Investigation Into the Role of Phosphatidylserine in Modifying the Susceptibility of Human Lymphocytes to Secretory Phospholipas
Total Page：16
File Type：pdf, Size：1020Kb
Biochimica et Biophysica Acta 1818 (2012) 1196–1204 Contents lists available at SciVerse ScienceDirect Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbamem Investigation into the role of phosphatidylserine in modifying the susceptibility of human lymphocytes to secretory phospholipase A2 using cells deficient in the expression of scramblase Jennifer Nelson, Lyndee L. Francom, Lynn Anderson, Kelly Damm, Ryan Baker, Joseph Chen, Sarah Franklin, Amy Hamaker, Izadora Izidoro, Eric Moss, Mikayla Orton, Evan Stevens, Celestine Yeung, Allan M. Judd, John D. Bell ⁎ Department of Physiology and Developmental Biology, Brigham Young University, Provo, Utah 84602, USA article info abstract Article history: Normal human lymphocytes resisted the hydrolytic action of secretory phospholipase A2 but became susceptible Received 8 September 2011 to the enzyme following treatment with a calcium ionophore, ionomycin. To test the hypothesis that this suscep- Received in revised form 22 November 2011 tibility requires exposure of the anionic lipid phosphatidylserine on the external face of the cell membrane, Accepted 9 January 2012 experiments were repeated with a human Burkitt's lymphoma cell line (Raji cells). In contrast to normal lympho- Available online 13 January 2012 cytes or S49 mouse lymphoma cells, most of the Raji cells (83%) did not translocate phosphatidylserine to the cell surface upon treatment with ionomycin. Those few that did display exposed phosphatidylserine were hydro- Keywords: Secretory phospholipase A2 lyzed immediately upon addition of phospholipase A2. Interestingly, the remaining cells were also completely Phosphatidylserine exposure susceptible to the enzyme but were hydrolyzed at a slower rate and after a latency of about 100 s. In contradis- Biophysical membrane change tinction to the defect in phosphatidylserine translocation, Raji cells did display other physical membrane changes Hydrolysis upon ionomycin treatment that may be relevant to hydrolysis by phospholipase A2. These changes were detected Merocyanine 540 by merocyanine 540 and trimethylammonium diphenylhexatriene fluorescence and were common among Trimethylammonium diphenylhexatriene normal lymphocytes, S49 cells, and Raji cells. The levels of these latter effects corresponded well with the relative rates of hydrolysis among the three cell lines. These results suggested that while phosphatidylserine enhances the rate of cell membrane hydrolysis by secretory phospholipase A2, it is not an absolute requirement. Other physical properties such as membrane order contribute to the level of membrane susceptibility to the enzyme independent of phosphatidylserine. © 2012 Elsevier B.V. All rights reserved. 1. Introduction the membranes of several types of cultured leukocytes have been reported to convert from being resistant to becoming susceptible to Secretory phospholipase A2 (sPLA2) hydrolyzes phospholipids at the the action of the enzyme during apoptosis and other forms of biochem- sn-2 position producing free fatty acid and lysophospholipid. It repre- ical cell death [4,5,7–10]. sents a family of isozymes and is thought to participate in a broad The nature of the biophysical changes in the cell membrane that range of functions including generation of pro-inflammatory mediators, trigger catalysis by sPLA2 during cell death is not yet established. Nev- protection against bacterial and fungal infections, digestion, participa- ertheless, important clues have been identified from biophysical tion in reproduction, nervous system development, and wound healing. studies of both artificial bilayers and living cells [6–8,10–18].Two Furthermore, sPLA2 has been implicated in several pathologies such as prominent candidates have emerged: 1) a reduction in the strength atherosclerosis, inflammatory diseases, septic shock, and cancer of interactions among adjacent phospholipids resulting in increased (reviewed in [1]). lipid mobility and greater interlipid spacing and 2) the presence of A major characteristic of sPLA2 is its extreme sensitivity to the bio- anionic lipids on the membrane surface [6,10,14,19]. The hypothesis physical properties of the bilayers on which it acts. This sensitivity is im- is that reduced interactions among neighboring phospholipids allow portant because it prevents damage to the membranes of normal substrates to migrate into the active site of adsorbed sPLA2, while healthy mammalian cells while allowing efficient catalysis of bacterial negative charge facilitates initial adsorption of the enzyme to the membranes and those of damaged or dying cells [2–6]. In particular, membrane surface [11,18,20–22]. The primary source of these surface anions during cell death is phosphatidylserine (PS). Ordinarily, this lipid is confined to the inner leaflet of the cell membrane, and several ⁎ Corresponding author at: 302A MSRB, Brigham Young University, Provo, Utah 84602, USA. Tel.: +1 801 422 2353; fax: +1 801 422 0263. enzymes are involved in maintaining that asymmetry [23]. However, E-mail address: [email protected] (J.D. Bell). during apoptosis and other forms of cell death, PS is translocated to 0005-2736/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.bbamem.2012.01.005 J. Nelson et al. / Biochimica et Biophysica Acta 1818 (2012) 1196–1204 1197 the outer leaflet [23–25]. Consequently, the exposed PS functions as a anisotropy or to polycarbonate tubes in a shaking water bath for flow marker for processes that eliminate the dying cell such as phagocytosis cytometry. Sample homogeneity and temperature were maintained in [24,26–28] and, perhaps, hydrolysis by sPLA2 [10,18,19]. Arguments for the spectrofluorometers by magnetic stirring and by circulating heated the importance of lipid-neighbor interactions and of surface PS are water through a jacket in the sample chamber. All experiments and accumulating, although the existing evidence from cells depends on incubations were performed at 37 °C. In each procedure where ionomy- temporal correlations rather than direct experimental manipulations cin was added to cell samples (600 nM final), the experiment was [6–8,10,14]. repeated with an equivalent volume of DMSO (0.5% v/v final) to control In order to distinguish more directly the potential roles of PS and for the effects of the solvent. membrane lipid interactions in capacitating hydrolysis during cell death, we have initiated a series of experiments with Raji Burkitt's 2.3. Membrane hydrolysis lymphoma cells. These cells are deficient in their ability to express the gene for scamblase, the enzyme primarily responsible for expo- As a generic model for sPLA2, we used enzyme purified from snake sure of PS during apoptosis [29–31]. Here, we report investigations venom as described above. This source of enzyme was selected because with a calcium ionophore, ionomycin, as the stimulator of cell of its previous use in biophysical studies of cell death by calcium iono- death. Ionomycin was chosen for our first study because it stimulates phore and because of its strong similarity to the human group X complete immediate exposure of PS and full hydrolytic susceptibility sPLA2, the isoform that responds best to ionophore-stimulated cells to sPLA2 in other cultured lymphoma cells [7,9]. Since Raji cells orig- [10,16,17].Weverified these assertions for human lymphocytes using inated from a human tumor, we used freshly-isolated normal human samples of human groups IIa, V, and X sPLA2 generously provided by lymphocytes as a control for comparison. Data were also compared to Michael Gelb (University of Washington) and Wonhwa Cho (University that attained with a cell line used in previous studies, S49 mouse of Illinois, Chicago). In these calibration experiments, the group IIa lymphoma cells, as a positive control. enzyme displayed 0.4% of the activity observed with snake venom sPLA2 toward human lymphocytes treated with ionomycin whereas 2. Materials and methods the groups V and X isozymes exhibited 25.7% and 82.8% respectively. These relative responses of the isozymes to ionomycin-treated lympho- 2.1. Reagents cytes were identical to those reported previously for S49 lymphoma cells and therefore justified the use of the snake venom enzyme as an The monomeric aspartate-49 phospholipase A2 from the venom of assay for cell susceptibility to sPLA2 [10,16]. the water moccasin, Agkistrodon piscivorus piscivorus was isolated accord- Fatty acid release from cell membranes was assayed in real time by ing to the procedure by Maraganore et al. [32]. Ionomycin, merocyanine fluorescence spectroscopy (Fluoromax 3, Horiba Jobin-Yvon, Edison, NJ 540 (MC540), propidium iodide, 1-(trimethylammoniumphenyl)- 6- ) using ADIFAB (excitation=390 nm, emission=432 and 505 nm, phenyl-1,3,5 hexatriene p-toluenesulfonate (TMA-DPH), and Alexa bandpass=4 nm) [33]. After data acquisition was initiated, ADIFAB Fluor 488 annexin V conjugate were acquired from Invitrogen (Carlsbad, (65 nM final) was added and allowed adequate time to equilibrate. CA). The acrylodan-labeled fatty acid-binding protein (ADIFAB) was The sample was then incubated with ionomycin for 10 min after purchased from FFA Sciences (San Diego, CA). Ionomycin and MC540 which sPLA2 (70 nM final) was added. The generalized polarization were dissolved as stock solutions in dimethylsulfoxide (DMSO), and (GP) was calculated from the fluorescence emission intensities at 505 TMA-DPH was suspended in dimethylformamide. Annexin V, ADIFAB, and 432 nm (I505 and I432) in order to quantify the amount of fatty and propidium