Porphyromonas gingivalis lipopolysaccharide is both agonist and antagonist for p38 mitogen-activated protein kinase activation

Abstract

Lipopolysaccharide (LPS) is a key inflammatory mediator. It has been proposed to function as an important molecule that alerts the host of potential bacterial infection. Although highly conserved, LPS contains important structural differences among different bacterial species that can significantly alter host responses. For example, LPS obtained from Porphyromonas gingivalis, an etiologic agent for periodontitis, evokes a highly unusual host cell response. Human monocytes respond to this LPS by the secretion of a variety of different inflammatory mediators, while endothelial cells do not. In addition, P. gingivalis LPS inhibits endothelial cell expression of E-selectin and interleukin 8 (IL-8) induced by other bacteria. In this report the ability of P. gingivalis LPS to activate p38 mitogen-activated protein (MAP) kinase was investigated. It was found that p38 MAP kinase activation occurred in response to P. gingivalis LPS in human monocytes. In contrast, no p38 MAP kinase activation was observed in response to P. gingivalis LPS in human endothelial cells or CHO cells transfected with human Toll-like receptor 4 (TLR-4). In addition, P. gingivalis LPS was an effective inhibitor of Escherichia coli-induced p38 MAP kinase phosphorylation in both endothelial cells and CHO cells transfected with human TLR-4. These data demonstrate that P. gingivalis LPS activates the LPS-associated p38 MAP kinase in monocytes and that it can be an antagonist for E. coli LPS activation of p38 MAP kinase in endothelial and CHO cells. These data also suggest that although LPS is generally considered a bacterial component that alerts the host to infection, LPS from P. gingivalis may selectively modify the host response as a means to facilitate colonization.

FIG. 1.

MAP kinase activation in response to various doses of either E. coli or P. gingivalis LPS. HUVECs were stimulated with various concentrations of LPS (Ec, E. coli; Pg, P. gingivalis) as indicated on the horizontal axis. Total cellular protein was harvested after 30 min and subjected to SDS-PAGE followed by immunoblotting with dual phosphospecific ERK (A) and p38 (B) antibodies. Gels were scanned for densitometry analysis, and the ratio of each LPS dose to an unstimulated control is shown. The data are presented as an averages from three separate experiments (each error bar indicates the standard error of the mean).

FIG. 2.

Time course of MAP kinase activation in response to either E. coli or P. gingivalis LPS. HUVECs were stimulated with E. coli LPS at a concentration of 0.1 μg/ml (triangles) or with P. gingivalis LPS at a concentration of 1 μg/ml (squares). Total cellular protein was harvested at the times indicated on the horizontal axis and subjected to SDS-PAGE followed by immunoblotting with dual phosphospecific ERK (A) and p38 (B) antibodies. Gels were scanned for densitometry analysis, and the ratio of each LPS dose to an unstimulated control is shown. The data are presented as an average from two (E. coli) or four (P. gingivalis) separate experiments (each error bar indicates the standard error of the mean).

FIG. 3.

Human monocyte p38 MAP kinase activation in response to various doses of either E. coli or P. gingivalis LPS. Human monocytes were stimulated with various concentrations of LPS (from E. coli or P. gingivalis) as indicated on the horizontal axis. Total cellular protein was harvested after 30 min and subjected to SDS-PAGE followed by immunoblotting with dual phosphospecific p38 antibody. Gels were scanned for densitometry analysis, and the ratio of each LPS dose to an unstimulated control is shown. The data are presented as averages from three separate experiments (each error bar indicates the standard error of the mean).

FIG. 4.

Inhibition of p38 MAP kinase phosphorylation by P. gingivalis LPS in response to E. coli LPS or TNF-α. (A) E. coli LPS (0.1 μg/ml) was mixed with various concentrations of P. gingivalis LPS (indicated on the horizontal axis) prior to the addition to HUVECs. E. coli (0.1 μg/ml) and P. gingivalis (1 μg/ml) LPSs were also added alone as indicated on the horizontal axis. (B) P. gingivalis LPS did not inhibit p38 MAP kinase phosphorylation in response to TNF-α. TNF-α (0.01 μg/ml) alone and premixed with P. gingivalis LPS (0.1 and 1 μg/ml) was added to HUVECs. Total cellular protein was harvested after 30 min and subjected to SDS-PAGE followed by immunoblotting with dual phosphospecific p38 antibodies. Gels were scanned for densitometry analysis, and the ratio of TNF-α alone and in combination with P. gingivalis LPS to an unstimulated control is shown. The data are presented as an average from four (A) or three (B) separate experiments (each error bar indicates the standard error of the mean).

FIG. 5.

P. gingivalis LPS is a TLR4 antagonist in CHO cells cotransfected with TLR4 and mCD14. CHO cells transfected with TLR4 and mCD14 were incubated with E. coli LPS (100 ng/ml) with or without 10 μg of P. gingivalis LPS/ml for 30 min in the presence of 1% normal human serum and lysed for determination of p38 by immunoblotting with phospho-specific p38 antibodies as described in the text. A representative blot was chosen from four identical experiments.