Ligation of Fc receptors for immunoglobulin G (FcgammaRs) inhibits lipopolysaccharide (LPS)-stimulated secretion of interleukin (IL)-12 by macrophages. FcgammaR activation of protein kinase C (PKC) contributes to several functions of this receptor including phagocytosis, activation of the reduced nicotinamide adenine dinucleotide phosphate oxidase, and secretion of certain cytokines. Therefore, we tested the hypothesis that PKC mediates the FcgammaR inhibition of IL-12 secretion by macrophages. In murine macrophages, FcgammaR ligation augmented LPS-stimulated activation of PKC-alpha and PKC-delta but reduced IL-12p40 secretion. Similarly, activation of PKC with phorbol 12-myristate 13-acetate (PMA) depressed LPS-stimulated IL-12p40 secretion, and depletion of PKC augmented LPS-stimulated IL-12p40 secretion. Antisense down-regulation of PKC-delta increased LPS-stimulated IL-12p40 secretion and fully prevented the effects of FcgammaR ligation or PMA on IL-12p40 secretion. In contrast, down-regulation of PKC-epsilon blocked LPS-stimulated secretion of IL-12p40. Down-regulation of PKC-alpha had no effect on LPS-stimulated IL-12p40 secretion. The results suggest a negative role for PKC-delta and a positive role for PKC-epsilon in the regulation of LPS-stimulated IL-12p40 secretion.

BackgroundStroke, caused by carotid plaque rupture, is a major cause of death in the United States. Whereas vulnerable human plaques have higher Fc receptor (FcγR) expression than their stable counterparts, how FcγR expression impacts plaque histology is unknown. We investigated the role of FcγRIIb in carotid plaque development and stability in apolipoprotein (Apo)e−/− and Apoe−/−FcγRIIb−/− double knockout (DKO) animals.Methods and ResultsPlaques were induced by implantation of a shear stress‐modifying cast around the carotid artery. Plaque length and stenosis were followed longitudinally using ultrasound biomicroscopy. Immune status was determined by flow cytometry, cytokine release, immunoglobulin G concentration and analysis of macrophage polarization both in plaques and in vitro. Surprisingly, DKO animals had lower plaque burden in both carotid artery and descending aorta. Plaques from Apoe−/− mice were foam‐cell rich and resembled vulnerable human specimens, whereas those from DKO mice were fibrous and histologically stable. Plaques from DKO animals expressed higher arginase 1 (Arg‐1) and lower inducible nitric oxide synthase (iNOS), indicating the presence of M2 macrophages. Analysis of blood and cervical lymph nodes revealed higher interleukin (IL)‐10, immune complexes, and regulatory T cells (Tregs) and lower IL‐12, IL‐1β, and tumor necrosis factor alpha (TNF‐α) in DKO mice. Similarly, in vitro stimulation produced higher IL‐10 and Arg‐1 and lower iNOS, IL‐1β, and TNF‐α in DKO versus Apoe−/− macrophages. These results define a systemic anti‐inflammatory phenotype.ConclusionsWe hypothesized that removal of FcγRIIb would exacerbate atherosclerosis and generate unstable plaques. However, we found that deletion of FcγRIIb on a congenic C57BL/6 background induces an anti‐inflammatory Treg/M2 polarization that is atheroprotective.

Atherosclerosis is responsible for the death of thousands of Americans each year. The carotid constriction model of plaque development has recently been presented as a model for unstable plaque formation in mice. In this study we 1) validate ultrasound biomicroscopy (UBM) for the determination of carotid plaque size, percent stenosis, and plaque development in live animals, 2) determine the sensitivity of UBM in detecting changes in blood flow induced by carotid constriction and 3) test whether plaque formation can be predicted from blood flow parameters measured by UBM. Carotid plaques were induced by surgical constriction in Apo E−/− mice. Arteries were imaged bi-weekly by UBM, at which time PW-Doppler measurements of proximal blood flow, as well as plaque length and percent stenosis were determined. Histology was performed 9 weeks post surgery. When compared to whole mount post-mortem measurements, UBM accurately reported carotid plaque length. Percent stenosis, based on transverse B-mode UBM measurements, correlated well with that calculated from histological sections. PW-Doppler revealed that constriction reduced maximum systolic velocity (vmax) and duration of the systolic velocity peak (ts/tt). Pre-plaque (2 week post-surgery) PW-Doppler parameters (vmax and ts/tt) were correlated with plaque length at 9 weeks, and were predictive of plaque formation. Correlation of initiating PW-Doppler parameters (vmax and ts/tt) with resulting plaque length established the degree of flow disturbance required for subsequent plaque development and demonstrated its power for predicting plaque development.

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