Figure 1.
CGRP affects cytokine productions in DCs via cAMP/PKA pathway.
(A) The percentage of CD11c+ BMDCs was analyzed by FACS. (B) BMDCs were incubated with or without CGRP in the presence of LPS stimulation. The cellular cAMP levels were determined after the indicated time periods. Values are presented as mean ± SD (n = 3). (C) The concentrations of each cytokine were determined by ELISA after 24 h stimulation with LPS. Values are presented as mean ± SD (n = 3). *P<0.05, **P<0.01 VS. LPS only. (D) The concentrations of each cytokine were determined by ELISA after 24 h stimulation with LPS. Values are presented as mean ± SD (n = 3). *P<0.05, **P<0.01. (E) The mRNA expression was determined by real-time PCR after 6 h stimulation with LPS. Values are presented as mean ± SD (n = 3). *P<0.05, **P<0.01 vs. LPS only. α: αCGRP (1 nM), β: βCGRP (1 nM), dbc: db-cAMP (100 µM), 6b: 6-bnz-cAMP (100 µM), and 8C: 8-CPT-cAMP (100 µM) were added at the start of stimulation.
Figure 2.
CGRP affects cytokine production in DC-Th cell interaction.
(A) The concentrations of each cytokine were determined by ELISA after 24 h stimulation with OVA and anti-CD40 mAb. Values are presented as means ± SD (n = 3). **P<0.01 VS. OVA+anti-CD40 mAb. (B) The mRNA expression was determined by real-time PCR after 6 h stimulation with OVA and anti-CD40 mAb. Values are presented as mean ± SD (n = 3). *P<0.05, **P<0.01 vs. OVA+anti-CD40 mAb. (C) BMDCs and DO 11.10 Th0 cells were co-cultured in the presence of 200 µg/ml OVA for 72 h. IFN-γ production was detected by FACS after intracellular staining. Data are represented of three independent experiments. α: αCGRP (1 nM), β: βCGRP (1 nM), dbc: db-cAMP (100 µM), 6b: 6-bnz-cAMP (100 µM), and 8C: 8-CPT-cAMP (100 µM) were added from start point of stimulation.
Figure 3.
CGRP physiologically inhibits DTH response.
(A) WT and RAMP1-deficient mice were sensitized with OVA, and ear swelling was measured after challenge. Values are represented as mean ± SD (n = 9). (B) Draining LNs were collected 24 h after challenge, and total lymphocytes were stimulated with anti-CD3 and anti-CD28 mAbs for 48 h. IL-4 and IFN-γ concentrations in the supernatants were measured by ELISA. Values are presented as means ± SD (n = 6). *P<0.05. (C) CD11c+ DCs were purified from dLNs 96 h after sensitization with OVA. The mRNA expression was measured by real-time PCR. Values are presented as means ± SD (n = 6). (D) Draining LNs were collected 24 h after challenge, and total lymphocytes were stimulated with PMA/ionomycin for 6 h. IL-12 production in CD11c+ cells was detected by FACS after intracellular staining. Values are presented as means ± SD (n = 4). (E) WT or RAMP1-deficient BMDCs were pulsed with OVA and injected into the tail base of BALB/c mice. After 7 days, we challenged the footpad of each mouse with OVA. Values are represented as mean ± SD (n = 9). (F) The serum was collected 72 h after challenge, and the levels of OVA-specific IgG subtypes were determined by ELISA. Values are represented as mean ± SD (n = 9). *P<0.05, **P<0.01.
Figure 4.
CGRP inhibits CCR2 expression in vitro and in vivo.
(A) The mRNA expression of CCR2 was determined by real-time PCR using untreated-, CD40-stimulated or LPS-stimulated BMDCs. Values are presented as mean ± SD (n = 3). *P<0.05, **P<0.01. (B) BMDCs were differentiated in the presence or absence of CGRP. The expression level of CCR2 protein at each time point was measured by FACS. (C) WT and RAMP1-deficient mice were sensitized with Alexa488-labeled OVA, and migrated DCs in dLNs were analyzed by FACS. Values are presented as mean ± SD (n = 3. *P<0.05.
Figure 5.
CCR2-positive DCs have a potent capacity to induce Th1 cell differentiation.
(A) FACS of CCR2+ or CCR2− DCs in OVA sensitized LNs. (B) Sorted DCs were co-cultured with DO11.10 Th0 cells for 72 h. Cytokines in the supernatant were measured by ELISA. Values are presented as mean ± SD (n = 4). *P<0.05, **P<0.01. (C) The expression of co-stimulation molecules in DCs was analyzed by FACS. Values are presented as mean ± SD (n = 3). **P<0.05.
Figure 6.
The cartoon depicts functions of CGRP in the DC-mediated immune responses.
CGRP physiologically regulates DC responses as described in ‘Wild type’. In the absence of RAMP1, the increased number of migrated DCs in dLN after immunization, the unusual CCR2 expression on migrated DCs, and the higher expression of inflammatory cytokines were observed. These result in severe DTH response.