A high-throughput chemotaxis assay for pharmacological characterization of chemokine receptors: Utilization of U937 monocytic cells

Ott, Thomas; Pahuja, Anil; Lio, Francisco M.; Mistry, Monica S.; Gross, Molly M.; Hudson, Sarah; Wade, Warren S.; Simpson, Pedro B.; Struthers, R. Scott; Alleva, David G.

doi:10.1016/j.vascn.2004.10.001

Cited by 14 publications

(11 citation statements)

References 22 publications

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“…To investigate the mechanisms of AIF-1-induced enhancement of T cell migration, the expression of chemokine receptor genes for CCR2 and CXCR4, functional receptors present on the cell surface of Jurkat T cells (21,39), was studied. CXCR4 gene expression was not affected by AIF-1 isoform 2 expression, whereas it was down-regulated by AIF-1 isoform 3 expression, compared with empty vector-transfected Figure 3A).…”

Section: Resultsmentioning

confidence: 99%

“…Type I collagen and type III collagen present in the culture media were quantified by Western blotting using specific anti-human type I or type III collagen polyclonal antibodies (Rockland, Gilbertsville, PA). For inhibition experiments, IL-4 neutralizing antibodies (BioLegend) were used as previously described (39). Briefly, conditioned supernatants (dilution 1:2) from AIF-1 isoform 2-expressing Jurkat T cells were preincubated for 1 hour at 37°C with or without anti-IL-4 antibodies at a 5-g/ml final concentration and then used for stimulation experiments.…”

Section: Ef070983 and Ef070982])mentioning

confidence: 99%

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T cells expressing allograft inflammatory factor 1 display increased chemotaxis and induce a profibrotic phenotype in normal fibroblasts in vitro

Galdo¹,

Jiménez²

2007

Arthritis & Rheumatism

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Objective. Allograft inflammatory factor 1 (AIF-1) was first identified in rat cardiac allografts undergoing chronic rejection. The vasculopathy of chronic allograft rejection is strikingly similar to that seen in patients with systemic sclerosis (SSc). We previously demonstrated AIF-1 expression in inflammatory cells infiltrating skin and lungs from SSc patients, but its role in SSc pathogenesis is unknown. The present study was undertaken to investigate the effects of AIF-1 on T cell migration and production of cytokines capable of modulating normal dermal fibroblast functions.Methods. Stably transfected Jurkat T cells expressing 2 AIF-1 splicing variants were prepared, and their migration toward fibroblast monolayers assayed in Transwell cultures. Cytokine production was assessed by real-time polymerase chain reaction (PCR) and multiplex enzyme-linked immunosorbent assay. Fibroblast gene expression was quantified by real-time PCR, and collagen production by Western blot analysis of culture media.Results. AIF-1 significantly increased Jurkat T cell migration toward fibroblast monolayers. Expression of AIF-1 isoform 2 in Jurkat T cells up-regulated their production of interleukin-4 (IL-4) and IL-17. Conditioned media from AIF-1-expressing clones stimulated synthesis of types I and III collagen and expression of IL-6, transforming growth factor ␤, endothelin receptor, and ␣-smooth muscle actin by normal dermal fibroblasts.Conclusion. These results suggest that AIF-1 may participate in the early pathogenesis of SSc by promoting tissue T cell infiltration and production of cytokines capable of inducing the expression of a fibrotic phenotype in normal fibroblasts.

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Section: Resultsmentioning

confidence: 99%

Section: Ef070983 and Ef070982])mentioning

confidence: 99%

T cells expressing allograft inflammatory factor 1 display increased chemotaxis and induce a profibrotic phenotype in normal fibroblasts in vitro

Galdo¹,

Jiménez²

2007

Arthritis & Rheumatism

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“…The Millipore MultiScreen-MIC assay (Millipore Corporation, Billerica, MA) was performed as previously described (Ott et al, 2004). Briefly, serial dilutions of 150-l chemokines (R&D Systems, Inc.) were prepared in phenol red-free DMEM supplemented with 2 mM L-glutamine, 10 mM HEPES, and 1 mM sodium pyruvate (Mediatech) and added to the 96-well MultiScreen-MIC receiver plate.…”

Section: Methodsmentioning

confidence: 99%

“…Next we investigated the specificity of NBI-74330 for CXCR3 in chemotaxis experiments using H9 cells, which express the chemokine receptors CXCR4 and CCR7 in addition to CXCR3 (Ott et al, 2004) (Fig. 6).…”

Section: Characterization Of Binding Affinities For Nbi-74330mentioning

confidence: 99%

Pharmacological Characterization of CXC Chemokine Receptor 3 Ligands and a Small Molecule Antagonist

Heise¹,

Pahuja²,

Hudson³

et al. 2005

J Pharmacol Exp Ther

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The CXC chemokine receptor 3 (CXCR3) is predominantly expressed on T helper type 1 (Th1) cells that are involved in inflammatory diseases. The three CXCR3 ligands CXCL9, CXCL10, and CXCL11 are produced at sites of inflammation and elicit migration of pathological Th1 cells. Here, we are the first to characterize the pharmacological potencies and specificity of a CXCR3 antagonist, N-1R- [3-(4-ethoxy-phenyl of 7 to 18 nM). NBI-74330 was selective for CXCR3 because it showed no significant inhibition of chemotactic responses to other chemokines and did not inhibit radioligand binding to a panel of nonchemokine G-protein coupled receptors. There was a striking difference in potencies among the three CXCR3 ligands, with CXCL11 Ͼ Ͼ CXCL10 Ͼ CXCL9. A comparison of the rank order of K i values with the rank order of monocyte production levels of these three ligands revealed a precise inverse correlation, suggesting that the weaker receptor affinities of CXCL9 and CXCL10 were physiologically compensated for by an elevated expression, perhaps to maintain effectiveness of each ligand under physiological conditions.

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“…The chemokine is placed in the lower chamber. The amount of cell migration can be determined by using automated fluorescence plate readers [126], microscopic quantification, or flow cytometric analysis [127], depending on initial labelling of cells. In developing such assays, it is worth noting that cell lines of leukocyte origin in general demonstrate a much higher ability to show directed chemotaxis in the presence of a chemokine than do cell lines derived from epithelial cells or solid cancers.…”

Section: The Discovery Of Ckr Antagonists By High Throughput Screeningmentioning

confidence: 99%

Chemokine Receptors as Targets for Cancer Therapy

Lee

Chevalier

et al. 2009

CPD

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Chemokines and their receptors play critical roles in leukocyte trafficking during inflammatory processes. Although the role of chemokine receptors (CKRs) in cancer biology is a relatively new field of study, a growing body of data suggest that a number of CKRs, including CXCR4, CCR4, CCR7, and CCR10, may play diverse of roles in cancer growth, cancer metastasis, cancer angiogenesis, or the composition of the cancer microenvironment. Preclinical models of cancer indicate that cancer antagonists, most notably those for CXCR4, can block cancer growth either directly or by altering the cancer stroma. Highthroughput screening methods to identify effective CKR antagonists have been developed, but specificity, potency, and drug-delivery of validated candidate compounds remain issues that result in the clinical failure of many initially promising candidates. The recent approval of a CCR5 receptor antagonist in HIV suggests that safe, effective small molecular antagonists for other CKRs may not be far away. There is still a clear need to extend our understanding of the signalling pathways by which CKRs facilitate cancer processes. Because of the role of CKRs in cancer cell survival, the combination of CKR antagonists with traditional chemotoxic agents or with immunotherapy is an alluring strategy since this increases the specificity of treatment to the cancer and potentially limits additional systemic side effects.

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A high-throughput chemotaxis assay for pharmacological characterization of chemokine receptors: Utilization of U937 monocytic cells

Cited by 14 publications

References 22 publications

T cells expressing allograft inflammatory factor 1 display increased chemotaxis and induce a profibrotic phenotype in normal fibroblasts in vitro

T cells expressing allograft inflammatory factor 1 display increased chemotaxis and induce a profibrotic phenotype in normal fibroblasts in vitro

Pharmacological Characterization of CXC Chemokine Receptor 3 Ligands and a Small Molecule Antagonist

Chemokine Receptors as Targets for Cancer Therapy

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