Selective loss of chemokine receptor expression on leukocytes after cell isolation

Abstract

Chemokine receptors are distinctively exposed on cells to characterize their migration pattern. However, little is known about factors that may regulate their expression. To determine the optimal conditions for an accurate analysis of chemokine receptors, we compared the expression of CCR2, CCR4, CCR5, CCR6, CXCR3 and CXCR4 on different leukocyte subsets using whole blood (WB) plus erythrocyte lysis and density gradient isolation (Ficoll). Most WB monocytes were CCR2+ (93.5 ± 2.9%) whereas 32.8 ± 6.0% of monocytes from Ficoll-PBMC expressed CCR2 (p<0.001). Significant reductions of CCR6 and CXCR3 on monocytes were also observed after Ficoll isolation (WB: 46.4 ± 7.5% and 57.1 ± 5.5%; Ficoll: 29.5 ± 2.2% and 5.4 ± 4.3% respectively) (p<0.01). Although comparable percentages of WB and Ficoll-PBMC monocytes expressed CCR4, CCR5 and CXCR4, Ficoll isolation significantly reduced the levels of CXCR4 (WB: MFI 5 ± 0.4 and Ficoll: MFI 3.3 ± 0.1) (p<0.05). Similarly to monocytes, CCR2, CXCR3 and CXCR4 were also reduced on lymphocytes. In addition, Ficoll isolation significantly reduced the percentage of CCR4 positive lymphocytes (WB: 90.2 ± 4.5% and Ficoll: 55 ± 4.1%) (p<0.01). The loss of expression of chemokine receptors after isolation of monocytes was not dependent on either the anticoagulant or the density gradient method. It was irreversible and could not be restored by LPS activation or in vitro macrophage differentiation. Experiments tagged with anti-CCR2 antibodies prior to density gradient isolation demonstrated that Ficoll internalized chemokine receptors. The method for cell isolation may alter not only the expression of certain chemokine receptors but also the respective functional migration assay. The final choice to analyze their expression should therefore depend on the receptor to be measured.

Figure 1. Levels of chemokine receptor expression on leukocytes from WB and Ficoll isolated PBMC.

Representative experiment with leukocytes from healthy donors (n = 12). Monocytes were stained with anti-CD14 mAbs and lymphocytes and neutrophils were selected by FS and SS parameters. Leukocytes were also stained with monoclonal antibodies for chemokine receptor CCR2, CCR4, CCR5 CCR6, CXCR3 and CXCR4. Profile of expression was assesed by flow cytometry (isotype control □ , chemokine receptor ▪).

Figure 2. Effect of anticoagulant and the cell separation method on monocyte chemokine receptors expression.

WB, Ficoll and Percoll isolated PBMC from healthy donors (n = 3) were collected in Vacutainer tubs containing sodium heparine or EDTA. Monocytes were CD14+ gated and CCR2 (clone 48607), CXCR3 and CXCR4 expression were determined. The results of a representative cytometric analysis were shown (n = 5) (isotype control □ , chemokine receptor ▪).

Figure 3. Chemokine receptor expression on LPS and seven-day cultured monocytes.

The results of a representative experiment are shown. a) Monocytes from WB or monocytes from Ficoll were culture 20 h in medium or LPS (0.01 µg/ml) and stained with anti-CCR2-PE or anti-CXCR3-PE. b) Monocytes from Ficoll were cultured in medium and seven days later, cells were harvested and stained with anti-CCR2-PE, anti-CXCR3-PE and anti-CCR5-PE (isotype control □ , chemokine receptor ▪).

Figure 4. Determination of fate of CCR2 downregulation on monocytes from Ficoll.

CCR2 expression was assessed after Ficoll isolation of untagged and tagged-CCR2 monocytes. Internalization of the complex was examined from one single layer of Z stacks by confocal microscopy. The results of a representative experiment are shown: a) CCR2 expression on monocytes from WB tagged with CCR2 prior to gradient separation; b) CCR2 expression on untagged monocytes after Ficoll isolation and the subsequent staining of CCR2; c) CCR2 expression in CCR2 tagged monocytes after Ficoll isolation, red signal within monocytes represents the CCR2-complex internalization (no aditional anti CCR2-PE was added to the cells after isolation with Ficoll); (isotype control □ , chemokine receptor ▪).

Figure 5. Migration capacity of monocytes from WB and Ficoll PBMC towards CCL2.

WB diluited 1∶5 in medium (100 µl per well) or Ficoll PBMC (2×10⁶ cells/ml per well) were subjected to a 4 h-chemotaxis assay towards CCL2 or fMLP (n = 5). The cells that had migrated into the lower chamber were collected and stained with anti-CD14-PEDy647 and analyzed by flow cytometry. Results are expressed as the mean of the number of migrated cells ± SEM.