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. 1998 Nov 10;95(23):13859–13864. doi: 10.1073/pnas.95.23.13859

Figure 4.

Figure 4

Inhibitors of NF-κB accelerate apoptosis in the inflamed rat synovium. (A) Proteasomal inhibitor MG132 induces apoptosis in the SCW arthritic joint. At day 1 after reactivation of the recurrence of SCW arthritis, ankle joints were injected either with MG132 dissolved in 10% DMSO (5 μg/joint) (a and c) or with the vehicle (b). One day later, synovial tissue was explanted and apoptosis was assessed by using TUNEL assay. Some of apoptotic cells are indicated by the arrowheads. Original magnification, ×100. (B) In vivo adenovirus-mediated gene transfer of srIκBα into the SCW arthritic joints. One day after reactivation of SCW arthritis, ankle joints were injected with the AdsrIκBα vector. Two days later, synovial tissue was explanted and srIκBα expression was assessed by HRP immunostaining for human IκBα (b). The specificity detection was confirmed by immunostaining the AdCMV-transduced joints (a). Data are representative of two experiments. Original magnification, ×200. (C) In vivo gene transfer of srIκBα induces apoptosis in the SCW arthritic joint. At day 1 after reactivation of SCW arthritis, ankle joints were injected either with the AdsrIκBα (a) or with the AdCMV (b) vectors. One day later, synovial tissue was explanted and apoptosis was assessed by using TUNEL assay. Some of apoptotic cells are indicated by the arrowheads. Note nuclear fragmentation in apoptotic cells. Data are representative of two experiments. Original magnification, ×1,000. (D) Quantitative assessment of apoptosis in SCW arthritic joints. A summary of the experiments is described in the legends to A and C. Each bar represents the average percentage of apoptotic cells counted in randomly chosen fields. (Bars = SD.) The frequency of apoptosis was counted at a low magnification in the indicated numbers of randomly chosen fields and compared with that in untransduced SCW arthritic rat synovium (the first bar in the row). MG 132, 12 fields in the explants of four joints; vehicle, 12 fields in the explants of four joints; AdsrIκBa, nine fields in the explants of three joints; AdCMV, nine fields in the explants of three joints. The significance of the difference between treated and untreated SCW joints was calculated by using unpaired two-tailed Student’s t test. (E) Time course of AdGFP (Upper) and AdsrIκBα (Lower) expression in rats with pristane-induced arthritis. Rats with established pristane arthritis received AdsrIκBα or AdGFP vectors into both ankle joints. Expression of the transgene was analyzed by Western blotting. (Upper) Immunodetection of Ad GFP in AdGFP-transduced joints. GFP, the arrow indicates the position of the band of recombinant GFP protein (not shown). (Lower) Immunodetection of srIκBα in AdsrIκBα-transduced joints. Pos., a positive control (in vitro AdsrIκBα-transduced cells); Neg., a negative control (in vitro AdCMV-transduced cells); srIκBα, the arrow indicates the position of the band of the srIκBα protein; n.s., a nonspecific band unrelated to human or rat IκBα, as determined by immunodetection with a peptide-blocked primary Ab (data not shown).