The influence of Dextran on CXCR2 expression and chemotactic function of isolated human neutrophils

Susan Summerhill, Gary Salmon, Simon Dales. Pfizer Global R&D, Sandwich, Kent, United Kingdom.

Dextran-induced red blood cell (RBC) agglutination is commonly used for removal of RBCs during neutrophil isolation. Here we report the surprising observation that different batches of Dextran influence CXCR2 expression level and associated chemotactic function of isolated human neutrophils.

Human whole blood was obtained from healthy volunteers following ethical approval. Neutrophils were isolated from the blood by Dextran sedimentation of RBCs followed by Ficoll density gradient centrifugation to generate granulocyte pellets. 3 batches of Dextran were employed for RBC sedimentation: 103K0053, 058K0083 (Sigma Aldrich) and D500 (Pharmacosmos). Isolated cell suspensions were typically 90-95% neutrophils, regardless of the Dextran reagent used. Cells were resuspended in Hanks Balanced Salt Solution (with Ca2+ and Mg2+) containing 10mM D-glucose and 0.1% bovine serum albumen. Chemotaxis to interleukin-8 (IL8) and growth related oncogene α (GROα) was determined in Millipore Transwell plates (3μm pore size) by fluorescent measurement of DNA of migrated cells in the bottom chamber using CyQuant dye. Cell surface expression of CXCR1 and CXCR2 receptors was measured by flow cytometry using fluorescently-labelled mouse antibodies to human CXCR1 and CXCR2 (R&D Systems). Chemotaxis data are reported as the chemokine EC50 and % of cells migrated to the lower chamber; CXCR1 and CXCR2 expression are reported as the % of the isolated neutophils positive for bound antibody. Data are summarised as geometric mean (EC50) or arithmetic mean (%Max and % cells) with 95% confidence intervals from (n) donors; statistical analysis was performed using analysis of variance (ANOVA).

Neutrophil chemotaxis to GROα and IL8 was significantly reduced (p<0.05) in cells isolated with Sigma Dextran batch 058K0083 compared to isolation with the other Dextran batches. This was associated with significantly reduced CXCR2 expression in neutrophils (p<0.05). In contrast, the CXCR1 receptor expression level was not influenced by Dextran reagent batch (p>0.05). Thus, Dextran isolation influences neutrophil CXCR2 expression and chemotactic function.