Figure 1.
IL-12 production by macrophage correlates with the frequency of LAB phagocytosis and ROS production.
(A, B) Peritoneal macrophages were cultured with FITC-labeled identical LAB strains (3 µg/ml) in a chamber slide. Following the culture for 8 hrs, macrophages were stained with CD11b antibody (red) and phagocytosis of LAB (green) was analyzed by confocal microscopy. Representative pictures (A) and mean phagocytosis index ± SD (B) are shown. (C) Peritoneal macrophages were cultured with 1 µg/ml LAB strains for 24 hr and the levels of IL-12p70 in the culture supernatants were determined by ELISA. (D) Peritoneal macrophages were cultured with 1 µg/ml KW3110, ATCC53103 or NRIC1942 for 8 hrs. ROS were measured using the luminescent dye L-012 at the end of culture. (E) Peritoneal macrophages were cultured with KW3110 (filled) or ATCC53103 (blank) with apocynin (0, 0.1, 0.5, 2 mM) and ROS production measured similarly. Values are average of duplicated culture except figure 1B. Representative data from more than three independent experiments yielding consistent results are shown.
Table 1.
IL-12 and ROS production from macrophages stimulated by various strains of LAB.
Figure 2.
Kinetic analysis of ROS production, IL-12 mRNA expression and IL-12 production from macrophage after LAB stimulation.
Peritoneal macrophages were cultured with 1 µg/ml KW3110 and IL-12p35 and IL-12p40 mRNA expression (A), IL-12p40 and IL-12p70 production (B) and ROS production (C) were measured over time. Values are mean of duplicate culture (C) or mean ± SD of triplicate culture (A, B). Data are representative of three independent experiments yielding similar results.
Figure 3.
ROS dependent IL-12 production from LAB stimulated macrophage.
(A) Peritoneal macrophages were cultured with 1 µg/ml KW3110, ATCC53103 or 1 µM CpG for 24 hrs in the presence of 0, 0.1, 0.5 or 2 mM apocynin (Apo) or 0, 0.01, 0.05 or 0.2 mM propyl gallate (PG). IL-12p40 and IL-12p70 in the cell culture supernatant were measured by ELISA. (C) Peritoneal macrophages were cultured with FITC-labeled identical KW3110 (3 µg/ml) in the presence of Apocynin or propyl gallate. Phagocytosis index was calculated in the same manner as described in Fig. 1A. Data are representative of three independent experiments.
Figure 4.
MyD88 dependent but TLR independent IL-12 production from peritoneal macrophages stimulated by LAB.
(A) Peritoneal macrophages from wild type or TLR2-, 4-, 9- or MyD88-deficient mice were cultured with 1 µg/ml KW3110 for 24 hrs. IL-12p70 production was determined by ELISA. Data value is the mean ± SD of three mice. (B) KW3110, French pressed KW3110 or positive ligand for each TLRs were cultured with HEK 293 cells expressing indicated TLR. TLR stimulation was analyzed by assessing alkaline phosphatase activity (OD650 nm) which is under the control NF-κB activation. Data are representative of two independent experiment yielding same results. (C) Peritoneal macrophages from wild type mice were cultured with 1 µg/ml KW3110, FSL-1, LPS or 1 µM CpG. Cells were recovered at the indicated time points (0, 0.5, 8, 24 hr) and analyzed by Western blot for IRAK-1 and actin. Data are representative of two independent experiments.
Figure 5.
MyD88-deficient macrophage shows impaired ROS production.
(A) Peritoneal macrophages were prepared from wild type and TLR2-, 4-, 9- and MyD88-deficient mice. Cells were cultured with FITC-labeled KW3110 (3 µg/ml) in a chamber slide and phagocytosis indices were measured as described in Fig. 1B. Data value is the mean ± SD of three mice. (B) Macrophage from wild type and TLR2-, 4-, 9- and MyD88-deficient mice were cultured with 1 µg/ml KW3110 for 8 hrs and ROS production was measured. Data are mean ROS intensity ± SD of three mice. * p<0.01 (Dunnett's test).