title: Interferon-gamma-secreting T-cell populations in rejecting murine cardiac allografts: assessment by flow cytometry.
abstract: Interplay between T-helper-1 (Th1) and T-helper-2 (Th2) cells is considered important in the development of acute allograft rejection and many other immune-mediated disease processes. Existing methods for evaluating expression of Th1 and Th2 cytokines, including reverse transcriptase polymerase chain reaction (RT-PCR), RNase protection assay (RPA), immunohistochemistry, and enzyme-linked immunosorbent assay (ELISA) all have limitations; alternate techniques to quantify cell populations expressing specific cytokine proteins, generate statistically analyzable data, and allow simultaneous identification of cytokine-secreting cell type are needed. To this end, we adapted a flow cytometric technique for intracellular cytokine immunofluorescence staining for use with cells isolated from solid tissue. To demonstrate the utility of the method, we determined the number of CD4+ and CD8+ cells secreting the prototypical Th1 and Th2 cytokines, interferon (IFN)-gamma, and interleukin (IL)-4 in acutely rejecting murine cardiac allografts. We also measured the cytokine production via ELISA, RPA, and semiquantitative competitive RT-PCR. The number of CD4+ cells producing IFN-gamma increased as rejection proceeded, in agreement with previous data; we detected no IL-4 production at any time, although relatively low numbers of IL-10-producing cells were identified. In addition, a high percentage of CD8+ cells, which outnumber CD4+ cells at day 6 after transplant, also produce IFN-gamma, suggesting that cytotoxic lymphocytes contribute significantly to the local cytokine milieu. This new application of intracellular cytokine staining provides a powerful methodology for studying transplantation immunology. The method may also be easily adapted to the study of other immune-mediated processes.
authors.affiliation: Department of Pathology, Brigham and Women's Hospital, Boston, Massachusetts 02115, USA.
authors.forename: J L
authors.initials: JL
authors.lastname: Stinn
authors.forename: M K
authors.initials: MK
authors.lastname: Taylor
authors.forename: G
authors.initials: G
authors.lastname: Becker
authors.forename: H
authors.initials: H
authors.lastname: Nagano
authors.forename: S
authors.initials: S
authors.lastname: Hasegawa
authors.forename: Y
authors.initials: Y
authors.lastname: Furakawa
authors.forename: K
authors.initials: K
authors.lastname: Shimizu
authors.forename: P
authors.initials: P
authors.lastname: Libby
authors.forename: R N
authors.initials: RN
authors.lastname: Mitchell
mesh_terms: D000818:Animals; D018414:CD8-Positive T-Lymphocytes / Q000187:drug effects / Q000276:immunology; D002469:Cell Separation / Q000379:methods*; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D004396:Coloring Agents; D005434:Flow Cytometry / Q000379:methods*; D006084:Graft Rejection / Q000276:immunology* / Q000473:pathology; D016027:Heart Transplantation / Q000276:immunology* / Q000473:pathology; D007371:Interferon-gamma / Q000378:metabolism*; D015847:Interleukin-4 / Q000378:metabolism; D015759:Ionomycin / Q000494:pharmacology; D007476:Ionophores / Q000494:pharmacology; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D013601:T-Lymphocytes / Q000187:drug effects / Q000276:immunology / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D018417:Th1 Cells / Q000187:drug effects / Q000276:immunology; D018418:Th2 Cells / Q000187:drug effects / Q000276:immunology; D014184:Transplantation, Homologous
chemical_list: D004396:Coloring Agents; D007476:Ionophores; D015847:Interleukin-4; D015759:Ionomycin; D007371:Interferon-gamma; D013755:Tetradecanoylphorbol Acetate