AD is a common inflammatory skin disease with lesions characterized by epidermal thickening and a prominent perivascular and dermal infiltrate, as well as high serum IgE levels. In this study, we used two well-characterized murine models of AD to examine the role of CRTH2 in cutaneous inflammation. Both of these models are CD4+
T lymphocyte dependent, as is AD (16
). In a model of repeated epicutaneous sensitization, the administration of a CRTH2 antagonist reduced inflammation in the dermis substantially, as well as prevented epidermal thickening (). Using a robust 25 day FITC model of contact hypersensitivity, the administration of the CRTH2 antagonist just prior to challenge substantially blocked ear swelling (~50% decrease). Additionally, administration of Compound A from days 14 to 25 inhibited inflammation further still, reducing ear thickness ~75% compared with untreated controls (). Importantly, an examination of serum Ig levels showed a decrease in total IgE in animals treated with the CRTH2 antagonist in both in vivo
models ( and ). Further, in the epicutaneous OVA sensitization model, where antigen-specific antibody levels could reliably be measured, there was a decrease in OVA-specific IgE, IgG1 and IgG2a levels. A closer examination of this was carried by epicutaneously sensitizing mice to OVA for a 1-week period while administering drug vehicle or Compound A. Again, an antigen-specific decrease was seen in the IgE, IgG1 and IgG2a Ig classes upon administration of the CRTH2 antagonist (). As antibody titers to protein antigens rise substantially with repeated immunizations, the effect of Compound A became more pronounced. Hence, we observed a much greater effect on Ig levels in the chronic OVA model, with Compound A administered during the second and third patching () than after the first patching (). It should also be pointed out that antibody production was not affected by the CRTH2 antagonist following intra-peritoneal immunization with same protein antigen, ovalbumin, emulsified in the adjuvant alum. This suggests that the route of antigen delivery and subsequent presentation may play a key role in determining the influence of a CRTH2 blockade. These findings confirm and extend the findings of Nakamura et al.
using a CRTH2−/−
mice bred to the BALB/c background (12
). They initially reported a modest decrease in total IgE levels in CRTH2−/−
mice following multiple cutaneous sensitizations with TNCB (12
). This slight decrease may be attributable to the fact that this model is not T cell dependent (32
). In a novel model of allergic rhinitis, intranasally sensitized CRTH2-deficient mice had a reduction in antigen-specific IgE and IgG1 (30
). However, in this model, very low levels of antigen-specific IgG2a were detected in the serum, in contrast to this report and studies by Geha et al.
using epicutaneous sensitization with ovalbumin (19
). These different results with respect to IgG2a may be due to different modes of antigen sensitization or the antigen itself (ovalbumin or Cry j 1 antigen). Taken together, however, these findings are of great importance given the role allergen-specific antibodies play in allergic inflammation diseases, such as AD, allergic rhinitis and asthma. This also implied that CRTH2 was not only having an effect on both acute inflammation (administration of Compound A at the time of FITC challenge; Stefen A. Boehme, unpublished results) but also having an effect on the underlying immune response resulting in antibody production to epicutaneously administered antigen.
Examination of gene expression levels from skin that had been epicutaneously sensitized to ovalbumin showed a robust pattern of gene up-regulation across the mouse genome (). In line with epicutaneous OVA sensitization generating a predominantly Th
2-type T cell response, a wide array of cytokines, chemokines and their cognate receptors and associated signaling molecules were found to be dramatically up-regulated. Much attention has been focused on the chemokine superfamily as the major stimulus for the directed migration of leukocytes during the elicitation phase of hypersensitivity reactions in skin. Additionally, much discussion has centered on the segregation of such mediators into their ability to promote Th
1-type or Th
2-type responses. While a large number of cytokines and chemokines were up-regulated, members of the MCP family of chemokines (CCL2/MCP-1, CCL8/MCP-2, CCL12/MCP-5) as well as CCL1/I-309, CCL3/MIP-1α, CCL4/MIP1β and the CXCL family members CXCL1/GRO-α and CXCL2/GRO-β, CXCL10/IP-10 and CXCL14/BRAK appeared to be the most relevant (). It is clear from this group of chemokines that there is no distinction of the Th
1- or Th
2-type responses since a wide array of cells including B cells, macrophages and neutrophils are stimulated by these chemokines. Studies in a different model of epidermal inflammation have shown that the CXCR2 ligands GRO-α, the murine ortholog of IL-8 and MIP-2, can induce a significant inflammatory state (33
). Both GRO-α and MIP-2 are up-regulated in our model and Compound A treatment significantly down-regulated the gene expression (). The gene for IP-10 was also noted to be up-regulated by OVA challenge and was significantly down-regulated by Compound A (34
). A number of T lymphocyte cell surface molecules, such as CD3ε, γ and δ, increase after OVA skin patching, but are strongly reduced by Compound A treatment (). As the inflammatory infiltrate into the dermis is primarily made up of T lymphocytes, both in this model and AD, the decrease in T cell gene expression is consistent with reduced inflammation (16
), and this may have important therapeutic implications.
Further investigations of the profiles of gene regulation have revealed interesting correlations with findings from assessment of the genetic risk factors associated with human atopy—specifically AD and asthma, two of the primary constituents of the ‘atopic march’ (26
). A large number of population studies have been conducted to elucidate genetic risk factors for atopy and its disease manifestations in different organs. With regard to AD in humans, several loci have been revealed including those encoding genes in the so-called epithelial differentiation complex (SPINK5, SPRR and S100A gene families) (35
). Our OVA challenge model reveals that Compound A treatment significantly down-regulated the SPRR2a, S100A8 and S100A9 (calgranulin) family members’ expression, a finding which may be relevant to the anti-inflammatory efficacy of the CRTH2-specific antagonism ().
Additional genes of interest in the atopy risk loci include the Th2 cytokines/receptors IL-13, TNF-α, IL-9, IL-17E, IL-18, IL-4 receptor α and the chemokine eotaxin (CCL11). In our hands, the cytokines TNF-α, IL-9, IL-17E as well as the receptors IL-4 receptor α and IL-18 receptor were shown to be up-regulated by OVA sensitization and significantly affected by Compound A treatment. Other miscellaneous risk factors include the proteases diprolylpeptidase 10, A disintegrin and metalloprotease family member 33 (ADAM33), the plant homeodomain finger protein 11 (PHF11) and the solute carriers SLC9A3R1 and SLC22 families. While a number of these factors are more relevant to the asthma phenotype, we have certainly noted an up-regulation in several of the SLC family members (data not shown). In the OVA-challenge model described here, expression of the monocyte CD antigen, CD14, increased, and Compound A treatment significantly down-regulated CD14 gene expression (). Similar findings have been shown for the dendritic and Langerhan’s cell markers CD207 and CD209c. Finally, in the treated skin sections, protein levels of IL-1β, IL-4 and MIP-1β (CCL4) were reduced by Compound A, a result consistent with the gene expression data. Taken together, this data suggested that the CRTH2 antagonist has a greater effect than just inhibiting the infiltration of CRTH2+ cells, but appears to act on a variety of genes involved in many aspects of the allergic inflammatory response.
An examination of cytokines produced by splenocytes from epicutaneously OVA-sensitized mice, 17 days after the initial OVA sensitization period and Compound A treatment period, showed a decrease in the IL-13, IFN-γ and IL-17A levels. The effect on IFN-γ and IL-17A suggests that Compound A is exerting an effect on more than just classical Th
2 cells. Perhaps, this could be explained by the observation that murine Th
1 cells also express CRTH2. Alternatively, expression patterns of CRTH2 show its expression in many unexpected tissues (7
). In the human system, we have observed that atopic and/or asthmatic patients express CRTH2 on numerous other leukocytes besides CCR4+
2 T lymphocytes (Stefen A. Boehme, unpublished results). Finally, it is possible that antagonism of CRTH2 may be exerting an indirect effect on Th
cell subsets besides Th
2 cells. Nonetheless, these observations demonstrate that the administration of the CRTH2 antagonist in vivo
appears to have a broad effect on the immune response to antigens delivered epicutaneously, and this effect extended significantly past the bioavailability of Compound A. Further, the observation that blockade of CRTH2 can impact cytokine production in multiple types of Th
cells, including Th
2 and Th
17 cells is novel, as this has not been fully explored in studies using CRTH2 gene-deficient mice (12
As epicutaneous antigen sensitization is thought to be critical for AD and to account for the effect of the CRTH2 antagonist on epicutaneously immunized antigen, we hypothesized that skin DC may play a role. Both dermal DC and Langerhans cells are able to take up antigen in the skin, migrate to the dLNs and initiate T cell-mediated immune responses (38
). Therefore, we examined the role that professional dermal APCs may play in shaping an immune response. After FITC was applied to dorsal skin, as opposed to OVA–FITC which gave a very weak response similar to observations of other groups (20
), we detected a slight but significant decrease in the number of FITChi+
DC that migrated from the skin to the dLN in Compound A-treated mice. This difference was not detected in earlier studies using CRTH2−/−
mice, as the percentage of FITC+
cells was compared with whole lymph node cell population, as opposed to just the CD11c+
). Further, our results show that a high percentage of the FITChi+
cells in the dLN express CD11c, strongly suggesting that these cells migrated from the skin, as opposed to acquiring FITC via the lymph or blood stream. Co-culturing of the different populations of CD11c+
DC, those expressing high levels of FITC, the resident CD11c+
DC in the lymph nodes that were FITC−
and splenic-derived CD11c+
DC, showed striking effects on their ability to activate naive T cells. As noted previously, FITChi+
DC elicited a much greater IL-17A response compared with splenic-derived CD11c+
DC co-cultured with naive T cells (; 20
). However, the FITChi+
DC isolated from Compound A-treated mice elicited substantially decreased levels of IL-17A, IFN-γ, IL-10 and IL-6 upon co-culture with naive DO11.10 TCR transgenic T cells compared with vehicle-treated animals. As IL-6 has been shown to play a role in the differentiation of Th
17 cells, perhaps this decrease in IL-17A may be linked to the decrease of IL-6 produced by naive T cells co-cultured with FITChi+
DC from Compound A-treated mice (39
). However, this decrease is already detected in cultures after 24 h and the CD11c+
cells do not make any IL-6 when cultured in the absence of T cells or antigen (). Additionally, no significant differences in the amount of TGF-β cytokine or IL-23 mRNA levels were detected between the various DC populations.
This decreased amount of cytokine production does not appear to be mediated by the induction of a regulatory T cell population, as we found no increase of FoxP3+ cells, or CTLA-4+ cells in the cultures after 72 h (data not shown). The levels of IL-10 were not increased in the cultures containing the FITChi+ CD11c+ DC from Compound A-treated mice. IL-6 was present in the same cultures and if a Treg population was induced, IL-6 levels would be expected to be greatly decreased. The FITChi+ DC derived from Compound A-treated mice did not appear to induce anergy in the naive T cell population, as the levels of IL-4 produced were not significantly different among the different DC populations examined (). Further, the percentage of cells in the S or G2/M phase of the cycle, or BrdU+, was not significantly changed between the different DC groups tested (data not shown). Finally, the levels of various cell surface proteins expressed by DC involved in T cell activation were very similar between the FITChi+ CD11c+ DC from both the Compound A-treated or untreated mice (). Collectively, this indicates that the Compound A-derived DC can process and present antigen efficiently to T cells. Additionally, various activation molecules were greatly increased, as expected, by the skin emigrant FITChi+ DC in the draining LN compared with resident DC ().
Thus, the mechanism by which DC, that migrated from the skin to the draining LN upon antigen capture from the CRTH2-antagonized mice, are able to suppress cytokine production when used to stimulate naive T cells is still unclear. Possible insight into a mechanism comes from our studies using a 1-week FITC-induced contact hypersensitivity model, which demonstrated that antagonism of CRTH2 led to a substantial decrease in TSLP and IL-1β protein levels (Boehme, S.A., Franz-Bacon, K., Chen, E.P., Šášik, R., Sprague, L.J., Ly, T.W., Hardiman, G and Bacon, K.B., submitted for publication). This decrease would be expected to have a profound effect on both skin DC and Langerhans cells as TSLP has been shown to trigger both activation and migration of these skin DC populations (41
). Consistent with this notion, we observed in our gene expression studies of epicutaneously sensitized skin, a decrease in CCL17/TARC mRNA levels upon Compound A treatment (). TSLP activation of DC has been shown to stimulate TARC production (41
). Taking this a step further, the effect of activated skin DC from Compound A-treated mice to suppress cytokine production in this in vitro
culture model appears to reproduce the in vivo
effect, as (1
) spleen cells from epicutaneously immunized and Compound A-treated mice also showed a marked decrease in cytokine production upon antigen re-stimulation (). And (2
) there is a profound decrease in various cytokine and chemokines mRNAs imparted by Compound A treatment of epicutaneously sensitized mice to OVA (). Additionally, the effect on both IFN-γ (Th
1) and IL-13 (Th
2) cytokines is consistent with a decrease in Ig levels of multiple classes. Taken together, ability of the CRTH2 antagonist to inhibit both cytokine and antibody levels induced by epicutaneous administration of antigen may very well account for the profound effect on the level of inflammation incurred by either repeated epicutaneous OVA-antigen sensitization or multiple FITC sensitizations and challenge. This could certainly cause the decreased inflammatory infiltrate and reduced gene and protein expression of pro-inflammatory mediators observed. These results also strongly suggest that inhibition of CRTH2 would have a greater effect on alleviating allergic inflammation than antagonism of the DP1 PGD2
receptor. Further, Satoh et al.
) report preliminary findings using the CRTH2−/−
mice that are consistent with our findings using a CRTH2 antagonist (22
). In two different models of murine allergic airway disease, the inflammatory infiltrate and pro-inflammatory cytokine and chemokine production are greatly reduced by deletion or antagonism of CRTH2. These findings are in sharp contrast to the results reported by Chevalier et al.
). Therefore, as Chevalier’s results point out, gene KO studies need to be evaluated with the possibility of influence from gene compensatory mechanisms. It may also be possible, though unlikely given Compound A specificity, that the effects we observed could be triggered by mechanisms independent of the PGD2
/CRTH2 pathway. We believe this unlikely, however, given the generally complementary results observed here and Nakamura et al.
using the CRTH2 knockout mice in comparable experiments.
In summary, the ability of potent and specific CRTH2 antagonist compound to decrease cutaneous inflammation and antigen-specific Ig levels strongly suggest that CRTH2 blockade may be powerful therapeutic course for the treatment of AD as well as for allergic rhinitis and asthma.