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Comparative Study
. 2004 Nov 15;200(10):1231-41.
doi: 10.1084/jem.20032152. Epub 2004 Nov 8.

Role of CCR8 and other chemokine pathways in the migration of monocyte-derived dendritic cells to lymph nodes

Chunfeng Qu  1 , Emmerson W Edwards, Frank Tacke, Véronique Angeli, Jaime Llodrá, Guzman Sanchez-Schmitz, Alexandre Garin, Nasreen S Haque, Wendy Peters, Nico van Rooijen, Carmen Sanchez-Torres, Jonathan Bromberg, Israel F Charo, Steffen Jung, Sergio A Lira, Gwendalyn J Randolph
Affiliations
Comparative Study

Role of CCR8 and other chemokine pathways in the migration of monocyte-derived dendritic cells to lymph nodes

Chunfeng Qu et al. J Exp Med. .

Abstract

Studying the influence of chemokine receptors (CCRs) on monocyte fate may reveal information about which subpopulations of monocytes convert to dendritic cells (DCs) and the migration pathways that they use. First, we examined whether prominent CCRs on different monocyte subsets, CCR2 or CX3CR1, mediated migration events upstream of the accumulation of monocyte-derived DCs in lymph nodes (LNs). Monocytes were labeled and traced by uptake of latex microspheres in skin. Unexpectedly, neither CCR2 nor CX3CR1 were required. However, absence of CCR2 led to an increased labeling of the minor Gr-1int monocyte population, and the number of latex+ DCs that emigrated to LNs was correspondingly increased. Characterization of Gr-1int monocytes revealed that they selectively expressed CCR7 and CCR8 mRNA in blood. CCR7 and CCR8 pathways were used by monocyte-derived DCs during mobilization from skin to LNs. The role of CCR8 in emigration from tissues also applied to human monocyte-derived cells in a model of transendothelial trafficking. Collectively, the data suggest that Gr-1int monocytes may be most disposed to become a lymphatic-migrating DCs. When these monocyte-derived DCs exit skin to emigrate to LNs, they use not only CCR7 but also CCR8, which was not previously recognized to participate in migration to LNs.

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Figures

Figure 1.
Figure 1.
Analysis of monocyte trafficking and microsphere transport to LNs in CX3CR1gfp/gfp mice. (A) Monocyte subsets were identified in blood of CX3CR1gfp/gfp mice as GFP+ cells with differing levels of Gr-1, revealing the presence of a small Gr-1int subpopulation in addition to previously described Gr-1hi and Gr-1lo populations. (B) GFP fluorescent intensities of these subsets, stable and noninterconverting for at least 1 d (4), were compared with the fluorescent intensities of cells that engulfed red fluorescent microspheres deposited in skin, examined 14 h after injection, and in cells that emigrated from skin to LNs by day 2. The same settings in flow cytometry were used to acquire all of these data, making them directly comparable. (C) Profiles of the cells in skin at the site of microsphere injection at 14 h are shown. (D) Quantification and profiles of LX-bearing cells that migrated to LNs were analyzed at day 2. To combine data from different experiments, the mean number of migrated cells in WT mice was set equal to 1.0 for each experiment, and relative values for all WT and knock-out individuals in that experiment were calculated. Six mice were studied for each part of the figure over the course of two experiments with three mice in each group.
Figure 2.
Figure 2.
Analysis of monocyte trafficking and microsphere transport to LNs in CCR2−/− mice. (A) The relative distribution of the various blood monocyte subsets was compared between CCR2+/+ and CCR2−/− mice. (B) The relative proportions of Gr-1hi and Gr-1int phagocytes bearing LX were also compared in the skin, 14 h after injection of green fluorescent microspheres. (A and B) Bold line profiles identify CCR2+/+ monocytes. and thin lines identify CCR2−/− monocytes. (C) The number of DCs bearing two or more LX particles in the draining LNs was quantified 2 d later. The increased number of LX+ cells in CCR2−/− LNs was statistically significant: P < 0.04.
Figure 3.
Figure 3.
Analysis of monocyte subsets in blood: expression of costimulatory activity and CCR mRNA. (A) Sorted Gr-1hi, Gr-1int, and Gr-1lo subsets were irradiated and used as APCs in an MLR with BALB/c T cells. This experiment is representative of two conducted experiments, both showing similar results. (B) Real-time PCR for CCR2, CX3CR1, CCR7, and CCR8 mRNA was conducted. Data are plotted as the percentage of expression relative to the subset with the highest expression of a particular CCR. These data were repeated in a similar pattern in three independent sorting experiments.
Figure 4.
Figure 4.
Analysis of cell-mediated microsphere transport to LNs in CCR8−/− and plt/plt mice. (A) Green fluorescent LX microspheres were injected into the skin of CCR8-deficient mice and plt/plt mice that were compared with age- and sex-matched WT C57BL/6 counterparts. The number of DCs bearing two or more LX particles in the draining LNs was quantified 3 d later. To combine data from different experiments, the mean number of migrated cells in WT mice was set equal to 1.0 for each experiment, and relative values for all WT and knock-out individuals in that experiment were calculated. Inset shows Gr-1 staining intensity among gated bead+ cells from CCR8−/− (bold line) and CCR8+/+ mice (thin line) in the skin 14 h after bead injection. (B and C) Plots show MHC II (I-Ab) and Gr-1 levels in LNs and skin of WT and CCR8−/− mice. LN plots (B) are quantitative comparisons, as they depict the entire population of LX-bearing cells recovered from pooled brachial LNs from individual mice. Skin plots (C) are not quantitative comparisons. Where available, plots depict all acquired events, although in some experiments (for MHC II staining in LN), LX+ cells were gated during acquisition to reduce file size. (D) High power magnification of WT skin section stained for LYVE-1 (green) and CCL1 (red). (E) LYVE-1 (green) and CCL1 (red) in LN subcapsular sinus (left, high power magnification). CCL1 staining in LN at low power (right). Outer subcapsule is indicated by an arrow.
Figure 5.
Figure 5.
Allostimulatory capacity of monocyte subsets sorted from the periphery and effect of an antagonist to the CCR8 ligand CCL1. (A) Gr-1hi and Gr-1int recruited monocytes from the peritoneal lavage of C57BL/6 CCR8+/+ mice were sorted to purity by flow cytometry using mAbs to F4/80 and Gr-1. The cells were cultured separately in GM-CSF for 2 d in the absence of added mAb, or in the presence of 5 μg/ml of neutralizing mAbs to chemokines CCL1 (TCA-3) or CCL19. These cells were washed to remove residual antibodies and cultured with BALB/c T cells for evaluation of their potential to support allogeneic T cell proliferation in a mixed lymphocyte reaction. These data depict one out of two experiments conducted with similar results.
Figure 6.
Figure 6.
Expression of CCR8 by human monocyte-derived cells and skin DCs, and its role in reverse transmigration. Monocytes were cocultured with endothelial cells grown on a type I collagen gel for 48 h, permitting the separation of the population into reverse-transmigrating DCs (RT) or subendothelial macrophages (SE). (A) Anti-CCR8 mAb 3B10 (thin solid line) reacted with both RT and SE monocyte-derived cells. Isotype-matched mAb was used to establish the baseline for negative staining (dotted line). Anti-CCR8 mAb 3B10 specifically recognized an ∼54 kD band by immunoblot, shown only for SE monocyte-derived cells (inset). (B) CD14+ DC precursors from human skin, but not CD14− DCs, were positive for CCR8 by immunoblot. Blotting for β-actin was conducted as a loading control. (C) Inclusion of neutralizing anti-CCR8 mAb 3B10 during the assay when monocytes traverse endothelium in the apical-to-basal direction had no effect (left), but 3B10 anti-CCR8 mAb and anti-CCR8 mAb 5B11 significantly (P < 0.005) inhibited reverse transmigration in more than five independent experiments. (D) CD16− monocytes were selected by magnetic depletion to remove CD16+ monocytes and cultured overnight in the presence or absence of TGFβ1 to induce CD16 (top). In some samples, 20 ng/ml TGFβ1 was added together with neutralizing mAbs to CCR1, CCR2, or CCR8. Expression of CD16 was monitored by flow cytometry. This result is representative of five experiments.
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