A dominant role for chemoattractant receptor-homologous molecule expressed on T helper type 2 (Th2) cells (CRTH2) in mediating chemotaxis of CRTH2+ CD4+ Th2 lymphocytes in response to mast cell supernatants
Shân L. Gyles,
Department of Drug Discovery, Oxagen Ltd, Abingdon, Oxon, United Kingdom
Dr Roy Pettipher, Department of Drug Discovery, Oxagen Ltd, 91 Milton Park, Abingdon, Oxfordshire, OX14 4RY, United Kingdom. Email: email@example.com Senior author: Dr Roy Pettipher
Human cultured mast cells, immunologically activated with immunoglobuin E (IgE)/anti-IgE, released a factor(s) that promoted chemotaxis of human CRTH2+ CD4+ T helper type 2 (Th2) lymphocytes. Mast cell supernatants collected at 20 min, 1 hr, 2 hr and 4 hr after activation caused a concentration-dependent increase in the migration of Th2 cells. The effect of submaximal dilutions of mast-cell-conditioned media was inhibited in a dose-dependent manner by ramatroban (IC50 = 96 nm), a dual antagonist of both the thromboxane-like prostanoid (TP) receptor and the chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2), but not by the selective TP antagonist SQ29548, implicating CRTH2 in mediating the chemotactic response of these Th2 cells. The effect of mast-cell-conditioned media was mimicked by prostaglandin D2 (PGD2) and this eicosanoid was detected in the conditioned media from activated mast cells in concentrations sufficient to account for the activity of the mast cell supernatants. Treatment of the mast cells with the cyclo-oxygenase inhibitor diclofenac (10 μm) inhibited both the production of PGD2 and the CRTH2+ CD4+ Th2-stimulatory activity, while addition of exogenous PGD2 to conditioned media from diclofenac-treated mast cells restored the ability of the supernatants to promote chemotaxis of these Th2 cells. The degree of inhibition caused by diclofenac treatment of the mast cells was concordant with the degree of inhibition of chemotactic responses afforded by CRTH2 blockade. These data suggest that PGD2, or closely related metabolites of arachidonic acid, produced from mast cells may play a central role in the activation of CRTH2+ CD4+ Th2 lymphocytes through a CRTH2-dependent mechanism.
chemoattractant receptor-homologous molecule expressed on T helper type 2 cells
T helper type 2
Mast cells are thought to play a central role in orchestrating the pathophysiological changes that occur in allergic disease.1,2 In particular, the characteristic pattern of T helper type 2 (Th2) lymphocyte recruitment seen in allergic disease may be influenced by immunoglobulin E (IgE)-dependent activation of mast cells. Immunological activation of mast cells can lead to recruitment of CD4+ T cells and enhanced Th2-mediated eosinophilic inflammation in the airways of experimental animals,3 while treatment with the anti-IgE antibody omalizumab reduces eosinophil and lymphocyte numbers in the airway mucosa of patients with allergic asthma.4 Mast cells are thought to be one of the principal cell types mediating IgE-dependent allergic responses and are a particularly rich source of mediators with the potential to regulate the function of Th2 lymphocytes and other cell-types involved in allergic disease.5,6
The major prostanoid produced by mast cells is prostaglandin D2 (PGD2), which has been detected in high concentrations in the airways of asthmatics challenged with antigen.7,8 In addition to its DP1 receptor-mediated effects, which include vasodilatation and inhibition of platelet aggregation,9 PGD2 can also cause activation of Th2 lymphocytes, eosinophils and basophils through action on a chemotactic receptor called CRTH2 (chemoattractant receptor-homologous molecule expressed on Th2 cells, also known as DP2).10 Elegant studies have shown that PGD2 can promote chemotaxis of Th2 lymphocytes through the activation of CRTH2 (an effect mimicked by the selective CRTH2 agonist 13,14-dihydro-15-keto-prostaglandin D2; DK-PGD2) and, furthermore, that PGD2 is the major CRTH2 agonist produced by mast cells.11,12 Recently, we have shown that PGD2 has the ability to stimulate the production of interleukin-4 (IL-4), IL-5 and IL-13 by Th2 cells in the absence of costimulation, providing further evidence that PGD2 may play a central role in mediating allergic responses.13 However, the relative importance of PGD2 in promoting the migration of Th2 cells compared to other mast cell-derived mediators is unclear. Investigations into the role of CRTH2 in allergic responses have been facilitated greatly by the discovery that ramatroban, a drug originally identified as a thromboxane-like prostanoid (TP) receptor antagonist,14 is also an effective antagonist of CRTH2 and can therefore be used to define CRTH2-dependent responses in vitro and in vivo.15
Mast cells produce a plethora of mediators that might promote the chemotaxis of Th2 lymphocytes but the relative importance of these mediators in mast cell-dependent activation of Th2 cells is unclear. We have conducted experiments to investigate the role of PGD2 and its receptor CRTH2 in the chemotactic response of Th2 cells to supernatants collected from immunologically activated human mast cells. Interestingly, in this in vitro setting we found that PGD2 is a dominant mast cell-derived mediator promoting chemotaxis of Th2 lymphocytes and that this response is mediated by CRTH2.
Materials and methods
PGD2 and PGD2-MOX enzyme immunoassay kits were purchased from Cayman Chemical (Ann Arbor, MI). Ramatroban (BAY u3405) was synthesized by Evotec OAI (Abingdon, Oxon, UK) and is also available commercially from Cayman Chemical. Mono-poly-resolving medium was purchased from Dainippon Pharmaceuticals (Osaka, Japan). Magnetic antibody cell sorting CD4+ isolation and anti-CRTH2 microbead kits were purchased from Miltenyi Biotec (Bisley, Surrey, UK). Human recombinant stem cell factor and human recombinant IL-6 were purchased from R & D Systems (Abingdon, Oxon, UK). CD34+ progenitors from human cord blood, Iscove's modified Dulbecco's medium and X-VIVO 15 medium were purchased from Cambrex BioScience (Wokingham, Berkshire, UK). Antibodies against human tryptase and chymase were purchased from Chemicon International (Chandlers Ford, Hampshire, UK). Human myeloma IgE was purchased from Biodesign International (Saco, ME). Human recombinant IL-2, human recombinant IL-4, goat anti-human IgE and diclofenac were purchased from Sigma (Poole, Dorset, UK). Ficoll–Hypaque was purchased from Amersham Biosciences (Amersham, Buckinghamshire, UK). The 96-well ChemoTx plates were purchased from Neuroprobe (Gaithersburg, MD).
Human mast cell culture and activation
Human mast cells were cultured from CD34+ progenitor cells as described by Nakahata and Toru.16 CD34+ progenitor cells from human cord blood were cultured at a density of 1 × 105 cells/ml with Iscove's modified Dulbecco's medium containing 10% human serum, 0.55 μm 2-mercaptoethanol, penicillin/streptomycin, human recombinant stem cell factor (100 ng/ml) and human recombinant IL-6 (50 ng/ml) in 5% CO2 at 37 °C for 8–10 weeks. Half of the culture medium was replaced twice a week with fresh medium containing the same concentration of cytokines. The expression of tryptase and chymase of the cells was tested with immunostaining using the method described by Craig et al.17 The cytospin smears were first air-dried for 2 hr at room temperature and then fixed with Carnoy's solution (ethanol : chloroform : glacial acetic acid, 6 : 3 : 1) for 1 min. The fixed smears were stained using monoclonal antibodies against human mast cell tryptase and human mast cell chymase. The mast cells used in this study were tryptase positive (> 95%) and chymase negative (< 1%). The cells were pretreated with 5 μg/ml purified human myeloma IgE and human recombinant IL-4 (10 ng/ml) for 4 days, washed and then passively sensitized with fresh IgE (5 μg/ml) for 2 hr. The cells were washed with medium for 20 min and then challenged with goat anti-human IgE (1 μg/ml) alone or goat anti-human IgE (1 μg/ml) in the presence or absence of diclofenac (10 μm). Conditioned media were collected 20 min and 1, 2 and 4 hr after challenge. The conditioned media were assayed for PGD2 using a PGD2-MOX enzyme immunoassay kit according to the manufacturer's instructions.
Human CRTH2+ CD4+ Th2 cell isolation and chemotaxis assay
CRTH2+ CD4+ Th2 cells were prepared using a modified method described previously.18 Briefly, peripheral blood mononuclear cells were isolated from buffy coats by Ficoll–Hypaque density gradient centrifugation. CD4+ cells were purified from the peripheral blood mononuclear cells by negative selection using a magnetic antibody cell sorting CD4+ isolation kit. After 7 days of culture in an X-VIVO 15 medium containing 10% human serum, human recombinant IL-2 (50 units/ml) and human recombinant IL-4 (100 ng/ml), CRTH2+ cells were isolated from the CD4+ culture by positive selection using an anti-human CRTH2 microbead kit. The harvested CD4+ CRTH2+ cells were expanded in an X-VIVO 15 medium containing 10% human serum and human recombinant IL-2 (50 units/ml) before use.
For measurement of chemotaxis, Th2 cells were resuspended in X-VIVO/10% human serum, respectively, at 3 × 106 cells/ml 25 μl cell suspension and test samples (29 μl) prepared in X-VIVO/10% human serum were applied to the upper and lower chambers of a 5-μm pore-sized 96-well ChemoTx plate. After incubation at 37 °C for 60 min, any cells remaining on top of the filter were wiped off and the plates were centrifuged at 300 g for 2 min to collect any cells on the underside of the filters. The upper membrane was carefully removed and cell migration was quantified by fluorescence-activated cell sorting. Background cell migration was determined by measuring the response to media alone.
All data involving multiple comparisons were analysed using one-way anova followed by the Newman–Keuls test. Probability values of P < 0.05 were considered statistically significant.
Release of CRTH2+ CD4+ Th2 cell stimulatory activity from IgE-activated human mast cells
At all the time-points tested supernatants collected from IgE/anti-IgE-treated mast cells contained activity that stimulated significantly greater chemotactic responses of CRTH2+ CD4+ Th2 cells than supernatants from unactivated mast cells (Fig. 1). The chemotactic activity was detectable as early as 20 min after treatment, increased with time, reached a maximum response at about 1–2 hr and was sustained for at least 4 hr. Supernatants from unactivated mast cells had a similar effect to the X-VIVO 15 medium negative control, which caused a background migration of 16.64 ± 0.01% maximum response (n = 6).
Effect of diclofenac on the production of CRTH2+ CD4+ Th2 cell stimulatory activity by human mast cells
Treatment of the mast cells with the cyclo-oxygenase inhibitor diclofenac (10 μm) led to a substantial inhibition of the production of CRTH2+ CD4+ Th2 cell stimulatory activity at all time-points (Fig. 1). Specificity experiments showed that direct treatment of CRTH2+ CD4+ Th2 cells with diclofenac did not affect the responses to the mast cell conditioned media (Fig. 2), suggesting that diclofenac affects the production, but not the action, of the factor which is likely to be a product of the cyclo-oxygenase pathway.
Effect of diclofenac on the production of PGD2 by mast cells
Negligible levels of PGD2 were detected in media from unactivated mast cells. Biologically active levels of PGD2 were detected as early as 20 min after treatment with IgE and anti-IgE antibody and peaked at 1–2 hr at approximately 18 ng/ml (35 ng/106 cells, Fig. 3). Production of PGD2 was substantially inhibited by treatment of the mast cells with diclofenac (10 μm).
Addition of PGD2 at concentrations of 1–100 nm (or 0.35–35 ng/ml) to the conditioned media from diclofenac-treated mast cells restored the ability of these supernatants to promote chemotaxis of CRTH2+ CD4+ Th2 cells (Fig. 4).
Effect of ramatroban on activation of CRTH2+ CD4+ Th2 cells by mast cell supernatants
The dual CRTH2/TP antagonist ramatroban was used to define the receptors mediating the chemotactic responses of CRTH2+ CD4+ Th2 cells to mast cell supernatants. Supernatants collected 1 hr after activation with IgE/anti-IgE were used to assess the effect of ramatroban. The medium showed a concentration-dependent (1/1000–1/10 dilutions) migration of CRTH2+ CD4+ Th2 cells and this effect was completely inhibited by the dual CRTH2/TP antagonist ramatroban (1 μm) (Fig. 5a). The inhibition was dose-dependent (IC50 = 0.096 ± 0.043 μm, n = 3) (Fig. 5b). In contrast to the effect of ramatroban, the selective TP antagonist, SQ29548 had no significant inhibitory effect on the migration of CRTH2+ CD4+ Th2 cells, suggesting that the action of ramatroban is mediated via CRTH2, not TP (Fig. 5c).
To further exclude the possibility that the action of ramatroban might be non-specific, we examined the effect of ramatroban on other factors that promote Th2-cell chemotaxis (Fig. 6). Ramatroban (1 μm) inhibited responses of CRTH2+ CD4+ Th2 to PGD2 but not responses to macrophage-derived chemokine (MDC), thymus activation regulated chemokine (TARC) or stromal cell-derived factor-1α (SDF-1α).
It is well established that PGD2 is produced in high concentrations from immunologically activated mast cells19,20 and this prostanoid can activate Th2 lymphocytes by a mechanism involving CRTH2.10,13 However, the relative importance of PGD2 in mediating this effect compared to other mediators produced from mast cells is unclear. Surprisingly, the results from the current study suggest that PGD2 is a dominant factor produced by mast cells that activates CRTH2+ CD4+ Th2 cells and this effect is mediated by CRTH2. Treatment of human mast cells with diclofenac blocked the production of PGD2 and this was associated with almost complete inhibition of the ability of conditioned media to cause migration of Th2 cells. Diclofenac did not have a direct effect on chemotactic responses of CRTH2+ CD4+ Th2 cells, indicating that the drug is acting at the level of production, not the level of action, of the factor in the conditioned medium. This concentration of diclofenac has been shown to cause close to maximal inhibition of PGD2 production without affecting production/release of other mediators,21 which suggests that the effect of diclofenac is related to cyclo-oxygenase inhibition and not some other non-specific action. The pattern of production of chemotactic activity peaking at 1–2 hr was similar to the time–course of PGD2 production and, furthermore, the ability of diclofenac-treated supernatants to promote migration of CRTH2+ CD4+ Th2 cells was restored by the addition of exogenous PGD2 at similar concentrations to those detected in supernatants from activated mast cells, further increasing confidence that the effects of diclofenac are related to specific inhibition of PGD2 production. The evidence that the effects of the mast cell supernatants are mediated by CRTH2 is based on the effect of ramatroban, a dual CRTH2/TP antagonist. The effect of ramatroban is unlikely to be the result of TP antagonism because the selective TP antagonist SQ29548 did not affect responses of CRTH2+ CD4+ Th2 cells to mast cell conditioned medium and its potency in blocking the effect of mast cell supernatants is in the range required for CRTH2 antagonism.15 Furthermore, the effect is likely to be a specific action on CRTH2 because ramatroban inhibited Th2 responses to exogenous PGD2 but not responses to SDF-1α, MDC or TARC.
Taken together, these data suggest that in this in vitro system PGD2 is a dominant mast cell product causing migration of Th2 cells through a CRTH2-dependent mechanism. While this is consistent with the earlier work of Hirai,10 who showed that PGD2 is the major CRTH2 agonist produced by immunologically activated mast cells in vitro, it is surprising that PGD2 plays such a dominant role in promoting chemotaxis of these cells given the large number of other mast cell products that could potentially affect the function of Th2 cells.5,6 The likely reason that PGD2 plays such a dominant role in this system is that PGD2 is produced in such high concentrations by mast cells and its effects on Th2 cells is so potent. In a more complex in vivo setting where other cell types, in addition to mast cells, may regulate leucocyte accumulation and activation, other chemotactic factors may play a more important role. It would be of great interest to determine whether selective CRTH2 blockade would reduce accumulation and/or activation of Th2 cells in an in vivo allergic response. In this context it is worth noting that studies involving exogenous PGD222 or over-expression of human PGD2 synthase23 have demonstrated exaggerated production of Th2 cytokines and enhanced eosinophil accumulation into the airways in response to antigen, illustrating that PGD2 has the potential to mediate Th2 activation in vivo. Furthermore, it has been shown more recently that the effects of PGD2 in exacerbating allergic inflammation in the lungs and skin of mice can be mimicked by the selective CRTH2 agonist DK-PGD2, implicating CRTH2 in this response.24 While the effect of a selective CRTH2 antagonist on Th2 cell accumulation in response to antigen has not yet been reported, studies with ramatroban in preclinical models indicate that CRTH2 is important in mediating eosinophil accumulation in a number of tissues in response to allergic challenge including the guinea-pig nasal mucosa,25 mouse airways26 and in the mouse skin during contact hypersensitivity reactions.27
The clinical relevance of the current findings remains to be determined but it is interesting to note that functional polymorphisms in the CRTH2 gene are strongly associated with severe asthma in both African-American and Chinese populations.28 The associated polymorphisms in the 3′-untranslated region of the CRTH2 gene lead to increased mRNA stability, suggesting that gain-of-function variants in CRTH2 are causally linked with asthma. Such individuals are likely to demonstrate an exaggerated chemotactic response to PGD2, leading to enhanced recruitment and activation of Th2 lymphocytes and other leucocytes involved in allergic responses. The DP1 receptor is also genetically associated with asthma29– in this case the mechanism is thought to be related to DP1-mediated inhibition of dendritic cell function30,31– leading to polarization of T cells towards the Th2 phenotype. Thus, it appears that PGD2 by acting on DP and CRTH2 may lead to both polarization and recruitment of Th2 lymphocytes.
In summary, these data suggest that PGD2, or a closely related metabolite of arachidonic acid, acting on CRTH2 may play a central role in mediating mast cell-dependent activation of Th2 cells and give further support to the view that CRTH2 antagonists may be useful in the treatment of asthma and other allergic diseases.