We have investigated the potential use of peroxisome proliferator-activated receptor γ (PPARγ) agonists as anti-inflammatory agents in cell-based assays and in a mouse model of endotoxemia. Human peripheral blood monocytes were treated with LPS or PMA and a variety of PPARγ agonists. Although 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) at micromolar concentrations significantly inhibited the production of TNF-α and IL-6, four other high affinity PPARγ ligands failed to affect cytokine production. Similar results were obtained when the monocytes were allowed to differentiate in culture into macrophages that expressed significantly higher levels of PPARγ or when the murine macrophage cell line RAW 264.7 was used. Furthermore, saturating concentrations of a potent PPARγ ligand not only failed to block cytokine production, but also were unable to block the inhibitory activity of 15d-PGJ2. Thus, activation of PPARγ does not appear to inhibit the production of cytokines by either monocytes or macrophages, and the inhibitory effect observed with 15d-PGJ2 is most likely mediated by a PPARγ-independent mechanism. To examine the anti-inflammatory activity of PPARγ agonists in vivo, db/db mice were treated with a potent thiazolidinedione that lowered their elevated blood glucose and triglyceride levels as expected. When thiazolidinedione-treated mice were challenged with LPS, they displayed no suppression of cytokine production. Rather, their blood levels of TNF-α and IL-6 were elevated beyond the levels observed in control db/db mice challenged with LPS. Comparable results were obtained with the corresponding lean mice. Our data suggest that compounds capable of activating PPARγ in leukocytes will not be useful for the treatment of acute inflammation.

This article is available online at http://www.jlr.org Coronary atherosclerosis is the most prevalent disease in industrialized societies. Although numerous advances have been made in understanding the underlying causes of atherosclerosis and treatment thereof, this condition still remains the leading cause of death in the Western world. The most important risk factor for atherosclerosis is hyperlipidemia ( 1 ). Development of atherosclerosis correlates with high levels of low density lipoprotein cholesterol (LDL). As a result, several therapies have been developed for management of LDL levels. Among these, statins are most widely used ( 2 ). However, there is a range of statin response in humans, and a subset of familial hyperlipidemia patients is unresponsive to statins, prompting the development of additional therapies.

Apolipoprotein B100 (apoB100) and apolipoprotein A1 (apoA1) are the primary protein components of low density lipoprotein (LDL) and high density lipoprotein (HDL) particles, respectively, and plasma levels of these proteins are associated with risks of cardiovascular disease. Existing apoB100 quantitation methods for animal models have been limited to affinity capture techniques such as enzyme-linked immunosorbent assay (ELISA) and Western blot which require specialized reagents for each species and in many cases are not readily available. Here we demonstrate a single translatable ultra-performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) assay that is fast and robust and can be used to measure apolipoprotein concentrations in plasma for six species. When possible, peptide sequences that are conserved across species were identified for this assay. The sample preparation is limited and can be carried out in 96-well microtiter plates and thus allows for multiplexed preparation of samples for analysis of large numbers of samples in a short time frame when combined with UPLC/MS/MS. Separation and quantitation of the tryptic peptides is carried out at 700 μL/min using a 1.7 µm core shell C18 column (2.1 × 50 mm). The chromatography is designed for the analysis of over 100 samples per day, and the UPLC run is less than 10 min. This assay is capable of supporting cardiovascular research by providing a single assay to measure critical biomarkers across multiple species without the need for antibodies, and does so in a high-throughput manner.

The leukocyte integrin complement receptor type 3 (CR3, Mac-1, CD11b/CD18) is the predominant ␤ 2 integrin receptor of polymorphonuclear leukocytes (PMNs). This cell surface receptor plays a central role in innate immunity against pathogens as well as being a major cellular effector of inflammation and tissue injury. Two small molecules, compounds 1 and 2, have been identified, that interact with CR3 and prevent CR3 from binding to its natural ligand, C3bi. Compounds 1 and 2 have IC 50 values of 0.14 and 0.33 M, respectively, for the inhibition of binding of monomeric C3bi-alkaline phosphatase to immobilized CR3. Both compounds also inhibit binding of CR3 to biotinylated sheep red blood cells opsonized with C3bi, with IC 50 values in the micromolar range. Inhibition of ligand binding by the compounds is not easily reversed and requires light, suggesting the formation of a covalent adduct through photoactivation. Compounds 1 and 2 also inhibit adhesion of human PMNs to fibrinogen in response to tumor necrosis factor (TNF) or PMA, with IC 50 values of 2.5 to Ͼ10 M. They block the adhesion-dependent production of H 2 O 2 stimulated by TNF or phorbol 12-myristate 13-acetate (PMA) with IC 50 values of 0.2 to 0.8 M and 1 to 3 M, respectively. Limited structure-activity relationship studies based on compound 2 indicate the importance of the two benzothiazole rings, an ethyl side chain, and the length of the carbon chain linking the rings. Further modification of these groups may help in making compounds appropriate for in vivo studies.Polymorphonuclear leukocytes (PMNs) represent an important constituent of the innate immune system. Under normal circumstances, they circulate in the vasculature in a quiescent state. However, in response to inflammatory stimuli, they adhere, transmigrate across the vascular endothelium, and enter areas of tissue inflammation where they participate not only in the destruction and removal of pathogens but also amplify the process of inflammation (Kishimoto et al., 1999). Migration of PMNs and phagocytosis of bacteria are tightly controlled, and adhesion receptors present on the cell surface of PMNs are crucial for initiating these responses. Upon activation of PMNs in response to stimuli such as TNF, the adhesion receptors, members of the integrin superfamily, are activated, and additional receptors present in intracellular granules translocate to the cell surface (Carlos and Harlan, 1994). The interaction of integrins with ligands present on vascular endothelial cells, stromal cells, and bacteria promotes adhesion and its sequelae.The major adhesion-promoting receptor present on the cell surface of activated PMNs is complement receptor type 3 (CR3), also called Mac-1, CD11b/CD18, or ␣ M ␤ 2 (Kishimoto et al., 1999). CR3 is a heterodimer belonging to the ␤ 2 integrin subfamily. The two subunits, ␣ M (M r 165,000) and ␤ 2 (M r 95,000), are held together by noncovalent interactions. Studies have been done on the structure and functions of ␤ 2 integrins and have been reviewed extensiv...

15-Deoxy-Δ12,14-PGJ2 (dPGJ2) is a bioactive metabolite of the J2 series that has been identified as a ligand for peroxisome proliferator-activated receptor γ (PPARγ) and has received attention for its potential antiinflammatory effects. Because neutrophils express cell-surface receptors for PGs, the effect of dPGJ2 was tested on an inflammatory response that should not require PPARγ, the oxidative burst made by adherent human neutrophils. dPGJ2 inhibited adhesion-dependent H2O2 production with an IC50 of 1.5 μM when neutrophils were stimulated with TNF, N-formylnorleucylleucylphenylalanine, or LPS. Inhibition by dPGJ2 occurred during the lag phase, before generation of peroxide, suggesting blockade of an early signaling step. Indeed, dPGJ2 blocked adhesion of neutrophils to fibrinogen in response to TNF or LPS with an IC50 of 3–5 μM. dPGJ2 was more potent at inhibiting the adhesion-dependent oxidative burst than several other PGs tested. Further, dPGJ2 did not appear to act through either the DP receptor or receptors for PGE2. PG receptors modulate cAMP levels, and the inhibition of adhesion and oxidative burst by dPGJ2 was enhanced in the presence of 3-isobutyl-1-methylxanthine, a cAMP phosphodiesterase inhibitor. A potent PPARγ agonist (AD-5075) did not inhibit peroxide production or adhesion, nor did it change the IC50 for dPGJ2 inhibition. These studies suggest that dPGJ2 may interact with an unknown receptor on neutrophils, distinct from PPARγ, to modulate the production of reactive oxygen intermediates.