Prostaglandin D2 Suppresses Human NK Cell Function via Signaling through D Prostanoid Receptor

Yingying Chen, Bice Perussia and Kerry S. Campbell
J Immunol September 1, 2007, 179 (5) 2766-2773; DOI: https://doi.org/10.4049/jimmunol.179.5.2766

Abstract

NK cells play critical roles in immune responses against tumors or virus infections by generating type 1 cytokine and cytotoxicity responses. In contrast, during type 2 dominant immune responses, such as allergic diseases, activities of NK cells are often impaired. These type 2 immune-mediated diseases have been reported to be closely associated with local production of PGD2. PGD2 is an eicosanoid primarily synthesized by mast cells and alveolar macrophages, and it functions through two major receptors, D prostanoid receptor (DP) and chemoattractant receptor-like molecule on the Th2 cell. Within the immune system, PGD2 binding to DP generally leads to suppression of cellular functions. In the current study, we show that: 1) DP is expressed in human NK cells as detected by mRNA analysis and Western blot; 2) PGD2 inhibits cytotoxicity, chemotaxis, and type 1 cytokine production of human NK cells via signaling through DP; 3) PGD2 signaling via DP elevates intracellular cAMP levels and the inhibitory effects on NK cells are cAMP dependent; 4) PGD2 binding to DP suppresses Ca2+ mobilization triggered by the cross-linking of the activating receptor, CD16. Together, these data uncover a novel mechanism by which PGD2 functions through DP to suppress type 1 and cytolytic functions of human NK cells, thus contributing to the promotion of a type 2 immune response.

The characteristics of an immune response are dictated by the nature of the antigenic stimulus, the types of immune cells participating in the response, and the cytokines produced by the cells. The majority of immune responses are classified in two groups; 1) type 1 immune responses that involve NK cells and type 1-biased T cells to produce cytokines, such as IFN-γ, IL-2, and IL-12, and 2) type 2 immune responses that involve eosinophils, basophils, and type 2-biased T cells to produce cytokines such as IL-4, IL-5, and IL-13 (1, 2, 3, 4, 5). A balance between type 1- and type 2-biased immune cells and cytokines must be maintained to mount an appropriate immune response. For example, development of a type 1 immune response generally involves recruitment of IFN-γ-producing NK cells to the site of inflammation and to draining lymph nodes (6, 7, 8), while functions of NK cells are often suppressed during type 2-dominated immune responses, such as asthma (9) and severe atopic eczema (10, 11, 12). Surface expression of adhesion molecules L-selectin and ICAM-1, which are critical for NK cell migration (13) and cytotoxicity (14), are reduced in asthmatic patients (15). Similarly, in a rat model of asthma induced by respiratory virus infection, NK cells from animals that are susceptible to asthma-like symptoms displayed reduced IFN-γ production during infection (16). It is therefore plausible that certain factors involved in type 2 immune responses have inhibitory effects on NK cell functions.

PGD2 is a critical factor in the development and pathogenesis of asthma (17, 18, 19), which is produced by alveolar macrophages and airway mast cells of asthmatic patients when they are exposed to allergen (17, 20, 21). PGD2 is a lipid mediator that has profound immunoregulatory functions (19, 22, 23) acting through binding to two receptors, D prostanoid receptor (DP)3 and chemoattractant receptor-like molecule on the Th2 cell (CRTH2) (19, 24). PGD2 binding to DP, a seven-transmembrane receptor coupled with the Gαs G protein subunit, elevates intracellular cAMP level and generates an inhibitory signal. By signaling through DP, PGD2 inhibits IL-12 production by dendritic cells (DC) (23, 25) and IFN-γ production by T cells (26). Moreover, PGD2 signaling through CRTH2, another seven-transmembrane receptor, coupled with the Gαi G protein subunit, induces type 2 cytokine production and migration of eosinophils and basophils (19, 22, 27). Through the downstream consequences of binding to these receptors, PGD2 may promote a type 2 immune response by suppressing type 1 and stimulating type 2 immune components.

It was also reported that PGD2 suppresses cytotoxicity of human PBMC against the K562 cell line, which is vulnerable to NK cell-mediated lysis (28, 29). However, it was not clear whether NK cells express receptors for PGD2 and whether PGD2 directly inhibits the cytotoxic function of NK cells or the inhibition is indirect and secondary to the impacts of PGD2 on other cell types in PBMC. In the current study, we provide the first evidence that human NK cells express DP, and PGD2 directly inhibits functions of NK cells upon signaling through this receptor. We speculate that inhibition of type 1 functions of NK cells by PGD2 could be a mechanism of skewing the immune response toward a type 2 dominant allergic disease.

Materials and Methods

Reagents

PGD2, BW245C (DP-specific agonist), DKPGD2 (CRTH2-specific agonist), and BWA868C (DP partial agonist) were purchased from Cayman Chemical. Adenosine 3′,5′-monophosphorothioate, 8-bromo-, Rp-isomer, sodium salt (8-Br-Rp-cAMP), and myristoylated protein kinase A inhibitor 14-22 amide (PKI14–22) were purchased from Calbiochem. Recombinant human CXCL12 was purchased from PeproTech. PMA, Ca2+ ionophore, etoposide, 3-isobutyl-1-methylxanthine, and brefeldin A were purchased from Sigma-Aldrich. Rabbit anti-DP1R polyclonal Ab was obtained from Cayman Chemical. FITC-conjugated annexin V, FITC-conjugated Abs (anti-CD161, -CD3, and -CXCR3), PE-conjugated Abs (anti-CCR7, -CXCR4, and -CXCR1), allophycocyanin-conjugated anti-CD56 Ab, allophycocyanin-conjugated anti-CRTH2 Ab, PerCP CyC5.5-conjugated-streptavidin (SA-PerCP-CyC5.5) and propidium iodide (PI) were purchased from BD Pharmingen. PE-conjugated anti-CX3CR1 was purchased from MBL International. Mouse anti-GAPDH Ab was obtained from Chemicon International. HRP-conjugated goat anti-mouse Ab was obtained from Jackson ImmunoResearch Laboratories. HRP-conjugated goat anti-rabbit Ab was obtained from Pierce. PE-conjugated Abs (anti-CD3, -CD56, -IFN-γ, -IL-13, -TNF-α, and mouse IgG1), FITC-conjugated Abs (anti-CD62L, -GM-CSF, and mouse IgG1), and goat anti-mouse IgG (H+L) were purchased from Caltag Laboratories. Fluo-3-penta-acetoxy-methylester (Fluo-3/AM) and pluronic F-127 were obtained from Molecular Probes/Invitrogen Life Technologies. Anti-CD16 (3G8) and -CD56 (B159.5.2) were produced in the laboratory as has been described (30). Anti-CD56 (Leu19) was purchased from BD Biosciences. Anti-NKp46 (9E2) and -2B4 (HD2) mAbs were provided by M. Colonna (Washington University, St. Louis, MO). Recombinant human (rh) IL-2 was obtained from Hoffman La Roche (provided by the Biological Response Modifiers Program, National Cancer Institute, Bethesda, MD). rhIL-12 was provided by Dr. S. Wolf (Genetics Institute, Andover, MA).

Cells

PBL were isolated from buffy coat samples obtained from the blood bank of Thomas Jefferson Hospital (Philadelphia, PA). Informed consent was obtained from all donors in accordance with protocols approved by the Institutional Review Board of Thomas Jefferson University (Philadelphia, PA). NK cells (>90% purity) were isolated from PBL by a negative selection method using MACS (Miltenyi Biotec). For RT-PCR, MACS-purified NK cells were further subjected to FACS in a high-speed MoFlo cell sorter (DakoCytomation) using FITC-conjugated anti-CD161, allophycocyanin-conjugated anti-CD3, and PE-conjugated anti-CD56 Abs to obtain highly purified (>99.5%) CD3−CD161+CD56+ NK cell populations. The human erythrocyte leukemia cell line, K562, and FcγRI-expressing mouse mast cell line, P815, were obtained from the Cell Culture Facility of Fox Chase Cancer Center. All cells were cultured in RPMI 1640 medium (Cellgro) containing 10% FCS (Atlanta Biologic) and l-glutamine (Invitrogen Life Technologies) at 37°C.

Evaluation of mRNA expression by RT-PCR

Total RNA was isolated from sorted NK cells using the RNeasy kit from Qiagen. cDNA was synthesized from 1 μg of RNA using Oligo dT primer (Promega), dNTP, and Moloney murine leukemia virus-derived reverse transcriptase. The resulting cDNA was further amplified with specific primers for DP, CRTH2, and β-actin using Taq polymerase (Fisher Scientific). Primers for DP (accession number: NM_000953) were: forward: 5′-GCTCTACTCCAGCCTCATGG-3′ (corresponding to nucleotides 705–724); reverse: 5′-CACCGGCTCCTGTACCTAAG-3′ (corresponding to nucleotides 1129–1148). Primers for CRTH2 (accession number: NM_004778) were: forward: 5′-AATCCTGTGCTCCCTCTGTG-3′ (corresponding to nucleotides 79–98); reverse: 5′-AAAGCACCAGGCAGACTTTG-3′ (corresponding to nucleotides 559–578). Primers for β-actin (accession number: NM_001101) were: forward: 5′-CCCAAGGCCAACCGCGAGAAG-3′ (corresponding to nucleotides 407–427); reverse: 5′-TCTTCATTGTGCTGGGTGCCA-3′ (corresponding to nucleotides 1032–1052). All primers were synthesized at the Fox Chase Cancer Center DNA Synthesis Facility. PCR was for a total of 30 cycles for DP, 35 cycles for CRTH2, and 20 cycles for β-actin. PCR products were separated on a 1.2% agarose gel.

Western blot

MACS-purified NK cells were lysed with cold lysis buffer (150 mM NaCl, 10 mM Tris (pH 7.4), 1 μg/ml soybean trypsin inhibitor, 1 μg/ml leupeptin, 1 μg/ml aprotinin, 10 nM NaF, 400 μM EDTA, 2 mM sodium orthovanadate, and 1 mM 4-(2-aminoethyl)benzenesulfonylfluoride) con-taining 1% Triton X-100 on ice for 30 min. The protein concentration of lysates was measured using a spectrometer (Bio-Rad Smartspec3000). Thirty micrograms of lysate was loaded in each lane and separated on 10% SDS-PAGE, transferred to polyvinylidene difluoride membrane (Millipore), and probed with 1 μg/ml rabbit anti-DP Ab followed by HRP-conjugated goat anti-rabbit IgG or 0.2 μg/ml mouse anti-GAPDH Ab followed by HRP-conjugated goat anti-mouse IgG. Immunoblotted proteins were visualized by chemiluminescence using the ECL detection reagents (Millipore).

Surface phenotyping and measurement of intracellular cytokine production

Total PBL were surface stained with FITC-conjugated anti-CD3 Ab, PE-conjugated anti-CD56 Ab, and allophycocyanin-conjugated anti-CRTH2 Ab. Viable cells, gated based on light scatter characteristics, were analyzed on a BD Biosciences FACSCalibur instrument. Data were analyzed using CellQuest software (BD Biosciences).

Total PBL or purified NK cells (5 × 106/ml) were stimulated either by incubating them on non-tissue-culture plates coated with anti-CD16 or anti-2B4 Abs (10 μg/ml) in the presence of rhIL-2 (100 U/ml) and rhIL-12 (10 ng/ml) or by incubating with PMA (2 × 10−9 M) and Ca2+ ionophore (0.1 μg/ml) in the presence of rhIL-2 (100 U/ml) for 5 h at 37°C. Brefeldin A (10 μg/ml) was added to the culture after the first 2 h of incubation to block cytokine secretion. After incubation, cells were stained with allophycocyanin-conjugated anti-CD56, PE-conjugated anti-CD3, or biotin-conjugated anti-CD3 Abs with SA-PerCP CyC5.5 followed by fixation and permeabilization as described previously (30). Subsequently, cells were stained with PE-conjugated Abs (anti-IFN-γ and -TNF-α), FITC-conjugated anti-GM-CSF, or corresponding PE- or FITC-conjugated mouse IgG1 isotype control. Intracellular cytokine expression was detected using flow cytometric analysis.

Cytotoxicity assay

Cytotoxicity of NK cells was assessed by a standard 4-h 51Cr-release assay as previously described (31). Briefly, K562 and P815 cell lines were used as target cells to assess the spontaneous killing and Ab-redirected killing by NK cells, respectively. For the Ab-redirected cytotoxicity assay, 51Cr-labeled P815 were preincubated with anti-CD56 (B159.2), anti-CD16 (3G8), and anti-NKp46 (9E2) Abs (1/10 dilution of supernatant collected from culture of hybridomas) for 10 min at room temperature before assay. Purified human NK cells, pretreated with either DMSO or ligands of PGD2 receptors at the indicated concentration for 15 min at 37°C, were used as effector cells. Specific lysis, as a measure of cytotoxicity of the NK cells, was calculated as percentage of lysis = (cpmsample − cpmspontaneous release)/(cpmmaximum release − cpmspontaneous release) × 100.

Chemotaxis assay

NK cells were suspended in RPMI 1640 supplemented with 2% FCS at a concentration of 5 × 106/ml. A total of 100 μl of cellular suspension was placed in the top chamber of 24-well plates separated with 5.0-μm pore polycarbonate transwell inserts (Corning). A total of 250 μl of RPMI 1640 plus 2% FCS, in the presence or absence of CXCL12 (100 ng/ml), was added in the lower chamber of the plates. The plates were incubated at 37°C for 2 h. Cells that migrated to the lower chamber were collected and counted under a microscope after the incubation period. Each assay was performed in triplicate and the results are expressed as the mean value ± SD of migrated cells.

Apoptosis assay

NK cells (3 × 106/ml) were incubated with DMSO, ligands of PGD2 receptors (100 nM), or etoposide (20 μM) at 37°C. At the end of incubation, cells were then washed with PBS and stained with FITC-conjugated annexin V and PI in annexin V-binding buffer (10 mM HEPES/NaOH (pH 7.4) and 140 mM NaCl and 2.5 mM CaCl2) according to the manufacturer’s instructions (BD Pharmingen) before flow cytometry analysis.

cAMP detection assay

cAMP was measured using a competitive protein-binding assay. NK cells were incubated with DMSO or PGD2 receptor ligands for various times at 37°C. 3-Isobutyl-1-methylxanthine (1 mM) was added to the culture 15 min before the treatment to prevent degradation of cAMP. Cells were then lysed by three rounds of freezing and thawing. Subsequently, proteins were removed by boiling samples for 3 min before centrifugation at 10,000 × g for 10 min. Supernatants were then collected and the concentrations of cAMP were determined using a cAMP detection kit following instructions from the manufacturer (Amersham Biosciences).

Ca2+ mobilization assay

PBL were washed and resuspended in serum-free HBSS at a concentration of 1 × 107/ml. Cells were labeled with 2 μM Fluo-3/AM in the presence of a 3 μl/ml 20% w/v stock solution of pluronic F-127 at room temperature for 20 min. Cells were then diluted to 2 × 106/ml with HBSS containing 1% FCS and incubated for another 40 min at 37°C. Fluo-3/AM-loaded cells were surface stained with biotin-conjugated anti-CD3 Ab, allophycocyanin-conjugated anti-CD56 Ab and SA-PerCP CyC5.5. Cells were then resuspended in HBSS containing 1% FCS and fluorescence acquisition, on a BD Biosciences FACSCalibur flow cytometry instrument, was performed. Ca2+ mobilization was induced by adding the indicated stimulus and monitored by FACS gating on CD3−CD56+ NK cells. Fluo-3/AM was detected by a blue laser with excitation at 488 nm. Data were analyzed using FlowJo software (Tree Star).

Results

DP is expressed on human NK cells

We first examined the expression of PGD2 receptors on NK cells. RT-PCR analysis on freshly isolated human primary NK detected moderate levels of mRNA transcript specific for DP, and a very low amount of transcript for the CRTH2 (Fig. 1⇓A). Compared with total PBL, NK cells express a higher level of DP transcript and a lower level of CRTH2 transcript.

FIGURE 1.
FIGURE 1.

Expression of DP in NK cells. A, mRNA of DP and CRTH2 was detected in PBL and sort-purified CD3−CD56+ NK cells using RT-PCR. Different dilutions of cDNA template (as indicated in the figure) were used for PCR to semiquantitatively compare the amounts of mRNA transcripts of DP and CRTH2 in each population. Levels of mRNA specific for housekeeping gene β-actin were also assessed to compare the amounts of cDNA in the samples. B, DP protein was detected in primary human NK cells by Western blot. NK cells from three donors were lysed and 30 μg of lysate from each sample was loaded and immunoblotted with anti-DP and -GAPDH Abs. Immunoblot of GAPDH was used as control to ensure that similar amounts of lysates were used. C, Fresh purified PBL was analyzed by FACS for surface expression of CRTH2. NK cells were gated as CD56+CD3− while T cells were gated as CD3+ (left panel). Histogram shows the staining of NK and T cells with specific mAb against CRTH2 in comparison with the isotype control. Each figure is representative of at least three independent experiments.

Western blot analysis further confirmed the presence of DP protein in primary NK cell lysates (Fig. 1⇑B). Unfortunately, the only available anti-DP Ab preparation is polyclonal and resulted in high background staining of NK cells when used for FACS analysis (data not shown). Therefore, it was impossible to establish whether all NK cells express DP. FACS analysis using a CRTH2-specific Ab showed that only a very minor fraction of NK cells (0.15 ± 0.12%, n = 7) express CRTH2 on the cell surface, while a small subset of T cells (1.5 ± 0.72%, n = 5) was stained positive for CRTH2, consistent with previous reports in the literature (32) (Fig. 1⇑). These results demonstrated substantial levels of DP expression in human NK cells while CRTH2 is only expressed on the surface of a minor fraction of NK cells.

PGD2 inhibits cytotoxicity of human NK cells via DP signaling

Because cytotoxicity is a major function of NK cells, next we investigated whether PGD2 has any direct effect on the cytotoxic function of NK cells. MACS-purified primary human NK cells were treated with different concentrations of PGD2 for 15 min before testing their capacity to spontaneously lyse target cells. Preincubation with PGD2 inhibited cytotoxicity of NK cells in a dose-dependent manner (Fig. 2⇓A). Similar inhibition of cytotoxicity was observed when PGD2 was added to NK cell cultures together with target cells. However, addition of PGD2 10 min after target cells did not have any effect (data not shown). These results suggest that PGD2 inhibits NK cell cytotoxicity directly and the inhibition might occur by affecting early functions of activating receptors and/or adhesion molecules.

FIGURE 2.
FIGURE 2.

Effects of PGD2 on cytotoxicity of NK cells. The effects of PGD2 receptor agonists on the cytotoxicity of purified NK cells in spontaneous killing of K562 target cells (A and B) and in the Ab redirected killing of P815 target cells (C). Cytotoxicity of NK cells treated with different concentrations of PGD2 or the vehicle DMSO, at fixed E:T cell ratios (10:1) (A) or with a fixed concentration 100 nM PGD2, BW245C, and DKPGD2 or 500 nM BWA868C but a varying E:T ratio using either K562 (B) or P815 target cells (C) was assessed as described in Materials and Methods. Mean values ± SD are shown, n = 3. ∗, p < 0.05, analyzed using one-way ANOVA. Data represent at least three independent experiments.

Next, we tested which PGD2 receptor, DP or CRTH2, mediated the inhibitory effect on NK cells. An agonist of DP (BW245C) inhibited the cytotoxicity of NK cells, but not an agonist of CRTH2 (DKPGD2) (Fig. 2⇑B, left panel). Moreover, pretreatment with a partial agonist of DP, BWA868C, inhibited cytotoxicity of NK cells to a lesser extent than that achieved with PGD2 alone, and partially reversed the PGD2-mediated inhibition (Fig. 2⇑B, right panel). Similar inhibition of killing was observed in Ab-redirected cytotoxicity assays, where anti-CD16 or anti-NKp46 Abs were used as surrogate ligands to engage NK cell surface receptors (Fig. 2⇑C).

PGD2 inhibits cytokine-producing function of NK cells via DP signaling

To further investigate the effects of PGD2 on human NK cells, we tested whether PGD2 affects the production of type 1 cytokines. Total PBL were pretreated with PGD2 or PGD2 receptor agonists and then stimulated with a combination of Ca2+ionophore and PMA to induce cytokine production. Pretreatment with PGD2 and DP agonist (BW245C), but not with the CRTH2 agonist (DKPGD2), significantly reduced the percent of IFN-γ-, TNF-α-, and GM-CSF-producing NK cells when compared with DMSO-pretreated cells (Fig. 3⇓A). Similar inhibition of IFN-γ production by NK cells was also observed when PGD2- and BW245C-pretreated cells were stimulated with immobilized Abs against the activating receptors CD16 or 2B4 (Fig. 3⇓B). In both cases, inhibition by PGD2 was partially reversed by the partial agonist, BWA868C (Fig. 3⇓B). Cytokine production by MACS-purified NK cells was similarly inhibited by PGD2 and DP agonist (BW245C) (data not shown).

FIGURE 3.
FIGURE 3.

PGD2 inhibits cytokine production by human NK cells through DP. Total PBL was incubated with PGD2, BW245C, DKPGD2 (all at 100 nM), and BWA868C (500 nM) or vehicle (DMSO) alone for 15 min at 37°C before stimulation with either PMA and Ca2+ ionophore in the presence of IL-2 (100 U/ml) (A) or plate-bound anti-CD16 (top row) or anti-2B4 (bottom row) Abs in the presence of IL-2 (100 U/ml) and IL-12 (10 ng/ml) (B) for 5 h. Cytokine-producing CD56+CD3− NK cells was detected by intracellular staining for IFN-γ, GM-CSF, and TNF-α as described in Materials and Methods. In A, percentage of cytokine-producing NK cells treated with PGD2 receptor agonists are normalized by taking the percent of cytokine-producing cells treated with DMSO as 100. Statistical significance of the differences were measured using one-way ANOVA. Mean ± SD are shown, n = 3. ∗, p < 0.05. Histograms inserted in A show cytokine production from DMSO-pretreated NK cells. Numbers in each inset indicate the percentage of cytokine-producing NK cells. Numbers in B are the percentage of cytokine-producing NK cells under each condition. All data are representative of three independent experiments.

PGD2 inhibits chemotaxis of NK cells via DP signaling

PGD2 signaling mediated through DP has been shown to inhibit chemotaxis in monocytes (24, 33). Therefore, we next tested whether PGD2 also affects the migration of NK cells. Chemotaxis of NK cells was determined by estimating the number of cells migrating along a chemokine CXCL12 concentration gradient across a porous membrane in 2 h. NK cells, either treated with CRTH2 agonist (DKPGD2) or DMSO, migrated across the membrane in the presence of a concentration gradient of the chemokine (Fig. 4⇓). Treatment with PGD2 or DP agonist (BW245C), however, blocked this CXCL12-driven migration of NK cells (Fig. 4⇓).

FIGURE 4.
FIGURE 4.

Effect of PGD2 on the chemotaxis of NK cells. NK cells were incubated with 100 nM PGD2, BW245C, DKPGD2, or vehicle DMSO and the numbers of cells that crossed a 5.0-μm pore polycarbonate membrane with or without the presence of a CXCL12 (100 ng/ml) gradient were counted to assess their migration as described in Materials and Methods. Mean values ± SD are shown, n = 3. ∗, p < 0.05, analyzed in one-way ANOVA. Data are representative of three independent experiments.

We tested whether the inhibition of chemotaxis is due to the altered expression level of chemokine receptors on NK cells after treatment with PGD2 or DP agonist. NK cells were incubated with 100 nM PGD2, DP agonist (BW245C), CRTH2 agonist (DKPGD2), or DMSO for a period of 2 h. At the end of incubation, we used flow cytometry to examine the surface expression of the CXCL12 receptor, CXCR4, some other chemokine receptors, and an adhesion molecule that are known to be present on NK cells (34, 35). No changes in the cellular surface expression of CXCR4, CXCR3, CCR7, CCR1, CX3CR1, or CD62L were detected after PGD2 or BW245C treatment (data not shown).

PGD2 does not inhibit NK cell functions through induction of apoptosis

PGD2 can inhibit cell growth and induce apoptosis in cells at concentrations higher than 10−5 M (36, 37). To test whether the inhibitory effects of PGD2 on NK cell functions observed above were the result of induction of apoptosis in these cells, we assessed the viability of NK cells after treatment with PGD2. Following 5 or 18 h of incubation with PGD2, BW245C, or DKPGD2 at the concentration of 10−7 M, the highest concentration used in our experiments, <10% of NK cells underwent apoptosis, similar to sham-treated control cells. In contrast, when cells were treated with an apoptosis-inducing agent, etoposide, 30–40% of NK cells underwent apoptosis within 18 h of incubation (Table I⇓).

View this table:
Table I.

Effect of PGD2 on viability of NK cellsa

PGD2 inhibits functions of NK cells via a cAMP-dependent pathway

DP is coupled to the G-protein subunit, Gαs, activation of which elevates cAMP levels in the cells (24). If the inhibitory effects of PGD2 on NK cells are mediated through DP signaling, PGD2 treatment of NK cells should increase the intracellular cAMP levels in these cells. To test this, we compared the intracellular cAMP level of control and PGD2 receptor agonist-treated NK cells. PGD2 increased cAMP levels of NK cells within 1 min of incubation. The level of cAMP peaked at 10 min and was sustained for at least 30 min (Fig. 5⇓A). Dose-dependent elevation of cAMP was observed in both PGD2 and DP agonist (BW245C) treated NK cells (Fig. 5⇓B).

FIGURE 5.
FIGURE 5.

DP mediates inhibition via a cAMP-dependent pathway. NK cells were treated with DMSO or 100 nM PGD2 in DMSO for 1, 5, 10, and 30 min (A) or PGD2 and BW245C at indicated concentration for 5 min at 37°C (B). Intracellular concentration of cAMP following stimulation was determined as described in Materials and Methods. In C, NK cells were incubated with various concentration of cAMP antagonist 8-Br-Rp-cAMP for 20 min at 37°C before addition of DMSO, 100 nM PGD2, or 100 nM BW245C in DMSO to these cells. After another 15 min of incubation, cells were used as effector cells in cytotoxicity assays against K562 cells at an E:T ratio of 10:1. The percentages of lysis were calculated and normalized to the DMSO-treated control. Data are representative of three independent experiments.

If the effects of PGD2 on NK cells are mediated through cAMP signaling, we predicted that inhibition of the cAMP pathway should reverse the functional inhibition caused by PGD2. 8-Br-Rp-cAMP is a membrane-permeable cAMP analog that competes with cAMP and inhibits activation of its downstream signaling molecules (38). Pretreatment with 8-Br-Rp-cAMP dose-dependently reversed the inhibitory effects of PGD2 or DP agonist (BW245C) on the cytotoxicity of NK cells (Fig. 5⇑C).

PGD2 binding to DP hinders Ca2+ mobilization triggered by CD16 ligation

PGD2 binding to DP has been shown to trigger an increase in the concentration of intracellular Ca2+ in certain cell types (24, 39). In the current study, PGD2, BW245C, or DKPGD2 treatment did not directly trigger any appreciable increase in the intracellular Ca2+ concentration in human NK cells over DMSO-treated cells (Fig. 6⇓A). We next tested, however, whether PGD2 signaling affects Ca2+ mobilization in NK cells that is induced by another stimulus. CD16 is an activating receptor present on human NK cells that can be stimulated by ligation with Abs (40). Cross-linking of CD16 triggered a rapid elevation in cytoplasmic Ca2+ concentration ([Ca2+]i), which is characteristic of the Ca2+ release from intracellular stores, followed by a sustained elevated level of [Ca2+]i, which is characteristic of Ca2+ influx from the extracellular medium (40). Treatment with PGD2 or DP agonist (BW245C) blunted the peak release of Ca2+ and significantly diminished the sustained phase, as compared with treatment with DMSO or CRTH2 agonist (DKPGD2) (Fig. 6⇓B).

FIGURE 6.
FIGURE 6.

Effects of PGD2 on CD16-stimulated Ca2+ mobilization in NK cells. A, PGD2, BW245C, or DKPGD2 (100 nM) in DMSO were added (arrow 1) to Fluo-3/AM-labeled NK cells and, after 7 min, Ca2+ ionophore (50 μg/ml) was added (arrow 2). Mean basal level of [Ca2+]i was measured for 1 min before any treatment. B, Fluo-3/AM-labeled NK cells were incubated with 100 nM PGD2, BW245C, DKPGD2 in DMSO or DMSO alone at 37°C. Followed by a 15-min incubation, the basal level of [Ca2+]i was measured for 1 min and then 10 μg/ml anti-CD16 (arrow 3) was added. A total of 50 μg/ml goat anti-mouse IgG (arrow 4) was added 4 min later to cross-link CD16. [Ca2+]i was continuously monitored for another 5 min. Results are representative of two independent experiments.

Discussion

PGD2 is known to induce a type 2 dominant immune response by stimulating immune cells such as type 2 T cells, eosinophils, and basophils (19). In this study, we have demonstrated that PGD2 also inhibits human NK cell functions that are involved in a type 1 immune response and this inhibition is mediated by DP expressed on human NK cells through a cAMP-dependent pathway. The suppressing effect of PGD2 on type 1 NK cells together with stimulatory effects on type 2 immune cells can lead to an overall bias toward a type 2 immune response. PGD2 can have pleiotropic effects on immune cells because different cell types express different receptors. Although inhibitory effects of PGD2 on NK cells are mediated by DP, stimulatory effects on type 2 cells are mediated via CRTH2 (19, 22).

Our results demonstrated that BW245C, a DP agonist, has similar inhibitory effects as PGD2 on the functions of NK cells. In contrast, the CRTH2 agonist DKPGD2 did not have any effect on these cells. This indicates that signaling via DP is dominant in human peripheral blood NK cells. We also found that BWA868C, a partial agonist of DP, inhibits the effect of PGD2 on NK cells to a certain extent. BWA868C binds to DP with similar affinity as PGD2 but only produces a partial physiologic response. Therefore, when used together, BWA868C competes with PGD2 to bind with DP and blocks the effect of PGD2. Currently, a DP-specific full antagonist is not available. The results that BWA868C slightly inhibits NK cell functions when used alone and reverses the inhibition by PGD2 when used in combination further confirm the involvement of DP in meditating the inhibition by PGD2.

The observation that inhibition only occurs if NK cells are treated with PGD2 before but not after encountering target cells allows us to speculate that PGD2 inhibits very early events associated with activation receptor signaling. Both spontaneous cytotoxicity against K562 target cells and Ab-redirected cytotoxicity triggered by activation of CD16 or NKp46 are mediated by release of cytolytic granules, a process that includes conjugation of NK and target cells, polarization of granules inside NK cells, and release of the granules at the NK-target cell interface (41, 42). Studies have suggested that elevation of intracellular cAMP in NK cells inhibits their binding to target cells (43). Consistent with these reports, our present findings show that PGD2 binding to DP enhances cAMP levels in NK cells (Fig. 5⇑) and inhibits the conjugation between NK and target cells (data not shown). At this point, we do not know whether PGD2 also influences other mechanisms of cytotoxicity by NK cells, such as Fas-FasL or TRAIL-TRAILR interactions.

We have also found that pretreatment with PGD2 suppressed cytokine production by NK cells stimulated either via activation of CD16 or 2B4 receptors on the NK cell surface or by addition of PMA and Ca2+ ionophore to these cells. Activation of CD16 or 2B4 receptors triggers cytokine production via ITAM-dependent and -independent pathways, respectively, while PMA and Ca2+ ionophore activate NK cells downstream from receptors at the levels of protein kinase C and Ca2+ signaling (44, 45, 46). This indicates that the inhibitory effect of PGD2 is not restricted to receptor desensitization and receptor proximal signals but may target a process that is involved in cytokine production in general. Other PGs, such as PGE2 and 15-deoxy-Δ(12, 14)-PGJ2, a metabolite of PGD2, have also been reported to inhibit IFN-γ production by NK cells (47, 48, 49). NK cells also produce two important inflammatory mediators TNF-α and GM-CSF that regulate maturation and activities of other immune cells, such as DCs and monocytes (50). Production of these two cytokines by NK cells was also inhibited by PGD2. Our study also showed that PGD2 inhibits chemotaxis of NK cells driven by CXCL12, a chemokine that directs homing as well as tissue infiltration of NK cells (51). Therefore, PGD2 does not only inhibit type 1 NK cell responses, but also suppresses migration of NK cells, which could effectively exclude them from the site of inflammation. This could be a mechanism to selectively enrich type 2 cells and skew the balance of the immune response in the local environment.

Our data also indicate that the inhibitory effects of PGD2 via DP signaling on cytotoxicity, cytokine production, and chemotaxis of NK cells is mediated by cAMP. We demonstrated here that signaling through DP, which is known to couple to the Gαs heterotrimeric G protein subunit, increases the intracellular cAMP level in NK cells. Moreover, 8-Rp-Br-cAMP, a cAMP antagonist, blocks the inhibitory effects of PGD2. Other cAMP-elevating agents, such as PGE2, are also known to inhibit the activities of NK cells (52, 53, 54). Although increase in cytosolic cAMP can result in the activation of protein kinase A (PKA), inhibition of the PKA pathway using a PKA inhibitor, PKI14–22, only slightly reversed the inhibitory effects of PGD2 in NK cells (data not shown). This result is consistent with a PKA-independent pathway of inhibitory signaling through DP, which has previously been reported in DCs (25). In primary human T cells and macrophages, gene expression and phagocytosis can also be regulated by exchange protein activated by cAMP (Epac), a downstream element of the cAMP pathway that is independent of PKA activation (55, 56, 57). A similar mechanism may also be involved in PGD2-induced inhibition of NK cell functions, which will require further investigation.

Depending on the source of signaling and cell type, cAMP can either enhance or suppress Ca2+-signaling responses (58, 59). In our study, PGD2 and DP agonist (BW245C) inhibited elevation in [Ca2+]i of NK cells triggered by cross-linking CD16. The general blunting of the response suggests that both the release of intracellular Ca2+ storage and influx of Ca2+ from the extracellular medium were inhibited by PGD2. Activation of CD16 induces tyrosine phosphorylation of phospholipase C-γ and subsequent hydrolysis of phosphatidylinositol 4,5-bisphosphate, which leads to the release of intracellular Ca2+ stores (60). An elevation of cytosolic Ca2+ is required for both granule release and cytokine production triggered via CD16 (40, 45). Suppression of Ca2+ signaling would be an important receptor-proximal mechanism to inhibit NK cell functions by PGD2. Besides inhibition in the receptor-proximal Ca2+ response, our results indicate that PGD2 also inhibits NK cell function at a level that is downstream from Ca2+ mobilization because cytokine production triggered by Ca2+ ionophore plus PMA was also reduced in PGD2-treated cells.

In summary, our study showed expression of DP on human NK cells and demonstrated that through binding to DP, PGD2 inhibits cytotoxicity, type 1 cytokine production, and chemotaxis of human NK cells. The level of PGD2 in bronchoalveolar lavage of asthmatic patients increases dramatically after acute exposure to allergen (61, 62). Clinical studies have shown that NK cells collected from asthmatic patients after acute allergen challenge exhibit decreased cytotoxicity (9). This reduction in cytotoxic function might be due to the inhibitory effect of PGD2-DP signaling on NK cells. In the present study, PGD2 inhibited functions of NK cells at a dose as low as 10 nM, which is close to the concentration of PGD2 in bronchoalveolar lavage (1 nM) from asthmatic patients after allergen challenge (62). Furthermore, studies have shown that DP knockout mice fail to develop an asthma-like phenotype (63). We speculate that PGD2-mediated suppression of type 1 NK cell activities and prevention of NK cells from migrating toward the site of inflammation may contribute to promoting a type 2-biased immune response in asthma. These findings define a novel immunoregulatory mechanism that may have considerable clinical significance in immune-mediated diseases.

Acknowledgments

We thank Dr. M. Colonna for reagents; Drs. L. Sigal and G. Rall for helpful comments on the manuscript. We also thank Dr. B. Mookerjee and the staff from the Blood Bank at Thomas Jefferson University for providing buffy coat samples; and the following facilities within Kimmel Cancer Center and Fox Chase Cancer Center for reagents and technical support: flow cytometry, DNA sequencing, DNA synthesis, and cell culture. This manuscript is dedicated to Dr. Bice Perussia: death took her away from us too soon, but her memories are still with us as a constant source of inspiration.

Disclosures

The authors have no financial conflict of interest.

Footnotes

  • The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

  • ↵1 This work was supported by U.S. Public Health Service Grant AI055842 (to B.P.) and National Institutes of Health Centers of Research Excellence Grant CA06927 (Fox Chase Cancer Center).

  • ↵2 Address correspondence and reprint requests to Dr. Kerry S. Campbell, Institute for Cancer Research, Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111. E-mail address: kerry.campbell{at}fccc.edu

  • ↵3 Abbreviations used in this paper: DP, D prostanoid receptor; CRTH2, chemoattractant-receptor-like molecule on the Th2 cell; DC, dendritic cell; SA, streptavidin; rh, recombinant human; PI, propidium iodide; Fluo-3/AM, fluro-3-penta-acetoxymethylester; [Ca2+]i, cytosolic Ca2+ concentration; PKA, protein kinase A.

  • Received April 5, 2007.
  • Accepted June 22, 2007.

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