Ann Dermatol.  2010 Aug;22(3):269-278. 10.5021/ad.2010.22.3.269.

The Roles of Reactive Oxygen Species Produced by Contact Allergens and Irritants in Monocyte-derived Dendritic Cells

• Byamba D
• Kim TG
• Kim DH
• Je JH
• Lee MG

• KMID: 2265359
• DOI: http://doi.org/10.5021/ad.2010.22.3.269

Abstract

BACKGROUND
Although reactive oxygen species (ROS) have been produced in both mouse bone marrow-derived dendritic cells (DCs) and XS-106 DCs by contact sensitizers and irritants in previous studies, the generation of ROS in human monocyte-derived DCs (MoDCs) and their role in contact hypersensitivity (CHS) has yet to be elucidated.
OBJECTIVE
The purpose of this study was to determine whether contact allergens and irritants induce ROS in MoDCs and, if so, to evaluate the role of contact allergen and irritant induced-ROS in MoDCs in CHS.
METHODS
Production of ROS was measured by 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate (CM-H2DCFDA) assay. Surface CD86 and HLA-DR molecules were detected by flow cytometry. Protein carbonylation was detected by Western blotting.
RESULTS
ROS were produced by contact allergens such as dinitrochlorobenzene (DNCB) and thimerosal and the irritant benzalkonium chloride (BKC). DNCB-induced, but not BKC-induced, ROS increased surface CD86 and HLA-DR molecules on MoDCs and induced protein carbonylation. These changes were reduced in the presence of antioxidant N-acetyl cysteine.
CONCLUSION
Our results suggest that DNCB-induced ROS may be different from those induced by irritant BKC. The DNCB-induced ROS may be associated with the CHS response, because they activate surface molecules on DCs that are important for generating immune reactions.

Keyword

Contact allergen; Contact dermatitis; Monocyte-derived dendritic cell; Reactive oxygen species

MeSH Terms

Allergens
Animals
Benzalkonium Compounds
Blotting, Western
Cysteine
Dendritic Cells
Dermatitis, Contact
Dinitrochlorobenzene
Flow Cytometry
HLA-DR Antigens
Humans
Irritants
Mice
Protein Carbonylation
Reactive Oxygen Species
Thimerosal
Allergens
Benzalkonium Compounds
Cysteine
Dinitrochlorobenzene
HLA-DR Antigens
Irritants
Reactive Oxygen Species
Thimerosal

Figure

• 

  Fig. 1 (A) Monocyte-derived dendritic cells (MoDCs) produce reactive oxygen species (ROS) after incubation with various chemicals including allergens and irritants. Immature MoDCs were incubated for 1 h with sublethal concentration of allergens, such as (a) dinitrochlorobenzene (DNCB), (b) thimerosal (Thim), (c) dinitrofluorobenzene (DNFB) and (d) trinitrobenzene sulfonic acid (TNBS); and irritants, such as (e) benzalkonium chloride (BKC) and (f) sodium dodecyl sulfate (SDS). ROS production was detected by CM-H2DCFDA assay. (A) Filled line represents control cells without CM-H2DCFDA staining; dotted line indicates physiologic ROS level in the cells (DCFDA staining only) without treatment; solid line indicates ATP synthase inhibitor (ASI)-treated cells as a positive control; solid thick line indicates ROS level induced by chemicals. Contact allergens DNCB, TNBS, Thim and DNFB increased ROS production 3.8-fold, 2.9-fold, 3.3-fold, and 1.5-fold, respectively, compared to the control. Irritants BKC and SDS induced ROS significantly (2.6-fold and 2.0-fold, respectively). (B) Relative increase of ROS after incubation with various chemicals including allergens and irritants. The relative increase in ROS was calculated by comparing the mean fluorescence intensity (MFI) of CM-H2DCFDA stained-treatment groups to that of the control group. Of these, the relative increases of ROS production by DNCB, Thim, and DNFB were statistically significant (p-values were 0.031, 0.029, and 0.034, respectively). Of the irritants used, BKC and SDS induced ROS significantly (p-values were 0.028 and 0.013, respectively).

• 

  Fig. 2 Contact allergen-DNCB, but not irritant-BKC, induced protein carbonylation. Immature day 6 MoDCs were incubated with 20µm DNCB and 10µM BKC for 6 h. Carbonylated groups were detected by immunoblotting with an anti-DNP antibody. DNCB treated cells but not BKC treated cells showed increased carbonylation compared with the control. Actin is included as a loading control. These data are representative of two independent experiments.

• 

  Fig. 3 Augmentation of CD86 and HLA-DR expression on MoDCs by DNCB but not by BKC. MoDCs were treated with 10µM DNCB or 5µM BKC for 24 h and then analyzed by flow cytometry. (A, B) Thick solid line indicates CD86 expression on MoDCs after treatment with DNCB and BKC, respectively; solid narrow line indicates positive control (treatment with cytokine cocktail); filled line indicates untreated DCs; dotted line indicates DCs stained with CD86 only. (C, D) Thick solid line indicates HLA-DR expression on MoDCs after treatment with DNCB and BKC, respectively; solid narrow line indicates positive control, filled line indicates untreated DCs, and dotted line indicates DCs stained with HLA-DR only. CD86 and HLA-DR expression on MoDCs cultured with DNCB was increased 2.5-fold and 1.2-fold respectively; p-values were 0.018 and 0.033, respectively, compared to that of the control.

• 

  Fig. 4 (A) Hapten and irritant-induced ROS diminished by N-acetyl L-cystein. MoDCs were pre-treated with 5 mM NAC for 30 min and then treated with 30µM DNCB or 20µM BKC. After 1 h they were stained with 3 mM CM-H2DCFDA and analyzed by flow cytometry. The histogram of chemical-treated MoDCs in the presence or the absence of NAC is expressed as a thick solid line or solid line, whereas a filled line or a dotted line indicates histograms of untreated DCs or of DCs stained with CM-H2DCFDA, respectively. Pre-treatment of MoDCs with 5 mM of NAC reduced the ROS production induced by DNCB significantly (the relative ROS increase with DNCB treatment was 3.8-fold while that with pre-treatment with NAC plus DNCB treatment was 0.56-fold. p≤0.0001). BKC-induced ROS production was decreased by pre-treatment with NAC (the relative ROS increase with BKC treatment was 2.6-fold while that with pre-treatment with NAC plus BKC treatment was 0.70-fold). Each experiment was repeated three times and one representative result is shown. (B) These carbonylated proteins were decreased slightly by NAC. Immature day 6 MoDCs were incubated with 20µM DNCB or 10µM BKC in the presence or absence of 5 mM NAC for 30 min. After 6 h, equal amounts of whole cell protein extracts were loaded in SDS gel and carbonylated proteins were detected using DNP antibody by Western blotting. Allergen DNCB but not irritant BKC induced protein carbonylation and the resulting carbonylated protein was decreased slightly by NAC. (C) NAC suppressed the augmentation of CD86 and HLA-DR molecules on MoDCs that were induced by contact allergen DNCB. MoDCs were cultured with DNCB in the presence or absence of pre-treatment 5 mM NAC. After 24 h their surface molecules were analyzed by flow cytometry. Thick solid line indicates the histogram of chemical-treated MoDCs with NAC; narrow solid line indicates treatment without NAC; filled line indicates non-treatment; dotted line indicates treatment with CD86 antibody only. Augmentation of CD86 and HLA-DR by DNCB was decreased by adding 10 mM NAC.

• 

  Fig. 5 Inhibition of ROS generating pathway. (A) DNCB induced-ROS blocked by NADPH synthase inhibitor DPI. MoDCs were treated with 20µM DNCB for 1 h in the presence (thick solid line) or absence (solid narrow line) of DPI (a specific blocker of NADPH oxidase). Filled line indicates no staining, while dotted line indicates staining with CM-H2DCFDA only. (B) BKC induced-ROS blocked slightly by iNOS inhibitor NMMA. MoDC treated with 10µM BKC in the absence (solid narrow line) or presence (thick solid line) of NMMA.

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