Absent mRNA accumulation of Th1 or Th2 cytokines in heart allografts with chimerism-based drug-induced tolerance.
OBJECTIVE
We recently described using cyclophosphamide (CP) plus busulfan (BU) to create drug-induced skin and heart allograft tolerance capable of regularly overcoming fully H-2 mismatched barriers in mice. The present study investigates the intragraft mRNA expressions of Th1 and Th2 cytokines.


METHODS
This method consists of the intravenous (i.v.) injection of 1 x 10(8) allogeneic spleen cells on day 0, the intraperitoneal injection of 200 mg/kg CP and 30 mg/kg BU on day 2, and the i.v. injection of 1 x 10(7) T cell-depleted allogeneic bone marrow cells from the same strain of mice on day 3. Heart grafting (HG) was performed on day 28. Chimerism in the peripheral blood was monitored by flow cytometric (FCM) analysis. The frequency of certain V(beta) families was determined by FCM to assess deletion of donor-reactive T cells. Th1 (interleukin [IL]-2, interferon [IFN]-gamma) and Th2 (IL-4, IL-10) cytokine expression in the heart grafts was analyzed with reverse transcription-polymerase chain reaction.


RESULTS
In a fully MHC mismatched combination of B10.D2 (H-2d, IE+) --> B10 (H-2b, IE-), B10.D2 heart grafts were accepted permanently in a donor-specific manner, mixed chimerism was observed, and IE-reactive V(beta)11+ T cells were specifically reduced in the periphery from the recipient B10 mice. In the donor B10.D2 heart grafts, there was no accumulation of Th1 (IL-2, IFN-gamma) or Th2 (IL-4, IL-10) cytokines.


CONCLUSIONS
These results show that the drug-induced tolerance we established can regularly induce long-lasting heart allograft tolerance without intragraft mRNA accumulation of Th1 or Th2.
Tomita
Yukihiro
Y
Department of Cardiovascular Surgery, Faculty of Medicine, Medical Institute of Bioregulation, Kyushu University, Fukuoka, 812-8582, Japan.
Zhang
Qi-Wei
QW
Matsuzaki
Goro
G
Shimizu
Ichiro
I
Iwai
Toshiro
T
Okano
Shinji
S
Nomoto
Kikuo
K
Yasui
Hisataka
H
D000818:Animals; D002066:Busulfan / Q000494:pharmacology; D003520:Cyclophosphamide / Q000494:pharmacology; D016207:Cytokines / Q000378:metabolism*; D005434:Flow Cytometry; D006085:Graft Survival / Q000276:immunology; D016027:Heart Transplantation / Q000276:immunology* / Q000473:pathology*; D006650:Histocompatibility Testing; D007108:Immune Tolerance / Q000187:drug effects; D016130:Immunophenotyping; D007165:Immunosuppression Therapy / Q000379:methods; D007166:Immunosuppressive Agents / Q000494:pharmacology; D017710:Lymphocyte Transfusion; D051379:Mice; D008810:Mice, Inbred C57BL; D008815:Mice, Inbred Strains; D012333:RNA, Messenger / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013154:Spleen / Q000166:cytology / Q000276:immunology / Q000637:transplantation*; D018417:Th1 Cells / Q000187:drug effects / Q000378:metabolism*; D018418:Th2 Cells / Q000187:drug effects / Q000378:metabolism*; D018183:Transplantation Chimera; D014184:Transplantation, Homologous
D016207:Cytokines; D007166:Immunosuppressive Agents; D012333:RNA, Messenger; D003520:Cyclophosphamide; D002066:Busulfan