In vitro characterization and micromechanics of tumor cell chemotactic protrusion,locomotion, and extravasation

The objective of this paper is to introduce some novel in vitro applications in characterizing human melanoma cell protrusion and migration in response to soluble extracellular matrix protein stimulation. Specifically, we describe two assay systems: (1) dual-micropipette manipulation and (2) flow-migration chamber. Applications of the dual-micropipet technique provided kinetic measure of cell movement, cyclic pseudopod protrusion, and subsequent cell locomotion governed by chemotactic molecular transport dynamics. Chemotactic concentration gradient was found to influence significantly pseudopod protrusion frequency and locomotion speed, but not the protrusion extension. To further characterize active tumor cell extravasation, a process that involves dynamic tumor cell adhesion to vascular endothelium under flow conditions and subsequent transendothelial migration in response to chemotactic signals from the interstitial space, we developed a flow-migration chemotaxis system. This assay enabled characterization of tumor cell transcellular migration in terms of chemotactic signal gradients, shear forces, and cell-substrate adhesion. Results suggest that shear flow plays significant roles in tumor cell extravasation that is regulated by both tumor cell motility and tumor cell adhesion to endothelial molecules in a cooperative process. © 2002 Biomedical Engineering Society. PAC2002: 8717Jj, 8719Xx     

The immunomodulator FTY720 interferes with effector functions of human monocyte-derived dendritic cells

The potent immunomodulator FTY720 elicits immunosuppression via acting on sphingosine 1-phosphate receptors (S1PR), thereby leading to an entrapment of lymphocytes in the secondary lymphoid tissue. To elucidate the potential in vitro effects of this drug on human monocyte-derived DC, we used low nanomolar therapeutic concentrations of FTY720 and phosphorylated FTY720 (FTY720-P) and investigated their influence on DC surface marker expression, protein levels of S1PR and DC effector functions: antigen uptake, chemotaxis, cytokine production, allostimulatory and Th-priming capacity. We report that both FTY720 and FTY720-P reduce chemotaxis of immature and mature DC. Mature DC generated in the presence of FTY720 or FTY720-P showed an impaired immunostimmulatory capacity and reduced IL-12 but increased IL-10 production. T cells cultured in the presence of FTY720- or FTY720-P-treated DC showed an altered cytokine production profile indicating a shift from Th1 toward Th2 differentiation. In treated immature and mature DC, expression levels for two S1PR proteins, S1P1 and S1P4, were reduced. We conclude that in vitro treatment with FTY720 affects DC features that are essential for serving their role as antigen-presenting cells. This might represent a new aspect of the overall immunosuppressive action of FTY720 and makes DC potential targets of further sphingolipid-derived drugs.     

The carboxyl terminus of the chemokine receptor CCR3 contains distinct domains which regulate chemotactic signaling and receptor down-regulation in a ligand-dependent manner

The chemokine receptor CCR3 regulates the chemotaxis of leukocytes implicated in allergic disease, such as eosinophils. Incubation of eosinophils with CCL11, CCL13 or CCL5 resulted in a rapid decrease of cell-surface CCR3 which was replicated using CCR3 transfectants. Progressive truncation of the CCR3 C terminus by 15 amino acids produced three constructs, Delta340, Delta325 and Delta310. Delta340 and Delta325 were able to bind CCL11 with affinities similar to wild-type CCR3. Delta340 transfectants exhibited enhanced migration and reduced receptor down-regulation in response to CCL11 and CCL13. Delta325 transfectants displayed chemotactic responses to CCL11 and CCL13 similar to wild-type CCR3, and had impaired down-regulation when stimulated with CCL13 but not CCL11. In contrast, neither the Delta325 nor Delta340 truncation affected chemotaxis or receptor down-regulation induced by CCL5. Delta310 transfectants bound CCL11 poorly and were biologically inactive. Inhibitors of p38 mitogen-activated protein kinase and PI3-kinase antagonized eosinophil shape change responses and chemotaxis of transfectants to CCL11 and CCL13. In contrast, shape change but not chemotaxis was sensitive to inhibition of the extracellular signal-regulated kinase kinase pathway suggesting differential regulation of the two responses. Thus, the CCR3 C terminus contains distinct domains responsible for the regulation of receptor desensitization and for coupling to chemotactic responses.     

Serum amyloid A chemoattracts immature dendritic cells and indirectly provokes monocyte chemotaxis by induction of cooperating CC and CXC chemokines

Serum amyloid A (SAA) is an acute phase protein that is upregulated in inflammatory diseases and chemoattracts monocytes, lymphocytes, and granulocytes via its G protein‐coupled receptor formyl peptide receptor like 1/formyl peptide receptor 2 (FPRL1/FPR2). Here, we demonstrated that the SAA1α isoform also chemoattracts monocyte‐derived immature dendritic cells (DCs) in the Boyden and μ‐slide chemotaxis assay and that its chemotactic activity for monocytes and DCs was indirectly mediated via rapid chemokine induction. Indeed, SAA1 induced significant amounts (≥5 ng/mL) of macrophage inflammatory protein‐1α/CC chemokine ligand 3 (MIP‐1α/CCL3) and interleukin‐8/CXC chemokine ligand 8 (IL‐8/CXCL8) in monocytes and DCs in a dose‐dependent manner within 3 h. However, SAA1 also directly activated monocytes and DCs for signaling and chemotaxis without chemokine interference. SAA1‐induced monocyte migration was nevertheless significantly prevented (60–80% inhibition) in the constant presence of desensitizing exogenous MIP‐1α/CCL3, neutralizing anti‐MIP‐1α/CCL3 antibody, or a combination of CC chemokine receptor 1 (CCR1) and CCR5 antagonists, indicating that this endogenously produced CC chemokine was indirectly contributing to SAA1‐mediated chemotaxis. Further, anti‐IL‐8/CXCL8 antibody neutralized SAA1‐induced monocyte migration, suggesting that endogenous IL‐8/CXCL8 acted in concert with MIP‐1α/CCL3. This explained why SAA1 failed to synergize with exogenously added MIP‐1α/CCL3 or stromal cell‐derived factor‐1α (SDF‐1α)/CXCL12 in monocyte and DC chemotaxis. In addition to direct leukocyte activation, SAA1 induces a chemotactic cascade mediated by expression of cooperating chemokines to prolong leukocyte recruitment to the inflammatory site.     

HMGN2分离制备及其趋化性研究

目的:建立分离天然HMGN2的方法并研究其抗菌性与对中性粒细胞的趋化活性。方法:5%高氯酸对子宫纤维囊腺瘤组织进行萃取,萃取物通过反相高效液相色谱(RP-HPLC)直接进行分离,Tricine-SDS-PAGE,AU-PAGE和Western-Blot对蛋白分子进行鉴定,采用Transwell实验检测HMGN2对中性粒细胞的趋化性。结果:分离出高纯度的HMGN2,HMGN2对大肠杆菌有杀菌活性但HMGN2在体外不能趋化中性粒细胞运动。结论:建立简单、直接分离高纯度的天然HMGN2的方法,有助于对其进一步研究,我们发现HMGN2对大肠杆菌有杀菌活性但它不能通过趋化中性粒细胞发挥作用。     

Quantitative analysis of eosinophil chemotaxis tracked using a novel optical device -- TAXIScan

We have reported previously the development of an optically accessible, horizontal chemotaxis apparatus, in which migration of cells in the channel from a start line can be traced with time-lapse intervals using a CCD camera (JIM 282, 1–11, 2003). To obtain statistical data of migrating cells, we have developed quantitative methods to calculate various parameters in the process of chemotaxis, employing human eosinophil and CXCL12 as a model cell and a model chemoattractant, respectively. Median values of velocity and directionality of each cell within an experimental period could be calculated from the migratory pathway data obtained from time-lapse images and the data were expressed as Velocity–Directionality (VD) plot. This plot is useful for quantitatively analyzing multiple migrating cells exposed to a certain chemoattractant, and can distinguish chemotaxis from random migration. Moreover precise observation of cell migration revealed that each cell had a different lag period before starting chemotaxis, indicating variation in cell sensitivity to the chemoattractant. Thus lag time of each cell before migration, and time course of increment of the migrating cell ratio at the early stages could be calculated. We also graphed decrement of still moving cell ratio at the later stages by calculating the duration time of cell migration of each cell. These graphs could distinguish different motion patterns of chemotaxis of eosinophils, in response to a range of chemoattractants; PGD₂, fMLP, CCL3, CCL5 and CXCL12. Finally, we compared parameters of eosinophils from normal volunteers, allergy patients and asthma patients and found significant difference in response to PGD₂. The quantitative methods described here could be applicable to image data obtained with any combination of cells and chemoattractants and useful not only for basic studies of chemotaxis but also for diagnosis and for drug screening.     

放创复合伤大鼠伤口液对真皮多能干细胞的趋化作用

目的观察放创复合伤大鼠伤口液对真皮多能干细胞(dMSCs)的趋化作用以及dMSCs对创面愈合的作用。方法采用Transwell培养体系,观察伤口提取液对dMSCs的趋化作用。用3HTdR标记dMSCs,经尾静脉输入大鼠体内,液闪法测量各组织内dMSCs的含量。用生物医学图像分析仪测量伤后创面残留面积。结果在伤口液的作用下,复合伤组Transwell培养小室内上层细胞迁移到下层的明显多于正常组织液组和生理盐水组(P<001)。伤后1周,复合伤组创面皮肤dMSCs含量明显高于正常对照组(P<001);伤后3和4周,复合伤组单位重量创面组织dMSCs含量明显高于同组的肺脏、肝脏等组织。伤后15d开始,dMSCs输注组创面残留面积明显小于复合伤对照组(P<005)。结论放创复合伤大鼠伤口提取液对dMSCs有很强的趋化作用,进而促进静脉输注的dMSCs偏向分布于创面,并促进其愈合。     

Human monocyte characteristics are altered in hypercholesterolaemia

Peripheral blood monocytes are involved during atherogenesis in adhering to endothelium, migrating into the subendothelial space and taking-up lipoproteins to become macrophage/foam cells. We have assessed whether peripheral blood monocyte characteristics are altered in human hyperlipidaemia in age/sex/smoking status matched pairs of patients and controls. Monocytes from the hypercholesterolaemic patients, as opposed to the controls, were more sensitive to stimulation by the agonist, N-formylmethionyl-leucyl-phenylalanine, with respect to chemokinesis (stimulation index 1.48 ± 0.17 vs. 1.10 ± 0.14), chemotaxis (4.05 ± 0.55 vs. 2.72 ± 0.24) and adhesion to porcine aortic endothelial monolayers (1.26 ± 0.05 vs. 1.17 ± 0.06). The patients' monocyte total surface expression of the adhesion glycoprotein CD11b/CD18 (37.5 ± 7.1 vs. 36.0 ± 7.1), but not CD11c/CD18 (31.6 ± 7.2 vs. 31.4 ± 6.8), was increased; however, the monocytes in hyperlipidaemia were larger (9.15 ± 0.11 μm vs. 8.98 ± 0.11 μm) such that the surface density of CD11b/CD18 was not altered (0.144 ± 0.029 vs. 0.142 ± 0.029). The data suggest that circulating monocytes are functionally different in hypercholesterolaemia. This may explain the increased involvement by monocytes in hypercholesterolaemia-related atherogenesis.     

Chemotaxis of the monocyte cell line U937: Dependence on cholesterol and early mevalonate pathway products

In the present study we investigated the influence of cholesterol depletion and hydroxymethylglutaryl-coenzyme A reductase (HMG-CoA reductase) inhibition on chemotaxis of the human monocytic cell line U937. Chemotaxis was nearly completely depressed after incubation for 24 h in the absence of lipoproteins. This was accompanied by a significant decrease in cellular cholesterol. Addition of 10 μg/ml low density lipoprotein (LDL) for 2 h to the cholesterol-depleted cells restored chemotaxis. Free cholesterol had no effect under these conditions. Inhibition of HMG-CoA reductase by pravastatin (0.01–1.0 mM) for 20 or 72 h also reduced chemotaxis. However, this effect was not accompanied by a decrease in cellular cholesterol when cells were grown in the presence of lipoproteins. The effect of pravastatin could be reversed by the addition of mevalonate. Addition of LDL did not change the response to pravastatin. We propose that the availability of cholesterol plays an important role in cellular chemotaxis. Furthermore, it can be suggested that other products of the mevalonate pathway apart from cholesterol may contribute to the regulation of chemotaxis.     

BMP-2 and -4 produced by vascular smooth muscle cells from atherosclerotic lesions induce monocyte chemotaxis through direct BMPRII activation

Objective

Monocytes and macrophages, together with vascular smooth muscle cells (VSMCs), play key roles at all stages of atherogenesis. There is also growing evidence that BMP signaling is involved in vascular diseases, including atherosclerosis. Here we evaluate the role played by the BMP agonist/antagonist axis in monocyte recruitment during atherogenesis.

Methods and results

Using ApoE−/− mice and BMPs, Gremlin and BMPRII siRNAs we show that BMPs (2 and 4) and their antagonist Gremlin are co-expressed in murine and human atherosclerotic vessels. Additionally, those genes are co-expressed and upregulated in cultured vascular smooth muscle cells early in atherosclerosis formation in ApoE−/− mice. Furthermore, we demonstrate that BMP-2 and -4 produced in atherosclerotic VSMCs promote, whereas Gremlin inhibits, monocyte chemoattraction. Finally, we demonstrate that chemotaxis induction occurs through direct BMP receptor II (BMPRII) activation.

Conclusion

These findings suggest that the balance between BMPs (2 and 4) and Gremlin levels modulate crosstalk processes between vascular and immune cells and ultimately the homeostasis in normal vasculature. They also indicate that under pro-atherogenic conditions, BMP signaling prevails, favoring monocyte recruitment and inflammation. Manipulation of BMP signaling may enable the identification of novel molecular approaches for preventing, stabilizing, and reverting atherosclerosis.