Structurally similar oxidized phospholipids differentially regulate endothelial binding of monocytes and neutrophils

OxPAPC was prepared as described (12). POVPC and PGPC were prepared by total synthesis as described (12). Alternatively, a preparative method was used involving ozonolysis of PAPC (obtained from Avanti Polar Lipids or Sigma). This method is a modification of a previously published method (15). Briefly, 50 mg of PAPC in hexane were cooled to −78°C by using a dry ice-acetone bath. Oxygen containing 2% ozone was bubbled through the solution at a rate of 100 ml/min until the solution turned faint blue in color. The hexane was evaporated under argon, and the residue was redissolved in chloroform (4 ml) and treated with triphenylphosphine (50 mg) at room temperature. The completion of ozonide reduction (about 1–2 h) was monitored by TLC, and the crude product was loaded onto preparative TLC (20 × 20 cm, 250 μm thick) and developed with chloroform/methanol/acetic acid/water (75:45:12:6). TLC was viewed by using p-anisaldehyde as developing agent and the POVPC region (R_ƒ = 0.35) of the silica was removed. The product was recovered from silica by using chloroform/methanol/water (65:25:4). Individual phospholipids were purified by using HPLC/MS as described (12). Individual phospholipids were quantitated by flow injection analysis-electrospray ionization MS using dimyristoyl-phosphatidylcholine as an internal standard (16). The concentration of endotoxin in the phospholipid solutions was less than 30 pg/ml (determined by a chromogenic assay), which is 30 times less than that required to induce endothelial cells to bind leukocytes.

Human aortic endothelial cells (HAEC) were cultured as described (4). For determination of E-selectin, VCAM-1 and CS-1 expression HAEC were plated into 96-well dishes, exposed to activators, and then incubated with antibodies to E-selectin (BioDesign, New York), VCAM-1 (BioDesign), or CS-1 (a gift from Cytel, San Diego). Peroxidase-labeled second antibody was used and detected with o-phenylene-diamine (Sigma). For leukocyte binding studies endothelial cells were cultured in 48-well dishes and incubated with or without activators for 4 h at 37°C. The test medium was removed, and a suspension of human monocytes or polymorphonuclear neutrophils was added for 12–15 min, then nonadherent cells were removed. Bound monocytes or neutrophils were visually counted. Monocytes were obtained from a large pool of healthy donors by modification of the Recalde procedure as described (17). Neutrophils were isolated by previously described methods (18). Cell survival experiments were performed by using a propidium iodide assay (19).

For measurement of cAMP, HAEC were cultured in 6-well dishes, and two wells were used for each determination of cAMP. Cells were pretreated for 10 min with 0.5 mmol/liter isobutyl-1-methylxanthine in medium 199 containing 5% FBS. Agonists then were added to the cells in the same medium and cells treated for 1 hr; the incubation was stopped by rinsing in PBS containing 4 mmol/liter EDTA. The cells then were removed from the dish by scraping into the same buffer. The cell pellet after centrifugation was resuspended in boiling water containing EDTA. The suspension was sonicated, placed in boiling water for 3 min, and centrifuged at 13,000 × g for 2 min to remove coagulated macromolecules. The supernatant was used to determine cAMP levels with a cAMP kit (Amersham Pharmacia, RPA 542).

HAEC were transfected with p(κB)₄-luciferase, which contains four κB sites cloned upstream of the minimal simian virus 40 promoter (20), by using the Superfect reagent (Qiagen, Chatsworth, CA) according to the manufacturer’s instructions. pSV-β- galactosidase (Promega) was used as a control plasmid to normalize the luciferase activity. Cells were incubated for 4 h at 37°C with the indicated agents 24 h posttransfection. In establishing the conditions for this method we used a green fluorescent protein (GFP) reporter and established conditions to routinely obtain a 15% transfection efficiency. In several experiments we also included a combination of GFP plasmid and plasmid of interest to be certain that transfection of several plasmids did not affect efficiency.

The cystic fibrosis transmembrane conductance regulator (CFTR)-containing plasmid (pACF23) obtained from J. Riordan (Mayo Clinic, Scottsdale, AZ) was linearized and transcribed in vitro into capped cRNA with SP6 RNA polymerase (Ambion, Austin, TX). The CFTR cRNA (40 ng) was injected into stage IV-V Xenopus laevis oocytes, which had been isolated by enzymatic digestion (2 mg/ml collagenase), then oocytes were incubated for 24–48 h at 19°C to allow for protein synthesis and transport of the protein to the cell membrane. Whole-oocyte currents were recorded at room temperature with the two-electrode voltage-clamp technique (21) using a Dagan Instruments (Minneapolis, MN) CA-1 oocyte clamp amplifier, a TL-1 DMA interface for data acquisition, and pclamp software (Axon Instruments, Foster City, CA). Phospholipids were dried under a stream of nitrogen and resuspended into the bath solution by vortexing before addition to clamped oocytes.

HAEC were incubated with oxidized phospholipids and/or lipopolysaccharide (LPS) as for cell adhesion experiments, and total RNA was isolated from cells by using the guanidine thiocyanate/phenol method. Northern blot analysis using E-selectin cDNA was performed as described (7). 36B4 or 28S cDNA were used to control for RNA loading.

Nuclear runoff experiments were performed essentially as described (22). For these studies cells were untreated or treated for 1 h with LPS (2 ng/ml), OxPAPC (100 μg/ml), or LPS plus OxPAPC. Briefly, nuclear pellets from these cells in a 75-cm² culture flask were resuspended in 90 μl of nuclear storage buffer (50 mmol/liter Tris⋅HCl, pH 8.3/40% glycerol/0.1 mmol/liter EDTA/0.1 mmol/liter DTT). To the nuclear preparation 100 μl of 2× reaction buffer (10 mmol/liter Tris⋅HCl, pH 7.5/5 mmol/liter MgCl₂/0.3 mol/liter KCl/5 mmol/liter DTT/1 mmol/liter each of ATP, GTP, and CTP/10 μl [α-³²P]UTP (3,000 Ci/mmol) were added and incubated at room temperature for 20 min. DNA was digested by 1 μl of 20,000 units/ml RNase-free DNase at room temperature for 5 min, then 10 μl of yeast tRNA (10 mg/ml) was added. The labeled RNA was isolated by using Trizol. RNA was redissolved in 500 μl of 20 mmol/liter Tris⋅HCl, pH 7.9, and 20 mmol/liter EDTA. Membranes were prepared by applying 2 μg of E-selectin cDNA or α-tubulin cDNA per slot and baking at 80°C for 2 h. Membranes were prehybridized at 65°C for 2 h and then hybridized with the isolated labeled RNA at 65°C for 48 h. Membranes were washed in 2× SSC and 0.1% SDS at room temperature for 15 min, in 2× SSC, 0.1% SDS and 10 μg/ml RNase A at 37°C for 20 min and in 0.2× SSC and 0.1% SDS at 65°C for 10 min before exposing to films.

Treatment of HAEC for 4 h with POVPC dose-dependently induced monocyte, but not neutrophil, adhesion without increasing the surface expression of VCAM-1 or E-selectin (Fig. 1A). However, POVPC caused an increase in surface deposition of CS-1-containing fibronectin, an alternative monocyte ligand. In contrast, treatment of endothelial cells for 4 h with PGPC increased both monocyte and neutrophil adhesion. PGPC also dose-dependently increased expression of E-selectin and VCAM-1 but had no effect on expression of CS-1 fibronectin (Fig. 1A). OxPAPC (150 μg/ml), which contains approximately 5 μg of POVPC and 5 μg of PGPC, showed a pattern similar to POVPC, inducing monocyte-specific binding and CS-1 fibronectin surface expression (Fig. 1B). Unoxidized PAPC at the same concentration neither increased leukocyte binding nor adhesion molecule expression (not shown). Control experiments showed that OxPAPC up to a concentration of 300 μg/ml was not toxic to HAEC (data not shown). The ability of POVPC, PGPC, and OxPAPC to stimulate leukocyte endothelial interactions did not depend on the concentration of serum in the medium, because serum concentrations from 0 to 15% gave similar results (data not shown).

Based on the above data, we hypothesized that POVPC and PGPC acted on two different receptors. To test this hypothesis, two-electrode ramped voltage-clamp recordings were performed in X. laevis oocytes perfused with PGPC or POVPC. PGPC activated a chloride ion conductance (Fig. 2A). This effect was reversible upon washout of the ligand and could be desensitized by prolonged treatment with PGPC (Fig. 2D), both characteristic of receptor-mediated events. In contrast, POVPC did not directly activate chloride currents in Xenopus oocytes (Fig. 2B). Previous work from our laboratory indicated that the effects of MM-LDL were mediated by a G-protein-coupled receptor (7) via a cAMP-dependent pathway (6). However, Xenopus oocytes do not endogenously express cAMP-activated ion channels (23), but can be made responsive to changes in cAMP by heterologous expression of the human CFTR, a cAMP-dependent chloride channel (24, 25). Xenopus oocytes were microinjected with cRNA encoding CFTR and incubated for 24–48 h, and then the effect of POVPC was examined. Functional expression of CFTR in oocytes was confirmed by using the cAMP-elevating agent forskolin (not shown). Only in oocytes heterologously expressing CFTR, a POVPC-inducible chloride current was observed (Fig. 2 B and C). The effect of POVPC was also reversible upon washout and could be homologously desensitized after an incubation for 5 min (data not shown). Conversely, PGPC did not activate a CFTR-mediated ion current, as evidenced by the similar activation pattern independent of CFTR-expression (Fig. 2A). Furthermore, the receptor for PGPC was found to be distinct from the high affinity lysophosphatidic acid (LPA) receptor (which is also present in Xenopus oocytes), because the action of LPA was not desensitized by treatment with PGPC (Fig. 2D). In addition, LPA receptor antagonists, which specifically block the effects of LPA (Fig. 2E), could not abrogate the PGPC-induced response (Fig. 2F). In addition, we examined the levels of cAMP in HAEC treated with oxidized phospholipids (Table 1). POVPC (5 μg/ml) but not PGPC (5 μg/ml) increased levels of cAMP by approximately 70% as compared with an approximate 300% increase seen with isoproteronol (5 μmol/liter). Thus, we conclude that different receptors are stimulated by PGPC and POVPC, the latter coupling to a cAMP-mediated pathway.

It has been shown by others that elevation of cAMP inhibits E-selectin expression (26–28), and we have demonstrated that MM-LDL and other cAMP-elevating agents inhibit LPS-induced neutrophil binding to HAEC (6, 7). We hypothesized that POVPC, because of its ability to increase cAMP, would inhibit agonist-induced E-selectin expression and neutrophil binding. Therefore we tested the effect of POVPC and OxPAPC on PGPC-induced E-selectin expression and neutrophil binding. POVPC inhibited neutrophil adhesion and E-selectin expression induced by PGPC (Fig. 3A), suggesting one mechanism whereby neutrophils may be excluded from MM-LDL- or OxPAPC- stimulated endothelium. POVPC, and to a greater extent OxPAPC, also dose-dependently inhibited LPS-induced neutrophil binding and E-selectin expression (Fig. 3B). Similar results were obtained when tumor necrosis factor was used as the stimulating agent (data not shown). Evidence for the presence of additional inhibitory lipids (in addition to POVPC) was obtained by testing HPLC fractions of OxPAPC that did not contain POVPC (data not shown).

To confirm that inhibition of E-selectin expression by OxPAPC and POVPC indeed depended on a protein kinase A (PKA)-mediated pathway, we examined whether H89, a specific inhibitor of cAMP-dependent PKA, could reverse the inhibitory effect. Pretreatment of HAEC with H89 (25 μmol/liter) for 1 h blocked the inhibitory activity of both OxPAPC and POVPC on LPS-induced E-selectin expression (Fig. 4). For these studies lower concentrations of POVPC and OxPAPC were used for proven nontoxic concentrations of H89. These data indicate that POVPC exerts its inhibitory activity via a cAMP/PKA-mediated pathway.

To further elucidate the mechanism of inhibition of E-selectin expression, the effects of POVPC (5 μg/ml) and OxPAPC (100 μg/ml) on LPS-induced E-selectin mRNA synthesis was examined. Northern blotting demonstrated that LPS-induced E-selectin mRNA levels were dramatically reduced by coincubation with POVPC (Fig. 5A) or OxPAPC (Fig. 5B). In addition, rates of transcription of E-selectin were examined by using nuclear runoff analysis (Fig. 5C). Transcription of E-selectin was increased by LPS and completely abrogated by coincubation with OxPAPC. NF-κB has been shown to play an important role in mediating E-selectin transcription (29–31). To determine the effects of POVPC and OxPAPC on NF-κB-mediated transcriptional activation, HAEC were transfected with p(κB)₄-luciferase, and the effects of POVPC and OxPAPC on LPS-induced reporter activation were determined. Addition of POVPC or OxPAPC stongly reduced LPS-induced NF-κB promoter activity to control levels (Fig. 6).