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Originally published online as doi:10.1189/jlb.0507292 on September 20, 2007

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(Journal of Leukocyte Biology. 2007;82:1501-1509.)
© 2007 by Society for Leukocyte Biology

Acyl chain-dependent effect of lysophosphatidylcholine on human neutrophils

P. J. Ojala*, T. E. Hirvonen*, M. Hermansson{dagger}, P. Somerharju{dagger} and J. Parkkinen*,{ddagger},1

* Finnish Red Cross Blood Service, Helsinki, Finland;
{dagger} Institute of Biomedicine, Department of Biochemistry, University of Helsinki, Helsinki, Finland; and
{ddagger} Department of Clinical Chemistry, Helsinki University Central Hospital, Helsinki, Finland

1Correspondence: Department of Clinical Chemistry, Helsinki University Central Hospital, Biomedicum, FIN-00290 Helsinki, Finland. E-mail: jaakko.parkkinen{at}helsinki.fi


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ABSTRACT
 
Lysophosphatidylcholine (LPC) is the most abundant lysophospholipid in plasma and tissues, and its level increases in ischemia and inflammation. LPC induces various proinflammatory actions in leukocytes, endothelial cells, and smooth muscle cells, but its effects may vary, depending on the acyl chain. In the present study, we identified the molecular species of LPC in human plasma and studied their effects on human neutrophils. Unsaturated LPC species over a wide concentration range (5–200 µM) induced long-lasting superoxide production in neutrophils. The response was preceded by a >10-min lag time and lasted for 60–90 min. Superoxide production was prevented when albumin was added together with LPC at a molar ratio of 1:2 or higher, and significant inhibition was observed even when albumin was added 4–8 min after LPC. Saturation of albumin by fivefold molar excess of stearic acid reduced the inhibitory effect significantly. Saturated LPCs, particularly the most abundant 16:0 species, induced significantly less superoxide production than the unsaturated species and only at 5–10 µM concentrations. Saturated LPC species elicited a several-fold higher increase in cytoplasmic calcium and at >20 µM, increased plasma membrane permeability. A mixture of LPCs mimicking the plasma LPC composition induced nearly similar superoxide production as the most active LPC18:1 alone. These results indicate remarkable acyl chain-dependent differences in the cellular effects of LPC. Elevation of LPC level may increase inflammation through activation of neutrophil NADPH oxidase, particularly when the simultaneous increase of free fatty acids diminishes the ability of albumin to scavenge LPCs.

Key Words: superoxide • NADPH oxidase • albumin • free fatty acids • ischemia


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INTRODUCTION
 
Lysophosphatidylcholine (LPC) is a proinflammatory phospholipid generated by phospholipase A2 (PLA2) from the phosphatidylcholine (PC) pools of cells and oxidized low-density lipoprotein (oxLDL) [1 2 3 ]. The cytoplasmic and secretory forms of PLA2 are up-regulated in ischemia and inflammation, and a lipoprotein-associated PLA2 is an independent risk factor for atherosclerotic complications [1 , 2 ]. LPC is also formed by the lecithin cholesterol acyl transferase in plasma [3 ]. LPC accumulates in tissues during ischemia and may contribute to reperfusion injury [4 ]. Plasma LPC level increases in rheumatoid arthritis [5 ] and to a lesser extent, in diabetes and obesity [6 , 7 ]. A considerable increase in LPC content during routine storage of cellular blood products has been reported [8 ] and is thought to play a causal role in transfusion-associated acute lung injury [9 ].

Most of the LPC in plasma is carried by albumin, and the level of free LPC is low. However, free fatty acids (FFA) have a higher affinity to albumin and can displace LPC from albumin [10 , 11 ]. LPC can also be transferred directly to cell membranes from oxLDL [12 ]. LPC elicits various proinflammatory and atherogenic phenomena, including up-regulation of adhesion molecules and permeability increase in vascular endothelium, as well as chemotaxis and secretion of cytokines by monocytes [1 , 13 ].

Neutrophils are major cellular mediators of innate immunity and tissue damage in acute inflammatory conditions [14 ]. As an essential part of the antimicrobial and inflammatory actions, neutrophils generate superoxide anions, which are converted into more reactive oxygen species (ROS). NADPH oxidase catalyzes superoxide formation by a single electron transfer to molecular oxygen, causing a huge increase in oxygen consumption (oxygen burst). The oxidase complex consists of a transmembrane glycoprotein, flavocytochrome b558, cytoplasmic regulatory subunits p40phox, p47phox, and p67phox, and a small GTPase, Rac [15 ]. During the activation process, the cytoplasmic subunits are phosphorylated, translocated to the phagosome or plasma membrane, and assembled with the flavocytochrome [15 ]. Translocation of Rac, a critical switch of NADPH oxidase activation, requires its dissociation from the inhibitory protein guanine nucleotide disassociation inhibitor and conversion into the GTP form [16 ].

Prior studies about effects of LPC on neutrophils have used mainly mixtures of tissue-derived LPCs or the saturated LPC16:0. In a previous study assessing synthetic LPC species, LPC, at concentrations of 0.1–1 µM, inhibited superoxide production in fMLP-stimulated neutrophils [17 ], whereas in another study, micromolar concentrations of LPC16:0 induced superoxide generation [18 ]. There is increasing evidence that LPC activates NADPH oxidase in nonphagocytic cells, particularly in vascular endothelial cells [19 20 21 ]. Among the different molecular species, the saturated LPC16:0 and LPC18:0 induced much higher elevations in intracellular calcium ([Ca2+]i) in neutrophils than the unsaturated LPC18:1, and fMLP-induced superoxide generation was primed when the LPCs were added with albumin [22 ].

In the present study, we sought to identify the primary determinants influencing proinflammatory effects of the different LPC species occurring in plasma. We exposed neutrophils to different LPC species in the absence and presence of albumin and found striking differences between saturated and unsaturated LPC species in their ability to activate NADPH oxidase and enhance membrane permeability.


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MATERIALS AND METHODS
 
Materials
Synthetic LPCs (LPC16:0, -17:0, -18:0, -18:1), lysophosphatidic acid (LPA16:0, -17:0, -18:0, -18:1), sphingosylphosphorylcholine (SPC18:1), sphingosine-1-phosphate (S1P18:1), and lysophosphatidylserine (LPS18:1) were from Avanti Polar Lipids (Alabaster, AL, USA). Diphenyleneiodonium (DPI), FFA, fMLP, and luminol were from Sigma-Aldrich (St. Louis, MO, USA). U0126 and SB203580 were from Promega (Madison, WI, USA). Platelet-activating factor (16:0PAF) and 16:0-lyso-PAF were from Biomol (Plymouth, PA, USA). The LPC species not available commercially (i.e., LPC16:1, -18:2, -18:3, -20:4, -22:5, -22:6) were synthesized from the corresponding PC species (Avanti Polar Lipids) by PLA2-catalyzed cleavage of the sn2-FA [23 ]. The concentrations of the phospholipid stock solutions in chloroform/methanol were determined by phosphate analysis [24 ]. Albumin was obtained from the manufacturing of pharmaceutical human plasma albumin prior to addition of caprylic acid and pasteurization [Finnish Red Cross (FRC) Blood Service, Helsinki, Finland]. Albumin concentration was determined by absorbance at 280 nm using the extinction coefficient of 0.530 L g–1cm–1.

For the experiments with cells, the phospholipids were dried from chloroform/methanol in silylated vials (Waters, Alltech, Milford, MI, USA) under nitrogen and dispersed in 10 mM Hepes, pH 7.4, by vortexing and tip sonication immediately before the experiments. In some experiments, the phospholipids were first dissolved in a small volume of ethanol and diluted in the Hepes buffer. Similar results were obtained with both phospholipid solutions in the neutrophils activation assay. When the influence of albumin was studied, albumin and the phospholipids or FA were first mixed in the Hepes buffer to allow complex formation before addition to the neutrophils suspension. In some experiments, albumin was added separately 4 or 8 min after the phospholipids to the neutrophil suspension.

Measurement of neutrophil superoxide production
Human polymorphonuclear leukocytes (PMNs) were isolated from buffy coats prepared from healthy blood donors at the FRC Blood Service by dextran sedimentation and density centrifugation on Ficoll-Paque (Amersham Healthcare, Uppsala, Sweden). Contaminating erythrocytes were lysed by hypotonic lysis in cold 0.2% NaCl. Cells were washed with and suspended in HBSS containing 10 mM Hepes, pH 7.4. The concentrations of Ca2+ and magnesium (Mg2+) in the HBSS were 1.26 mM and 0.9 mM, respectively. For monitoring of real-time ROS generation, PMNs at different densities and luminol (5-amino-2,3-dihydro-1,4-phthalazinedione; final concentration, 63 µM) were added in HBSS to Opti-Plate F96 microplate wells (Perkin Elmer, Boston, MA, USA), and after addition of the test compounds, luminescence was monitored at 37°C using a Victor 2 multilabel counter with a 700-nm cut-off filter (Wallac-PerkinElmer, Turku, Finland). To determine intracellular production of ROS, PMNs (2x106/ml) were loaded with 10 µM H2-2',7'-dichlorodihydrofluorescein (DCF) diacetate (Molecular Probes-Invitrogen, The Netherlands) in HBSS for 30 min at 37°C and washed with HBSS by centrifugation. The PMN suspension (100 µl 107/ml) was added to 96-well plates containing the test compounds dispersed in 100 µl HBSS. Fluorescence was measured at 2 min intervals.

Measurement of [Ca2+ ]i
PMNs were suspended in Ca2+-free HBSS and loaded with 5 µM Fluo-3/AM (Molecular Probes-Invitrogen) for 30 min at room temperature. The cells were washed with Ca2+-free HBSS and incubated further for 30 min. The PMN suspension (100 µl 4x106/ml in HBSS) was added to 96-well plates containing the test compounds in 100 µl HBSS, and the fluorescence intensity was measured at 2 min intervals with excitation and emission wavelengths set at 485 nm and 535 nm, respectively.

Measurement of lactate dehydrogenase (LDH) release and trypan blue exclusion
The release of LDH was determined by the cytotoxicity detection kit (Roche Applied Science, Germany). Total LDH activity was determined by lysing the cells with 1% Triton X-100. Trypan blue-positive cells were counted in a hemocytometer after incubation of the PMNs in HBSS containing 0.1% trypan blue for 5–15 min at room temperature.

Determination of phospholipids of plasma and blood cells
Citrated blood from individual donors was chilled immediately on ice, centrifuged at 5000 g for 10 min, and the separated plasma was frozen. During melting, 10 mM EDTA and 1 mM Pefabloc (PAF-acetyl hydrolase inhibitor) were added, and thereafter, the samples were spiked with a mixture of phospholipid standards. Extraction was done in silane-treated screw-cap tubes according to Folch et al. [25 ], except that the solvent contained 0.1 M HCl. The extracts were dried under nitrogen stream and dissolved in chloroform/methanol 1:2 (v/v). The extracts were evaporated under a nitrogen stream, dissolved in chloroform/methanol 1:2 (v/v), and transferred to silylated vials (Waters, Alltech) for storage at –20°C.

The identification and quantification of the lipid species were carried out with a Micromass Quattro Micro triple-quadrupole instrument (Micromass, Manchester, UK). NH4OH (1%) was added to the lipid extracts just prior to the analysis, and samples were injected into the ion source at the flow rate of 6 µl/min. Nitrogen was used as the nebulizer (500 l/h at 130°C) and cone gas (50 l/h). The source temperature was set at 90°C, and the potential of the cone, extractor, and Rangefinder lens was 40, 2, and 0.3 V, respectively. The capillary voltage was 3.8 kV. The different phospholipid classes were detected selectively by using head group-specific precursor ion or neutral-loss scanning. LPC, PC, and sphingomyelin were detected by scanning for the precursors of 184 in the positive ion mode; phosphatidylethanolamine and PS by scanning for the neutral loss 141 and 185, respectively, in the positive ion mode; and LPA by scanning for the precursors of 153 in the negative ion mode [26 ]. The collision energy was set to 20–55 eV, and argon was used as the collision gas. The spectra were smoothed and transferred to Microsoft Excel, and the relevant peaks were quantified using the LIMSA add-on as described [27 ].

Statistical analysis
The statistical significance of the results was assessed with the nonparametric Mann-Whitney U-test using Statsdirect software v. 2.5.6. (StatsDirect Ltd., Cheshire, UK).


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RESULTS
 
Identification of the molecular species of LPC in human plasma
The most abundant LPC species in fresh plasma samples was LPC16:0, which comprised ~40% of the total LPC pool. The next abundant species was LPC18:2, corresponding to ~20%, and LPC18:1 and LPC18:0 constituted 10–15% of the total (Table 1 ). The average concentration of LPC in fresh plasma was 190 µM, and it increased more than twofold during incubation of plasma at 37°C for 24 h. Most of the increase was a result of the saturated species LPC16:0 and LPC18:0. The ratio of LPC:PC in fresh plasma was ~1:8 but increased to ~1:2 during incubation (Table 1) .


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  		Table 1. Relative Concentrations of Different LPC Species in Plasma and Blood Cell Membranes

A large industrial plasma pool from 3500 blood donors (FRC Blood Service) contained a similar distribution of the LPC species as the fresh plasma samples, but its total LPC content was ~50% higher (Table 1) . This was probably a result of storage of the whole blood overnight at 20°C before separation of plasma. Although the molar ratio of LPC:albumin in the plasma pool was ~1:2, pharmaceutical albumin purified by the Cohn process of cold ethanol fractionation contains only 2–5 mol% LPC [5 ] and has thus nearly full binding capacity for LPC. The ratio of LPC:PC was lower in red cell membranes and platelets than in plasma, and the cell-associated LPC contained proportionally more saturated LPC species (Table 1) .

Effect of different LPC species on ROS production of PMNs
We first measured the effect of different plasma LPC species on the superoxide production by PMNs using luminol, which detects extra- and intracellular ROS generation. Above a threshold concentration of 2–5 µM, unsaturated LPC species induced considerable ROS production over a wide concentration range, up to 200 µM. ROS production by LPC18:1 was preceded by a long lag-phase of ~10 min and started much later than that elicited by fMLP (Fig. 1 ). ROS production induced by LPC18:1 also lasted longer, reaching its peak after 30–40 min, depending on the LPC concentration.


Figure 1
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  		Figure 1. Activation of neutrophil ROS production by LPC18:1. Different concentrations of LPC18:1 indicated or 1 µM fMLP were added to 107/ml PMNs. Real-time ROS production was monitored with luminol. Means of three parallel determinations are shown.

The dose-response relationship of LPC-induced ROS generation was dependent on PMN density. At 106 cells/ml, LPC18:1 elicited ROS production at concentrations ranging from 5 µM up to 200 µM (Fig. 2A ). The peak response was obtained at 10–15 µM concentration. In contrast, the saturated species LPC16:0 and LPC18:0 elicited clearly less ROS production and only in a narrow concentration range, approximately 5 µM (Fig. 2A) . At a tenfold higher cell density, LPC18:1 induced proportionally higher ROS generation with a peak response at 20–50 µM and only slightly lower response at 100 µM (Fig. 2B) . At this higher cell density, LPC18:0 elicited ROS generation also over a wider concentration range but not at concentrations in excess of 50 µM, and LPC16:0 elicited ROS production only at approximately 10 µM. LPC18:2, the most abundant unsaturated species in plasma, induced a somewhat lower ROS response than LPC18:1 (Fig. 2B) . Likewise, the less abundant, unsaturated LPCs, i.e., the 16:1, 18:3, and 20:4 species, elicited lower responses than LPC18:1 (data not shown).


Figure 2
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  		Figure 2. Differential effect of LPC species on neutrophil ROS production and the influence of neutrophil density and extracellular Ca2+. Synthetic LPC species indicated were added to (A) 106/ml PMNs and (B–E) 107/ml PMNs. Ca2+-free medium was used in C. "Plasma LPC-mix" was a reconstituted mixture shown in Table 1 . ROS production was monitored with luminal, and the area under the curve (AUC) was measured. Mean ± SD of three independent experiments is shown.

A mixture of synthetic LPCs mimicking the plasma acyl chain composition (cf., Table 1 ) induced only a somewhat lower ROS response than the most active LPC18:1 alone (Fig. 2C) . When peak amplitude was used instead of the AUC, the responses relative to the background level were about twice as high.

The LPC18:1 concentration required for peak response of PMNs at cell densities of 1–25 x 106/ml indicated that 2–6 x 109 LPC18:1 molecules per one PMN cell were needed for optimal activation. The EC50 value for LPC18:1 increased from 5 µM at 106 cells/ml to 50 µM at 25 x 106/ml. Omission of Ca2+ from the medium decreased but did not abolish ROS generation completely (Fig. 2D) , whereas the omission of Ca2+ and Mg2+ abolished ROS generation completely (not shown). Addition of 5 µM DPI, an inhibitor of NADPH oxidase, prevented LPC-induced ROS generation completely (Fig. 3 ). This confirmed that the ROS derived from superoxide produced by the NADPH oxidase.


Figure 3
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  		Figure 3. Inhibition of LPC-induced ROS production by DPI. The LPC species indicated were added to PMNs (107/ml). When indicated, 5 µM DPI was added before LPC. ROS production was monitored with luminal, and the AUC was measured. Mean ± SD of three parallel experiments is shown. DPI significantly (*, P<0.05) inhibited LPC-induced ROS generation. In the presence of DPI, the responses did not differ significantly from the vehicle-treated control.

Other lysoglycerophospholipids and lysosphingolipids having a different polar group and/or tail structure were also tested for their ability to elicit ROS generation in neutrophils (Fig. 2E) . Apart from the rapid, receptor-mediated, oxidative burst generated by PAF16:0, only SPC18:1 triggered ROS generation with a signal amplitude similar to LPC18:1. However, this required nearly 50 µM SPC concentrations, which were about three orders of magnitude greater than the reported plasma concentration [28 ]. Similarly, the responses obtained with the other lysophospholipids required concentrations higher than the physiological plasma levels. LPS18:1 and lyso-PAF16:0 elicited a four- to six-fold increase in ROS generation at 25 µM concentration, which disappeared when their concentration was increased to 100 µM. S1P18:1 and LPA18:1 displayed moderate responses, which reached a maximum of three- to fivefold at 100 µM.

The differences in response to unsaturated versus corresponding saturated LPC species were even more striking when intracellular ROS production was monitored with the fluorescent probe DCF. LPC16:0 and -18:0 decreased the background ROS generation at 10–100 µM concentrations, whereas LPC16:1, -18:1, and -18:2 induced intracellular ROS production in a dose-dependent manner (Fig. 4 ).


Figure 4
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  		Figure 4. Differential effect of LPC species on intracellular ROS production. The LPC species indicated were added to PMNs (5x106/ml) at 10, 30, and 100 µM, without or with 150 µM albumin. Accumulation of intracellular ROS was monitored with DCF. Means of three parallel determinations are shown.

Effect of albumin on LPC-induced ROS generation
ROS production by neutrophils was inhibited effectively when albumin was added together with LPC at a molar ratio of 1:2 or higher. Albumin prevented LPC-induced intracellular ROS production completely and even decreased the background ROS generation in control cells without added LPC (Fig. 4D) . Albumin also prevented the induction of luminol-detectable ROS generation by LPC (Fig. 5 ). When albumin was preincubated with fivefold molar excess of stearate together with LPC, its ability to scavenge LPC was diminished significantly (Fig. 5) . Stearate alone had no significant effect on ROS generation at the corresponding concentration. The polyunsaturated FA DHA was less effective in repressing the LPC scavenging effect of albumin than stearate (data not shown). Scavenging of LPC by albumin was impaired only moderately when albumin was added 4 or 8 min after LPC18:1 to the PMN suspension (Fig. 5) . Other macromolecules, hydroxylethyl starch, and polyethylene glycol 20,000 had no significant effect on LPC18:1-induced ROS generation (data not shown).


Figure 5
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  		Figure 5. Effect of albumin and FFA on LPC-induced ROS production. LPC18:1, albumin [human serum albumin (HSA)], and stearic acid were added to the final concentrations indicated to PMNs (107/ml). When indicated, albumin was added 4 min or 8 min after LPC. ROS production was monitored with luminal, and the AUC was determined. Mean ± SD of three independent experiments is shown.

Differential effects of LPC species on [Ca2+]i in PMNs
The effect of LPC species on [Ca2+]i was monitored with the Fluo-3 probe. In agreement with previous reports [12 ], 10 µM LPC18:0 induced a rapid increase in [Ca2+]i, which then decreased slowly over 60 min. LPC16:0 induced a similar, although somewhat lower increase, whereas LPC18:1 evoked a significantly lower increase in [Ca2+]i (Fig. 6 ).


Figure 6
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  		Figure 6. Differential effect of LPC species on [Ca2+]i. PMNs (2x106/ml) loaded with Fluo-3 were incubated with the LPC species indicated, and the AUC of fluorescence traces was determined. Mean ± SD of three parallel determinations is shown. n.s., Not significant. **, P < 0.01.

Differential effects of LPC species on PMN plasma membrane permeability
The influence of the different LPC species on plasma membrane integrity of PMNs was studied using LDH release and trypan blue staining. Control cells incubated in HBSS for 30 min at 37°C without added lipids released, on average, 26% of the cellular LDH. At 10 µM, LPC16:0 and -18:0 hardly increased LDH release, whereas at 25 µM, both LPC species enhanced LDH release significantly, i.e., 75% and 60% of total LDH, respectively (Fig. 7A ). Significantly less (40%) LDH was released in the presence of 25 µM LPC18:1. The EC50 of LDH release was ~20 µM for LPC16:0, 40 µM for LPC18:0, and 200 µM for LPC18:1. Albumin prevented the release of LDH by LPC18:1 when added at molar ratios of 1:2 or higher (Fig. 7B) .


Figure 7
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  		Figure 7. LDH release from PMNs caused by LPC species. (A) PMNs (107/ml) were incubated in HBSS containing different concentrations of LPC16:0 ({circ}), LPC18:0 ({square}), or LPC18:1 ({blacksquare}) for 30 min at 37°C, after which LDH release was determined. LPC16:0 differed significantly (P<0.05) from LPC18:1 at 25–100 µM and LPC18:0 at 50–100 µM. (B) Prevention of LDH release caused by 100 µM LPC18:1 by increasing concentrations of albumin. PMNs (107/ml) were incubated for 2 h at 37°C, after which the LDH release was determined. Mean ± SD of three independent experiments is shown. *, P < 0.05, compared with control.

Control cells kept in HBSS remained >98% trypan blue-negative for 2 h at 37°C. After the addition of 30 µM LPC16:0 or LPC18:0, the proportion of trypan blue-positive cells increased within 5 min to 84% and 38%, respectively (Fig. 8 ). The unsaturated LPC species increased the proportion of trypan blue-positive cells significantly less, which was not more than 20% at 30 µM LPC concentrations. It is interesting that the increase in membrane permeability induced by LPC16:0 was transient, as the proportion of trypan blue-positive cells decreased significantly during 90 min. At concentrations in excess of 30 µM, LPC16:0 lysed the cells. In contrast, LPC18:1 did not influence PMN viability at concentrations up to 100 µM (data not shown).


Figure 8
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  		Figure 8. Trypan blue positivity induced by LPC in PMNs. The LPC species at 30 µM concentration were incubated for the time periods indicated with PMNs (107/ml), and the proportion of trypan blue-positive cells was counted. Mean ± SD of three independent experiments is shown. *, P < 0.05, compared with control.


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DISCUSSION
 
The present study shows remarkable acyl chain-dependent differences in the effects of LPC on neutrophils in vitro. Among the major LPC species in human plasma, those with an unsaturated acyl chain induced a long-lasting superoxide generation over a wide concentration range of 5–200 µM. In contrast, the saturated species, particularly LPC16:0, induced clearly less superoxide production and within a much narrower concentration range, i.e., 5–10 µM. A mixture of LPCs, mimicking the plasma LPC composition, activated superoxide production similarly to the unsaturated species. At higher concentrations, the saturated species increased the permeability of the plasma membrane, whereas the unsaturated ones had little effect.

Other lysophospholipids with the same acyl chain but a different head group were much less effective than LPC18:1 in activation of neutrophil NADPH oxidase. It is interesting that SPC18:1, a lysosphingolipid with the same acyl chain and head group structure as LPC18:1, induced considerable superoxide generation but only at concentrations, which were about two orders of magnitude higher than occur in plasma. LPC was thus the only lysolipid, which activated neutrophil superoxide production at concentrations prevailing in plasma.

Compared with neutrophil activation elicited by activation of the formylpeptide receptor, LPC acted much more slowly, i.e., after a significant lag, and produced more persistent superoxide production. Further, a relatively large number of LPC molecules were required for the activation, i.e., approximately 4 x 109/cell. This suggests that the response was not mediated by interaction with a (high-affinity) receptor. The previous publications, proposing a high-affinity interaction of LPC with the G protein-coupled receptors G2A (GPR132) and GPR4, have been formally retracted recently [29 , 30 ], and currently, no proven receptors for LPC are known. A recent study showed that LPC and other lysophospholipids bearing various head groups mobilized latent G2A within secretory vesicles to the surface of neutrophils and resulted in G2A receptor/G{alpha}i/PLC signaling for Ca2+ flux [31 ]. This rapid response was similar to the Ca2+ influx observed with the various LPC species in the present study. The wide variety of lysophospholipids triggering the Ca2+ influx and its rapid kinetics suggests that the activation of NADPH oxidized by LPC is mediated at least partially by different molecular mechanism.

Regarding the mechanisms of LPC-induced activation of the NADPH oxidase, it has been demonstrated that exogenous LPC incorporates into the plasma membrane rapidly and slowly moves to the inner leaflet [32 ]. The transbilayer movement of lysophospholipids is slow (t1/2>12 h) in phospholipid membranes but is accelerated by membrane proteins [32 ]. As albumin has been shown to remove LPC from the outer but not from the inner leaflet of the plasma membrane [32 ], the observation that albumin inhibited LPC-induced oxygen burst significantly, even when added 4–8 min after LPC, implies that transbilayer movement of LPC is necessary for the initiation of the mechanisms, which lead to activation of NADPH oxidase.

LPC incorporation may influence functions of integral membrane proteins by changing, e.g., the bilayer thickness and/or leaflet curvature balance [33 , 34 ]. Already 2–3 mol% of LPC has been shown to change the order parameter of spin-labeled FA in membranes and produce aberrant electrophysiological effects in myocardial cells [35 , 36 ]. LPC could also affect membrane protein function by modulating their interactions with membrane lipids and causing conformational changes in their membrane-spanning domains [37 ]. A possible example of such interaction of LPC with membrane proteins is the highly specific activation of the H+-ATPase activity in the plant cell plasma membrane by long-chain LPCs [38 ], and LPC was also shown recently to activate the canonical transient receptor potential-5 Ca2+ channel, independently of G-protein signaling [39 ]. In these studies, LPC mixtures or LPC16:0 were used, and the effective concentrations were in the range of 10–20 µM.

Considering the differential activation of NADPH oxidase by various LPC species, it is unlikely that it would be explained barely by their different critical micelle concentrations (CMC). The CMC values for different saturated LPC species decrease systematically with increasing hydrophobicity, to ~7 µM for LPC16:0 [40 ]. Addition of a double bond to a saturated acyl chain decreases hydrophobicity nearly equally as the removal of two methylene units [41 ], and thus, the CMC of, e.g., LPC18:1 should be similar to that of LPC16:0. We confirmed this by fluorescence shift by using diphenylhexatriene as a probe (unpublished results). As these LPC species activated NADPH oxidase to a different extent, the activation cannot be explained by simply the concentration of the monomeric lysolipid.

Prior studies have demonstrated activation of various kinase pathways by LPC, including protein kinase C (PKC) [13 ] and PI-3K{gamma}/Jak2/MEK-1/ERK1/2 cascade [42 ] in endothelial cells, PI-3K in neutrophils [18 ], p38 and p42/44 MAPKs in monocytic THP-1 cells [43 ], and PLC{gamma}-1, PKC, tyrosine kinase-Ras, and p42/p44 in mesangial cells [44 ]. It is interesting that the activation of PKC and RhoA signals in endothelial cells [13 ] began at 5 min after addition of LPC, remained elevated for 15 min, and decreased toward baseline by 60 min. This resembles the time course of activation of NADPH oxidase by LPC in the present study.

Under normal conditions, plasma albumin binds most of LPC and thus maintains the level of "free" LPC too low to activate neutrophils. Another plasma protein capable of scavenging LPC is {alpha}1-acid glycoprotein [11 ]. Considering the possible pathophysiological significance of LPC as an activator of neutrophil NADPH oxidase, the potential concentration of nonprotein-bound LPC in plasma and tissues is of critical importance. We found that fivefold molar excess of FA impaired LPC scavenging significantly by albumin. LPC level increases in inflammation and ischemia and can rise, e.g., to 200 µM in the lymph of ischemic dog hearts [45 ]. As the level of nonalbumin-bound, FFA also increases in ischemia and inflammation [46 ], it seems plausible that the level of free LPC becomes high enough (~5 µM) to activate superoxide production in neutrophils. This is particularly relevant concerning extravasated neutrophils in inflamed and ischemic tissues, as the concentration of the binding proteins is lower in interstitial fluid than plasma, and LPC is formed locally. A further clinical situation, where the LPC-induced superoxide production should be considered, is blood banking, where the storage of whole blood and cellular blood products has been shown to result in considerable accumulation of LPC [8 , 9 ].


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ACKNOWLEDGEMENTS
 
We are grateful to professor Gabor Tigyi for constructive criticism of an earlier draft of the manuscript. The skilled technical assistance of Ms. Tarja Grundström, Teija Kupari, and Sisko Lehmonen is recognized.

Received May 9, 2007; revised July 28, 2007; accepted August 24, 2007.


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