PURPOSE: Previous reports demonstrated that nitric oxide (NO) plays immuno-regulatory role in immune responses including allograft rejection response. However, its possible role in xenograft rejection has not been examined. The purpose of this study is to elucidate possible immunoregulatory role of NO in skin xenograft rejection by determining the expressions of chemokines and cytokines in the presence or absence of iNOS inhibitors. METHODS: C57BL/6J mice were grafted with Lewis rat tail skin. The mice were injected intraperitoneally with potent inhibitor of iNOS, aminoguanidine (AMG, 200 mg/kg). Graft survival was monitored and cytokine and chemokine mRNA expressions were measured by real-time RT-PCR in context with iNOS expression on day 3, 5, 7 and 9. These data were compared with those of control mice (saline injected). RESULTS: Compared with the control mice, the AMG treated mice showed delayed xenograft rejection by approximately 3 days (8.9+/-0.7 days vs 11.7+/-1.2 days). Infiltrations of CD11b+, MOMA-2+ cells and neutrophils were significantly reduced but not CD4+ and CD8+ cells in AMG treated graft. The expression of cytokines such as IL-1beta, IL-2, IL-6, IL-12, IFN-gamma in AMG treated graft significantly decreased (P<0.01) whereas IL- 10, TNF-alpha and TGF-beta1 were not changed or enhanced. Additionally, the expression of CC-chemokines such as RANTES and MIP-1alpha significantly reduced (P<0.01) whereas CXC-chemokines such as IP-10 and MIG did not change. CONCLUSION: These data imply that NO suppression by iNOS inhibitor may prolong rat to mouse skin xenograft survival through a selective inhibition of pro-inflammatory cytokines and chemokines. The possible role of NO in transplant rejection can be, therefore, extended to regulation of cytokine and chemokine expressions.
Allografts ; Animals ; Chemokine CCL3 ; Chemokine CCL5 ; Chemokines ; Cytokines ; Graft Rejection ; Graft Survival ; Heterografts* ; Interleukin-12 ; Interleukin-2 ; Interleukin-6 ; Mice ; Neutrophils ; Nitric Oxide ; Rats ; RNA, Messenger ; Skin* ; Tail ; Transforming Growth Factor beta1 ; Transplantation, Heterologous ; Transplants ; Tumor Necrosis Factor-alpha

PURPOSE: Stem cells are considered promising candidates for cell replacement therapy in many devastating diseases. However, it is assumed that stem cells may be rejected on transplantation. Therefore, we introduced human cytomegalovirus (hCMV) US genes, which are known to be able to reduce MHC class I expression on the cell surface after infection, into two known stem cell lines in order to test the feasibility of modifying these cells to reduced MHC class I antigens by the introduction of hCMV US genes. METHODS: The MHC class I expressions of mock-transfected or hCMV US gene-transfected human embryonic neural stem cell line (HB1.F3) and human breast epithelial stem cell line (M13SV1) were examined by FACS. RESULTS: MHC class I expressions in HB1.F3 and M13SV1 cells were dramatically induced by IFN-gamma treatment. In FACS analysis, cells transfected with the hCMV US2, 3, 6 or 11 genes exhibited a dramatic reduction (40~60%) of MHC class I expression compared with mock-transfected cells. CONCLUSION: Our results suggest that human stem cells express high levels of MHC class I antigens, and thus may be rejected on transplantation unless they are odified. In addition introduction of hCMV US genes can be exploited for stemcell transplantation.
Breast ; Cytomegalovirus ; Down-Regulation* ; Histocompatibility Antigens Class I ; Humans* ; Neural Stem Cells ; Stem Cells*

PURPOSE: The host immune responses to skin xenografts are known to be much stronger than those to allografts. The possible reasons for that, however, are unclear. We hypothesized that chemokines trafficking leukocytes may be involved in stronger xenograft rejection process as compared to allograft rejection. METHODS: Thus, we evaluated the profiles of chemokine expression and cellular infiltration in the skin xenografts and compared to those in fully allogeneic skingrafts. RESULTS: At day 5, post-transplantation the heavier cellular infiltration of CD4+, CD8+ T cells and neutrophils into xenografts was found as compared to allografts. Similarly, more CD8+ T, CD11b+ and MOMA-2+ cells were found to infiltrate into xenografts than allografts at day 7. The xenograft showed earlier and stronger mRNA expression of chemokines such as MCP-1, IP-10 and MIG. In the late phase of xenograft rejection, strong expression of RANTES and MIP-1a was noted. In xenografts, the time of expression of IP-10 and MIG was correlated with the time of infiltration of CD4+, CD8+ cells and neutrophils whereas, the expression patterns of RANTES and MIP-1a were similar to the infiltration patterns of CD11b+ and MOMA-2+ cells. CONCLUSION: These results suggest that the prompt xenograft cellular rejection as compared to the allograft rejection may be related to the rapid induction of pro-inflammatory chemokines such as MCP-1, IP-10 and MIG in the early phase and resultant significant infiltration of CD4+ and CD8+ T cells and neutrophils.
Allografts ; Chemokine CCL5 ; Chemokines ; Heterografts* ; Leukocytes ; Neutrophils ; RNA, Messenger ; Skin* ; T-Lymphocytes ; Transplantation, Heterologous

PURPOSE: Human embryonic stem (ES) cell is pluripotent cell derived from a group of cells called the inner cell mass and has the ability to reproduce itself for long periods and give rise to types of cells that develop from the three germ layers. Due to its pluripotency, ES cell holds the promise of being able to replace cells that are damaged or destroyed by many devastating diseases. However, the potential for the recipient of an ES cell transplant to reject this cell as foreign is very high. Thus, it is essential to determine whether human ES cells express MHC antigens. The purpose of this study is to characterize the stem cell properties of our cell line (SNUhES1) and the expression profile of MHC antigens on the surface of these cells and their differentiated derivatives, embryoid bodies (EBs). METHODS: The ES cells were grown on STO fibroblast in DMEM-F12. The EBs were grown in the same medium with exception that it lacked LIF and bFGF. The expression of self-renewal-associated genes and three germ layer cell-specific genes in ES cells and EBs were measured by RT-PCR at varying time point of incubation (1, 7, 14 and 28 day). The expression of MHC molecules were measured by RT-PCR and FACS analysis. RESULTS: The SNUhES1 cells expressed all self-renewal- associated genes (Fgf4, FoxD3, Oct4, Sox2 and TERT) we tested. During the differentiation three germ layer cell-specific genes in EBs were expressed as following order: ecto-, meso- and endodermal cell-specific genes. MHC class I proteins (HLA-ABC and beta2m) on the surfaces of ES cells and EBs were expressed in very low levels. MHC class II proteins (HLA-DP, -DQ and -DR) and HLA-G were not expressed on the surface of these cells. However, the expression of MHC class II proteins were detected in 1% more or less cells of 28-day-old EBs which were hardly detected in the population of 1-day-old EBs. CONCLUSION: These data imply that SNUhES1 cells and EBs have stem cell properties. Although they express very low MHC antigens, further investigation determining whether the MHC expression in the ES cells and EBs may alter under inflammatory condition which can be occurred in damaged tissue or through surgical process.
Cell Line ; Embryoid Bodies* ; Embryonic Stem Cells* ; Endoderm ; Fibroblasts ; Germ Layers ; HLA-G Antigens ; Humans* ; Stem Cells ; Transplants