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Selective loss of chemokine receptor expression on leukocytes after cell isolation.

Nieto JC, Cantó E, Zamora C, Ortiz MA, Juárez C, Vidal S - PLoS ONE (2012)

Bottom Line: 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 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Institut Recerca Hospital de Sant Pau, Barcelona, Spain.

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.

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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.
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pone-0031297-g005: 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.

Mentions: To directly test the influence of the isolated method on the chemokine receptor function, we compared the chemotactic responses of monocytes from WB or isolated by Ficoll to CCL2. For this purpose, we measured the chemotaxis capacity of monocytes from WB or isolated by Ficoll from five healthy donors (n = 5) in presence of CCL2 at 2 ng/ml We observed that the number of monocytes isolated by density gradient Ficoll (1701±581) exhibited a significantly reduced chemotaxis toward CCL2 (p<0.05) compared to monocytes from WB (3082±806) (Fig. 5). To further analyze the extend of the migratory defect of Ficoll-isolated monocytes regardeless of different interfering factors in whole blood and MNC culture, we compared the migration to fMLP (a standard chemoattractant) [18] and CCL2 . The chemotactic response of WB monocytes to CCL2 was significantly higher than to 10−6 M fMLP (2.08 fold, p<0.01). However, the chemotactic response of Ficoll-isolated monocytes to CCL2 was comparable to fMLP (1.05 fold). Regardless of different soluble factors or cell interactions that could take place in WB and MNC 4 h culture, the down-regulation of monocyte CCR2 expression by Ficoll may be associated with a decreased ability of monocytes to migrate towards CCL2.


Selective loss of chemokine receptor expression on leukocytes after cell isolation.

Nieto JC, Cantó E, Zamora C, Ortiz MA, Juárez C, Vidal S - PLoS ONE (2012)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3293867&req=5

pone-0031297-g005: 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.
Mentions: To directly test the influence of the isolated method on the chemokine receptor function, we compared the chemotactic responses of monocytes from WB or isolated by Ficoll to CCL2. For this purpose, we measured the chemotaxis capacity of monocytes from WB or isolated by Ficoll from five healthy donors (n = 5) in presence of CCL2 at 2 ng/ml We observed that the number of monocytes isolated by density gradient Ficoll (1701±581) exhibited a significantly reduced chemotaxis toward CCL2 (p<0.05) compared to monocytes from WB (3082±806) (Fig. 5). To further analyze the extend of the migratory defect of Ficoll-isolated monocytes regardeless of different interfering factors in whole blood and MNC culture, we compared the migration to fMLP (a standard chemoattractant) [18] and CCL2 . The chemotactic response of WB monocytes to CCL2 was significantly higher than to 10−6 M fMLP (2.08 fold, p<0.01). However, the chemotactic response of Ficoll-isolated monocytes to CCL2 was comparable to fMLP (1.05 fold). Regardless of different soluble factors or cell interactions that could take place in WB and MNC 4 h culture, the down-regulation of monocyte CCR2 expression by Ficoll may be associated with a decreased ability of monocytes to migrate towards CCL2.

Bottom Line: 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 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Institut Recerca Hospital de Sant Pau, Barcelona, Spain.

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.

Show MeSH
Related in: MedlinePlus