Epicutaneous Natural Rubber Latex Sensitization Induces T Helper 2-Type Dermatitis and Strong Prohevein-Specific IgE Response Maili Lehto, Minna Koivuluhta,

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Epicutaneous Natural Rubber Latex Sensitization Induces T Helper 2-Type Dermatitis and Strong Prohevein-Specific IgE Response Maili Lehto, Minna Koivuluhta, Guoying Wang, Iman Amghaiab, Marja-Leena Majuri, Kai Savolainen, Kristiina Turjanmaa, Henrik Wolff, Timo Reunala, Antti Lauerma, Timo Palosuo, Harri Alenius Journal of Investigative Dermatology Volume 120, Issue 4, Pages 633-640 (April 2003) DOI: 10.1046/j.1523-1747.2003.12104.x Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Histologic features of epicutaneous sensitized skin sites in BALB/C mice. Marked infiltration of inflammatory cells can be seen in NRL-sensitized skin sites compared with PBS-treated sites. Scale bar=100μm. Journal of Investigative Dermatology 2003 120, 633-640DOI: (10.1046/j.1523-1747.2003.12104.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Thickness of epidermal and dermal layers of mice skin after epicutaneous sensitization with NRL and PBS. A significant 3.5-fold thickening of epidermis (A) and a 1.5-fold thickening of dermis (B) were observed in NRL-sensitized sites. **p<0.01, ***p< 0.001. Journal of Investigative Dermatology 2003 120, 633-640DOI: (10.1046/j.1523-1747.2003.12104.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Infiltration of total inflammatory cells (A) and eosinophils (B) in skin after epicutaneous sensitization with NRL and treatment with PBS. The skin infiltrating cells were counted in 15–20 HPF at×1000 and expressed as cells per HPF. ***p< 0.001. Journal of Investigative Dermatology 2003 120, 633-640DOI: (10.1046/j.1523-1747.2003.12104.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 The number of mast cells in epicutaneous sensitized skin sites. (A) The number of intact mast cells and (B) degranulated mast cells in NRL-sensitized and PBS-treated skin sites. The skin infiltrating mast cells were counted in 15–20 HPF at×400 and expressed as cells per HPF. ***p< 0.001. Journal of Investigative Dermatology 2003 120, 633-640DOI: (10.1046/j.1523-1747.2003.12104.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Morphologic features of mast cells after epicutaneous sensitization with NRL and PBS. (A) Intact mast cells (black arrows) in the PBS-treated skin site. Densely stained granules are seen within the cell membrane. (B) Degranulated mast cells (red arrows) in the NRL-treated skin site. Some granules of mast cells are leaking through the cell membrane. Journal of Investigative Dermatology 2003 120, 633-640DOI: (10.1046/j.1523-1747.2003.12104.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 The number of CD4-positive cells (A) and CD3-positive cells (B) in mice skin sensitized with NRL and treated with PBS. The CD3- and CD4-positive cells were counted in 10 HPF at×1000 and expressed as cells per HPF. **p<0.01, ***p<0.001. Journal of Investigative Dermatology 2003 120, 633-640DOI: (10.1046/j.1523-1747.2003.12104.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 Real-time, quantitative reverse transcription-PCR analysis of IL-1β (A), IL-4 (B), and IFN-γ (C) mRNA expression levels in skin biopsies of BALB/C mice after epicutaneous sensitization with NRL or PBS. RU are expressed as fold differences relative to the calibrator. The columns and error bars represent mean±SEM. n=9 for NLR group and n=5 for PBS group. ***p< 0.001, ns=not significant. Journal of Investigative Dermatology 2003 120, 633-640DOI: (10.1046/j.1523-1747.2003.12104.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 8 Real-time, quantitative reverse transcription-PCR analysis of CCL3 (A), CCL4 (B), CCL2 (C), and CCL11 (D) mRNA expression levels in skin biopsies from BALB/C mice after epicutaneous sensitization with NRL or PBS. RU are expressed as fold differences relative to the calibrator. The columns and error bars represent mean±SEM. n=9 for NLR group and n=5 for PBS group. *p< 0.05, ***p< 0.001. Journal of Investigative Dermatology 2003 120, 633-640DOI: (10.1046/j.1523-1747.2003.12104.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 9 Total IgE and NRL-specific antibody responses in BALB/C mice after epicutaneous or IP sensitization with NRL or treatment with PBS. (A) Total serum IgE levels. (B) NRL-specific IgE serum levels. (C) NRL-specific IgG2a serum levels. Specific IgE and IgG2a antibody levels of the sample are expressed as absorbance units. The columns and error bars represent mean±SEM. PBS group n=5, epicutaneous NLR group n=9, IP NLR group n=4. **p< 0.01, ***p< 0.001. Journal of Investigative Dermatology 2003 120, 633-640DOI: (10.1046/j.1523-1747.2003.12104.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 10 Hev b6.01 and Hev b1 specific antibody responses in BALB/C mice after epicutaneous or IP sensitization with NRL or PBS. (A) Hev b6.01-specific IgE serum levels. (B) Hev b6.01-specific IgG2a serum levels. (C) Hev b1-specific IgE serum levels. (D) Hev b1-specific IgG2a serum levels. Specific IgE and IgG2a antibody levels of the sample are expressed as absorbance units. The columns and error bars represent mean±SEM. PBS group n=5, epicutaneous NLR group n=9, IP NLR group n=4. *p< 0.05, **p< 0.01. Journal of Investigative Dermatology 2003 120, 633-640DOI: (10.1046/j.1523-1747.2003.12104.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions