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1.
Biochemistry ; 57(5): 852-860, 2018 02 06.
Article in English | MEDLINE | ID: mdl-28994588

ABSTRACT

The relationship between the oligomeric status and functions of chemokine receptor CCR3 is still controversial. We use total internal reflection fluorescence microscopy at the single-molecule level to visualize the oligomeric status of CCR3 and its regulation of the membrane of stably transfected T-REx-293 cells. We find that the population of the dimers and oligomers of CCR3 can be modulated by the binding of ligands. Natural agonists can induce an increase in the level of dimers and oligomers at high concentrations, whereas antagonists do not have a significant influence on the oligomeric status. Moreover, monomeric CCR3 exhibits a stronger chemotactic response in the migration assay of stably transfected CCR3 cells. Together, these data support the notion that CCR3 exists as a mixture of monomers and dimers under nearly physiological conditions and the monomeric CCR3 receptor is the minimal functional unit in cellular signaling transduction. To the best of our knowledge, these results constitute the first report of the oligomeric status of CCR3 and its regulation.


Subject(s)
Microscopy, Fluorescence/methods , Receptors, CCR3/metabolism , Single Molecule Imaging/methods , Cell Line , Cell Membrane/metabolism , Chemotaxis , Dose-Response Relationship, Drug , Humans , Ligands , Photobleaching , Protein Multimerization , Receptors, CCR3/agonists , Receptors, CCR3/antagonists & inhibitors , Recombinant Proteins/metabolism , Signal Transduction
2.
BMC Immunol ; 18(1): 54, 2017 12 28.
Article in English | MEDLINE | ID: mdl-29281969

ABSTRACT

BACKGROUND: Chemokines and their cognate receptors play important role in the control of leukocyte chemotaxis, HIV entry and other inflammatory diseases. Developing an effcient method to investigate the functional expression of chemokines and its interactions with specific receptors will be helpful to asses the structural and functional characteristics as well as the design of new approach to therapeutic intervention. RESULTS: By making systematic optimization study of expression conditions, soluble and functional production of chemokine C-C motif ligand 8 (CCL8) in Escherichia coli (E. coli) has been achieved with approx. 1.5 mg protein/l culture. Quartz crystal microbalance (QCM) analysis exhibited that the purified CCL8 could bind with C-C chemokine receptor type 3 (CCR3) with dissociation equilibrium constant (K D) as 1.2 × 10-7 M in vitro. Obvious internalization of CCR3 in vivo could be detected in 1 h when exposed to 100 nM of CCL8. Compared with chemokine C-C motif ligand 11 (CCL11) and chemokine C-C motif ligand 24 (CCL24), a weaker chemotactic effect of CCR3 expressing cells was observed when induced by CCL8 with same concentration. CONCLUSION: This study delivers a simple and applicable way to produce functional chemokines in E. coli. The results clearly confirms that CCL8 can interact with chemokine receptor CCR3, therefore, it is promising area to develop drugs for the treatment of related diseases.


Subject(s)
Chemokine CCL8/metabolism , Escherichia coli/genetics , Receptors, CCR3/agonists , Chemokine CCL8/genetics , Chemotaxis , Datasets as Topic , Gene Expression , HEK293 Cells , Humans , Isopropyl Thiogalactoside , Ligands , Plasmids , Protein Binding , Recombinant Proteins/genetics
3.
J Cell Physiol ; 232(11): 2996-3005, 2017 Nov.
Article in English | MEDLINE | ID: mdl-28387445

ABSTRACT

The focus of this study was to determine which chemokine receptors are present on oral fibroblasts and whether these receptors influence proliferation, migration, and/or the release of wound healing mediators. This information may provide insight into the superior wound healing characteristics of the oral mucosa. The gingiva fibroblasts expressed 12 different chemokine receptors (CCR3, CCR4, CCR6, CCR9, CCR10, CXCR1, CXCR2, CXCR4, CXCR5, CXCR7, CX3CR1, and XCR1), as analyzed by flow cytometry. Fourteen corresponding chemokines (CCL5, CCL15, CCL20, CCL22, CCL25, CCL27, CCL28, CXCL1, CXCL8, CXCL11, CXCL12, CXCL13, CX3CL1, and XCL1) were used to study the activation of these receptors on gingiva fibroblasts. Twelve of these fourteen chemokines stimulated gingiva fibroblast migration (all except for CXCL8 and CXCL12). Five of the chemokines stimulated proliferation (CCL5/CCR3, CCL15/CCR3, CCL22/CCR4, CCL28/CCR3/CCR10, and XCL1/XCR1). Furthermore, CCL28/CCR3/CCR10 and CCL22/CCR4 stimulation increased IL-6 secretion and CCL28/CCR3/CCR10 together with CCL27/CCR10 upregulated HGF secretion. Moreover, TIMP-1 secretion was reduced by CCL15/CCR3. In conclusion, this in-vitro study identifies chemokine receptor-ligand pairs which may be used in future targeted wound healing strategies. In particular, we identified the chemokine receptors CCR3 and CCR4, and the mucosa specific chemokine CCL28, as having an predominant role in oral wound healing by increasing human gingiva fibroblast proliferation, migration, and the secretion of IL-6 and HGF and reducing the secretion of TIMP-1.


Subject(s)
Chemokines, CC/pharmacology , Fibroblasts/drug effects , Gingiva/drug effects , Receptors, CCR3/agonists , Receptors, CCR4/agonists , Wound Healing/drug effects , Cell Line, Transformed , Cell Movement/drug effects , Cell Proliferation/drug effects , Dose-Response Relationship, Drug , Fibroblasts/metabolism , Fibroblasts/pathology , Gingiva/metabolism , Gingiva/pathology , Hepatocyte Growth Factor/metabolism , Humans , Interleukin-6/metabolism , Ligands , Receptors, CCR3/metabolism , Receptors, CCR4/metabolism , Signal Transduction/drug effects , Tissue Inhibitor of Metalloproteinase-1/metabolism
4.
Clin Exp Rheumatol ; 28(4): 462-7, 2010.
Article in English | MEDLINE | ID: mdl-20659406

ABSTRACT

OBJECTIVES: To evaluate the expression of CCR3 receptors as well as CCR3 agonists, including eotaxin-2 and RANTES, among patients suffering from rheumatoid arthritis and healthy controls, as a possible pathogenetic mechanism in inflammatory joint disease. METHODS: Twenty-two patients and 13 healthy controls were recruited and clinically evaluated. CCR3 expression on CD4+ lymphocytes and mononuclear cells was evaluated by FACS analysis after staining with human CD4 APC (bioscience) and human CCR3 (CD193)PE. Levels of eotaxin-2 and RANTES were analysed by ELISA. RESULTS: A significant decrease was observed in the level of CD4+ cells expressing the CCR3 receptor in serum of RA patients (0.96+/-0.5) as compared with healthy controls (1.48+/-0.6) (p<0.05). A significant decrease in serum eotaxin-2 levels was evident among RA patients suffering from active disease, defined by a DAS-28 score above 5.5, compared with RA patients with lower activity scores (2.1+/-1.6 vs. 7.0+/-5.1; p=0.01). A significant decrease was evident in the number of CCR3 expressing Monocytes among RA patients treated with steroids and anti TNF-a medications as compared with RA patients not receiving such treatment. CONCLUSIONS: CCR3 is differentially expressed on inflammatory cells in RA, while eotaxin-2, a potent CCR3 agonist, is differentially expressed in active disease. Anti-inflammatory medications may down-regulate CCR3 expression in RA. The CCR3-CCR3 agonist pathway may thus have a pathogenic role in RA and may be a future target for novel treatment modalities.


Subject(s)
Arthritis, Rheumatoid/blood , CD4-Positive T-Lymphocytes/metabolism , Receptors, CCR3/blood , Adult , Aged , Arthritis, Rheumatoid/drug therapy , Arthritis, Rheumatoid/pathology , CD4-Positive T-Lymphocytes/pathology , Case-Control Studies , Chemokine CCL24/blood , Chemokine CCL5/blood , Female , Humans , Male , Middle Aged , Receptors, CCR3/agonists , Steroids/therapeutic use , Tumor Necrosis Factor-alpha/antagonists & inhibitors
5.
J Leukoc Biol ; 86(2): 327-36, 2009 Aug.
Article in English | MEDLINE | ID: mdl-19414538

ABSTRACT

Eosinophils express the chemoattractant receptors CCR3 and FPR. CCR3 binds several agonists such as eotaxin-1, -2, and -3 and RANTES, whereas the FPR binds the formylated tripeptide fMLP and a host of other ligands. The aim of this study was to investigate if there is interplay between these two receptors regarding the elicitation of migration and respiratory burst in human blood-derived eosinophils. Inhibition of the FPR with the antagonists CyH and boc-MLP abrogated the migration of eosinophils toward all of the CCR3 agonists. Similar results were seen when the FPR was desensitized with its cognate ligand, fMLP. In contrast, the respiratory burst triggered by eotaxin-1 was not inhibited by CyH. Thus, signals evoked via the FPR caused unidirectional down-regulation of CCR3-mediated chemotaxis but not respiratory burst in human eosinophils. The underlying mechanism was neither reduced ability of the CCR3 ligand eotaxin-1 to bind to CCR3 nor down-regulation of CCR3 from the cell surface. Finally, confocal microscopy and adFRET analysis ruled out homo- or heterodimer formation between FPR and/or CCR3 as an explanation for the reduction in chemotaxis via CCR3. Pharmacologic inhibition of signal transduction molecules showed that the release of free oxygen radicals in response to eotaxin-1 compared with fMLP is relatively more dependent on the p38 MAPK pathway.


Subject(s)
Eosinophils/immunology , Eosinophils/metabolism , Receptor Cross-Talk/immunology , Receptors, CCR3/metabolism , Receptors, Formyl Peptide/metabolism , Signal Transduction/immunology , Cell Movement/drug effects , Cell Movement/immunology , Chemokine CCL11/metabolism , Chemotaxis, Leukocyte/immunology , Cyclohexenes/pharmacology , Eosinophils/drug effects , Free Radicals/metabolism , Humans , Ligands , Microscopy, Confocal , Oligopeptides/pharmacology , Protein Binding/immunology , Receptor Cross-Talk/drug effects , Receptors, CCR3/agonists , Receptors, Formyl Peptide/agonists , Receptors, Formyl Peptide/antagonists & inhibitors , Respiratory Burst/drug effects , Respiratory Burst/immunology , Signal Transduction/drug effects , p38 Mitogen-Activated Protein Kinases/metabolism
6.
J Biomol Screen ; 13(1): 40-53, 2008 Jan.
Article in English | MEDLINE | ID: mdl-18227225

ABSTRACT

High-content screening, typically defined as automated fluorescence microscopy combined with image analysis, is now well established as a means to study test compound effects in cellular disease-modeling systems. In this work, the authors establish several high-content screening assays in the 384-well format to measure the activation of the CC-type chemokine receptors 2B and 3 (CCR2B, CCR3). As a cellular model system, the authors use Chinese hamster ovary cells, stably transfected with 1 of the respective receptors. They characterize receptor stimulation by human monocyte chemoattractant protein-1 for CCR2B and by human eotaxin-1 for CCR3: Receptor internalization and receptor-induced phosphorylation of ERK1/2 (pERK) were quantified using fluorescence imaging and image analysis. The 4 assay formats were robust, displayed little day-to-day variability, and delivered good Z' statistics for both CCRs. For each of the 2 receptors, the authors evaluated the potency of inhibitory compounds in the internalization format and the pERK assay and compared the results with those from other assays (ligand displacement binding, Ca(2+) mobilization, guanosine triphosphate exchange, chemotaxis). Both physiological agonists and test compounds differed significantly with respect to potencies and efficacies in the various profiling assays. The diverse assay formats delivered partially overlapping and partially complementary information, enabling the authors to reduce the probability of test compound-related technology artifacts and to specify the mode of action for individual test compounds. Transfer of the high-content screening format to a fully automated medium-throughput screening platform for CCR3 enabled the profiling of large compound numbers with respect to G protein signaling and possible tolerance-inducing liabilities.


Subject(s)
Drug Evaluation, Preclinical/methods , Receptors, Chemokine/drug effects , Animals , CHO Cells , Chemokine CCL11/pharmacology , Chemokine CCL2/pharmacology , Cricetinae , Cricetulus , Humans , In Vitro Techniques , MAP Kinase Signaling System/drug effects , Receptors, CCR2/drug effects , Receptors, CCR2/genetics , Receptors, CCR2/metabolism , Receptors, CCR3/agonists , Receptors, CCR3/drug effects , Receptors, CCR3/genetics , Receptors, CCR3/metabolism , Receptors, Chemokine/genetics , Receptors, Chemokine/metabolism , Recombinant Proteins/drug effects , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Transfection
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