Fig 1.
Effect of CpG-DNA on the expression of CD11b and CD18 in RAW 264.7 cells.
RAW 264.7 cells were treated with MB-ODN 4531 (4531(O), 4531(S)), CpG-DNA 1826 (1826(O), 1826(S)), non-CpG-DNA 2041 (2041(O), 2041(S)) or LPS for 24 h. After stimulation, RAW 264.7 cells were harvested and the expressions of CD11b (A) and CD18 (B) were analyzed by flow cytometry. Each bar indicates the Mean ± SD of average mean fluorescence intensities (log scale) from three independent experiments. (O), phosphodiester CpG-DNA, (S) phosphorothioate-modified backbone CpG-DNA. For example, the phosphorothioate version of MB-ODN 4531(O) is MB-ODN 4531(S).
Fig 2.
Extracellular release of CD11b in macrophages by CpG-DNA stimulation.
RAW 264.7 cells were pretreated with 2 μl GolgiStop™, a protein transport inhibitor. The cells were then stimulated with the indicated CpG-DNAs for 24 h. Then, the cell culture media and cell lysates were collected, and the CD11b was monitored by Western blotting. Lipoplex(O), complex of MB-ODN 4531(O) encapsulated with DOPE:CHEMS (1:1 ratio). Lipoplex(S), complex of MB-ODN 4531(S) encapsulated with DOPE:CHEMS (1:1 ratio). β-actin expression level was used as a control. One representative experiment of three is shown.
Fig 3.
Induction of CD11b release by CpG-DNA stimulation is associated with exosome.
(A) RAW 264.7 cells were treated with PBS, CpG-DNA 1826(S) (1826(S)), or non-CpG-DNA 2041(S) (2041(S)) for 24 h. The cell culture media and cell lysates (left panel) were collected, and the CD11b was monitored by Western blotting. β-actin expression level was used as a control. Exosome precipitant (right panel) was collected with ExoQuick-TC™ exosome precipitation solution, and the CD11b was monitored by Western blotting. CD81 was used as an exosome control protein. (B) Macrophages obtained from the peritoneal cavity of BALB/c mice and TLR9 knockout mice were treated with PBS, CpG-DNA 1826(S) (1826(S)), or non-CpG-DNA 2041(S) (2041(S)) for 24 h. The cell lysates (left panel) and exosome precipitant (right panel) were collected, and the CD11b was monitored by Western blotting. (C) BALB/c mice and TLR9 knockout mice were i.p. injected with CpG-DNA 1826(S) (1826(S)), non-CpG-DNA 2041(S) (2041(S)), incomplete Freund’s adjuvants, or alum. After 24 h, peritoneal cavity fluids and peritoneal cavity macrophages were collected. CD11b expression in the cell lysates and exosome precipitant was monitored by Western blotting. One representative experiment of three is shown.
Fig 4.
CpG-DNA-induced release of CD11b is linked to p38 MAP kinase activation, lysosomal degradation, and vacuolar acidification.
RAW 264.7 cells were pretreated with each inhibitor. The cells were then stimulated for 24 h with CpG-DNA 1826(S) (5 μg/ml). Then, the cell culture media and cell lysates were collected, and the amount of CD11b was monitored by Western blotting. β-actin expression level was used as a control. One representative experiment of three is shown. (A, graph) The band intensity of CD11b in the cell lysates of the media control was taken as 1.0 and relative intensity of the CD11b protein bands in the supernatant and cell lysates compared with the control was calculated. * p < 0.01 (Significant decrease).