Article Figures & Data
Figures
- FIG. 1.
Concentrations of MMP-9 and sCD14 in serum from patients with acute Lyme disease. (A) Concentration of MMP-9 in serum from patients with acute Lyme disease (LD) (n = 43) compared to that for healthy controls (n = 29), determined by using ELISA. *, P < 0.05. (B) Concentration of sCD14 from the patients with acute Lyme disease (n = 8) compared to that for healthy controls (n = 8), determined by using ELISA. *, P < 0.05. ELISAs were repeated twice in duplicate.
- FIG. 2.
Upregulation of MMP-9 by stimulation of B. burgdorferi in U937 cells. U937 cells were incubated in the presence or absence of B. burgdorferi (106 organisms/ml) for 0 to 72 h or of B. burgdorferi (0 to 106 organisms/ml) for 48 h. The activities of 92-kDa and 72-kDa proteins with stimulation by B. burgdorferi were analyzed by zymography in a time (A) or concentration (B) pattern (St, standard). Western blotting was performed with monoclonal antibody against human MMP-9 (92 kDa) or with monoclonal antibody against human MMP-2 (72 kDa; data not shown) in the supernatants (equal volume, 10 μl/well) (C) or in the cell lysates (D). β-Actin was used as a control (E). mRNA expression of MMP-9 in U937 cells with B. burgdorferi was assayed using real-time RT-PCR (F) or a targeted gene array of MMPs (G). *, P < 0.01; n = 3. Zymography and Western blot experiments were repeated three times, and representative experiments are shown. Real-time RT-PCR and targeted gene array experiments were repeated three times, and data presented are mean values for three separate experiments performed in duplicate.
- FIG. 3.
mRNA expression of MMP-9 in fibroblasts. Fibroblasts were incubated in the presence or absence of supernatants of cultured U937 cells with B. burgdorferi (CM, 0.5 ml/ml) in the presence or absence of B. burgdorferi. mRNA expression of MMP-9 was analyzed by real-time RT-PCR. HF, human fibroblasts; Bb, Borrelia burgdorferi. For HF plus CM versus HF only, *, P < 0.05; for HF plus CM plus Bb versus HF plus CM or HF plus Bb, **, P < 0.01; n = 5. The real-time RT-PCR experiments were repeated five times, and data presented are mean values for five separate experiments performed in duplicate.
- FIG. 4.
Upregulation of CD14 by stimulation of B. burgdorferi in U937 cells. Western blotting was performed with cell lysates of U937 with or without B. burgdorferi (106 organisms/ml) using monoclonal antibody against human CD14 (55 kDa; membrane CD14) (A) or β-actin (B). Western blotting was also performed with supernatants of U937 cells with or without B. burgdorferi with a time-dependent (C) or concentration-dependent (D) pattern (48 kDa; sCD14) (equal volume of 10 μl/well of supernatants). RNA from treated U937 cells was isolated. mRNA expression of CD14 (E) or TLR2 (F) was determined in U937 cells with or without B. burgdorferi, using real-time RT-PCR. *, P < 0.01; n = 3. Western blot experiments were repeated three times, and representative experiments are shown. Real-time RT-PCR experiments were repeated three times, and data presented are mean values of three separate experiments performed in duplicate.
- FIG. 5.
Effect of anti-CD14 antibody on production of MMP-9. (A) Gelatinolytic activity of MMP-9 in U937 cells. Lane 1, medium control; lanes 2 to 7, Borrelia burgdorferi (Bb), 106 organisms/ml (lane 2, Bb only; lane 3, Bb plus mouse IgG [3 μg/ml]; lane 4, Bb plus monoclonal anti-CD14 antibody [50 ng/ml]; lane 5, Bb plus monoclonal anti-CD14 antibody [100 ng/ml]; lane 6, Bb plus monoclonal anti-CD14 antibody [1 μg/ml]; lane 7, Bb plus monoclonal anti-CD14 antibody [3 μg/ml]). Zymography experiments were repeated three times, and representative experiments are shown. (B) Quantitative analysis of activity of MMP-9 from values in all three separate experiments, using Kodak digital image analysis software. For 6 and 7 versus 2, *, P < 0.01; n = 3.
- FIG. 6.
Recombinant sCD14 directly activated MMP-9 enzymatic activity in human fibroblasts (HF). (A) Gelatinolytic activity of MMP-9 in HF in the presence or absence of recombinant CD14 (rCD14) and/or anti-CD14 antibody. Lane 1, HF medium control; lane 2, HF plus Borrelia burgdorferi (Bb); lane 3, HF plus Bb plus rsCD14 (1 μg/ml) plus anti-CD14 (3 μg/ml); lane 4, HF plus rCD14 (1 μg/ml); lane 5, HF plus rCD14 (1 μg/ml) plus Bb; lane 6, HF plus rCD14 (1 μg/ml) plus Bb plus mouse IgG (3 μg/ml). Zymography experiments were repeated four times, and representative experiments are shown. (B) Quantitative analysis of activity of MMP-9 from values in all four separate experiments, using Kodak digital image analysis software. 5 versus 4 or 1, **, P < 0.01; 4 versus 1, *, P < 0.05; n = 4. (C) mRNA expression of MMP-9 in fibroblasts with or without recombinant CD14 and/or anti-CD14 antibody. HF plus rsCD14 plus Bb versus HF plus rsCD14 or HF only, **, P < 0.01; HF plus rCD14 versus HF only, *, P < 0.05; n = 5. Real-time RT-PCR experiments were repeated five times, and data presented are mean values for five separate experiments performed in duplicate.
