Figure 1.
TLR2/4 knockout mice are unable to mount CHS responses to TNCB.
(A) WT or TLR2/4 double knock out mice were sensitized with TNCB (3%) or acetone as solvent control and challenged with TNCB (1%) 5 days later. Data show mean increase in ear thickness +/− SD with n = 3 mice/group. One of two independent experiments is shown. (B) Germ-free C57BL/10 mice were treated as in A. Data show mean increase in ear thickness +/− SD of 6 (acetone) or 7 (TNCB) mice. One experiment was performed.
Figure 2.
Contact sensitizers induce ROS production in murine Pam212 and L929 cell lines in vitro.
(A) Pam212 or L929 cells were incubated with CM-H2DCFDA (5 µM) for 15 min before addition of RF-40s, solvent or NAC. 15 min later TNCB (300 µM) was added. OD485/528 was measured every 10 min for 1 h. Results show the calculated mean OD +/− SD of triplicate wells of all time points analyzed. One representative of three independent experiments is shown. (B) Mean fluorescence was calculated comprising all time points after incubation of Pam212 cells with CM-H2DCFDA and treatment with different concentrations of the pro-hapten paraphenylenediamine (PPD), the extreme sensitizer oxazolone or the moderate sensitizer mercaptobenzothiazole (MBT) as in (A). DMSO and H2O2 served as solvent and positive control, respectively.
Figure 3.
Contact sensitizers induce ROS production in vivo and in mitochondria in vitro.
(A) Mice were pre-treated by topical application of antioxidants (NAC 5 mM or PBS as solvent, RF-40s 5.24 mM or solvent) on the ears. NAC/PBS was applied 1 h before and RF-40s/solvent 15 min before induction of ROS production by topical application of TNCB (7%). Acetone treatment served as solvent control for TNCB. 15 min later, ears were taken after euthanasia and incubated ex vivo with DHE (5 mM) in DMSO for 30 min before analysis of ROS production by fluorescence microscopy. Fluorescence was set for minimal background staining with the acetone control to optimize visualization of differences in ROS production in the other samples. Same acquisition times were used for all samples of one experiment. Results shown are representative of three independent experiments. Magnification = 200×, scale bar = 50 µm. (B) Pam212 or L929 cells were incubated with TNCB, LPS or left untreated for 1 h before addition of MitoSOX™. ROS production was observed by red/orange fluorescence of MitoSOX™ by fluorescence microscopy. Nuclei were visualized by DAPI staining (blue). Pictures shown are representative of three independent experiments. Magnification = 400×, scale bar = 20 µm. (C) Primary human fibroblasts or keratinocytes were incubated with DNCB or left untreated for 1 h before addition of MitoSOX™. ROS production was observed by red/orange fluorescence of MitoSOX™ by fluorescence microscopy. Pictures shown are representative of three independent experiments. Magnification = 1000×, scale bar = 50 µm.
Figure 4.
Source and kinetics of TNCB induced ROS production in vitro.
(A) DCF fluorescence after incubation of BMDC with different concentrations of TNCB for 2 h either with or without NAC treatment was analyzed by flow cytometry. Data show mean fluorescence intensity of DCF +/− SD of triplicate stimulations. One of two independent experiments is shown. (B) Pam212 or L929 cells were left untreated (−) or stimulated with TNCB (300 µM) after pre-treatment with antioxidants NAC or RF-40s for 1 h. In addition, the influence of the mitochondria specific antioxidant APDC and the NADPH oxidase specific antioxidant DPI was analyzed under the same conditions. DCF fluorescence was analyzed 5, 10, 15, 20, 25, 30, 35, 40 and 45 min after TNCB addition. Data show mean fluorescence as calculated comprising all timepoints (B) or kinetics of fluorescence (C) +/− SD of quadruplicate wells from one representative experiment out of three. (D) gp91phox −/− mice were sensitized with TNCB (3%) or mock treated with acetone. 5 days later, mice were challenged with TNCB (1%) and increase in ear thickness was measured 24 h later. Data shows mean increase in ear thickness +/− SD of n = 3 mice/group. One representative of three independent experiments is shown.
Figure 5.
Contact sensitizers induce HA degradation in vivo.
(A) Staining of HA in TNCB or acetone treated murine skin. Biopsy samples from murine abdominal skin were fixed as described and paraffin sections (3 µm) were stained with bHABP with subsequent AEC (3-Amino-9-ethylcarbazole) staining and haematoxylin counterstaining. HA is stained brown/red and cell nuclei in blue. Pictures are representative of three independent experiments with three mice each. Magnification = 400×, scale bar = 50 µm. (B) Staining of HA in TNCB treated human skin. Biopsy samples were treated with indicated concentrations of TNCB for 24 h and afterwards treated like the murine samples above. Pictures are representative of samples from three independent donors. Magnification = 200×, scale bar = 50 µm. (C) Abdominal skin was excised either untreated (lane 1), or 4 h (lane 2) or 24 h (lane 3) after application of TNCB (3%). Samples (8 mm diameter) were digested and the molecular weight of HA of the samples was determined by agarose gel electrophoresis. In lane 4 and 5 molecular weight markers (5 µl/lane of HiLadder or LoLadder) were loaded. The picture shows one representative gel out of three. (D) Abdominal mouse skin was treated with sensitizers (TNCB, DNFB or oxazolone) and HA staining was performed 4 or 24 h later. Respective solvent controls (acetone, acetone/olive oil (AOO), ethanol) are shown below. Biopsy samples from murine abdominal skin were fixed as described and paraffin sections (3 µm) were stained with bHABP with subsequent AEC (3-Amino-9-ethylcarbazole) staining and haematoxylin counterstaining. HA is stained brown/red and cell nuclei in blue. Pictures are representative of three independent experiments with three mice each. Magnification = 200×, scale bar = 50 µm.
Figure 6.
Antioxidant treatment inhibits HA degradation by ROS in vitro and in vivo.
(A) High molecular weight HA was incubated with ROS inducing compounds in combination with RF-40s (R) or solvent controls (S) at corresponding concentrations. MW of HA after incubation was analyzed by SDS gel electrophoresis and staining with Stains all. The untreated HA control is shown in lane 1, CuSO4 or H2O2 only treated HA controls are shown in lanes 2 and 3. One representative gel out of three is shown. (B) High molecular weight HA was incubated with ROS inducing compounds as in (A) with or without addition of different concentrations of the antioxidant NAC. MW of HA after incubation was analyzed by SDS gel electrophoresis and staining with Stains all. One representative gel of three is shown. (C) Staining of HA in murine ears treated topically with acetone, TNCB or TNCB and NAC on the back side of the ear skin (upper side in panels). Samples from ears were fixed as described and paraffin sections (3 µm) were stained with bHABP with subsequent AEC (3-Amino-9-ethylcarbazole) staining and haematoxylin counterstaining. HA is stained brown/red and cell nuclei in blue. Pictures are representative of three independent experiments with three mice each. Magnification = 200×; scale bars = 50 µm.
Figure 7.
Effects of treatment with different antioxidants before and after sensitization or challenge on CHS responses.
(A) Ears of mice were pre-treated by topical application of the antioxidants NAC, the hydrophilic Vitamin E analogon Trolox, the hydrophobic Vitamin Eα-Tocopherol or RF-40s or the respective solvent controls PBS, acetone or solvent before sensitization with TNCB (3%). 5 days later, increase in ear thickness was measured after challenge with TNCB (1%). Data represent the mean increase in ear thickness +/− SD of groups of five mice. One representative of two independent experiments is shown. (B) Mouse ear skin was treated 20 min before sensitization (TNCB 3%) with either RF-40s, ethanol dissolved RF-40 (RF-40e) or ethanol dissolved pure Luteolin (<98% HPLC) with the same molar concentrations of Luteolin. 24 h after challenge (TNCB 1%), increase in ear thickness was measured. The data represent the mean increase in ear thickness of groups of n = 5 mice +/− SD. One of two independent experiments is shown. (C) Ears of mice were treated with RF-40s at different times before or after sensitization (left) or challenge (right) with TNCB and increase in ear thickness was measured 24 h later. Data show mean increase in ear thickness +/− SD from one of two independent experiments with n = 5 mice/group. (D) Mice sensitized to oxazolone (3%) were either left untreated or were pre-treated with RF-40s or solvent 15 min before challenge with oxazolone (1%). Data represent mean increase in ear thickness of groups of n = 5 mice +/− SD. One representative of three independent experiments is shown.
Figure 8.
ROS induce increased hyaluronidase activity and blocking hyaluronidases prevents IL-6 production and CHS.
(A) The abdomen of mice was topically pre-treated with NAC or PBS before application of TNCB (3%) for 24 h. Hyaluronidase activity was detected by hyaluronidase (HAdase) zymography and fold increase in density over untreated controls from inverted gels is shown as bars. One representative experiment of three is shown. (B) Ears of mice were pre-treated by injection of either PBS as solvent or the hyaluronidase inhibitor aristolochic acid (AA) with or without addition of hyaluronidase (660 U/ml) or hyaluronidase alone. 15 min later, mice were sensitized by topical application of TNCB on the pre-treated ears. 5 days later, ears were challenged by topical application of TNCB. Data show mean increase in ear thickness of groups of three mice +/− SD. One representative of three independent experiments is shown. (C) Ears of mice were topically pre-treated with RF-40s, solvent or NAC or by intracutaneous injection of hyaluronidase inhibitor AA or p38 MAPK inhibitor SB203580 or with acetone (solvent control for TNCB). Indicated groups were additionally injected with active or heat inactivated hyaluronidase. 15 min later, mice were sensitized. Challenged was done 5 days later as in (B). Data show mean increase in ear thickness of groups of three mice +/− SD. One representative of two independent experiments is shown. (D) Ear sheets of C57BL/6 mice were incubated with PBS in combination with AA (50 µM) or with TNBS (3 mM) and AA for 24 h. Samples were analyzed for IL-6 production by ELISA. Data show mean concentrations of IL-6+/− SD of one representative experiment out of two with 9 mice each. (E) Ear sheets of NMRI mice were incubated with TNBS (3 mM) either in combination with hyaluronidase (HAdase; 660 U/ml) or AA (50 µM) or with AA and HAdase for 24 h. Samples were analyzed for IL-6 production by ELISA. Data show mean concentrations of IL-6+/− SD of two independent experiments with n = 3 mice each. (F) Mice were pre-treated by injection of PBS or different concentrations of hyaluronidase (HAdase) into the ear pinna. Afterwards ears were treated with TNCB (3%) for sensitization and increase in ear thickness was measured 5 days later after challenge with TNCB (1%) for 24 h. Data show mean +/− SD of n = 3 mice.