Effects of CO2 and filter pore size on bovine neutrophil chemotaxis

Abstract Text:

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.

Keywords:

neutrophils, chemotaxis, bovine