Accumulation of Metal-specific T Cells in Inflamed Skin in a Novel Murine Model of Chromium-induced Allergic Contact Dermatitis
Chromium (Cr) causes delayed-type hypersensitivity reactions possibly mediated by accumulating T cells into allergic inflamed skin, which are called irritants or allergic contact dermatitis (ICD or ACD). However, accumulating T cells during development of metal allergy are poorly characterized because a suitable animal model is not available. This study aimed to elucidate the skewing of T-cell receptor (TCR) repertoire and cytokine profiles in accumulated T cells in inflamed skin during elucidation of Cr allergy.
Materials and Methods
Ethics Statement
This study was performed in a strict accordance with recommendations in the Guidelines for Care and Use of Laboratory Animals set by the Clinical Research Center for Rheumatology and Allergy, Sagamihara National Hospital, Japan.
Sensitization: A total of 125ml of 10 mM CrCl2with 10 mg/ml LPS in saline was injected twice intradermally (i.d.) into the left and right groin of BALB/cAJcl mice.
Challenge for elicitation: Non-sensitized mice (ICD mice, n=5) or sensitized mice (ACD mice, n=5) were challenged for elicitation with 25 ml of 10 mM CrCl2in saline into the left and right footpad by i.d. injection.
Quantitative PCR (qPCR)
The expression levels of mRNA for immune response-related genes including T cell-related CD antigens and cytokines were measured by qPCR.
Immunohistochemical (IHC) analyses
Footpads were obtained from Cr-induced ICD and ACD mice for histology and IHC analyses (F4/80, CD3, CD4, and CD8).
TCR repertoire analysis
TCR repertoire analysis was performed using samples from Cr induced ACD mice and control mice by adaptor ligation-mediated PCR and microplate hybridization assay. In addition, determination of CDR3 nucleotide sequences analyzed by the CEQ8000 Genetic Analysis System.
Statistical analysis
Differences were analyzed statistically using the Student’s unpaired t-test. A Pvalue < 0.05 was determined to be statistically significant.
Results
The footpad swelling at 1 day after challenge was similar in the ICD and ACD mice. Footpad swelling was reduced to basal levels 7 days after challenge in the ICD mice. In contrast, footpad swelling continued for 14 days after challenge in the ACD mice. In the ACD mice, CD3 and CXCR3 levels were significantly higher than the other mice at 1, 3, 7 days after challenge. Epidermal keratinocytes were partially separated, creating spongiotic dermatitis by H&E staining in the ACD mice. IHC staining showed that CD3+CD4+ T cells were also present in the epithelial basal layer and the upper dermis, but not in the ICD or control mice. The percentage usage of VA11-1, VA14-1, VB8-2, and VB14-1 was significantly higher in the footpads of Cr-induced ACD mice compared with spleens from the control mice. Sequence analyses showed that identical TCR clonotypes bearing VA11-1, VA14-1, and VB8-2 were obtained from the footpads of different mice while diverse TCR clonotypes with VB14-1 were obtained from the footpads of all mice. Surprisingly, CDR3 sequences of VA14-1 cDNA clones was most frequently utilized in the accumulated natural killer (NK)T cells and a clonotype (CVV-G-DRGSALGRLHFG) of CDR3 was obtained from the footpads in all Cr-induced ACD mice.
Conclusions
This study demonstrated the successful establishment of a murine model of Cr-induced ICD and ACD with appropriate pathology. In addition, accumulated T cells included NKT cells and Cr-specific T cells with TCRVA11-1/VB14-1 usage, suggesting metal-specific T cells driven by invariant NKT cells might contribute to the pathogenesis of Cr allergy.
References
1.Shigematsu H, Kumagai K, Kobayashi H, et al. Accumulation of metal-specific T cells in inflamed skin in a novel murine model of Chromium-induced allergic contact dermatitis. PLoS One 9: e85983, 2014.
2.Eguchi T, Kumagai K, Shigematsu H, et al. Accumulation of invariant NKT cells into inflamed skin in a novel murine model of nickel allergy. Cell Immunol 284: 163-171, 2013.