Lipopolysaccharide-induced maturation of bone marrow-derived dendritic cells is regulated by notch signaling through the up-regulation of CXCR4

Abstract

Dendritic cells (DCs) are professional antigen presenting cells to initiate immune response against pathogens, but mechanisms controlling the maturation of DCs are unclear. Here we report that, in the absence of recombination signal binding protein-Jkappa (RBP-J, the transcription factor mediating Notch signaling), lipopolysaccharide-stimulated monocyte-derived DCs are arrested at a developmental stage with few dendrites, low major histocompatibility complex II (MHC II) expression, and reduced motility and antigen presentation ability. RBP-J null DCs had lower expression of CXCR4. Transduction with a CXCR4-expressing lentivirus rescued developmental arrest of RBP-J-deficient DCs. Activation of Notch signaling in DCs up-regulated CXCR4 expression and increased the outgrowth of dendrites and the expression of MHC II. These effects were abrogated by a CXCR4 inhibitor. Therefore, Notch signaling is essential for DCs to transit from a dendrite(low)MHC II(low) immature state into a dendrite(high)MHC II(high) mature state, during the lipopolysaccharide-induced DC maturation, most likely through the up-regulation of CXCR4.

FIGURE 1.

RBP-J-deficient DCs showed maturation defects upon LPS stimulation. A, cells (2 × 10⁶) were obtained from BM and cultured in 24-well plates in the presence of GM-CSF and IL-4. Aliquots of cells were stimulated with LPS for 12 h on day 3, 6, and 9 of the culture, and were analyzed by FACS. B, number of CD11c⁺ DCs with low SSC (R1) and high SSC (R2). The number of cells per well in R1 and R2 in A was calculated and compared between RBP-J knockout and control mice. C, typical appearance of DCs from RBP-J knockout and control mice under an SEM. D, comparison of dendrite number between DCs from RBP-J knockout and control mice. DCs were cultured as above, and the number of dendrites of each DC was counted under an SEM. The average dendrite number was compared between RBP-J knockout and control DCs (n = 25). E, RBP-J knockout and control cells were cultured as in Fig. 1A. DCs with >50 dendrites and DCs with <50 dendrites were counted under an SEM. F, LPS-stimulated DCs were analyzed by FACS using anti-MHC II. The result represents three independent experiments. Bars represent means ± S.D. (n = 5). **, p < 0.01.

FIGURE 2.

RBP-J knockout reduced the expression of CXCR4 in DCs. A, BM-derived DCs were stimulated with LPS and were analyzed by FACS using anti-CD11c plus anti-CD184 (CXCR4). B, total RNA was prepared from DCs, reverse-transcribed, and then analyzed by real-time PCR for relative levels of CXCR4 mRNA, with β-actin as a reference control. C, Western blot. Total cell lysates were prepared from cultured DCs and were subjected to Western blot analysis using an anti-CXCR4 antibody, with tubulin as a control. D, single cell suspension was prepared from BM and peripheral blood and was analyzed by FACS. E, real-time RT-PCR. Total RNA was prepared from magnetically isolated DCs and was analyzed as in B. F, cell migration assay. In vitro cultured DCs were seeded in the upper chamber of a Transwell culture system, with HeLa cells expressing SDF1α in the lower chamber. CD11c⁺ DCs migrating into the lower chamber were analyzed by FACS and cell counting. G, in vivo migration assay. DCs cultured from RBP-J knockout and control mice were labeled with CFSE and were injected into the hind foot pads of normal mice. Cells migrating into the draining lymph nodes (LN) were analyzed by FACS. H, contact sensitization assay. FITC solution was painted on mouse ears. CD11c⁺ FITC⁺ cells at the draining lymph nodes (LN) were analyzed by FACS 48 h later. The results represent three independent experiments. Bars represent means ± S.D. (n = 5).

FIGURE 3.

RBP-J-deleted DCs showed reduced antigen presentation ability. A, mixed lymphocyte reaction. BM-derived DCs (2 × 10⁵) were stimulated with LPS, and were co-cultured with syngeneic T-cells (2 × 10⁶) loaded with CFSE and stimulated with irradiated allogenic BM-derived DCs. Five days later, the proliferation of T-cells was detected by FACS. N.C., negative control without stimulator. B and C, the hind footpads of normal mice were injected with DCs pulsed with E. coli super-antigens. The mice were injected with BrdUrd to label proliferating T-cells. Three days later, the draining lymph nodes were photographed (B), and T-cells in draining lymph nodes were analyzed for BrdUrd by FACS (C). The result represented three independent experiments. D, DTH assay. Mice were injected at hind footpads with E. coli antigen-pulsed DCs, and were re-challenged with E. coli super-antigen or phosphate-buffered saline 7 days later. Twenty-four hours after the re-challenge, footpad swelling was measured using a Vernier caliper. The magnitude of the DTH responses was determined by the differences in the thickness between the Ag- and phosphate-buffered saline-injected footpads. Bars represent means ± S.D. (n = 5). *, p < 0.05.

FIGURE 4.

Overexpression of CXCR4 rescued the RBP-J deficiency-induced blockade of DC differentiation. A, overexpression of the mouse CXCR4 by lentivirus infection. HeLa cells or BM-derived DCs were infected with different virons, and the CXCR4 expression was assessed by FACS. B and C, RBP-J^−/− and control BM cells were infected with lentivirus-expressing GFP (as control) or CXCR4 plus GFP, and were induced for DC differentiation as above. Cells were collected after 9-day culture and were analyzed by FACS. The data represent three independent experiments.

FIGURE 5.

Notch stimulation up-regulated CXCR4 expression and promoted DC differentiation. A, BM cells of normal mice were cultured in the presence of GM-CSF and IL4, with recombinant hDll1^DSL or Trx for 9 days. CXCR4 expression on DCs was assayed by FACS. B, total RNA was prepared from the in vitro cultured DCs in Fig. 5A, reverse-transcribed, and was analyzed by real-time PCR for CXCR4 mRNA with β-actin as a reference control. C, DC2.4 was cultured in the presence of recombinant hDll1^DSL or Trx for 48 h and analyzed by FACS. D, total RNA was prepared from DC2.4 in Fig. 5C, reverse-transcribed, and analyzed by real-time PCR for CXCR4 mRNA, with β-actin as a reference control. E, BM cells of normal mice were cultured in the presence of GM-CSF and IL4, with recombinant hDll1^DSL or Trx for 9 days. CD11c⁺ DCs were counted by cell counting and FACS analysis. F, the number of dendrites of DCs cultured in the presence of hDll1^DSL or Trx. DCs were cultured in vitro as above, and were examined on day 9 under an SEM. The number of dendrites was counted and compared (n = 20). G, SSC change of CD11c⁺ DCs during their differentiation in the presence of hDll1^DSL or Trx. The number of CD11c⁺ DCs with low SSC (in R1) and high SSC (in R2) at different time points was calculated and compared between cultures with hDll1^DSL or Trx. H, MHC II expression in DCs cultured in the presence or absence of hDll1^DSL. DCs were cultured in vitro as above, collected on day 9, and analyzed by FACS. I, OP9-Dll1 or OP9-GFP cells (2 × 10⁵) were seeded in 24-well plates. BM cells (2 × 10⁶) were then seeded and co-cultured in the presence of GM-CSF and IL4 for 9 days, and stimulated with LPS 12 h before the end of the culture. The number of DCs was analyzed by FACS and cell counting. J and K, CXCR4 and MHC II expression was analyzed by FACS. The result represents three independent experiments. Bars represent means ± S.D. (n = 5). *, p < 0.05; **, p < 0.01.

FIGURE 6.

A CXCR4 inhibitor abrogated Notch activation-promoted DC maturation. DCs were cultured from normal BM cells in the presence or absence of hDll1^DSL. The CXCR4 inhibitor AMD3100 was added as indicated. Cells were cultured for 9 days and analyzed by FACS. A, CD11c⁺ DCs were plotted for SSC and forward scattering. B, the number of CD11c⁺ DCs in R1 and R2 was calculated and compared. C, expression of MHC II was analyzed by FACS. Bars represent means ± S.D. (n = 5). **, p < 0.01.

FIGURE 7.

The CXCR4 promoter was activated by Notch-RBP-J signaling. A, reporter assay. DC2.4, HEK293, and HeLa cells were transfected with pGL-CXCR4 and increasing amounts (0, 50, 100, and 150 ng) of pEFBOS-NIC (NIC). Cells were collected 48 h after the transfection, and the luciferase activity in lysates was assayed. B, different truncates of the mouse CXCR4 promoter were fused with the basic promoter and luciferase gene, and were used for reporter assay as in A. The amounts of pEFBOS-NIC added were 0, 100, and 150 ng. Bars represent means ± S.D. (n = 5).