To test this hypothesis, we measured by circulation cytometry the migration of main human T cells towards CCR5-binding chemokine CCL5 gene, and observed that this CCR5 over-expression induced an over-migration towards CCL5

To test this hypothesis, we measured by circulation cytometry the migration of main human T cells towards CCR5-binding chemokine CCL5 gene, and observed that this CCR5 over-expression induced an over-migration towards CCL5. chemotactic response of peripheral blood CD8+ T cell to CCL5 and their level of surface CCR5 expression. T-cell surface CCR5 density, which is constant Rabbit Polyclonal to TIGD3 over time for a given individual, but varies drastically among individuals, might therefore be an important personal determinant of T-cell migration in many biological situations where CCR5-binding chemokines play a role, such as graft rejection, T helper 1-mediated auto-immune diseases, and infectious diseases involving CCR5. Moreover, our data spotlight the therapeutic potential of CCR5 antagonists in these situations. gene resulting in the production of a truncated CCR5 molecule that is not expressed at the cell surface.15,16 CCR5-32 homozygotes do not express the receptor at the cell surface, while heterozygous individuals for CCR5-32 express intermediate levels of CCR5 on their T-cell surface.15,16 In RA patients, the frequency of the CCR5-32 allele is reduced.17C19 Moreover, heterozygous individuals develop less aggressive disease than homozygous wild type gene carriers.19C21 In patients with Sj?gren’s syndrome, the frequency of 32/CCR5 genotype is significantly decreased and suggests that carrier status for the CCR5-32 allele may contribute to protection from the development of this disease.22 We hypothesized that, if the level of expression of the CCR5 receptor at the surface of T cells determines their migratory capacity in response to CCR5 ligands, there might be a polymorphism in the capacity of individuals to respond to C-C chemokines, and thereby in the occurrence and/or the course of the diseases where these chemokines are involved. Therefore, we wondered whether the functional efficiency of CCR5 function as a chemokine receptor might be influenced by its cell surface density, and tested this hypothesis on human peripheral blood T cells. Materials and methods Pyrogallol Cell culture and Pyrogallol reagentsPeripheral blood mononuclear cells (PBMC) from healthy donors were isolated by Ficoll-Paque density centrifugation and cultured at 2 106 cells/ml in RPMI-1640 medium supplemented with 2 mm glutamax-1, 10% fetal calf serum (FCS), 100 U/ml penicillin and 100 g/ml streptomycin (Gibco, Grand Island, NY). Recombinant human CCL5/RANTES was purchased from R & D Systems (Minneapolis, MN). Purified anti-human CCR5 antibody, clone 2D7, was purchased from PharMingen (Becton Dickinson, San Jose, CA). Anti-CCR5 monoclonal antibody (mAb) PA12 was purchased from Progenics Pharmaceuticals, Inc (Tarrytown, NY, USA). Fluorescein isothiocyanate (FITC)-conjugated anti-CD4 mAb and anti HLA-DR (anti-CD4 FITC and anti-HLA-DR FITC), phycoerythrin (PE)-conjugated anti-CD8 and anti-CD3 mAb (anti-CD8 PE and anti-CD3 PE), phycoerythrin cyanin 5-conjugated anti-CD69 (anti-CD69 PC5), and their respective isotype controls were purchased from Beckman-Coulter (Roissy, France). FITC-conjugated goat anti-mouse (GAM) immunoglobulin probe (H + l) (GAM FITC) was purchased from Jackson (West Grove, PA). Fluorescent beads for the cytometry quantification were purchased from Beckman-Coulter. Chemotaxis assayThe chemotaxis of peripheral blood lymphocytes (PBL) in response to recombinant human CCL5 was measured across 3 m pore-size cell culture inserts incorporating polyethylene terephthalate membranes (Falcon, Becton Dickinson) in 24-well companion plates (Falcon cell culture place system). Five hundred l of culture medium (RPMI-1640 as explained above), supplemented or not with recombinant human CCL5 were placed in the lower chamber of the plate. Peripheral blood from healthy volunteers was collected into an ethylenediamine tetra-acetic acid (EDTA) tube, diluted 1 : 5 in culture medium and placed into culture inserts (total volume of 100 l per place). The plates were then incubated for 4 hr at 37 (5% CO2). Then, Pyrogallol the cells that experienced crossed the membrane were collected from the lower chamber, pelleted down, resuspended in 150 l of culture medium, and incubated for 20 min at 20 with 10 l of anti-CD4 FITC mAb and 10 l of anti-CD8 PE mAb. After washing in phosphate-buffered saline (PBS), reddish blood cells were lysed, and the remaining cells were fixed (fluorescence-activated cell sorting (FACS) lysing answer, Becton Dickinson). After an additional washing, cells were resuspended in 250 l of PBS and 30 l of fluorescent beads were added. Circulation cytometric analysis was performed on a FACScalibur circulation cytometer (Becton Dickinson). Lymphocytes were gated on the basis of forward- and side-scatter, and the percentage of CD4+ and CD8+ T cells was decided according to thresholds for positive cell surface staining set at 1% using unfavorable isotype-matched controls. Numeration of the absolute quantity of T cells was normalized after the quantity of fluorescent beads counted by the cytometer. To quantify the specific migration of T cells towards CCL5, the number of T cells that migrated in absence of the chemokine was subtracted from the number of T cells that migrated in presence of the chemokine. Inhibition of migration with the anti-CCR5 2D7Cells were preincubated at room temperature with the anti-CCR5 mAb 2D7 at indicated concentrations or with an immunoglobulin G2a (IgG2a) isotype control antibody (Beckman-Coulter) for 1 hr in RPMI-1640 supplemented with 10% FCS and were then subjected to a chemotaxis assay as explained above..