The effects of CO2 and HEPES buffer on in-vitro chemotaxis assays of bovine neutrophils

Abstract Text:

Neutrophil (PMN) chemotaxis can be measured in vitro with or without 5% CO₂. The production of carbonic acid during incubation with 5% CO₂ may reduce pH, possibly affecting cellular migratory function. The goals of this study were to determine whether incubation with 5% CO₂ impacts PMN chemotactic function and if the addition of HEPES buffer affects response to CO₂. Neutrophils were isolated from 12 lactating cows over 5 different test dates and cell concentration adjusted to 2×10⁶ cells/mL. Media consisted of HBSS containing 5% FBS with or without the addition of 10 mM HEPES. The bottom wells of a 48-well chemotaxis chamber (Neuro Probe Inc., Gaithersburg, MD) contained 28 µL of media supplemented with 50 ng/mL of complement component 5a (C5a) or 100 ng/mL of Interleukin-8 (IL-8). A 5 µm polycarbonate membrane separated the top and bottom wells. Neutrophil suspension (50 µL) was added to the top wells, and the chambers were incubated at 37°C for one hour, in the presence or absence of 5% CO₂. Negative controls did not contain chemoattractant in either well while positive controls contained 50 ng/mL C5a in both top and bottom wells. 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 HEPES, CO₂, chemoattractant and all interactions and random effects of date and cow within date. RawAd and RelAd were both affected by CO₂ (P = 0.03; 0.002, respectively) and chemoattractant (P = <.0001; 0.0002, respectively). The presence of CO₂ caused a decrease in both RawAd and RelAd. For both RawAd and RelAd, chemotaxis towards C5a or IL-8 did not differ but both were greater than chemotaxis to controls. RawAd was also affected by CO₂×chemoattractant (P = 0.02) and HEPES×CO₂ (P = 0.0001). RawAd for C5a or IL-8 wells was approximately twice that of control wells in the absence of CO₂, but was not significantly different from controls in the presence of CO₂. RawAd decreased in the presence of CO₂ when HEPES was not present but was unaffected by CO₂ in the presence of HEPES. These results suggest that incubating PMN with CO₂ reduces the chemotactic response and that HEPES partially ameliorated this effect.

Keywords:

neutrophils, chemotaxis, bovine