RESUMEN
INTRODUCTION: Higher-throughput chemotaxis assays have had limited use in chemokine receptor pharmacology studies mainly because of the unavailability of optimal assay formats in addition to an incompatibility of chemotactic cell backgrounds with other pharmacological assays. Here, we developed a high-throughput 96-well chemotaxis assay for leukocytic cell lines and identified the human U937 monocytic line as an excellent cell background for both chemotaxis and the high-throughput calcium mobilization Fluorescent Imaging Plate Reader (FLIPR) assay. METHODS: Optimal chemotactic conditions were developed using the Neuroprobe MBA96 nondisposable and the Millipore MultiScreen-MIC disposable apparatuses with responses to CXC chemokine receptor (CXCR)-4 endogenously expressed on the human H9 T lymphoma line, and confirmed with Jurkat T cell and U937 monocytic cell lines. RESULTS: The U937 cell line was chosen for site-directed mutagenesis studies with CC chemokine receptor (CCR)-7 because this cell line did not endogenously express this receptor, it demonstrated a good chemotaxis index, and it showed an exceptional ability to mobilize calcium measured via FLIPR. Using the Millipore MultiScreen-MIC and FLIPR assays, alanine substitutions at K130 and Q227 caused threefold shifts in potency for the CCR7 ligand, CCL19, whereas that at K137 had no effect. DISCUSSION: Because these CCR7 mutations have previously been shown not to affect ligand binding, our results here show that these residues are specifically involved in receptor activation signals critical to chemotaxis and underscore the importance of using the U937 cell background to confirm results of chemotaxis with those of the FLIPR assay.
Asunto(s)
Quimiocinas CXC/farmacología , Quimiotaxis de Leucocito/efectos de los fármacos , Monocitos/efectos de los fármacos , Receptores de Quimiocina/fisiología , Linfocitos T/metabolismo , Secuencia de Aminoácidos , Calcio/metabolismo , Línea Celular Tumoral , Quimiocinas CXC/metabolismo , Relación Dosis-Respuesta a Droga , Citometría de Flujo , Fluorometría , Humanos , Procesamiento de Imagen Asistido por Computador , Células Jurkat , Cinética , Ligandos , Monocitos/citología , Mutación Puntual , Receptores de Quimiocina/química , Receptores de Quimiocina/genética , Reproducibilidad de los Resultados , Células U937RESUMEN
APCs of the nonobese diabetic (NOD) mouse have a genetically programmed capacity to overexpress IL-12p40, a cytokine critical for development of pathogenic autoreactive Th1 cells. To determine whether a diabetes-associated NOD chromosomal locus (i.e., Idd) was responsible for this defect, LPS-stimulated macrophages from several recombinant congenic inbred mice with Idd loci on a C57BL/6 background or with different combinations of NOD and CBA genomic segments were screened for IL-12p40 production. Only macrophages from the congenic strains containing the Idd4 locus showed IL-12p40 overproduction/expression. Moreover, analysis of IL-12p40 sequence polymorphisms demonstrated that the Idd4 intervals in these strains contained the IL-12p40 allele of the NOD, although further analysis is required to determine whether the IL-12p40 allele itself is responsible for its overexpression. Thus, the non-MHC-associated Idd4 locus appears responsible for IL-12p40 overexpression, which may be a predisposing factor for type 1 diabetes in NOD mice.