Phagocytic clearance of electric field induced 'apoptosis-mimetic' cells

Abstract

Cells undergoing apoptosis lose lipid asymmetry that is often manifested by the exposure of phosphatidylserine (PS) to the outer surface of the cell membrane. Macrophages and other cell types recognize externalized PS to signal phagocytosis, thereby eliciting a non-inflammatory response. PS exposure is obligatory in the recognition and clearance of apoptotic cells. Here, we find that externally applied moderate electric field induces PS externalization in a mouse B-cell (FOX-NY) membrane without procaspase-3 activation, a major characteristic of apoptotic cells. The field-induced PS inversion is caused as a result of electroporation and/or a process involving membrane reorganizations and recovery that ensues following field exposure. Using a mouse macrophage cell line (J7444A.1) from the same strain, we show phagocytic clearance of PS expressing B-cells and demonstrate that this is in part due to the apoptosis mimicry of the field exposed cells.

Fig 1

Phase and fluorescence (Annexin (+) and EthD-1 (−) ) microscope images of electric field induced PS externalization in B-cells. (A) Control – No Electric field. (B) Electroporated B-cells. E ~ 2.1 kV/cm, ~ 200 μSec pulse. (C) Addition of 0.2% Triton X-100 to the sample shown in (B) to permeabilize the membrane.

Fig 2

Blebbing and morphological changes in B cells exposed to ~200 μSec, ~2.1 kV/cm electroporating pulse. (A) Control – No Electric field applied. (B) Cells exposed to external field showing bleb formation. White arrows point to blebs in some of the cells. Image was taken ~10 min after field application.

Fig 3

Macrophage uptake of PS expressing cells. (A) Control. No Electric field (B) Field treated B-cells. E ~ 2.1 kV/cm, ~ 200 μSec. See Results section for description of frames in each panel. (C) Percentage of macrophages positive for uptake under conditions shown: Lanes 1- Control; 2-Field exposed; 3-Field exposed + Annexin I; 4-Field exposed + Annexin V blocked; 5-Ca⁺² ionophore treated for 12 hrs; 6-Field exposed + 12 hrs incubation. Error bars represent the standard error.

Fig 4

Schematic for electric field induced PS (red head group) externalization. Pathway-A: Lipid inversion could occur either through field driven transport of the negatively charged PS and/or via PS diffusion along the walls of hydrophilic membrane pores. Pathway-B: Electroporation leads to bleb formation similar to those shown in Fig. 2B. The bleb geometry constrains lipid packing due to curvature, thus favoring the scrambling of the inner and out lipid leaflets resulting in PS exposure. Exposed PS diffuses along the outer membrane periphery and remains exposed for extended time till bleb retraction and eventual sequestration to the cytoplasmic side.