Figure 1.

Submerged normal human bronchial epithelial (NHBE) cells were treated with heat-killed Staphylococcus aureus (HKSA; 5 × 10⁸ particles/ml, except dose dependency) for 24 hours. Cell lysates were collected to assess expression of (A) matrix metalloproteinase (MMP) -1, -2, -9, and -10 and tissue inhibitors of metalloproteinase (TIMP) -1 mRNA by real-time RT-PCR. (B) The concentration dependency and (C) time course of expression of HKSA-induced MMP-1 mRNA were analyzed by RT-PCR. (D) Cycloheximide (CHX; 1 or 10 μg/ml) was applied 1 hour before HKSA stimulation for 24 hours, and expression of MMP-1 mRNA was analyzed by RT-PCR. NHBE cells were transfected with 10 nM small interfering RNA (siRNA) against nonspecific control RNA or Toll-like receptor (TLR) 2. (E) Knockdown efficiency of TLR2 was analyzed by RT-PCR. After transfection with siRNA against control RNA or TLR2, cells were incubated with HKSA for 24 hours, cell lysates were collected to assess (F) MMP-1 mRNA by RT-PCR, and supernatants were collected to assess the expression of (G) MMP-1 protein by ELISA. NHBE cells were pretreated with dexamethasone (DEX; 100 nM) 1 hour before HKSA stimulation. Cell lysates were collected to assess expression of (H) MMP-1 mRNA by RT-PCR, and supernatants were collected for (I) MMP-1 protein detection by ELISA. Data represent mean ± SEM of three independent experiments in three donors. *P < 0.01 by t test when compared with media only or dimethyl sulfoxide (DMSO) control; ^#P < 0.01 by t test when compared with HKSA-stimulated cells; NS, not significant by t test when compared with media-only control or HKSA-stimulated cells.