Early expression of interferon-gamma inducible protein 10 and monokine induced by interferon-gamma in cardiac allografts is mediated by CD8+ T cells

BACKGROUND: Our goal was to test the intragraft mRNA expression and production of two chemokines that are potent chemoattractants for antigen-primed T cells, interferon-gamma inducible protein 10 (IP-10) and monokine-induced by IFN-gamma, (Mig), in allogeneic heart grafts. METHODS: Syngeneic or allogeneic A/J (H-2a) hearts were heterotopically transplanted to wild-type, CD4-/-, CD8alpha-/-, or IFN-gamma-/- C57BL/6 (H-2b) recipients. To test expression of IP-10 and Mig, grafts were removed 1-8 days posttransplant for RNA isolation and Northern blot analysis. To test the potential recipient leukocyte populations mediating intraallograft expression of IP-10 and Mig, recipients were treated with anti-NK 1.1, anti-CD4, and/or anti-CD8 monoclonal antibodies before transplantation. RESULTS: Allogeneic heart grafts transplanted to wild-type, but not IFN-gamma-/-, recipients expressed IP-10 and Mig at day +2 posttransplant that increased thereafter until rejection was completed. Expression of IP-10 and Mig in isografts was low or undetectable. Cardiac allografts from CD8+ T cell depleted, but not NK cell or CD4+ T cell depleted, recipients had low to undetectable expression of IP-10 and Mig on day +2 posttransplant. Similarly, cardiac allografts from CD8-/-, but not CD4-/-, recipients had low to undetectable expression of IP-10 and Mig on day +2 posttransplant. CONCLUSIONS: Early intraallograft expression of Mig and IP-10 during primary rejection of cardiac allografts is dependent on the activities of recipient CD8+ T cells.     

The graft helps to define the character of the alloimmune response

Introduction: In mice, kidney and liver allografts may be spontaneously accepted, whereas cardiac and skin allografts in the same strain combinations are rapidly rejected. The reasons for this dichotomy in murine response outcomes remains to be determined. Methods and results: When DBA/2 (H-2d) cardiac allografts were placed in C57BL/6 (H-2b) recipients, they were rejected within 10 days, unless the allograft recipients were transiently treated with gallium nitrate (GN), at which time the allografts were accepted for > 150 days. The cardiac allograft rejector mice displayed DBA/2-reactive DTH responses, whereas the cardiac allograft acceptor mice displayed both TGFbeta- and IL10-mediated inhibition of DTH responses. In contrast, DBA/2 kidney allografts placed at the same location in C57BL/6 mice were spontaneously accepted without immunosuppression. These kidney allograft acceptor mice displayed TGFbeta-mediated, but not IL10-mediated inhibition of donor-reactive DTH responses. Conclusions: In the DBA/2-> C57B1/6 strain combination, cardiac allografts induce pro-inflammatory immunity and allograft rejection, while kidney allografts induce anti-inflammatory immunity and allograft acceptance despite the fact that both organs display the same strong MHC disparities and are implanted at the same location. Anti-inflammatory immunity and allograft acceptance are displayed by cardiac allograft recipients when they are transiently treated with select immunosuppressants. Thus, multiple immune response options are available to the organ allograft recipient, and the choice is determined, to some degree, by the allograft, itself.     

MHC-identical heart and hepatocyte allografts evoke opposite immune responses within the same host

BACKGROUND: Purified allogeneic hepatocytes are highly antigenic and elicit immune responses that are not easily controlled. However, it is not clear whether hepatocytes are not capable of protective immune mechanisms or whether they are not to protection by immune mechanisms that permit long-term survival of other allografts. The purpose of the current study was to determine whether donor-matched allogeneic hepatocytes are protected from rejection in mice that have been induced to accept heart allografts. METHODS: Transient treatment with anti-CD4 monoclonal antibody (mAb) or gallium nitrate (GN) was used to induce acceptance of heterotopic FVB/N (H-2(q)) heart allografts by C57BL/6 (H-2(b)) mice. Transgenic hA1AT-FVB/N hepatocytes were sequentially transplanted into C57BL/6 mice that had accepted FVB/N heart allografts more than 60 days (heart acceptor mice), CD8 depleted C57BL/6 heart acceptor mice, or B-cell knockout (BCKO, H-2(b)) heart acceptor mice. Hepatocyte survival was determined by the detection of secreted transgenic product hA1AT by enzyme-linked immunosorbent assay (ELISA). RESULTS: FVB/N hepatocytes were rejected by day 10-14 posttransplant, while FVB/N heart allografts continued to function in C57BL/6, BCKO, and CD8 depleted heart acceptor mice. When FVB/N hepatocytes and heart allografts were transplanted into C57BL/6 or BCKO mice under short-term cover of anti-CD4 mAb or GN, hepatocyte rejection occurred by day 10 posttransplant, while most heart allografts survived for more than 60 days. CONCLUSIONS: Hepatocyte rejection does not appear to interfere with the of mechanisms that permit heart allograft acceptance. However, immune responses to allogeneic hepatocytes are not to regulation by mechanisms induced in heart acceptor mice. The simultaneous rejection of FVB/N allogeneic hepatocytes and continued acceptance of FVB/N-matched heart allografts is independent of host CD8+ T cells and humoral immunity.     

CCR5 Is Required for Regulation of Alloreactive T-Cell Responses to Single Class II MHC-Mismatched Murine Cardiac Grafts

The effector CD4 T-cell response in wild-type C57BL/6 recipients of single class II MHC-disparate B6.H-2^bm12 cardiac allografts is restricted by CD4⁺CD25⁺ regulatory T cells (Tregs) resulting in long-term allograft survival. To investigate the role chemokine receptors might play in Treg function, this study tested the requirement for CCR5 on Tregs to suppress the alloimmune response in C57BL/6 recipients of B6.H-2^bm12 cardiac allografts. In contrast to the long-term survival of B6.H-2^bm12 allografts in wild-type recipients (>100 days), the allografts were acutely rejected within 25 days in CCR5^−/− recipients with intense infiltration of CD4 T cells. Numbers and duration of donor-reactive CD4 T cells producing IFN-γ and IL-4 were markedly increased in spleens of B6.CCR5^−/− versus wild-type recipients. Wild-type and B6.CCR5^−/− mice had equivalent numbers of splenic FoxP3⁺ Tregs before and following transplantation, and these Tregs were equivalently suppressive in vitro . However, diminished numbers of FoxP3⁺ Tregs infiltrated B6.H-2^bm12 allografts in B6.CCR5^−/− recipients. Adoptive transfer of wild-type, but not CCR5-deficient, CD4⁺CD25⁺ Tregs to CCR5^−/− recipients restored long-term survival of B6.H-2^bm12 cardiac grafts. Collectively, these results indicate that CCR5 expression is required for the regulatory functions of Tregs that restrict alloreactive CD4 T-cell responses to single class II MHC-mismatched cardiac allografts.     

Early increased chemokine expression and production in murine allogeneic skin grafts is mediated by natural killer cells

BACKGROUND: Increased expression of chemokine mRNA is observed in allogeneic but not syngeneic skin grafts 3-4 days after transplantation. The recipient cells mediating this early inflammatory response in allografts remain unidentified. METHODS: Isogeneic and allogeneic skin grafts were transplanted to euthymic and athymic nude mice. mRNA expression and protein production of macrophage inflammatory protein-1alpha (MIP-1alpha), MIP-1beta, and the murine homolog of Gro(alpha), i.e. KC, from graft homogenates retrieved 3-4 days posttransplantation was tested by Northern blot hybridization and ELISA. To deplete NK cells, recipients were treated with antiasialo GM1 (ASGM1) antisera or with anti-NK1.1 mAb before transplantation. RESULTS: Expression of KC, MIP-1alpha, and MIP-1beta mRNA was equivalent in C57BL/6 allogeneic skin grafts and BALB/c isografts at day 2 posttransplant. At day 3 posttransplant, chemokine mRNA levels decreased in isografts but were maintained at high levels in the allografts. Increased early chemokine mRNA was also observed in C57BL/6, but not BALB/c++ grafts on BALB/c athymi(nu/nu) recipients. Treatment of allograft recipients with ASGM1 or with anti-NK1.1 antibody eliminated NK cells from the spleen and allograft infiltrating cell populations and decreased early chemokine mRNA levels in allografts 60-70%. Analyses of allograft homogenates indicated increased levels of KC, MIP-1alpha, and MIP-1beta protein at day 4 posttransplant that were decreased in recipients depleted of NK cells. Early chemokine mRNA levels were equivalent in isogeneic and semiallogeneic F1 grafts. CONCLUSIONS: Early chemokine mRNA expression and protein production in allogeneic skin grafts is amplified by recipient natural killer (NK) cells. These results indicate a novel function for infiltrating NK cells in mediating early increased intra-allograft chemokine production and inflammation during the initiation of acute rejection.     

Organ-specific differences in the function of MCP-1 and CXCR3 during cardiac and skin allograft rejection

BACKGROUND: Chemokines are well-established to function in the recruitment of leukocytes into allografts in the course of rejection. Moreover, some studies have indicated that there are organ-specific differences in chemokine function, but the mechanism accounting for this difference is not known. METHODS: Fully major histocompatibility complex-mismatched vascularized cardiac transplants or skin transplants were performed using BALB/c (H-2d), C57BL/6 (H-2b), MCP-1-/- (H-2b) and CXCR3-/- (H-2b) mice as donors or recipients. Also, skin grafts (H-2b) were placed onto SCID mice (H-2d) that received BALB/c splenocytes (H-2d) by adoptive transfer either at the time of transplantation, or after a period of 28 days. RESULTS: Cardiac allografts in MCP-1-/- recipients survived significantly longer (P<0.0005) than wild-type (WT) controls. However, there was no prolongation of survival when MCP-1-/- grafts were used a donors in WT mice. In contrast, the absence of donor but not recipient MCP-1 prolonged skin allograft survival. WT donor cardiac grafts in CXCR3-/- recipients had a modest prolongation of survival (P<0.0005), whereas CXCR3-/- donor cardiac grafts in WT recipients were rejected similar to controls. Also, while recipient CXCR3 had no effect on the rejection of skin, CXCR3-/- donor skin grafts survived significantly longer than WT controls. This survival advantage was lost when vascularized CXCR3-/- skin grafts were used as donors in the SCID model of rejection. CONCLUSION: Recipient derived MCP-1 and CXCR3 are functional in the rejection of vascularized, but not nonvascularized, allografts. In contrast, donor-derived MCP-1 and CXCR3 are functional in nonvascularized, but not vascularized grafts.     

Chronic antagonism of Mig inhibits cellular infiltration and promotes survival of class II MHC disparate skin allografts

BACKGROUND: The goal of the current study was to test the ability of monokine induced by IFN-gamma (Mig)-specific antibodies to inhibit long-term T cell infiltration into class II major histocompatibility complex (MHC) disparate skin allografts and to test cellular and molecular changes in the graft during the rejection observed following cessation of treatment. METHODS: C57BL/6 recipients of B6.H-2bm12 skin grafts were treated with normal rabbit serum (NRS) or rabbit Mig antiserum (Mig AS) every other day from day 7 until day 21 posttransplant and then weekly thereafter. Allografts were retrieved during the course of treatment and following cessation. Tissue sections were prepared and stained to compare infiltration by macrophages and CD4+ and CD8+ T cells and to assess collagen deposition in the grafts. RNA was prepared and tested by ribonuclease protection assay for intragraft levels of Mig, monocyte chemotactic protein-1 (MCP-1) and regulated on activation normal T-cell expressed and secreted (RANTES). RESULTS: T cell and macrophage infiltration into allografts was inhibited and graft survival maintained as long as Mig-specific antibodies were given. Following cessation of treatment, T cells and macrophages infiltrated the allografts. In contrast to the histology of acute rejection observed in allografts from NRS-treated recipients, the resulting rejection of the allografts from Mig AS-treated recipients was accompanied by dense collagen deposition and high level expression of Mig and RANTES. CONCLUSIONS: Mig directs T cell infiltration into B6.H-2bm12 skin allografts on C57BL/6 recipients. Delayed T cell and macrophage infiltration and rejection of the grafts following cessation of Mig AS treatment results in rejection that is histologically and molecularly distinct from acute rejection.     

阻断ICOS/B7h信号的供体特异性输血对移植受体CD4+CD25+调节性T细胞的影响

目的 观察阻断ICOS/B7h信号的供体特异性输血(DST)对异基因小鼠心脏移植术后体内CD4+CD25+调节性T细胞(Treg)的影响.方法 按陈氏方法建立小鼠颈部异位心脏移植模型,实验分3组,异基因组及同基因组:供心分别来源于BALB/C和C57BL/6小鼠,受体均为C57BL/6小鼠,未予治疗.治疗组:移植当天给予受体鼠(C57BL/6)尾静脉注射5×106 ICOS-Fc靶定的供体(BALB/C)脾B淋巴细胞,d0～6连续给予受体鼠尾静脉注射ICOS-Fc 200 μg/d.术后统计各组移植物的存活时间,通过流式细胞术检测受体鼠外周血中CD4+CD25+Treg的亚群比例,利用逆转录-聚合酶链反应(RT-PCR)检测移植物中FOXP3的mRNA表达,在混合淋巴细胞反应中检测CD4+CD25+Treg对CD4+CD25-效应T细胞(Teff)的增殖抑制效率.结果 与异基因组比较,治疗组心脏移植物存活时间明显延长[(84.38±29.14)d比(7.00±0.76)d,P＜0.01].各组中,治疗组受体外周血中CD4+CD25+Treg亚群比例显著上调[(15.60±5.69)%,P＜0.01].与其他两组比较,治疗组心脏移植物中FOXP3 mRNA表达显著上调.以正常鼠为对照,耐受鼠脾脏中获取的CD4+CD25+Treg能够更高效地抑制CD4+CD25-Teff在混合淋巴细胞培养中的增殖效应.结论 通过阻断ICOS/B7h信号的DST可以诱导异基因小鼠心脏移植耐受,CD4+CD25+Treg在耐受的形成与维持中均起着重要作用.     

Airway Epithelium is the Primary Target of Allograft Rejection in Murine Obliterative Airway Disease

Murine heterotopic tracheal allografts develop obliterative airway disease (OAD), a suitable model of chronic lung allograft rejection. This model, however, fails to account for the behavior of the allograft when adjacent to recipient airway tissues, particularly the epithelium. This study was performed to determine the immunologic role of the epithelium in development of OAD. BALB/c (H2d) tracheal allografts were transplanted orthotopically into C57BL/6 (H2b) mice and harvested 14-150 days post-transplantation. The phenotype of the allograft epithelium after orthotopic transplantation was determined with immunofluorescent staining. Orthotopic BALB/c tracheal allografts harvested at 28 days were re-transplanted heterotopically into BALB/c or C57BL/6 mice, harvested after 28 days, and assessed for OAD. Orthotopic allografts displayed mild cellular infiltration, no fibrosis and preserved epithelium at 28 days post-transplant. The presence of recipient-derived epithelium within the allograft was demonstrated with immunofluorescent staining at day 14. Significantly, BALB/c orthotopic allografts re-transplanted heterotopically into BALB/c mice developed OAD by day 28, whereas BALB/c orthotopic allografts re-transplanted heterotopically into C57BL/6 mice did not. Repopulation of orthotopic tracheal allografts with recipient-derived epithelium confers a protective effect against OAD after heterotopic re-transplantation. This indicates that the airway epithelium plays a crucial role in OAD development.     

Acceptance of Third-Party Cardiac but not Skin Allografts Induced by Neonatal Exposure to Semi-Allogeneic Lymphohematopoietic Cells

Neonatal tolerance is exclusively donor-specific when assessed by skin allograft survival and in vitro alloreactivity assays. In contrast, we reported previously that acceptance of primarily vascularized cardiac allografts was not donor-specific in C3H/He (C3H, H-2(k)) mice treated as neonates with BALB/c-derived (BALB, H-2(d)) lymphohematopoietic cells, but included third-party C57BL/10 (B10, H-2(b)) allografts. The present study examined whether this unusual pattern is limited to heart grafts in this strain combination, and defined the relative importance of the donor cell H-2(d) haplotype for third-party cardiac allograft acceptance. C3H neonates were injected with (C3HxBALB)F1 bone marrow and spleen cells. Tolerance was assessed at age 8-10 weeks by transplantation of heart or skin allografts from several donor strains, and by in vitro assays of proliferation and cytotoxicity. Additionally, cells from H-2(d) and H-2(b)-expressing strains on BALB or non-BALB minor histocompatibility (miH) antigen backgrounds were tested as tolerizing inocula. Prolonged survival of cardiac grafts from all donor strains was observed in neonatally treated mice, whereas skin grafting and in vitro assays demonstrated donor-specific hyporesponsiveness. Both H-2(d) haplotype and non-H-2 miH background of graft donor and tolerizing cell donor were important to third-party cardiac allograft acceptance. These results suggest that the functional alteration in alloreactivity induced by neonatal alloantigen exposure depends partly on method of assessment.     

Adoptive transfer of transplantation tolerance mediated by CD4+CD25+ and CD8+CD28- regulatory T cells induced by anti-donor-specific T-cell vaccination

Previous studies have shown that vaccinating rodents with anti-donor-specific T cells significantly prolonged allograft survival; however, the putative mechanism of the tolerance remains unclear. In this study, we used the model of heterotopic heart transplantation between the C57BL/6 donor mice and BALB/c recipient mice vaccinated with anti-donor (C57BL/6) or anti-third party (C3H)-specific T cells to determine whether T cells prolong survival of mouse heart allografts and which cells were involved in induction of allograft tolerance. We observed that the mean survival time (MST) of C57BL/6 heart grafts in BALB/c mice vaccinated with anti-C57BL/6 specific T cells (43.1 +/- 4.7 days) was prolonged from that in untreated BALB/c mice (9.5 +/- 1.1 days) or BALB/c mice receiving anti-C3H-specific T cells (10.4 +/- 1.9 days). These results suggested that alloantigen-specific T-cell vaccination significantly prolonged cardiac allograft survival. The CD4+CD25+ or CD8+CD28- T cells purified from splenocytes of BALB/c mice vaccinated with anti-donor-specific T cells proliferated markedly in response to irradiated anti-C57BL/6-specific T cells in vitro. Adoptive transfer of these CD4+CD25+ or CD8+CD28- T cells to na?ve syngenic mice significantly prolonged the survival of heart allografts. These data suggested that anti-donor-specific T-cell vaccination induced development of CD4+CD25+ or CD8+CD28- regulatory T cells, which in turn mediated allogeneic-specific tolerance.     

Prolonged survival of heart allografts from p53-deficient mice

BACKGROUND: Acute rejection of the heart allograft is the major cause of heart failure in the first month after transplantation. Most studies on the prevention of acute rejection have concentrated on immune suppression of the recipients, whereas little is known about the effects of genetically manipulated donor organs on heart allograft survival. Herein, we describe a mouse model of heart allografts donated by p53-/- mice that can prolong the survival time of the grafts. METHODS: Hearts of p53-/- or p53+/+ C57BL/6J mice were grafted to the neck carotid artery and jugular vein of BALB/c mice using a cuff technique. The graft survival was observed daily. The hearts were analyzed using several techniques, including histology, immunofluorescence, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL), and Western blot analysis. RESULTS: p53+/+ allografts ceased beating at 7.6+/-0.5 days, whereas p53-/- hearts were beating at 10.5+/-1.1 days after transplantation (P<0.01). Mean histological rejection scores were significantly lower in allografts donated by p53-deficient mice. Furthermore, apoptotic cells, determined by TUNEL and a reagent kit for detection of cardiac apoptosis, were of high numbers in the allograft sections from wild-type hearts but rare in p53-/- allografts (4.2+/-1.3 vs. 0.7+/-0.5/250x field). Immunofluorescence staining and Western blot analysis revealed that high levels of p53 and proapoptotic protein Bax were expressed in wild-type grafts but not p53-/- allografts. Interestingly, Bcl-2, an antiapoptotic protein, was abundant in cardiac allografts from p53-/- mice and almost undetectable in grafts from wild-type mice. CONCLUSIONS: Thus, p53 is involved in cardiac apoptosis induced by alloimmune reaction, and prolonged survival of heart allografts can be achieved when p53 is lacking.     

A comparison of gene expression in murine cardiac allografts and isografts by means DNA microarray analysis

BACKGROUND: Acute rejection of allografts remains a significant problem in clinical transplantation, and the fundamental mechanism underlying this rejection are as yet only poorly elucidated. Recently, DNA microarrays have come into use for the study of gene expression profiles, and we have taken advantage of this new technology to investigate acute rejection. We compared mRNA profiles in murine cardiac allografts with isografts using DNA microarrays with probe sets corresponding to more than 11,000 mice genes. METHODS: We screened for gene expression changes in murine cardiac allografts between fully incompatible mice strains (BALB/c H2d to C3H/He H2k) using a DNA microarray. The heart was heterotopically transplanted. Allografts (BALB/c to C3H/He) were removed on days 1, 3, and 5. As a control, isografts (C3H/He to C3H/He) harvested on days 1, 3, and 5 and native hearts of both strain mice (C3H/He and BALB/c) were obtained. RESULTS: On day 5, interferon-gamma (IFN-gamma) and many IFN-gamma-inducible genes were profoundly induced in the allograft relative to isograft. Monokine induced by IFN-gamma was most profoundly induced followed by inducibly expressed GTPase and Lmp-2. IFN-gamma was also profoundly induced. The induction was detectable from day 3. In contrast, genes regulated by other cytokines exhibited only modest changes. CONCLUSION: IFN-gamma-inducible genes are specifically up-regulated in murine cardiac allografts, suggesting that signaling mediated by IFN-gamma may play an important role in the late phase of acute rejection in vivo.     

A novel tool for B-cell tolerance research: characterization of mouse alloantibody development using a simple and reliable cellular ELISA technique

In animal-based transplantation research, the measurement of anti-donor antibodies in transplant recipients is limited by lack of an appropriate technique. We have developed a novel immunoassay capable of quantifying antibody bound to cell-surface major histo- compatability complex (MHC) and non-MHC antigens, using splenocytes from wild-type and MHC-deficient mice as antigen-bearing target cells. We utilized our "cellular ELISA" (CELISA) technique to study the development of tolerance versus immunity in the B-cell compartment in response to neonatal exposure to allogeneic fetal liver cells (FLC). This neonatal tolerance protocol typically induces permanent acceptance of donor-type and third-party cardiac allografts, but rejection of both donor-type and third-party skin grafts occurs. C3H/He (C3H; H-2(k)) mice were injected as neonates with BALB/c (BALB; H-2(d)) FLC and transplanted as adults with C57BL/6 (B6; H-2(b)) cardiac grafts. Despite long-term acceptance of third-party B6 cardiac grafts, serum contained increased anti-B6 IgG and IgM levels as measured by CELISA; IgM production was elevated by 2 weeks posttransplant and remained stable, while IgG production increased rapidly between 2 and 5 weeks posttransplant. In another experimental setting, CELISA assays were able to detect that neonatal injection of C3H mice with FLC from wild-type B6 mice or from MHC class II-deficient or class I/II-deficient (B6 background) mice (CI(+)CII(+), CI(+)CII(-), CI(-)CII(-), respectively) prevented sensitization to B6 antigens by subsequent skin transplants but did not induce graft acceptance, whereas FLC from MHC class I-deficient-only (CI(-)CII(+)) did not prevent B6 sensitization. The CELISA technique is a simple and sensitive means for quantifying alloantibodies in mice and will assist in further delineating the role of the B-cell compartment in neonatally induced cardiac allograft acceptance.     

Role of ve-cadherins, beta-1 integrins and endothelin-1 in endothelial dysfunction after cardiac transplantation

Introduction: Endothelial dysfunction is a major cause of chronic allograft vasculopathy in cardiac transplantation. The dysfunction of the endothelium in coronary vasculature can be due to the loss of endothelial contacts with the extracellular matrix, endothelial cell-to-cell contact. We investigated the expression of VE-cadherins, integrins and endothelin-1 in a heart transplant model in mice to determine if endothelial dysfunction is from the underexpression of these endothelial specific molecules. Methods: Nonvascularised neonatal heart allografts from C57 BL/6 (H-2b) were transplanted to ear pinna of Balb/c (H-2d) recipient mice. Isografts from C57 BL/6 (H-2b) mice were performed as control. Allo and isografts were harvested at P.O day 1,3,5,7 & immunostaining for endothelin-1, VE-cadherin and Beta-1 integrin was performed with monoclonal antibodies specific for each molecules. Results: In normal neonatal hearts, VE-cadherin and Beta-1 integrin were detected in endothelial cells only, while ET-1 was detected in both endothelial and myocardial cells. Following transplantation, VE-cadherin became undetectable until P.O day 7 in isografts and P.O.day 5 in allografts at which time it became detectable at low levels on endothelial cells. Beta-1 integrin showed a pattern similar to VE-cadherin, except that it became barely detectable in either iso or allografts after transplantation. ET-1 did not show any detectable changes in either allo or isografts after transplantation. Conclusion: VE-cadherin and Beta-1 integrin expression on endothelial cells is diminished after cardiac transplantation, while ET-1 expression continues after transplantation. These data provide molecular insights into the loss of vascular endothelial integrity in cardiac allografts.     

Islet allograft survival induced by costimulation blockade in NOD mice is controlled by allelic variants of Idd3

NOD mice develop type 1 autoimmune diabetes and exhibit genetically dominant resistance to transplantation tolerance induction. These two phenotypes are genetically separable. Costimulation blockade fails to prolong skin allograft survival in (NOD x C57BL/6)F1 mice and in NOD-related strains made diabetes-resistant by congenic introduction of protective major histocompatibility complex (MHC) or non-MHC Idd region genes. Here, we tested the hypothesis that the genetic basis for the resistance of NOD mice to skin allograft tolerance also applies to islet allografts. Surprisingly, costimulation blockade induced permanent islet allograft survival in (NOD x C57BL/6)F1 mice but not in NOD mice. After costimulation blockade, islet allograft survival was prolonged in diabetes-resistant NOD.B6 Idd3 mice and shortened in diabetes-free C57BL/6 mice congenic for the NOD Idd3 variant. Islet allograft tolerance could not be induced in diabetes-resistant NOD.B10 Idd5 and NOD.B10 Idd9 mice. The data demonstrate that 1) NOD mice resist islet allograft tolerance induction; 2) unlike skin allografts, resistance to islet allograft tolerance is a genetically recessive trait; 3) an Idd3 region gene(s) is an important determinant of islet allograft tolerance induction; and 4) there may be overlap in the mechanism by which the Idd3 resistance locus improves self-tolerance and the induction of allotolerance.     

Seven Japanese Herbals Prolonged Cardiac Allograft Survival

Japanese herbal medicines have long been used as alternative therapy because of their immunomodulatory effects. In recent years, use herbal medicines is rapidly increasing worldwide. In this study, we investigated the effect of 17 components of traditional Japanese herbal medicines on alloimmune responses in a murine model of cardiac allograft transplantation. Fully vascularized heterotopic hearts from C57BL/6 donors were transplanted into CBA mice by using microsurgical techniques. Artemisiae capillaris herba (Inchinko) was given to CBA recipients at a dosage of 1 g/kg/day from the day of transplantation until 7 days afterward. The other 16 components were given at a dosage of 2 g/kg/day for the same time period. Naïve CBA mice rejected C57BL/6 cardiac grafts acutely (median survival time [MST] of 7 days). CBA transplant recipients given 2 g/kg/day of Glycyrrhizae radix (Kanzou), Poria sclerotium (Bukuryo), Pinellia tuber (Hange), Cnidii rhizome (Senkyu), Paeoniae radix (Shakuyaku), and Scutellariae radix (Ogon) had prolonged C57BL/6 allograft survival significantly (MSTs were 18, 18, 17, 14, 12, and 12 days, respectively). Moreover, CBA transplant recipients given 1g/kg/day of Artemisiae capillaris herba had prolonged C57BL/6 allograft survival (MST >100 days); however, none of other 10 components prolonged allograft survival. In conclusion, administration of 7 components of traditional Japanese herbal medicines might induce prolongation of fully major histocompatibility complex–mismatched cardiac allografts.     

Non-Complement- and Complement-Activating Antibodies Synergize to Cause Rejection of Cardiac Allografts

Alloantibodies (AlloAbs) are a clinically significant component of the immune response to organ transplants. In our experimental model, B10.A (H-2a) cardiac transplants survived significantly longer in C57BL/6 (H-2b) immunoglobulin knock-out (IgKO) recipients than in their wild-type (WT) counterparts. Passive transfer of a single 50-200-microg dose of complement-activating IgG2b AlloAbs to IgKO recipients reconstituted acute rejection of cardiac allografts. Although passive transfer of a subthreshold dose of 25 microg of IgG2b or a single 100-200-microg dose of non-complement-activating IgG1 AlloAbs did not restore acute rejection to IgKO recipients, a combination of these AlloAbs did cause acute graft rejection. Histologically, rejection was accompanied by augmented release of von Willebrand factor from endothelial cells. IgG1 AlloAbs did not activate complement on their own and did not augment complement activation by IgG2b AlloAbs. However, IgG1 AlloAbs stimulated cultured mouse endothelial cells to produce monocyte chemotactic protein 1 (MCP-1) and neutrophil chemoattractant growth-related oncogene alpha (KC). TNF-alpha augmented IgG1 induced secretion of MCP-1 and KC. These findings indicate that non-complement-activating AlloAbs can augment injury to allografts by complement-activating AlloAbs. Non-complement-activating AlloAbs stimulate endothelial cells to produce chemokines and this effect is augmented in the milieu of proinflammatory cytokines.