Tag: AP48

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BLTR mediates leukotriene B(4)-induced chemotaxis and adhesion and plays a dominant role in eosinophil accumulation in a murine model of peritonitis.

Leukotriene B(4) (LTB(4)) is a potent chemoattractant active on multiple leukocytes, including neutrophils, macrophages, and eosinophils, and is implicated in the pathogenesis of a variety of inflammatory processes. A seven transmembrane-spanning, G protein-coupled receptor, called BLTR (LTB(4) receptor), has recently been identified as an LTB(4) receptor. To determine if BLTR is the sole receptor mediating LTB(4)-induced leukocyte activation and to determine the role of LTB(4) and BLTR in regulating leukocyte function in inflammation in vivo, we generated a BLTR-deficient mouse by targeted gene disruption. This mouse reveals that BLTR alone is responsible for LTB(4)-mediated leukocyte calcium flux, chemotaxis, and firm adhesion to endothelium in vivo. Furthermore, despite the apparent functional redundancy with other chemoattractant-receptor pairs in vitro, LTB(4) and BLTR play an important role in the recruitment and/or retention of leukocytes, particularly eosinophils, to the inflamed peritoneum in vivo. These studies demonstrate that BLTR is the key receptor that mediates LTB(4)-induced leukocyte activation and establishes a model to decipher the functional roles of BLTR and LTB(4) in vivo.

http://jem.rupress.org/content/192/3/439.long

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Decreased neutrophil function as a cause of retained placenta in dairy cattle.

It is unclear why some cows fail to expel the placenta following calving. One theory suggests the fetal placenta must be recognized as “foreign” tissue and rejected by the immune system after parturition to cause expulsion of the placenta. We hypothesized that impaired neutrophil function causes retained placenta (RP). We examined the ability of neutrophils to recognize fetal cotyledon tissue as assessed by a chemotaxis assay, which utilized a placental homogenate obtained from a spontaneously expelled placenta as the chemoattractant. Neutrophil killing ability was also estimated by determining myeloperoxidase activity in isolated neutrophils. Blood samples were obtained from 142 periparturient dairy cattle in two herds. Twenty cattle developed RP (14.1%). Neutrophils isolated from blood of cows with RP had significantly lower neutrophil function in both assays before calving, and this impaired function lasted for 1 to 2 wk after parturition. The addition of antibody directed against interleukin-8 (IL-8) to the cotyledon preparation used as a chemoattractant inhibited chemotaxis by 41%, suggesting that one of the chemoattractants present in the cotyledon at parturition is IL-8. At calving, plasma IL-8 concentration was lower in RP cows (51 +/- 12 pg/ml) than in cows expelling the placenta normally (134 +/- 11 pg/ml). From these data, we suggest that neutrophil function is a determining factor for the development of RP in dairy cattle. Also, depressed production of IL-8 may be a factor affecting neutrophil function in cows developing RP.

pdf available at: ddr.nal.usda.gov/bitstream/10113/13056/1/IND23327452.pdf

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Homocysteine induces monocyte chemoattractant protein-1 expression by activating NF-kappaB in THP-1 macrophages.

Homocysteinemia is an independent risk factor for cardiovascular disorders. The recruitment of monocytes is an important event in atherogenesis. Monocyte chemoattractant protein-1 (MCP-1) is a potent chemokine that stimulates monocyte migration into the intima of arterial walls. The objective of the present study was to investigate the effect of homocysteine on MCP-1 expression in macrophages and the underlying mechanism of such effect. Human monocytic cell (THP-1)-derived macrophages were incubated with homocysteine. By nuclease protection assay and ELISA, homocysteine (0.05-0.2 mM) was shown to significantly enhance the expression of MCP-1 mRNA (up to 2.6-fold) and protein (up to 4.8-fold) in these cells. Homocysteine-induced MCP-1 expression resulted in increased monocyte chemotaxis. The increase in MCP-1 expression was associated with activation of nuclear factor (NF)-kappaB due to increased phosphorylation of the inhibitory protein (IkappaB-alpha) as well as reduced expression of IkappaB-alpha mRNA in homocysteine-treated cells. In conclusion, our results demonstrate that homocysteine, at pathological concentration, stimulates MCP-1 expression in THP-1 macrophages via NF-kappaB activation.

http://ajpheart.physiology.org/content/280/6/H2840.long

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Angiotensin II stimulates migration of retinal microvascular pericytes: involvement of TGF-beta and PDGF-BB.

We studied the promigratory effect of angiotensin II (ANG II) on cultured bovine retinal microvascular pericytes. ANG II stimulated migration of pericytes by 86% at 10(-8) M, but this effect was lost at 10(-4) M. Migratory responses were inhibited by the ANG II type 1 (AT(1)) receptor antagonist losartan but not by PD-123319, an AT(2) antagonist. Addition of PD-123319 to the 10(-4) M ANG II dose restored migratory responses. The promigratory effect of ANG II (10(-7) M) was reduced by 59% in absence of gradient. Although ANG II augmented the latent matrix metalloproteinase-2 (MMP-2) activity of the pericyte by 35%, it also doubled tissue inhibitors of MMPs. ANG II-induced migration was not altered by a broad-spectrum MMP inhibitor (GM6001); it was inhibited by ~50% by antibodies against transforming growth factor (TGF)-beta(1/2/3) and was abolished by antibodies against platelet-derived growth factor (PDGF)-BB. We conclude that ANG II induces chemotactic responses on retinal microvascular pericytes acting through the AT(1) receptor. This effect is opposed by the AT(2) receptor. ANG II-induced chemotaxis is mediated by PDGF-BB and involves TGF-beta, but it is independent of MMP activity. It is also independent of vascular endothelial growth factor (VEGF) because VEGF did not stimulate pericyte migration. ANG II can contribute to the regulation of retinal neovascularization by stimulating pericyte migration.

link to pdf at: ajpheart.physiology.org/content/282/2/H739.full.pdf

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CXC Chemokine Receptor 5 Expression Defines Follicular Homing T Cells with B Cell Helper Function

Leukocyte traffic through secondary lymphoid tissues is finely tuned by chemokines. We have studied the functional properties of a human T cell subset marked by the expression of CXC chemokine receptor 5 (CXCR5). Memory but not naive T cells from tonsils are CXCR5+ and migrate in response to the B cell–attracting chemokine 1 (BCA-1), which is selectively expressed by reticular cells and blood vessels within B cell follicles. Tonsillar CXCR5+ T cells do not respond to other chemokines present in secondary lymphoid tissues, including secondary lymphoid tissue chemokine (SLC), EBV-induced molecule 1 ligand chemokine (ELC), and stromal cell–derived factor 1 (SDF-1). The involvement of tonsillar CXCR5+ T cells in humoral immune responses is suggested by their localization in the mantle and light zone germinal centers of B cell follicles and by the concomitant expression of activation and costimulatory markers, including CD69, HLA-DR, and inducible costimulator (ICOS). Peripheral blood CXCR5+ T cells also belong to the CD4+ memory T cell subset but, in contrast to tonsillar cells, are in a resting state and migrate weakly to chemokines. CXCR5+ T cells are very inefficient in the production of cytokines but potently induce antibody production during coculture with B cells. These properties portray CXCR5+ T cells as a distinct memory T cell subset with B cell helper function, designated here as follicular B helper T cells (TFH).

http://jem.rupress.org/content/192/11/1553.long

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Loss of responsiveness to chemotactic factors by deletion of the C-terminal protein interaction site of angiomotin

We have recently identified a novel protein, named angiomotin, by its ability to bind the angiogenesis inhibitor angiostatin in the yeast two-hybrid system. Angiomotin belongs to a family with two other members, AmotL-1 and -2 characterized by coiled-coil and C-terminal PDZ binding domains. Here we show that the putative PDZ binding motif of angiomotin serves as a protein recognition site and that deletion of three amino acids in this site results in inhibition of chemotaxis. Furthermore, endothelial cells expressing mutant angiomotin failed to migrate and form tubes in an in vitro tube formation assay. To study the effect of angiomotin on embryonic angiogenesis, we generated transgenic mice expressing wild-type angiomotin and the C-terminal deletion mutant driven by the endothelial cell-specific receptor tyrosine kinase (TIE) promoter. Expression of mutant angiomotin in endothelial cells inhibited migration into the neuroectoderm and intersomitic regions resulting in death at embryonic day 9.5. In contrast, mice expressing wild-type angiomotin developed normally and were fertile. These results suggest that the putative PDZ binding motif of angiomotin plays a critical role in regulating the responsiveness of endothelial cells to chemotactic cues.

http://jcs.biologists.org/content/116/18/3803.long

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Effect of Molybdenum-Induced Copper Deficiency on In Vivo and In Vitro Measures of Neutrophil Chemotaxis both Before and Following an Inflammatory Stressor

Twelve Angus Hereford heifers (avg wt=183.6kg) were allotted by initial liver copper (Cu) concentrations into one of two treatments. Control (n=6) heifers were fed a basal diet supplemented to provide a dietary Cu level of 10ppm. Molybdenum (Mo)-induced Cu-deficient heifers (n=6) were fed an identical basal diet supplemented with sodium molybdate (Cu:Mo ratio = 1:2.5), with dietary sulfur at .3% of the total diet. Dietary treatments were delivered for 120d, at which time Mo-supplemented heifers were considered Cu-deficient (286 and 49ppm liver Cu for control and Mo-induced Cu-deficient, respectively). Peripheral blood neutrophils were enumerated both before and after the administration of an inflammatory stressor, a subcutaneous injection (1.5mL) of Freund’s complete adjuvant. In vitro and in vivo measures of neutrophil chemotaxis were evaluated and the expression of two adhesion molecules, CD18 and L-selectin, were analyzed by flow cytometric procedures. Molybdenum-induced Cu deficiency increased (P<.01) the number of peripheral blood neutrophils; however, in vitro neutrophil chemotaxis was not affected. In vivo neutrophil chemotaxis tended (P<.08) to be increased in Mo-induced Cu-deficient heifers (1.55 vs 2.26 x 106 cells/sponge for control and Mo-supplemented, respectively). No differences in CD18 or L-selectin expression were detected between treatments. However, CD18 expression was decreased (P<.05) in both treatments following adjuvant injection. These data suggest that Mo-induced Cu deficiency results in an increase in peripheral blood neutrophil number, without altering chemotactic ability and adhesion molecule expression.

link to pdf at: http://www.ncbi.nlm.nih.gov/pubmed/8923191

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Prostaglandin D2 is a potent chemoattractant for human eosinophils that acts via a novel DP receptor

Prostaglandin D2 (PGD2) is released following exposure of asthmatics to allergen and acts via the adenylylcyclase–coupled receptor for PGD2 (DP receptor). In this study, it is reported that human eosinophils possess this receptor, which would be expected to inhibit their activation. In contrast, it was found that prostaglandinD2 is a potent stimulator of eosinophil chemotaxis, actin polymerization, CD11b expression, and L-selectin shedding. These responses are specific for eosinophils, as neutrophils display little or no response to prostaglandinD2. They were not due to interaction with receptors for other prostanoids, as prostaglandins E2 and F2a, U46619 (a thromboxane A2 analogue), and carbaprostacyclin (a prostacyclin analogue) displayed little or no activity. Furthermore, they were not shared by the selective DP receptor agonist BW245C and were not prevented by the selective DP receptor antagonist BWA868C, indicating that they were not mediated by DP receptors. In contrast, the prostaglandin D2 metabolite 13,14-dihydro-15-oxoprostaglandin D2 induced eosinophil activation but did not stimulate DP receptor–mediated adenosine 3’,5’–cyclic monophosphate (cAMP) formation. These results indicate that in addition to the classic inhibitory DP1 receptor, eosinophils possess a second, novel DP2 receptor that is associated with PGD2-induced cell activation. These 2 receptors appear to interact to regulate eosinophil responses to PGD2, as blockade of DP1 receptor–mediated cAMP production by BWA868C resulted in enhanced DP2 receptor–mediated stimulation of CD11b expression. The balance between DP1 and DP2 receptors could determine the degree to which prostaglandin D2 can activate eosinophils and may play a role in eosinophil recruitment in asthma.

link to pdf at bloodjournal.hematologylibrary.org/content/98/6/1942.full.pdf

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Leptin Stimulates Rat Aortic Smooth Muscle Cell Proliferation and Migration

Leptin, a peptide secreted from adipose tissue, plays an important role in the regulation of food intake and energy expenditure. In obese patients, plasma leptin levels are elevated and obesity is one of the major risk factors for cardiovascular diseases. Therefore, in this study, we investigated the effect of leptin on vascular smooth muscle cell (VSMC) functions. Cultured rat aortic VSMC expressed 130-kDa short form of leptin receptor. Leptin stimulated both proliferation and migration of VSMC. Leptin stimulated phosphorylation and activation of mitogen-activated protein (MAP) kinases, and also increased phosphatidylinositol (PI) 3-kinase activity. Further, two distinct PI 3-kinase inhibitors, wortmannin and LY294002 inhibited the migratory effect of leptin. These results demonstrate that leptin is a proliferative and migratory factor for VSMC, implying that leptin may play a role in the formation and development of vascular lesions.

link to pdf at http://www.ncbi.nlm.nih.gov/pubmed/11729375

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Murine Astrocytes Express a Functional Chemokine Receptor

Elevated levels of chemokines have been observed in various diseases of the CNS. Little is known, however, about how these chemokines affect parenchymal cells of the CNS. The  current studies examine astrocyte chemotaxis to the mouse chemokine macrophage inflammatory protein-1 (MIP-1a). Murine astrocytes demonstrate directed migration along a chemical gradient in response to 10e-10 – 10e-8M MIP-1a. Peak chemotactic responses are noted at 1e-09M. MIP-1a-induced astrocyte migration is specifically inhibitable with pertussis toxin, suggesting a role for G1a proteins in the signaling process. RT-PCR and insitu hybridization were used to identify expression of the murine CCR1MIP-1a receptor on astrocytes. Astrocytes contain mRNA for CCR1, but messages for CCR4 and the orphan chemokine receptorMIP-1aR-like #1 were not detected. The combined results suggest that a functional chemokine receptor is expressed on resident cells of the CNS. We speculate that the interactions of chemokines with astrocytes are involved in inflammatory reactions of the CNS.

www.jneurosci.org/content/17/17/6522.full.pdf