Essential role for hematopoietic prostaglandin D2 synthase in the control of delayed type hypersensitivity

Abstract

Hematopoietic prostaglandin D(2) synthase (hPGD(2)S) metabolizes cyclooxygenase-derived prostaglandin (PG) H(2) to PGD(2), which is dehydrated to cyclopentenone PGs, including 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)). PGD(2) acts through two receptors (DP1 and DP2/CRTH2), whereas 15d-PGJ(2) can activate peroxisome proliferator-activated receptors or inhibit a range of proinflammatory signaling pathways, including NF-kappaB. Despite eliciting asthmatic and allergic reactions through the generation of PGD(2), it is not known what role hPGD(2)S plays in T helper (Th)1-driven adaptive immunity. To investigate this question, the severity and duration of a delayed type hypersensitivity reaction was examined in hPGD(2)S knockout and transgenic mice. Compared with their respective controls, knockouts displayed a more severe inflammatory response that failed to resolve, characterized histologically as persistent acute inflammation, whereas transgenic mice had little detectable inflammation. Lymphocytes isolated from inguinal lymph nodes of hPGD(2)S(-/-) animals showed hyperproliferation and increased IL-2 synthesis effects that were rescued by 15d-PGJ(2), but not PGD(2), working through either of its receptors. Crucially, 15d-PGJ(2) exerted its suppressive effects through the inhibition of NF-kappaB activation and not through peroxisome proliferator-activated receptor signaling. In contrast, lymph node cultures from transgenics proliferated more slowly and synthesized significantly less IL-2 than controls. Therefore, contrary to its role in driving Th2-like responses, this report shows that hPGD(2)S may act as an internal braking signal essential for bringing about the resolution of Th1-driven delayed type hypersensitivity reactions. Consequently, hPGD(2)S-derived cyclopentenone PGs may protect against inflammatory diseases, where T lymphocytes play a pathogenic role, as in rheumatoid arthritis, atopic eczema, and chronic rejection.

Fig. 1.

hPGD₂S controls the severity and duration of DTH responses. C57BL/6 mice lacking hPGD₂S (■) and wild-type controls (□) (A) along with FVB mice overexpressing this enzyme (♦) and their appropriate controls (□) (B) were sensitized at the base of the tail and challenged in the left paw 14 d later with mBSA, which induced a DTH reaction. Contralateral paws received saline. Paw swelling measurements were made by water-displacement plethysmometry at selected time points after challenge. n = 6–8 animals per group; ∗, P ≤ 0.05; ∗∗, P ≤ 0.01, as determined by ANOVA, followed by Bonferroni t test, with data expressed as mean ± SEM.

Fig. 2.

hPGD₂S acts as a braking signal for lymphocyte proliferation and IL-2 synthesis. Inguinal lymph nodes from hPGD₂S^−/− mice previously sensitized to mBSA to trigger a DTH reaction were incubated with the specific antigen (mBSA) for 72 h (A), revealing significantly increased proliferation of these cells, and for 24 h (B), showing enhanced IL-2 synthesis. Similar experiments were carried out on mice overexpressing hPGD₂S, where proliferation (C) and IL-2 synthesis (D) were found to be significantly reduced compared with controls. n = 5 animals per group; ∗, P ≤ 0.05; ∗∗, P ≤ 0.01, as determined by t test, with data expressed as mean ± SEM.

Fig. 3.

Synthesis of hPGD₂S-derived PGD₂ and 15d-PGJ₂ increases during inflammatory resolution. Inguinal lymph nodes were excised from groups of mBSA-sensitized wild-type and hPGD₂S^−/− mice 8, 24, 48, and 96 h after mBSA challenge and cultured for 24 h to allow for accumulation of PGs within the culture medium. Synthesis of PGD₂ (A) and its metabolite 15d-PGJ₂ (B) was found to increase during resolution in wild-type (□) animals, with levels barely detectable in hPGD₂S^−/− (■) samples. (C) PGE₂ was also measured in culture supernatants and found not to change substantially through the time course of the reaction in wild types (□), and no significant difference was detectable in PGE₂ levels between wild types and knockouts (■). n = 3–5 animals per group. Data are expressed as mean ± SEM.

Fig. 4.

The 15d-PGJ_2, but not PGD₂, reverses lymphocyte hyperproliferation and IL-2 synthesis in hPGD₂S^−/− mice. Inguinal lymph nodes were taken from mBSA-sensitized animals 24 h after in vivo challenge and incubated ex vivo for 24 h with BW245C (DP1 receptor), 15R-PGD₂ (DP2 receptor agonist), and 15d-PGJ₂. Only 15d-PGJ₂ was found to reverse the hyperproliferative phenotype (A) and IL-2 synthesis (B) of cells deficient in hPGD₂S, whereas DP1 and DP2 agonists were without effect. In the experiments shown in B, BW245C and 15R-PGD₂ were also used at 30 nM and 2 μM, respectively, without detectable effect (data not shown). Experiments with a second DP2 receptor agonist, 15(R)-15 methyl PGD₂ at 1.25, 2.5, and 5.0 nM confirmed original findings (data not shown). Importantly, the neutralizing property of 15d-PGJ₂ was effective at doses that did not cause lymphocyte apoptosis. n = 3–5 animals per group; ∗, P ≤ 0.05; ∗∗, P ≤ 0.01, as determined by ANOVA, followed by post hoc Dunnett’s test, with data expressed as mean ± SEM

Fig. 5.

The inhibitory property of 15d-PGJ₂ on lymphocyte proliferation is not mediated through PPAR signaling but, at least in part, through inhibition of NF-κB. (A) Inguinal lymph nodes were taken from mBSA-sensitized hPGD₂S^−/− and wild-type animals 24 h after in vivo challenge and incubated for 24 h with a PPARγ agonist (rosiglitazone) or antagonist (GW9662) as well as 15d-PGJ₂ alone or with GW9662, where GW9662 was preincubated for 1 h before adding the cyPG. As before (Fig. 4), only 15d-PGJ₂ altered proliferation, whereas PPARγ agonists and antagonists were without effect. Even prior blocking of PPARγ did not alter the rescuing effects of 15d-PGJ₂ on knockout cells when compared with corresponding controls. A PPARα agonist (GW7647, 0.1 and 1.0 μg/ml) as well as a PPARβ/δ agonist (L-165041, 0.01–1 μM) were also without effect on proliferation (data not shown). In contrast, NF-κB binding was found to be greatly enhanced in lymph node extract from hPGD₂S^−/− animals (B), including freshly isolated cells from sensitized mice (labeled C) as well as those stimulated for 24 h with either the specific antigen (mBSA, labeled M) or Con A (nonspecific, labeled A). The converse also held true for hPGD₂S transgenic mice (C), where NF-κB binding was reduced in all treatment groups. n = 3–5 animals per group; ∗∗, P ≤ 0.01, as determined by ANOVA, followed by post hoc Dunnett’s test, with data expressed as mean ± SEM.