Dataset Information

Pharmacological characterization of the chemokine receptor, hCCR1 in a stable transfectant and differentiated HL-60 cells: antagonism of hCCR1 activation by MIP-1beta.

ABSTRACT: C-C chemokine receptor-1 (CCR1) has been implicated in mediating a variety of inflammatory conditions including multiple sclerosis and organ rejection. Although originally referred to as the MIP-1alpha/RANTES receptor, CCR1 is quite promiscuous and can be activated by numerous chemokines. We used radioligand binding and [35S]-GTPgammaS exchange assays in membranes from a cell line transfected to express CCR1 (Ba/F3-hCCR1) to characterize a panel of chemokines (HCC-1, MIP-1alpha, MIP-1beta, MIP-1delta, MPIF-1, MCP-2, MCP-3, and RANTES) as CCR1 ligands. In this recombinant model, these chemokines displaced 125I-MIP-1alpha with a wide range of potencies and, with the exception of MCP-2, acted as full agonists in stimulating [35S]-GTPgammaS exchange. We then assessed the utility of HL-60 cells cultured with known differentiating agents (PMA, DMSO, dibutyryl-cAMP or retinoic acid) for investigating CCR1 pharmacology. In [35S]-GTPgammaS exchange assays, membranes from cells cultured with retinoic acid (4-6 days) were the most responsive to activation by MIP-1alpha and MPIF-1. FACS analysis and comparative pharmacology confirmed that these activities were mediated by CCR1. Using [35S]-GTPgammaS exchange assays, intracellular calcium flux and/or whole cell chemotaxis assays in HL-60(Rx) cells, we validated that MIP-1alpha was the most potent CCR1 ligand (MIP-1alpha>MPIF-1>RANTES>or=MIP-1beta) although the ligands differed in their efficacy as agonists. MPIF-1 was the more efficacious (MPIF-1>RANTES=MIP-1alpha>>MIP-1beta). 125I-MIP-1beta binding in Ba/F3-hCCR1 and HL-60(Rx) membranes was competitively displaced by MIP-1alpha, MPIF-1 and MIP-1beta. The binding K(i) for these chemokines with 125I-MIP-1beta were essentially identical in the two membrane systems. Lastly, MIP-1beta antagonized [35S]-GTPgammaS exchange, Ca2+ flux and chemotaxis in HL-60(Rx) cells in response to robust agonists such as MIP-1alpha, RANTES and MPIF-1. Based on our results, we propose that MIP-1beta could function as an endogenous inhibitor of CCR1 function.

SUBMITTER: Chou CC

PROVIDER: S-EPMC1573530 | biostudies-literature | 2002 Nov

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Pharmacological characterization of the chemokine receptor, hCCR1 in a stable transfectant and differentiated HL-60 cells: antagonism of hCCR1 activation by MIP-1beta.

Chou Chuan-Chu CC Fine Jay S JS Pugliese-Sivo Catherine C Gonsiorek Waldemar W Davies Liza L Deno Gregory G Petro Mary M Schwarz Martin M Zavodny Paul J PJ Hipkin R William RW

British journal of pharmacology 20021101 5

C-C chemokine receptor-1 (CCR1) has been implicated in mediating a variety of inflammatory conditions including multiple sclerosis and organ rejection. Although originally referred to as the MIP-1alpha/RANTES receptor, CCR1 is quite promiscuous and can be activated by numerous chemokines. We used radioligand binding and [35S]-GTPgammaS exchange assays in membranes from a cell line transfected to express CCR1 (Ba/F3-hCCR1) to characterize a panel of chemokines (HCC-1, MIP-1alpha, MIP-1beta, MIP-1 ...[more]

PMID: 12381680

Similar Datasets

Project description:Toll-like receptor 2 (TLR2) is an important sensor for innate immune cells, including neutrophils, for the recognition of pathogen infection. Lipoteichoic acid (LTA), a cell wall component of gram-positive bacteria, is a TLR2 ligand. LTA-induced TLR2 signaling pathways are well established in neutrophils. However, experimental studies regarding transcriptional regulation and the molecular mechanisms in primary human neutrophils are limited due to their short lifespan. The promyelocytic leukemia cell line, HL-60, can differentiate into a neutrophil-like phenotype following treatment with dimethyl sulfoxide. The aim of the present study was to investigate whether differentiated HL-60 (dHL-60) cells induced a similar gene expression profile upon LTA treatment as that previously determined for primary human neutrophils. After 4 or 24 h of Staphylococcus aureus LTA treatment, undifferentiated HL-60 (uHL-60) and dHL-60 cells were collected for RNA sequencing. The results demonstrated that hundreds of identical differentially expressed genes (DEGs) were observed in 1 and 10 µg/ml LTA-treated dHL-60 cells following 4 and 24 h of incubation, while almost no DEGs between LTA-treated HL-60 and dHL-60 cells were observed. Using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes analyses (KEGG), it was noted that the pathways of shared DEGs between the 1 and 10 µg/ml LTA-treated dHL-60 cells at both time points were significantly enriched in immune and inflammatory response-related pathways, such as cellular response to tumor necrosis factor, interleukin-1, interferon γ, neutrophil chemotaxis, the NF-κB signaling pathway and the Toll-like receptor signaling pathway. In addition, when comparing the effect of 1 and 10 µg/ml LTA treatment on dHL60 cells, it was found that all enriched GO and KEGG pathways were associated with the TLR signaling pathways of neutrophils. The results of the present study provided important information for the implementation of mRNA profiling in LTA-treated dHL-60 cells and may indicate the feasibility of using dHL-60 cells as a research model for TLR2 signaling in human neutrophils.

Project description:Synthetic lipopeptides activate superoxide-anion (O2-) formation in human neutrophils in a pertussis-toxin (PTX)-sensitive manner, suggesting the involvement of G-proteins of the Gi family in the signal-transduction pathway. We compared G-protein activation by lipopeptides and the chemotactic peptide N-formylmethionyl-leucyl-phenylalanine (fMLP) in dibutyryl-cyclic-AMP-differentiated HL-60 cells. The lipopeptide (2S)-2-palmitoylamino-6-palmitoyloxymethyl-7-palmitoyloxy heptanoyl-SK4 (Pam3AhhSK4) and fMLP activated high-affinity GTPase, i.e. the enzymic activity of G-protein alpha-subunits, in HL-60 membranes in a time- and protein-dependent manner, but they had no effect on Mg(2+)-ATPase and Na+/K(+)-ATPase. Pam3AhhSK4 and fMLP increased Vmax. of GTP hydrolysis. Pam3AhhSK4 activated GTP hydrolysis with half-maximal and maximal effects at about 2 microM and 10 microM respectively. Other lipopeptides activated GTP hydrolysis as well. Lipopeptides were less effective than fMLP to activate GTPase. In membranes from PTX-treated cells, the stimulatory effects of lipopeptides and fMLP on GTPase were abolished. In N-ethylmaleimide-treated membranes, the relative stimulatory effect of Pam3AhhSK4 on GTP hydrolysis was enhanced, whereas that of fMLP was diminished. fMLP and Pam3AhhSK4 activated GTPase in an over-additive manner in N-ethylmaleimide-treated membranes. Unlike fMLP, Pam3AhhSK4 did not enhance incorporation of GTP azidoanilide into, and cholera-toxin-catalysed ADP-ribosylation of Gi-protein alpha-subunits in, HL-60 membranes and did not induce rises in cytosolic Ca2+ concentration. Pam3AhhSK4 and fMLP stimulated phosphatidic acid formation in a PTX-sensitive manner. Pam3AhhSK4 itself did not activate O2- formation, but potentiated the stimulatory effects of fMLP. Our data suggest that (i) lipopeptides activate the GTPase of Gi-proteins, (ii) lipopeptides and fMLP activate Gi-proteins differently, (iii) lipopeptides stimulate phospholipase D via Gi-proteins, and (iv) phosphatidic acid formation is not sufficient for activation of O2- formation.

Project description:The production of chemotactic cytokines (chemokines) and other cytokines by macrophages in response to fungal infection is thought to be critical during the course of candidiasis. However, the mechanism of cytokine synthesis by macrophages in response to fungi is not well understood. Therefore, the response of macrophages to Candida albicans was examined in terms of receptor-mediated chemokine and other cytokine mRNA induction. Attachment of C. albicans to murine thioglycollate-elicited peritoneal macrophages induced increased mRNA levels of the cytokines interleukin-1beta (IL-1beta), IL-6, and granulocyte-macrophage colony-stimulating factor (GM-CSF) and the chemokines macrophage inflammatory protein 1beta (MIP-1beta), MIP-2, and KC (a member of the platelet factor 4 neutrophil chemoattractant family), as determined by quantitative reverse transcription-PCR. However, treatment of macrophages with alpha-methyl-D-mannoside significantly reduced the cytokine GM-CSF response to C. albicans but did not affect the chemokine MIP-2 response. Antisense oligodeoxynucleotide (ODN) to mannose receptor (MR) mRNA inhibited the expression and function of MR in macrophages as determined by Western blot analysis and 125I-labeled mannose-bovine serum albumin (BSA) binding, and also inhibited the elevation of cytokine IL-1beta, IL-6, and GM-CSF mRNA levels induced by C. albicans attachment. Elevation of chemokine MIP-1beta, MIP-2, and KC mRNA levels induced by C. albicans was not affected in macrophages whose MR expression was suppressed by antisense ODN treatment. Furthermore, IL-4 treatment of macrophages, which up-regulated MR expression as determined by Western blot analysis and fluorescein isothiocyanate-labeled mannose-BSA uptake, enhanced the level of cytokine GM-CSF mRNA induced by C. albicans but not the level of the chemokine MIP-2 mRNA. These results indicate that selected cytokine responses of macrophages to C. albicans are mediated by MR, while some chemokine responses may be mediated by other receptors.

Project description:HL-60 cells differentiated with DMSO increased their rates of uptake of ascorbate when they were activated with PMA. The rates observed after this activation were essentially the same as those with dehydroascorbic acid as the original transport substrate. The effect of activation was sensitive to the antioxidant enzymes superoxide dismutase and catalase. When ascorbate was oxidized in situ by chemical or enzymic oxidation, the rates of uptake were similar to those after activation of the cells by phorbol ester; however, in the latter case the extracellular vitamin remained largely in the reduced form and there was very little loss by degradation, whereas after immediate oxidation no more reduced ascorbate could be found outside the cells after a few minutes and a significant part of the total vitamin was lost. The generation of superoxide by xanthine/xanthine oxidase stimulated the uptake of ascorbate much less than the activation by phorbol ester; H(2)O(2) was even less effective. Stimulation of the uptake by phorbol ester was also insensitive to GSH, in contrast with stimulation by the chemical oxidation of ascorbate. Stimulation of ascorbate uptake by phorbol ester was sensitive to the respiratory-burst inhibitor diphenyliodonium as well as the protein kinase C inhibitor staurosporine, indicating the respiratory burst as the cause of stimulation. Activation of the cells by the phorbol ester also stimulated the uptake of dehydroascorbate as the original substrate, in a manner insensitive to antioxidants or inhibitors of the respiratory burst. In all cases the intracellular vitamin was completely in the reduced form. Kinetic characterization by the calculation of maximal velocities and apparent K(m) values and assaying for the dependence of uptake rates on the ionic milieu and for inhibition by glucose analogues and inhibitors of glucose transport revealed that after treatment with phorbol ester the uptake of total vitamin C in differentiated HL-60 cells was largely due to the low-affinity high-capacity glucose transporter. In contrast, in non-stimulated cells reduced ascorbate was taken up by the Na(+)-dependent high-affinity low-capacity ascorbate transporter. This change was probably due to the oxidation of ascorbate and, simultaneously, the recruitment of additional transporter molecules to the cell surface.

Dataset Information

Pharmacological characterization of the chemokine receptor, hCCR1 in a stable transfectant and differentiated HL-60 cells: antagonism of hCCR1 activation by MIP-1beta.

Publications

Pharmacological characterization of the chemokine receptor, hCCR1 in a stable transfectant and differentiated HL-60 cells: antagonism of hCCR1 activation by MIP-1beta.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets