,
* Bonfils Blood Center and Departments of
Pediatrics and
Surgery, University of Colorado School of Medicine, Denver; and
Department of Laboratory Medicine, National Jewish Center for Allergy and Respiratory Medicine, Denver, Colorado
Correspondence: Christopher C. Silliman, M.D., Ph.D., Associate Medical Director, Bonfils Blood Center, 717 Yosemite Circle, Denver, CO 80230. E-mail: Christopher.Silliman{at}uchsc.edu
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Key Words: adhesion • chemotaxis • morphology
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In previous studies, we have demonstrated the generation of an effective PMN priming activity during routine storage of cellular blood components [16 , 17 ]. This activity has been identified by gas chromatography/mass spectroscopy as a mixture of lysophosphatidylcholines (lyso-PCs) in micromolar concentrations [16 ]. Moreover, we have implicated these lipids in the pathogenesis of transfusion-related acute lung injury (TRALI) in humans and have demonstrated that lyso-PCs are etiologic in a two-event animal model of PMN-mediated TRALI [18 , 19 ]. However, a controversy exists regarding the activity of lyso-PCs, as a number of investigators have asserted that lyso-PCs are inactive with respect to leukocytes and platelets [20 21 22 23 24 ]. We hypothesize that lyso-PCs directly prime the PMN oxidase and stimulate multiple PMN functions through signaling mechanisms that require activation of a receptor linked to a pertussis toxin-sensitive G-protein and increases in cytosolic Ca2+.
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Lyso-PC preparation
The individual lyso-PCs and those used in the mixture were solubilized in 1.25–5% essentially fatty acid-free, globulin-free, human albumin with 3-min pulses using a bath sonicator, model W-220F (Heat Systems–Ultrasonics, Plainview, NY) set at 30% maximal voltage. To determine the role of albumin as a lipid carrier, the lyso-PC mix was dissolved in 1.25% albumin, 95% ethanol, or 0.9% saline for injection, USP. The lyso-PC mixture contained purified, individual lyso-PCs in the following molar ratios as previously published [16
]: 1-O-palmitoyl:24; 1-O-oleoyl:10; 1-O-stearoyl:10; 1-O-hexadecyl (C16) lyso-PAF:0.65; and 1-O-octadecyl (C18) lyso-PAF:0.35. Other purified lyso-PCs, including 1-O-lauroyl, 1-O-heptadecanoyl, and 1-O-myristoyl, were obtained to test their ability to prime the PMN oxidase.
PMN isolation and oxidase priming
PMNs were isolated from whole blood drawn from healthy donors under the auspices of a protocol approved by the Colorado Multiple Institutional Review Board at the University of Colorado School of Medicine (Denver). The isolation used standard techniques including dextran sedimentation, ficoll-hypaque gradient centrifugation, and hypotonic lysis of contaminating red blood cells [16
]. PMN priming assays were completed as follows: PMNs (3.75x105) were incubated with vehicle controls (saline, 1.25–5% albumin or 1% ethanol), lyso-PCs (0.045–25 µM), or 2 µM platelet-activating factor (PAF) for 5 min at 37°C. The lyso-PCs and the PAF were dissolved in saline, albumin, or ethanol. After the 5-min incubation, the PMNs were activated with 1 µM fMLP or 200 ng/ml phorbol myristate acetate (PMA), and the maximal rate of superoxide anion was measured as the reduction of cytochrome c at 550 nm as described [25
]. Priming activity was measured as the augmentation of the maximal rate of O2- in response to fMLP or to PMA.
Cellular association of 3H–C18 lyso-PAF and 7-nitrobenz-2-oxa-1,3'-diazol-4-yl (NBD)-labeled 1-O-lauroyl lyso-PC
Whole blood (20 ml) was obtained from healthy donors and mixed for 5 min at 37°C, and 10 ml was incubated for 15 min with 14.5 µM lyso-PCs with 2 nM 3H–C18 lyso-PAF tracer bound to albumin. The other 10 ml whole blood underwent identical separation, and the cells were used to obtain cell counts using a Coulter blood Plus IV cell counter (Beckman Coulter, Houston, TX). Following incubation, the blood was spun at 3000 g for 5 min to isolate platelet-rich plasma, which was separated from the red cells and the leukocytes, and the platelets were isolated from the plasma by centrifugation [26
, 27
]. The remaining cells underwent dextran sedimentation, which isolated the majority of the red blood cells from the leukocytes, and the supernatant was separated into mononuclear and polymorphonuclear leukocytes by ficoll-hypaque gradient centrifugation. All samples were centrifuged to yield cellular pellets, and the numbers of counts were determined using a tritium scintillation counter. For the leukocytes and platelets, all of the cells were counted and in the case of the leukocytes, 1.0 x 107 mononuclear cells and 2.5 x 107 polymorphonuclear cells. For the red blood cells, 5 x 107 cells were counted, and the number of counts was multiplied to represent the total number of red blood cells in 10 ml whole blood. Isolated PMNs were incubated with NBD-labeled 1-O-dodecanoyl-sn-glycero-PC (10 pM–100 µM) for 60 min at 4°C, fixed with 4% paraformaldehyde, and examined by flow cytometry. Controls consisted of PMNs incubated with unlabeled 1-O-lauroyl lyso-PC at the two highest concentrations used and free NBD. These experiments were repeated three times on PMNs isolated from healthy donors.
Inhibition of the PAF receptor
PMNs were incubated with 400 µM WEB 2170, 5 µM A-79981.0, or 10 µM WEB 2347 PAF receptor antagonists for 5 min at 37°C with gentle agitation. These PMNs were then used in priming assays with 0.45–14.5 µM lyso-PCs or 2 µM PAF as described previously [16
]. These concentrations were determined to have maximal inhibitory capacity by performing the concentration: response relationship for each of the compounds on PAF priming of the fMLP-activated respiratory burst in isolated PMNs (results not shown).
Changes in cytosolic Ca2+ concentration
Isolated PMNs (2.5x107) were loaded with indo-1 and washed, and the changes in cytosolic Ca2+ for 2 x 106 PMNs were measured at 37°C over real time as previously reported [25
, 28
].
Pertussis toxin inhibition
Isolated PMNs were exposed to 2 µg/ml pertussis toxin for 2 h at 37°C and 7.5% CO2 with gentle rocking. Following incubation, the PMNs were primed with lyso-PCs, PAF, or albumin control and were activated with PMA or fMLP, and the maximal amount of superoxide anion was measured as described previously. In addition, the PAF-, fMLP-, and lyso-PC-mediated changes in cytosolic Ca2+ were measured over real time as described previously [25
, 28
].
MAP kinase activation and changes in tyrosine and serine phosphorylation
Isolated PMNs (1.25x106) were exposed to 4.5–14.5 µM lyso-PC for 15 s–5 min, the PMNs were lysed using Laemmli sample buffer, and the proteins were separated using 5–15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), as described previously [29
]. The proteins were transferred to nitrocellulose and incubated with a rabbit polyclonal antibody to phosphorylated (Tyr180/Thr182) p38 MAP kinase or a rabbit antibody to phosphorylated (Thr202/Tyr204) p42/44 MAP kinase or to a mAb to phosphotyrosine. PAF (2 µM) was used as a positive control, and 10 µM 1,2 diolein, a diacylglycerol, was used as a negative control. In addition, control immunoblots for total p38 and p42/44 MAP kinase were completed, which demonstrated an identical band of immunoreactivity for all treatment groups (results not shown).
PMN adhesion to fibrinogen-coated microtiter plates
Fibrinogen (50 µl, 1 mg/ml) was added to individual wells of a 96-well microtiter plate and was allowed to dry overnight [30
, 31
]. Freshly isolated PMNs (1x106) were placed into each well and allowed to settle for 30 min. The settled PMNs were then stimulated with buffer control, 200 ng/ml PMA, 1 µM fMLP, 2 µM PAF, or varying concentrations of the mixture of lyso-PCs (0.045 µM–15 µM) and were allowed to incubate for 30 min at 37°C. Following incubation, the plates were centrifuged inverted for 5 min at 200 g, the supernatants were discarded, and 0.1% Triton X was added to each well. From these lysates, the percentage of adherent PMNs was determined by measurement of the cellular elastase from firmly adherent PMNs divided by the cellular elastase from the total number of PMNs added to each well. The elastase was measured spectrophotometrically as described previously [32
]. It is important to note that this assay may underestimate the number of firmly adherent PMNs, as do all assays that use granule constituents as a marker of PMN presence including myeloperoxidase (MPO). To authenticate the results of this assay, we performed analogous experiments using 51Cr-loaded PMNs as described previously [32
]. Briefly, PMNs were incubated with 1 µCi 51Cr/2.5 x 107 PMNs for 30 min at 37°C. The PMNs were washed, resuspended in warmKrebs-Ringers-phosphate with 2% dextrose, ph 7.35 (KRPD), and incubated in fibrinogen-coated plates exactly as described above. PMN adherence was determined by using Triton X lysis of the identical number of 51Cr-loaded cells not placed into the wells and calculating the percentage of counts from lysates of the various wells from the fibrinogen-coated plates as previously reported [32
].
Measurement of CD11b and fMLPr surface expression
The surface expression of the ß2-integrin subunit CD11b and the fMLPr was measured using mAb to CD11b and the fMLPr and flow cytometry, as previously reported [25
].
PMN chemotaxis
Directed chemotaxis of isolated PMNs was determined in a Boyden chamber as described previously [33
]. Directed PMN chemotaxis to differing concentrations of lyso-PCs was compared with directed PMN migration to zymosan-activated, normal human serum (positive control) and to Hanks’ buffered saline with 2% albumin, pH 7.4 (negative control). Results were expressed as the distance migrated, in microns, of the leading front of PMNs [33
].
Digital microscopy
Isolated PMNs were incubated with albumin or lyso-PCs (1.45–14.5 µM) for 1–10 min, fixed with 4% paraformaldehyde, and smeared onto slides. All manipulations with these slides, unless otherwise indicated, occurred at room temperature. Slides were washed three times with phosphate-buffered saline (PBS), pH 7.4, for 10 min, and the PMNs were porated with 30% acetone–70% methanol for 3 min at -20°C. After washing with PBS for 10 min, the slides were stained for 60 min with bis-benzimide and wheat germ agglutinin (WGA) and then washed, mounted in antiquenching media, and examined for lyso-PC-induced changes in cellular morphology. PMNs from four healthy donors were treated and processed identically and analyzed as a replicate.
Determination of elastase, lactoferrin (LF), and MPO release in isolated PMNs
PMNs (1.25x106) were warmed to 37°C in a shaking water bath and treated with 5 µM cytochalasin B or buffer control for 5 min. The PMNs were then incubated for 5 min with buffer, 4.5 µM lyso-PCs, or 2 µM PAF and then activated with buffer, 1 µM fMLP, or 200 ng/ml PMA. After a 5-min reaction time, the PMNs were pelleted, and the supernatants were removed. These supernatants were used to determine the amount of elastase, MPO, and LF released. Thus, the measurements of released proteases were performed on the same samples with the same number of cells under virtually identical conditions.
Elastase release was determined spectrophotometrically by the reduction of the specific elastase substrate methoxysuccinyl-alanyl-alanyl-prolyl-valyl p-nitroanilide (AAPVNA) at 405 nm in duplicate [32 ]. To ensure the reduction of AAPVNA was secondary to elastase, identical wells containing 5 µM of the specific elastase inhibitor methoxysuccinyl-alanyl-alanyl-prolyl-valyl chloromethyl ketone were run in conjunction with each treatment. MPO release was determined by the oxidation of o-dianisidine at 405 nm as described previously [34 ]. LF release was quantified by a modification of a competitive enzyme-linked immunosorbent assay as described previously [35 , 36 ]. Elastase, MPO, and LF release is reported as the percentage of total cellular elastase, MPO, and LF, as determined by 0.1% Triton X-paired treatment of an identical number of PMNs.
Cytosolic Ca2+ chelation with BAPTA-AM
PMNs were incubated with 50 µM BAPTA or dimethyl sulfoxide (DMSO) for 30 min at 37°C as described previously [25
, 29
]. Following incubation, the PMNs were washed and placed into warm KRPD buffer at the identical concentration (2.5x107 PMNs/ml). The BAPTA concentration was determined by measuring BAPTA inhibition of cytosolic Ca2+ flux over a range of concentrations [25
, 29
]. PMNs loaded with 50 µM BAPTA did not exhibit a cytosolic Ca2+ in response to 2 µM PAF or 1 µM fMLP, and PMNs loaded with lesser concentrations of BAPTA, 10–25 µM, exhibited an attenuated cytosolic Ca2+ flux in response to these agonists (results not shown) [29
].
Statistical analysis
The means, standard deviations, and SEM were calculated using standard techniques. Statistical differences among groups were determined by a paired or an independent ANOVA followed by a Tukey post hoc analysis for multiple comparisons. Statistical significance was determined at the P< 0.05 level.
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Lyso-PC priming of the fMLP-activated respiratory burst was evaluated over a range of concentrations from 0.01 to 25 µM (Table 1 ). As compared with albumin-treated controls, the priming activity of the lyso-PC mix, found in stored blood, became statistically significant at 0.45 µM and reached a relative maximum at 14.5 µM. This priming activity at 14.5 µM was even greater than PAF-primed positive controls (lyso-PCs: 15.3±2.0 vs. 2 µM; PAF: 12.1±1.6 nmol O2-/3.75x105 PMNs/min, n=8, P<0.05). At all concentrations of the lyso-PC mix used (0.01–25 µM), there was no evidence of PMN lysis, and cell viability following lyso-PC incubation was >99% by trypan blue exclusion, similar to controls.
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Table 1. Concentration-Dependent Lyso-PC Priming of the fMLP-Activated Respiratory Burst and Their Effects on PMN Viability
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In addition, to determine if the solvent for the priming agent affected PMN function, PAF and the lyso-PC mix were dissolved in higher concentrations of albumin, 2.5% and 5%, 95% ethanol, and aqueous solutions without albumin. Higher albumin concentrations augmented PAF priming but did not affect lyso-PC priming as compared with lipid-priming agents solubilized in the 1.25% albumin (Table 2 ). Dissolving the lipid stocks in ethanol, final concentrations of 0.1–1%, inhibited fMLP activation of the oxidase by 60 ± 22% to 67 ± 20%, respectively, and no lipid priming was observed (Table 2) . When PAF or lyso-PCs were added to aqueous solutions without albumin, the solutions were turbid and inhibited the fMLP activation of the respiratory burst at all concentrations tested. Lipids dissolved in albumin did not affect viability of PMNs; however, when lyso-PCs were solubilized in ethanol or added to aqueous solutions without a protein carrier, PMN viability was compromised (Table 2) . Lyso-PC mediated changes in cytosolic Ca2+
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Table 2. The Effects on Lyso-PC Priming of the fMLP-Activated Respiratory by Different Solvents
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Figure 1. Lyso-PC (LPC)-mediated changes in cytosolic Ca2+ concentration. The elicited changes in cytosolic Ca2+ concentration of indo-1-loaded PMNs are shown in response to (A) the lyso-PC mixture from stored blood, (B) 1-O-palmitoyl lyso-PC, (C) 1-O-stearoyl lyso-PC, (D) 1-O-oleoyl lyso-PC, (E) C16 lyso-PAF, and (F) C18 lyso-PAF. The various lipids were injected into the cuvette at 20 s. This figure is a representation of three separate experiments, which used different donors for each lyso-PC compound and each concentration.
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As PAF and lyso-PCs elicited rapid changes in cytosolic Ca2+, and previous reports from this laboratory have demonstrated that PAF receptor antagonists may inhibit lyso-PC priming activity, we investigated if PAF receptor desensitization would affect the lyso-PC-mediated increase in cytosolic Ca2+ and the converse. In addition, we explored the effects of the lyso-PC mix on the cytosolic Ca2+ flux in response to the individual lyso-PC compounds as well as the effects of the individual lyso-PCs on the cytosolic Ca2+ flux of the lyso-PC mix and other individual lyso-PC compounds. As demonstrated in Figure 2 , PAF pretreatment had little effect on the Ca2+ flux of lyso-PCs, and lyso-PCs had little effect on the increase in cytosolic Ca2+ to PAF. As expected, pretreatment of the lyso-PC mix totally abrogated any Ca2+ flux in response to the 1-O-stearoyl lyso-PC, 1-O-palmitoyl lyso-PC, or C16 lyso-PAF (Fig. 2) . Pretreatment with the individual compounds 1-O-stearoyl, 1-O-palmitoyl, or C16 lyso-PAF abrogated the Ca2+ flux in response to the lyso-PC mix or to the individual lyso-PC compounds tested (Fig. 2 ; results not shown).
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Figure 2. Receptor desensitization. The changes in cytosolic Ca2+ were determined in indo-1-loaded PMNs in a dual-wavelength spectrofluorimeter over real time. PAF (2 µM) and lyso-PCs (4.5 µM) elicited changes in cytosolic Ca2+ concentration despite pretreatment with one another (A and B). Pretreatment of PMNs with the lyso-PC mix (4.5 µM) inhibited the Ca2+ response of C16 lyso-PAF (C). Pretreatment with 1 µM 1-O-palmitoyl (D), 1 µM 1-O-stearoyl (E), or 1 µM C16 lyso-PAF (F) inhibited the Ca2+ response to the lyso-PC (L-PC) mix or C16 lyso-PAF. The figure represents the data from at least three different experiments using different, healthy donors for each lyso-PC or PAF treatment.
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Figure 3. Cellular association of NBD-labeled 1-O-lauroyl lyso-PC. Albumin (Alb)-treated PMNs were incubated with differing concentrations of NBD-labeled 1-O-lauroyl lyso-PC for 60 min at 4°C. The PMNs were fixed and examined by digital microscopy (A, upper) and flow cytometry (A, lower) and digital microscopy (B). (A) The green cellular fluorescence, visualized by digital microscopy, which is directly related to the concentration of labeled lyso-PC added as quantitated by flow cytometry in the lower portion of the panel. (B) The results of PMNs incubated for 60 min with 10-4 M NBD-labeled 1-O-lauroyl lyso-PC alone (left) or the identical PMNs incubated concomitantly with 10-4 M-unlabeled 1-O-lauroyl lyso-PC + 10-4 M NBD-labeled 1-O-lauroyl lyso-PC. This figure is representative of three separate experiments using healthy donors.
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Table 3. Plasma and Cellular Association of 3H-C16 Lyso-PAF Incubations of Whole Blood
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Table 4. Lipid Receptor and Pertussis Toxin Inhibition of Lyso-PC Priming
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As pertussis toxin inhibited lyso-PC priming and fMLP activation of the oxidase, we investigated its effects on the lyso-PC-mediated increase in cytosolic Ca2+ as compared with fMLP. Pertussis toxin pretreatment inhibited the initial rise and duration of the fMLP, and lyso-PC elicited changes in cytosolic Ca2+ concentration (Fig. 4A and 4B ). In contrast, pertussis toxin inhibited the duration of the PAF-mediated change in cytosolic Ca2+ but had little effect on the rate of rise or the peak level of cytosolic Ca2+ (Fig. 4C) .
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Figure 4. Pertussis toxin (PTX) inhibition of changes in cytosolic Ca2+. The PMNs were treated for 2 h with 2 µg/ml pertussis toxin at 37°C, 5% CO2, and were loaded with 5 µM indo-1 for 5 min, and the changes in cytosolic Ca2+ were measured over real time (sec) in a dual-wavelength spectrofluorimeter. The effects of pertussis toxin on the rise in cytosolic Ca2+ are shown in response to 1 µM fMLP (A), 4.5 µM lyso-PCs (B), and 2 µM PAF (C). The figure is representative of three identical experiments.
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Figure 5. Lyso-PC-mediated changes in protein serine phosphorylation. PMNs were incubated with lyso-PCs for 0.5–1 min (L.5 and L1) at 37°C, the cells were lysed, the proteins were separated by SDS-PAGE, and the proteins were transferred to nitrocellulose and immunoblotted with an antibody to phosphoserine. A serine-phosphorylated protein of 68 kD was found in PMNs stimulated with lyso-PCs. This phosphorylation was inhibited with BAPTA (50 µM) pretreatment. The figure is representative of three separate experiments, and the numbers on the left are the locations of the molecular weight markers. C, albumin-treated control PMNs.
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Figure 6. Lyso-PC-mediated PMN adhesion to fibrinogen-coated plates. The figure represents the total percentage of adherent cells as measured by total cellular elastase in Triton X-treated cells as a function of treatment groups. The data are presented as the mean ± SEM. The controls consist of fibrinogen-coated wells treated with 1.25% albumin and wells incubated with PMNs treated with albumin. Three different positive controls were used (hatched bars): 200 ng/ml PMA, 2 µM PAF, and 1 µM fMLP, for comparison with PMNs treated with lyso-PCs (solid bars) over a range of concentrations, 0.045–14.5 µM. The figure represents a sample size of nine; *, statistical differences (P<0.05) as compared with the albumin-treated controls.
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Figure 7. Lyso-PC-mediated changes in CD11b surface expression. The figure represents the changes in CD11b surface expression as a function of the concentration of the lyso-PC mixture, and the data are expressed as the mean ± SEM. The open bar represents the albumin-treated control PMNs, the solid bars depict the effects of differing concentrations of lyso-PCs (LPC) on CD11b surface expression, and the hatched bars demonstrate the PMNs incubated with positive controls: 1 µM fMLP and 2 µM PAF. The figure represents the data from eight separate experiments; *, statistical differences as compared with albumin-treated controls (P<0.05).
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Table 5. Lyso-PC-Mediated Chemotaxis of Isolated PMNs
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Figure 8. Lyso-PC-mediated changes in PMN morphology. PMNs were incubated with albumin (control) lyso-PCs (1.45–14.5 µM) and PAF (2 µM) for 3–15 min at 37°C. Immediately at the end of the incubation period, the PMNs were fixed with paraformaldehyde and then doubly stained with a blue nuclear stain (bis-benzamide) and a green membrane stain (WGA). The PMNs were examined at 100x (original magnification), and the bar in the first control panel is 5 µm (original magnification). This figure is representative of five separate experiments.
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Figure 9. Lyso-PC-mediated release of PMN granule constituents. (A–C) Elastase, MPO, and LF release from isolated PMNs as a function of treatment group, and the data are represented as the mean ± SEM. The open bars are DMSO-pretreated controls (pre-TX), the solid bars represent PMNs pretreated with 5 µM cytochalasin B for 30 min at 37°C, and the hatched bars represent PMNs primed with 4.5 µM lyso-PC (LPC) mix or 2 µM PAF for 5 min followed by activation with 1 µM fMLP. *, Statistical significance as compared with albumin-treated, control PMNs (P<0.05). +, Statistical significance (P<0.05) in cytochalasin B-pretreated PMNs as compared with paired DMSO-pretreated PMNs. #, Statistical significance (P<0.05) of lyso-PC- or PAF-pretreated PMNs activated with fMLP as compared with lyso-PC-, PAF-, or fMLP-treated PMNs. The figure represents the results of seven separate experiments.
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LF release from specific granules was also quantified, and we demonstrated that lyso-PC elicited a direct, reproducible release of LF from PMNs, similar to PAF-, fMLP-, and PMA-mediated LF release and significantly greater than buffer-treated controls (n=7, P<0.05; Fig. 9C ). Pretreatment with cytochalasin B significantly enhanced the amount of LF release to fMLP, PAF, and PMA. In addition, lyso-PC and PAF pretreatment did not enhance the amount of fMLP-induced LF release.
BAPTA inhibition of lyso-PC activity
As changes in cytosolic Ca2+ were elicited by lyso-PC priming of the PMN oxidase, we examined the effects of cytosolic Ca2+ chelation with BAPTA (50 µM), which buffers cytosolic Ca2+, rendering it biologically unavailable, regardless of the Ca2+ source [42
]. PMNs were pretreated with 50 µM BAPTA or DMSO, primed with 1.25% albumin, lyso-PCs (4.5 µM or 14.5 µM), or PAF (2 µM) for 5 min, and then activated with 200 ng/ml PMA. BAPTA inhibited the lyso-PC priming by 85 ± 6% and 88 ± 7%, respectively, and inhibited PAF priming by 67 ± 12%. In addition, BAPTA chelation inhibited lyso-PC-mediated increases in CD11b expression by 52 ± 11% and changes in PMN morphology (results not shown).
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450 nM must be used for PMN stimulation [20
21
22
23
24
, 43
]. Moreover, the addition of these compounds to fresh, human plasma resulted in priming of the PMN oxidase by 1.7 ± 0.2-fold as compared with albumin-pretreated controls [16
]. Recent studies have demonstrated that lyso-PCs not only rapidly prime human PMNs but also rapidly prime PMNs from healthy rats [19
]. Thus, lyso-PCs, solubilized in albumin solutions, effectively prime human and rodent PMNs at physiologically relevant concentrations in the context of blood transfusions [16
]. The initial observations that a mixture of lyso-PCs primes the reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase have been extended by documentation of a range of priming concentrations (three logs); by demonstration of the activity of individual lyso-PC compounds and identification of the requirement of albumin as a carrier for PMN stimulation, identical to human plasma; and by explanation of conflicting reports that deemed lyso-PCs inactive as a result of the use of suboptimal concentrations or the deletion of the albumin carrier [16 , 19 20 21 22 23 24 , 43 44 45 46 47 ]. These experiments also demonstrated that PAF required an albumin carrier for activity, as reported previously, and that the activity of lyso-PCs was not affected by incubation with sPLA2, an enzyme that inactivates the activity of PAF [48 ]. With respect to the priming activity of the individual lyso-PC compounds, the 1-O-alkyl lyso-PAF moieties and the 1-O-stearoyl lyso-PC showed increasing augmentation of the fMLP-activated respiratory burst at concentrations greater than 4.5 µM, similar to the lyso-PC mixture. The other two acyl compounds, 1-O-palmitoyl and 1-O-oleoyl, did cause rapid priming of the NADPH oxidase, but their activity did not increase above the levels demonstrated at 4.5 µM. In addition, the concentrations of the lyso-PCs, the individual species and the mix, which induced changes in cytosolic Ca2+, parallel those concentrations that prime the NADPH oxidase. Thus, the lyso-PC concentrations, which elicited a rapid rise in cytosolic Ca2+, also primed the NADPH oxidase. It is also important to note that the highest concentrations of lyso-PCs used in these experiments, including the lyso-PC mix and the individual species, did not compromise PMN cellular integrity. Furthermore, the activity of the lyso-PC mixture was similar to the activity of the three most effective, individual lyso-PC compounds, 1-O-stearoyl lyso-PC, C16 lyso-PAF, and C18 lyso-PAF; however, the two lyso-PAF compounds comprise only 100–400 nM of the total concentration of the lyso-PC mixture at concentrations ranging from 4.5 to 15 µM, and the 1-O-stearoyl lyso-PC accounts for an additional 1–3.5 µM of the total. The 1-O-oleoyl and 1-O-palmitoyl lyso-PC compounds did display activity that should not be discounted, as there may be some additive or synergistic activity among the lyso-PCs that comprise the mixture and influence its relative activity, and further experimentation is required to elucidate the relative participation of the individual species that comprise the mixture.
Lyso-PCs demonstrated selective association with blood cells, mononuclear leukocytes, polymorphonuclear leukocytes, and platelets, which are affected by lipid stimuli. If such a cell membrane association were nonspecific, there should have been 3H–C18 lyso-PAF associated with red blood cells. Moreover, one would have expected that more of the total radioactive lyso-PC tracer would have become cell-associated, but virtually 97% was free in the plasma bound to an albumin carrier. A concentration-dependent membrane association of an active NBD-labeled 1-O-lauroyl lyso-PC provided supportive evidence of the existence of a lyso-PC receptor on the PMN surface [22 ]. In addition, receptor desensitization studies showed that when PMNs were desensitized to PAF, lyso-PCs could still cause an increase in cytosolic Ca2+, and when PMNs were desensitized to lyso-PCs, the PAF response was also not affected. Such results are not surprising, as we have previously shown that lyso-PCs could activate the oxidase of PAF-primed but not vehicle-treated PMNs, and PAF could activate the oxidase of lyso-PC-primed PMNs but not vehicle-treated controls [49 ]. Moreover, pertussis toxin dramatically affected lyso-PC-induced changes in cytosolic Ca2+ and priming of the oxidase, whereas pertussis toxin had little effect on PAF-mediated changes in cytosolic Ca2+ and PMN priming. Taken together with WEB 2347 data, these results suggest that PAF and lyso-PCs activate PMNs through disparate receptors, and the observed activity was not a result of contamination of the purified lipid by acetylated PAF analogs.
The cytosolic Ca2+ desensitization studies demonstrated that the individual lyso-PC compounds, 1-O-alkyl and 1-O-acyl, could inhibit the activity of the lyso-PC mix and other individual moieties. These results implicate a receptor that recognizes 1-O-alky and 1-O-acyl lyso-PC analogs, much like the PAF receptor and chemokine receptors [50 51 52 ]. In addition, pertussis toxin inhibition of the lyso-PC priming implicates a receptor-linked heterotrimeric G-protein in the signaling pathway. Downstream from the pertussis toxin-sensitive G-protein, lyso-PCs caused rapid, concentration-dependent increases in cytosolic Ca2+ concentration that were linked to an increase in serine phosphorylation of a 68-kD protein. Chelation of cytosolic Ca2+ in intact PMNs abrogated lyso-PC-mediated effects in PMNs, including priming the PMA-activated respiratory burst, increased surface expression of CD11b, changes in PMN morphology, and serine phosphorylation of a 68-kD protein. However, unlike most chemoattractants, lyso-PCs did not cause changes in whole-cell protein tyrosine phosphorylation or activation of MAP kinases.
The lyso-PC mixture directly caused changes in multiple PMN functions. At concentrations 0.45 µM, lyso-PCs elicited firm adhesion of PMNs to the RGD ligands inherent to fibrinogen, without significant differences among the effective lyso-PC concentrations (0.45–14.5 µM) [53
]. As the adhesion assay used subjects the adherent PMNs to 200 g, only firmly adherent PMNs remain attached to the Arg-Gly-Asp ligands via ß2integrins [32
]. Such a stringent assay does not invite comparisons with other adhesion assays that subject adherent PMNs to substantially less detachment force. The lyso-PC mixture also directly caused concentration-dependent increases in surface expression of CD11b and the fMLPr, similar to a number of PMN-priming agents [15
, 25
, 54
, 55
]. Moreover, the lyso-PC-mediated changes in chemotaxis were modest as compared with zymosan-activated serum but were statistically significant as compared with the albumin-treated controls. Furthermore, lyso-PC-induced changes in cellular morphology were similar to those of PAF, and such morphologic changes are typical of chemotactic agents that cause increases in F-actin [56
57
58
]. In addition, the lyso-PC mix also elicited rapid (5 min) degranulation of elastase, MPO, and LF, similar to other agonists including PAF. The lyso-PC mixture also augmented the amount of elastase and MPO released by fMLP; however, cytochalasin B pretreatment did not significantly affect lyso-PC-mediated degranulation, the significance of which is not entirely clear. Thus, lyso-PC treatment resulted in the direct release of the contents from azurophilic and specific granules and was able to augment the fMLP-mediated release of contents from primary granules.
Recent work has delineated the importance of lyso-PCs and other lysophospholipids in diverse organisms from yeast to humans [59 ]. A "subfamily of orphan receptors recognize lyso-PCs and other lysophospholipids and are G-protein-linked cellular receptors present on fibroblasts, ovarian cancer cells, and leukocytes" [59 ]. Although their roles in human disease are poorly characterized, there appears to be a linkage among lyso-PCs, their receptors, and autoimmunity [60 , 61 ]. This line of research is in its infancy, and much remains unknown at the present time; however, some years ago, a receptor that recognized two types of alkyl lyso-PAF, the C16 and C18, was localized on the surface of PMNs [22 , 59 ]. Although the presence of this receptor may provide an attractive candidate for the lyso-PC stimulation of PMNs, these studies were marred by the use of ethanol as the lipid solvent and lyso-PC concentrations not exceeding 100 nM [22 ]. Thus, it was of little surprise that the authors were unable to find lyso-PC-mediated changes in PMN function despite receptor occupancy [22 ]. Moreover, there are no data regarding the activation of PMNs through this receptor, its avidity for the 1-O-acyl lyso-PC compounds, nor its antagonism by PAF receptor antagonists [22 ].
In conclusion, lyso-PCs affect multiple PMN functions in a concentration-dependent manner. The relatively high concentrations used appear to have physiologic significance, because of the millimolar concentrations generated during the routine storage of blood [16 ]. Moreover, lyso-PCs have been reported to cause activation of vascular endothelium, an increase in adhesion molecule expression, and adherence of PMNs to these activated cells via a protein kinase C-dependent mechanism [62 63 64 65 ]. Thus, previous work has documented the activity of lyso-PCs in other primary cells or cell lines [62 63 64 65 ]. Recent findings from our laboratory have documented that lyso-PCs may serve as one of two insults in a two-event animal model of acute lung injury, and lyso-PCs have been implicated in clinical cases of transfusion-related, acute lung injury [18 , 19 ]. Thus, lyso-PCs may not only affect individual cells in vivo or in vitro, e.g., PMNs or vascular endothelium, but also have the capacity to influence the physiology of the lung.
Received April 10, 2002; revised November 21, 2002; accepted December 18, 2002.
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