Susceptibility of lysosomes to rupture is a determinant for plasma membrane disruption in tumor necrosis factor alpha-induced cell death

Abstract

Since a release of intracellular contents can induce local inflammatory responses, mechanisms that lead to loss of plasma membrane integrity in cell death are important to know. We showed previously that deficiency of the plasma membrane Ca2+ ATPase 4 (PMCA4) in L929 cells impaired tumor necrosis factor alpha (TNF-alpha)-induced enlargement of lysosomes and reduced cell death. The lysosomal changes can be determined by measuring the total volume of intracellular acidic compartments per cell (VAC), and we show here that inhibition of the increase in VAC due to PMCA4 deficiency not only reduced cell death but also converted TNF-alpha-induced cell death from a process involving disruption of the plasma membrane to a cell demise with a nearly intact plasma membrane. The importance of the size of lysosomes in determining plasma membrane integrity during cell death was supported by the observations that chemical inhibitors that reduce VAC also reduced the plasma membrane disruption induced by TNF-alpha in wild-type L929 cells, while increases in VAC due to genetic mutation, senescence, cell culture conditions, and chemical inhibitors all changed the morphology of cell death from one with an originally nearly intact plasma membrane to one with membrane disruption in a number of different cells. Moreover, the ATP depletion-mediated change from apoptosis to necrosis is also associated with the increases of VAC. The increase in lysosomal size may due to intracellular self-digestion of dying cells. Big lysosomes are easy to rupture, and the release of hydrolytic enzymes from ruptured lysosomes can cause plasma membrane disruption.

FIG. 1.

PMCA4 deficiency changes the morphology of TNF-α-induced cell death in L929 cells. (A) Parental and PMCA^mut L929 cells were untreated (−) or treated with 100 ng of TNF-α/ml (+) for 10 h. Cells were analyzed by microscopy. The genomic DNA was isolated and electrophoresed on a 2% agarose gel. Parental cells died with swelling (Sw), while PMCA^mut cells were shrunken (Sh). DNA laddering was detected in TNF-α-treated PMCA^mut cells but not in parental cells. (B) Parental, PMCA^mut, PMCA4-reconstituted, and empty vector-transfected PMCA^mut cells were untreated (−) or treated with TNF-α (+) for 10 h, and the cells were analyzed by PI exclusion plus FSC. The samples were analyzed by flow cytometry (FACSan flow cytometer [Becton Dickinson]) with CellQuest acquisition and analysis software. Different populations of dead and live cells were gated as R1, R2, and R3. R1 cells, necrotic cells that lost plasma membrane integrity, as indicated by PI-positive stains; R2 cells, apoptotic cells that had reduced size and low level of PI stains; R3 cells, healthy cells of normal size with PI-negative stains. The percentages of these three groups of cells are marked. (C) Parental and PMCA^mut cells were treated with TNF-α for various times and analyzed as described for panel B. The percentages of R1 and R2 cells are shown. (D) Parental and PMCA^mut cells were treated with staurosporine (1 μM), etoposide (10 μM), or lonidamine (10 μM) for 24, 24, and 4 h, respectively, and analyzed as described for panel B. The percentages of R1 and R2 cells are shown.

FIG. 2.

R1 and R2 types of cell death in different cell death systems. (A) Jurkat cells were treated with the Fas MAb (Fas Ab; 0.5 μg/ml) for various time periods. (B) MCF-7 cells were treated with TNF-α and CHX (5 μg/ml) for various time periods. (C) L929 cells were treated with TNF-α for various time periods. The cells were analyzed by microscopy and by PI exclusion plus FSC as described for Fig. 1. Sh and Sw are as defined for Fig. 1.

FIG. 3.

Increases of the VAC due to sucrose can lead to the loss of plasma membrane integrity in cell death. PMCA^mut L929 cells (A), parental L929 cells (B), MCF-7 cells (C), and Jurkat cells (D) were untreated (−) or treated with 30 mM glucose (Glu) or 30 mM sucrose (Suc). The relative value of the VAC was determined by LysoTracker staining. The L929 cells and MCF-7 cells were treated with TNF-α or TNF-α plus CHX, respectively, for 10 h. The Jurkat cells were treated with the Fas MAb for 3 h. The results are means ± standard errors (n = 3 to 6). The cells were then analyzed by PI exclusion plus FSC as described for Fig. 1. The percentages of R1 and R2 cells are shown.

FIG. 4.

The loss of plasma membrane integrity is associated with a large VAC in cells. Fibroblasts of beige mice (Lyst^bg) or control mice (C57BL/6) (A) and C57BL/6 fibroblasts of different passages (B) were treated with or without TNF-α plus CHX for 10 h. Half of each sample was analyzed by LysoTracker staining, and the other half was analyzed by PI exclusion plus FSC. The relative value of the VAC was determined by the intensity of LysoTracker staining. The results represent means ± standard errors (n = 3). The percentages of R1 and R2 cells are shown. (C) Jurkat cells were cotransfected with the BAX expression vector or an empty vector and a green fluorescent protein (GFP) expression vector. The cells were left untreated or treated with zVAD (50 μM) for 12 h after transfection. GFP expression was used to identify transfected cells by using a fluorescence-activated cell sorter. The relative value of the VAC in transfected cells was measured by staining the cells with LysoTracker at 12, 18, 24, and 30 h after transfection. The viability of cells was analyzed 30 h after transfection by PI exclusion plus FSC. Percentages of R1 and R2 are shown.

FIG. 5.

ATP depletion-mediated conversion of apoptosis to necrosis is associated with an increase of VAC. (A) Jurkat cells with or without the depletion of ATP were treated with the Fas MAb for different periods of time, stained with LysoTracker, and analyzed by microscopy. (B) Jurkat cells with or without depletion of ATP were treated with or without the Fas MAb. Relative values of the VAC were determined at different time points after Fas MAb addition. The percent increases of the VAC are shown. (C) Jurkat cells were treated as described for panel B and analyzed by PI exclusion plus FSC. The percentages of R1 and R2 cells are shown. (D) MCF-7 cells with or without the depletion of ATP were treated withor without TNF-α plus CHX. Relative values of the VAC were determined at different time points after TNF-α-CHX addition. The percent increases of the VAC are shown. (E) MCF-7 cells were treated as described for panel D and analyzed by PI exclusion plus FSC. The percentages of R1 and R2 cells are shown. (F) MCF-7 cells with or without the depletion of ATP were treated with TNF-α plus CHX in the presence or absence of forskolin (FSK; 100 μM). The viability of cells was analyzed by PI exclusion plus FSC. The percentages of R1 and R2 cells are shown. Intracellular ATP levels before and after ATP depletion were measured and are shown. Olig, oligomycin.

FIG. 6.

Inhibition of TNF-α-induced increases in the VAC increases the percentage of R2 cells, and enhancement of TNF-α-induced VAC increases the percentage of R1 cells. Parental L929 cells (A) and MCF-7 cells (B) were untreated or treated with TNF-α or TNF-α plus CHX in the presence or absence of A-23187 (1 μM), forskolin (FSK), nocodazole (Noc; 10 μM), jasplakinolide (Jas; 0.3 μM), or MDL-12330A (MDL; 50 μM) for 10 h. The VAC (left) and percentages of R1 and R2 cells (right) were measured.

FIG. 7.

Large lysosomes are easy to disrupt, and lysosomal leakage can lead to plasma membrane disruption. (A) MCF-7 cells were cultured in sucrose- or glucose-containing medium for 12 h. The cells were loaded with acridine orange for 15 min and then exposed to blue light (10 × 10⁴ lx) for different periods of time. Lysosome leakage was determined by the decrease of red fluorescence measured by flow cytometry. (B) Fibroblasts isolated from beige or control (C57BL/6) mice were used to perform the same experiments as described for panel A. (C) Viability of MCF-7 cells was measured 4 h after exposure to blue light. The percentages of R1 and R2 cells are shown. (D) Viability of fibroblasts was determined as described for panel C).