Plasmacytoid dendritic cells in multiple sclerosis: chemokine and chemokine receptor modulation by interferon-beta

Abstract

Plasmacytoid dendritic cells (pDCs) are present in peripheral blood, leptomeninges and demyelinating lesions in patients with multiple sclerosis (MS). The ability of pDCs to produce chemokines and express the chemokine receptor CCR7 in MS is not known. We studied pDCs in MS patients and healthy subjects. The ability of pDCs to up-regulate CCR7 was significantly increased in untreated MS patients as compared to healthy subjects. IFN-beta treatment significantly inhibited TLR9 agonist-specific secretion of chemokines, which are ligands for CCR5-positive Th1 cells (CCL3, CCL4, and CCL5), and impaired TLR9 agonist-induced up-regulation of CCR7 and IFN-alpha in MS patients. This finding represents a new immunomodulatory effect of IFN-beta in patients with multiple sclerosis.

Copyright © 2010 Elsevier B.V. All rights reserved.

Figures

Figure 1. Intracellular secretion of IFN-α in pDCs

PBMC (1×106 /ml) were separated from healthy subjects (HD), non-treated MS patients (MS: No Rx) and MS patients treated with IFN-beta (MS: IFN-β) as described in Table 1. Cells were stimulated for 4 hours with TLR9 agonist CpGA. Cell surface staining with anti-CD123 and anti-BDCA-2 mAbs followed by intracellular staining with anti-IFN-α mAb were performed as described in Materials and Methods. Figure 1a: The population of pDCs was defined as double-positive BDCA-2+CD123+ cells and was gated (small square in the right upper quadrant of exemplary Dot Plot A (unstimulated cells) or C (cells stimulated with TLR9 agonist)) by flow cytometry analysis. The gated population of pDCs was analyzed for frequency (in %) of IFN-α producing pDCs (the number (%) in right upper quadrant of exemplary Dot Plot B (unstimulated pDCs) or D (stimulated cells). The frequency of pDCs producing IFN-alpha without stimulation with TLR9 agonist was always less than 1.7%. Figure 1b: The final results for TLR9-ligand induced IFN-alpha secretion by pDCs (% of IFN-α producing pDCs) for healthy donors (HD), untreated MS patients (MS: No Rx) and IFN-beta treated MS patients (MS: IFN-β) are shown. MFI ratio for HD was 22.53 ± 4.59. MFI ratio for IFN-beta treated patients was 14.21± 1.22, p < 0.0001 as compared to untreated MS patients (35.01 ± 3.61). Statistical analysis was done with unpaired t-test.

Figure 1. Intracellular secretion of IFN-α in pDCs

PBMC (1×106 /ml) were separated from healthy subjects (HD), non-treated MS patients (MS: No Rx) and MS patients treated with IFN-beta (MS: IFN-β) as described in Table 1. Cells were stimulated for 4 hours with TLR9 agonist CpGA. Cell surface staining with anti-CD123 and anti-BDCA-2 mAbs followed by intracellular staining with anti-IFN-α mAb were performed as described in Materials and Methods. Figure 1a: The population of pDCs was defined as double-positive BDCA-2+CD123+ cells and was gated (small square in the right upper quadrant of exemplary Dot Plot A (unstimulated cells) or C (cells stimulated with TLR9 agonist)) by flow cytometry analysis. The gated population of pDCs was analyzed for frequency (in %) of IFN-α producing pDCs (the number (%) in right upper quadrant of exemplary Dot Plot B (unstimulated pDCs) or D (stimulated cells). The frequency of pDCs producing IFN-alpha without stimulation with TLR9 agonist was always less than 1.7%. Figure 1b: The final results for TLR9-ligand induced IFN-alpha secretion by pDCs (% of IFN-α producing pDCs) for healthy donors (HD), untreated MS patients (MS: No Rx) and IFN-beta treated MS patients (MS: IFN-β) are shown. MFI ratio for HD was 22.53 ± 4.59. MFI ratio for IFN-beta treated patients was 14.21± 1.22, p < 0.0001 as compared to untreated MS patients (35.01 ± 3.61). Statistical analysis was done with unpaired t-test.

Figure 2. Chemokine production by activated pDCs

pDCs were isolated from peripheral blood of patients described in Table 2 before and 3 month after treatment with IFN-beta. Cells were stimulated with or without TLR9 agonist CpGA for 16 hours and concentration of CCL3 (A), CCL4 (B), CCL5(C), CXCL10 (D) in cell supernatants was measured as described in Materials and Methods. The figure depicts the difference between the level of chemokines produced by pDCs activated with TLR9 agonist and the level of chemokines produced by non-activated pDCs (baseline). Statistical analysis was done with paired t-test.

Figure 3. Up-regulation of CCR7 in activated pDCs

PBMC were separated from healthy subjects (HD), non-treated MS patients (MS: No Rx) and MS patients treated with IFN-beta (MS: IFN-β) as described in Table 3. Cells were stimulated for 4 hours with or without TLR9 agonist CpG-A. The population of pDCs was selected based on gating of BDCA-2 and CD123 double-positive cells. The frequency of pDCs expressing surface CCR7 was measured by three-color flow cytometry as described in Materials and Methods. The level of CCR7 up-regulation in stimulated pDCs was obtained by subtracting the baseline level of CCR7 expression for each individual. MFI ratio for HD was 1.65± 0.21. MFI ratio for IFN-beta treated patients was 1.41± 0.09 which was significantly decreased compared to untreated MS patients (1.97± 0.22), p = 0.0355. Statistical analysis was done with unpaired t-test.