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

Abstract

Conventional assays of polymorphonuclear cell (PMN, neutrophil) function such as oxidative burst (OB) and phagocytosis (PC) are widely used to evaluate innate immunity in the transition period of dairy cows. Oxidative burst is commonly evaluated by measuring PMN median fluorescence intensity (MFI) involving the release of reactive oxygen species after in vitro stimulation. Phagocytosis can be measured by engulfment of fluorescent beads by PMN. DQ-ovalbumin (DQ-OVA) is a molecule suitable for the assessment of intracellular proteolytic degradation, so it might be informative about an additional pathway of pathogen handling by PMN. In this study, we evaluated the use of the DQ-OVA assay for the assessment of PMN function and the relationships among OB, PC, and DQ-OVA results in PMN isolated from blood of dairy cows between 5 and 21 d post partum. Results of the DQ-OVA validation assay were assessed with mixed linear regression models. Pearson correlation tests and kappa values for agreement were used to associate the MFI between each PMN function assay (OB, PC, and DQ-OVA). For the validation assay (9 cows in 3 replicates), PMN incubated with DQ-OVA were stimulated with IFN-γ or inhibited with cytochalasin D, and fluorescence was compared with untreated PMN. Stimulated and inhibited PMN had greater (970 ± 160 units) and lesser (593 ± 55 units) MFI relative to untreated PMN (791 ± 154 units), respectively, indicating that DQ-OVA fluorescence reflected enhanced or reduced endocytic and proteolytic function. To associate the MFI outcomes among OB, PC, and DQ-OVA, 153 samples from 40 cows were analyzed. Results showed significant, although weak association between DQ-OVA and PC MFI (Pearson r = 0.16). When values of MFI were categorized according to the first ("high" PMN functionality), second and third ("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 and PC. The DQ-OVA assay may complement traditional PMN functional assays because it provides additional information regarding the combination of endocytosis and proteolytic degradation, but it is not a substitute for assessment of OB or PC.

Effect of anti-inflammatory treatment on systemic inflammation, immune function, and endometrial health in postpartum dairy cows

“…Blood samples were collected (between 0800 and 0900 h) by coccygeal venipuncture into tubes containing acid citrate dextrose (Vacutainer, Becton Dickinson, Franklin Lakes, NJ) and processed within 3 hours of collection. The PMN isolation protocol was performed as described by Pascottini et al 37 . In the final isolation step (sample free of visible hemoglobin) the cell pellet was resuspended in 500 µL of 1 × PBS.…”

“…For the oxidative burst assay 37 , 200 µL of the PMN suspension and 2 µL of 1 mM 2′,7′-dih ydro-dichlorofluorescein-diacetate (Molecular Probes, Eugene, OR) were incubated in two flow cytometry tubes for 15 min in the dark at 37 °C under gentle agitation. Then, 200 µL of phorbol myristate acetate (Sigma-Aldrich, St. Louis, MO; 25 ng/mL in PBS/FBS) was added to the treatment tube and 200 µL of PBS (1×) containing 10% FBS was added to the control tube.…”

“…For the phagocytosis assay 37 , activated normal cow serum was produced for the whole experiment using serum from 20 healthy lactating Holstein cows. Next, 100 mg of Zymosan A from Saccharomyces cerevisiae (Sigma-Aldrich) was added per 10 mL of pooled serum, incubated at 37 °C for 60 min under gentle rotation, centrifuged at 2,500 × g for 15 min, filtered, and stored at −80 °C.…”

“…For the DQ-ovalbumin assay, to measure proteolytic degradation following endocytosis 37 , two tubes containing 1 × 10 6 PMN in 120 µL of Dulbecco's modified Eagle's medium (Gibco/Thermo Fisher Scientific, Waltham, MA) supplemented with 10% FBS were incubated at 37 °C under gentle agitation for 45 min in the dark. One of the tubes served as control and the other was supplemented with 10 µL of DQ-ovalbumin (10 μg/mL; Thermo Fisher Scientific, Eugene, OR).…”

Flow Cytometric Assessment of the Viability and Functionality of Uterine Polymorphonuclear Leukocytes in Postpartum Dairy Cows

“…Traditionally, PMN function has been tested on PMN isolated from blood. After their isolation and in vitro stimulation with either bacteria or chemokines, flow cytometric assessment of their per-cell capacity of phagocytosis or production of reactive oxygen species (ROS) is routinely performed [25][26][27]. Though, to date, it remains unclear whether the outcome of in vitro function tests of circulating PMN correlates with the functionality of (activated) PMN at their target site.…”