We used a well-characterized model of FITC-induced contact hypersensitivity to examine the role of the PGD2
receptor, CRTH2, in mediating the inflammatory response. In this model, mice are sensitized to FITC by two topical applications to the ventral skin on days 1 and 2, and this results in FITC-specific IgE antibody production. Six days following the second abdominal painting, the mice are challenged by a topical FITC application to the right ear. The ensuing inflammatory response and skin lesions in many ways reflect acute lesions observed in human AD (11
). Using this murine model, we established that much of the inflammatory response observed is dependent upon the CRTH2–PGD2
receptor, and not on the DP1 receptor, as the DP1 antagonist, BW868c, did not inhibit ear swelling post-FITC challenge (). Further, this response was both dose dependent and not due to delayed kinetics of the inflammation ().
An investigation into the inflammatory mediators thought to play a role in orchestrating the inflammatory response proved insightful. One hour after challenge, we did not detect any IL-1β or TSLP in protein lysates from the FITC-treated ears (data not shown). However, 4 h post-challenge, both these mediators were readily detectable (). Interestingly, we noticed that the addition of the FITC Veh, acetone:dibutyl phthalate, induced TSLP in the absence of FITC. It has previously been noted that dibutyl phthalate has an adjuvant effect in this model of contact hypersensitivity and can increase trafficking of dendritic cells from the skin to the draining lymph nodes (32
). Further, it has recently been shown that TSLP-treated epidermal Langerhans cells secrete increased amounts of the Th
2 T cell-attracting chemokine CCL17 (TARC) and promote the differentiation of naive CD4+
T cells into pro-allergic Th
2 lineage T cells (33
). The ability of dibutyl phthalate to act as an adjuvant may in part be attributable to our observation of its ability to induce TSLP production, thereby activating the skin dendritic cells (6
) and Langerhans cells (33
Investigating TSLP production in FITC-challenged ears showed that TSLP levels increase significantly over the levels observed with just Veh treatment. This increase upon FITC addition was almost completely abrogated by the oral administration of Cmpd A prior to challenge, suggesting that skin allergen-induced TSLP release was dependent upon CRTH2 activation, whereas the dibutyl phthalate-mediated TSLP production was not. The major producer of PGD2
is activated mast cells, and these cells are present throughout the dermis and surrounding blood vessels. Because this model of contact hypersensitivity is both CD4+
T cell and mast cell dependent (11
), we hypothesized that IgE-mediated activation of mast cells, which results in the release of PGD2
, could directly stimulate keratinocytes or epithelial cells to produce TSLP. This, in turn, could initiate the inflammatory cascade consistent with the idea that TSLP is a ‘master switch’ for allergic inflammation (29
). We also observed that human basal epidermal cells, and possibly keratinocytes which secrete TSLP (34
), express the CRTH2 receptor (). To test whether CRTH2-mediated TSLP production was responsible for the allergic inflammation, we treated mice prior to ear challenge with a neutralizing anti-TSLP antibody in the presence or absence of a suboptimal dose of Cmpd A. No TSLP was detected in protein lysates of challenged ears from mice receiving the neutralizing antibody (data not shown). Together with the observation that anti-TSLP-treated animals displayed a reduced level of ear swelling, this suggested, but did not rule out, that TSLP was effectively neutralized. Animals that received the anti-TSLP antibody and the suboptimal dose of Cmpd A had a further reduction in ear swelling and a reduced inflammatory infiltrate. Additionally, levels of IL-4, a key pro-inflammatory cytokine in this model (11
), were reduced to a greater degree by the combination of anti-TSLP and 0.1 mg kg−1
Cmpd A than either alone. This suggested that TSLP played a role in coordinating the inflammatory response to FITC challenge, but other non-TSLP-mediated pathways were also involved. This was confirmed by histological analysis, which showed a decreased inflammatory infiltrate in dually treated mice compared with mice that received either anti-TSLP antibody or 0.1 mg kg−1
Cmpd A alone, as well as the observation that anti-TSLP antibody treatment had no effect on GRO-α levels, but whose levels were greatly reduced by Cmpd A treatment ( and ). These observations also suggest, although not directly tested, that PGD2
-mediated activation of CRTH2 may also positively regulate TSLP expression. Alternatively, PGD2
–CRTH2 may act via another rapidly induced cytokine, such as IL-1β.
Examining gene expression levels from FITC-challenged ears showed a dynamic pattern of up-regulation across many pro-inflammatory gene families. For instance, the transcription of many cytokines was dramatically increased within 4–8 h of FITC application and were negatively affected by Cmpd A, such as IL-4, IL-1α and β and IFN-γ. Similarly, many FITC-induced chemokines were negatively regulated by Cmpd A, including CCL7/MCP-3, CCL17/TARC, CCL5/RANTES, CCL24/eotaxin-2, CXCL9/MIG and CXCL1/GRO-α. There was no distinction of Th1- or Th2-type cytokines or chemokines being singularly modulated, and similarly, there is a wide array of cells including T and B cells, macrophages, eosinophils and neutrophils that could be stimulated by these cytokines and chemokines. Thus, consistent with the protein data, the gene expression analysis demonstrates that a wide range of inflammatory mediators are elicited by FITC challenge and are down-regulated by antagonism of CRTH2 via Cmpd A.
It is of interest to note that the major therapeutic effect seen with Cmpd A administration at the gene expression level occurred at 8 h with some (but not all) genes becoming up-regulated again at 24 h. This may be a result of the predicted T1/2 of Cmpd A being ~3 h in mice. Not all genes were back up, however, such as CCR4 and CCR6, the receptors for CCL17/TARC, CCL22/MDC and CCL20/MIP-3α, respectively, as well as a number of genes from other families, for example ALOX 15. While this may be reflective of the regulation specificities of the genes themselves, it is intriguing that in many examples, these same genes were rapidly and more strongly up-regulated at times later than 8 h following Dex treatment when compared with Cmpd A. This is particularly evident within the entire CC chemokine cluster of genes, suggesting that steroid treatment may not be as effective as Cmpd A in the long-term down-regulation of these genes in this model. Mechanistically, this effect may be attributed to the observation that Cmpd A administration strongly inhibited the inflammatory influx, which would in turn translate to a reduction in the mRNA levels of genes expressed by these cell types. Additionally, as PGD2-mediated CRTH2 activation occurs early in the allergic inflammatory cascade, blockade of this interaction would be expected to have broad downstream effects, inhibiting both the recruitment of inflammatory cell types as well as mediators produced. This is in contrast to the mechanism of action of the steroid Dex, which inhibits the transcription factor NF-AT. Along these same lines, GPR44/CRTH2 did not appear to be down-regulated by Dex treatment at all in this FITC model compared with Cmpd A.
In summary, an examination of cytokine and chemokine RNA and protein levels suggests that multiple pathways underlie the FITC-induced inflammation and are regulated, in part, the CRTH2 activation. These reductions in cytokines and chemokines are consistent with the gross reduction in ear thickness observed, as well as the histology, which shows a rather limited inflammatory infiltrate upon treatment with the higher doses (1 and 10 mg kg−1) of Cmpd A. Taken together, these observations underscore the pivotal role of the CRTH2–PGD2 interaction in regulating allergen-induced cutaneous inflammation. However, a complete reduction in pro-inflammatory mediators was not observed with Cmpd A administration. This may be due, in part, to other pro-inflammatory mediators released by activated mast cells or the pharmacokinetic/pharmacodynamic profile of Cmpd A in mice. Nonetheless, these observations suggest that an overall reduction, but not necessarily a complete abrogation, of pro-inflammatory cytokine and chemokine production may be sufficient to severely dampen the cutaneous inflammatory response and the ensuing tissue pathology. Further, these studies suggest that antagonism of CRTH2 may be a potentially useful strategy in the therapeutic intervention of allergic disease.