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Conversion of the CD4+ T cell profile from TH2-dominant type to TH1-dominant type after varicella-zoster virus infection in atopic dermatitis  Takao Fujimura,

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Presentation on theme: "Conversion of the CD4+ T cell profile from TH2-dominant type to TH1-dominant type after varicella-zoster virus infection in atopic dermatitis  Takao Fujimura,"— Presentation transcript:

1 Conversion of the CD4+ T cell profile from TH2-dominant type to TH1-dominant type after varicella-zoster virus infection in atopic dermatitis  Takao Fujimura, PhDa, Rika Yamanashi, BSca, Mikio Masuzawa, MD, PhDa, Yuhsuke Fujita, MDa, Kensei Katsuoka, MD, PhDa, Sigeo Nishiyama, MD, PhDa, Masao Mitsuyama, MD, PhDb, Kikuo Nomoto, MD, PhDc  Journal of Allergy and Clinical Immunology  Volume 100, Issue 2, Pages (August 1997) DOI: /S (97) Copyright © 1997 Mosby, Inc. Terms and Conditions

2 Fig. 1 Cytokine profiles of AD lesions. Erythematous skin lesions of AD were examined for cytokine mRNA expression by means of RT-PCR. Total RNA extracted from each biopsy specimen was reverse transcribed to cDNA. cDNA for β-actin, CD3δ, IL-2, IFN-γ, IL-4, and IL-5 was amplified by PCR. DNA size markers (øX174/HaeIII digest) are shown in last lane on right in both panels. Predicted sizes of PCR products for β-actin, CD3 δ, IL-2, IFN-γ, IL-4, and IL-5 were 548, 316, 266, 356, 317, and 405 bp, respectively. Journal of Allergy and Clinical Immunology  , DOI: ( /S (97) ) Copyright © 1997 Mosby, Inc. Terms and Conditions

3 Fig. 2 Cytokine profiles in atopic skin lesions after these patients contracted varicella. Atopic skin lesions subsequently improved. Total RNA extracted from each biopsy specimen was reverse transcribed to cDNA. cDNAs for β-actin, CD3δ, IL-2, IFN-γ, IL-4, and IL-5 were amplified by PCR. Predicted sizes of PCR products for β-actin, CD3 δ, IL-2, IFN-γ, IL-4, and IL-5 were 548, 316, 266, 356, 317, and 405 bp, respectively. Expression patterns of cytokines before contracting varicella are shown in lanes 1 to 3 of Fig. 1. Journal of Allergy and Clinical Immunology  , DOI: ( /S (97) ) Copyright © 1997 Mosby, Inc. Terms and Conditions

4 Fig. 3 Enumeration of IL-4– and IL-5–producing cells by ELISPOT assay. PBMCs obtained from A, three AD patients showing improvement after varicella (cases 1 to 3) and from B, three healthy volunteers as controls (cont 1 to 3) were stimulated with 10 μg mite antigen/ml for 48 hours. After cells were washed, serial dilutions of cell suspension were transferred to nylon-based plates that had been coated with anti-IL-4 or anti-IL-5 mAb, and further incubated for 24 hours. Plates were then washed and incubated with anti-IL-4 or anti-IL-5 polyclonal antibodies. Plates were again washed and incubated with peroxidase-conjugated 2nd antibody. Spots representing IL-4– or IL5–producing cells were visualized by adding 3-amino-9-ethylcarbazole in 0.1 mol/L phosphate-citrate buffer. Data are expressed as mean of three wells ± SD. *p < 0.05, **p < p-value compared with control (none) every individual case. Journal of Allergy and Clinical Immunology  , DOI: ( /S (97) ) Copyright © 1997 Mosby, Inc. Terms and Conditions

5 Fig. 3 Enumeration of IL-4– and IL-5–producing cells by ELISPOT assay. PBMCs obtained from A, three AD patients showing improvement after varicella (cases 1 to 3) and from B, three healthy volunteers as controls (cont 1 to 3) were stimulated with 10 μg mite antigen/ml for 48 hours. After cells were washed, serial dilutions of cell suspension were transferred to nylon-based plates that had been coated with anti-IL-4 or anti-IL-5 mAb, and further incubated for 24 hours. Plates were then washed and incubated with anti-IL-4 or anti-IL-5 polyclonal antibodies. Plates were again washed and incubated with peroxidase-conjugated 2nd antibody. Spots representing IL-4– or IL5–producing cells were visualized by adding 3-amino-9-ethylcarbazole in 0.1 mol/L phosphate-citrate buffer. Data are expressed as mean of three wells ± SD. *p < 0.05, **p < p-value compared with control (none) every individual case. Journal of Allergy and Clinical Immunology  , DOI: ( /S (97) ) Copyright © 1997 Mosby, Inc. Terms and Conditions

6 Fig. 4 Enumeration of IL-4– and IL-5–producing cells by ELISPOT assay. PBMCs obtained from three AD patients showing improvement after varicella (cases 1 to 3) were stimulated with 100 PFU VZV/ml for 48 hours, and IL-4– and IL-5–producing cells were counted as described for Fig. 3. Data are expressed as mean of three wells ± SD. *p < 0.05, **p  < p-value compared with control (none) every individual case. Journal of Allergy and Clinical Immunology  , DOI: ( /S (97) ) Copyright © 1997 Mosby, Inc. Terms and Conditions

7 Fig. 4 Enumeration of IL-4– and IL-5–producing cells by ELISPOT assay. PBMCs obtained from three AD patients showing improvement after varicella (cases 1 to 3) were stimulated with 100 PFU VZV/ml for 48 hours, and IL-4– and IL-5–producing cells were counted as described for Fig. 3. Data are expressed as mean of three wells ± SD. *p < 0.05, **p  < p-value compared with control (none) every individual case. Journal of Allergy and Clinical Immunology  , DOI: ( /S (97) ) Copyright © 1997 Mosby, Inc. Terms and Conditions

8 Fig. 5 Enumeration of IFN-γ–producing cells by ELISPOT assay. PBMCs obtained from three AD patients showing improvement after varicella (cases 1 to 3) were stimulated with 10 μg/ml mite antigen or 100 PFU/ml VZV or mixing both for 48 hours. IFN-γ producing cells were counted as described for Fig. 3. Data are expressed as mean of three wells ± SD. *p < 0.05, **p < p-value compared with control (none) every individual case. Journal of Allergy and Clinical Immunology  , DOI: ( /S (97) ) Copyright © 1997 Mosby, Inc. Terms and Conditions

9 Fig. 6 PCR detection of mRNA for IL-12 p35 and p40 in varicella lesions. Skin biopsy specimens were obtained from lesions within 4 days after appearance of vesicles (lanes 1 to 8) and from crusting lesions 7 to 10 days after appearance of vesicles (lanes 9 and 10). RNA from each varicella lesion was reverse transcribed into cDNA and amplified by PCR with primers specific for β-actin and IL-12 p35 and p40. DNA size markers (øX174/HaeIII digest) are shown in last lane on right. PCR products for IL-12 p35 and p40 cDNA were transferred to nylon membranes and hybridized to an internal 32P-labeled probe. Journal of Allergy and Clinical Immunology  , DOI: ( /S (97) ) Copyright © 1997 Mosby, Inc. Terms and Conditions

10 Fig. 7 IL-12 p40 mRNA production induced by VZV. PBMCs from atopic or nonatopic donors were cocultured for 5 hours in medium alone or medium containing VZV. RNA was reverse transcribed into cDNA and amplified by PCR with primers specific for β-actin and IL-12 p40. DNA size markers (øX174/HaeIII digest) are shown in last lane on right. PCR products for IL-12 p40 cDNA were transferred to nylon membranes and hybridized to an internal 32P-labeled probe. Even-numbered lanes show results for VZV-stimulated cells. Odd-numbered lanes, show results for nonstimulated cells. A, PBMCs from atopic donors. B, PBMCs from nonatopic donors. Journal of Allergy and Clinical Immunology  , DOI: ( /S (97) ) Copyright © 1997 Mosby, Inc. Terms and Conditions

11 Fig. 7 IL-12 p40 mRNA production induced by VZV. PBMCs from atopic or nonatopic donors were cocultured for 5 hours in medium alone or medium containing VZV. RNA was reverse transcribed into cDNA and amplified by PCR with primers specific for β-actin and IL-12 p40. DNA size markers (øX174/HaeIII digest) are shown in last lane on right. PCR products for IL-12 p40 cDNA were transferred to nylon membranes and hybridized to an internal 32P-labeled probe. Even-numbered lanes show results for VZV-stimulated cells. Odd-numbered lanes, show results for nonstimulated cells. A, PBMCs from atopic donors. B, PBMCs from nonatopic donors. Journal of Allergy and Clinical Immunology  , DOI: ( /S (97) ) Copyright © 1997 Mosby, Inc. Terms and Conditions

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