Langerhans cells arise from monocytes in vivo

Abstract

Langerhans cells (LCs) are the only dendritic cells of the epidermis and constitute the first immunological barrier against pathogens and environmental insults. The factors regulating LC homeostasis remain elusive and the direct circulating LC precursor has not yet been identified in vivo. Here we report an absence of LCs in mice deficient in the receptor for colony-stimulating factor 1 (CSF-1) in steady-state conditions. Using bone marrow chimeric mice, we have established that CSF-1 receptor-deficient hematopoietic precursors failed to reconstitute the LC pool in inflamed skin. Furthermore, monocytes with high expression of the monocyte marker Gr-1 (also called Ly-6c/G) were specifically recruited to the inflamed skin, proliferated locally and differentiated into LCs. These results identify Gr-1(hi) monocytes as the direct precursors for LCs in vivo and establish the importance of the CSF-1 receptor in this process.

Figure 1

LC development in CSF-1- and CSF-1R-deficient mice. (a,b) Immunofluorescence of epidermal sheets (a) and flow cytometry of cell suspensions (b) from 3-week-old Csf1r^−/− or Csf1r^+/+ control littermate FVB/NJ mice and stained with monoclonal ‘pan-anti–MHC class II’. Original magnification, ×10 (a). Dot plots (b) show propidium iodide–negative CD45⁺ epidermal cells that are positive for MHC class II. Results are representative of two separate experiments. (c) Flow cytometry of LCs repopulating the skin of 3-week-old Csf1^op/op mice (op/op) or Csf1^op/+ control littermates (op/+) with (UV) or without (No UV) exposure to ultraviolet light for 30 min, assessed 2 or 4 weeks after exposure. Data represent the mean percent of LCs (CD45⁺I-A^b+) among the total propidium iodide–negative CD45⁺ population in three individual mice.

Figure 2

Function of CSF-1R in LC differentiation in vivo. Lethally irradiated CD45.1⁺ recipient mice were reconstituted with mixed chimera bone marrow and were exposed 3 weeks later to ultraviolet light. (a) Mean percent peripheral blood Gr-1 (Ly-6G)⁺ granulocytes and B220⁺ B lymphocytes in control and Csf1r^−/− mice 3 weeks after transplantation in six individual mice. (b) Wild-type versus Csf1r^−/− leukocyte ratio, calculated as percent CD45.2⁺ Csf1r^+/+ leukocytes divided by percent CD45.2⁺ Csf1r^−/− leukocytes in six individual mice. (c) Propidium iodide–negative peritoneal, kidney and dermal F4/80⁺ CD11b⁺ macrophages that are CD45.1⁺ or CD45.2⁺ in control and Csf1r^−/− chimeric mice, 3 weeks after exposure to ultraviolet irradiation. (d) Mean percent CD45.2⁺ cells among total CD45⁺ (CD45.1⁺ and CD45.2⁺) cells in the corresponding macrophage populations in control and Csf1r^−/− mice. (e) Propidium iodide–negative–gated LCs that are CD45.1⁺ or CD45.2⁺ in control and Csf1r^−/− mice, 3 weeks after exposure to ultraviolet irradiation. (f) Mean percent CD45.2⁺ LCs in control and Csf1r^−/− mice 3 and 6 weeks after exposure to ultraviolet irradiation. (g) Reduction in cells, calculated as percent CD45.2⁺ Csf1r^+/+ LCs or spleen DCs divided by percent Csf1r^−/− LCs or spleen DCs at 3 weeks after exposure to ultraviolet irradiation. Results in c–g are representative of three or more separate experiments. Numbers in quadrants (c,e) indicate percentage of propidium iodide–negative total CD45⁺ gated cells that are CD45.1⁺ (bottom right) or CD45.2⁺ (top left).

Figure 3

Gr-1^hi monocytes infiltrate and proliferate in situ in ultraviolet irradiation–inflamed skin. Mice were treated as described in Supplementary Figure 1 online. (a,b) At 1 d after bead injection, all monocytes positive for fluorescein isothiocyanate–labeled beads are mainly Gr-1^lo in the absence of clodronate treatment (a) but are Gr-1^hi when beads are injected after mice are treated with clodronate-loaded liposomes (b). Data are representative of at least five separate experiments. (c) At 4 d after exposure to ultraviolet irradiation, bead-positive Gr-1^hi but not bead-positive Gr-1^lo monocytes are present in the skin. (d) Percent propidium iodide–negative CD45.1⁺ bead-positive cells that infiltrate the epidermis in Gr-1^lo and Gr-1^hi groups after exposure to ultraviolet irradiation. (e) Gr-1^hi monocytes labeled with fluorescein isothiocyanate–positive beads before exposure to ultraviolet light are recruited to the skin, whereas Gr-1^hi monocytes labeled with phycoerythrin-positive beads 4 d after exposure to ultraviolet light fail to enter the skin, suggesting that Gr-1^hi monocytes do not enter the skin 4 d after skin injury. Numbers in quadrants, beside outlined areas and above bracketed lines indicate percentage of cells in each area. FITC, fluorescein isothiocyanate; FSC, forward scatter; PE, phycoerythrin; UV, ultraviolet. Data in c–e are representative of three separate experiments.

Figure 4

Bead-positive Gr-1^hi monocytes differentiate into dermal macrophages and epidermal LCs in inflamed skin. (a) Epidermal sheets stained for CD45.1 (red) and DAPI (blue) at 7 d and 10 d after ultraviolet irradiation. Fluorescein isothiocyanate–positive beads are bright green dots (arrows). Scale bars, 10 μm. Images are representative of at least four separate experiments. (b) Percent BrdU⁺ cells in recruited CD45.1⁺ bead-negative or CD45.1⁺ bead-positive populations in the epidermis after a 72-hour pulse of BrdU. Data are representative of three separate experiments. (c) Percent CD45.1⁺ bead-negative BrdU⁺ cells and CD45.1⁺ bead-positive BrdU⁺ cells after BrdU pulses of varying times. Data are one representative experiment (n = 3). (d) Frozen sections of skin stained for Ki67 (red), CD45.1 (green) and DAPI (blue), 7 d after ultraviolet treatment. Right, higher magnification. Fluorescein isothiocyanate–positive beads are bright green dots (arrows). Scale bars, 10 μm. Images are representative of two separate experiments.

Figure 5

Skin-infiltrating Gr-1^hi monocytes differentiate into LCs. (a) Gr-1 and I-A^b expression patterns in bead-negative CD45.1⁺ and bead-positive CD45.1⁺ populations recruited into inflamed epidermis at days 7 and 10 after exposure to ultraviolet irradiation. Data are representative of three separate experiments. (b,c) Mean percent bead-negative or bead-positive CD45.1⁺ cells that are Gr-1⁺ (b) or I-A^b+ (c) at various times after exposure to ultraviolet irradiation (n = 3). (d) Epidermal sheets stained for CD45.1 (red), langerin (green) and DAPI (blue) at various times after exposure to ultraviolet irradiation. Scale bars, 10 μm. Images are representative of two individual mice. (e) Gr-1 and F4/80 expression patterns in bead-negative and bead-positive propidium iodide–negative CD45.1⁺ populations recruited into inflamed dermis at days 4 and 7 after exposure to ultraviolet irradiation (n = 2). (f) Frozen sections of skin stained for CD68 (red) and DAPI (blue), 7 d after ultraviolet treatment. Beads are bright green dots (arrows). Scale bar, 10 μm. Images are representative of two individual mice. Numbers in quadrants (a,e) indicate percentage of cells positive or negative for the marker.

Figure 6

Adoptive transfer of Gr-1^hi monocytes in vivo. (a,b) Top, percent propidium iodide–negative–gated epidermal cells that are CD45.1⁺ or CD45.2⁺ in mice injected with monocytes or control (I-A^b+ CD11c⁺) populations. Bottom, Gr-1 and I-A^b expression patterns of gated epidermal CD45.1⁺ cells in mice injected with monocytes or control (I-A^b+ CD11c⁺) populations, 5 d (a) or 21 d (b) after exposure to ultraviolet irradiation. Results are representative of three separate experiments. (c) Percent CD45.1⁺ LCs derived from adoptively transferred CD45.1⁺ cells among total LCs. Data represent percent LCs derived from 3 × 10⁶ or 9 × 10⁶ monocytes or 9 × 10⁶ enriched bone marrow I-A^b+CD11c⁺ cells (Control) and are from one representative experiment (n = 3). (d) Langerin expression pattern of gated CD45.2⁺ I-A^b+ and CD45.1⁺ I-A^b+ epidermal cells in mice 6 weeks after adoptive transfer of CD45.1⁺ monocytes. Results are representative of two individual mice. (e) Epidermal sheet stained with CD45.1 (red), langerin (green) and DAPI (blue). Epidermis is from a CD45.2⁺ mouse, 6 weeks after adoptive transfer of 9 × 10⁶ CD45.1⁺ monocytes. Scale bar, 10 μm. Results are representative of two individual mice.