Functional redundancy of Langerhans cells and Langerin+ dermal dendritic cells in contact hypersensitivity

Abstract

The relative roles of Langerhans cells (LC), dermal dendritic cells (DC), and, in particular, the recently discovered Langerin(+) dermal DC subset in the induction and control of contact hypersensitivity (CHS) responses remain controversial. Using an inducible mouse model, in which LC and other Langerin(+) DC can be depleted by injection of diphtheria toxin, we previously reported impaired transport of topically applied antigen to draining lymph nodes and reduced CHS in the absence of all Langerin(+) skin DC. In this study, we demonstrate that mice with a selective depletion of LC exhibit attenuated CHS only upon sensitization with a low hapten dose but not with a high hapten dose. In contrast, when painting a higher concentration of hapten onto the skin, which leads to increased antigen dissemination into the dermis, CHS is still diminished in mice lacking all Langerin(+) skin DC. Taken together, these data suggest that the magnitude of a CHS reaction depends on the number of skin DC, which have access to the hapten, rather than on the presence or absence of a particular skin DC population. LC and (Langerin(+)) dermal DC thus seem to have a redundant function in regulating CHS.

Figure 1. Steady state repopulation of Langerin⁺ dendritic cell (DC) populations

(a) Anti-Langerin (red fluorescence) and anti-major histocompatibility complex II (MHCII) (green fluorescence) staining of ear cross-sections of wild type (WT) (left panel) and Langerin-diphtheria toxin receptor (DTR) mice (“Dutch mice”) injected with diphtheria toxin (DT) at 48 hours (middle panel) or 14 days (right panel) earlier. The arrowhead identifies one of the rare Langerin⁺ dermal DC (yellow fluorescence) that had recovered from the toxin at 2 weeks. Bars = 50 μm. (b) Recovery of Langerin⁺ cells in epidermis, dermis, and cutaneous lymph node (LN). Mice were injected with DT at day 0. Single-cell suspensions were prepared at different time points and analyzed for the presence of Langerin⁺ cells. Values of untreated mice (day 0) were set equal to 100%. The graph shows the percentages of MHCII⁺ Langerin⁺ cells (mean±SD). In dermal suspensions, epidermal Langerhans cells (LC) in transit and Langerin⁺ dermal DC were distinguished by differential expression of EpCam and CD103 (not shown). Only numbers of CD103⁺ EpCam^neg dermal Langerin⁺ cells are considered in the columns for dermis. Four mice per time point were analyzed. (c) At 15 days after injection of phosphate-buffered saline (PBS) or DT, whole-skin explants were prepared from the ears of French or Dutch Langerin-DTR mice, and further cultured for 4 days. Migratory cells from 4–6 skin explants were pooled and stained for CD11c, Langerin and CD103. At least 3,000 live migratory cells were acquired by fluorescence activated cell sorting. Dot plots display migratory cells gated for Langerin expression. Percentages represent the proportions of CD11c⁺ CD103^neg LC and CD11c⁺ CD103⁺ Langerin⁺ dermal DC. Results are representative of two independent experiments.

Figure 2. Contact hypersensitivity reactions to low-dose hapten

Wild type (WT) or Langerin-diphtheria toxin receptor (DTR) mice (“Dutch mice”) injected with diphtheria toxin (DT) at day −2 or −10 (n=6–8) were (a) sensitized on the abdomen with 0.5% oxazolone and challenged 5 days later with 0.25% oxazolone on one ear, or (b) sensitized with 0.5% 2,4-dinitrofluorobenzene (DNFB) and challenged with 0.25% DNFB. Ear swelling was measured as the difference between before and after challenge. One representative experiment out of three is shown; symbols represent individual mice. Ear swelling responses were compared using a Student’s t-test; WT versus DTR + DT: *P≤0.05. LC, Langerhans cells.

Figure 3. Contact hypersensitivity responses elicited by high-dose hapten

Wild type (WT) or Langerin-diphtheria toxin receptor (DTR) mice (“Dutch mice”) injected with diphtheria toxin (DT) at day − 2 or −10 (n=6–8) were (a) sensitized on the abdomen with 2% oxazolone and challenged 5 days later with 0.5% oxazolone on one ear, or (b) sensitized with 1% 2,4-dinitrofluorobenzene (DNFB) and challenged with 0.5% DNFB on one ear. Ear swelling was measured as the difference between before and after challenge. One representative experiment out of three is shown; symbols represent individual mice. Ear swelling responses were compared using a Student’s t-test; WT versus DTR + DT: *P≤0.05. LC, Langerhans cells.

Figure 4. Comparison of low-dose contact hypersensitivity in French and Dutch Langerin-diphtheria toxin receptor (DTR) mice

Wild type (WT), French or Dutch Langerin-DTR mice injected with diphtheria toxin (DT) at (a)day −1 or (b) day −10 (n=6–9) were sensitized on the abdomen with 0.5% oxazolone and challenged 5 days later with 0.25% oxazolone on one ear. Swelling was measured as the difference between challenged (right) and unchallenged (left) ear. The summary of 2–3 experiments is shown; symbols represent individual mice. Ear swelling responses were compared using a Student’s t-test; WT versus DTR + DT: *P≤0.05. LC, Langerhans cells.

Figure 5. High-dose contact hypersensitivity in French and Dutch Langerin-diphtheria toxin receptor (DTR) mice

Wild type (WT), French or Dutch Langerin-DTR mice injected with diphtheria toxin (DT) at (a) day −1 or (b) day −10 (n=8–10) were sensitized on the abdomen with 2% oxazolone and challenged 5 days later with 0.5% oxazolone on one ear. Ear swelling was measured as the difference between challenged (right) and unchallenged (left) ear. The summary of two experiments is shown; symbols represent individual mice. Ear swelling responses were compared using a Student’s t-test; WT versus DTR + DT: *P≤0.05. LC, Langerhans cells.