Microarray analysis of peroxisome proliferator-activated receptor-gamma induced changes in gene expression in macrophages

We used a combination of expression microarray and Northern blot analyses to identify target genes for peroxisome proliferator-activated receptor (PPAR) gamma in RAW264.7 macrophages. PPARgamma natural ligand 15-deoxy-Delta(12,14) prostaglandin and synthetic ligands ciglitazone and rosiglitazone increased the expression of scavenger receptor CD36 and ATP-binding cassette transporter A1, as well as adipophilin (a lipid droplet coating protein involved in intracellular lipid storage and transport), calpain (a protease implicated in ABCA1 protein degradation), and ADAM8 (a disintegrin and metalloprotease protein involved in cell adhesion). These findings are relevant to understanding the effect of PPARgamma activation on gene expression and cognate pathways in macrophages.     

Electron microscopic study of the in vivo erythrophagocytosis by alveolar macrophages of the cat

Summary Erythrophagoeytosis in vivo by cat alveolar macrophages was studied under the electron microscope by collecting the macrophages at 2 hours and 48 hours following the intratracheal injection of autologous blood. Considering the progressive ultrastructural modifications of the red blood cell plasma membrane, different successive stages were observed, corresponding to the hemolysis of the erythrocytes: 1. A recently engulfed erythrocyte appears unaltered within the phagocytic vacuole. 2. A dense layer, surrounding the plasma membrane of the red cell, is observed within the phagocytic vacuole. 3. The content of the vacuole is uniformly dense and the plasma membrane of the red cell exhibits discontinuous thickenings. 4. The whole vacuole appears very dense (hyperdense stage) and the plasma membrane is shown altered. The whole process of erythrophagocytosis is accompanied by an active fusion of the phagocytic vacuole with typical lysosomes and lysosomes containing crystal-like material. It is suggested that hemolysis may be explained in terms of enzymic digestion of the proteinic part of the plasma membrane of the erythrocyte.The authors wish to thank Miss Gabrielle Audet for her technical assistance, and Mr. Gaston Chevalier for revision of the English text.     

Evaluation of foam cell formation in cultured macrophages: an improved method with Oil Red O staining and DiI-oxLDL uptake

Macrophage-derived foam cell formation elicited by oxidized low-density lipoprotein (oxLDL) is the hallmark of early atherogenesis. Detection of foam cell formation is conventionally practiced by Oil Red O (ORO) staining of lipid-laden macrophages. Other methods include 1,1′-dioctadecyl-3,3,3′3′-tetra-methylindocyanide percholorate (DiI)-labeled oxLDL (DiI-oxLDL) uptake and Nile Red staining. The purpose of the present study is to report an optimized method for assessing foam cell formation in cultured macrophages by ORO staining and DiI-oxLDL uptake. After incubation with oxLDL (50 μg/ml) for 24 h, the macrophages were fixed, stained with ORO for just 1 min, pronounced lipid droplets were clearly observed in more than 90% of the macrophages. To test the in vivo applicability of this method, lesions (or foam cells) of cryosections of aortic sinus or primary mouse peritoneal macrophages from ApoE deficient mice fed a high cholesterol diet were successfully stained. In another set of experiments, treatment of macrophages with DiI-oxLDL (10 μg/ml) for 4 h resulted in significant increase in oxLDL uptake in macrophages as demonstrated by confocol microscopy and flow cytometry. We conclude that the optimized ORO staining and fluorescent labeled oxLDL uptake techniques are very useful for assessing intracellular lipid accumulation in macrophages that are simpler and more rapid than currently used methods.     

Hypoxia inhibits the expression of the CCR5 chemokine receptor in macrophages

Hypoxia, a decrease in oxygen tension occurring in pathological tissues, has a profound effect on macrophage functions. Here, we provide the first evidence that hypoxia inhibits CCR5 chemokine receptor expression in mouse macrophages. CCR5 was constitutively expressed in macrophages and upregulated by IFNgamma. Hypoxia downregulated both constitutive and IFNgamma-induced CCR5 mRNA and protein. Reoxygenation of hypoxic cells reverted CCR5 inhibition. CCR5 upregulation by IL-10, LPS, and IL-4 was also antagonized by hypoxia. CCR5 inhibition may be a way to retain/concentrate recruited macrophages at hypoxic sites or a feedback mechanism to control the autocrine activation of macrophages which produce CCR5 ligands.     

Expression of adiponectin receptors in human macrophages and regulation by agonists of the nuclear receptors PPARalpha, PPARgamma, and LXR

Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors expressed in macrophages where they control cholesterol homeostasis and inflammation. In an attempt to identify new PPARalpha and PPARgamma target genes in macrophages, a DNA array-based global gene expression profiling experiment was performed on human primary macrophages treated with specific PPARalpha and PPARgamma agonists. Surprisingly, AdipoR2, one of the two recently identified receptors for adiponectin, an adipocyte-specific secreted hormone with anti-diabetic and anti-atherogenic activities, was found to be induced by both PPARalpha and PPARgamma. AdipoR2 induction by PPARalpha and PPARgamma in primary and THP-1 macrophages was confirmed by Q-PCR analysis. Interestingly, treatment with a synthetic LXR agonist induced the expression of both AdipoR1 and AdipoR2. Furthermore, co-incubation with a PPARalpha ligand and adiponectin resulted in an additive effect on the reduction of macrophage cholesteryl ester content. Finally, AdipoR1 and AdipoR2 are both present in human atherosclerotic lesions. Moreover, AdipoR1 is more abundant than AdipoR2 in monocytes and its expression decreases upon differentiation into macrophages, whereas AdipoR2 remains constant. In conclusion, AdipoR1 and AdipoR2 are expressed in human atherosclerotic lesions and macrophages and can be modulated by PPAR and LXR ligands, thus identifying a mechanism of crosstalk between adiponectin and these nuclear receptor signaling pathways.     

安徽省宁国县畲族红细胞血型分布

调查１６０名安徽省宁国县畲族村民的ＡＢＯ、Ｒｈ、Ｐ、ＭＮ系统红细胞血型,结果显示ＡＢＯ血型表型频率分布为Ｏ（０．４６８７）〉Ｂ（０．２３７５）〉Ａ（０．２２５０）〉ＡＢ（０．０６８８）,基因频率ｐ＝０．１５００,ｑ＝０．１５７５,ｒ＝０．６９２５;Ｒｈ血型表型频率分布为ＣＣｄｅｅ（０．５３８５）〉ＣＣＤＥ（０．１６６７）〉ＣｃＤＥ（０．１４７４）〉ＣｃＤｅｅ（０．０９６１）〉ｃｃＤＥ（０．０３２１     

A stereological ultrastructural study of stimulated peritoneal macrophages in the germ-free mouse

Peritoneal macrophage ultrastructure was analysed stereologically in germ-free mice given a single intraperitoneal injection of sterile, pyrogen-free saline. Thus the stimulant was non-particulate, non-antigenic and inorganic, and effects of immune reactions were minimal. Macrophages were recovered 1, 6, 24 and 72 h after stimulation. A sequence of structural alterations is reported which may be fundamental to macrophage activation. The plasma membrane and nuclear envelope increased in area within only 1 h of saline injection. During the next 5 h loss of plasma membrane, probably by pinocytosis, caused cellular "rounding" and clear-cut alteration in surface configuration. At the same time lysosome-like granules enlarged but decreased in number. By 24 h most cellular structures and compartments (including the plasma membrane) were enlarged. Morphological evidence of nuclear activation accompanied a rather modest enlargement of the nucleus at this stage. The RER hypertrophied last and must, therefore, be judged sufficient in resident macrophages to support the initial growth response which results after stimulation. Thus hypertrophy was observed eventually in every structure examined. Even the minimally activated macrophages resident in the peritoneum of germ-free mice respond readily to stimulation.     

云南阿昌族的红细胞血型分布

调查了１０２名云南阿昌族的ＡＢＯ,ＮＮＳｓ,Ｒｈ和Ｐ系统的红细胞血型,结果表明,阿昌族的基因频率ｐ（０．３８７４）是迄今国内调查过人群中的最高值,Ｅ（０．２４５９）和ＣＤｅ（０．６９３６）基因或染色体频率较高,而Ｓ（０．０６８６）和Ｐ１（０．１０８９）频率较低;Ｍｓ（０．５９５０）连锁率高于Ｎｓ（０．３３５３）;未发现ＳＳ和Ｒｈ（－Ｄ）阴性表现型。     

24种红细胞血型抗原在黑龙江省赫哲族人群中的分布

对３代均为赫哲族的黑龙江省同江县街津口乡和八叉乡的９４人进行了１０个红细胞血型系统（ＡＢＯ、Ｒｈ、ＭＮＳｓ、Ｄｕｆｆｙ、Ｋｉｄｄ、Ｌｕｔｈｅｒａｎ、Ｄｉｅｇｏ、Ｐ、Ｋｅｌｌ、Ｌｅｗｉｓ）共２３种抗原分布的调查,结果显示,基因频率ｑ（０．２２２３）〉ｐ（０．１６９２）,ｍ（０．６０１１）〉ｎ（０．３９８９）,为我国北方人群特点。其它基因频率为,ｒ＝０．６０８５;Ｓ＝０．０６９１,ｓ＝０．９３０９,Ｍ     

A physiological role for DNA supercoiling in the anaerobic regulation of colicin gene expression

Summary The mechanism of anaerobic regulation of synthesis of colicins E1, E2, E3, K and D was studied. It was found that anaerobiosis significantly increases expression of the genes for colicins E1, E2, E3, K, and D. Experiments with novobiocin (a DNA gyrase inhibitor) showed that colicin synthesis in minicells and derepressed colicin synthesis in cells are dramatically reduced by relaxation of DNA supercoiling. A good correlation was observed between the levels of colicin synthesis and plasmid DNA supercoiling and the degree of aeration of the cultures. Thus, the regulation of colicin gene expression in response to a change in aeration appears to be mediated by environmentally induced variations in DNA supercoiling.     

An optical-manipulation technique for cells in physiological flows

We have developed a technique to manipulate human red blood cells (RBCs) in hydrodynamic flows. This method applies optical tweezers to trap and move microbead-attached RBCs in a liquid medium at various speeds, while it significantly minimizes laser heating and photon-induced stress for normal operation with laser-trapped cells. Computational fluid dynamics is applied to simulate flow-induced shear stress over the cell membrane and to correlate quantitatively the forces with the cell deformations. RBCs can be manipulated under physiological conditions by this approach, which may open an avenue to design principles for the next generation of cell sorting and delivery.     

Enhanced phagocytosis of CD47-deficient red blood cells by splenic macrophages requires SHPS-1

The interaction of CD47 on red blood cells (RBCs) with SHPS-1 on macrophages is implicated to prevent the phagocytosis of the former cells by the latter cells. Indeed, the rate of clearance of transfused CD47-deficient (CD47(-/-)) RBCs from the bloodstream of wild-type mice was markedly increased compared with wild-type RBCs. Conversely, the rate of clearance of transfused wild-type RBCs was markedly increased in mice that expressed a mutant form of SHPS-1 lacking most of the cytoplasmic region of the protein. However, we here found that the clearance of CD47(-/-) RBCs in SHPS-1 mutant mice was minimal. In addition, the phagocytosis of CD47(-/-) RBCs by splenic macrophages from SHPS-1 mutant mice was markedly reduced compared with wild-type macrophages. These results thus suggest an additional role for CD47 on RBCs in the negative regulation of phagocytosis by macrophages and in determination of the life span of circulating RBCs.     

Visualization study of motion and deformation of red blood cells in a microchannel with straight,divergent and convergent sections

The size of red blood cells (RBC) is on the same order as the diameter of microvascular vessels. Therefore, blood should be regarded as a two-phase flow system of RBCs suspended in plasma rather than a continuous medium of microcirculation. It is of great physiological and pathological significance to investigate the effects of deformation and aggregation of RBCs on microcirculation. In this study, a visualization experiment was conducted to study the microcirculatory behavior of RBCs in suspension. Motion and deformation of RBCs in a microfluidic chip with straight, divergent, and convergent microchannel sections have been captured by microscope and high-speed camera. Meanwhile, deformation and movement of RBCs were investigated under different viscosity, hematocrit, and flow rate in this system. For low velocity and viscosity, RBCs behaved in their normal biconcave disc shape and their motion was found as a flipping motion: they not only deformed their shapes along the flow direction, but also rolled and rotated themselves. RBCs were also found to aggregate, forming rouleaux at very low flow rate and viscosity. However, for high velocity and viscosity, RBCs deformed obviously under the shear stress. They elongated along the flow direction and performed a tank-treading motion.     

客家人的红细胞血型分布

对父母双方上溯三代均为客家人的广东梅县200名 (其中男89人,女111人) 健康学生进行了红细胞血型ABO,MNSs,Rh,Kidd,Duffy,Diego,Xg,Lewis及P等系统的分布调查。结果显示,客家人的基因频率S=0.0250,NS=0,pl=0.0917和Fyb=0.0300,都是汉族人群中最低的。其它基因频率为r=0.6632,p=0.1863,q=0.1505;m=0.5250,n=0.4750,MS=0.0250,Ms=0.5000,Ns=0.4750,s=0.9750;C=0.6575,D=1.0000,E=0.1515,CDe=0.6226,cDE=0.1200,cDe=0.2189,CDE=0.0389;JKa=0.4642,JKb=0.4881,JK=0.0477;Fya=0.9700;Dia=0.0202,Dib=0.9798;Xga=0.3633,Xg=0.6367;P2=0.9083。发现了国内第二例Jk(a-b-)表型,未发现MNS型,NS型,NSs型,CCDEE型,CcDEE型,Fy(a-)型和Rho(-)型。Le(a+b-)型29人,Le(a+b+)型2人,Le(a-b+)型67人,Le(a-b-)型102人。客家人与国内19个群体的遗传距离计算结果表明,与客家人遗传距离最近的是福建汉族、湖南苗族、贵州汉族及广西侗族,其次为河南汉族、黑龙江汉族、陕西汉族,福建畲族及上海汉族,而与云南白族、辽宁满族、甘肃汉族、广西瑶族、广西壮族、内蒙汉族及四川彝族的遗传距离较远。与客家人遗传距离最远的是湖南土家族、海南苗族及海南黎族。     

云南“本人”的红细胞血型分布及其与契丹人血缘关系的探讨

调查了云南省施甸县木老元乡哈寨村１０４名“本人”的４个细胞血型系统分布。结果表明,“本人”的ＡＢＯ血型系统分布特点是,基因频率ｐ（０．３０６９）〉基因频率ｑ（０．１７３９）;在ＭＮＳｓ血型系统中,基因频率ｍ（０．６５３８）〉基因频率ｎ（０．３４６２）;在Ｐ血型系统中,基因频率Ｐ１（０．１７９８）较低。这些均与我国南方少数民族的分布特点基本相符。与我国大部分地区一样。Ｒｈ因型系统的单倍型频率中最高的