), the p75 soluble TNF receptor (C), and IL-11 (D). Error bars indicate SEM (n = 6).

Figure 5

IκBα over-expression potently inhibits MMP 1 and MMP 3 production but slightly potentiates the production of TIMP-1. Shown is the effect of infection of rheumatoid synovial cells with 40:1 of either Adv0 or AdvIκBα on the spontaneous production of MMP 1 (A), MMP 3 (B), and TIMP-1 (C). Error bars indicate SEM (n = 6).

Figure 6

Effect of AdvIκBα infection on the balance between MMP production versus the production of TIMP-1 over time. Shown is the effect of infection of rheumatoid synovial cells with 40:1 of either Adv0 or AdvIκBα on the spontaneous production MMP 1 (A), MMP 3 (B), and TIMP-1 (C) over time. Symbols denote uninfected (■), Adv0-infected (♦), or AdvIκBα-infected (●) cells. Shown is a representative of three independent experiments.

Figure 7

Effect of AdvIκBα infection on MMP and TIMP production in rheumatoid synovial cell cultures in which IL-1 and/or TNFα were neutralized. Rheumatoid synovial cells were either left uninfected or infected with 40:1 of Adv0 or AdvIκBα. After the virus was taken off and culture medium was added, the cells in each of the three categories either were left untreated or were treated with 20 μg/ml of the IL-1 receptor antagonist and/or 20 μg/ml of the A2 anti-TNFα antibody. The spontaneous production of MMP 1 (A), MMP 3 (B), and TIMP-1 (C) was assayed after 2 days. Shown is a representative of three complete experiments.