Langerhans cells facilitate epithelial DNA damage and squamous cell carcinoma

Abstract

Polyaromatic hydrocarbons (PAHs) are prevalent, potent carcinogens, and 7,12-dimethylbenz[a]anthracene (DMBA) is a model PAH widely used to study tumorigenesis. Mice lacking Langerhans cells (LCs), a signatory epidermal dendritic cell (DC), are protected from cutaneous chemical carcinogenesis, independent of T cell immunity. Investigation of the underlying mechanism revealed that LC-deficient skin was relatively resistant to DMBA-induced DNA damage. LCs efficiently metabolized DMBA to DMBA-trans-3,4-diol, an intermediate proximal to oncogenic Hras mutation, and DMBA-treated LC-deficient skin contained significantly fewer Hras mutations. Moreover, DMBA-trans-3,4-diol application bypassed tumor resistance in LC-deficient mice. Additionally, the genotoxic impact of DMBA on human keratinocytes was significantly increased by prior incubation with human-derived LC. Thus, tissue-associated DC can enhance chemical carcinogenesis via PAH metabolism, highlighting the complex relation between immune cells and carcinogenesis.

Figure 1

Resistance to two-stage chemical carcinogenesis is αβand γδ T cell–independent. Chemical carcinogenesis was induced in (A) Tcrβ^−/− Tcrδ^−/− Lang-DTA mice using 50 µg DMBA to initiate and 6.25 µg TPA/week to promote, as well as in (B) Tcrd^−/− Lang-DTA mice using 100 µg DMBA to initiate and 25 µg TPA/week to promote. The numbers of tumors per mouse (left) and individual tumor area (right) 14 weeks postinitiation are shown (mean ± SEM). ***P < 0.0001 (Student’s t test); n = 8 to 12 mice per group. NS, not significant. (C) Representative images from (B).

Figure 2

LCs enhance DMBA induced DNA damage. Confocal microscopy of epidermal sheets isolated from the ears of (A) NLCs or (B) Lang-DTA mice 24 hours post-DMBA application shows LCs (CD207+) in blue and nuclei containing DNA damage (γH2AX+) in red. (C) The ears of NLCs and Lang-DTA mice were treated with 8.75 µg DMBA or DMBA-t-3,4-diol and gH2AX⁺ cells enumerated 24 hours post treatment. Each dot represents an individual mouse. **P < 0.001 (Student’s t test). Similar results were obtained from vehicle (acetone)– treated and –untreated mice. (D) XS106 was treated with 64 µM DMBA and changes in gene expression monitored by quantitative real-time fluorescence PCR (qPCR). Cyp1a1 expression is not shown because it was undetectable at all time points. (E) NLC mice were untreated or treated with 100 µg DMBA, and, 24 hours later, epidermal Langerhans cells were purified by cell sorting and analyzed for the expression of the indicated genes by qPCR. Purified LCs did not express Cyp1a1 either before or after DMBA treatment (n.d., not detected). All graphs depict mean ± SEM.

Figure 3

Reduced levels of cutaneous Hras codon 61 mutations in LC-deficient mice detected by qPCR of gDNA. Lang-DTA, Tcrβ^−/− Tcrδ^−/− Lang-DTA, and appropriate NLC animals were treated with DMBA [200 µg for D+T(2), 100 µg for all others], followed by TPA (25 µg per week, number of weeks in parenthesis). Hras codon 61 mutations in the treated skin were quantified by qPCR of gDNA. *P < 0.01 (Student’s t test), n = 5 to 6 mice per group. Error bars indicate SEM.

Figure 4

LCs have the capacity to metabolize DMBA to DMBA-t-3,4-diol, and initiation with this metabolite abrogates the tumor resistance seen in LC-deficient mice. XS106 cells were cultured with 64 µM DMBA for 24 hours, the supernatant was then collected, and cells were pelleted to prepare a lysate. (A and C) HPLC analysis of the lysate. (B,D, and E) HPLC analysis of the supernatant. (F) LC/MS/MS analysis of XS106 cells in the presence or absence of DMBA. (G) Tumor induction after treatment with DMBA (12.5 µg) or DMBA-t-3,4-diol (100 µg). 25 µg/week TPA promotion was used in both groups, and initiator doses were chosen on the basis of predicted diol instability, reduced cutaneous penetrance, and preliminary studies. Each dot represents an individual mouse. (H) Human keratinocytes or LCs were cultured in the presence (right) or absence (left) of 64 µM DMBA for 24 hours, then Cyp1a1 (1A1) and Cyp1b1 (1B1) expression was quantified relative to β-actin by qPCR. (I and J) Human keratinocytes (in EpiLife medium) or LCs [in RPMI^w(–)medium] were cultured 24 hours in the presence (or absence, not shown here) of 64 µM DMBA. Supernatants (sup.) were then harvested and transferred onto fresh keratinocyte cultures for an additional 24 hours, then DNA damage was assessed by γH2AX staining. Confocal microscopy (I) and quantification of the data (J). **P < 0.001, ***P < 0.0001 (Student’s t test).