Technical note: Assessment of neutrophil endocytosis and proteolytic degradation and its relationship with phagocytosis and oxidative burst in dairy cows

摘要

Conventional assays of polymorphonuclear cell (PMN,neutrophil) function such as oxidative burst (OB) andphagocytosis (PC) are widely used to evaluate innateimmunity in the transition period of dairy cows. Oxidativeburst is commonly evaluated by measuring PMNmedian fluorescence intensity (MFI) involving therelease of reactive oxygen species after in vitro stimulation.Phagocytosis can be measured by engulfment offluorescent beads by PMN. DQ-ovalbumin (DQ-OVA)is a molecule suitable for the assessment of intracellularproteolytic degradation, so it might be informativeabout an additional pathway of pathogen handling byPMN. In this study, we evaluated the use of the DQOVAassay for the assessment of PMN function andthe relationships among OB, PC, and DQ-OVA resultsin PMN isolated from blood of dairy cows between 5and 21 d post partum. Results of the DQ-OVA validationassay were assessed with mixed linear regressionmodels. Pearson correlation tests and kappa values foragreement were used to associate the MFI betweeneach PMN function assay (OB, PC, and DQ-OVA).For the validation assay (9 cows in 3 replicates), PMNincubated with DQ-OVA were stimulated with IFN-γor inhibited with cytochalasin D, and fluorescence wascompared with untreated PMN. Stimulated and inhibitedPMN had greater (970 ± 160 units) and lesser (593± 55 units) MFI relative to untreated PMN (791 ± 154units), respectively, indicating that DQ-OVA fluorescencereflected enhanced or reduced endocytic and proteolyticfunction. To associate the MFI outcomes amongOB, PC, and DQ-OVA, 153 samples from 40 cows wereanalyzed. Results showed significant, although weakassociation between DQ-OVA and PC MFI (Pearson r= 0.16). When values of MFI were categorized accordingto the first (“high” PMN functionality), second andthird (“moderate” PMN functionality), or fourth (“low”PMN functionality) quartiles, agreement beyond chance(κ) was moderate: κ = 0.38 for DQ-OVA and OB, κ =0.43 for DQ-OVA and PC, and κ = 0.43 for OB andPC. The DQ-OVA assay may complement traditionalPMN functional assays because it provides additionalinformation regarding the combination of endocytosisand proteolytic degradation, but it is not a substitutefor assessment of OB or PC.