Indomethacin inhibits eosinophil migration to prostaglandin D2 : therapeutic potential of CRTH2 desensitization for eosinophilic pustular folliculitis

Abstract

Indomethacin is a cyclo-oxygenase inhibitor, and shows therapeutic potential for various eosinophilic skin diseases, particularly eosinophilic pustular folliculitis. One of the unique characteristics of indomethacin is that, unlike other non-steroidal anti-inflammatory drugs, it is a potent agonist of chemoattractant receptor-homologous molecule expressed on T helper type 2 cells (CRTH2), a receptor for prostaglandin D2 (PGD2 ). This study investigated the pharmacological actions of indomethacin on eosinophil migration to clarify the actual mechanisms underlying the therapeutic effects of indomethacin on eosinophilic pustular folliculitis. Eosinophils exhibited chemokinetic and chemotactic responses to both PGD2 and indomethacin through CRTH2 receptors. Pre-treatment of eosinophils with indomethacin greatly inhibited eosinophil migration to PGD2 and, to a much lesser extent, to eotaxin (CCL11); these effects could be mediated by homologous and heterologous desensitization of eosinophil CRTH2 and CCR3, respectively, by agonistic effects of indomethacin on CRTH2. Indomethacin also cancelled a priming effect of Δ(12) -PGJ2 , a plasma metabolite of PGD2 , on eosinophil chemotaxis to eotaxin. Indomethacin down-modulated cell surface expression of both CRTH2 and CCR3. Hair follicle epithelium and epidermal keratinocytes around eosinophilic pustules together with the eccrine apparatus of palmoplantar lesions of eosinophilic pustular folliculitis were immunohistochemically positive for lipocalin-type PGD synthase. Indomethacin may exert therapeutic effects against eosinophilic skin diseases in which PGD2 -CRTH2 signals play major roles by reducing eosinophil responses to PGD2 .

Keywords: CCR3; Lipocalin-type prostaglandin D synthase; chemoattractant receptor-homologous molecule expressed on T helper type 2 cells; desensitization; eotaxin.

Figure 1

Indomethacin and prostaglandin D₂ (PGD₂) induced chemotaxis in eosinophils through chemoattractant receptor-homologous molecule expressed on T helper type 2 cells (CRTH2) receptors. Cells were mixed with either indomethacin, PGD₂, or medium, and then applied to the upper chamber. Eosinophil migration to the lower chamber containing either indomethacin, PGD₂, or medium was assessed by photometric assay using eosinophil peroxidase activity (a, b). Eosinophil migration in response to indomethacin and PGD₂ (lower chambers) was assessed in the presence of CAY10471 (3 nm), a CRTH2 antagonist, in the upper chamber (c). Representative results of two independent experiments are shown.

Figure 2

Eosinophil chemotaxis is inhibited by pre-treatment with indomethacin, but not diclofenac. Eosinophils were pre-treated with indomethacin or diclofenac for 90 min at 37°, followed by centrifugation and application to the upper chamber. Eosinophil migration to prostaglandin D₂ (PGD₂) (a, c) and eotaxin (CCL11) (b, d) in the lower chambers was assessed by eosinophil peroxidase photometric assay. *P < 0·05. Representative results of at least three independent experiments are shown.

Figure 3

Effects of indomethacin on chemotaxis to prostaglandin D₂ (PGD₂) in combination with eotaxin. Eosinophils were pre-treated with indomethacin for 90 min at 37°, followed by centrifugation and application to the upper chamber. In lower chambers, both PGD₂ and eotaxin (CCL11) were added, and eosinophil migration was assessed. *P < 0·05. Representative results of at least three independent experiments are shown.

Figure 4

Chemoattractant receptor-homologous molecule expressed on T helper type 2 cells (CRTH2) -mediated eosinophil priming by Δ¹²-prostaglandin J₂ (PGJ₂) was cancelled by indomethacin. (a) Eosinophils were pre-treated with CAY10471 for 10 min at 37°. After centrifugation, they were mixed with Δ¹²-PGJ₂, then applied to the upper chamber. Chemotaxis to eotaxin (lower chamber) was assessed in the presence of Δ¹²-PGJ₂. Pre-treatment with CAY10471 cancelled the priming effects of Δ¹²-PGJ₂ on eosinophil migration to eotaxin. (b) Eosinophils were pre-treated with indomethacin for 90 min at 37°. After centrifugation, they were mixed with Δ¹²-PGJ₂, then applied to the upper chamber, and subjected to chemotactic assay for eotaxin. *P < 0·05. Representative results of at least three independent experiments are shown.

Figure 5

Down-modulation of chemoattractant receptor-homologous molecule expressed on T helper type 2 cells (CRTH2) and CCR3 by indomethacin. Eosinophils were incubated with indomethacin (10⁻⁷ or 10⁻⁶ m) for 90 min. Cell surface expression of CRTH2 and CCR3 was assessed by flow cytometry. Representative results of three independent experiments are shown.

Figure 6

Effects of continuous exposure to indomethacin on eosinophil migration in response to prostaglandin D₂ (PGD₂) and eotaxin. Indomethacin was added to both upper and lower chambers. Migration to PGD₂ (a), eotaxin (b) and PGD₂/eotaxin (c) was assessed. Chemokinesis (eosinophil migration to medium, open column) markedly increased under exposure to indomethacin. On the other hand, eosinophil migration to PGD₂ significantly decreased, and eotaxin-induced migration was enhanced at high doses of indomethacin along with increased chemokinesis. (c) Eosinophil migration to PGD₂/eotaxin was dose-dependently suppressed under continuous exposure to indomethacin. *P < 0·05. Representative results of at least three independent experiments are shown.

Figure 7

Immunohistochemical localization of lipocalin-type prostaglandin D synthase (L-PGDS) in skin lesions of eosinophilic pustular folliculitis (EPF). (a) Hair follicle epithelium with eosinophil infiltration was positive for L-PGDS. (b) Isotype control of (a). (c) Palmar lesions of EPF. Epidermal keratinocytes around pustules and eccrine ostia in the stratum corneum showed positive results for L-PGDS. (d) L-PGDS in eccrine ducts and glands. Basal (clear) cells in secretory portion and ductal cells were positive for L-PGDS.