Protein kinase C-⑀ (PKC-⑀) translocates to phagosomes and promotes uptake of IgG-opsonized targets. To identify the regions responsible for this concentration, green fluorescent protein (GFP)-protein kinase C-⑀ mutants were tracked during phagocytosis and in response to exogenous lipids. Deletion of the diacylglycerol (DAG)-binding ⑀C1 and ⑀C1B domains, or the ⑀C1B point mutant ⑀C259G, decreased accumulation at phagosomes and membrane translocation in response to exogenous DAG. Quantitation of GFP revealed that ⑀C259G, ⑀C1, and ⑀C1B accumulation at phagosomes was significantly less than that of intact PKC-⑀. Also, the DAG antagonist 1-hexadecyl-2-acetyl glycerol (EI-150) blocked PKC-⑀ translocation. Thus, DAG binding to ⑀C1B is necessary for PKC-⑀ translocation. The role of phospholipase D (PLD), phosphatidylinositol-specific phospholipase C (PI-PLC)-␥1, and PI-PLC-␥2 in PKC-⑀ accumulation was assessed. Although GFP-PLD2 localized to phagosomes and enhanced phagocytosis, PLD inhibition did not alter target ingestion or PKC-⑀ localization. In contrast, the PI-PLC inhibitor U73122 decreased both phagocytosis and PKC-⑀ accumulation. Although expression of PI-PLC-␥2 is higher than that of PI-PLC-␥1, PI-PLC-␥1 but not PI-PLC-␥2 consistently concentrated at phagosomes. Macrophages from PI-PLC-␥2؊/؊ mice were similar to wild-type macrophages in their rate and extent of phagocytosis, their accumulation of PKC-⑀ at the phagosome, and their sensitivity to U73122. This implicates PI-PLC-␥1 as the enzyme that supports PKC-⑀ localization and phagocytosis. That PI-PLC-␥1 was transiently tyrosine phosphorylated in nascent phagosomes is consistent with this conclusion. Together, these results support a model in which PI-PLC-␥1 provides DAG that binds to ⑀C1B, facilitating PKC-⑀ localization to phagosomes for efficient IgGmediated phagocytosis.

Stroke is a leading cause of death in the United States. As ∼60% of strokes result from carotid plaque rupture, elucidating the mechanisms that underlie vulnerability is critical for therapeutic intervention. We tested the hypothesis that stable and vulnerable human plaques differentially express genes associated with matrix degradation. Examination established that femoral, and the distal region of carotid, plaques were histologically stable while the proximal carotid plaque regions were vulnerable. Quantitative RT-PCR was used to compare expression of 22 genes among these tissues. Distal carotid and femoral gene expression was not significantly different, permitting the distal carotid segments to be used as a paired control for their corresponding proximal regions. Analysis of the paired plaques revealed differences in 16 genes that impact plaque stability: matrix metalloproteinases (MMP, higher in vulnerable), MMP modulators (inhibitors: lower, activators: higher in vulnerable), activating Fc receptors (FcγR, higher in vulnerable) and FcγR signaling molecules (higher in vulnerable). Surprisingly, the relative expression of smooth muscle cell and macrophage markers in the three plaque types was not significantly different, suggesting that macrophage distribution and/or activation state correlates with (in)stability. Immunohistochemistry revealed that macrophages and smooth muscle cells localize to distinct and non-overlapping regions in all plaques. MMP protein localized to macrophage-rich regions. In vitro, treatment of macrophages with immune complexes, but not oxidized low density lipoprotein, C-reactive protein, or TNF-α, induced a gene expression profile similar to that of the vulnerable plaques. That ligation of FcγR recapitulates the pattern of gene expression in vulnerable plaques suggests that the FcγR → macrophage activation pathway may play a greater role in human plaque vulnerability than previously appreciated.