Prostaglandin D2 and leukotriene E4 synergize to stimulate diverse TH2 functions and TH2 cell/neutrophil crosstalk

Abstract

Background: Prostaglandin D2 (PGD2) and cysteinyl leukotrienes (cysLTs) are lipid mediators derived from mast cells, which activate TH2 cells. The combination of PGD2 and cysLTs (notably cysteinyl leukotriene E4 [LTE4]) enhances TH2 cytokine production. However, the synergistic interaction of cysLTs with PGD2 in promoting TH2 cell activation is still poorly understood. The receptors for these mediators are drug targets in the treatment of allergic diseases, and hence understanding their interaction is likely to have clinical implications.

Objective: We aimed to comprehensively define the roles of PGD2, LTE4, and their combination in activating human TH2 cells and how such activation might allow the TH2 cells to engage downstream effectors, such as neutrophils, which contribute to the pathology of allergic responses.

Methods: The effects of PGD2, LTE4, and their combination on human TH2 cell gene expression were defined by using a microarray, and changes in specific inflammatory pathways were confirmed by means of PCR array, quantitative RT-PCR, ELISA, Luminex, flow cytometry, and functional assays, including analysis of downstream neutrophil activation. Blockade of PGD2 and LTE4 was tested by using TM30089, an antagonist of chemoattractant receptor-homologous molecule expressed on TH2 cells, and montelukast, an antagonist of cysteinyl leukotriene receptor 1.

Results: PGD2 and LTE4 altered the transcription of a wide range of genes and induced diverse functional responses in TH2 cells, including cell adhesion, migration, and survival and cytokine production. The combination of these lipids synergistically or additively enhanced TH2 responses and, strikingly, induced marked production of diverse nonclassical TH2 inflammatory mediators, including IL-22, IL-8, and GM-CSF, at concentrations sufficient to affect neutrophil activation.

Conclusions: PGD2 and LTE4 activate TH2 cells through different pathways but act synergistically to promote multiple downstream effector functions, including neutrophil migration and survival. Combined inhibition of both PGD2 and LTE4 pathways might provide an effective therapeutic strategy for allergic responses, particularly those involving interaction between TH2 cells and neutrophils, such as in patients with severe asthma.

Keywords: Prostaglandin D(2); T(H)2 cells; chemoattractant receptor-homologous molecule expressed on T(H)2 cells; leukotriene E(4); neutrophils.

Fig E1

Analysis of cell phenotype. A and B, Expanded T_H2 cells were CD3⁺CD4⁺CRTH2⁺CD45RO⁺GATA3⁺CCR6⁻CCR7⁻CD45RA⁻RORγt⁻ effector memory cells (Fig E1, A), which produce the type II cytokines IL-8 and IL-22 after stimulation with PMA (5 ng/mL) and ionomycin (500 ng/mL; Fig E1, B), as detected by means of flow cytometry. Blue lines represent staining of indicated cell markers, and red lines represent unstained controls. C, The same cytokine profile was observed in freshly isolated ex vivo T_H2 cells by using qPCR (mRNA) and Luminex (protein) assays (high background IL-8 levels in the unstimulated sample precluded accurate analysis of this cytokine in the Luminex assay; n = 6 for Fig E1, A and B; n = 2 for mRNA and n = 1 for protein in Fig E1, C).

Fig E2

Network diagram of genes depicting pathways involved in activation of T_H2 cells induced by PGD₂ and LTE₄ based on microarray data. A, PI3K pathway. B, Apoptosis pathway. Red color shows gene upregulation, and green color shows downregulation.

Fig E3

Phosphorylation of Akt in T_H2 cells after treatment with PGD₂ and LTE₄ in the presence or absence of TM30089 and montelukast. The intensity of the bands for phospho-Akt was quantified after normalization with the bands for total Akt.

Fig E4

Effect of TM30089 and montelukast on transcriptional regulation of cytokine genes induced by PGD₂ and LTE₄ in T_H2 cells determined by using qPCR. The control sample was treated as 1-fold (n = 1).

Fig E5

Effect of IL-8 on neutrophil migration. A, Neutrophil migration to various concentrations of rhIL-8 in chemotaxis assay. B, Effect of increasing concentration of anti–IL-8 antibody on neutrophil migration induced by 50 nmol/L rhIL-8.

Fig E6

Effect of GM-CSF on the decrease in CD16 levels in neutrophils, a biomarker of apoptosis, induced by serum deprivation. A, Decrease of CD16^high neutrophils with the time of serum deprivation. B, Inhibitory effect of various concentration of rhGM-CSF on the decrease of CD16^high neutrophils induced by serum deprivation for 12 hours. C, The inhibitory effect of rhGM-CSF (1 ng/mL) was reversed by anti–GM-CSF antibody in a dose-dependent manner. IC₅₀, Inhibitory concentration of 50%.

Fig 1

Gene regulation in T_H2 cells by PGD₂ and LTE₄ detected by using a microarray. A, Venn diagram representing total numbers of genes regulated significantly. B, Venn diagrams and heat map showing numbers of genes downregulated or upregulated significantly. P < .05.

Fig 2

Effects of PGD₂ and LTE₄ on the apoptosis and migration of T_H2 cells. A, Detection of Annexin V in T_H2 cells treated with IL-2 deprivation in the presence of compounds, as indicated. B, Cell migration in response to PGD₂, LTE₄, or LTD₄(left panel) or the combination of indicated compounds (right panel). *P < .05 between indicated treatments (Fig 2, A) or between PGD₂ plus LTE₄ and other treatments (Fig 2, B). n = 3.

Fig 3

Involvement of integrins in CAIA of T_H2 cells induced by PGD₂ and LTE₄. Cell aggregation after incubation with indicated treatments for 2 hours (A), for 2 or 9 hours in the presence of EDTA or MnCl₂(B), or for 1 hour in the presence of the indicated antibodies (C). Scale bar = 0.5 mm. *P < .05 between PGD₂ plus LTE₄ and other treatments or between indicated conditions (n = 2-5).

Fig 4

PGD₂ and LTE₄ modulate gene transcription of cytokines, chemokines, and surface receptors in T_H2 cells determined by means of microarray (A and B) or PCR array (C). Fig 4, A, Venn diagram and heat map showing the number and distribution of genes significantly regulated. Fig 4, B and C, Strongly upregulated genes. P < .05 (n = 3).

Fig 5

Effects of PGD₂ and LTE₄ on production of selected cytokines in T_H2 cells. A, Levels of mRNA measured by using qPCR. The mRNA levels in control samples were treated as 1-fold. B, Protein levels were detected with the Luminex assay. *P < .05 between PGD₂ plus LTE₄ and other conditions or the indicated condition (n = 3).

Fig 6

T_H2-derived cytokines activate neutrophils. A, IL-8 and GM-CSF levels in stimulated T_H2 cell supernatants assigned as supernatants 1 (white bars), 2 (gray bars), and 3 (black bars, n = 4). B, Effect of supernatants (left panel), IL-8, PGD₂, LTE₄(right panel), and anti–IL-8 antibody on neutrophil migration. C, Effect of supernatants and anti–GM-CSF antibody on expression of CD16 (left panel) and Annexin V (right panel) in neutrophils determined by using fluorescence-activated cell sorting. *P < .05 between the indicated treatment and other conditions (n = 2-7).