Methods for defining distinct bioenergetic profiles in platelets, lymphocytes, monocytes, and neutrophils, and the oxidative burst from human blood

Abstract

Peripheral blood mononuclear cells and platelets have long been recognized as having the potential to act as sensitive markers for mitochondrial dysfunction in a broad range of pathological conditions. However, the bioenergetic function of these cells has not been examined from the same donors, yet this is important for the selection of cell types for translational studies. Here, we demonstrate the measurement of cellular bioenergetics in isolated human monocytes, lymphocytes, and platelets, including the oxidative burst from neutrophils and monocytes from individual donors. With the exception of neutrophils, all cell types tested exhibited oxygen consumption that could be ascribed to oxidative phosphorylation with each having a distinct bioenergetic profile and distribution of respiratory chain proteins. In marked contrast, neutrophils were essentially unresponsive to mitochondrial respiratory inhibitors indicating that they have a minimal requirement for oxidative phosphorylation. In monocytes and neutrophils, we demonstrate the stimulation of the oxidative burst using phorbol 12-myristate 13-acetate and its validation in normal human subjects. Taken together, these data suggest that selection of cell type from blood cells is critical for assessing bioenergetic dysfunction and redox biology in translational research.

Figure 1

The bioenergetic profiles of human leukocytes and platelets. The bioenergetic profiles of monocytes (a), lymphocytes (b), platelets (c), and neutrophils (d) were determined using the extracellular flux analyzer. The basal measurements of oxygen consumption rates (OCR) were followed by sequential injections of 0.5 μg/ml oligomycin (0.75 μg/ml for platelets), 0.6 μM FCCP, and 10 μM antimycin A to determine the indices of mitochondrial function. Data expressed as mean±s.e.m. from representative donor profiles, n=3–5 assay replicates per sample.

Figure 2

Indices of mitochondrial respiratory function from monocytes, lymphocytes, and platelets. The critical indices of mitochondrial function were calculated from the cellular bioenergetic profiles for each cell type. Basal (basal rate—antimycin A rate) (a), maximal (FCCP rate—antimycin A rate) (b), ATP-linked (basal rate—oligomycin rate) (c), reserve capacity (FCCP response—basal rate) (d), proton leak (oligomycin response—antimycin A rate) (e), and non-mitochondrial (f) OCR of monocytes, lymphocytes, and platelets were plotted. Comparison of the percentage oxygen consumption (g) demonstrates distinct oxygen utilization pattern of each cell type. Cumulative data from eight healthy donors expressed as mean±s.e.m. *P≤0.05, **P≤0.005.

Figure 3

The oxygen consumption rate (OCR) and PPR of each cell type. (a) The mean basal OCR vs basal PPR. (b) The fold increase in PPR after treatment with 0.5 μg/ml oligomycin (0.75 μg/ml for platelets). Cumulative data from eight healthy donors expressed as mean±s.e.m. **P≤0.005.

Figure 4

Levels of electron transport chain proteins in peripheral blood cells isolated from freshly drawn human blood. Monocytes, lymphocytes, and platelets isolated from blood were lysed with RIPA buffer, and 50 μg of protein was separated on a SDS-PAGE gel followed by western blot for (a) Complex IV subunit I and (b) RIS protein. Protein expression was quantified using AlphaView SA software. Data presented as mean±s.e.m., n=3. *P≤0.05, **P≤0.005.

Figure 5

Activating the oxidative burst in monocytes and neutrophils. (a) The OCR of CD15⁺ neutrophils before and after injection of 100 ng/ml PMA±10 μM DPI, PMA-dependent response quantified in (b). (c) CD14⁺ monocyte OCR before and after injection of 100 ng/ml PMA±10 μM DPI, PMA-dependent response quantified in (d). The mean values from a single donor±s.e.m. n=3–5 assay replicates per sample. **P≤0.005.

Figure 6

Activating the oxidative burst in monocytes and neutrophils following bioenergetic profiling. (a) The representative bioenergetic profile of CD14⁺ monocytes followed by an injection of 100 ng/ml PMA. (b) CD14⁺ monocyte PPR response to PMA from the same donor before and after 10 μM antimycin A. (c) The representative bioenergetic profile of CD15+ neutrophils followed by an injection of 100 ng/ml PMA. (d) CD15+ neutrophil PPR response to PMA from the same donor before and after 10 μM antimycin. The mean values±s.e.m. n=3–5 assay replicates per sample.

Figure 7

PMA response by cell type. (a) The PMA-stimulated OCR of monocytes, lymphocytes, neutrophils, and platelets are shown. (b) The PMA-stimulated PPR of monocytes, lymphocytes, neutrophils, and platelets are shown. Cumulative data from 6–8 healthy donors expressed as mean±s.e.m. **P≤0.005.