Increased PAF Acetylhydrolase Expression Following LPS Exposure in MM6 Cells

Objectives:

Platelet-activating factor (PAF), a potent inflammatory mediator, has been implicated in the pathogenesis of periodontal disease. The objective of the current research was to examine the level of the major PAF degradative enzyme, PAF acetylhydrolase, after exposure to lipopolysaccharide (LPS) in a human monocyte/macrophage cell line.

Methods:

Human Mono-Mac 6 cells (MM6) were cultured in RPMI with L-glutamine containing 10% FBS, 1 mM oxaloaxetate, 0.45 mM pyruvate, 0.2 U/ml insulin, 100 units/ml penicillin, and 100 µg/ml streptomycin and grown at 37 ^oC in a humidified 5% CO ₂ incubator. 2 X 10 ⁵ cell/ml were seeded into 24-well tissue culture plates, allowed to recover for three days and then treated with various concentrations of LPS (10-1000 ng/ml) and several inhibitors of various LPS-induced signaling pathways. PAF acetylhydrolase expression levels were examined by Ribonuclease Protection Assays with RNA isolated from the treated cells.

Results:

LPS treatment of MM6 cells induced a 5 to 10-fold increase in PAF acetylhydrolase mRNA levels and the increase detected was LPS dose-dependent. The LPS-induced increase in PAF-acetylhydrolase was not altered in the presence of the protein synthesis inhibitor cyclohexamide (1 µg/ml). Administration of a Janus kinase 2 inhibitor (AG490) and an inhibitor of p38 mitogen-activated protein kinase (SB203580) suppressed the LPS-stimulated upregulation of PAF acetylhydrolase by 100% and 50%, respectively. An inhibitor of the c-jun N-terminal kinase (SP600125) had little to no effect on elevated PAF acetylhydrolase levels.

Conclusion:

Monocyte/macrophages exposed to bacterial LPS increase expression of the major PAF degradative enzyme, PAF acetylhydrolase. This increased expression is the result of activation of preformed signaling proteins and likely occurs through both a JAK/STAT and p38 MAP kinase dependent pathway. Upregulation of PAF acetylhydrolase by activated monocytes/macrophages likely represents a compensatory mechanism to control local PAF levels in inflammatory situations. Research Supported by NIH Grant HL66130-02.