Efficient adenoviral infection with IκBα reveals that macrophage tumor necrosis factor α production in rheumatoid arthritis is NF-κB dependent

Abstract

Tumor necrosis factor (TNF) ␣ has been shown to be a major therapeutic target in rheumatoid arthritis with the success of anti-TNF␣ antibody clinical trials. Although signaling pathways leading to TNF␣ expression have been studied in some detail, there is evidence for considerable differences between individual cell types. This prompted us to investigate the intracellular signaling pathways that result in increased TNF␣ synthesis from macrophages in the diseased synovial joint tissue. Using an adenoviral system in vitro we report the successful delivery of genes to more than 95% of normal human macrophages. This permitted us to show, by using adenoviral transfer of IB␣, the natural inhibitor of NF-B, that induction of TNF␣ in normal human macrophages by lipopolysaccharide, but not by some other stimuli, was inhibited by 80%. Furthermore the spontaneous production of TNF␣ from human rheumatoid joint cell cultures was inhibited by 75%, indicating that the NF-B pathway is an essential step for TNF␣ synthesis in synovial macrophages and demonstrating that NF-B should be an effective therapeutic target in this disease.

Heterogeneous requirement of IκB kinase 2 for inflammatory cytokine and matrix metalloproteinase production in rheumatoid arthritis: Implications for therapy

“…This paralleled the results with AdI B␣ that confirmed our previous observations that NF-B is required for IL-1␤, IL-6, and IL-8 production (17,35). In addition, we found that IKK-2 and NF-B are essential for the expression of the angiogenic cytokine VEGF and the extracellular matrix-degrading enzymes MMPs 1, 3, 9, and 13.…”

“…In addition, we found that IKK-2 and NF-B are essential for the expression of the angiogenic cytokine VEGF and the extracellular matrix-degrading enzymes MMPs 1, 3, 9, and 13. Inhibition was not due to increased cell death of AdIKK2dn-infected cells, a finding that was consistent with our previous work in RA synovial membrane cells using AdI B␣ (17,35). On the other hand, IKK-1 was not involved in the release of any of these mediators, with the exception of VEGF.…”

“…Two days after infection, the effect of these molecules on spontaneous cytokine production was studied. As we previously reported (17,35) AdI B␣ significantly inhibited TNF␣ (61 Ϯ 10% inhibition; P Ͻ 0.01), IL-1␤ (58 Ϯ 5%; P Ͻ 0.05), IL-6 (96 Ϯ 3%; P Ͻ 0.05), and IL-8 (86 Ϯ 2%; P Ͻ 0.05) production, whereas a control Ad␤-gal virus had no effect ( Figures 4C-G). Unexpectedly, however, we found that neither AdIKK2dn nor AdIKK1dn had any significant effect on TNF␣ production (30 Ϯ 17% [P Ͼ 0.05] and 4 Ϯ 19% [P Ͼ 0.05], respectively) in RA synovial membrane cultures.…”

“…For passaged synovial fibroblasts, an MOI of 500 was used because these cells are more resistant to adenovirus infection. These MOIs have been previously shown by us and others to result in Ͼ90% of these cells expressing the transgene of interest (17,35,41,42,45).…”

“…Primary human dermal fibroblasts, HUVECs, peripheral blood monocytes, RA synovial membrane cells, and rheumatoid synovial fibroblasts from patients undergoing joint replacement surgery were isolated as previously described (32)(33)(34)(35)(36)(37)(38). Macrophages were derived from monocytes after differentiation for 2-3 days with 100 ng/ml of M-CSF (35).…”