Nitric oxide and cGMP protein kinase (cGK) regulate dendritic-cell migration toward the lymph-node-directing chemokine CCL19

Abstract

Dendritic-cell (DC) migration to secondary lymphoid organs is crucial for the initiation of adaptive immune responses. Although LPS up-regulates CCR7 on DCs, a second signal is required to enable them to migrate toward the chemokine CCL19 (MIP-3beta). We found that the nitric oxide (NO) donor NOR4 provides a signal allowing LPS-stimulated DCs to migrate toward CCL19. NO affects DC migration through both the initial activation of the cGMP/cGMP kinase (cGMP/cGK) pathway and a long-term effect that reduced cGK activity via negative feedback. Indeed, migration of DCs toward CCL19, unlike migration toward CXCL12 (SDF-1alpha), required inhibition of cGK. LPS increased both cGK expression and cGK activity as measured by phosphorylation of the key cGK target vasodilator-stimulated phosphoprotein (VASP). Because cGK phosphorylation of VASP can disrupt focal adhesions and inhibit cell migration, LPS-induced VASP phosphorylation may prevent DCs from migrating without a second signal. Long-term NOR4 treatment inhibited the increase in cGK-dependent VASP phosphorylation, releasing this brake so that DCs can migrate. NO has been implicated in the regulation of autoimmunity through its effect on T cells. Our results suggest that NO regulation of DC migration and cytokine production may contribute to the protective effects of NO in autoimmune disorders.

Figure 1.

NO reduces the LPS-induced release of IL-12p70 without affecting DC maturation. (A-B) Intracellular expression of DC-LAMP and surface expression of CD83 and CD86 (A) and CD54 (B) in DCs matured for 24 hours with 0.3 μg/mL LPS only (bold open histograms) or plus 100 μM NOR4 (open histograms). iDCs treated for 24 hours in medium only are represented by filled histograms. Isotype controls are indicated by dotted histograms. Data shown are representative of more than 10 independent experiments for DC-LAMP, CD83, and CD86 and 3 experiments for CD54. (C) iDCs were stimulated or not (□) for 24 hours with LPS (0.3 μg/mL) in the presence () or absence (▪) of graded doses of NOR4 (25, 50, 100 μM) or the vehicle control DMSO (). Supernatants were collected and analyzed for IL-12p70 and IL-6 by ELISA. ^*P < .001 compared with control LPS-treated DCs (▪), unpaired Student t test. Data are from 1 representative experiment of 9 independent experiments performed using cells from different donors. A statistical analysis of the 9 experiments using the paired t test gave a value of P = .01, confirming that the inhibition of IL-12p70 production by NOR4 is statistically significant

Figure 2.

NO induces DC migration toward CCL19/MIP-3β. (A) Surface CCR7 expression of iDCs matured or not for 24 hours with LPS (0.3 μg/mL) in the presence or absence of 100 μM NOR4 or DMSO. The mean fluorescence intensity (M) and percentages of positive cells (%) are indicated. Data shown are representative of more than 10 independent experiments. (B) iDCs matured or not for 24 hours with 0.3 μg/mL LPS in the presence or absence of 100 μM NOR4 or DMSO were tested for their migration toward CCL19. Data shown are mean of duplicate cultures ± SEM and are representative of 9 independent experiments. (C) iDCs were matured for 24 hours with graded doses of LPS with medium only (•), 100 μM NOR4 (♦), or 0.001% DMSO (▴) and tested for migration toward CCL19. Data are from 1 representative experiment of 2 performed using cells from different donors.

Figure 3.

NO enhances LPS-induced expression of CXCR4 and migration toward CXCL12/SDF-1α. iDCs were treated or not for 24 hours with 0.3 μg/mL LPS in the absence or presence of graded doses of NOR4. (A) Surface expression of CXCR4 or CCR7. The mean fluorescence intensity (M) and percentages of positive cells (%) are indicated. (B) Cells treated with medium only (•) or 0.3 μg/mL LPS plus graded doses of NOR4 (▴) were tested for their chemotactic response toward CXCL12 or CCL19. The receptor levels after stimulation with LPS plus graded doses of NOR4 are shown in the inserts; the percentages of cells expressing CXCR4 (▪) and CCR7 (♦) are shown in the top and bottom panels, respectively. Data shown are representative of 3 independent experiments.

Figure 4.

NO induction of migration toward CCL19 is only partially dependent on the cGMP/cGK pathway. (A-B) iDCs were treated or not for 24 hours with 0.3 μg/mL LPS in the presence or absence of graded doses of NOR4 or 8B-cGMP. Surface expression of CCR7, percentages of positive cells (%) are indicated. Chemotactic responses toward CCL19 (A) or CXCL12 (B) are shown as the mean of duplicate cultures ± SEM. Data shown are representative of 3 independent experiments. (C) iDCs were treated or not for 24 hours with 0.3 μg/mL LPS in the presence or absence of 100 μM NOR4 with or without 500 nM DT-3 (cGK inhibitor) and tested for their chemotactic response toward CCL19 or CXCL12. Data shown are representative of 3 independent experiments.

Figure 5.

Inhibition of either cGK or cAK allows LPS-matured DCs to migrate toward CCL19, while migration toward CXCL12 requires cAK activity. iDCs were treated (▪) or not (□) with 0.3 μg/mL LPS for 24 hours. Cells were harvested, incubated with graded doses of either the cGK inhibitor DT-3 (A) or the cAK inhibitor myrPKI (B), and then tested for their chemotactic response toward CCL19 or CXCL12. Data shown are representative of 3 experiments using different donors.

Figure 6.

LPS induces cGK-dependent phosphorylation of VASP in DCs, which is inhibited by NOR4. iDCs were treated or not for 24 hours with 0.3 μg/mL LPS in the presence or absence of 100 μM NOR4. Cells from each group were then stimulated for the indicated times with either CCL19 or CXCL12. (A,B) Cell extracts were prepared and immunoblotted with anti–phospho-VASP antibody and then with anti-VASP antibody. (A) Antiphospho-VASP immunoblot of 1 representative experiment of 3 performed with cells from different donors. (B) Quantitative densitometric analysis of 3 different immunoblot experiments. The activated phospho-VASP expression was normalized to the total VASP. ^*P < .001, paired Student t test. (C) mRNA levels of cGKIβ determined by quantitative real-time RT-PCR. The relative expression of cGKIβ was normalized to the endogenous β-actin. Data shown are from 1 representative of 2 independent experiments giving the same results. (D) cGMP levels in cell extracts were measured with cGMP EIA assay. Data shown are representative of 2 independent experiments.

Figure 7.

NO promotes LPS-mediated DC migration toward CCL19. LPS alone induces expression of cGK and phosphorylation of VASP, thereby preventing DCs from responding to CCL19 and migrating. NO releases DCs from this inhibition via a feedback inhibition pathway that blocks cGK-dependent phosphorylation of VASP.