These authors contributed equally to this work.
CD64 distinguishes macrophages from dendritic cells in the gut and reveals the Th1-inducing role of mesenteric lymph node macrophages during colitis
Version of Record online: 17 OCT 2012
© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
European Journal of Immunology
Volume 42, Issue 12, pages 3150–3166, December 2012
How to Cite
Tamoutounour, S., Henri, S., Lelouard, H., de Bovis, B., de Haar, C., van der Woude, C. J., Woltman, A. M., Reyal, Y., Bonnet, D., Sichien, D., Bain, C. C., Mowat, A. McI., Reis e Sousa, C., Poulin, L. F., Malissen, B. and Guilliams, M. (2012), CD64 distinguishes macrophages from dendritic cells in the gut and reveals the Th1-inducing role of mesenteric lymph node macrophages during colitis. Eur. J. Immunol., 42: 3150–3166. doi: 10.1002/eji.201242847
See accompanying Commentary: http://dx.doi.org/10.1002/eji.201243061
- Issue online: 18 DEC 2012
- Version of Record online: 17 OCT 2012
- Accepted manuscript online: 31 AUG 2012 05:01AM EST
- Manuscript Accepted: 28 AUG 2012
- Manuscript Revised: 26 AUG 2012
- Manuscript Received: 20 JUL 2012
- European Communities Framework Program 7. Grant Number: HEALTH-F2–2008-223404
- Marie Curie Fellowship from the European Communities. Grant Number: 237109
- Leukaemia and Lymphoma Research. L.F.P.
- CNRS/INSERM ATIP/AVENIR program
- Medical Research Council and Wellcome Trust (UK)
Disclaimer: Supplementary materials have been peer-reviewed but not copyedited.
Figure 1. Characterization of intestinal CD8⟨+-type and CD11b+-type DCs. Cells were prepared from the LP of the small and large intestine and from the MLN of Flt3L-deficient (A), B6 mice (A and B) or of CX3CR1-EGFP mice (C). After excluding NK cells, B cells, T cells, eosinophils and neutrophils, the remaining MHCII+ cells were divided into CD11chiCD64– Int-DCs and CD11c– to intCD64+ MΦs (see Fig. 1). (A) CD11chiCD64– DCs Int-DCs are absent in the LP of Flt3L-deficient mice. (B) CD11chiCD64–-gated Int-DCs present in the intestinal LP were analyzed for CD24, CD11b, CD172⟨ and CD103 expression and categorized into CD24+CD11b–CD103+or– CD8⟨+-type DCs and CD11b+CD64–CD103+or– CD11b+-type DCs. The MLN contained a migratory counterpart for each of the Int-DC subsets. (C) Expression of CX3CR1 and F4/80 on CD24+CD11b– CD8⟨+-type DCs, CD11b+CD64– CD11b+-type DCs and CD11c– to intCD64+ MΦs from the specified tissues. In the MLN, Mig-DCs were identified as MHCIIhiCD11cinter-to-hiCCR7+ cells. The percentages of cells found in each of the specified gates are indicated. Data shown are representative of 3 independent experiments.
Figure 2. CD64 marks monocyte-derived MΦs in humans. (A) Human CD34+ cord-blood progenitor cells were infected with lentivirus coding for the GFP fluorescent protein and then injected into NSG mice. 8 weeks later, cells were prepared from the LP of the small and large intestine of humanized mice and GFP+ cells were analyzed by flow cytometry. After excluding B cells, T cells and NK cells, HLA-DR+ cells were analyzed for CD11c, BDCA1, BDCA3 and CD64 expression. (B) Cells were prepared from non-inflamed and inflamed sections of colon biopsy specimens obtained from patients with a history of IBD. After excluding B cells, NK cells and T cells, HLA-DR+ cells were analyzed for CD11c, CD14, BDCA1 and BDCA3 expression. Histograms correspond to CD64 expression on BDCA1+CD11c+, BDCA3+CD11c+, and CD14+CD11c+ cells. Note that human LP MΦs expressed higher levels of CD11c than those found on mouse LP MΦs. The percentages of cells found in each of the specified gates are indicated. Data in (A) are representative of 2 groups of 3 humanized mice. Due to the small amount of GFP+ cells recovered per mouse, the small and the large intestines were pooled for the analysis. Data in (B) are representative of biopsies from 4 individual patients.
Figure 3. A model of the developmental pathway that unfolds in the healthy intestinal LP and leads from blood Ly-6Chi monocytes to MΦs. The expression of CD11c, MHCII, CD64, CX3CR1, Ly-6C, CD172⟨ and CD11b is schematized. Four stages can be distinguished: P1 (tissue monocytes: Ly-6ChiMHCII–CD64lowCX3CR1int), P2 (Intermediates: Ly-6ChiMHCII+CD64lowCX3CR1int), P3 (CX3CR1int MF: Ly-6ClowMHCII+CD64+CX3CR1int) and P4 (CX3CR1hi MF: Ly-6ClowMHCII+CD64+CX3CR1hi).
Figure 4. T cell mediated colitis in Cd3e⊗5/⊗5 mice. (A) Four weeks after transfer of CD4+Foxp3– T cells into Cd3e⊗5/⊗5 hosts, CD4+ T cells were isolated from the MLN and the LP of the large intestine and restimulated in vitro with PMA plus ionomycin. IFN-©, and IL-17 expression was assessed by intracellular staining 6 hours later. The percentages of IFN-©+ and IL-17+ cells among CD4+ T cells are shown. (B) Evolution of the weight of Cd3e⊗5/⊗5 mice transferred with or without CD4+ Foxp3– T cells. In (A) and (B), data are representative of three independent experiments involving at least 3 (A) and 6 (B) mice per group or time-point. The error bars correspond to the SEM. *, p < 0.05.
Figure 5. Gating strategy used for the identification of CD64+ MΦs and of CD24+CD11b– and CD11b+CD64– DCs in the large intestine LP and the MLN of healthy and colitic mice. Cells were prepared from the LP of the large intestine (A) and the MLN (B) of steady-state B6 mice (B6 steady-state) and of Cd3e⊗5/⊗5 mice that had received CD4+Foxp3– T cells (IBD). After excluding NK cells, B cells, T cells, eosinophils and neutrophils, the remaining MHCII+ cells of the large intestine LP were analyzed for CD64, CD11c, CD24, CD11b and CCR7 expression and divided in the following subsets: MΦs (MHCII+CD11c– to intCD64+CD11b+), CD8⟨+-type (MHCII+CD11chiCD64–CD24+CD11b–) and CD11b+-type (MHCII+CD11chiCD64–CD11b+) Int-DCs. In the MLN, MΦs were identified as MHCII+CCR7–CD11c– to intCD64+CD11b+ cells and the MHCIIhiCCR7+CD64– Mig-DCs were subdivided into CD24+CD11b– CD8⟨+-type and CD11b+CD64– CD11b+-type Mig-DCs. Data are representative of at least 3 independent experiments.
Figure 6. CD64+ MΦs excel in phagocytosis and do not display a dendritic morphology. Cd3e⊗5/⊗5 mice were sacrificed 3 weeks after transfer of CD4+Foxp3– T cells. Light density cells were isolated from the MLN and FACS-sorted into MHCIIhiCD64–CD24+CD11b– CD8⟨+-type Mig-DCs, MHCIIhiCD64–CD11b+ CD11b+-type Mig-DCs and MHCIIhiCD64+CD11b+ CD64+ MΦs. The distinct subsets were then subjected to a microsphere phagocytosis assay and uptake of fluorescent microspheres was analyzed by confocal microscopy. Scale bars, 20 μm. Data are representative of 3 independent experiments.
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