Analysis of sphingosine kinase activity in single natural killer cells from peripheral blood

Abstract

Sphingosine-1-phosphate (S1P), a lipid second messenger formed upon phosphorylation of sphingosine by sphingosine kinase (SK), plays a crucial role in natural killer (NK) cell proliferation, migration, and cytotoxicity. Dysregulation of the S1P pathway has been linked to a number of immune system disorders and therapeutic manipulation of the pathway has been proposed as a method of disease intervention. However, peripheral blood NK cells, as identified by surface markers (CD56(+)CD45(+)CD3(-)CD16) consist of a highly diverse population with distinct phenotypes and functions and it is unknown whether the S1P pathway is similarly diverse across peripheral blood NK cells. In this work, we measured the phosphorylation of sphingosine-fluorescein (SF) and subsequent metabolism of S1P fluorescein (S1PF) to form hexadecanoic acid fluorescein (HAF) in 111 single NK cells obtained from the peripheral blood of four healthy human subjects. The percentage of SF converted to S1PF or HAF was highly variable amongst the cells ranging from 0% to 100% (S1PF) and 0% to 97% (HAF). Subpopulations of cells with varying levels of S1PF formation and metabolism were readily identified. Across all subjects, the average percentage of SF converted to S1PF or HAF was 37 ± 36% and 12 ± 19%, respectively. NK cell metabolism of SF by the different subjects was also distinct with hierarchical clustering suggesting two possible phenotypes: low (<20%) or high (>50%) producers of S1PF. The heterogeneity of SK and downstream enzyme activity in NK cells may enable NK cells to respond effectively to a diverse array of pathogens as well as incipient tumor cells. NK cells from two subjects were also loaded with S1PF to assess the activity of S1P phosphatase (S1PP), which converts S1P to sphingosine. No NK cells (n = 41) formed sphingosine, suggesting that S1PP was minimally active in peripheral blood NK cells. In contrast to the SK activity, S1PP activity was homogeneous across the peripheral blood NK cells, suggesting a bias in the SK pathway towards proliferation and migration, activities supported by S1P.

Fig. 1

Optimization of the automated single-cell CE system for NK cells. (a) Schematic (top view) of the channel system used for automated singlecell CE analysis. (b) Schematic (cross section) through a section of the cell traps (not drawn to scale). Surface tension prevents the electrophoretic (green) and physiologic (purple) buffers from spilling into the air gap. The orange spheres represent cells within the traps. (c) Brightfield image of an NK cell trapped in each of three microwells. (d) The percentage of microwells entrapping a single cell is shown for microwells of varying diameter. Three replicates were obtained for each histogram (*p ≤ 0.05; **p ≤ 0.01).

Fig. 2

Single-cell analysis of peripheral blood NK cells. (a) Representative electropherogram demonstrating separation of SF, S1PF, and U₅₀ (HAF) from 19 cells sequentially analyzed. (b) An expanded region of the electropherogram shown in panel (b) demonstrating the contents of 5 cells. (c) The S1P pathway in NK cells. Abbreviations: ceramidase (CDase) and ceramide synthase (CS).

Fig. 3

Metabolism of SF in single NK cells. (a) A 3D plot comparing the percentage of S1PF and HAF formed from the total SF loaded into a cell. The lower x axis displays the total concentration of reporter loaded into the cell (SF_mol + S1PF_mol + HAF_mol). (b) Histograms expressing the number of cells with different percentages of S1PF for each subject. (c) Histograms comparing the percent of reporter converted to HAF for each subject. (d) The tree diagram showing hierarchical clustering comparing the overall distribution of percent S1PF formation between subjects 1–4.

Fig. 4

S1PF loading and metabolism in NK cells from two subjects (n = 36 cells). (a) The total amount of S1PF loaded into cells (S1PF_mol + HAF_mol) plotted against the diameter of the cells. (b) The total amount of S1PF loaded into cells (S1PF_mol + HAF_mol) was plotted against the reporter exposure time. (c) The percent of S1PF and HAF formed in a cell (relative to the total reporter loaded) was plotted against the total amount of S1PF reporter loaded into that cell.