Chemokine Receptor CCR3 Function Is Highly Dependent on Local pH and Ionic Strength

Abstract

The CC chemokine receptor 3 (CCR3) plays an important role in the regulation of the migration of eosinophils, a leukocyte population involved in many inflammatory pathologies including asthma. CCR3 binds to the CC chemokine eotaxin, a promigratory cytokine originally isolated as the key component in a model of eosinophil-induced airway inflammation. We show here that eotaxin/CCR3 binding interactions exhibit a marked sensitivity to relatively small changes in the extracellular environment. In particular, modest variations in the pH and the level of sodium chloride over a range of physiologic and near physiologic conditions had dramatic effects on eotaxin binding and CCR3-mediated cytoplasmic Ca 2؉ mobilization. These biochemical indices were reflected at the functional level as well; small changes in pH and salt also resulted in striking changes in the migration of primary human eosinophils in vitro. These results reveal that relatively small perturbations in extracellular buffer conditions can yield widely disparate interpretations of CCR3 ligand binding and affinities and suggest that modulation of the tissue microenvironment might be utilized to control the affinity and efficacy of chemokine-mediated cell migration.Eosinophils are involved in many inflammatory pathologies including asthma, urticaria, and hypereosinophilic syndrome (1-3). The CC chemokine receptor 3 (CCR3) 1 and its ligands, most notably eotaxin, have been shown to play a central role in controlling eosinophil migration (4 -12). Eotaxin was originally isolated as a protein responsible for eosinophil chemoattraction in the bronchoalveolar fluid of allergen-sensitized guinea pigs (9). Subsequently, the human and mouse homologs were identified and also shown to be chemoattractants for eosinophils (10,12). Eotaxin has been shown to be a primary ligand for the seven-transmembrane G protein-coupled receptor CCR3. The genes for CCR3 and the related receptors CCR1-CCR5 are encoded within a 130-kilobase region of human chromosome 3 (13) indicating recent gene duplication and divergence and perhaps explaining the partial overlap of chemokine ligand repertoires. CCR3 is highly expressed on primary human eosinophils (50,000 -400,000 sites/cell) (5-7), consistent with the potent ability of eotaxin to selectively influence the migration of these cells.The mechanisms by which specific cells are attracted by specific chemokines have been a topic of intense interest and clinical relevance. The question is complicated by the fact that a number of closely related chemokine receptors with overlapping specificities are expressed on many different classes of leukocytes. In addition, the affinity of a specific receptor/ligand interaction can vary depending upon the specific cell type expressing the chemokine receptor, perhaps due to posttranslational modification events or G protein coupling. Thus it appears that cells can modulate their responses to chemokines, but how this is done is not yet clear. While most investigations have focused on factors intrinsic to the...

Receptor reserve analysis of the human CCR3 receptor in eosinophils and CCR3-transfected cells

“…The reason for this is unknown but could relate to differences in buffer conditions used in the latter study, which are known to have an effect on these parameters [26].…”

“…Important to this analysis is that all measurements be done under similar conditions. This applies to CCR3, where small changes in salt and pH alter eotaxin affinity and functional responses in CREM3 cells [26]. In these studies, chemotaxis and calcium assays were conducted in the same buffer (HBSS/0.5% BSA), which is very similar but not identical to that used in the competition binding assays.…”

“…To examine CCR3 agonists for differences in potency and intrinsic efficacy, a high-capacity intracellular calcium assay in a 96-well format was developed using a FLIPR and the human CCR3-transfected, adherent CREM3 cell line [26]. All known CCR3 agonists of human origin were simultaneously tested, as were mouse and guinea pig eotaxin.…”

“…CREM3 cells [26] or human eosinophils were added to 96-well plates (200,000-300,000 in 140 µL/well). 125 I-Eotaxin (40 µL/well) was added at a final concentration of 0.1 nM.…”

“…CREM3 cells, in which the human CCR3 receptor is stably expressed in the rat Y3 cell line [26], were cultured in Dulbecco's modified Eagle's medium (DMEM) high glucose containing 10% FBS, 50 U/mL penicillin, 50 µg/mL streptomycin, 2 mM L-glutamine, 1 mg/mL G418 (Gemini Bioproducts). Cells were cultured in fresh medium every 2 days.…”