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Published ahead of print on October 13, 2005, doi:10.1165/rcmb.2005-0126OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 34, pp. 233-241, 2006
© 2006 American Thoracic Society
DOI: 10.1165/rcmb.2005-0126OC

Neutrophil Sphingosine 1-Phosphate and Lysophosphatidic Acid Receptors in Pneumonia

Matiur Rahaman, Richard W. Costello, Kristen E. Belmonte, Samir S. Gendy and Marie-Thérèse Walsh

Department of Medicine, Royal College of Surgeons in Ireland, Beaumont Hospital, Dublin, Ireland; and Respiratory and Inflammation Centre of Excellence for Drug Discovery, GlaxoSmithKline, King of Prussia, Pennsylvania

Correspondence and requests for reprints should be addressed to Marie-Thérèse Walsh, Department of Medicine, RCSI, Beaumont Hospital, Dublin 9, Ireland. E-mail: mtwalsh{at}rcsi.ie


   Abstract
Top
 Abstract
Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
The phospholipids sphingosine 1-phosphate (S1P) and lysophosphatidic acid (LPA) act via transmembrane receptors S1P 1–5 and LPA 1–3, respectively. Both have been implicated in inflammatory responses. S1P and LPA receptor profiles on neutrophils of patients with pneumonia compared with healthy subjects were determined by PCR and Western blotting. Chemotaxis studies were performed to assess functional differences. S1P or LPA receptors were immunoprecipitated from neutrophils to assess receptor heterodimerization with CXCR1, an IL-8 receptor, by Western blotting. Receptors S1P 1, 4, and 5 and LPA 2 were expressed on neutrophils from both subject groups, but S1P 3 and LPA 1 receptor expression was mainly confined to neutrophils of patients with pneumonia. Chemotaxis of neutrophils from patients with pneumonia compared with control subjects was significantly increased in response to S1P and LPA. Pretreatment with S1P or LPA reduced IL-8–induced neutrophil chemotaxis and transcriptional expression of the CXCR1 receptor. Receptors S1P 3 and 4 and LPA 1 formed constitutive heterodimers with CXCR1. LPA treatment reduced the amount of LPA 1/CXCR1 heterodimer. Therefore, profiles of S1P and LPA receptors differ between neutrophils of patients with pneumonia and control subjects, with consequences for neutrophil function.

Key Words: heterodimerization • lysophosphatidic acid • neutrophils • receptor • sphingosine 1-phosphate


   Introduction
Top
 Abstract
 Introduction
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Sphingosine 1-phosphate (S1P) and lysophosphatidic acid (LPA) are structurally related phospholipids with pleiotropic cellular effects exerted mainly via a family of eight G-protein–coupled receptors, five of which (S1P 1–5) are specific for S1P. Three, termed LPA 1–3, are specific for LPA (1). Both phospholipids are found in micromolar amounts in normal human serum (2, 3). Their receptors display varying tissue distribution, ligand sensitivity, and G-protein–coupling properties and activate multiple intracellular signaling pathways (4–9). Thus, the cell- or tissue-specific effects of S1P and/or LPA on specific cell types depends on receptor profile and how the receptors cross-talk with each other and other mediators, including potentially other G-protein–coupled receptors.

Activated platelets are the major source of S1P and LPA (4, 10). Roles for both phospholipids and their receptors have been postulated in inflammatory responses (e.g., in attraction and activation of T and B cells) (8, 9). Administration of S1P, or phosphorylated FTY720 (an S1P receptor ligand) induces blood and thoracic duct lymphopenia in rodents by sequestration of T and B cells (12) and inhibition of Th2-cell–induced bronchoalveolar lavage fluid eosinophilia in a murine asthma model (13). Knockout mice studies have established that receptor S1P 1 is essential for lymphocyte recirculation (14). S1P has also been recently implicated in allergic inflammation (11, 15, 16). LPA induces proinflammatory gene expression in endothelial cells (17), induces chemotaxis and reactive oxygen species generation in human eosinophils (18), and accelerates mast cell development (19). On the other hand, it has been implicated in the reduction of gastrointestinal inflammation and in the promotion of wound healing (20).

S1P and LPA have specific neutrophil effects. S1P reduces neutrophil chemotaxis in response to IL-8 or FMLP (21), antagonizes neutrophils apoptosis (22), and induces pertussis toxin–sensitive calcium signals in neutrophils (23). Neutrophil priming agents, such as FMLP or platelet-activating factor, may activate sphingosine kinase in neutrophils leading to production of S1P, which acts via its receptors on neutrophils to induce further priming (24, 25). LPA augments FMLP-induced superoxide production in neutrophils (26). There are conflicting reports on the LPA-induced effects on neutrophil migration. LPA reduces renal neutrophil influx in a mouse model of renal injury (27) but enhances infiltration of neutrophils and eosinophils into guinea pig lavage fluid (28) and induces chemotaxis of neutrophils under agarose (29).

Despite these multiple effects of S1P and LPA, which imply the expression of S1P and LPA receptors on neutrophils, there has been no definition of the receptor expression profiles of these mediators on neutrophils or a comparison of such a profile in healthy subjects to subjects with diseases characterized by neutrophil infiltration. We hypothesized that neutrophils would express multiple receptors for S1P and LPA, that this profile might vary in healthy subjects compared with patients with conditions characterized by neutrophil infiltration of airways (e.g., pneumonia), and that differences in receptor expression might influence neutrophil function in response to S1P or LPA.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Materials
Dulbecco's modified Eagle's medium plus Glutamax and penicillin/streptomycin solution were purchased from GIBCO/BRL Life Technologies (Paisley, UK). PMSF, leupeptin, S1P, LPA, pepstatin A, TRI reagent, IL-8, and all common buffer constituents were obtained from Sigma (Poole, UK). An AA96 chemotaxis chamber and 5-µm pore size framed filters were purchased from Neuro Probe, Inc. (Gaithersburg, MD). I-Block for Western blot blocking was purchased from Tropix (Bedford, MA). Dulbecco's PBS was purchased from Invitrogen, Ltd. (Paisley, UK). Polyclonal rabbit anti-human anti-S1P 1, anti-S1P 2, anti-S1P 3, anti-S1P 4, and anti-S1P 5 and anti-LPA 1, anti-LPA 2, and anti-LPA 3 were kind gifts from Dr. Kristen Belmonte (GlaxoSmithKline, Philadelphia, PA). Polyclonal goat anti-human anti-S1P 1, anti-S1P 3, anti-S1P 4, anti-S1P 5, anti-LPA 1, anti-LPA2, rabbit anti-human anti-CXCR1, mouse anti-human CXCR1, rabbit anti-human anti-beta-actin, rabbit anti-goat IgG horseradish peroxidase (HRP) conjugate, and protein A/G agarose beads were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). LumiGlo Reagent A and Peroxide Reagent B for HRP detection were obtained from Cell Signaling Technology (Beverly, MA). Anti-rabbit IgG HRP and anti-mouse IgG HRP conjugates were obtained from Promega (Madison, WI). Ficoll-Paque PLUS was purchased from Amersham Pharmacia Biotech (Little Chalfont, UK). First-strand cDNA synthesis kit was purchased from Roche Diagnostics (Mannheim, Germany). Primer pairs for S1P 1, S1P 2, S1P 3, S1P 4, S1P 5, LPA 1, LPA 2, LPA 3, and beta-actin were purchased from MWG Biotech, Ltd. (Cork, Ireland) and are listed in Table 1.


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  		TABLE 1. PRIMERS USED FOR GENE AMPLIFICATION

 
Subjects
Sixteen pneumonia patients (eight men and eight women, 62.6 ± 19.2 yr of age (mean ± SD), range 30–92 yr; 10 smokers and six nonsmokers) were recruited from the Accident and Emergency Department and wards of Beaumont Hospital, Dublin, Ireland. Diagnosis was confirmed by taking appropriate history and by x-ray findings. Patients had suffered symptoms such as chest pain, cough, elevated temperature, and shortness of breath for 4.2 ± 2.9 d upon recruitment. Patients had been treated with antibiotics for 1.6 ± 2.1 d on recruitment. White cell count was 14.0 ± 4.6, and temperature was 37.9 ± 1.1°C upon recruitment. Three of the patients (30, 58, and 69 yr of age) were retested between 1 and 5 wk postrecovery when all symptoms were absent. Ten healthy control subjects (six men and eight women, 34.5 ± 8.3 yr of age, range 22–45 yr; five smokers and five nonsmokers) with no history of pneumonia or other serious inflammatory conditions were recruited from the laboratory and hospital community. The Beaumont Hospital Ethics Committee approved the study.

Neutrophil Isolation
Peripheral blood neutrophils were prepared from 20 ml of peripheral blood drawn from patients or healthy human volunteers as previously described by Ficoll density gradient and hypotonic lysis of erythrocytes (30). Neutrophil viability and purity was determined by Trypan Blue and Speedy-Diff staining. Only populations containing neutrophils that were > 98% pure and > 95% viable were used in experiments.

RNA Extraction and RT-PCR
Neutrophils were harvested and washed in ice-cold PBS. Cells were lysed at room temperature in TRI-reagent and RNA and protein extracted according to the manufacturer's guidelines. RNA (1 µg) was reverse transcribed with avian myeloblastosis virus reverse transcriptase and oligo-dT primers using a first-strand cDNA synthesis kit. RT-PCR analysis of cDNA preparations was performed in 50-µl reactions with Taq-DNA polymerase; primers sets are outlined in Table 1. PCR conditions were 94°C, 4 min (1 cycle); 94°C, 1.5 min, 54°C, 1.5 min, 72°C, 2 min (25–40 cycles); 72°C, 10 min (1 cycle). PCR products were separated by 1.5% agarose gel electrophoresis and photographed under UV illumination. Results presented are for 30 cycles of PCR (S1P 1, 2, 3, and 5; LPA 1–3), 27 cycles (S1P 4), or 25 cycles (beta-actin). Up to 40 cycles of PCR were performed to verify the absence of S1P 2 or 3 and LPA 1 or 3 as relevant. Band intensities were quantified by laser densitometry scanning. The results were expressed as a ratio of the band intensity relative to the corresponding beta-actin band obtained by amplification of the same template cDNA.

Western Blotting
Total protein was extracted from neutrophils from patients or control subjects using TRI reagent according to the manufacturer's instructions. Protein concentration was determined by the Bradford method (31), and 10 µg were heated to 95°C in sample buffer (100 mM Tris [pH 6.8], 2% [wt/vol] SDS, 0.002% [wt/vol] bromophenol blue, 20% [vol/vol] glycerol, and 10% [vol/vol] beta-mercaptoethanol) and separated by SDS-PAGE on 10% polyacrylamide separating gel overlaid with 4% stacking gel at 500 V for 1 h. The separated proteins were transferred onto nitrocellulose membranes in transfer buffer (20 mM Tris, 150 mM glycine, 0.01% [wt/vol] SDS, and 20% [vol/vol] methanol) at 500 V overnight. For immunodetection with rabbit anti-human anti-S1P 1, 2, 3, 4, or 5 or anti-LPA 1, 2, or 3, goat anti-human anti-S1P 1, anti-S1P 4, or anti-S1P 5; rabbit anti-human anti-beta-actin or rabbit anti-human anti-CXCR1 antibodies, membranes were incubated in blocking buffer (Dulbecco's PBS containing 0.2% [wt/vol] I-block and 0.1% [vol/vol] Tween-20) for 1 h at room temperature and incubated for 2 h in blocking buffer containing the individual respective antibody (1:500 for each). After six 5-min washes in washing buffer (PBS [pH 7.4], 0.1% [vol/vol] Tween-20), membranes were incubated for 1 h in blocking buffer containing the appropriate goat anti-rabbit (1:2,000) or rabbit anti-goat (1:10,000) IgG HRP conjugate. Membranes were washed six times for 5 min each and exposed to LumiGlo substrate solution (Cell Signaling Technology) for 1 min at room temperature according to the manufacturer's instructions. Blots were exposed to X-OMAT light-sensitive film to obtain an image.

Immunoprecipitation of Sphingosine 1-Phosphate, Lysophosphatidic Acid, or CXCR1 Receptors from Neutrophils
Before beginning the cell lysis procedures, microfuge tubes containing protein A/G agarose beads (20 µl), the immunoprecipitating antibody or control IgG (2 µg in 10 µl), and 70 µl immunoprecipitation buffer (50 mM Tris [pH 8.0], 150 mM NaCl, 1 mM EDTA, 1% IgePal [vol/vol], 1 µg/ml leupeptin, 1 µg/ml pepstatin, and 0.5 mM PMSF) were set up with continual rotation for 1 h at 4°C. Meanwhile, cell lysis was performed. Neutrophils were washed twice in ice-cold PBS before lysis in immunoprecipitation buffer on ice for 15 min. Lysed cells were passed sequentially through 19-, 23-, and 25-gauge needles three or four times each and centrifuged at 10,000 x g for 5 min at 4°C to pellet nuclear material and undisrupted membrane aggregates. Supernatants were transferred to tubes containing the immunoprecipitating antibody-Protein A/G complexes, and the tubes were set up with continual rotation overnight at 4°C. Agarose beads were centrifuged at 10,000 x g for 5 min at 4°C before two washes in immunoprecipitation buffer, and the beads were resuspended in sample buffer (100 mM Tris [pH 6.8], 2% [wt/vol] SDS, 0.002% [wt/vol] bromophenol blue, 20% [vol/vol] glycerol, and 10% [vol/vol] beta-mercaptoethanol) before boiling for 5–10 min and separating by SDS-PAGE on 10% polyacrylamide separating gel overlaid with 4% stacking gel at 500 V for 1 h.

Chemotaxis of Neutrophils
Chemotaxis of neutrophils was performed on an AA96 96-well chemotaxis chamber fitted with a polycarbonate filter (pore size: 5 µm). Putative chemoattractants were resuspended in 1x PBS + 0.1% BSA (vehicle) at appropriate concentrations and loaded into the wells of the lower compartment of the chemotaxis chamber. In each experiment, vehicle alone was used as control with no chemoattractant. Neutrophils for chemotaxis were resuspended in serum-free Dulbecco's modified Eagle's medium + Glutamax (Gibco BRL) at a concentration of 1.28 x 106 cells/ml, and a total of 5 x 105 cells were loaded into each experimental well of the upper compartment of the chamber. In some experiments, neutrophils were pretreated for 1 h with 0.1 µM LPA or S1P. In chemokinesis controls, an equal concentration of chemoattractant was added to the eosinophils in the upper well as in the bottom chemoattractant chamber. Chemotaxis was allowed to proceed for 2.5 h at 37°C, 5% CO2, after which the upper compartment was removed. Fifty microliters were removed from respective experimental wells in the bottom compartment, added to 400 µl 1x PBS, and subjected to counting by flow cytometry (Beckman Coulter Epics XL-MCL) for 30 s to quantify cells that had migrated into the bottom well. Filters were also fixed and stained with Speedy-Diff stain after removal of cells from the top side of the filter and viewed under a light microscope for counting the number of cells migrating to the lower side of the filter in five random fields to confirm consistency with the more readily quantifiable flow cytometry method. Cell migration to different putative chemoattractants was assessed as a percentage of migration in the absence of any chemoattractant.

Statistical Analysis
Values are expressed as mean ± SD or mean ± SEM. The statistical significance of differences between patient and control samples, between treated samples and the appropriate time point control, or between treated and untreated samples was evaluated by analysis of variance followed by Tukey-Kramer pairwise multiple comparison as appropriate. A P value of 0.05 or less was taken as significant. Row-column association for expression or not of a given receptor in the control versus the patient group was assessed by Fisher's exact test.


   RESULTS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
S1P 1, 4, and 5 Are the Predominantly Expressed Neutrophil Sphingosine 1-Phosphate Receptors at the cDNA Level, but Additional S1P 3 Expression Is Associated with Pneumonia
We defined the expression profile of the S1P receptors S1P 1, 2, 3, 4, and 5 on cDNA from neutrophils of 16 subjects with pneumonia and 10 healthy control subjects who had no history of pneumonia (Figure 1, Table 2). Receptor S1P 4 was expressed on the neutrophils of all subjects (Figure 1, Table 2). S1P 1, and to a lesser extent S1P 5, were also expressed on neutrophils from both subject groups (Figure 1, Table 2). S1P 2 was expressed on neutrophils of only a few subjects (Figure 1, Table 2). The S1P 3 receptor was expressed on neutrophils of 93.8% of patients with pneumonia but was expressed in only 1 of 10 control subjects (Fisher exact test: P < 0.0001 for row-column association for expression of S1P3) (Figure 1, Table 2). A potentially confounding factor was the advanced age of some of the patients with pneumonia. However, five of the patients were less than 50 yr of age, and all of these patients had S1P 3 expression on their neutrophils; the one patient not to express S1P 3 was an 85-yr-old man. Three of the patients were retested between 1 and 5 wk postrecovery; all three (30, 58, and 69 yr of age) still had S1P3 expression on their neutrophils (data not shown).


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  		TABLE 2. NUMBERS OF SUBJECTS EXPRESSING S1P RECEPTOR SUBTYPES AT RNA LEVEL

 

Figure 1
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  		Figure 1. Expression of the S1P3 receptor is associated with pneumonia. RNA from neutrophils from 10 patients with pneumonia and 10 healthy control subjects was converted to cDNA, which was subjected to RT-PCR with primers specific to S1P 1, 2, 3, 4, or 5 or to beta-actin. Gels show S1P receptor cDNA expression profiles for a representative control subject and a patient with pneumonia or control PCR with the housekeeping gene beta-actin and negative control PCR for all primer pairs.

 
LPA 2 Is the Predominantly Expressed Neutrophil Lysophosphatidic Acid Receptor at the cDNA Level, but Additional LPA 1 Expression Is Associated with Pneumonia
We defined the expression profile of the LPA receptors LPA 1, 2, and 3 on cDNA from neutrophils of 10 patients with pneumonia and 10 healthy control subjects (Figure 2, Table 3). LPA 2 was expressed on neutrophils from 90–100% of all subjects (Figure 2, Table 3). By contrast, LPA 1 expression was observed in 93.8% of patients with pneumonia but in only 2 of 10 control subjects (Fisher exact test P < 0.0002 for row–column association for expression of LPA1) (Figure 2, Table 3). LPA3 was not represented on neutrophils from any subjects. Three of the patients were retested between 1 and 5 wk postrecovery; all three (30, 58, and 69 yr of age) still had LPA1 expression on their neutrophils (data not shown).


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  		TABLE 3. NUMBERS OF SUBJECTS EXPRESSING LPA RECEPTOR SUBTYPES AT RNA LEVEL

 

Figure 2
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  		Figure 2. Expression of the LPA1 receptor is associated with pneumonia. RNA from neutrophils from 10 patients with pneumonia and 10 healthy control subjects was converted to cDNA, which was subjected to RT-PCR with primers specific to LPA 1, 2, or 3, or to beta-actin. Gels show LPA receptor cDNA expression profiles for a representative control subject and a patient with pneumonia and negative control PCR for all primer pairs.

 
S1P 3 and LPA 1 Protein Are Expressed on Neutrophils of Patients with Pneumonia
Neutrophils from a greatly increased proportion of patients with pneumonia compared with control subjects expressed S1P 3 and LPA 1 receptors at the cDNA level. Therefore, protein expression of these receptors was examined for these two groups by Western blotting with antibodies against S1P 3 or LPA 1 or, as controls, against the commonly expressed S1P 1, S1P 4, or LPA 2. For a typical patient with pneumonia, strong expression of S1P 1, 3, and 4 and of LPA 1 and 2 was observed, which is consistent with cDNA/RNA expression profiles (Figures 3A–3C). By contrast, for typical control subjects, no S1P 3 or LPA 1 expression could be observed within the limits of detection of the respective antibodies, whereas S1P 1, S1P 4, and LPA 2 expression was readily detected (Figures 3A–3C). Protein expression profiles were consistent with cDNA/RNA expression profiles for each individual.


Figure 3
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  		Figure 3. Neutrophils from patients with pneumonia but not control subjects express S1P3 and LPA1 receptor protein. Neutrophil total protein (10 µg) from patients with pneumonia or control subjects was subjected to Western blotting with antibodies to S1P 1, 3, or 4 and LPA 1 or 2. (A) Western blots of total protein (10 µg) from a representative control subject and a patient with pneumonia probed with anti-S1P receptor antibodies against S1P 1, 3, or 4, or (B) with anti-LPA receptor antibodies against LPA 1 or 2. (C) The graph shows the number of subjects within control (filled bars) and pneumonia (open bars) groups expressing S1P 1, S1P 3, S1P 4, LPA 1, or LPA 2 receptor protein.

 
S1P and LPA Exert Significantly Greater Chemoattraction for Neutrophils of Patients with Pneumonia Compared with Control Subjects
To determine if S1P or LPA have a functional effect on neutrophils from control subjects, neutrophils were subjected to chemotaxis using increasing doses of S1P (Figure 4A) or LPA (Figure 4C) from 0.01–10 µM as chemoattractant. S1P and LPA are present in normal human serum at micromolar levels (2, 3). S1P and LPA were chemoattractant for neutrophils; however, S1P was a stronger chemoattractant than LPA (Figure 4A and 4C). The strongest chemoattraction was observed for S1P at 0.1 or 1 µM.


Figure 4
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  		Figure 4. S1P is a more effective neutrophil chemoattractant than LPA, and chemotaxis induced by S1P or LPA is significantly greater for neutrophils from patients with pneumonia than for control subjects. Peripheral blood neutrophils from control subjects (A–E) or patients with pneumonia (B, D, E) (5 x 105) were subjected to chemotaxis to the indicated concentrations of S1P (A, B) or LPA (C, D) or IL-8 (E) in vehicle (1x PBS, 0.1% BSA) for 2.5 h at 37°C, 5% CO2. The graphs show fold increase in chemotaxis over basal (vehicle only) levels. The graphs represent the results from three or four independent experiments. Data are mean ± SD. *P < 0.05 compared with vehicle with no chemoattractant (basal) (A, C) or mean ± SEM (B, D, E). *P < 0.05 compared with control subjects.

 
Because neutrophils from patients with pneumonia commonly expressed S1P 3 and LPA 1 receptor in contrast to control subjects (Figures 1–3 GoGo), it was determined whether S1P or LPA had different effects on the chemotaxis of neutrophils from patients with pneumonia compared with those from control subjects. For neutrophils used in these studies, it was confirmed that the patients with pneumonia expressed S1P 3 and LPA 1, whereas the control subjects did not (data not shown). Neutrophils were subjected to chemotaxis using increasing doses of S1P (Figure 4B) or LPA (Figure 4D) from 0.01–1 µM as chemoattractant. For all doses of S1P and LPA tested, significantly increased chemotaxis was observed for neutrophils from patients with pneumonia compared with neutrophils from control subjects (Figures 4B and 4D). Increases ranged from ~ 50- to 200-fold for S1P and from ~ 20- to 100-fold for LPA. This was in contrast to observations for IL-8–induced chemotaxis (Figure 4E), which was marginally but not significantly reduced for neutrophils of patients with pneumonia compared with control subjects.

Sphingosine 1-Phosphate or Lysophosphatidic Acid Pretreatment Reduced IL-8–induced Neutrophils Chemotaxis for Control Subjects and Patients with Pneumonia
To further elucidate the functional effects of S1P or LPA on neutrophils, cells isolated from control subjects or patients with pneumonia were pretreated with 0.1 µM S1P or LPA for 1 h in serum-free media before being subjected to chemotaxis to IL-8 (10 ng/ml). S1P and LPA significantly reduced IL-8–induced chemotaxis of control (Figure 5A) or pneumonia (Figure 5B) neutrophils to an average of 60% of that for non-pretreated cells.


Figure 5
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  		Figure 5. Pretreatment with S1P or LPA reduces chemotaxis of neutrophils to IL-8 in control subjects and in patients with pneumonia. Peripheral blood neutrophils (5 x 105 per well) from control subjects (A) or patients with pneumonia (B) (5 x 105) were not pretreated or were pretreated for 1 h with 0.1 µM S1P or LPA in vehicle before being subjected to chemotaxis to 10 ng/ml IL-8 for 2.5 h at 37°C, 5% CO2. The graphs show the percentage of IL-8–induced chemotaxis observed in LPA- or S1P-pretreated cells. The graphs represent the results from three to five independent experiments. Data are mean ± SD. *P < 0.05 compared with no pretreatment.

 
S1P and LPA Receptors Heterodimerize with the IL-8 Receptor CXCR1
To further define mechanisms of interaction of IL-8 and S1P- or LPA-induced signaling in neutrophils, cells from control subjects or patients with pneumonia were subjected to immunoprecipitation with an anti-CXCR1 antibody and subsequently to Western blotting with antibody against CXCR1 or against S1P 4 (all subjects), S1P 3, or LPA 1 (patients with pneumonia). CXCR1 formed constitutive homodimers with itself in neutrophils (Figure 6A) and constitutively heterodimerized with S1P 4 (Figure 6A). When studies were performed on neutrophils from patients with pneumonia, heterodimerization was observed between CXCR1 and S1P 3 and LPA 1 (Figure 6B). LPA pretreatment (1 µM, 15 min) reduced the formation of CXCR1-LPA 1 heterodimers to ~ 30% of constitutive levels in the patients with pneumonia.


Figure 6
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  		Figure 6. The IL-8 receptor CXCR1 forms heterodimers with S1P receptors 3 and 4 and LPA receptor LPA 1. Peripheral blood neutrophils (1.5 x 106) from control subjects (A) or patients with pneumonia (B) were immunoprecipitated with normal rabbit IgG (A and B), rabbit anti-human CXCR1 (A and B), rabbit anti-human S1P 3 (B), rabbit anti-human S1P 4 (A), or rabbit anti-human LPA 1 (B) and subjected to Western blotting with anti-CXCR1. Where indicated, cells were pretreated with S1P (1 µM) or LPA (1 µM) for 15 min at 37°C, 5% CO2. Two subjects were examined in each group. The figure shows one representative subject for each group. Open arrowheads indicate expected migration points of monomers; closed arrowheads indicate expected dimers. The graph shows the reduction of LPA1/CXCR1 heterodimerization compared with CXCR1 monomer after LPA (1 µM) pretreatment for 15 min at 37°C, 5% CO2.

 
Sphingosine 1-Phosphate and Lysophosphatidic Acid Reduce Transcription of the IL-8 Receptor CXCR1
To further examine how S1P or LPA reduce IL-8–induced neutrophil chemotaxis, the effects of both lysophospholipids on expression of CXCR1 was examined. Neutrophils from control subjects were incubated for 1 or 24 h in serum-free conditions in the presence of S1P (0.1 or 1 µM), LPA (0.1 or 1 µM), or vehicle (DMSO). RNA was extracted from cells and converted to cDNA for RT-PCR with primers specific to CXCR1 or the "housekeeping" gene beta-actin. Expression of CXCR1 was normalized to beta-actin, and the levels in S1P- or LPA- treated cells were compared with the corresponding vehicle-treated control. At the 1-h and 24-h time points, S1P induced a significant reduction in CXCR1 expression compared with vehicle (Figure 7A). LPA induced a significant reduction in CXCR1 expression compared with vehicle at 24 h (Figure 7B).


Figure 7
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  		Figure 7. S1P and LPA pretreatment reduce transcription of the IL-8 receptor CXCR1; S1P has a more rapid effect. Peripheral blood neutrophils (2.5 x 106) from control subjects were pretreated for the indicated times with 0.1 or 1 µM S1P (A) or 0.1 or 1 µM LPA (B) in serum-free conditions for 1 h or 24 h. Control cells were treated with vehicle (DMSO) for corresponding times. Cells were lysed in TRI reagent and RNA and cDNA made for RT-PCR using primers specific to the IL-8 receptor CXCR1 or to the housekeeping gene beta-actin. The level of CXCR1 was normalized to beta-actin, and normalized levels in LPA or S1P-treated cells are expressed as a percentage of the corresponding vehicle-treated cells. Results represent the mean of three or four independent experiments. Results are mean ± SD. *P < 0.05; **P < 0.005; ***P < 0.0005.

 

   DISCUSSION
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
In this study, the profile of S1P receptors S1P 1–5 and LPA receptors LPA 1–3 on peripheral blood neutrophils of patients with pneumonia and healthy control subjects was elucidated at both the cDNA and protein levels. Expression of p2y9, a fourth LPA receptor phylogenetically distant from LPA 1–3 (32), was not considered in this study. S1P 4 was ubiquitously expressed, whereas S1P 1 and 5 were commonly expressed on neutrophils of all subjects. S1P 3 expression was predominantly confined to peripheral blood neutrophils from patients with pneumonia. LPA 2 was expressed on the neutrophils of most subjects in both groups, whereas LPA 3 was not observed at the cDNA or protein level. LPA 1 expression was associated predominantly with peripheral blood neutrophils obtained from patients with pneumonia. S1P and LPA induced chemotaxis of neutrophils in serum-free medium, although S1P was a more effective neutrophil chemoattractant than LPA. S1P and LPA exerted stronger chemoattraction for neutrophils from patients with pneumonia than for control subjects. However, pretreatment of neutrophils from patients with pneumonia or control subjects with S1P or LPA at 0.1 µM strongly reduced chemotaxis of neutrophils to IL-8. Analysis of expression of the IL-8 receptor CXCR1 suggested that S1P and LPA reduced the transcription of CXCR1. Furthermore, S1P 3, S1P 4, and LPA 1 formed constitutive heterodimers with CXCR1. These data suggest a mechanism whereby S1P and/or LPA influence neutrophil chemoattractant responses.

In patients with pneumonia, expression of S1P 3 and LPA 1 on neutrophils was observed in addition to the more commonly expressed S1P and LPA receptors. This suggests that S1P and LPA may have unique effects on neutrophil migration and survival in pneumonia. Increased chemotaxis in response to S1P and to LPA was observed for neutrophils from patients with pneumonia compared with healthy subjects. S1P 3 is a high-affinity S1P receptor (33) that can couple via Gi, Gq, or G12/13 (5). S1P 3 activation has been associated with enhanced cell migration in transfected Chinese hamster ovary cells (34–36). S1P 3 is essential for S1P-induced human umbilical vein endothelial cell adhesion and migration (37), and decreased expression of S1P 3 in breast cancer cells is associated with decreased cell migration (38). These findings of S1P 3 receptor association with enhanced cell migration are consistent with our observation of increased S1P-induced chemotaxis in neutrophils from patients with pneumonia compared with neutrophils from healthy subjects. Similarly, LPA is consistently observed to induce cell migration via the LPA 1 receptor. In the DLD1 human colon carcinoma cell line, which expresses high levels of LPA 1, lysophosphatidic acid stimulates cell migration, whereas in other human colon carcinoma cell lines, which express LPA 2 but not LPA 1, LPA enhances cell proliferation and secretion but not migration or adhesion (39). In neuroblastoma cells, LPA binds to LPA 1 and activates Rac via the guanine nuclear exchange factor Tiam1 and stimulates cell motility (40). Pancreatic cancer cells with high migration activity to ascites express high levels of LPA 1, and their migration response to ascites is inhibited by small interfering RNA against LPA 1 (41). Thus, S1P 3 and LPA 1, which are consistently expressed on neutrophils of patients with pneumonia but not on neutrophils from control subjects, are associated with cell migration, consistent with our observations of enhanced S1P- and LPA-induced chemotaxis of neutrophils from patients with pneumonia versus neutrophils from healthy subjects. However, our results do not prove that expression of S1P3 and LPA1 directly causes the observed increases in pneumonia patient neutrophil chemotaxis to S1P and LPA; such proof requires access to specific blocking antibodies or inhibitors.

It is not known what causes the induction of S1P 3 and LPA 1 expression on neutrophils of patients with pneumonia. However, we observed that S1P3 and LPA1 were expressed on neutrophils of three patients with pneumonia 1–5 wk after resolution of their infection, suggesting that the presence of S1P3 and/or LPA1 on neutrophils may be a predisposing factor for pneumonia. These data are preliminary, and further studies are needed to determine if these receptors are present months after resolution and if, for example, polymorphisms in the S1P3 and/or LPA1 genes exist that may be linked to predisposition to pneumonia or if a prolonged expression of S1P3 and LPA1 is due in the first instance to factors present during pneumonia, such as induction of transcription factor upregulation by inflammatory cytokines (e.g., IL-1 and TNF) or neutrophil priming by factors such as GM-CSF. Elucidation of the mechanism of receptor expression induction will be the subject of future studies.

Consistent with the findings of others (21), we found that neutrophil pretreatment with S1P reduced chemotaxis to IL-8. Similarly, LPA pretreatment reduced IL-8–induced neutrophil migration. The reductions were of a similar magnitude for healthy and control subjects. This interference with IL-8–induced chemotaxis implies "cross-talk" between S1P and/or LPA receptors and the IL-8 receptors CXCR1 and/or CXCR2. We observed that S1P and LPA reduced the transcriptional expression of the IL-8 receptor CXCR1, which could contribute to the S1P- and LPA-mediated decreases in IL-8–induced chemotaxis. The effect of S1P was more rapid: A significant decrease in CXCR1 expression was evident after 1 h of S1P treatment.

We observed that the S1P receptors S1P 3, 4, and 5 and the LPA receptor LPA 1 could form endogenous heterodimers with the CXCR1 receptor. Physical dimerization between G-protein–coupled receptors has mainly been demonstrated in transfected cell lines (42–44), including homo- and heterodimerization of S1P 1, 2, and 3 (43). However, it was considered likely that endogenously expressed receptors would also form dimers and that this would influence receptor signaling and function (42–44). Our observed heterodimerization between CXCR1 and S1P 3, 4, and 5 is likely to contribute to the cross-talk in neutrophil chemotaxis between S1P and IL-8 in control subjects and in patients with pneumonia. Furthermore, the heterodimerization between LPA 1 and CXCR1 but not LPA 2 and CXCR1 suggests that this process is important in cross-talk between LPA and IL-8 in pneumonia, where LPA 1 was also observed to be prominently expressed on neutrophils but not in healthy subjects whose neutrophils generally express LPA 2 but not LPA 1. LPA treatment reduced the formation of LPA 1/CXCR1 heterodimers. This suggests that the presence of these heterodimers has a positive effect on IL-8–induced neutrophil migration and that reduction in their expression could contribute to a reduction in IL-8–induced chemotaxis, as we observed when neutrophils were pretreated with LPA. By contrast, in control subjects, whose neutrophils generally lack LPA 1, LPA-mediated reduction in IL-8–induced chemotaxis is more likely to be exclusively due to other mechanisms, such as reduced CXCR1 transcription. Ongoing studies in our laboratory are focusing on the examination of neutrophil migration over endothelial cell layers under flow conditions and the observation of the effects of S1P and LPA.

It is unclear whether S1P and/or LPA would promote or inhibit neutrophil chemotaxis in vivo in pneumonia. S1P 3 and LPA 1 are cell migration-enhancing receptors (34–38, 40, 41), and we have shown that S1P and LPA strongly induce chemotaxis of neutrophils of patients with pneumonia. Furthermore, we have demonstrated that S1P and LPA reduce IL-8–induced neutrophil chemotaxis. These factors could suggest that if S1P and LPA are present in serum, then they may promote intravascular retention of neutrophils. On the other hand, it is unknown what local levels of S1P and LPA are present in airways during episodes of pneumonia and whether these could tend to promote neutrophil chemotaxis or whether S1P or LPA receptor expression levels are changed during cell migration to the inflammatory site. Also, the cell lysis protocol used in this study would be unlikely to yield information on possible intracellular stores of receptor, such as the perinuclear stores of LPA 1 recently shown in porcine endothelial cells (45). These questions will be a subject of further research.

In summary, we have shown an altered pattern of S1P and LPA receptor expression on neutrophils of patients with pneumonia in comparison to healthy subjects. Our results suggest that S1P and LPA influence neutrophil recruitment in these inflammatory conditions. We propose a mechanism whereby these phospholipids inhibit IL-8 chemotaxis by demonstrating heterodimerization and transcriptional effects on chemokine receptors.


   Footnotes
 
This work was supported by the Health Research Board of Ireland and The Wellcome Trust.

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Originally Published in Press as DOI: 10.1165/rcmb.2005-0126OC on October 13, 2005

Received in original form April 7, 2005

Accepted in final form September 1, 2005


   References
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

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