Constructing a bone marrow chimera prior to graft transplantation can induce donor-specific immune tolerance. Mixed chimerism containing hematopoietic cells of both recipient- and donor-origin has advantages attributed from low dose of total body irradiation. In this study, we explored the mechanism of mixed chimerism supplemented with depletion of Natural Killer cells. Mixed chimerism with C57BL/6 bone marrow cells was induced in recipient BALB/c mice which were given 450 cGy of gamma-ray irradiation (n = 16). As revealed by reduced proliferation and cytokine production in mixed leukocyte reaction and ELISpot assay (24.6 vs 265.5), the allo-immune response to bone marrow donor was reduced. Furthermore, the induction of transferable immunological tolerance was confirmed by adoptive transfer and subsequent acceptance of C57BL/6 skin graft (n = 4). CD4+FoxP3+ regulatory T cells were increased in the recipient compartment of the mixed chimera (19.2% --> 33.8%). This suggests that regulatory T cells may be therapeutically used for the induction of graft-specific tolerance by mixed chimerism.
Animals ; Bone Marrow Cells/cytology ; *Bone Marrow Transplantation ; Cell Proliferation ; Chimerism ; Cytokines/metabolism ; Gamma Rays ; Graft Survival ; *Immune Tolerance ; Killer Cells, Natural/immunology/radiation effects ; Leukocytes/immunology/radiation effects ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Models, Animal ; Skin Transplantation ; T-Lymphocytes, Regulatory/cytology/*immunology/metabolism ; Whole-Body Irradiation

The aim of this study was to explore the effect of hyperbaric oxygen (HBO) preconditioning on the rejection of skin allograft in mice and its molecular mechanism. BALB/c donor mice and C57BL/6 recipients received hyperbaric oxygen preconditioning once a day for 7 days. After skin transplantation, the recipients were treated with cyclosporine A (CsA) intraperitoneally. Immunofluorescent staining technique and flow cytometry were used to observe the influence HBO on percentage of spleen lymphocytes CD3+, CD4+, CD8+ and cell adhesion molecule LFA-1 (CD11a/CD18). The results showed that as compared with control, the numbers of CD3+, CD4+, CD8+, CD4+CD11a+, CD4+ CD18+, CD8+CD11a+, CD8+CD18+ lymphocytes of spleen decreased in HBO preconditioning groups and CsA group, and decreased markedly in HBO preconditioning combined with CsA group (p<0.05); the general state of recipient mice in HBO preconditioning combined with CsA group was better than that of recipient mice received HBO preconditioning or CsA only. It is concluded that the method of HBO preconditioning combined with traditional immunosuppressive agent CsA has remarkable advantage in inhibiting the rejection of skin graft. Its molecular protective mechanism is correlated with the expression of adhesive molecules on T cell subsets.
Animals ; Cell Adhesion ; Cell Adhesion Molecules ; pharmacology ; Cyclosporine ; pharmacology ; Female ; Graft Rejection ; prevention & control ; Graft Survival ; Hyperbaric Oxygenation ; Lymphocyte Count ; Lymphocytes ; cytology ; metabolism ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Skin Transplantation ; immunology ; Spleen ; cytology ; Transplantation Conditioning ; methods

Down syndrome critical region 1 (DSCR1), an oxidative stress-response gene, interacts with calcineurin and represses its phosphatase activity. Recently it was shown that hydrogen peroxide inactivates calcineurin by proteolytic cleavage. Based on these facts, we investigated whether oxidative stress affects DSCR1-mediated inactivation of calcineurin. We determined that overexpression of DSCR1 leads to increased proteolytic cleavage of calcineurin. Convertsely, knockdown of DSCR1 abolished calcineurin cleavage upon treatment with hydrogen peroxide. The PXIIXT motif in the COOH-terminus of DSCR1 is responsible for both binding and cleavage of calcineurin. The knockdown of overexpressed DSCR1 in DS fibroblast cells also abrogated calcineurin proteolysis by hydrogen peroxide. These results suggest that DSCR1 has the ability to inactivate calcineurin by inducing proteolytic cleavage of calcineurin upon oxidative stress.
Adenoviridae/genetics ; Adult ; Animals ; Calcineurin/antagonists & inhibitors/*metabolism ; Cells, Cultured ; Chromatin Immunoprecipitation ; Down Syndrome/*metabolism/pathology ; Fibroblasts/metabolism/pathology ; Humans ; Hydrogen Peroxide/pharmacology ; Immunoglobulin G/immunology ; Intracellular Signaling Peptides and Proteins/*physiology ; Male ; Mice ; Mice, Inbred ICR ; Muscle Proteins/*physiology ; Neuroblastoma/genetics/metabolism/pathology ; Neurons/cytology/metabolism ; Oxidants/pharmacology ; Oxidative Stress ; Peptide Fragments/immunology ; RNA, Messenger/genetics/metabolism ; RNA, Small Interfering/pharmacology ; Rabbits ; Reverse Transcriptase Polymerase Chain Reaction ; Skin/pathology ; Young Adult

The study was aimed to explore the roles of exosomes derived from regulatory dendritic cells of mice in the induction of immune tolerance. Immature DC (iDC) from mouse bone marrow cells and regulatory DCs (rDC) were induced by treating iDC with TGF-beta1 and IL-10. The phenotype of regulatory DCs and normal DCs were assayed by flow cytometry. Exosomes from immature DCs (iDex) and regulatory DCs (rDex) were isolated by ultracentrifugation and ultrafiltration. A skin transplantation model was established with the recipients BALB/c mice and the donor C57BL/6 mice. Recipients were divided into PBS control group, iDex group (injection 10 microg iDex of donor C57BL/6 mice via tail vein at days 7 and 3 before skin transplantation), rDex group (injection 10 microg rDex of donor C57BL/6 mice via tail vein at days 7 and 3 before skin transplantation). The capacity of the donor mice and the unrelated allogeneic donor mice to stimulate allogeneic T lymphocyte proliferation was examined by mixed lymphocyte culture (MLR). The results showed that TGF-beta1 and IL-10 could down-regulate the expressions of costimulatory molecules, including CD80, CD86 and CD40. The graft mean survival time (MST) in control group, iDex group and rDex group was 7.8, 10.7 and 18.8 days, respectively. There was significant difference in MST between iDex group and control group (p<0.05), and between rDex group and iDex group (p<0.01). The results of MLR assays indicated donor-specific hyporeactivity especially in rDex group, while the tolerant B/C mice were still immunocompetent to unrelated allogeneic DBA mouse. It is concluded that injection iDex or rDex of donor mice via tail vein before skin transplantation induces immunotolerance, and the effect of rDex is more significant.
Animals ; Dendritic Cells ; cytology ; immunology ; transplantation ; Exosomes ; immunology ; transplantation ; Female ; Graft Enhancement, Immunologic ; methods ; Graft Survival ; Immune Tolerance ; immunology ; Lymphocyte Culture Test, Mixed ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Mice, Inbred DBA ; Skin Transplantation ; Transplantation Immunology ; Transplantation, Homologous

OBJECTIVETo investigate the synergic effects of rapamycin and donor bone marrow-derived immature dendritic cells (DCs) in inducing skin allograft tolerance in mice.

METHODSThe recipient BALB/c mice receiving transplantation of skin allograft from C57BL/6 mice were divided into control group (without perioperative treatments), rapamycin group (receiving rapamycin at 1 mg.kg(-1).d(-1) by gavage for 7 consecutive 7 days after skin transplantation), immature DC group (receiving an injection of donor bone marrow-derived immature DCs of 2 x 10(6) via tail vein before skin transplantation), combined group (receiving an injection of the DCs of 2 x 10(6) before transplantation and rapamycin at 1 mg.kg(-1).d(-1) for 7 consecutive days after transplantation). The survival time of the skin allograft was observed in each group.

RESULTSThe survival time of the skin allograft in the control, rapamycin, immature DC and immature DC +rapamycin groups were 6.9-/+1.9, 12.3-/+3.0, 17.0-/+3.4 and 20.8-/+3.6 days, respectively, showing significant differences among the groups (P<0.05), and SNK test also indicated significant differences between every two groups.

CONCLUSIONSRapamycin and donor bone marrow-derived immature DCs have synergic effects in inducing skin allograft tolerance in mice.

Animals ; Bone Marrow Cells ; cytology ; immunology ; Dendritic Cells ; immunology ; Graft Survival ; drug effects ; immunology ; Immunosuppressive Agents ; pharmacology ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Sirolimus ; pharmacology ; Skin Transplantation ; immunology ; methods ; Transplantation, Homologous

Animals ; Collagen ; chemistry ; Goats ; Humans ; Mice ; Skin ; cytology ; immunology ; Skin, Artificial ; Swine

Blockade of signal 1 or 2 for T-cell activation by the use of anti-CD45RB and anti-CD154 monoclonal antibodies (mAb) (two-signal blockade) has been proven effective in preventing or delaying graft rejection. However, the mechanisms of its immunomodulatory effects are clearly unknown and the present studies were performed to determine how the two-signal blockade modulate allogeneic immune responses, especially T-cell mediated cellular immunity, in a murine skin allograft model. We now report on the profound inhibition of alloreactive T cells by two-signal blockade via CD4-dependent mechanisms. C57BL/6 mice of BALB/c skin allograft were treated with anti-CD45RB, anti-CD154, CTLA4-Ig, or their combinations. For depletion of CD4 or CD8 T cells, the recipients received CD4-depleting or CD8-depleting mAb. We confirmed that survival of skin allograft was markedly prolongated in the two-signal blockade-treated group. In depletion study, anti-CD45RB, anti-CD154 and CD4-depleting mAb-treated group showed acute rejection of skin allograft in contrast to CD8-depleting group treated with the two-signal blockade. In the group treated with the two-signal blockade, the proportions of CD4+CD45RB(low)and CD8+CTLA-4 regulatory T cells were increased while effector CD8+ T cells, including IFN-gamma-secreting and CD8+CD62L(low)T cells, were decreased when compared with non-treated group. In contrast, the CD4-depleted group treated with the two-signal blockade resulted in recovery from immunoregulatory effects of two-signal blockade. In addition, results of IL-4 and IL-10 production were also showed CD4-dependence. Therefore, the two-signal blockade is accompanied by CD4-dependent mechanisms in allogeneic skin transplantation.
Transplantation, Homologous ; T-Lymphocytes, Regulatory/cytology/immunology ; Skin Transplantation/*immunology ; Signal Transduction/drug effects/immunology ; Mice, Inbred C57BL ; Mice, Inbred BALB C ; Mice ; Male ; Lymphocyte Depletion ; Lymphocyte Activation/immunology ; Interleukin-4/biosynthesis ; Interleukin-10/biosynthesis ; Graft Rejection/*immunology/prevention & control ; Flow Cytometry ; Cytotoxicity, Immunologic/immunology ; CD8-Positive T-Lymphocytes/cytology/immunology/metabolism ; CD40 Ligand/*immunology ; CD4-Positive T-Lymphocytes/cytology/immunology/metabolism ; Antigens, CD45/*immunology ; Antigens, CD4/*immunology ; Antibodies, Monoclonal/administration & dosage/*pharmacology ; Antibodies, Blocking/administration & dosage/pharmacology ; Animals

This study was aimed to investigate the migration and distribution processes of allogeneic donor T lymphocytes in the organs of recipient mice. GVHD model was established by transfusion of the splenocytes of eGFP transgeneic C57BL/6 mice together with born marrow cells harvested from C57BL/6 mice into BALB/c mice underwent 8.0 Gy total body irradiation. The migration and homing of eGFP(+) cells were tracked by stereo-fluorescent microscopy or inverted fluorescent microscopy and flow cytometry. The enzyme linked immunosorbent assay (ELISA) was performed on supernatants from the tissue homogenates to detect the amount of MIP-1alpha. The results indicated that GVHD clinical manifestation and pathological changes of organs appeared on day 8 post transplantation. eGFP-positive donor T cells in recipient organs were observed by inverted fluorescence microscope in frozen section, or by stereo-fluorescence microscopy in living organs, such as liver, spleen, skin, lungs, bowels, and tongue. The highest expression of MIP-1alpha was on day 7 post transplantation in the liver (491.3 +/- 32.1 pg/ml), and day 3 post transplantation in the spleen (881.5 +/- 45.2 pg/ml), respectively (P < 0.05). It is concluded that GVHD was induced by splenocytes of eGFP transgeneic C57BL/6 mice. eGFP(+) cells in the organs can be observed by fluorescent microscopy. In this GVHD model, donor T cells proliferate and infiltrate in liver, skin, bowels, as well as lungs and tongue. MIP-1alpha may be in relation with the infiltration of T lymphocytes in liver and spleen.
Animals ; Bone Marrow Transplantation ; adverse effects ; Cell Movement ; Female ; Graft vs Host Disease ; immunology ; pathology ; Green Fluorescent Proteins ; Liver ; immunology ; pathology ; Lung ; immunology ; pathology ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Skin ; immunology ; pathology ; Spleen ; cytology ; T-Lymphocytes ; immunology

It was previously thought that keratinocytes did not express the CD80 and CD86 which provide the most important costimulatory signals for the antigen-specific T-cell activation. The cultured keratinocytes allografts were initially accepted, but eventually, all grafted donor cells were gradually replaced by recipient cells. The precise mechanisms are not very clear. In this study, neonatal murine keratinocytes were cultured for 7 days, the results of flow cytometry and confocal microscopy showed that CD80 could be detected on keratinocytes, while CD86 could not be detected all the time. RT-PCR analysis confirmed this result. The expression level of the CD80 mRNA amplified significantly from day 1 to day 7, as expression of the control beta-actin, but CD86 was not detected. Mixed Lymphocyte Reaction (MLR) showed that keratinocytes cultured with 10% serum for 7 d stimulated effectively allogeneic rather than syngeneic T cell proliferation. This study demonstrated for the first time that costimulatory molecule CD80 can be expressed on keratinocytes in vitro. These data provided an alternative explanation for the ultimate rejection of allogeneic keratinocytes in which keratinocytes act as antigen-presenting cells.
Animals ; Animals, Newborn ; Antigen-Presenting Cells ; cytology ; B7-1 Antigen ; biosynthesis ; genetics ; B7-2 Antigen ; biosynthesis ; genetics ; Cells, Cultured ; Graft Rejection ; Keratinocytes ; cytology ; Lymphocyte Activation ; Lymphocytes ; immunology ; Mice ; Mice, Inbred BALB C ; RNA, Messenger ; biosynthesis ; genetics ; Skin ; cytology

The role of lung mast cells in exercise-induced asthma (EIA) is controversial. To investigate whether the skin mast cell releasability is increased after exercise in EIA, 49 young atopic men with or without asthma took part in a free-running test for 6 min and were given skin prick tests using morphine, a mast cell secretagogue, before and after the exercise. The mean diameters of the wheal induced by morphine in patients with EIA were not significantly different from those in patients without EIA before exercise, although the baseline lung function was significantly lower and the airway hyperresponsiveness, the peripheral blood eosinophil count, and the size of the wheal in response to Dermatophagoides pteronyssinus were significantly higher in patients with EIA. However, the differences of the morphine-induced wheal diameter between patients with EIA and those without EIA became significant at 120 min after exercise (p<0.05), while the responses to histamine were not significantly different. These results suggest that exercise increases the releasability of skin mast cells in EIA patients whose asthma/allergy are relatively severe.
Adolescent ; Adult ; Analgesics, Opioid/diagnostic use ; Asthma/*immunology/physiopathology ; *Exercise ; Histamine/diagnostic use ; Humans ; Male ; Mast Cells/drug effects/*immunology ; Morphine/diagnostic use ; Skin/cytology/*immunology ; Skin Tests