Abstract
PGs play key regulatory roles in inflammation and immunity. PGD2, released from mast cells and Th2 cells during allergic responses, has recently been shown to target a novel receptor, chemoattractant receptor-homologous molecule expressed TH2 cells (CRTH2), in addition to the classic PGD (DP) receptor. CRTH2 is expressed on Th2 cells and eosinophils and mediates chemotaxis of these cells to PGD2. Thus, CRTH2 is thought to be a key receptor mediating eosinophil and Th2 cell recruitment during allergic responses. To examine the role of CRTH2 in this context in vivo, we generated CRTH2 knockout mice. Surprisingly, in an allergic inflammatory model of asthma, CRTH2 knockout mice showed enhanced eosinophil recruitment into the lung compared with wild-type littermate mice. This is consistent with our observation that CRTH2 knockout cells produce significantly higher amounts of IL-5 and IL-3 in vitro. These results suggest a nonredundant role of CRTH2 in restricting eosinophilia and allergic response in vivo.
The arachidonic acid metabolite PGD2 is an inflammatory mediator released from mast cells and Th2 cells (1). A number of properties of PGD2, including induction of bronchoconstriction, and chemotactic recruitment of inflammatory cells such as Th2 cells, eosinophils, and basophils (2), have implicated PGD2 in allergic diseases. The PGD2 level in bronchoalveolar lavage (BAL) 2 fluid increases in response to Ag provocation and is used as a marker for activation of mast cells in vivo (3, 4). The cellular infiltration and impaired lung function in a mouse asthma model are attenuated in PGD (DP) receptor null mice (5). This observation is consistent with the concept that the DP receptor mediates the effect of PGD2 in allergic responses.
The recent discovery of the second PGD2 receptor, chemoattractant receptor-homologous molecule expressed on TH2 cells (CRTH2), has made the issue more complex (6). In humans, CRTH2 is selectively expressed on cells important in allergic responses: Th2-type T cells (Th2 and type 2 cytotoxic T), eosinophils, basophils, and a subset of monocytes (7). CRTH2-mediated signals induce migration and activation (2, 8, 9) of these cells. These studies and others (10) rely on a series of PGD2 analogues that are selective for CRTH2 over the DP receptor. Whether these compounds modulate additional receptors that might affect cell migration is unknown. More importantly, whether CRTH2 plays any nonredundant role in mediating eosinophil and Th2 cell chemotaxis in vivo remains unclear in light of other known chemokine receptors that mediate these activities.
We unexpectedly found that CRTH2−/− mice produce a higher amount of IL-5 than control littermate mice and showed enhanced eosinophilia in an OVA model. Our findings suggest a nonredundant role of PGD2 and CRTH2 in restricting eosinophil recruitment during allergic responses.
Materials and Methods
Targeting vector construction and generation of CRTH2-deficient mice
The CRTH2 targeting vector was constructed by cloning 5.0 kb of the 5′ homology region and 1.8 kb of the 3′ homology region of the murine CRTH2 (mCRTH2) gene derived from a 129SvJ genomic λ phage library (Stratagene) into pJNS2 (PGK-Neo/TK vector). The targeting vector was designed to replace 1.8 kb of the CRTH2 genomic locus, containing bps 1–1085 of mCRTH2 cDNA, with the PGK-Neo cassette. The correctly targeted embryonic stem (ES) cell clones were identified by Southern analysis using a 600-bp 3′ probe and microinjected in C57BL/6J blastocysts. Male chimeras were backcrossed with C57BL/6J females to generate heterozygous germline offspring. Heterozygous males and females were intercrossed to generate homozygous CRTH2−/− mice along with their +/+ littermate controls. All CRTH2 offspring were initially genotyped using either Southern analysis or PCR (data not shown).
OVA model
Ten ± 4-wk-old male CRTH2-deficient mice and control littermates were immunized and boosted by i.p. injection of 50 μg/ml OVA (albumin chicken eggs, grade F V, A-5503; Sigma-Aldrich) complexed with 20 mg/ml Al(OH)3 powder (Merck) in 0.2 ml of 0.9% NaCl (saline; Meram) at days 0 and 7. Mice were then challenged with a single OVA aerosol (2.5% during 1 h) or saline for controls using a nebulizer SPAG2–6000 (Valeant Pharmaceuticals) at day 14. All experiments were approved by the Pfizer Fresnes Laboratories Ethical Committee.
Sampling of BAL fluid (BALF)
Seventy-two hours postchallenge, mice were anesthetized by i.p. injection (0.3 ml/mice) of 6% Pentobarbital (Sanofi-Synthelabo); the lungs were flushed via the cannulated trachea with PBS. Total cell numbers were automatically counted. BALF were cytocentrifuged with Cytospin (Thermo Electron) and slides were stained with a May-Grünwald Giemsa method and a total of 100 cells was counted for each sample by light microscopy, the percentage of each cell population was determined. The BALF was centrifuged for 10 min at 1900 × g, 4°C, and supernatant was removed and stored at −80°C until used.
Cytokine measurement
Splenocytes from naive knockout and littermate mice were stimulated at 1 × 106/ml with 10 μg/ml plate-bound anti-CD3 (2C11) alone or plate-bound anti-CD3 plus 1 μg/ml soluble anti-CD28. At various times after stimulation, supernatant was harvested and cytokine/chemokine concentrations were measured using the Bioplex (Bio-Rad) multiplex cytokine measurement system.
TaqMan
Cell culture and quantitative RT-PCR for Th1 and Th2 cells were done as previously described (11). Quantitative RT-PCR analysis of CRTH2 was performed using the following primer set: CRTH2 forward primer, 5′-TGGCCTTCTTCAACAGCGT-3′; CRTH2 reverse primer, 5′-ACGCAGTTGGGGAATTCG-3′. G3PDH mRNA was used as an internal control.
Statistics
Data are expressed as mean ± SEM and statistical significance was determined using the unpaired Student’s t test, values of p < 0.05 were considered as statistically significant (∗).
Results and Discussion
Generation of CRTH2−/− mice
CRTH2 has recently been identified as a highly selective human Th2 marker, and it appears to mediate PGD2-elicited eosinophil migration. Recruitment of eosinophils to the site of inflammation is a critical event in asthma and many allergic diseases. To directly examine the role of PGD2 acting through the CRTH2 receptor in physiological responses, including eosinophil recruitment, we generated CRTH2-deficient mice (Fig. 1⇓A). Eight correctly targeted ES cell clones were identified by Southern blot analysis using a 3′ 600-bp NheI/BglII genomic fragment as a probe (Fig. 1⇓B).
Targeted disruption of the CRTH2 gene. A, Strategy to inactivate CRTH2 by homologous recombination in RW4–129SvJ ES cells. Diagram depicts the CRTH2 targeting vector (top), endogenous CRTH2 locus (middle), and the targeted CRTH2 locus (bottom). The 1.8-kb fragment deleted in the knockout animal contains most of the single CRTH2 exon, bps 1–1085. B, Southern blot analysis of ES cell clones electroporated with the CRTH2 targeting vector. ES cell DNA was digested with EcoRV and BglII and Southern blot analysis was done using a 600-bp 3′ probe. This blot depicts three clones containing both the 5.2-kb endogenous fragment along with the 3.6-kb predicted size targeted fragment.
ES cells from two CRTH2-targeted lines were microinjected into C57BL/6J blastocysts, generating male chimeras. These chimeras were crossed with C57BL/6J females and germline CRTH2+/− offspring were identified. CRTH2−/− mice were generated by intercrossing CRTH2+/− mice. Normal 1:2:1 Mendelian segregation was observed for offspring generated from these CRTH2+/− mating. Both +/− and −/− mice appear normal. CRTH2−/− mice and CRTH2+/+ littermate mice, then crossed with −/− and +/+ mice, respectively, were used for all following experiments. To initially verify that the targeted mutation introduced into the CRTH2 locus resulted in complete inactivation of the gene, we examined expression of CRTH2 mRNA by RT-PCR in all offspring. CRTH2 mRNA was not detected in all tissues tested from −/− mice and no compensatory expression of DP was observed in various tissues examined (data not shown).
Increased eosinophil recruitment into the BAL in CRTH2−/− mice
PGD2 was shown to be a chemoattractant for eosinophils in vitro (2) and CRTH2 selective analogues of PGD2 that has CRTH2 agonist activity were shown to attract eosinophils in vivo (10, 12). Because the broader spectrum selectivity is unclear for these agonist compounds, CRTH2−/− mice would provide an ideal tool to examine the role in CRTH2 in eosinophil migration. Surprisingly, when these mice were tested in an OVA-elicited allergic airway inflammatory model, dramatically increased eosinophils were observed in CRTH2−/− BALF, and in lungs (data not shown) compared with CRTH2+/+ littermates (Fig. 2⇓). Small increases in macrophages were also seen in CRTH2−/− mice.
Enhanced eosinophil recruitment in CRTH2 knockout mice. Cellular infiltration in BALF of immunized and challenged CRTH2−/− and CRTH2+/+ mice. Cells were counted and differentiated at +72 h postsaline (controls) or OVA aerosol challenge. Results are expressed as mean ± SEM of 12 mice. Three data points (total cell count, macrophages, and eosinophils) in the −/− OVA sample are statistically different from control samples (p < 0.05 vs time-matched saline-treated mice), and are marked with an asterisk.
Enhanced IL-5 production in CRTH2−/− mice
Eosinophils rely on a number of cytokines and chemokines for activation, maturation, and migration. To examine whether the production of cytokines and chemokines are affected in CRTH2−/− mice in comparison to +/+ mice, we measured 13 cytokines and 3 chemokines produced by splenocytes stimulated in vitro (Table I⇓). Upon 3 days of in vitro stimulation, the most dramatic changes detected were increases in IL-5 production by cells stimulated with either anti-CD3 alone or with the combination of anti-CD3 and anti-CD28. Under anti-CD3 stimulation alone, a 5-fold increase in IL-5 production in CRTH2−/− vs wild type was observed, while stimulation with the combination of anti-CD3 and anti-CD28 caused a 2.5-fold increase in IL-5 in CRTH2−/− splenocytes. Small increases in IL-3 and IL-17 were also observed from CRTH2−/− splenocytes. Similar to IL-5, IL-3 was also known to stimulate eosinophil generation in vivo (13). In addition, a 3-fold reduction in IL-2 was also observed when cells were stimulated with anti-CD3 and anti-CD28.
Cytokine and chemokine production from splenocytes
Measurement of IL-5 production at 24, 48, 72, and 96 h post-in vitro stimulation of wild-type and CRTH2−/− splenocytes confirmed enhanced IL-5 production in CRTH2−/− mice throughout the time course (Fig. 3⇓). Enhanced IL-5 production could provide a feasible explanation for enhanced eosinophil recruitment. IL-5 is required for eosinophil differentiation and recruitment, as shown in both knockout and neutralizing Ab studies (14, 15). Thus, in wild-type mice, CRTH2 controls eosinophil recruitment through limitation of IL-5 production. This finding is surprising, because CRTH2 was thought to mediate the chemotactic effect of PGD2 on eosinophils (10, 12).
Time course of IL-5 production from T cells of CRTH2 knockout mice. Splenocytes from CRTH2−/− and CRTH2+/+ control were stimulated in vitro with anti-CD3 alone or anti-CD3 plus anti-CD28. At various time after stimulation, IL-5 concentration in the supernatant was determined using Bioplex.
Our results suggest that the eosinophil recruitment effect of PGD2 (2) and CRTH2 may be redundant (with eotaxin and CCR3 for example), while the inhibitory effect on IL-5 production and eosinophil maturation/recruitment is nonredundant. To examine whether PGD2 and analogues have a direct inhibitory effect on IL-5 production, we stimulated wild-type and CRTH2−/− splenocytes with anti-CD3+/− anti-CD28, adding PGD2, BW245C, or DK-PGD2. No significant changes in IL-2, IL-5, and IFN-γ production were seen (data not shown). Because T cells are known to produce a significant amount of PGD2 (1), it is likely that in these cultures, endogenously produced PGD2 has already reached saturating level.
Down-regulation of CRTH2 upon T cell activation
Our finding of enhanced IL-5 production in CRTH2−/− T cells raised the question of whether the selective effect on IL-5 is a result of differential expression of CRTH2 on Th1 vs Th2 cells. CRTH2 is a well-validated human Th2 cell surface marker (7, 16) while it has not been shown to be Th2 selective in murine cells (17). We then differentiated murine Th1 and Th2 cells in vitro from purified naive CD4 T cells for four days using polarizing cytokines and cytokine neutralizing Abs, and restimulated these differentiated cells for 4 h at the end of the 4-day culture period. The differentiation of Th1 and Th2 cells was confirmed by exclusive expression and Th1 and Th2 cytokines and transcription factors in respective cultures (11). Expression levels of CRTH2 in Th1 and Th2 cells at days 0–4 and upon restimulation were examined using quantitative RT-PCR. CRTH2 was expressed at similar levels between Th1 and Th2 cells at all time points examined (Fig. 4⇓). No differential expression of CRTH2 was seen at any stage of T cell activation/differentiation. Interestingly, upon stimulation (both primary and secondary stimulation), the expression of CRTH2 decreases significantly in both Th1 and Th2 cultures. At days 3 and 4 poststimulation, the expression level of CRTH2 comes back to normal. Throughout this time course, IL-5 and IL-3 were not expressed at days 1 and 2, were expressed at low levels at days 3 and 4, and were expressed at high levels upon restimulation. The expression kinetics of CRTH2 and IL-5/IL-3 suggest a scenario where down-regulation of CRTH2 on T cells upon TCR stimulation provides a permitting environment for IL-5 and IL-3 production. During primary stimulation of naive cells, other factors required for Th2 differentiation take several days to accumulate, while IL-5/IL-3 production in primed cells is immediate upon restimulation. In this scenario, elevated CRTH2 expression at later time points during the height of IL-5 and IL-3 production serves to keep the expression of these cytokines in check.
Down-regulation of CRTH2 upon T cell activation. Expression of CRTH2 mRNA in Th1 and Th2 cells at various time points after primary and secondary stimulation was analyzed using quantitative RT-PCR. Sample labeling is as follows: naive, naive CD4 T cells; Th1day1–Th1day4, naive cells differentiated under Th1 polarizing condition for 1–4 days. Th1d4restim, naive cells differentiated under Th1 polarizing condition for 4 days and then restimulated with plate bound anti-CD3 for 4 h. Th2 samples follow similar labeling convention.
A recent study (18) showed that PGD2 enhanced the production of IL-5 and other Th2 cytokines by human Th2 cells via CRTH2. Although our data demonstrated a role of CRTH2 in limiting IL-5 production, this does not preclude a scenario where CRTH2 stimulation transmits signals to both promote IL-5 production and inhibit IL-5 production, a feature reminiscent of the negative feedback mechanisms increasingly observed for many biological systems. One possible situation is that CRTH2 promotes IL-5 production early on and the inhibitory pathway is turned on later. The cytokine measurement in the Tanaka study (18) was done at 6 h poststimulation. In our murine system, adding PGD2, DK-PGD2, or BW245C did not cause any changes in IL-5 production at day 3 poststimulation. An interesting paper from Hirai et al. (19) indicated that CCR3+ splenocytes from IL-5 transgenic mice expressed high levels of CRTH2 but did not bind PGD2 raising the possibility that sustained exposure to IL-5 may have caused down-regulation of the surface expression of CRTH2. This observation and our results suggest cross-regulation between IL-5 and CRTH2. More study is needed to further elucidate these correlations.
PGD2 is a prominent inflammatory mediator, with known biological actions including vasodilatation and bronchoconstriction; its precise role in complex allergic and inflammatory responses is not clear. In transgenic mice expressing hemopoietic type PGD synthase, increased expression of Th2 cytokines and pronounced eosinophilia were observed (20). Studies using DP knockout mice indicated the requirement of DP receptor in mediating the effect of PGD2 on eosinophilia and airway inflammation (5).
Studies using hemopoietic type PGD synthase knockout mice indicated a role of PGD2 to bring about resolution of inflammation in vivo (21). Our study using CRTH2−/− mice revealed an inhibitory role of CRTH2 on cytokine production and allergic inflammation. Taken together, these data suggest that PGD2 plays a complex role during inflammation and allergic response, with redundant roles in stimulating cytokine production and allergic inflammation, and nonredundant roles in limiting IL-5 production and eosinophil recruitment.
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 Address correspondence and reprint requests to Dr. Baiyong Li, Department of Antibacterials, Immunology and Cancer, Pfizer Global Research and Development, Eastern Point Road, Groton, CT 06340. E-mail address: Baiyong.li{at}pfizer.com
-
↵2 Abbreviations used in this paper: BAL, bronchoalveolar lavage; CRTH2, chemoattractant receptor-homologous molecule expressed on TH2 cells; mCRTH2, murine CRTH2; ES, embryonic stem; BALF, BAL fluid.
- Received August 9, 2004.
- Accepted March 2, 2005.
- Copyright © 2005 by The American Association of Immunologists
References
In this issue
