The A2B adenosine receptor impairs the maturation and immunogenicity of dendritic cells

Abstract

The endogenous purine nucleoside adenosine is an important antiinflammatory mediator that contributes to the control of CD4(+) T cell responses. While adenosine clearly has direct effects on CD4(+) T cells, it remains to be determined whether actions on APC such as dendritic cells (DC) are also important. In this report we characterize DC maturation and function in BMDC stimulated with LPS in the presence or absence of the nonselective adenosine receptor agonist NECA (5'-N-ethylcarboxamidoadenosine). We found that NECA inhibited TNF-alpha and IL-12 in a concentration-dependent manner, whereas IL-10 production was increased. NECA-treated BMDC also expressed reduced levels of MHC class II and CD86 and were less effective at stimulating CD4(+) T cell proliferation and IL-2 production compared with BMDC exposed to vehicle control. Based on real-time RT-PCR, the A(2A) adenosine receptor (A(2A)AR) and A(2B)AR were the predominant adenosine receptors expressed in BMDC. Using adenosine receptor subtype selective antagonists and BMDC derived from A(2A)AR(-/-) and A(2B)AR(-/-)mice, it was shown that NECA modulates TNF-alpha, IL-12, IL-10, and CD86 responses predominantly via A(2B)AR. These data indicate that engagement of A(2B)AR modifies murine BMDC maturation and suggest that adenosine regulates CD4(+) T cell responses by selecting for DC with impaired immunogencity.

FIGURE 1

The nonselective adenosine analog NECA modulates LPS-induced BMDC maturation. A, MACS CD11c⁺ BMDC were treated with 1 μM NECA or DMSO vehicle control in the presence or absence of 500 ng/ml LPS, and after 18 h supernatant was collected. TNF-α, IL-12p70, and IL-10 were assayed by ELISA. Data represent the mean ± SEM from three or more experiments B, Representative histogram of CD86 expression 24 h after BMDC treated in the presence or absence of LPS and 250 nM NECA or vehicle control. C, IL-2 from cocultures with CD4⁺ T cells. Before coculture, BMDC were treated in the presence or absence of LPS and NECA overnight and then washed thoroughly. In the syngeneic coculture BMDC were pulsed with OVA during overnight incubation and then cultured with OT-II CD4⁺ T cells for 24 h. Data represent the mean ± SEM from three experiments. In the allogeneic study BMDC from C57BL/6 mice were cultured with CD4⁺ T cells from BALB/cJ mice for 48 h. Data represent the mean ± SEM from two experiments conducted in duplicate. *, p < 0.05.

FIGURE 2

IL-10 is an important mediator in the regulation of BMDC by NECA. A, BMDC were treated in presence or absence of LPS and 250 nM NECA. At various time points, cells were lysed, RNA was isolated, and real-time RT-PCR was conducted to determine IL-10 mRNA levels. Transcripts were normalized to the 1-h media control treatment group. Data represent the mean ± SEM from three or more experiments. B, Representative histogram of CD86 expression 24 h after BMDC treated in the presence or absence of LPS, 250 nM NECA, or 10 μg/ml anti-IL-10 neutralizing Ab. C, BMDC were treated with 1 μM NECA or DMSO vehicle control in the presence or absence of 500 ng/ml LPS, and after 18 h supernatant was collected. Anti-IL-10 neutralizing Ab (10 μg/ml) was added where indicated. TNF-α and IL-12p70 were assayed by ELISA. Data represent the mean ± SEM from two experiments conducted in duplicate. *, p < 0.05.

FIGURE 3

DC expression of adenosine receptors as measured by real-time RT-PCR and cAMP functional studies. A, Adenosine receptor mRNA in resting BMDC. Data represent the mean ± SEM from 10 or more experiments B, Kinetics of adenosine receptor mRNA expression in LPS-stimulated BMDC. Each gene transcript was compared with resting transcript levels. Data represent the mean ± SEM from three experiments. C, cAMP produced by BMDC treated with 250 nM NECA or DMSO vehicle control for 15 min in presence of 1 μM rolipram. In some cases cells had been treated overnight with 500 ng/ml LPS. Data represent the mean ± SEM from three or more experiments. D, MACS CD11c-enriched cells were isolated from intestinal lamina propria as described in Materials and Methods, and adenosine receptor mRNA was evaluated. Data represent the mean ± SEM from three experiments.*, p < 0.05.

FIGURE 4

NECA modulates LPS-induced TNF-α, IL-12, and IL-10 in an A_2BAR-dependent manner. BMDC were treated with LPS in the presence or absence of varying concentrations of NECA. Supernatants were collected after 18 h and cytokines were measured by ELISA. A, BMDC derived from wild-type, A_2AAR^−/−, or A_2BAR^−/− mice. B, Wild-type BMDC were pretreated for 15 min with 100 nM ZM241385, SCH58261, or ATL692 before addition of NECA and LPS. Data represent the mean ± SEM from three or more experiments. *, p < 0.05.

FIGURE 5

NECA inhibits LPS-induced CD86 expression in an A_2BAR-dependent manner. BMDC were treated with LPS in the presence or absence of 250 nM NECA. Cells were harvested after 24 h, stained with anti-CD86-PE or isotype control, and assayed by flow cytometry. A, Representative histograms of BMDC derived from wild-type, A_2AAR^−/−, A_2BAR^−/−, or A_2AAR^−/−/A_2BAR^−/− mice. B, Representative histograms of wild-type BMDC pretreated for 15 min with 1 μM ZM241385, SCH58261, or ATL692 before addition of NECA and LPS. All histograms are representative of at least three independent experiments.

FIGURE 6

NECA-treated CD86^low BMDC express more A_2BAR and altered cytokine response as compared with NECA-treated CD86^high or vehicle control-treated BMDC. NECA-treated, LPS-stimulated BMDC were harvested after 16 h and sorted into CD86^low and CD86^high populations. These sorted cells were compared with unsorted BMDC that were untreated or with LPS-stimulated cells in the presence of the vehicle control. A, Representative histograms showing NECA-treated, LPS-stimulated BMDC sorted for CD86 expression. B, A_2AAR and A_2BAR mRNA in BMDC. Data represent the mean ± SEM from three independent experiments. C, Cytokine mRNA in BMDC. Transcripts were standardized to the media-treated control group and represent the mean ± SEM from three independent experiments. *, p < 0.05.

FIGURE 7

The CD86^low subset of NECA-treated, LPS-stimulated BMDC have impaired immunogenicity. A, MLR with BMDC derived from C57BL/6 mice and CD4⁺ T cells from BALB/c mice. Sorted CD86^low and CD86^high NECA-treated BMDC and unsorted control BMDC were irradiated and then cocultured with CD4⁺ T cells for 96 h at indicated ratios, and 1 μCi of [³H]thymidine was added for the final 16 h. This is representative of three experiments conducted. B, MLR using 1:10 ratio of BMDC to CD4⁺ T cells. Supernatant was harvested at 48 h and assayed for IL-2. Data represent the mean ± SEM from three independent experiments. C, Syngeneic coculture with sorted CD86^low and CD86^high ADO-BMDC and unsorted control cells. BMDC were irradiated and then cocultured with CD4⁺ T cells for 72 h with 500 ng/ml soluble anti-CD3 Ab at indicated ratios with 1 μCi of [³H]thymidine added for the final 16 h. This is representative of three experiments conducted. D, Syngeneic coculture using nonirradiated BMDC and conducted at 1:10 ratio of BMDC to CD4⁺ T cells. Supernatants were harvested at 24 h and assayed for IL-2. Data represent the mean ± SEM from three independent experiments. *, p < 0.05.