Loss of lipopolysaccharide receptor CD14 from the surface of human macrophage-like cells mediated by Porphyromonas gingivalis outer membrane vesicles
Introduction
Periodontitis is an inflammatory disorder that results in the destruction of the supporting structures of the teeth, including the alveolar bone. It is the most important cause of tooth loss among adults and is initiated by an accumulation of predominantly anaerobic Gram-negative bacteria in subgingival sites. Among the various bacterial species associated with the development of periodontitis, the Gram negative anaerobic bacterium Porphyromonas gingivalis is suspected to be one of the most important causative agents of the chronic form of this disease [1].
P. gingivalis produces a broad spectrum of virulence factors, including outer membrane vesicles, adhesins, lipopolysaccharides (LPS), hemolysins and proteinases [2], [3], [4], [5]. Arg- and Lys-gingipain cysteine proteinases are the main endopeptidases produced by P. gingivalis and have received considerable attention due to their strong ability to degrade a broad range of host proteins [5]. Two genes code for Arg-gingipain (rgpA and rgpB), and one codes for Lys-gingipain (kgp) [5]. Gingipains and LPS are present in large quantities on the surface of outer membrane vesicles, which are extracellular structures believed to contribute to host colonization, evasion of immune defense mechanisms and destruction of periodontal tissues [6].
P. gingivalis vesicles may be involved in shedding and cleavage processes of surface receptors present on host cells of various types, a phenomenon that may modify the capacity of these cells to respond to stimuli. Shedding implies the release of the ectodomain of a cell surface molecule that will keep its biological activity, whereas cleavage or hydrolysis is associated with production of several fragments usually resulting in inactivation of the molecule. Several types of human cell surface molecules may constitute a substrate for gingipains including CD14, the specific receptor for LPS. This 55 kDa glycoprotein is anchored in the plasma membrane by a phospholipid called glycosylphosphatidyl inositol and is strongly expressed on monocytes/macrophages [7]. Once present in the extracellular environment in a soluble and biologically active form, CD14 can interact with cells that do not normally express CD14, such as endothelial and epithelial cells [8], [9]. This enables LPS activation of these cells, which in turn increases proinflammatory cytokine production, amplifying the inflammatory process [8], [9]. On the other hand, shedding and cleavage of CD14 receptors result in a decreased capacity of these cells to respond to stimuli. In this study, we investigated the capacity of P. gingivalis outer membrane vesicles to promote the shedding or cleavage of the LPS receptor CD14 from the surface of human macrophage-like cells.
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Results
In the first part of the study, gingival crevicular fluid (GCF) samples from patients with various periodontal conditions were analyzed by SDS-PAGE/Western immunoblotting in order to support the hypothesis of an in vivo shedding or cleavage of CD14 during periodontitis (Fig. 1). Three out of eight GCF samples from healthy periodontal sites showed traces of soluble CD14 whereas 9 out of 16 samples from patients affected by moderate or advanced periodontitis contained higher amount of soluble
Discussion
Outer membrane vesicles are produced by P. gingivalis and can act as a virulence factor [6]. More specifically, LPS as well as Arg- and Lys-gingipain cysteine proteinases, found in large quantities on the vesicle surface, may contribute to the pathogenic process of periodontitis by disrupting host defense mechanisms and promoting tissue destruction. In this study, we hypothesized that P. gingivalis vesicles may mediate the shedding or cleavage of the LPS receptor CD14 from the surface of human
Gingival crevicular fluid (GCF) collection and analysis for the presence of soluble CD14 receptor and CD14-derived fragments
GCF samples were obtained from 24 patients attending the dental clinic at Université Laval. The pocket depth of each site was measured using a Michigan periodontal probe. Patients were distributed into three categories: healthy (pocket depth <3 mm), moderate periodontitis (pocket depth between 4 and 6 mm) and advanced periodontitis (pocket depth >7 mm). Paper strips (2×8 mm; 3MM; Whatman, Clifton, NJ) were inserted into the subgingival site for 30 s. The strips were then placed in 250 μl of 100
Acknowledgements
We wish to thank C. Duchaı̂ne (Université Laval) for her assistance in the flow cytometric analysis. We also thank K. Nakayama (Nagasaki University) for providing the gingipain-deficient mutants of P. gingivalis and G. Grenier (Université Laval) for collecting GCF samples. This study was supported by the Canadian Institutes of Health Research.
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