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[The synergistic effects of lipopolysaccharide, bacterial lipoprotein and bacterial DNA on mouse alveolar macrophage activation].

OBJECTIVE
To investigate the synergistic effects of lipopolysaccharide (LPS), bacterial lipoprotein (BLP), and bacterial DNA on the expression of pattern recognition receptors (PRRs) on the cell surface of mouse alveolar macrophages and cellular activation at the level of receptor and its possible mechanism.


METHODS
Mouse alveolar macrophages were isolated, cultivated and randomly divided into 7 groups: control group, LPS group, CpG oligonucleoetide (CpG-ODN) group, BLP group, LPS + BLP group, LPS + CpG-ODN group, and LPS + BLP + CpG-ODN group. Six hours later the supernatants were collected to detect the level of tumor growth factor alpha (TNFalpha) by ELISA. RT-PCR was used to detect the expression of the main PRRs: CD14, SR, TLR2, TLR4, and TLR9.


RESULTS
The TNFalpha levels in the supernatant were 234 pg/ml +/- 30 pg/ml in the LPS group, 274 pg/ml +/- 30 pg/ml in the BLP group, and 308 pg/ml +/- 28 pg/ml in the CpG-ODN group, all significantly higher than that in the control group (92 pg/ml +/- 27 pg/ml, P < 0.01 or P < 0.01). The TNFalpha levels in the supernatant were 483 pg/ml +/- 31 pg/ml in the LPS + BLP group, and 511 pg/ml +/- 46 pg/ml in the LPS + CpG-ODN group, both significantly higher than those of the groups of the 3 factor alone (all P < 0.05). And the TNFalpha levels in the supernatant was 665 pg/ml +/- 24 pg/ml in the LPS + BLP + CpG-ODN group, significantly higher than those of the LPS + ODN group and LPS + BLP group (both P < 0.05). LPS, BL, and CpG-ODN alone, combinations of any 2 of them, and the combination of the three all up-regulated the expression of CD14 mRNA more and more strongly in sequence. LPS, BLP, and CpG-ODN alone all up-regulated the expression of SR mRNA (all P < 0.01), however, the combinations of any 2 factors or of the 3 factors failed to further up-regulate the expression of SR. LPS and BLP up-regulated the expression of TLR2 mRNA (both P < 0.05), LPS combined with BLP showed a stronger up-regulation of TLR2 mRNA (P < 0.05) than those by LPS and BLP alone. CPG-ODN alone failed to up-regulate the expression of TLR2 mRNA (P > 0.05) but significantly increased the up-regulation by LPS (P < 0.05). In comparison with the combinations of any 2 factors, LPS and BLP with CPG-ODN together up-regulated the expression of TLR2 mRNA more strongly (all P < 0.05). LPS, BLP, and CpG-ODN alone did not significantly up-regulate the expression of TLR4 mRNA (P > 0.05), LPS + BLP significantly regulated the expression of TLR4 mRNA than the groups of any factor alone (all P < 0.05). LPS + BLP + CpG-ODN further up-regulated the expression of TLR4 mRNA. LPS and CpG-ODN, especially LPS + CpG-ODN significantly up-regulated the expression of TLR9 mRNA (all P < 0.05). BLP failed to up-regulate the expression of TLR9 mRNA (P > 0.05) and did not coordinate the upregulation by LPS, however, in comparison with any combinations of the 3 factors, the combination of LPS, BLP, and ODN up-regulated the expression of TLR9 mRNA the most strongly (all P < 0.05).


CONCLUSION
Bacterial LPS, BLP and bacterial DNA not only up-regulate the expression of PRRs of each other, but also synergistically increase the each other's effects on the cell surface of mouse alveolar macrophages.