Mechanical regulation of calcium signaling of HL-60 on P-selectin under flow

Abstract

Background: Binding of P-selectin to P-selectin glycoprotein ligand-1 (PSGL-1) makes neutrophils roll on and adhere to inflammatory site. Intracellular calcium bursting of adhered neutrophils is a key event for subsequent arresting firmly at and migrating into the injured tissue. But, it remains unclear how the cytoplasmic calcium signaling of the cells were modulated by the fluid shear stress. Here, we focus on mechanical regulation of P-selectin-induced calcium signaling of neutrophil-like HL-60 cells under flow.

Methods: HL-60 cells were loaded with Fluo-4 AM for fluorescent detection of intracellular calcium ion, and then perfused over P-selectin-coated bottom of parallel-plate flow chamber. The intracellular calcium concentration of firmly adhered cell under flow was observed in real time by fluorescence microscopy.

Results: Force triggered, enhanced and quickened cytoplasmic calcium bursting of HL-60 on P-selectin. This force-dependent calcium signaling was induced by the immobilized P-selectin coated on substrates in absence of chemokine. Increasing of both shear stress and P-selectin concentration made the calcium signaling intensive, through quickening the cytosolic calcium release and upregulating both probability and peak level of calcium signaling.

Conclusions: Immobilized P-selectin-induced calcium signaling of HL-60 cells is P-selectin concentration- and mechanical force-dependent. The higher both the P-selectin concentration and the external force on cell, the more intensive the calcium signaling. It might provide a novel insight into the mechano-chemical regulation mechanism for intracellular signaling pathways induced by adhesion molecules.

Keywords: Calcium signaling; Flow chamber; HL-60 cell line; P-selectin; Shear stress.

Fig. 1

Number of firmly adhered HL-60 on substrates with five different treatments under shear stress of 2 dyn/cm². The substrate were treated by coating with blank, BSA only or plus P-selectin (0.1, 1 and 10 μg/mL). The data represent the mean plus SEM from three independent experiments. The significant level of difference from blank substrate group was shown by p value, * for p < 0.05 and ** for p < 0.01

Fig. 2

Calcium bursting of firmly adhered HL-60 on P-selectin under wall shear stress. a Two series of typical fluorescence images of firmly adhered HL-60 cells on P-selectin at different times, and b the time-course of the normalized fluorescence intensity of the cells over the observation time. Here red and black expressed the cases with or without external force (wall shear stress) of 2 dyn/cm² on cells

Fig. 3

Variation of activation ratio, peak intensity and delay time for calcium signaling of firmly adhered HL-60 under shear stress of 2 dyn/cm² versus immobilized P-selectin concentration. a The time-course, b the activation ratio, c the peak intensity and d the delay time of calcium signaling with different of immobilized P-selectin concentrations under wall shear stress of 2 dyn/cm². The data represent the mean plus SEM from at least 20 cells in three experiments. The significant level of difference from blank substrate group was shown by p value, * for p < 0.05 and ** for p < 0.01

Fig. 4

Variation of activation ratio, peak intensity and delay time for calcium signaling of firmly adhered HL-60 against shear stress. The plot illustrates the time-course (a), activation ratio (b), peak intensity (c) and delay time (d) for calcium signaling of firmly adhered HL-60 on substrate coated with 10 μg/mL P-selectin under shear stresses of 0.0, 0.2, 0.6 and 2 dyn/cm². The data represent the mean plus SEM from at least 20 cells in three experiments. Each inset in b, c or d presents the calcium signaling of HL-60 on blank substrates under shear stresses of 0 and 2 dyn/cm². The significant level of difference from blank substrate group was shown by p value, * for p < 0.05 and ** for p < 0.01