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Originally published In Press as doi:10.1194/jlr.M500006-JLR200 on February 1, 2005

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Journal of Lipid Research, Vol. 46, 1027-1037, May 2005
Copyright © 2005 by American Society for Biochemistry and Molecular Biology

RAW264.7 cells lack prostaglandin-dependent autoregulation of tumor necrosis factor-{alpha} secretion

Carol A. Rouzer1,*,{dagger},§,**, Aaron T. Jacobs*,{dagger},§,**, Chetan S. Nirodi*,{dagger},§,**, Philip J. Kingsley*,{dagger},§,**, Jason D. Morrow§,{dagger}{dagger} and Lawrence J. Marnett*,{dagger},§,**

* Departments of Biochemistry and Chemistry, Vanderbilt University School of Medicine, Nashville, TN 37232-0146
{dagger} Vanderbilt Institute for Chemical Biology, Vanderbilt University School of Medicine, Nashville, TN 37232-0146
§ Center for Pharmacology and Drug Toxicology, Vanderbilt University School of Medicine, Nashville, TN 37232-0146
** Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, TN 37232-0146
{dagger}{dagger} Department of Clinical Pharmacology, Vanderbilt University School of Medicine, Nashville, TN 37232-0146

1 To whom correspondence should be addressed. e-mail: c.rouzer{at}vanderbilt.edu

Studies of the response of RAW264.7 cells (RAW) to lipopolysaccharide (LPS) were carried out to determine why these cells do not demonstrate the prostaglandin (PG)-dependent autocrine regulation of tumor necrosis factor-{alpha} (TNF-{alpha}) secretion observed in primary resident peritoneal macrophages (RPMs). The major cyclooxygenase (COX) product of LPS-stimulated RAW was PGD2, with lesser amounts of PGE2. LPS-treated RAW produced PGs more slowly and reached their maximal PG synthetic rate later than did LPS-treated RPMs, as a result of lower constitutive COX-1 expression and a slower rate of COX-2 induction. Cytosolic phospholipase A2 and levels of free arachidonic acid were similar in RAW and RPMs. In contrast to RPMs, LPS-treated RAW produced high quantities of TNF-{alpha}, which were not altered in the presence of COX inhibitors. This failure of endogenous PGs to suppress TNF-{alpha} secretion was explained by the absence of the prostaglandin D2 receptor and the low levels of PGE2 produced during the first 2 h of the LPS response.

These studies demonstrate that autocrine regulation of TNF-{alpha} secretion in response to LPS is greatly facilitated by a COX-1-mediated rapid accumulation of PGs as well by a correspondence between the PGs produced and the receptors expressed by the cells.

Abbreviations: ATCC, American Type Culture Collection; COX, cyclooxygenase; cPGI2, carbaprostacyclin; cPLA2, cytosolic phospholipase A2; LC/MS/MS, liquid chromatography/tandem mass spectrometry; LPS, lipopolysaccharide; mPGES-1, microsomal prostaglandin E synthase-1; PG, prostaglandin; PGI2, prostacyclin; RAW, RAW264.7 cells; RPM, resident peritoneal macrophage; TNF-{alpha}, tumor necrosis factor-{alpha}; 20:4, arachidonic acid

Supplementary key words macrophage • lipopolysaccharide • cyclooxygenase-1 • cyclooxygenase-2 • arachidonic acid • cytosolic phospholipase A2


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