Innate immune-stimulatory activity of Porphyromonas gingivalis fimbriae is eliminated by phase separation using Triton X-114.

Fimbriae are virulence factors of Porphyromonas gingivalis (P. gingivalis). In this study, the action of fimbriae on neutrophil respiratory burst and cytokine production by mononuclear cells (MNC) were investigated. Native or denatured form of purified P. gingivalis fimbriae contained endotoxin at an equivalence of 1-3µglipopolysaccharides(LPS)/mg protein. The endotoxin could be reduced to the equivalent of 1ng-LPS/mg protein by phase separation using Triton X-114. Unfractionated fimbriae caused serum-dependent priming of neutrophils for enhanced respiratory burst, but both native and denatured forms of Triton X-114-fractionated fimbriae were not active at 100µg/mL. Unfractionated fimbriae induced serum-dependent production of IL-1ß by MNC. Triton X-114-fractionated fimbriae (10µg/mL)-induced production of IL-1ß, IL-8 or TNF-α was much lower than that induced by unfractionated fimbriae or 10ng/mL P. gingivalis-LPS preparation. Triton X-114-fractionated fimbriae immobilized on polystyrene tubes induced adhesion-stimulated superoxide release by LPS-primed neutrophils in a ß2 integrin-dependent manner. P. gingivalis cells caused priming of neutrophils; however, Toll-like receptor (TLR) 4 antagonists did not affect this response. Thus, P. gingivalis fimbriae were ineffective in inducing innate immune response in leukocytes; however, they induced ß2 integrin-mediated response by neutrophils. Immune-stimulatory components of P. gingivalis might be recognized by receptors other than TLR4.

Mechanisms of the Macrolide-Induced Inhibition of Superoxide Generation by Neutrophils.

The effect of macrolides on the superoxide (O2 (-)) production by neutrophils was studied. Resting neutrophils become primed by lipopolysaccharide (LPS) or N-formyl-methionyl-leucyl-phenylalanine (fMLP), and primed neutrophils generate O2 (-) in response to fMLP or adhesion, respectively. Both LPS-primed fMLP-stimulated O2 (-) generation by macrolide-treated neutrophils and adhesion-stimulated O2 (-) generation by macrolide-treated fMLP-primed neutrophils were inhibited. Macrolide inhibition of O2 (-) generation was dependent on serum or pH. Serum could be substituted by NaHCO3. The intensity of inhibition was azithromycin = roxithromycin > clarithromycin > erythromycin, in that order. Non-antimicrobial derivatives of erythromycin, that is, EM703 and EM900, inhibited O2 (-) generation at pH 7.4. NH4Cl abolished the activity of azithromycin (AZ) only when added to neutrophils with AZ but not after incubation with AZ, suggesting that NH4Cl prevented the influx of AZ. AZ did not affect the expression of alkaline phosphatase, CD11b, and cytochrome b558 in both resting and LPS-primed neutrophils. These results suggested that macrolides did not affect granule mobilization but inhibited O2 (-) generation selectively.

Local anesthetics inhibit priming of neutrophils by lipopolysaccharide for enhanced release of superoxide: suppression of cytochrome b558 expression by disparate mechanisms.

Local anesthetics have anti-inflammatory effects in vivo and inhibit neutrophil functions in vitro, but how these agents act on neutrophils remains unclear. Phagocytosis and bactericidal activity of neutrophils are enhanced by exposure to bacterial components such as lipopolysaccharide (LPS); this process is termed priming, which for enhanced release of superoxide (O2-) causes mobilization of intracellular granules that contain cytochrome b558, a component of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. We studied whether local anesthetics affected LPS priming for enhanced release of O2- in response to triggering by the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP), and we investigated which element in the LPS signaling pathway might be the target of local anesthetics. Neutrophils were incubated with 10 ng/ml LPS and 1% plasma+/-local anesthetics, washed, and triggered with fMLP. Local anesthetics all inhibited LPS priming, and 50% inhibition was at 0.1 mM tetracaine, 0.5 mM bupivacaine, 3.0 mM lidocaine, or 4.0 mM procaine. Local anesthetics inhibited LPS-induced mobilization of specific granules and secretory vesicles. Local anesthetics inhibited LPS-induced up-regulation of cytochrome b558 but not LPS-induced translocation of p47phox. Inhibition of priming by local anesthetics was reversed by washing and incubating for 5 min. Tetracaine alone, but not the other local anesthetics, inhibited LPS activation of p38 mitogen-activated protein kinase (MAPK) and MAPK kinase 3 (kinases in the LPS signaling pathway). The p38 MAPK inhibitors SB203580 and PD169316 also blocked LPS priming. Thus, tetracaine and the other local anesthetics inhibit by disparate mechanisms, but all the local anesthetics impaired up-regulation of cytochrome b558 and all impaired priming of NADPH oxidase by LPS.