title: A rapid flow cytometric assay to detect CD4+ and CD8+ T-helper (Th) 0, Th1 and Th2 cells in whole blood and its application to study cytokine levels in atopic dermatitis before and after cyclosporin therapy.
abstract: BACKGROUND
  The immune response in atopic dermatitis (AD) is thought to be driven by T-helper (Th) 2 cytokines. Using flow cytometry, higher frequencies of peripheral blood CD4+ and CD8+ T cells producing interleukin (IL)-4 and correspondingly lower frequencies of CD4+ T cells producing interferon (IFN)-gamma have been found in patients with AD compared with healthy controls. It would be of interest to know whether other Th1 and Th2 cytokines such as IL-5, IL-13 and tumour necrosis factor (TNF)-alpha are similarly skewed in patients with AD and whether this immune skewing, detected via a simple blood assay, can be correlated with other clinical measurements or treatments in AD.
  
  
  OBJECTIVE
  To use a rapid (4-h) flow cytometric assay to study a wide range of Th1 and Th2 cytokine patterns in peripheral blood lymphocytes from patients with AD, comparing them with non-atopic healthy controls. To correlate cytokine patterns with the degree of eosinophilia observed and in the case of one patient with severe disease, to observe the effect of cyclosporin therapy on peripheral blood cytokine patterns.
  
  
  METHODS
  Peripheral blood from eight patients with AD and 23 healthy controls was examined for the frequencies of CD4+ and CD8+ T cells expressing IL-2, IL-4, IL-5, IL-13, IFN-gamma and TNF-alpha using flow cytometry.
  
  
  RESULTS
  Significantly higher frequencies of CD4+/IL-4+ (P < 0.005) and CD4+/IL-13+ (P < 0.0001) and lower frequencies of CD4+/IFN-gamma+ (P < 0.002) and CD8+/TNF-alpha+ (P < 0.05) T lymphocytes were found in patients with AD compared with controls. There were significant positive correlations with the increased percentages of CD4+/IL-4+ and CD4+/IL-13+ T lymphocytes and the degree of eosinophilia observed (P < 0.05, P < 0.001) and a negative correlation between the percentage of CD4+/IFN-gamma+ T lymphocytes and eosinophilia (P < 0.05). In one patient examined before and 8 days after cyclosporin therapy, 50% or greater reductions were observed in percentages of peripheral blood CD8+/IL-5+, CD8+/IL-13+, CD4+/IL-4+ and CD4+/IL-5+ T lymphocytes following cyclosporin therapy. A smaller reduction of 15% after cyclosporin therapy was found in percentages of CD4+/IL-13+ T cells.
  
  
  CONCLUSIONS
  These data strongly support a Th2 predominance in the peripheral blood of AD. The results suggest that administration of cyclosporin therapy in patients with AD may help to restore the Th2 cytokine imbalance seen in these patients.
authors.affiliation: Department of Dermatology, Oxford Radcliffe Hospital, The Churchill Hospital, Oxford OX3 7LJ, UK.
authors.forename: A M
authors.initials: AM
authors.lastname: Farrell
authors.forename: P
authors.initials: P
authors.lastname: Antrobus
authors.forename: D
authors.initials: D
authors.lastname: Simpson
authors.forename: S
authors.initials: S
authors.lastname: Powell
authors.forename: H M
authors.initials: HM
authors.lastname: Chapel
authors.forename: B L
authors.initials: BL
authors.lastname: Ferry
mesh_terms: D000328:Adult; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology; D016572:Cyclosporine / Q000627:therapeutic use; D016207:Cytokines / Q000096:biosynthesis; D003876:Dermatitis, Atopic / Q000188:drug therapy / Q000276:immunology*; D003879:Dermatologic Agents / Q000627:therapeutic use; D004804:Eosinophils / Q000276:immunology; D005260:Female; D005434:Flow Cytometry / Q000379:methods; D006801:Humans; D007166:Immunosuppressive Agents / Q000627:therapeutic use; D007958:Leukocyte Count; D008297:Male; D008875:Middle Aged; D016176:T-Lymphocyte Subsets / Q000276:immunology*; D006377:T-Lymphocytes, Helper-Inducer / Q000276:immunology*; D018417:Th1 Cells / Q000276:immunology; D018418:Th2 Cells / Q000276:immunology
chemical_list: D016207:Cytokines; D003879:Dermatologic Agents; D007166:Immunosuppressive Agents; D016572:Cyclosporine