title: The direct cellular target of topically applied pimecrolimus may not be infiltrating lymphocytes.
abstract: BACKGROUND
  Topically applied calcineurin inhibitors have been shown to be effective in the treatment of atopic dermatitis. When systemically administered, these agents cause immunosuppression via inhibition of calcineurin in lymphocytes. As topical agents, the mechanism of action is poorly defined.
  
  
  OBJECTIVE
  To test the hypothesis that skin-infiltrating lymphocytes are directly targeted when calcineurin inhibitors are applied to the skin.
  
  
  METHODS
  Ten patients with atopic dermatitis were treated with 1% pimecrolimus cream twice daily to target lesions. Skin biopsies were performed before and 48 h after beginning therapy. We assessed the cellular localization of NFAT1 and NFAT2 as a surrogate measure of intracellular calcineurin activity (e.g. increasing cytoplasmic localization with increasing calcineurin inhibition).
  
  
  RESULTS
  All patients showed a clinical response, at both 48 h and 2 weeks. As previously described, NFAT2 localized to the follicular keratinocytes, and its activation was partially inhibited by topical pimecrolimus. NFAT1 was found to be expressed by follicular and interfollicular keratinocytes, and its mostly nuclear localization was not affected by topical pimecrolimus therapy. Both NFAT1 and NFAT2 were found in the infiltrating lymphocytes. However, using both manual counting as well as an automated method to assess nuclear intensity of NFAT staining, we found that the proportion of infiltrating leucocytes with nuclear ('activated') NFAT did not change following therapy with pimecrolimus.
  
  
  CONCLUSIONS
  Our results suggest that topical pimecrolimus does not act primarily by inhibiting the calcineurin/NFAT axis in lymphocytes but may instead act by other mechanisms, possibly by decreasing NFAT2 activity in follicular keratinocytes.
authors.affiliation: Department of Dermatology, Stanford University School of Medicine, Stanford, CA 94305, USA. fiorentino@stanford.edu
authors.forename: D F
authors.initials: DF
authors.lastname: Fiorentino
authors.forename: R O
authors.initials: RO
authors.lastname: Chen
authors.forename: D B
authors.initials: DB
authors.lastname: Stewart
authors.forename: K K
authors.initials: KK
authors.lastname: Brown
authors.forename: U N
authors.initials: UN
authors.lastname: Sundram
mesh_terms: D000287:Administration, Topical; D001706:Biopsy; D065095:Calcineurin Inhibitors; D003876:Dermatitis, Atopic / Q000188:drug therapy* / Q000473:pathology; D005455:Fluorescent Antibody Technique; D006801:Humans; D007150:Immunohistochemistry; D007166:Immunosuppressive Agents / Q000494:pharmacology* / Q000627:therapeutic use; D008214:Lymphocytes / Q000187:drug effects* / Q000378:metabolism; D050778:NFATC Transcription Factors / Q000378:metabolism; D016559:Tacrolimus / Q000031:analogs & derivatives* / Q000494:pharmacology / Q000627:therapeutic use
chemical_list: D065095:Calcineurin Inhibitors; D007166:Immunosuppressive Agents; D050778:NFATC Transcription Factors; C117268:pimecrolimus; D016559:Tacrolimus