Stimulation of GITR induces the NF-κB activation, which is required for MMP-9 induction. (a) THP-1 cells were preincubated with or without human IgG (20 μg/ml) for 30 min to block FcRs. These cells were then stimulated with immobilized anti-GITR mAb (20 μg/ml). Whole cell lysates were then collected 0, 1 or 2 hr after the activation and IκB levels were measured by Western blot analysis. Actin levels were measured as a protein loading control. (b–d), THP-1 cells were activated with immobilized anti-GITR mAb in the presence or absence of NF-κB inhibitors TPCK (b), ethyl pyruvate (c), and sulphasalazine (d). Culture supernatants were collected after 24 hr for the measurement of MMP-9 activity using gelatin zymogram. Con, no treatment control; LPS, treatment with 1 μg/ml lipopolysaccharide as a positive control.