Peter M. Morganelli

Recombinant immune interferon increases immunoglobulin G Fc receptors on cultured human mononuclear phagocytes.

Although recent studies suggest that interferons can increase the number of IgG Fc receptor (FcR gamma) sites on mouse macrophages, direct assessment of similar effects on human mononuclear phagocytes is lacking. We therefore measured the specific binding of 125I- and fluorescein-labeled IgG1 to human monocytes and leukemic cell lines after culture in vitro with highly purified human interferons. We report that natural and recombinant human gamma-interferon causes a dramatic (nearly 10-fold) increase in the number of FcR gamma on normal human monocytes and on the human cell lines HL-60 and U-937. Alpha and beta-interferons cause a modest but significant increase in these receptors. This report demonstrates that gamma-interferon acts directly on human mononuclear phagocytes to increase FcR gamma sites, it identifies a qualitative difference in the physiologic actions of human type I and type II interferons, and it suggests that HL-60 and U-937 cells will be important models for further study of the molecular mechanisms of interferon action. The results reported here could also be the basis for a bioassay to assess the pharmacokinetics and variability of gamma-interferon action on monocytes of individual patients during treatment in vitro and in vivo.

Endotoxin induces rapid metalloproteinase-mediated shedding followed by up-regulation of the monocyte hemoglobin scavenger receptor CD163

CD163, a monocyte and macrophage‐specific surface glycoprotein, which is increased by interleukin‐10 and glucocorticoids, is a scavenger receptor for hemoglobin/haptoglobin complexes. We report a rapid and highly reproducible rise in soluble CD163 in the plasma of human volunteers given intravenous lipopolysaccharide (LPS). We also show that LPS induces shedding of CD163 from the surface of isolated monocytes, identifying shedding from monocytes and macrophages as a likely mechanism for the endotoxemia‐associated rise in plasma CD163 in vivo. Studies using the inhibitor TAPI‐0 indicate that a metalloproteinase is responsible for LPS‐mediated shedding of CD163. Finally, we demonstrate a marked increase in surface CD163 expression on circulating monocytes 24 h following experimental endotoxemia. These findings show that CD163 is rapidly mobilized in response to bacterial endotoxin. As hemoglobin can bind LPS and enhance its toxicity, it will be important to determine how cell surface and soluble CD163 influence inflammatory processes during sepsis.

Expression of ACAT-1 Protein in Human Atherosclerotic Lesions and Cultured Human Monocytes-Macrophages

The acyl coenzyme A:cholesterol acyltransferase (ACAT) gene was first cloned in 1993 (Chang et al, J Biol Chem. 1993;268:20747-20755; designated ACAT-1). Using affinity-purified antibodies raised against the N-terminal portion of human ACAT-1 protein, we performed immunohistochemical localization studies and showed that the ACAT-1 protein was highly expressed in atherosclerotic lesions of the human aorta. We also performed cell-specific localization studies using double immunostaining and showed that ACAT-1 was predominantly expressed in macrophages but not in smooth muscle cells. We then used a cell culture system in vitro to monitor the ACAT-1 expression in differentiating monocytes-macrophages. The ACAT-1 protein content increased by up to 10-fold when monocytes spontaneously differentiated into macrophages. This increase occurred within the first 2 days of culturing the monocytes and reached a plateau level within 4 days of culturing, indicating that the increase in ACAT-1 protein content is an early event during the monocyte differentiation process. The ACAT-1 protein expressed in the differentiating monocytes-macrophages was shown to be active by enzyme assay in vitro. The high levels of ACAT-1 present in macrophages maintained in culture can explain the high ACAT-1 contents found in atherosclerotic lesions. Our results thus support the idea that ACAT-1 plays an important role in differentiating monocytes and in forming macrophage foam cells during the development of human atherosclerosis.

Lipoprotein lipase links dietary fat to solid tumor cell proliferation.

Many types of cancer cells require a supply of fatty acids (FA) for growth and survival, and interrupting de novo FA synthesis in model systems causes potent anticancer effects. We hypothesized that, in addition to synthesis, cancer cells may obtain preformed, diet-derived FA by uptake from the bloodstream. This would require hydrolytic release of FA from triglyceride in circulating lipoprotein particles by the secreted enzyme lipoprotein lipase (LPL), and the expression of CD36, the channel for cellular FA uptake. We find that selected breast cancer and sarcoma cells express and secrete active LPL, and all express CD36. We further show that LPL, in the presence of triglyceride-rich lipoproteins, accelerates the growth of these cells. Providing LPL to prostate cancer cells, which express low levels of the enzyme, did not augment growth, but did prevent the cytotoxic effect of FA synthesis inhibition. Moreover, LPL knockdown inhibited HeLa cell growth. In contrast to the cell lines, immunohistochemical analysis confirmed the presence of LPL and CD36 in the majority of breast, liposarcoma, and prostate tumor tissues examined (n = 181). These findings suggest that, in addition to de novo lipogenesis, cancer cells can use LPL and CD36 to acquire FA from the circulation by lipolysis, and this can fuel their growth. Interfering with dietary fat intake, lipolysis, and/or FA uptake will be necessary to target the requirement of cancer cells for FA. Mol Cancer Ther; 10(3); 427–36. ©2011 AACR.

Acyl-Coenzyme A:Cholesterol Acyltransferase 2 (ACAT2) Is Induced in Monocyte-Derived Macrophages: In Vivo and In Vitro Studies

To test the possibility that acyl-coenzyme A:cholesterol acyltransferase 2 (ACAT2) may be expressed in human macrophages under pathologic conditions, we employed specific anti-ACAT2 antibodies and found clear ACAT2 signals in lipid-laden as well as lipid-free macrophages under various disease conditions, including atherosclerosis. However, no ACAT2 signal was detectable in macrophages under normal physiologic conditions. Using cultured human macrophages derived from blood-borne monocytes, immunoblot and RT-PCR analyses demonstrated that immature macrophages expressed only ACAT1, but the fully differentiated macrophages expressed both ACAT1 and ACAT2. Furthermore, RT-PCR clearly revealed the presence of both ACAT1 and ACAT2 mRNAs in human atherosclerotic aorta. Double immunohistochemical staining indicated that in human atherosclerotic aorta, all macrophages expressed ACAT1, while approximately 70% to 80% of macrophages also expressed ACAT2. In congenital hyperlipidemic mice, immunohistochemistry and RT-PCR demonstrated that ACAT2 was also present in lipid-laden cells of the atheromatous plaques. Our results suggest that in atherosclerotic plaque, the ability of macrophage foam cell transformation may be augmented by the dual expressions of ACAT1 and ACAT2. Additional immunoblot and RT-PCR experiments showed that the ACAT2 signal was clearly detectable in thioglycollate-elicited exudate mouse macrophages but not in peritoneal resident macrophages. We conclude that under various pathologic conditions, fully differentiated macrophages express ACAT2 in addition to ACAT1.

Expression of "Spot 14" (THRSP) predicts disease free survival in invasive breast cancer: immunohistochemical analysis of a new molecular marker.

SummaryMost breast cancers are “lipogenic”, defined by high fatty acid synthase (FAS) content and dependence on fatty acid synthesis for growth and survival. S14 (Spot 14; THRSP) is a nuclear protein that activates genes required for fatty acid synthesis. The S14 gene is amplified in ~15% of breast cancers, but clinical correlates of its expression were unknown. We analyzed 131 breast cancers by immunohistochemistry for S14 and FAS. Staining was graded 0, 1, or 2+, and scores were correlated with traditional tumor markers, histological features, and outcome. S14 and FAS staining were related to tumor size (p=0.05 for S14, p=0.038 for FAS), but not to stage. S14 but not FAS scores correlated with tumor grade in both DCIS (p=0.003) and invasive cases (p<0.001). Invasive cases (pooled node − and +) with weak S14 staining (n=21) showed no recurrence over 3000 d follow-up, including 10 cases with lymph node involvement, whereas 32% of 67 strongly-staining tumors recurred (log rank p<0.0001). S14 scores did not cosegregate with sex steroid receptors, Her2/neu, or cyclin D1. Low level S14 expression is associated with prolonged disease-free survival in invasive cases, including those with nodal metastasis. High-level expression of S14 identifies a subset of high-risk breast cancers that is not specified by analysis of sex steroid receptors, Her2/neu, or cyclin D1, and provides a molecular correlate to histologic features that predict recurrence.

Inhibition of arterial lesion progression in CD16-deficient mice: evidence for altered immunity and the role of IL-10

AIMS Given the importance of IgG Fc receptors in immune regulation, we hypothesized that Fcg receptor type III (FcgRIII, CD16) plays an important role in atherogenesis. We therefore analysed the formation of arterial lesions in LDL receptor-deficient (LDLR(-/-)) and FcgRIII(-/-)xLDLR(-/-) double knockout mice at three different points up to 24 weeks of exposure to a high-fat diet. METHODS AND RESULTS Analysis of Oil Red-O-stained sections revealed no difference in lesion formation between strains after 6 weeks of a high-fat diet, and a modest decrease after 14 weeks in double knockouts relative to LDLR(-/-) controls. After 24 weeks, lesion formation was decreased in the aortic root (30%) and innominate artery (50%) in FcgRIII double knockouts relative to LDLR(-/-) controls. Analysis of peripheral CD4+ T-cells by intracellular flow cytometry from double knockouts after 24 weeks of a high-fat diet revealed statistically significant increases in the percentages of cells producing interferon-gamma, interleukin (IL)-10, and IL-4 relative to controls, differences that were also observed by analyses of whole aortas for cytokine mRNA levels. As determined by flow cytometry, FcgRIII deficiency resulted in an expansion of CD4+ cells and an increase in the CD4 to CD8 ratio. Analysis of plasma anti-oxidized LDL (OxLDL) antibodies by chemiluminescent assay revealed that IgG1 and IgG2c titers to OxLDL were increased in FcgRIII (-/-)xLDLR(-/-) double knockouts relative to LDLR(-/-) controls, while total IgG levels were similar. CONCLUSION These results reveal altered immunity in FcgRIII(-/-)xLDLR(-/-) mice and a reduction in lesion formation associated with increased production of IL-10 by an expansion of CD4+ T-cells. The reduction in lesion formation was manifest well after evidence of an immune response to OxLDL, suggesting that FcgRIII contributes to lesion progression in murine atherosclerosis.

Levels of expression of Fcγ receptor IIA (CD32) are decreased on peripheral blood monocytes in patients with severe atherosclerosis

To obtain information in vivo concerning the role of Fcgamma receptors (FcgammaR) in atherosclerosis, we used quantitative flow cytometry to measure the levels of expression of FcgammaRI and FcgammaRIIA on peripheral monocytes in patients with severe atherosclerosis. Expression of several other markers was also measured. We found that differences in the levels of expression of FcgammaRI were not statistically significant when compared between patients and control subjects. For FcgammaRIIA, levels of expression were decreased in the patient group, a difference that was statistically significant. Levels of expression of CD14 and CD36 were also significantly decreased in the patient group. The decrease in expression of FcgammaRIIA was statistically significant when the effects of current cigarette smoking status or medication use, including statins, were taken into account. There was also a positive and statistically significant correlation between high-density lipoprotein-cholesterol and levels of expression of FcgammaRIIA for all subjects. In contrast, decreased levels of expression of CD14 and CD36 were strongly associated with current smoking status or statin use. In summary, levels of expression of FcgammaRIIA on peripheral blood monocytes were significantly decreased in patients with clinical atherosclerosis. Additional studies are warranted to determine if levels of expression of FcgammaRIIA have utility as a phenotypic marker for assessing relative risk of atherosclerotic disease.

The renin-angiotensin system during pregnancy in chronically instrumented, conscious rats

Whether the renin-angiotensin system is activated during rat gestation is controversial. Therefore we serially assessed plasma renin activity in unrestrained, chronically instrumented conscious rats during pregnancy and the postpartum period. Plasma renin activity was 3.26 +/- 0.30, 2.80 +/- 0.31, and 2.70 +/- 0.26 ng.ml-1.hr-1 on gestational days 6, 12, and 20, respectively. When the same rats were studied after delivery, plasma renin activity was 1.87 +/- 0.29, 1.81 +/- 0.09, and 2.31 +/- 0.35 ng.ml-1.hr-1 on postpartum days 3, 6, and 11, respectively. Levels measured during pregnancy were significantly greater than in the postpartum period (p less than 0.05 or less than 0.01). We then evaluated potential functional consequences of the renin-angiotensin system in gravid rats. Near term, renal hemodynamics fall from the peak levels of midgestation; we tested whether angiotensin II mediates this apparent vasoconstriction. Captopril (1.5 mg/kg, 1.5 mg.kg-1.hr-1) was acutely administered to lower circulating angiotensin II. The drug produced an 80% inhibition of angiotensin I pressor response, a tenfold elevation in plasma renin activity, but caused the same degree of mild renal vasodilation in rats whether they were virgin or pregnant. We also tested whether prior occupancy of receptors by endogenous hormone or receptor downregulation mediates the attenuated pressor response to angiotensin II observed during late pregnancy. Acute administration of captopril failed to augment refractory pressor responsiveness. Chronic treatment with enalaprilat (2.0 mg.kg-1.day-1 by osmotic minipump) also did not restore pressor responsiveness. But, in our hands, chronic administration of enalaprilat most likely failed to lower plasma angiotensin II. In summary, we suggest that during rat gestation (1) the renin-angiotensin system is activated, (2) angiotensin II does not mediate the apparent renal vasoconstriction observed near term, (3) prior receptor occupancy by endogenous hormone is not responsible for the attenuated pressor response to angiotensin II, and (4) long-term treatment with enalaprilat can produce hypotension without reducing plasma concentrations of angiotensin II.

AUF1/hnRNP D represses expression of VEGF in macrophages.

Vascular endothelial growth factor (VEGF) expression is regulated by sequence elements in the 3′ UTR of VEGF mRNA. AUF1/hnRNP D suppresses VEGF 3′ UTR–dependent expression. Peptides with arginine–glycine–glycine motifs derived from AUF1 also suppress VEGF expression.