The Retinoic Acid Receptor-alpha mediates human T-cell activation and Th2 cytokine and chemokine production

Abstract

Background: We have recently demonstrated that all-trans-retinoic acid (ATRA) and 9-cis-retinoic acid (9-cis RA) promote IL-4, IL-5 and IL-13 synthesis, while decreasing IFN-gamma and TNF-alpha expression by activated human T cells and reduces the synthesis of IL-12p70 from accessory cells. Here, we have demonstrated that the observed effects using ATRA and 9-cis RA are shared with the clinically useful RAR ligand, 13-cis retinoic acid (13-cis RA), and the retinoic acid receptor-alpha (RAR-alpha)-selective agonist, AM580 but not with the RAR-beta/gamma ligand, 4-hydroxyphenylretinamide (4-HPR).

Results: The increase in type 2 cytokine production by these retinoids correlated with the expression of the T cell activation markers, CD69 and CD38. The RAR-alpha-selective agonist, AM580 recapitulated all of the T cell activation and type 2 cytokine-inducing effects of ATRA and 9-cis-RA, while the RAR-alpha-selective antagonist, RO 41-5253, inhibited these effects.

Conclusion: These results strongly support a role for RAR-alpha engagement in the regulation of genes and proteins involved with human T cell activation and type 2 cytokine production.

Figure 1

The effects of various clinically-utilized retinoid compounds on Th2-associated cytokine production by anti-CD3e-activated PBMCs. Supernatants of anti-CD3e-activated PBMCs treated with EtOH or 10^-9 to 10^-6 M ATRA (○), 9-cis-RA (□), 13-cis-RA (●), or 4-HPR (■) for 48 h were examined for IL-4 or IL-5. The values shown represent the average fold change obtained from 4 donors.

Figure 2

The effects of various clinically-utilized retinoid compounds on Th1-associated cytokine production by anti-CD3e-activated PBMCs. Supernatants of anti-CD3e-activated PBMCs treated with EtOH or 10^-9 to 10^-6 M ATRA (○), 9-cis-RA (□), 13-cis-RA (●), or 4-HPR (■) for 48 h were examined for IFN-γ or IL-12 protein levels by ELISA analysis. The values shown represent the average fold change obtained from 4 donors.

Figure 3

Correlation between retinoid-induced IL-5 expression and T cell-associated CD38 and CD69 expression in anti-CD3, -activated PBMC. IL-5 protein levels were quantitated by ELISA in supernatants of 48 h, anti-CD3,-activated PBMC treated with ETOH or ATRA (○), 9-cis-RA (□), 13-cis-RA (●), or 4-HPR (■). The values for the level of expression (mean channel number) of CD38 and CD69 on T cells were obtained by flow cytometry of cells from the same culture. Simple regression was performed on these values as described in Materials and Methods. Data shown is average fold change calculated from three experiments.

Figure 4

A RAR-α agonist specifically induces Th2-associated cytokine production by anti-CD3,-activated PBMC. IL-4, IL-5, and IL-13 proteins were quantitated by ELISA in supernatants of 48 h, anti-CD3,-activated PBMC treated with EtOH, ATRA (10^-7 M), the RAR-α agonist, AM580 (10^-7 M), or an RAR- α antagonist, RO 41-5254 (10^-7 M). The values shown represent the average obtained from 4 donors ± SEM. Means that express different superscripts are significantly different by at least p < 0.02.

Figure 5

A RAR-α agonist specifically inhibits Th1-associated cytokine production by anti-CD3,-activated PBMC. IFN-γ (panel A) and IL-12p70 (panel B) proteins were quantitated by ELISA in supernatants of 48 h, anti-CD3,-activated PBMC treated with control EtOH, ATRA (10^-7 M), AM580 (10^-7 M), or an RAR- α antagonist, RO 41–5254 (10^-7 M). The values shown represent the average obtained from 4 donors ± SEM. Means that express different superscripts are significantly different by at least p < 0.03.

Figure 6

ATRA and an RAR- -α agonist upregulate and an RAR-α antagonist downregulates the expression of IL-4 and IL-5 mRNA in anti-CD3, -activated PBMC. Taqman^® semi-quantitative PCR for IL-4 and IL-5 transcripts was performed using total cellular RNA of 48 h, anti-CD3,-activated PBMC treated with EtOH, ATRA (10^-7 M), an RAR- α agonist, AM580 (10^-7 M), or an RAR- α antagonist, RO 41–5254 (10^-7 M). Values obtained for each cytokine message was normalized to that obtained for 18S rRNA in the same sample as described in "Materials and Methods". The normalized values were then expressed as a function of the ETOH control sample. The data are representative of all of the donors tested.

Figure 7

A RAR- α agonist induces and the RAR- α antagonist, R0 41–5254, reduces the expression of CD69 and CD38 on human T cells during activation. PBMC were activated with anti-CD3, mAb in the presence or absence of control ETOH or 10^-7 M of ATRA, AM580, or RO41-5254 for 48 h. After activation, the cells were harvested and the cell surface levels of CD69 and CD38 were assessed by flow cytometric analysis as described in the Materials and Methods. The data are representative of all of the donors tested.

Figure 8

The effects of ATRA on Th1 and Th2 cytokine production are primarily mediated through RAR-α with minimal involvement of liganded RXRs. IL-12p70 and IFN-γ (Panel A) and IL-4 and IL-5 (Panel B) proteins were quantitated by ELISA in supernatants of 48 h, anti-CD3,-activated PBMC treated in the absence or presence of EtOH, ATRA (10^-8 M), or the RAR-α agonist, AM580 (10^-8 M) and in the presence or absence of EtOH (□) or the RXR agonist (10^-8 M, ■). The above data are representative of the results from two different donors examined.