Multiple actions of lysophosphatidylcholine in human Jurkat T cells¹

Introduction

Lysophospholipids regulate a variety of biological processes including cell proliferation, tumor cell invasiveness and inflammation^[1,2]. lysophosphatidylcholine (LPC) (Figure 1)^[3,4], produced by the action of phospholipase A₂ (PLA₂) on phosphatidylcholine, promotes inflammatory effects, including the increased expression of endothelial cell adhesion molecules and growth factors, monocyte chemotaxis^[5], and macrophage activation^[6]. As a component of oxidized low-density lipoprotein (LDL), LPC plays an etiological role in atherosclerosis^[7], and is implicated in the pathogenesis of the autoimmune disease, systemic lupus erythematosus (SLE)^[8]. LPC was initially discovered as a ligand for G protein-coupled receptors, G2A and GPR4, and could stimulate PTX-sensitive intracellular Ca²⁺ transients and extracellular signal-related kinase (ERK) phosphorylation in transfected cells^[9,10]. Expression of G2A also caused a decrease of MMP in HeLa cells^[11].

Figure 1 Structures of LPC and other lysophospholipids.

Reactive oxygen species (ROS) exert numerous effects on cell functions, including induction of growth and regulation of kinase activity^[12–14]. Oxidative stress plays a prominent role in the functioning of the immune system at both physiological and pathological levels, and is also known to be associated with reduced or abnormal immune function with aging^[15], AIDS^[16], diabetes^[17], smoking-related pathologies^[18], and some autoimmune and inflammatory diseases^[19].

Jurkat T-cell line is made up of homogeneous T lymphocytes and possess membrane markers that characterize normal T lymphocytes^[20]. In the present study, LPC was found to induce ROS generation in human lymphocytic Jurkat T cells, and its mechanism was investigated. In particular, LPC-stimulated depolarization of MMP and [Ca²⁺]_i increase were investigated in relationship with ROS generation.

Materials and methods

Materials 1-Hexanoyl (C6:0) LPC, 1-octanoyl (C8:0) LPC, 1-decanoyl (C10:0) LPC, 1-lauroyl (C12:0) LPC, 1-myristoyl (C14:0) LPC, 1-palmitoyl (C16:0) LPC, 1-stearoyl (C18:0) LPC, 1-oleoyl (C18:1) LPC, 1-nonadecanoyl (C19:0) LPC, 1-arachi-doyl (C20:0) LPC, 1-lignoceroyl (C24:0) LPC, lysophospha-tidic acid (LPA), lysophosphatidylserine (LPS), lysophospha-tidylethanolamine (LPE), sphingosylphos-phorylcholine (SPC), sphingosine 1-phosphate(S1P), and lysophospha-tidylglycerol (LPG) were purchased from Avanti Polar Lipids (Alabaster, AL, USA). Fura 2/acetoxymethyl ester was from Calbiochem (Darmstadt, Germany). 2',7' dichlorofluorescin diacetate(DCFDA), antimycin A, N-acetyl cystein (NAC), indomethacin, diphenyleneiodonium (DPI), cis-N-(2-phenyl-cyclopentyl)-azacyclotridec-1-en-2-amine (MDL 12330A), pertussis toxin (PTX), ethylene glycol-bis(2-aminoethyl-ether)-N,N,N',N'-tetraacetic acid (EGTA), α-tocopherol and cyclosporine A were from Sigma-Aldrich (St Louis, MO, USA). 5,5',6,6' bensimidazolyl tetrachloro 1,1',3,3'-tetraethyl-carbocyanine iodide (JC-1) was from Biotium (Hayward, CA, USA). 2-[1-(3-(Amidinothio)propyl)-1H-indol-3-yl]-3-(1-methylindol-3-yl)maleimide· methanesulfonate (Ro 31-8220), bisindolymaleimide 1/2-[1-(3-dimethyl aminopropyl)indol-3-yl]maleimide (GF 109203X), and U73122 were from Biomol (Plymouth Meeting, PA, USA). Chelerythrine, rottlerin, staurosporin, trans-4-[(1R)-1-Aminoethyl]-N-4-pyridinyl-cyclohexane carboxamide (Y27632), carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), and A23187 were from Tocris Cookson (Avonmouth, Bristol, UK).

Cell culture Jurkat T cells were maintained in RPMI 1640 media containing 10% (v/v) fetal bovine serum, 100 U/mL penicillin, 50 µg/mL streptomycin, 2 mmol/L glutamine, and 1 mmol/L sodium pyruvate at 37 ºC in a humidified 5% CO₂ incubator.

Measurement of ROS The cells were sedimented, resuspended in Hepes-buffered medium consisting of 20 mmol/L Hepes (pH 7.4), 103 mmol/L NaCl, 4.8 mmol/L KCl, 1.2 mmol/L KH₂PO₄, 1.2 mmol/L MgSO₄, 0.5 mmol/L CaCl₂, 25 mmol/L NaHCO₃ and 15 mmol/L glucose, and then incubated for 20 min with 10 µmol/L DCFDA. Fluorescence was measured in DCFDA-loaded cells. Fluorescence emission at 520 nm wavelength from 488 nm excitation wavelength was measured every 0.1 s by F4500 fluorescence spectrophotometer(Hitachi, Tokyo, Japan). Individual histogram was made by the percent of control level, and ROS was measured more than twice.

Reverse Transcriptase-PCR To confirm the expression of GPCR in Jurkat T cells by RT-PCR, first-strand cDNA was synthesized with total RNA isolated by the ImProm-II Reverse Transcription System (Promega, Madison, WI, USA). Synthesized cDNA product was used for PCR with the primers for OGR1, GPR4, G2A, and TDAG8 by Go-Taq DNA polymerase (Promega, Madison, WI, USA). Specific primers for human OGR1 (sense 5'-TTC CTG CCC TAC CAC GTG TTG C-3', antisense 5'-TGG CGA GTT AGG GGT CTG GAA G-3'), human GPR4 (sense 5'-ACC TCT ATC GGG TGT TCG TG-3', antisense 5'-TTC CTC TTG GAG GTG AGT GG-3'), human G2A (sense 5'-CCG CCT ACA TCT TCT TCT GC-3', antisense 5'-ACA GGC ACA GAA ACA CCA CA-3'), or TDAG8 (sense 5'-TGC AAA GGG AGT GCT TTT CT-3', antisense 5'-CAC CTC CCA GTG CAG AAT TT-3') were used to amplify 341, 400, 500, and 600 bps of fragments of OGR1, GPR4, G2A, and TDAG8, respectively. The PCR reaction was performed by 30 cycles of denaturation at 95 ^oC for 30 s, annealing at 55 ^oC for 1 min, and elongation at 72 ^oC for 2 min^[21].

Measurement of mitochondrial membrane potential (ΜΜP) Mitochondrial membrane potential was measured using JC-1. JC-1 is a cationic mitochondrial vital dye that is lipophilic and becomes concentrated in the mitochondria in proportion to the membrane potential; the more dye that accumulates in mitochondria, the greater MMP and ATP generating capacity. The dye exists as a monomer at low concentrations (emission, 530 nm, green fluorescence), but forms J aggregates at higher concentrations (emission, 590 nm, red fluorescence). Cells were labeled for 20 min with 1 µmol/L JC-1 at 37°C, washed and resuspended in the Hepes-buffered medium. Fluorescence was monitored at two different wavelengths, and the ratio of the reading at 590 nm to the reading at 530 nm (590:530 ratio) was considered as the relative MMP value.

Measurement of [Ca²⁺]_i The cells were sedimented, resuspended in the Hepes-buffered medium, and then incubated for 40 min with 5 µmol/L Fura 2/acetoxymethyl ester. Fura 2-loaded cells were washed twice with the Hepes-buffered media and resuspended in the same media. Fluorescence emission at 510 nm wavelength from two excitation wavelengths (340 nm and 380 nm) were measured every 0.1 s (F4500, Hitachi). The ratio of fluorescence intensities from the two wavelengths was monitored as an estimate of [Ca²⁺]_i.

Statistical analysis Student’s t-test was conducted to analyze significant differences between measured ROS levels.

Results

LPC stimulated ROS generation in Jurkat T cells 20 µmol/L 16:0 LPC rapidly and significantly increased ROS level in Jurkat T cells compared with vehicle-treated control. LPC-induced ROS generation continued up to 40 min (Figure 2). 16:0 LPC generated the highest level of ROS increase, compared with other structurally related lysolipids, including LPA, LPS, LPE, SPC, S1P, and LPG (Figure 3A). The optimum chain length of LPC for ROS generation was found to be 16 (Figure 3B), and LPC-induced ROS production was observable with 20 µmol/L or higher concentrations (Figure 3C).

Figure 2 ROS generation induced by LPC. After loading DCFDA into Jurkat T cells (1.5×10⁶ cells/mL), cells suspended in HKRB without BSA were divided into 2 mL cuvette. Each trace was monitored and saved in F4500 fluorescent spectrophotometer. A, Vehicle was added in control, and 20 µmol/L 16:0 LPC was added at arrow-indicated time point. B, Time course of ROS generation by LPC. All traces were measured for 45 s and measurement was repeated after 20 min and 40 min (arrow-indicated).

Figure 3 ROS generation by lysophospholipids. A, All reagents were added at the first trace and measurements were repeated after 10 min. The first ten points of each step were averaged, and individual histogram was made by the percent of control level (fluorescence level of control was 250±40 at 10 min). The concentration of lipids was 20 µmol/L. B, The concentration of synthetic LPC was 20 µmol/L. C, Indicated concentrations of 16:0 LPC were tested. ^bP<0.05, ^cP<0.01 vs control.

PKCδ dependency of LPC-induced ROS production In order to elucidate which enzymes and signaling molecules were involved in LPC-induced ROS generation, antioxidants (NAC and α-tocopherol), NAD(P)H oxidase inhibitors (DPI and apocynine), xanthine oxidase inhibitor (allopurinol), cyclooxygenase inhibitor (indomethacin), mitochondrial ATP synthase inhibitor (oligomycin) and PKC inhibitors (chelerythrine, GF 109203X, Ro 31-8220, and Go 6976), and mitochondrial complex inhibitors (I: rotenone, II: thenoyl-trifluoroacetone (TTFA), III: sodium azide and IV: antimycin A) were tested together with 16:0 LPC on ROS measurement. Antioxidant α-tocopherol and PKCδ inhibitor rottlerin were found to be inhibitory on 16:0 LPC-induced ROS generation, and the inhibitory effect of rottlerin was concentration-dependent (Figure 4). Because mRNA expression of G2A family GPCR, of which G2A and GPR4 were initially reported as GPCR for LPC, were detected in Jurkat T cells (Figure 5A), inhibitors of PLC, G_i/o proteins, adenylyl cyclase and rho-dependent kinase, were also tested (Figure 5B). However, U73122 (a specific inhibitor of PLC), PTX (a specific inhibitor of G_i/o proteins), MDL 12330A (a specific inhibitor of adenylyl cyclase), and Y27632 (a specific inhibitor of rho-dependent kinase) did not exert any inhibitory effect on LPC-induced ROS generation (Figure 5B). Therefore, GPCR expressed in Jurkat T cells may not appear to be involved in LPC-induced ROS production.

Figure 4 Effects of specific inhibitors on LPC-induced ROS production. All inhibitors were preincubated with the cells for 10 min before addition of 16:0 LPC (20 µmol/L). A, Antioxidants NAC (20 mmol/L), α-tocopherol (20 µmol/L), NAD(P)H oxidase inhibitors DPI (10 µmol/L), apocynine (200 µmol/L), xanthine oxidase inhibitor allopurinol (100 µmol/L), cyclooxygenase inhibitor indomethacin (100 µmol/L), mitochondrial ATP synthase inhibitor oligomycin (5 µg/mL), PKC inhibitors including GF 109203X (200 nmol/L), Ro 31-8220 (100 nmol/L), chelerythrine (1 µmol/L), Go6976 (20 mmol/L), mitochondrial complex inhibitors rotenone (40 μmol/L), TTFA (500 µmol/L), soium azide (100 µmol/L), and antimycin A (2 µmol/L) were used. B, Indicated concentrations of rottlerin were preincubated for 10 min before the addition of 16:0 LPC (20 µmol/L). n=3. Mean±SD. ^bP<0.05, ^cP<0.01 vs LPC-induced ROS production.

Figure 5 mRNA expression of G2A family GPCRs and effects of inhibitors for GPCR signaling on LPC-induced ROS production. A, mRNA expression of OGR1 (300 bp), GPR4 (400 bp), G2A (500 bp) and TDAG (600 bp) in unstimulated Jurkat T cells. B, Effects on 16:0 LPC (20 µmol/L)-induced ROS production by inhibitors of PLC (U73122, 5 µmol/L), G_i/o proteins (PTX, 100 ng/mL), adenylyl cyclase (MDL 12330A, 10 µmol/L) and rho-dependent kinase (Y27632 0.2 µmol/L). All inhibitors, except PTX (24 h) were preincubated for 10 min before the addition of 16:0 LPC (20 µmol/L). n=3. Mean±SD.

LPC-induced depolarization of mitochondrial membrane potential Recently, changes of MMP were found to result from the inhibition of mitochondrial complex and this inhibition caused ROS generation^[22]. Furthermore, the expression of G2A caused a decrease of MMP and LPC-stimulated T lymphocytes showed a reduction of MMP^[11]. Therefore, MMP was measured along with LPC in Jurkat T cells: 20 µmol/L LPC significantly decreased JC-1 fluorescence compared with control, indicating depolarization of MMP (Figure 6A). Respiratory chain complexes are located in the mitochondria and maintain MMP through protons generated during electron transfer. Therefore, it is expected that any interference with electron transport would influence MMP. As seen in Figure 6C, mitochondrial complex IV inhibitor, antimycin A and H⁺ ionopore FCCP, caused significant depolarization of MMP (Figure 6B). However, antimycin A and FCCP did not increase ROS level (Figure 6C). In order to further elucidate whether LPC-induced ROS production caused depolarization of MMP, antioxidant α-tocopherol was used and found to have no inhibitory effect on LPC-induced depolarization of MMP (data not shown).

Figure 6 Depolarization of MMP (Δψ_m) induced by LPC. After loading JC-1 into Jurkat T cells (1.5×10⁶ cells/mL), cells suspended in HKRB without BSA were divided into 2 mL cuvette. Each trace was monitored and saved for F4500 fluorescent spectrophotometer. A, Vehicle was added in control and 20 µmol/L 16:0 LPC was added at arrow-indicated time point. B, Mitochondrial complex IV inhibitor, antimycin A (2 µmol/L) or H⁺ ionopore FCCP (1 µmol/L), was added at arrow-indicated time point. C, Mitochondrial complex inhibitor, antimycin A (2 µmol/L) and H⁺ ionopore FCCP (1 µmol/L), was tested on ROS measurement.

One of the changes associated with dissipation of MMP in some cell systems is the opening of the mitochondrial megachannel or permeability transition pore (PTP)^[23]. Therefore, cells were preincubated with cyclosporin A, a blocker of the PTP^[24], before exposure to LPC to see whether cyclosporine A-sensitive channels were operative during LPC-induced dissipation of MMP. Cyclosporin A induced the slow decrease of MMP by itself, however, it did not prevent MMP reduction by LPC (data not shown).

LPC-induced [Ca²⁺]_i increase and ROS generation Recent data show that LPC elevate the [Ca²⁺]_i in Jurkat T cells^[25]. Thus, the effect of LPC on [Ca²⁺]_i and its involvement with ROS were studied. As shown in Figure 7A, the same concentration of LPC as tested on ROS measurement sharply increased [Ca²⁺]_i and this increase was eliminated by an extracellular calcium chelator (EGTA) (Figure 7B), implying that LPC-induced [Ca²⁺]_i increase resulted from calcium influx across the plasma membrane. To determine whether LPC-induced [Ca²⁺]_i increase caused ROS production, the cells were pretreated with extracellular Ca²⁺ chelator EGTA; however, EGTA pretreatment did not prevent LPC-induced ROS production (Figure 7C). And Ca²⁺ ionophore A23187, which increased [Ca²⁺]_i (Figure 7D), also could not generate ROS (Figure 7C). We also examined whether this LPC-induced intracellular ionic change such as Ca²⁺ influx could influence MMP, however, EGTA could not abrogate LPC-induced decrease of MMP (data not shown).

Figure 7 [Ca²⁺]_i increase by LPC and its inhibition by EGTA. After loading Fura-2/AM into Jurkat T cells (1.5×10⁶/mL cells), cells suspended in HKRB without BSA were divided into 2 mL cuvette. Each trace was monitored and saved for F4500 fluorescent spectrophoto-meter. A, 20 µmol/L LPC was added at arrow-indicated time point. B, Extracellular Ca²⁺ chelator EGTA (5 mmol/L) was added at arrow-indicated time point. C, A23187 (0.2 µmol/L) and 16:0 LPC (20 µmol/L) were added at the first trace, and measurements after 10 min and 20 min were repeated. Ca²⁺ chelator EGTA (5 mmol/L) was preincubated for 10 min before the addition of 16:0 LPC (20 µmol/L). D, [Ca²⁺]_i increase was induced by Ca²⁺ ionopore A23187 of 0.2 µmol/L.

Discussion

LPC is one of the major phospholipid components that is increased during the oxidation of LDL. LPC-induced ERK1/2 activation is mediated by ROS that are generated through the NAD(P)H oxidase in PKC-dependent or -independent pathway in vascular smooth muscle cells (VSMC)^[26]. In human umbilical vein endothelial cells (HUVEC), oxLDL and LPC induce proliferation through activation of NAD(P)H oxidase that generates O₂^{– [27]}.

Cellular sources of ROS production include xanthine/xanthine oxidase, NAD(P)H oxidase, lipoxygenase, cyclooxygenase, and mitochondrial electron transport components. In VSMC and endothelial cells, NAD(P)H oxidases represent the most important source of O₂^{– [28,29]}. NAD(P)H oxidase catalyzes the NADPH-dependent reduction of oxygen to O₂^–, which in turn leads to the production of secondary derivatives such as OH· and H₂O₂. Mitochondria are also one of the most important sites of ROS production under physiological conditions^[30,31]. In this study, to elucidate the source of LPC-induced ROS generation, inhibitors of NAD(P)H oxidase, xanthine oxidase, cyclooxygenase, mitochondrial ATP synthase and mitochondrial complex were tested; however, their inhibitory effects were not observed.

ROS was produced by 16:0 LPC and 18:0 LPC, compared with other synthetic LPC. The optimum length of LPC acyl chain for ROS generation was 16 and even oleoyl LPC (18:1), that has an unsaturation on the acyl chain, couldn’t produce ROS, suggesting that structural bending by the double bond of unsaturated acyl chain of 18:1 LPC might prevent ROS generation. SPC is the counterpart structure of LPC in sphingolipids (SPC structure in Figure 1). However, SPC couldn’t produce ROS in Jurkat T cells and this might be due to the positive charge of amine group on the C2 position of SPC. The choline moiety of LPC seemed to be essential in ROS generation as other lysolipids with modified choline group, such as LPA, LPE and LPS, couldn’t produce ROS.

In VSMC, LPC induced ERK1/2 activation through ROS in PKC-dependent or -independent pathway and subsequent growth promotion^[26]. It has also been reported recently that SPC treatment generated ROS in MS1 endothelial cells and the SPC-induced ROS production was inhibited by pretreatment of the cells with PKCδ specific inhibitor rottlerin^[32]. In this study, rottlerin inhibited LPC-induced ROS generation in Jurkat T cells in a concentration-dependent manner, suggesting that LPC generates ROS through a PKCδ-dependent pathway.

G2A, named for its ability to cause accumulation of cells in G₂/M phase of the cell cycle, is a GPCR with tumor suppressor-like properties^[33]. Expression of G2A is transcriptionally upregulated by stress-inducing and cell-damaging agents, and the ectopic expression of G2A leads to growth inhibition. GPR4 is a member of the G2A subfamily, and stimulation of GPR4-overexpressing cells with its high affinity ligand, SPC, enhances DNA synthesis^[10]. Furthermore, LPC could stimulate [Ca²⁺]_i increase in Jurkat T cells^[25]. In the present study, we observed that LPC induced [Ca²⁺]_i increase; however, this event seemed to not be caused through GPCR, because inhibitors of PLC (U73122), G_i/o protein (PTX), adenylyl cyclase (MDL 12330A) and Rho-dependent kinase (Y27632) could not prevent LPC-induced [Ca²⁺]_i increase (data not shown), as observed in LPC-induced ROS generation. Furthermore, [Ca²⁺]_i increase through GPCR has usually been shown to release Ca²⁺ from intracellular calcium stores and subsequent Ca²⁺ influx; however, LPC-induced [Ca²⁺]_i increase observed in this study was totally inhibited by extracellular Ca²⁺ chelator EGTA, suggesting that Ca²⁺ influx across the plasma membrane was the only Ca²⁺ source. Legradi et al reported that LPC-induced Ca²⁺ increase was dependent on PTX-sensitive G proteins in Jurkat T cells^[25], but in our experiment it was not abrogated by PTX treatment. Two differences between their experiment and ours are Ca²⁺ increase patterns and used LPC concentrations. LPC-induced Ca²⁺ increase was observed approximately 1 min after the addition of LPC in the report of Legradi et al^[25]. However, in our experiment the increase was observed within several seconds. Legradi et al used 90 µmol/L, which is higher than the 20 µmol/L used in our experiment^[25]. Several different methods were also found, for example, different fluorescent dyes (Fluo-3 and FuraRed vs Fura-2), different sera (calf vs bovine), and assay media (RPMI 1640 vs Hepes-buffered solution). Currently, we are not able to explain why the results are not compatible. However, our data in ROS production experiment and with other inhibitors support our observation, which is GPCR-independent Ca²⁺ increase. The lag time of 1 min in their observation is unusual, because Ca²⁺ increase by GPCR activation is normally observed within several seconds.

A23187, a Ca²⁺ ionopore, quickly increased [Ca²⁺]_i, but couldn’t increase ROS. Furthermore, pretreatment with EGTA couldn’t abrogate LPC-induced ROS generation, therefore, LPC-induced [Ca²⁺]_i increase seemed to be not a cause of LPC-induced ROS generation. Expression of G2A caused a decrease of MMP and T lymphocytes that were stimulated with nanomolar concentrations of LPC (0.01 or 0.5 µmol/L) showing depolarization of MMP^[11]. Jurkat T cells have the intrinsic G2A receptor expression and showed dramatic depolarization of MMP. Mitochondrial respiratory chain is also one of the most important sites of ROS production under physiological conditions, and mitochondrial complex inhibition caused dissipation of MMP and generated ROS^[22]. In this study, antimycin A, complex IV inhibitor, and FCCP, a H⁺ ionopore, caused depolarization of MMP, but both failed to generate ROS. Therefore, mitochondrial dysfunction does not seem to be related to ROS generation in Jurkat T cells. Furthermore, because α-tocopherol couldn’t prevent LPC-induced MMP depolarization, LPC-induced reduction of mitochondrial membrane potential seems to not be involved in LPC-induced ROS generation. By using cyclosporin A, PTP inhibitor, we found that PTP is also not involved in LPC-induced MMP depolarization.

In summary, LPC-induced cellular responses in Jurkat T cells were found to include ROS generation, [Ca²⁺]_i increase, and depolarization of mitochondrial membrane potential. 16:0 LPC induced the maximal responses among structurally related lysophospholipids and LPC with different lengths of acyl chain. LPC-induced ROS generation was dependent on PKCδ, that independent of GPCR, although four members of G2A family GPCR were expressed in Jurkat T cells. This study suggests novel action mechanisms of LPC that are not mediated through GPCR and are independent from each other.

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