Effects of CO2 and filter pore size on bovine neutrophil chemotaxis
In vitro chemotaxis assays are an efficient and cost-effective way to assess neutrophil (PMN) function, but variations in the procedure may impact assay results. The aim of this study was to evaluate the effects of CO2 and membrane pore size on chemotaxis of PMN from lactating cows (n=9; 4-163 DIM). Neutrophils were isolated and adjusted to 2×106 cells/mL. Media consisted of HBSS supplemented with 5% FBS. The bottom wells of 48-well chemotaxis chambers (Neuro Probe Inc., Gaithersburg, MD) contained media supplemented with 50 ng/mL of complement component 5a (C5a) or 100 ng/mL of Interleukin 8 (IL-8). Polycarbonate membranes with 3, 5, or 8 µm pores separated the bottom and top wells. Neutrophil suspension (50 µL) was added to the top wells, and chambers were incubated at 37°C for one hour in the presence or absence of 5% CO2. Negative controls contained no chemoattractant in either well, and positive controls contained 50 ng/mL of C5a in both top and bottom wells. All combinations of cow PMN, CO2, pore size, and chemoattractant were evaluated in triplicate wells over 6 different test dates. Raw adherence (RawAd) was determined by counting PMN adhered to the bottom of the membrane in 5 microscope fields per well. Relative adherence (RelAd) was calculated as (RawAd test well) / (RawAd negative control well) × 100%. Data were analyzed using the Glimmix procedure of SAS with the fixed effects of CO2, pore size, chemoattractant, and all interactions and random effects of date and cow within date. Both RawAd and RelAd were affected by CO2, pore size, chemoattractant, and CO2×pore size (P <0.001), and RawAd was affected by CO2×chemoattractant (P <0.001). Both RawAd and RelAd decreased in the presence of CO2. RawAd increased and RelAd decreased with increasing pore size. For both RawAd and RelAd, chemotaxis to C5a and IL-8 did not differ but both were greater than chemotaxis to controls. The CO2×pore interaction for both RawAd and RelAd was driven by a reduced increase in chemotaxis with increasing pore size when CO2 was present. The CO2×chemoattractant interaction for RawAd was due to muted effects of C5a or IL-8 on chemotaxis in the presence of CO2. Of the conditions evaluated, we propose that the use of a 3 µm membrane and incubation without CO2 may be preferable because this combination resulted in the greatest increase in adherence relative to the negative control.
neutrophils, chemotaxis, bovine