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Basophils regulate the recruitment of eosinophils in a murine model of irritant contact dermatitis  Chisa Nakashima, MD, Atsushi Otsuka, MD, PhD, Akihiko.

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Presentation on theme: "Basophils regulate the recruitment of eosinophils in a murine model of irritant contact dermatitis  Chisa Nakashima, MD, Atsushi Otsuka, MD, PhD, Akihiko."— Presentation transcript:

1 Basophils regulate the recruitment of eosinophils in a murine model of irritant contact dermatitis 
Chisa Nakashima, MD, Atsushi Otsuka, MD, PhD, Akihiko Kitoh, MD, PhD, Tetsuya Honda, MD, PhD, Gyohei Egawa, MD, PhD, Saeko Nakajima, MD, PhD, Satoshi Nakamizo, MD, Makoto Arita, PhD, Masato Kubo, PhD, Yoshiki Miyachi, MD, PhD, Kenji Kabashima, MD, PhD  Journal of Allergy and Clinical Immunology  Volume 134, Issue 1, Pages e12 (July 2014) DOI: /j.jaci Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

2 Fig 1 Eosinophils play some role in the development of ICD. A, Histology of the skin of patients with ICD (I; n = 10) and healthy donors (H; n = 6). The number of eosinophils per field is shown in the left panel. Scale bars = 50 μm. B and C, Ear swelling of WT and ΔdblGATA mice (n = 9 per group; Fig 1, B) and WT (n = 10) and IL-5 Tg (n = 7) mice (Fig 1, C) after application of croton oil. D, H&E staining of ears 6 hours after application. Histologic scores of the skin before and 6 hour after application are shown. Scale bars = 100 μm. *P < .05. Journal of Allergy and Clinical Immunology  , e12DOI: ( /j.jaci ) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

3 Fig 2 ICD is mediated by eosinophil-derived ROS. A, Ear swelling of WT (n = 11) and ΔdblGATA (n = 7) mice pretreated with or without antioxidant NAC measured 24 hours after application. B and C, Histogram (Fig 2, B) and mean fluorescence intensity (MFI) of 2′,7′-dichlorofluorescein (DCF; Fig 2, C) on Siglec-F+CD11b+ eosinophils and CD4+ T cells before (ie, steady states; 0 hours) or 6 hours after application. *P < .05. Journal of Allergy and Clinical Immunology  , e12DOI: ( /j.jaci ) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

4 Fig 3 Basophils accumulate in the skin before eosinophil infiltration in ICD. A, Mcp8+ basophils (black arrowheads) and Papanicolaou staining–positive eosinophils (red arrowheads) 24 hours after croton oil application. Scale bars = 50 μm. B and C, Fluorescence-activated cell sorting plots (Fig 3, B) and numbers (Fig 3, C) of infiltrating eosinophils (Eo), basophils (Baso), and neutrophils (Neu) of WT and ΔdblGATA mice per square millimeter of surface of the skin 6 hours after croton oil application. D, Kinetics of the numbers of eosinophils and basophils in the skin. E and F, Ear swelling (Fig 3, E) and cell infiltration (Fig 3, F) in ICD determined by means of depletion of basophils by MAR-1 antibody (n = 5 per group). G and H, Ear swelling in ICD (Fig 3, G) and cell infiltration (Fig 3, H) of WT (n = 5) and Bas TRECK Tg (n = 4) mice 24 hours after croton oil application. *P < .05. Journal of Allergy and Clinical Immunology  , e12DOI: ( /j.jaci ) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

5 Fig 4 Basophils promote the activation of eosinophils through direct cell interaction. Histogram (A) and mean fluorescence intensity (MFI; B) of CD69, CD86, and intercellular adhesion molecule 1 (ICAM-1) expression on the eosinophil subset in BM cells of IL-5 Tg mice cultured with or without BMBas directly or indirectly. *P < .05. Journal of Allergy and Clinical Immunology  , e12DOI: ( /j.jaci ) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

6 Fig 5 Basophils promote eosinophil recruitment directly or indirectly through fibroblasts. A, Migration of eosinophils and basophils. BMBas or BMEos were applied to the upper or lower chambers with or without IL-3, and cell numbers were evaluated. B, Amount of RANTES in the supernatants of BMBa cultures with or without IL-3. C and D, Amount of RANTES (Fig 5, C) and eotaxin (Fig 5, D) in supernatants of MEFs, BMBas (Baso), or MEFs plus BMBas for 24 hours with or without neutralizing anti–IL-4 or anti–TNF-α antibodies. E, Amount of TNF-α and IL-4 in supernatants of BMBa or MEF cultures. F, Number of migrating eosinophils. Chemotaxis of eosinophils in the lower chamber that were incubated with MEFs, BMBas, or MEF plus BMBas in the presence or absence of IL-3 was evaluated. ND, Not determined. *P < .05. Journal of Allergy and Clinical Immunology  , e12DOI: ( /j.jaci ) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

7 Fig E1 Increased RANTES and eotaxin protein levels in ICD. For measurement of RANTES and eotaxin levels in skin, ear skin was collected and homogenized in PBS. RANTES and eotaxin protein levels were measured before (0 hours) or 24 hours after croton oil application. ND, Not determined. *P < .05. Journal of Allergy and Clinical Immunology  , e12DOI: ( /j.jaci ) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

8 Fig E2 Coincidental presence of eosinophils and basophils in inflamed skin of human patients with ICD. Immunohistochemistry with 2D7 and H&E staining was performed by using inflamed skin of human patients with ICD. 2D7+ basophils (black arrowheads) and eosinophils (red arrowheads) were present coincidently. The representative histologic findings of 2 patients with ICD were shown. Scale bars = 50 μm. Journal of Allergy and Clinical Immunology  , e12DOI: ( /j.jaci ) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

9 Fig E3 Intact mast cells in the skin determined by using anti-FcεRIα (MAR-1) antibody treatment. A, Basophils can be efficiently depleted by anti-FcεRIα (MAR-1) antibody treatment. BALB/c mice were injected twice daily for 3 days with 5 μg of isotype-matched control or anti-FcεRIα (MAR-1) antibody. On day 4, the depletion efficiency in blood was examined by using fluorescence-activated cell sorting. B, Preservation of mast cell population in the skin detected by using toluidine blue staining after MAR-1 treatment. Journal of Allergy and Clinical Immunology  , e12DOI: ( /j.jaci ) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

10 Fig E4 Basophils were efficiently depleted by DT-treated Bas TRECK Tg mice. A, Control BALB/c mice and Bas TRECK Tg mice (BALB/c background) were injected intraperitoneally with 100 ng of DT per mouse. On day 2, the depletion efficiency in blood was examined by means of fluorescence-activated cell sorting. B, Numbers of eosinophils in BM were preserved equivalently in DT-treated Bas TRECK Tg mice. Journal of Allergy and Clinical Immunology  , e12DOI: ( /j.jaci ) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

11 Fig E5 Impaired ICD in the absence of mast cells. A, Ear swelling of WBB6F1-KitW/Wv mice (W/Wv) and congenic normal WBB6F1-Kit+/+ mice (+/+; n = 5 per group). B, Numbers of infiltrating eosinophils (Eo), basophils (Baso), and neutrophils (Neu) of congenic normal WBB6F1-Kit+/+ and WBB6F1-KitW/Wv mice. *P < .05. Journal of Allergy and Clinical Immunology  , e12DOI: ( /j.jaci ) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

12 Fig E6 Eosinophils were recruited through CCR3, and CCR3 ligands were produced in skin in a basophil-dependent manner. A, Administration of anti-CCR3 antibodies significantly suppressed infiltration of eosinophils in skin. B, Production of the CCR3 ligands RANTES and eotaxin in skin was reduced in DT-treated Bas TRECK Tg mice using skin organ culture. *P < .05. Journal of Allergy and Clinical Immunology  , e12DOI: ( /j.jaci ) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

13 Fig E7 RANTES and eotaxin mRNA expression in BMBas and MEFs, Quantitative RT-PCR analysis of RANTES and eotaxin mRNA in BMBas and MEFs. ND, Not determined. Journal of Allergy and Clinical Immunology  , e12DOI: ( /j.jaci ) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

14 Fig E8 Depletion of basophils reduced TNF-α and IL-4 protein levels in skin. Amounts of TNF-α and IL-4 were reduced in DT-treated Bas TRECK Tg mice by using organ culture. ND, Not determined. *P < .05. Journal of Allergy and Clinical Immunology  , e12DOI: ( /j.jaci ) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

15 Fig E9 Roles for basophils and eosinophils in ICD. When chemical agents, including irritants, are exposed to skin, basophils are recruited into the skin as an early phase (step 1). Activated basophils secrete cytokines, including TNF-α and IL-4 (step 2), which in turn act on dermal fibroblasts and are induced to produce chemokines, such as eotaxin (step 3). In addition, basophils themselves produce RANTES in a TNF-α–dependent manner (step 3). These chemokines recruit eosinophils into skin (step 4). Accumulated eosinophils are activated in interaction with basophils and release inflammatory mediators, including ROS, and establish skin inflammation (step 5). Journal of Allergy and Clinical Immunology  , e12DOI: ( /j.jaci ) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

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