Novel Function of CRTH2 in Preventing Apoptosis of Human Th2 Cells through Activation of the Phosphatidylinositol 3-Kinase Pathway

Prostaglandin D₂ is the major prostanoid released from mast cells (1) and is also produced in substantial quantities by Th2 lymphocytes (2, 3). It has been detected in high concentrations at sites of allergic inflammation and mediates its effects through high affinity interactions with D prostanoid receptor (DP)² 1 (2) and chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2; also named DP₂). DP₁ is expressed by vascular smooth muscle and some leukocyte populations, and activation of this receptor leads to increased blood flow, consequent congestion in allergic tissue (4, 5), and modulation of both eosinophil and basophil functions (6, 7, 8). Through activation of CRTH2, PGD₂ promotes the migration of eosinophils, basophils, and Th2 cells (6, 9, 10) and has the unusual ability to stimulate Th2 cytokine production (11) even in the absence of allergen recognition or costimulation (12). Initial studies with CRTH2 knockout mice (13) suggested that CRTH2 might constrain Th2 cytokine production and promote Th1 cytokine production, but this is likely to be due to CRTH2 expression by Th1 cells in mice and the particular immunization procedures used. More recent studies with selective CRTH2 antagonists and genetically deficient mice have highlighted an important role for CRTH2 in promoting the accumulation of lymphocytes and eosinophils (14, 15) and the production of Th2 cytokines (16) and IgE (14, 16) at sites of allergic inflammation. The PI3K and calcineurin pathways are involved in mediating the responses of Th2 cells to CRTH2 activation (17). It is well known that, in addition to its roles in cell motility and activation (18), the PI3K pathway is critical in regulating T cell survival and proliferation (19, 20). T cell survival and death, especially apoptosis, are believed to be important for lymphocyte homeostasis and self-tolerance in the immune system (21, 22). Too little cell death of activated lymphocytes can result in autoimmune or allergic disorders, whereas too much apoptosis can lead to immunodeficiency. We have therefore investigated the role of CRTH2 in the apoptosis of Th2 cells that may be relevant to the pathogenesis of asthma where a defect in T cell apoptosis has been proposed as a central feature resulting in chronic airway inflammation (23, 24, 25).

We found that PGD₂ significantly suppressed the apoptosis of Th2 cells induced by IL-2 deprivation. This effect was mediated by CRTH2-dependent activation of the PI3K pathway.

PGD₂ was purchased from Biomol. 13,14-Dihydro-15-keto-PGD₂ (DK-PGD₂), BW245C, BW868C, SQ29548, and ramatroban were from Cayman Chemical. TM30089 was supplied by ChemieTek. LY294002, recombinant human Fas ligand (FasL), t-butoxycarbonyl-Asp-fluoromethyl ketone (BAF), N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (Z-VAD), N-benzyloxycarbonyl-Ile-Glu-Thr-Asp-fluoromethyl ketone (Z-IETD), and a cytochrome c (cyt c) release apoptosis assay kit were obtained from Calbiochem. The human CD4⁺ T cell isolation kit II, an anti-human CRTH2 microbead kit, a human CD4⁺CD25⁺ regulatory T cell isolation kit, a T cell activation/expansion kit, anti-CD4-PE, anti-CD25-allophycocyanin, anti-CRTH2-PE, anti-FoxP3-PE, and a FoxP3 staining buffer set were from Miltenyi Biotec. X-VIVO 15 medium was purchased from Lonza. Recombinant human IL-2 and recombinant human IL-4 were from eBioscience. PE-annexin V, propidium iodide (PI), and annexin-binding buffer were obtained from Invitrogen. All Abs were supplied by New England Biolabs except for the mAb to CD95 Fas (clone 7C11), which was from Immunotech, the Abs to p-BAD (Ser¹³⁶) and β-tubulin, which were from Santa Cruz Biotechnology, and Alexa Fluor 647 mouse anti-cyt c and rabbit anti-caspase-8, which were from BD Pharmingen. Other chemicals were from Sigma-Aldrich.

Human CRTH2⁺CD4⁺ Th2 cells were prepared using a modified method described previously (3). Briefly, PBMC were isolated from buffy coats from healthy blood donors whose atopic status was unknown (National Blood Service, Bristol, U.K.) by Ficoll Hypaque (Amersham Biosciences) density gradient centrifugation, followed by CD4⁺ cell purification using MACS CD4⁺ T cell isolation kit II (Miltenyi Biotec). After 7 days of culture in X-VIVO 15 medium containing 10% human serum, 50 U/ml IL-2, and 100 ng/ml IL-4, CRTH2-positive cells were isolated from the CD4⁺ cultures by positive selection using an anti-human CRTH2 microbead kit. The harvested CD4⁺ CRTH2⁺ cells were treated as Th2 cells and were further amplified by stimulation with a T cell activation/expansion kit (Miltenyi Biotec) and grown in X-VIVO 15 medium containing 10% human serum and 50 U/ml IL-2 before use.

Human CD4⁺CD25⁺ regulatory T cells (Treg) were isolated with buffy coats by Ficoll Hypaque density gradient centrifugation, followed by Treg cell separation using a human CD4⁺CD25⁺ regulatory T cell isolation kit (Miltenyi Biotec). The cells were cultured in X-VIVO 15 medium containing 10% human serum and 50 U/ml IL-2 before use.

The purity of the Th2 and Treg cells was confirmed by fluorescent staining with anti-CD4-PE, anti-CD25-allophycocyanin, anti-CRTH2-PE, or anti-FoxP3-PE according to the supplier’s instructions (Miltenyi Biotec) followed by analysis using a FACSArray flow cytometer (BD Bioscience).

Th2 or Treg cells were harvested after different treatments and transferred to annexin-binding buffer followed by incubation with PE-annexin V/PI at room temperature for 15 min according to the manufacturer’s instructions (Invitrogen). The stained cells were analyzed by a FACSArray flow cytometer. The cells with annexin V positive and PI negative were counted as apoptotic cells.

Th2 cells, after treatment, were washed with PBS and loaded into a poly-l-lysine-coated, 8-well chamber slide (Lab-Tek). After a short spin to attach the cells, the cells were fixed with 5% formaldehyde in PBS, permeabilized with precold (−20°C) 90% methanol, blocked with 3% BSA in PBS and then probed with Alexa Fluor 647 mouse anti-cyt c followed by staining with 2.5 μg/ml Hoechst 33342 in PBS. The slides were observed on a Leica DMRXA fluorescence microscope and images were recorded with Openlab 3.0.9 software (Improvision).

The assay was performed using a cyt c release apoptosis assay kit according to the manufacturer’s protocol (Calbiochem). Briefly, Th2 cells, after treatment as indicated in the results, were washed and resuspended in a cytosol extraction buffer. After 10 min of incubation in ice, the cells were homogenized using a tissue grinder. Supernatants were collected after 10 min of centrifugation at 700 × g for a further centrifugation at 10,000 × g for 30 min. The supernatants after the second centrifugation were treated as cytosolic fractions, and the pellets resuspended in a mitochondrial extraction buffer were treated as mitochondrial fractions. The fractions were analyzed with Western blotting for cyt c.

Human Th2 cells were treated under different conditions as indicated in Results for 20 min (for Akt), 30 min (for BAD), 7 h (for caspase 8), or 16 h (for caspase 3 or poly (ADP-ribose) polymerase (PARP)). The cells were washed once with PBS and then solubilized in lysis buffer (20 mM Tris-HCl (pH 7.4), 250 mM sucrose, 1 mM EDTA, 1 mM EGTA, 1 mM sodium orthovanadate, 10 mM sodium glycerophosphate, 50 mM sodium fluoride, 5 mM sodium pyrophosphate, 0.1% 2-ME, protease inhibitor mixture, and 1% Triton X-100). The samples from the above treatments or from cyt c release apoptosis assay were fractionated by SDS-PAGE and then electrophoretically transferred to a nitrocellulose membrane, and probed with Abs as indicated in the results. Immunocomplexes were detected using ECL, and immunopositive bands were recorded using x-ray film.

Data were analyzed using one-way ANOVA followed by the Newman-Keuls test. Probability values of p < 0.05 were considered as statistically significant.

Th2 cells grown in normal medium containing 50U/ml IL-2 consistently demonstrated normal nuclear morphology, intact mitochondria filled with cyt c, and a low degree of annexin V binding (<7%) (Fig. 1, A–C). Removal of IL-2 led to an increase in the number of cells exhibiting chromatin condensation, nuclear shrinkage, DNA degradation, and cyt c release from mitochondria into cytoplasm and a decrease in the number of cells with normal morphology (Fig. 1, A and B). The proportion of annexin V-positive cells was increased to ∼31% at 24 h (Fig. 1,C). The increase in cellular morphological changes and annexin V binding was suppressed markedly by PGD₂ (Fig. 1, A–C). The addition of 100 nM PGD₂ reduced the number of annexin V-positive cells from ∼26.2 to ∼11% at 16 h and from ∼28.7 to ∼12.8% at 20 h. The reduction was decreased thereafter, suggesting that the inhibition of apoptosis diminished after 20 h. However, due to the degradation of early apoptotic cells and the instability of PGD₂ over prolonged periods of time, the precise potency of PGD₂ on Th2 apoptosis after 24 h could not be determined (data not shown). The inhibitory effect of PGD₂ was concentration dependent (IC₅₀ = 11.4 ± 3.1 nM at 16 h) (Fig. 1 D).

To confirm that the annexin V binding was indicative of the apoptotic process in human Th2 cells, we examined the caspase activities in the same samples (Fig. 2). IL-2 withdrawal promoted the cleavage of caspase 3 and PARP in the Th2 cell cultures. Cleaved caspase 3 (∼17 kDa) and cleaved PARP (∼89 kDa) fragments increased significantly after IL-2 deprivation (Fig. 2,A). An extra band smeared below the major bands and in proportion with the major bands in the blot to PARP is likely caused by incomplete denaturation of the protein during sample preparation. The cleavages of caspase 3 and PARP were almost completely abolished by 100 μM BAF, a pan-caspase inhibitor (26). PGD₂ (100 nM) also inhibited cleavage of caspase 3 and PARP. Addition of the PI3K inhibitor LY294002 (100 μM) inhibited the antiapoptotic effect of PGD₂ and also significantly enhanced the cleavage of caspase 3 and PARP (Fig. 2,A). Furthermore, the pan-caspase inhibitors, BAF and Z-VAD also suppressed the increase of annexin V binding induced by IL-2 deprivation (Fig. 2 B).

To elucidate the receptor mediating the above effects of PGD₂, we compared effects of BW245C, a specific DP₁ agonist, and DK-PGD₂, a specific CRTH2 agonist, with that of PGD₂ on the apoptosis of Th2 cells (Fig. 3,A). DK-PGD₂ exhibited a similar potency (IC₅₀ = 10.2 ± 0.7 nM) to that of PGD₂ in mediating this effect. In contrast, BW245C was without effect. The inhibitory effect of PGD₂ on Th2 apoptosis was completely inhibited by the CRTH2 antagonists ramatroban and TM30089, but not by the DP₁ antagonist BW868C or by the thromboxane-like prostanoid receptor (TP) antagonist SQ29548 (Fig. 3 B).

To further confirm the receptor mediating the effect of PGD₂ on Th2 cell apoptosis, we also investigated the effect of PGD₂ on the apoptosis of human CD4⁺CD25⁺ Treg cells. Although, like Th2 cells, Treg cells express both CD4 and CD25 and their growth is IL-2-dependent in culture, Treg cells are CRTH2 negative (Table I) and PGD₂ did not prevent their apoptosis induced by IL-2 deprivation (Fig. 3 C).

Reduced phosphorylation of Akt, a substrate for PI3K, and phosphorylation of BAD at Ser¹³⁶, a substrate for Akt, was detected in Th2 cells shortly after IL-2 withdrawal (1–2 h) and was restored by the addition of IL-2 or PGD₂ (Fig. 4,A). PGD₂ stimulated a dose-dependent increase in phospho-Akt (Fig. 4,B), and the intensity of Akt phosphorylation was highly correlated with the antiapoptotic effect of PGD₂ (Fig. 1,C). In contrast, BAF did not enhance the levels of phospho-Akt and phospho-BAD (Fig. 4,A). The effect of PGD₂ on both Akt and BAD phosphorylation was completely blocked by 100 μM LY294002 (Fig. 4,A). Furthermore, inhibition of PI3K with LY294002 prevented the inhibitory effect of PGD₂ on annexin V binding of Th2 cells and increased annexin V binding even in the presence of IL-2 (Fig. 4 C).

Treatment of Th2 cells with increasing concentrations of FasL (Fig. 5,A) or 7C11, an Ab against human Fas (Fig. 5,B), for 16 h increased annexin V-positive cells in a concentration-dependent manner, and this response was not affected by addition of PGD₂ (100 nM). To confirm the apoptotic mechanism induced by the Fas reaction in Th2 cells, caspase activities in the cells were analyzed using Western blotting. Treatment with 7C11 induced significant cleavage of caspases 3 and 8 in Th2 cells (Fig. 5 C). Z-IETD, a caspase 8 inhibitor, blocked the cleavage of caspase 3 but not caspase 8 cleavage. However, PGD₂ had no effect on the cleavage of either caspase induced by Fas activation.

PGD₂ is able to promote recruitment and activation of Th2 cells through a high affinity interaction with its receptor, CRTH2 (9, 11, 12). However, in addition to enhanced recruitment, the inflammatory burden of Th2 cells within inflamed tissues is likely to be also influenced by the clearance of these cells (27). Apoptosis is critically required for the clearance of Th2 cells, and a defect in apoptosis has been proposed as a feature of asthma pathology (23, 24, 25, 28). Indeed, induction of T cell apoptosis has been proposed as an important component of the action of both inhaled corticosteroids and immunotherapy in asthma (29, 30, 31). We have therefore investigated whether the apoptosis of Th2 cells can be influenced by PGD₂ and the signaling mechanisms involved. It was found that PGD₂ suppressed the cellular morphological changes, annexin V binding, cyt c release, and caspase activities in Th2 cells undergoing apoptosis as a result of IL-2 deprivation. This effect is likely to be due to a direct effect of PGD₂ rather than secondary effect on the production of IL-2, because IL-2 levels were not increased by PGD₂ treatment (data not shown). The inhibitory effect of PGD₂ was concentration dependent and mimicked by the selective CRTH2 agonist DK-PGD₂, but not by the selective DP₁ agonist BW245C. Ramatroban, a dual TP/CRTH2 antagonist, and TM30089, a selective CRTH2 antagonist, blocked the antiapoptotic effect of PGD₂ whereas the selective TP antagonist SQ29548 was without effect. Further evidence for the involvement of CRTH2 in this response is provided by the absence of effect of PGD₂ on the apoptosis of CD4⁺CD25⁺ Treg cells that do not express CRTH2. PI3K activity is clearly involved in the inhibition of apoptosis induced by PGD₂, because IL-2 deprivation resulted in the dephosphorylation of Akt and its downstream substrate BAD and induced cyt c release from mitochondria into cytoplasm, whereas PGD₂ restored phosphorylation of these proteins and prevented the release of cyt c. The PI3K inhibitor LY294002 prevented the effect of PGD₂ on both the signaling events and the apoptosis of Th2 cells. Therefore, PGD₂/CRTH2 interaction prevents the apoptosis of Th2 cells induced by cytokine deprivation through stimulation of the PI3K signal pathway.

T cell homeostasis can be controlled by several processes including two key apoptotic mechanisms (27, 32): 1) activation-induced cell death triggered by the restimulation of TCR involving death receptors, e.g., Fas (Apo-1/CD95) (33, 34, 35); and 2) activated T cell autonomous death induced by the disappearance of survival signals involving Bcl-2 family members in the mitochondria (36, 37). Both mechanisms are thought to be important in avoiding the overexpansion of activated cell clones after Ag stimulation and to avoid attack by autoreactive lymphocytes (21, 22, 38, 39). It has also been reported recently that Treg cells can suppress immune responses by inducing IL-2 deprivation-mediated apoptosis of effector CD4⁺ T cells (40). Pathophysiological concentrations of PGD₂ prevented the apoptosis of Th2 cells induced by IL-2 deprivation, a form of activated T cell autonomous death, suggesting that this may contribute to the survival of Th2 cells at sites of allergic inflammation and impede the resolution of disease because PGD₂ is found in high concentrations in a number of allergic diseases (41, 42, 43). We did not test the effect of PGD₂ on activation-induced cell death responses, but interestingly PGD₂ has no effect on the apoptosis of Th2 cells induced by Fas ligation. Considering that Bcl-2 regulation and Fas regulation are two different apoptotic mechanisms but share the same downstream effector pathway involving caspase 3 activation (27, 36), it is likely that PGD₂ does not interfere directly with the cellular apoptotic machinery but rather acts as a survival factor through the activation of PI3K.

In T cells, PI3K can be activated by a number of different receptors, including the TCR, costimulatory receptors, cytokine receptors, and chemokine receptors (44). We have reported that PI3Kγ, a class IB PI3K isoform, is involved in CRTH2-mediated proinflammatory responses of Th2 cells to PGD₂ (17). Considerable evidence suggests that activation of the PI3K pathway is required for T cell survival and proliferation (19, 45). Overactivity of PI3K activity leads to lymphoproliferative disorders and autoimmune disease (46), and inhibition of PI3K increases cell apoptosis (20, 47). It also has been reported that activation of class IA PI3K (p85) by the ligation of CD3 was able to protect activated Th2 clones from Fas-mediated apoptosis through blockade of Fas aggregation (48, 49). However, no such an effect was observed in our system when the PI3K pathway was activated by PGD₂/CRTH2, suggesting that the signaling via PI3Kγ induced by G protein-coupled receptors is probably not sufficient to interfere with Fas-dependent apoptosis (50).

One of the mechanisms used by PI3K to protect cells from apoptosis is regulation of the activity of proteins of the Bcl-2 family. It promotes Bcl-x_L expression in response to CD28 costimulation (51) and catalyzes BAD phosphorylation at Ser¹³⁶ through its downstream kinase Akt (52, 53, 54). Bcl-x_L is a prosurvival member of Bcl-2 family that can heterodimerize with proapoptotic members of the family to neutralize their activity (55), while phospho-BAD can be sequestered in the cytosol by 14-3-3 proteins, thereby preventing BAD-mediated inhibition of Bcl-x_L/Bcl-2 (56). Both of these effects will result in the protection of mitochondria from membrane permeabilization and the release of apoptotic factors, including cyt c. The effect of PGD₂/CRTH2 on phospho-BAD and cyt c in Th2 cells suggests that PI3K-mediated protection of mitochondria plays a central role in the antiapoptotic effects observed.

In conclusion, this study highlights an antiapoptotic role for PGD₂ mediated by CRTH2 in human Th2 cells. PGD₂ inhibits Th2 cell apoptosis in response to the disappearance of growth factor but not in response to Fas ligation. The summary scheme in Fig. 6 shows the proposed survival signals induced by PGD₂/CRTH2 interaction that leads Th2 cells to become resistant to apoptosis. Antagonism of CRTH2 has been proposed as an attractive approach to treat allergic diseases (57). Other mediators in addition to PGD₂ may contribute to Th2 cell survival through the activation of PI3K and possibly other pathways. IL-2 itself has been reported to be present in asthmatic airways (58) and may prevent the apoptosis of Th2 cells. However, the high levels of PGD₂ produced in a chronic allergic setting combined with its known dominant role in driving other aspects of Th2 cell activation (59, 60) suggest that it may contribute to survival signaling in Th2 cells. Further studies are required to define the precise pathophysiological role of PGD₂ in promoting Th2 cell survival. The identification of an inhibitory effect of CRTH2 activation on Th2 apoptosis suggests that the activation of CRTH2 may impede the resolution of allergic inflammation in addition to promoting the recruitment and activation of Th2 cells. These findings lend further support to the view that antagonism of CRTH2 may find utility in the treatment of allergic disease.

The authors have no financial conflict of interest.