The selectin family of molecules (L-, P-, and E-selectin) mediates adhesive interactions between leukocytes and endothelial cells required for recruitment of leukocytes to inflammatory sites. Soluble E-selectin levels are elevated in inflammatory diseases and act to promote neutrophil β2-integrin–mediated adhesion by prolonging Ca2+ mobilization. Although soluble E-selectin alone was unable to initiate Ca2+ signaling, it allowed a novel “permissive” store-operative calcium entry (SOCE) following the initial platelet-activating factor (PAF)–induced release of Ca2+ from inositol 1,4,5-trisphosphate (IP3)–sensitive stores. This induction of permissive SOCE in response to soluble E-selectin and PAF was shown to act through a G protein-coupled receptor (GPCR) coupled to pertussis toxin-insensitive Gq/11. Furthermore, we demonstrated that permissive SOCE was mediated by canonical transient receptor potential channel (TRPC) due to its sensitivity to specific inhibition by MRS1845 and Gd3+ and that TRPC6 was the principal TRPC family member expressed by human neutrophils. In terms of mechanism, we demonstrated that soluble E-selectin activated Src family tyrosine kinases, an effect that was upstream of phosphatidylinositol 3′-kinase in a signaling pathway that regulates permissive SOCE following exposure of neutrophils to PAF. In summary, this report provides the first evidence for communication between an inflammatory mediator and adhesion receptors at a molecular level, through selectin receptor ligation allowing permissive SOCE to occur following PAF stimulation of human neutrophils.

Dysregulation of neutrophil granulocyte function has been implicated as having a key role in the initiation and progression of a number of inflammatory diseases. The control of events involved in leukocyte recruitment is critical for development of effective antimicrobial defenses and also for efficient wound healing. However, excessive inflammatory-cell recruitment or inappropriate cell activation leads to the development of chronic inflammation that favors fibrotic repair and, ultimately, loss of organ function. The selectin family of molecules (L-, P-, and E-selectin) mediate adhesive interactions between leukocytes and endothelial cells, representing one of the earliest events in the recruitment of inflammatory cells. Studies in vitro and in vivo have revealed the critical role of the selectin family of molecules in the initial capture and subsequent rolling adhesion on vascular endothelial ligands1  before neutrophils firmly adhere and undergo diapedesis at sites of tissue injury and inflammation. In terms of structure, the selectins are type I transmembrane receptors that contain an amino terminal Ca2+-dependent (C-type) lectin domain that has been shown to be important in ligand recognition and is directly involved in mediating cell-to-cell contact through Ca2+-dependent interactions with cell-surface carbohydrates.2 

Several ligands for E-selectin, which all contain sialyl Lewisx-type glycans, have been identified including P-selectin glycoprotein ligand 1 (PSGL-1),3  L-selectin,4  CD66,5  CD44,6,7  and E-selectin ligand 1 (ESL-1).8  However, E-selectin ligands on neutrophils have not been fully characterized to date. The best characterized selectin ligand is PSGL-1, which is found on leukocytes and platelets. PSGL-1 binds to P-, E-, and L-selectins in vitro and represents an important functional ligand for all of these molecules.9 

Recent studies using double or triple selectin knockout mice revealed that selectins have both overlapping and distinct functions.10  Single-selectin knockout mice showed only minor deficiencies in leukocyte recruitment in response to tumor necrosis factor α (TNF-α) or thioglycollate, suggesting distinct roles for the selectins in the inflammatory process. In contrast, double-mutant mice displayed more profound defects in neutrophil recruitment. For example, E- and P-selectin double-mutant mice showed an increased susceptibility to bacterial infection, with the majority of animals developing chronic inflammatory lesions of the oral mucosa and skin, suggesting that E- and P-selectin may function cooperatively.11  The most severe deficiencies in neutrophil recruitment were found in E-, L-, and P-selectin triple-knockout mice, which had impaired neutrophil emigration, neutrophil rolling, and significant leukocytosis.12 

Although the role of selectins in leukocyte recruitment is well established, it is now becoming clear that pathways engaged in response to E-selectin receptor engagement may trigger cell activation even though the molecular mechanisms remain to be defined. Engagement of selectin receptors has been reported to activate the mitogen-activated protein kinase (MAPK) pathway or activate cell-surface receptor-associated protein tyrosine kinases.13  Recent findings from a number of studies suggest that soluble E-selectin may also engage selectin receptors. Soluble E-selectin levels are elevated in many chronic inflammatory conditions, including rheumatoid arthritis and asthma.2,14  In addition, soluble forms of selectins are rapidly released from activated endothelial cells.14  One possibility is that receptor shedding may represent a mechanism for limiting further inflammatory-cell recruitment by decreasing the availability of endothelial ligands for inflammatory cells. However, it is becoming clear that soluble E-selectin may activate inflammatory cells15  and exert potentially proinflammatory effects.16 

Early studies have suggested that selectin and platelet-activating factor (PAF) signaling act cooperatively to induce neutrophil adhesion to the endothelium.17  E-selectin is known to reduce the rolling velocity of neutrophils in vitro.18  In vivo work by Kanwar et al19  found that low concentrations of exogenous PAF induced an increase in neutrophil adhesion in slow-rolling cells, whereas fast-rolling cells were unresponsive to the same concentration of PAF. Similarly, P-selectin has been shown to slow the rolling cells so that they are able to firmly adhere in the presence of lower concentrations of PAF.20  These findings would suggest that reduced neutrophil rolling velocity following adhesion to selectins confers a higher ability to adhere in the presence of an appropriate stimulus. However, one possibility is that selectin receptor engagement may facilitate integrin-mediated “firm” adhesion following exposure to a second stimulus and that there could be communication between receptors for inflammatory mediators and those involved in adhesion at a molecular level.

We have previously demonstrated that Ca2+ mobilization induced by PAF in neutrophils, an early key event in the control of motility, respiratory burst, and degranulation, is prolonged in the presence of soluble E-selectin.21  Neutrophil adhesion to β2-integrin ligands (albumin-coated latex beads) induced by PAF but not by leukotriene B4 (LTB4) or formyl-Met-Leu-Phe (fMLP) was promoted by soluble E-selectin and this adhesion required PAF-induced Ca2+ mobilization from inositol 1,4,5-trisphosphate (IP3)–sensitive intracellular stores. In this paper, we provide biochemical evidence for molecular cross-talk between these structurally distinct receptor pathways.

Reagents

Hanks balanced salt solution (HBSS) was obtained from Life Technologies (Paisley, United Kingdom). Dextran T500 was obtained from Amersham Pharmacia Biotech (Buckinghamshire, United Kingdom). PAF, pertussis toxin, gadolinium(III) chloride, and wortmannin were obtained from Sigma-Aldrich (Poole, United Kingdom). ENA2 was purchased from Abcam (Cambridge, United Kingdom) and MRS1845 and ruthenium red from Tocris (Bristol, United Kingdom). U73122, U73343, LY294002 hydrochloride, LY303511, Fura2-am, PP2, PP3, SB203580, SB202474, and PD98059 were purchased from Calbiochem (Nottingham, United Kingdom). Enhanced chemiluminescence (ECL) Western blotting detection reagents were obtained from Amersham Pharmacia Biotech.

Antibodies

Anti-TRPC6 was purchased from Alamone Labs (Jerusalem, Israel). Phospho-Src family (Tyr416) was purchased from Cell Signaling (Hertfordshire, United Kingdom) and anti–phopho-Akt1/PKBα (Ser473), clone 11E6, was obtained from Upstate Biotechnology (Milton Keynes, United Kingdom). Monoclonal anti–β-actin antibody was purchased from Sigma-Aldrich. Goat anti–mouse and –rabbit horseradish peroxidase (HRP)–conjugated antibodies were obtained from Dako (Ely, United Kingdom).

Expression and purification of E-selectin

Recombinant proteins of E-selectin were obtained using a baculovirus expression construct kindly provided by Dr Mike Bird (GlaxoSmithKline, Stevenage, United Kingdom). Recombinant human E-selectin, lacking the last 2 consensus repeats, was produced in a baculovirus insect-cell expression system as a C-terminal chimera with 2 protein A domains in tandem. High Five cells (BT1-TN-5B1-4 cell line, Invitrogen, Paisley, United Kingdom) were used to express recombinant E-selectin. High Five cells were cultured in Express Five serum-free media supplemented with l-glutamine and penicillin/streptomycin. High Five cells (9 × 106/75-cm2 flask) were seeded into cell-culture flasks and left to adhere for 20 minutes. After attachment of the cells, the medium was removed and the cells were infected with recombinant virus at 2 PFU/cell. Three hours later, the medium was replaced with fresh medium. After 72 hours of incubation at 27°C, the culture supernatant was collected and stored at 4°C for further purification.

Recombinant proteins were then purified from High 5 insect-cell culture supernatants using IgG affinity column chromatography using the protein A domain in the recombinant protein. A column containing IgG-agarose was equilibrated with 5 column volumes of 0.1 M phosphate buffer, pH 8.0. The supernatant was applied to the affinity column, the column was washed with 2 column volumes of 0.1 M phosphate buffer, and eluted with 100 mM glycine in 500-μL fractions. Fractions were analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) for protein content. The eluted proteins were then dialyzed against PBS (Ca2+ and Mg2+ free) overnight. A typical yield was 1.5 to 2.0 mg protein/250 mL supernatant.

Neutrophil isolation

Polymorphonuclear leukocytes were isolated from peripheral blood of healthy donors as described previously.22  After centrifugation of citrated whole blood at 300g for 20 minutes and removal of platelet-rich plasma, leukocytes were separated from erythrocytes by dextran sedimentation using 0.6% (wt/vol) dextran T500. Polymorphonuclear leukocytes were then separated from mononuclear leukocytes using discontinuous isotonic Percoll gradients. Polymorphonuclear leukocytes were 95% to 98% neutrophils using morphologic criteria and cell viability was assessed by trypan blue exclusion.

Measurement of [Ca2+]i

Freshly isolated leukocytes were resuspended at 107/mL in HBSS (Ca2+ and Mg2+ free) and were incubated with 2 μM Fura2-am at 37°C for 30 minutes in the dark. The cells were then washed twice to remove Fura2-am and resuspended at 4 × 107/mL in HBSS (containing Ca2+ and Mg2+). Intracellular calcium was monitored by Fura-2 emission fluorescence at 510 nm using 340/380-nm dual wavelength excitation in a Perkin Elmer (Beaconsfield, CA) luminescence spectrometer at 37°C with constant stirring. Calibration was performed after each experiment using 100 μL 0.1% (vol/vol) Triton X (Rmax) and 10 mM EGTA (Rmin). [Ca2+]i was calculated from the 340:380-nm fluorescence ratio.23 

RT-PCR analysis

Total RNA was extracted from freshly isolated neutrophils using the TRIzol reagent method per the manufacturer's instructions (Invitrogen, Life Technologies). Oligo(dT)–primed first-strand cDNA synthase was performed with Moloney murine leukemia virus reverse transcriptase (RT; SuperScript II, Promega, Madison, WI) using 500 ng mRNA as template in a total volume of 20 μL. The cDNA was then used for polymerase chain reaction (PCR) with Taq polymerase (Promega). Two pairs of specific primers each were used to detect the canonical transient receptor potential channel (TRPC) 1, 3, 4, and 6 and one primer pair was used for TRPC2 and 5; primers were synthesized by MWG Biotech (Ebersberg, Germany). The sequence of the primer pairs used along with the predicted size of their expected amplicons are as follows: TRPC1: forward 5′-ATGTATACAACCAGCTCTATCTTG-3′ and reverse 5′-AGTCTTTGGTGAGGGAATGATG-3′ (525 bp); TRPC1: forward 5′-TTCTGTGGATTATTATTGGGATGA-3′ and reverse 5′-CAGAACAAAGCAAAGCAGGTG-3′ (505 bp); TRPC2: forward 5′-TCTGGACCATGTTCGGTATG-3′ and reverse 5′-GCTACCTCGCTTTGCAGTC-3′ (565bp); TRPC3: forward 5′-CTGCAAATGAGAGCTTTGGC-3′ and reverse 5′-AACTTCCATTCTACATCACTGTC-3′ (388 bp); TRPC3: forward 5′-GCGAATTGTTAACTTTCCCAAATGC-3′ and reverse 5′-TCTTCCAAAAGTTCATAACGAAGGC-3′ (300 bp); TRPC4: forward 5′-ATTCATATACTGCCTTGTGTTG-3′ and reverse 5′-GGTCAGCAATCAGTTGGTAAG-3′ (329 bp); TRPC4: forward 5′-TTGCCTCTGAAAGACATAACATAAG-3′ and reverse 5′-CTACTAACACACATTGTTCACTGAG-3′ (300 bp); TRPC5: forward 5′-ACTTCTATTATGAAACCAGAGC-3′ and reverse 5′-GCATGATCGGCAATAAGCTG-3′ (289 bp); TRPC6: forward 5′-AAGACATCTTCAAGTTCATGGTC-3′ and reverse 5′-TCAGCGTCATCCTAATTTCCC-3′ (322 bp); TRPC6: forward 5′-ACAGATAATGCAAAACAGCTG-3′ and reverse 5′-ATGATGCTCTGGGCTTTG -3′ (244 bp); and GAPDH: forward 5′-TGCCTCCTGCACCACCAAGTG-3′ and reverse 5′-AATGCCAGCCCCAGCGTCAAAG-3′ (450 bp).

The reaction solution (50 μL) contained 0.4 μM of each primer (forward and reverse), 2 mM MgCl2, 0.2 mM dNTPs, 1.25 U Taq polymerase, and 1 μL cDNA. The PCR conditions were: 95°C for 10 minutes, followed by 35 cycles, each consisting of denaturation at 95°C for 1.5 minutes, annealing at 63°C for 2 minutes and extension at 72°C for 2 minutes, and a final extension at 72°C for 10 minutes. After PCR amplification, the reaction mixtures were applied to 1% (wt/vol) agarose gel for electrophoresis and DNA fragments were detected by ethidium bromide staining.

Western blot analysis

Neutrophils (5 × 1010 cells/L/sample) were lysed, following stimulation as detailed in the figure legends, in lysis buffer containing Tris HCl (100 mM, pH 8.0), NaCl (100 mM), EDTA (2 mM), Nonidet NP-40 (1% vol/vol), Na3VO4 (5 mM), NaF (50 mM), and protease inhibitor cocktail (Sigma-Aldrich) for 30 minutes at 4°C. Samples were centrifuged for 20 minutes at 15 000g at 4°C and supernatants were reduced with electrophoresis sample buffer containing Tris HCl (0.25 M, pH 6.8), SDS (8% wt/vol), β-mercaptoethanol (10% wt/vol), glycerol (30% vol/vol), and bromophenol blue (0.02% wt/vol). Each sample was loaded onto a 10% SDS-polyacrylamide gel and proteins were separated and electrophoretically transferred to nitrocellulose. The membranes were then blocked for 1 hour in 5% (wt/vol) dried milk and probed with primary antibody overnight. After washing with Tris-buffered saline containing 0.1% (vol/vol) Tween 20 (TBST), blots were incubated with goat anti–mouse HRP (1:2500) or with goat anti–rabbit HRP (1:2500) antibodies for 1 hour at room temperature. The membranes were incubated with ECL reagent (Amersham Biosciences, Buckinghamshire, United Kingdom), placed under BioMax MS-1 x-ray–sensitive film, and processed through an x-ray developer (X-Ograph Imaging Systems, Wilts, United Kingdom).

Statistical analysis

Statistical analysis was carried out using the one-way ANOVA test with a Newman-Keuls multiple comparison posttest analysis, with statistical significance being achieved when P was below .05.

Soluble E-selectin prolongs PAF-induced Ca2+ mobilization

Incubation of human neutrophils in the presence of soluble E-selectin did not induce Ca2+ mobilization (Figure 1A). However, in agreement with our previous studies,21  preincubation of neutrophils with soluble E-selectin caused a subsequent sustained increase in [Ca2+]i in response to PAF without affecting the initial increase of [Ca2+]i (Figure 1A). To facilitate comparison of experimental data, we have calculated the area under the Ca2+ curve to provide a measure of the Ca2+ mobilization observed. Inhibition of soluble E-selectin interaction with neutrophils by ENA2, an anti–E-selectin monoclonal antibody that binds to the lectin domain of E-selectin, inhibited the sustained Ca2+ levels without affecting the initial rapid rise in [Ca2+]i observed in response to PAF treatment (Figure 1B). These data demonstrate that binding of soluble E-selectin via the lectin domain to a counterreceptor present on neutrophils is required for the prolongation of Ca2+ mobilization in neutrophils in response to PAF.

Figure 1.

Soluble E-selectin prolongs PAF-induced Ca2+ mobilization. (A) Freshly isolated neutrophils were loaded with Fura2-am (2 μM) for 30 minutes at 37°C in Ca2+- and Mg2+-free HBSS, then washed and resuspended in HBSS containing Ca2+ and Mg2+, and then preincubated with or without soluble E-selectin (E-sel, 5 μg/mL) for 15 minutes at 37°C as indicated. For blockade of CD62E, ENA2 (1:50) and soluble E-selectin were preincubated for 1 hour before being added to neutrophils. Neutrophils were stimulated with PAF (100 nM) after recording “baseline” Ca2+ levels for 60 seconds. Data are shown as a representative trace of 5 separate experiments showing similar results and are expressed as percent of peak [Ca2+]i following PAF stimulation (typically from 50 nM control levels to 2.5 μM following stimulation). (B) Traces from panel A have been integrated to calculate area under each curve using GraphPad Prism software (GraphPad, San Diego, CA) to compare the effects of various inhibitors. Data from 5 separate experiments that were performed and expressed as mean ± SEM. *Statistically different (P < .05) from PAF-treated controls.

Figure 1.

Soluble E-selectin prolongs PAF-induced Ca2+ mobilization. (A) Freshly isolated neutrophils were loaded with Fura2-am (2 μM) for 30 minutes at 37°C in Ca2+- and Mg2+-free HBSS, then washed and resuspended in HBSS containing Ca2+ and Mg2+, and then preincubated with or without soluble E-selectin (E-sel, 5 μg/mL) for 15 minutes at 37°C as indicated. For blockade of CD62E, ENA2 (1:50) and soluble E-selectin were preincubated for 1 hour before being added to neutrophils. Neutrophils were stimulated with PAF (100 nM) after recording “baseline” Ca2+ levels for 60 seconds. Data are shown as a representative trace of 5 separate experiments showing similar results and are expressed as percent of peak [Ca2+]i following PAF stimulation (typically from 50 nM control levels to 2.5 μM following stimulation). (B) Traces from panel A have been integrated to calculate area under each curve using GraphPad Prism software (GraphPad, San Diego, CA) to compare the effects of various inhibitors. Data from 5 separate experiments that were performed and expressed as mean ± SEM. *Statistically different (P < .05) from PAF-treated controls.

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Using a range of preincubation times with soluble E-selectin, it was demonstrated that the effects of soluble E-selectin on prolongation of [Ca2+]i in neutrophils were maximal by 15 minutes (Figure 2A), suggesting that downstream signaling pathways may be involved to induce prolonged Ca2+ mobilization in response to PAF rather than a rapid and direct physical interaction causing conformational changes. Furthermore, the effects of soluble E-selectin on prolongation of [Ca2+]i levels were maintained whether soluble E-selectin was present or removed by washing; no significant difference was evident between samples (Figure 2B). Thus, the effects of soluble E-selectin are unlikely to be attributed to nonspecific effects such as sequestration/buffering of extracellular Ca2+ or changes in associated molecules that affect Ca2+ ion movements. These data suggest that soluble E-selectin triggers intracellular signaling pathways to modulate Ca2+ entry. We have previously shown that soluble E-selectin promotes Ca2+ mobilization and adhesion selectively to PAF but not to stimulation by fMLP or LTB4.21  Pretreatment of neutrophils with pertussis toxin, which ADP ribosylates Gi and G0, was found to inhibit fMLP- and LTB4-induced Ca2+ mobilization but had no effect on Ca2+ responses (Figure 3A), thereby suggesting a role for a pertussis toxin-insensitive G protein in mediating the effects of PAF.

Figure 2.

Effect of soluble E-selectin is time-dependent. (A) Freshly isolated neutrophils were loaded with Fura2-am (2 μM) as for Figure 1 and preincubated with E-selectin (5 μg/mL) for various times as indicated. The cells were then stimulated with 100 nM PAF after 60 seconds of recording. Data from 3 separate experiments that were performed are expressed as mean area under the curve ± SEM. *Statistically different (P < .05) from PAF-treated controls. (B) Freshly isolated neutrophils were preincubated with soluble E-selectin and samples were either washed to remove E-selectin (WO) or used after 15 minutes of incubation. Data shown are expressed as mean area under the curve ± SEM from 3 independent experiments.

Figure 2.

Effect of soluble E-selectin is time-dependent. (A) Freshly isolated neutrophils were loaded with Fura2-am (2 μM) as for Figure 1 and preincubated with E-selectin (5 μg/mL) for various times as indicated. The cells were then stimulated with 100 nM PAF after 60 seconds of recording. Data from 3 separate experiments that were performed are expressed as mean area under the curve ± SEM. *Statistically different (P < .05) from PAF-treated controls. (B) Freshly isolated neutrophils were preincubated with soluble E-selectin and samples were either washed to remove E-selectin (WO) or used after 15 minutes of incubation. Data shown are expressed as mean area under the curve ± SEM from 3 independent experiments.

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E-selectin causes Ca2+ influx through a TRPC

PAF-induced Ca2+ mobilization is dependent on activation of phospholipase C and release of IP3 because this response is sensitive to complete inhibition by U73122, a specific phospholipase C inhibitor (Figure 3B), whereas the inactive analog U73343 had no effect. Our previous studies also demonstrated that the initial PAF-induced [Ca2+]i spike was abolished by TMB-8, which blocks Ca2+ release from intracellular stores, and that a relatively small second phase of Ca2+ mobilization was sensitive to inhibition by the receptor-operated channel inhibitor SKF96365.21  Importantly, soluble E-selectin–induced promotion of Ca2+ mobilization in this latter phase was insensitive to SKF96365,21  suggesting it occurs through distinct ion channels. We therefore sought to further define the molecular mechanism by which soluble E-selectin induced prolonged elevation of [Ca2+]i. Neutrophils treated with ruthenium red, an inhibitor of Ca2+-induced Ca2+ release from ryanodine-sensitive stores, had no effect on mobilization of Ca2+ in response to PAF in the presence of E-selectin (Figure 3C). Chelation of extracellular Ca2+ by EGTA showed that only the E-selectin–mediated prolongation of [Ca2+]i was sensitive to blockade and that PAF-induced Ca2+ release from stores was unaffected, indicating that soluble E-selectin affected Ca2+ influx rather than release from an intracellular store. The initial elevation of [Ca2+]i in response to PAF, known to be due to release of Ca2+ from IP3-sensitive stores, was unaffected by MRS1845, a store-operated channel inhibitor; however, soluble E-selectin–induced sustained [Ca2+]i following stimulation with PAF was sensitive to inhibition by MRS1845 (Figure 3D). These results indicate that soluble E-selectin was allowing activation of a store-operated channel by PAF-induced Ca2+ store emptying, an effect we have termed “permissive” store-operated Ca2+ entry (SOCE). We next tested whether Gd3+, a specific transient receptor potential family of cation channels (TRPC) inhibitor, would affect the E-selectin–induced [Ca2+]i response to PAF. As shown in Figure 3C, the prolonged rise in [Ca2+]i observed in the presence of soluble E-selectin was sensitive to Gd3+, which is consistent with a role for TRPC in this response.

Figure 3.

TRPCs mediate soluble E-selectin prolongation of PAF-induced Ca2+ mobilization. (A) Freshly isolated neutrophils were incubated with pertussis toxin (2 μg/mL) for 1 hour, washed, and then loaded with Fura2-am (2 μM) for 30 minutes at 37°C prior to stimulation with 100 nM PAF. Data shown are expressed as mean area under each curve ± SEM from 3 separate experiments. *Statistically different (P < .05) from PAF/LTB4- or fMLP-treated controls. n.s. indicates not significant. (B) U73122 (5 μM) and U73343 (5 μM) were added 5 minutes and EGTA (1.25 mM) was added 10 minutes before stimulation with 100 nM PAF. A representative Ca2+ trace from 3 separate experiments that were performed is shown. (C) Calcium traces showing the effect of calcium channel inhibitors on the prolonged [Ca2+]i elevation induced by soluble E-selectin. Ruthenium red (RR; 20 nM), MRS1845 (2 μM) were added 5 minutes and Ga3+Cl3 (10 μM) was added 3 minutes before stimulation with 100 nM PAF. The calcium trace shown is representative of 3 separate experiments with similar results. (D) Bar graph representing area under the curves of the graph in panel C, calculated using GraphPad Prism software. Data shown are expressed as mean ± SEM from 3 separate experiments that were performed. *Statistically different (P < .05) from PAF/E-selectin–treated controls.

Figure 3.

TRPCs mediate soluble E-selectin prolongation of PAF-induced Ca2+ mobilization. (A) Freshly isolated neutrophils were incubated with pertussis toxin (2 μg/mL) for 1 hour, washed, and then loaded with Fura2-am (2 μM) for 30 minutes at 37°C prior to stimulation with 100 nM PAF. Data shown are expressed as mean area under each curve ± SEM from 3 separate experiments. *Statistically different (P < .05) from PAF/LTB4- or fMLP-treated controls. n.s. indicates not significant. (B) U73122 (5 μM) and U73343 (5 μM) were added 5 minutes and EGTA (1.25 mM) was added 10 minutes before stimulation with 100 nM PAF. A representative Ca2+ trace from 3 separate experiments that were performed is shown. (C) Calcium traces showing the effect of calcium channel inhibitors on the prolonged [Ca2+]i elevation induced by soluble E-selectin. Ruthenium red (RR; 20 nM), MRS1845 (2 μM) were added 5 minutes and Ga3+Cl3 (10 μM) was added 3 minutes before stimulation with 100 nM PAF. The calcium trace shown is representative of 3 separate experiments with similar results. (D) Bar graph representing area under the curves of the graph in panel C, calculated using GraphPad Prism software. Data shown are expressed as mean ± SEM from 3 separate experiments that were performed. *Statistically different (P < .05) from PAF/E-selectin–treated controls.

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TRPC expression in polymorphonuclear leukocytes

To determine the profile of TRPC expression in human neutrophils, multiple specific primer pairs were used to screen for the presence of TRPC1-TRPC6 mRNA species in highly purified human neutrophils. The expression profile for members of the TRPC family is illustrated in a representative gel of RT-PCR products shown in Figure 4A. PCR products for TRPC6 were found in all neutrophil samples (n = 10), whereas TRPC3 was only found in 20% of samples. We did not observe signals for TRPC1, 2, 4, and 5 in any of the preparations, despite positive RT-PCR controls demonstrating that these PCR conditions were optimal. To confirm TRPC6 protein expression, we assayed crude membrane preparations from freshly isolated human neutrophils using Western blotting techniques. A specific antibody for TRPC6 revealed a strong band in the appropriate 90- to 100-kDa range, which could be blocked by a TRPC6-blocking peptide (Figure 4B), confirming the presence of protein and the RT-PCR data.

Figure 4.

TRPC6 expression in human neutrophils. (A) Expression of TRP family members in isolated human neutrophils. Representative results of RT-PCR analysis with mRNA from a single isolation and cDNA preparation of neutrophils with each of the indicated primers are given and GAPDH was used as a positive control. Lanes are indicated as: M, markers; 1, TRPC1; 2, TRPC2; 3, TRPC3; 4, TRPC4; 5, TRPC5; and 6, TRPC6. (B) Western blotting of neutrophil (N) or mononuclear cell (M) membrane preparations stained with anti-TRPC6 antibody (1:400) revealed a strong band at the predicted molecular weight (100 kDa) as indicated. Specificity was demonstrated by incubation of the TRPC6 antibody with a 4-fold excess of the antigenic peptide exhibited no signal. Nonspecific binding was assessed using an IgG control stained with rabbit anti-IgG antibody (1:400). A representative immunoblot of 3 different experiments is shown.

Figure 4.

TRPC6 expression in human neutrophils. (A) Expression of TRP family members in isolated human neutrophils. Representative results of RT-PCR analysis with mRNA from a single isolation and cDNA preparation of neutrophils with each of the indicated primers are given and GAPDH was used as a positive control. Lanes are indicated as: M, markers; 1, TRPC1; 2, TRPC2; 3, TRPC3; 4, TRPC4; 5, TRPC5; and 6, TRPC6. (B) Western blotting of neutrophil (N) or mononuclear cell (M) membrane preparations stained with anti-TRPC6 antibody (1:400) revealed a strong band at the predicted molecular weight (100 kDa) as indicated. Specificity was demonstrated by incubation of the TRPC6 antibody with a 4-fold excess of the antigenic peptide exhibited no signal. Nonspecific binding was assessed using an IgG control stained with rabbit anti-IgG antibody (1:400). A representative immunoblot of 3 different experiments is shown.

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E-selectin–induced SOCE is regulated by Src and PI-3K

It is now clear that critical regulatory elements that control TRPC activity include phosphorylation and Src family tyrosine kinases in particular.24  We therefore used specific protein kinase inhibitors to test their involvement in soluble E-selectin–mediated SOCE. Inhibition of p38 MAPK by SB203580 (10 μM) or the negative control SB202474 (10 μM) had no effect on soluble E-selectin–induced Ca2+ influx following stimulation of neutrophils with PAF (Figure 5B). Similarly, a lack of effect by PD98059 (10 μM) on soluble E-selectin–induced modulation of Ca2+ influx identified that MEK1 was not involved in mediating these responses (Figure 5B). However, the specific Src family tyrosine kinase inhibitor PP2 (5 μM) selectively inhibited the soluble E-selectin–induced SOCE to levels observed in PAF-only stimulated neutrophils (Figure 5A-B), whereas PP3, the inactive analog, had no effect on soluble E-selectin–induced Ca2+ influx. These data suggest either a direct role for Src in modulating TRPC6 channel activity or potentially a role for Src in the downstream signaling events following soluble E-selectin binding to its putative receptor on neutrophils. Because PI 3-kinase is known to be a key regulator of ion channels in a variety of other cell types, we pretreated neutrophils with the specific PI 3-kinase inhibitors wortmannin (100 nM) or LY294002 (10 μM) and LY303511, an inactive structural analog. PI 3-kinase inhibition also inhibited the soluble E-selectin–induced SOCE to controls levels (Figure 5C-D), the inactive analog having no effect, thus identifying PI 3-kinase as a key regulator in the signaling pathway, which mediates the effects of soluble E-selectin on Ca2+ influx.

Figure 5.

PI 3-kinase and Src kinase activity are required for soluble E-selectin Ca2+ mobilization. (A) PD98059 (10 μM), SB203580 (10 μM), SB202474 (10 μM), PP2 (5 μM), and PP3 (5 μM) were added concurrently with E-selectin for 15 minutes prior to stimulation with 100 nM PAF. The Ca2+ trace shown is representative of 3 separate experiments that were performed with similar results. (B) Bar graph representing area under the curves of graph in panel A, calculated using GraphPad Prism software. Data expressed as mean ± SEM of 3 independent experiments. *Statistically different (P < .05) from PAF/E-selectin–treated controls. (C) LY294002, LY303511 (10 μM, 5-minute preincubation), and wortmannin (100 nM, 15-minute preincubation) were added prior to stimulation with 100 nM PAF. The Ca2+ trace shown is representative of 3 independent experiments that were performed. (D) Bar graph representing area under the curves of graph in panel C, calculated using GraphPad Prism software. Data expressed as mean ± SEM from 3 separate experiments is shown. *Statistically different (P < .05) from PAF/E-selectin–treated controls.

Figure 5.

PI 3-kinase and Src kinase activity are required for soluble E-selectin Ca2+ mobilization. (A) PD98059 (10 μM), SB203580 (10 μM), SB202474 (10 μM), PP2 (5 μM), and PP3 (5 μM) were added concurrently with E-selectin for 15 minutes prior to stimulation with 100 nM PAF. The Ca2+ trace shown is representative of 3 separate experiments that were performed with similar results. (B) Bar graph representing area under the curves of graph in panel A, calculated using GraphPad Prism software. Data expressed as mean ± SEM of 3 independent experiments. *Statistically different (P < .05) from PAF/E-selectin–treated controls. (C) LY294002, LY303511 (10 μM, 5-minute preincubation), and wortmannin (100 nM, 15-minute preincubation) were added prior to stimulation with 100 nM PAF. The Ca2+ trace shown is representative of 3 independent experiments that were performed. (D) Bar graph representing area under the curves of graph in panel C, calculated using GraphPad Prism software. Data expressed as mean ± SEM from 3 separate experiments is shown. *Statistically different (P < .05) from PAF/E-selectin–treated controls.

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Western blot analysis of neutrophil protein lysates using a phosphorylation state-specific antibody (Tyr(P)416), which correlates with Src activation, showed significant phosphorylation above control levels with soluble E-selectin treatment (Figure 6A). Interestingly, stimulation of neutrophils with PAF alone had no effect on the levels of phospho-Src. Pretreatment of neutrophils with PP2, prior to stimulation with soluble E-selectin, inhibited active phospho-Src to below control levels. In addition, LY294002 (10 μM) had no significant effect on soluble E-selectin–induced phosphorylation and activation of Src (Figure 6A), indicating that PI 3-kinase may be involved in a parallel pathway or acts downstream of Src in regulating Ca2+ influx. Soluble E-selectin caused activation of PI 3-kinase as assessed by a significant increase in phosphorylated Akt, a downstream target of PI 3-kinase, compared with control untreated cells, whereas PAF did not induce any Akt phosphorylation or activation (Figure 6B). Soluble E-selectin–induced increases in phospho-Akt levels could be inhibited completely by LY294002, confirming that it is a target of PI 3-kinase, and interestingly the Src tyrosine kinase inhibitor PP2 also showed complete inhibition of phospho-Akt accumulation following treatment with soluble E-selectin (Figure 6B). These data would support the hypothesis that the pathway that regulates permissive SOCE induced by soluble E-selectin is mediated primarily by Src with PI 3-kinase acting downstream.

The selectin family of receptors is critical for the appropriate recruitment of neutrophils to sites of infection or tissue injury and the initiation and progression of the inflammatory response. We have previously shown that soluble E-selectin acts to promote neutrophil adhesion, inhibit migration, and amplify destructive responses,21  raising the possibility that elevated levels of soluble E-selectin in patients with inflammatory diseases, such as rheumatoid arthritis, and associated with tumor growth, have a proinflammatory effect.25 

Figure 6.

Soluble E-selectin induces Src and Akt activation in neutrophils. Freshly isolated neutrophils were incubated in the presence or absence of PP2 (5 μM, 15 minutes) or LY294002 (10 μM, 15 minutes), with soluble E-selectin (5 μg/mL, 15 minutes). Western blots of neutrophil lysates were carried out as described in “Materials and methods,” and probed with (A) phospho-Src (Tyr 116) antibody (1:500) or (B) antiphospho-Akt1/PKBα antibody (Ser473; 1:200). To verify equal loading, the blots were probed with β-actin (1:10 000) or total Akt (1:2000). This figure is a representative blot from 3 independent experiments that were performed with similar results.

Figure 6.

Soluble E-selectin induces Src and Akt activation in neutrophils. Freshly isolated neutrophils were incubated in the presence or absence of PP2 (5 μM, 15 minutes) or LY294002 (10 μM, 15 minutes), with soluble E-selectin (5 μg/mL, 15 minutes). Western blots of neutrophil lysates were carried out as described in “Materials and methods,” and probed with (A) phospho-Src (Tyr 116) antibody (1:500) or (B) antiphospho-Akt1/PKBα antibody (Ser473; 1:200). To verify equal loading, the blots were probed with β-actin (1:10 000) or total Akt (1:2000). This figure is a representative blot from 3 independent experiments that were performed with similar results.

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In leukocytes, PAF acts through a specific G protein-coupled receptor to induce chemotaxis. Neutrophil activation by PAF has been shown to be insensitive to pertussis toxin, implicating a Gq/11 family member. Neutrophil responses to stimulation by LTB4 and fMLP are sensitive to pertussis toxin, suggesting Go or Gi involvement.26-28  β2-Integrin-mediated neutrophil adhesion to albumin-coated latex beads induced by PAF but not fMLP and LTB4 was promoted by soluble E-selectin.21  Furthermore, we have also shown that soluble E-selectin specifically prolongs elevation of [Ca2+]i in response to PAF but not fMLP or LTB4.21  We have demonstrated that pertussis toxin does not affect PAF-induced Ca2+ mobilization but abolishes fMLP- and LTB4-induced Ca2+ mobilization. Taken together, this would indicate that only signals from a pertussis toxin-insensitive Gq/11-coupled receptor such as the PAF receptor are able to communicate through a G protein-derived signal to allow prolonged Ca2+ signaling to occur in the presence of soluble E-selectin.

We provide important new information relating to the mechanism by which soluble E-selectin prolongs PAF-induced Ca2+ signaling in neutrophils. Stimulation of neutrophils with PAF causes primarily a rapid release of Ca2+ from IP3-sensitive stores and a relatively minor influx of Ca2+ through SKF96365-sensitive channels, most likely receptor-operated channels.21  Soluble E-selectin alone does not cause release of Ca2+ from intracellular stores or via Ca2+ influx but acts to prolong Ca2+ signals induced by PAF receptor ligation in a novel manner by allowing “permissive” SOCE. We have demonstrated that prolongation of PAF-induced Ca2+ signaling by E-selectin is due to Ca2+ influx due to its sensitivity to blockade by EGTA rather than further release from ryanodine-sensitive intracellular stores. Furthermore, an obligate requirement for IP3-mediated release of Ca2+ from intracellular stores to act as a trigger for the Ca2+ influx permitted by soluble E-selectin was demonstrated by inhibition of phospholipase C causing a complete loss of any Ca2+ signaling. In addition, the susceptibility of E-selectin–permitted Ca2+ influx to blockade by MRS1845, a store-operated channel (SOC) inhibitor, identified this as SOCE. A role for TRPCs as candidates for mediating this novel SOCE was proposed based on the ability of Gd3+ to cause selective inhibition of prolonged Ca2+ entry following PAF stimulation in the presence of soluble E-selectin. Proteins homologous to the Drosophila transient receptor potential gene (trp) Ca2+ channels that assemble into tetrameric ion channels are known to be involved in the generation of store-operated Ca2+ entry (SOCE). Our RT-PCR studies found that only TRPC3 and TRPC6 mRNA were expressed in polymorphonuclear leukocytes (Figure 4A), in agreement with Heiner et al.29  TRPC6 appears to represent the principal TRPC family member present, being detected at both the level of mRNA and protein.

Thus, soluble E-selectin acts to promote a novel form of molecular cross-talk involving TRPCs that allow a putative E-selectin receptor to influence PAF-induced signaling pathways. Our data also suggest that both Gq/11- and soluble E-selectin–mediated signals are required to communicate with TRPC6 before release of Ca2+ from intracellular stores can trigger SOCE. We are currently investigating potential mechanisms for this effect, for example, whether a regulatory protein becomes recruited to TRPCs to permit SOCE, or promotion of a physical interaction between IP3 channels in the endoplasmic reticulum (ER) with TRPC in the plasma membrane, or alternatively, TRPCs may become sensitized to the signals that mediate SOCE, such as calcium influx factor (CIF).30  It has recently been shown that TRPC6 is externalized to the plasma membrane by the stimulation of a Gq protein-coupled receptor,31  and it has also been shown that expression of TRPC6 in COS cells increases Ca2+ entry in response to stimulation of a Gq protein-coupled receptor.32  We are therefore currently investigating whether soluble E-selectin preincubation leads to the up-regulation of TRPC6 on the cell surface and if this is sensitive to activation via Gq-coupled receptor-induced signals specifically.

The recent finding that diacylglycerol directly activates TRPC3 and TRPC6 may represent an alternative mechanism for activation of these channels via phospholipase C-linked receptors,33  allowing regulation to occur through a lipid mediator. Recent studies have shown that tyrosine phosphorylation by Src family protein tyrosine kinases represents a potential regulatory mechanism of TRPC6 activity.24  It has been suggested that 2 simultaneous events, opening of the channel by DAG and modulation by Src-induced tyrosine phosphorylation, contribute to the efficient influx of calcium through TRPC6. We found that PP2 specifically inhibited soluble E-selectin–mediated SOCE without affecting PAF-induced responses. In parallel, soluble E-selectin caused phosphorylation and activation of Src, which was sensitive to inhibition by PP2 but was unaffected by PI 3-kinase inhibition. These findings strongly suggest that Src activity is involved in modulating TRPC6 activity to regulate Ca2+ influx in human neutrophils.

Inhibition of PI 3-kinase selectively blocked the soluble E-selectin–induced SOCE in neutrophils. Several potential intracellular regulatory motifs have been identified on TRPC6 including PI3K-SH2 recognition domains, suggesting a mechanism by which these channels might interact with the PI 3-kinase signaling pathway.34  Several groups35,36  have discovered that the PI 3-kinase lipid product phosphatidylinositol 3,4,5-trisphosphate (PIP3) mediates calcium influx through a mechanism independent of phospholipase C (PLC) activity or store depletion in several cell lines. The activation of receptor tyrosine kinase cascades leads to the membrane colocalization of PLCγ and PI 3-kinase, both of which use phosphatidylinositol 4,5-bisphosphate (PIP2) as a substrate to generate IP3 and PIP3, respectively. These 2 signaling intermediates trigger the activation of calcium channels at different cellular compartments, giving rise to elevated levels of [Ca2+]i. Soluble E-selectin was demonstrated to cause an increase in phospho-Akt, a downstream target of PI 3-kinase. We would speculate that PI 3-kinase acts to modulate TRPC6 activity and that PI 3-kinase lies downstream of Src in the regulation of soluble E-selectin–mediated permissive SOCE.

Several putative glycoprotein selectin ligands have been isolated from hematopoietic cells using in vitro affinity purification techniques, but the exact identity and contribution of physiologic E-selectin ligands on neutrophils is unknown.37  In this paper, we have demonstrated that E-selectin binds via the lectin domain to cause permissive SOCE in neutrophils in response to PAF, presumably through a putative E-selectin receptor present on neutrophils. A cell-adhesion molecule suggested to play a role in E-selectin adhesion is CD66 or carcinoembryonic antigen (CEA). Neutrophils are known to express several CEA family members, which are all highly glycosylated molecules with multiple sialyl and fucosyl residues. In preliminary experiments, CD66 ligation with antibodies caused prolonged PAF-induced Ca2+ mobilization in a similar manner to that caused by soluble E-selectin (data not shown). We are currently investigating the possibility that CD66 and other adhesion receptors need to coengage via soluble E-selectin to regulate Ca2+ signaling in response to PAF.

Receptor-mediated activation of leukocytes by inflammatory stimuli requires Ca2+ mobilization and influx as a critical common activation mechanism. Selective modulation of distinct components of these Ca2+ signals may represent potentially attractive strategies for developing anti-inflammatory drugs to attenuate leukocyte activation. Our report is the first demonstration of soluble E-selectin causing permissive SOCE to occur following activation of neutrophils by PAF and that this SOCE most likely occurs through TRPC6. We have identified a novel form of permissive SOCE induced by soluble E-selectin in human neutrophils, which occurs through a Src/PI 3-kinase–dependent pathway and also requires a Gq/11-derived signal to sensitize or prime TRPCs to open on increased intracellular Ca2+ and depletion of Ca2+ stores, but the precise order of these molecular events is yet to be fully explored (Figure 7). This novel mechanism of molecular cross-talk integrates signals from pertussis toxin-insensitive Gq/11-coupled receptors and TRPCs and could be critical for fine tuning adhesion and migratory responses during neutrophil recruitment during inflammation.

Figure 7.

Schematic model of intracellular communication between E-selectin receptors and PAF receptor. PAF binds to its Gq/11 protein-coupled receptor resulting in activation of PLC, leading to cleavage of PIP2 and generation of membrane-retained DAG and cytosolic IP3. DAG can directly activate TRPC6.34  Soluble InsP3 activates the IP3R on the endoplasmic reticulum to release intracellular Ca2+. These responses result in the initial rapid increase of [Ca2+]i. E-selectin interacts with E-selectin receptors on the neutrophil surface, permitting PAF-induced Ca2+ mobilization to communicate with TRPC6 and allowing permissive SOCE to occur. Modulation of this Ca2+ channel involves Src and PI 3-kinase pathways. ROC indicates receptor-operated channel; G, G protein-coupled receptor.

Figure 7.

Schematic model of intracellular communication between E-selectin receptors and PAF receptor. PAF binds to its Gq/11 protein-coupled receptor resulting in activation of PLC, leading to cleavage of PIP2 and generation of membrane-retained DAG and cytosolic IP3. DAG can directly activate TRPC6.34  Soluble InsP3 activates the IP3R on the endoplasmic reticulum to release intracellular Ca2+. These responses result in the initial rapid increase of [Ca2+]i. E-selectin interacts with E-selectin receptors on the neutrophil surface, permitting PAF-induced Ca2+ mobilization to communicate with TRPC6 and allowing permissive SOCE to occur. Modulation of this Ca2+ channel involves Src and PI 3-kinase pathways. ROC indicates receptor-operated channel; G, G protein-coupled receptor.

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Prepublished online as Blood First Edition Paper, March 2, 2006; DOI 10.1182/blood-2005-09-3803.

Supported by the Medical Research Council (United Kingdom) and Arthritis Research Campaign.

The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 U.S.C. section 1734.

We are very grateful to Dr M. Bird and Dr Girish Shah (GlaxoSmithKline, Stevenage, United Kingdom) for their generous gift of the recombinant-selectin baculovirus expression constructs and to Dr Gavin Nicoll for his kind help with the baculovirus expression technique.

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