Intestinal graft versus native liver cytokine expression in a rat model of intestinal transplantation: effect of donor-specific cell augmentation

INTRODUCTION: Immunomodulation by portal vein delivery of donor antigen reduces intestinal graft rejection. We investigated the impact of portal venous donor-specific cell augmentation (blood versus bone marrow) on cytokine expression in intestinal grafts versus native livers. METHODS: Ten groups of intestinal transplants (brown Norway male to Lewis female rats) varied by (1). the type of donor-specific cell augmentation and (2). the use and dose of tacrolimus-based immunosuppression. Tissue samples for histologic analysis and cytokine mRNA analysis were obtained at designated time points. RESULTS: Without immunosuppression, no type of cell augmentation reduced the rate of rejection. With immunosuppression, outcome was significantly better after portal donor-specific blood transfusion (versus bone marrow infusion). Irrespective of the type of cell augmentation, severe rejection caused strong intragraft expression of IL-1alpha, IL-1beta, IFN-gamma, and TNF-alpha; liver expression mainly involved TNF-alpha. Of note, nonimmunosuppressed, cell-augmented rats showed hardly any differences in cytokine expression in their grafts versus significant increases in their native livers. With immunosuppression, bone marrow infusion (versus blood transfusion) increased intragraft cytokine expression of IL-1alpha, IL-1beta, IFN-gamma, as well as TNF-alpha, and liver expression of IL-1beta. CONCLUSIONS: (1). Rejection and donor-specific cell augmentation independently caused differences in intragraft versus native liver cytokine expression after intestinal transplants. (2). Portal donor-specific blood transfusion (versus bone marrow infusion) lowered the incidence of rejection and diminished intragraft cytokine up-regulation. (3). In our study, TNF-alpha appeared to be the cytokine most strongly associated with rejection.     

Cytokine and T cell receptor gene expression at the site of allograft rejection.

Intragraft cytokine and T cell receptor gene expression was analyzed in rejecting renal allografts by polymerase chain reaction (PCR). Message for IL-1 beta, IL-6, and TNF-alpha was detected in nephrectomy tissue with pathological evidence of acute or chronic rejection. Similarly, mRNA for both IL-6 and TNF-alpha was present in renal biopsies from acute rejecting kidneys. IL-2R, IL-4, and IL-5 mRNA was present in both rejecting and rejected kidney allografts, indicating that these cytokines may play a role in ongoing renal allograft rejection. Conversely, IL-2, IL-7, and IFN-gamma message was detected infrequently. In order to address the diversity of T cells in rejecting kidneys, we have analyzed the clonality of the TcR present within the allograft tissue. Rearranged TcR genes were identified in all allografts examined (n = 16) indicating the presence of T cells bearing the alpha/beta TcR. We have determined that there is a heterogeneous infiltration of T cells in the rejected allograft with TcR representing x = 7.47 +/- 2.4 families rearranged in samples obtained from nephrectomies, whereas x = 5.33 +/- 0.58 families were detected in samples obtained from biopsy tissue. These data indicate that (1) cytokines are produced locally which may contribute to graft cell destruction, (2) the heterogeneity of intragraft T cells during kidney allograft rejection may exist because nonspecific lymphocytes have been recruited to the site by locally produced cytokines or because T cells are responding to multiple epitopes or multiple donor antigens. Detection of intragraft cytokines and TcR may prove useful in elucidating the mechanism of rejection and therefore lead to improved immunosuppression.     

Early accumulation of interferon-gamma in grafts tolerized by donor-specific blood transfusion: friend or enemy?

BACKGROUND: We previously documented an early (day-2) interferon (IFN)-gamma accumulation in cardiac allografts of rats made tolerant by donor-specific blood transfusion (DSBT) but not in rejecting controls. This contrasted with the IFN-gamma peak seen later (day 5) in rejecting but not in tolerant rats. METHODS: To further examine the role of early intragraft IFN-gamma in DSBT-induced tolerance, we studied whether IFN-gamma up-regulation correlates with the magnitude of the DSBT effect and how IFN-gamma is influenced by interventions abrogating tolerance. RESULTS: The protective effect of DSBT depended upon the timing of administration: day-12 DSBT induced indefinite graft survival; day-6 DSBT gave a moderate, and day-0 DSBT, no graft prolongation. IFN-gamma up-regulation correlated with the DSBT effect: it was maximal after day-12 DSBT, intermediate after day-6 DSBT, and absent after day-0 DSBT. Tolerant splenocytes transferred tolerance into naive rats in a donor-specific manner, indicating that alloantigen-specific regulatory cells operate. Thymectomy prevented regulatory cells development, caused further amplification of intragraft IFN-gamma, and led to rejection, although graft survival was still prolonged. CONCLUSIONS: Day 2 intragraft IFN-gamma correlates with the DSBT protective effect. Thymectomy abrogates DSBT-induced tolerance, prevents regulatory cell development, and paradoxically causes further accumulation of intragraft IFN-gamma. These data indicate that DSBT has a stimulatory and a (thymus-dependent) inhibitory effect on early intragraft IFN-gamma. Intragraft IFN-gamma is beneficial, providing it occurs early and remains moderate. The role of intragraft IFN-gamma in tolerance and rejection depends upon the timing and the degree of production and perhaps the type of IFN-gamma producing cells (regulatory or effector).     

Cytokine regulation of ICAM-1 expression on human renal tubular epithelial cells in vitro.

Regulation of the intercellular adhesion molecule-1 (ICAM-1) expression on human renal tubular epithelial cells in culture (hKEC-1) was investigated. A large proportion of hKEC-1 cells from the primary cultures expressed the ICAM-1 antigen. Supernatants from mixed lymphocyte reaction (MLR) of both specific and third-party combinations augmented the expression of the ICAM-1 antigen, in a dose-dependent manner. A kinetic study revealed maximal augmentation by MLR supernatant on the first day, with a gradual decrease thereafter. Among several recombinant human cytokines tested, i.e., interferon-gamma, tumor necrosis factor-alpha, interleukin 1 alpha and beta, and IL-4, IFN-gamma, TNF-alpha, and IL-1 alpha/beta were shown to augment the expression of ICAM-1. MLR supernatants and IFN-gamma were more effective in augmenting ICAM expression than TNF-alpha and IL-1 alpha/beta. IFN-gamma upregulated ICAM-1 expression in a dose-dependent manner, and maximal augmentation was achieved on the first day. The MLR supernatants were shown to contain IFN-gamma and TNF-alpha, and the activity of the MLR supernatant was partially inhibited by neutralizing antibody against IFN-gamma. These data suggest that cytokines, especially IFN-gamma, TNF-alpha, and IL-1 alpha/beta, released by T cells and antigen-presenting cells upon recognition of alloantigens upregulate ICAM-1 expression on renal tubular epithelial cells. This may result in an increase in the attachment of graft-infiltrating T cells to the renal tubular cells, by the ICAM-1-LFA-1 interaction.     

DNA microarray-based gene expression profiles of cytomegalovirus infection and acute rejection in liver transplants

An association between cytomegalovirus (CMV) infection and alloresponse has been suggested. CMV increases inflammation and adhesion molecule expression in graft, and induces cytokines and growth factors, linked with transplant vasculopathy and chronic rejection. We have investigated the gene expression of various inflammatory factors in the CMV-associated immune response and compared this with the immune response of acute rejection in liver transplants by using DNA microarray technology. Gene expression was studied at mRNA level in biopsies from liver transplant patients experiencing CMV infection or acute rejection. RNA extracted from liver grafts after reperfusion was used as control material. Among the strongly upregulated genes in the specimens obtained from liver transplants during CMV infection were IFN-gamma, caspases 1 and 3, granzymes A and B, TGF-beta receptors II and III, IL-10 receptor alpha, VCAM-1, TNF receptor, IL-4, TNF-alpha, IL-10, IL-2 receptor beta, IL-1beta, PDGF-receptor beta, vascular adhesion protein-1, TGF-beta2, and ICAM-1. In biopsies with acute liver allograft rejection, the most significantly upregulated genes were MHC class II, IFN-gamma, caspases 1 and 3, IL-2R beta and gamma, granzymes A and B, VLA-4, L-selectin, E-selectin, VCAM-1, and IL-1beta. Upregulated genes common for CMV and alloresponse were granzyme A and B, E-selection, IFN-gamma, VCAM-1, VLA-4, TNF, caspases 1, 3, and 8, and PDGF. Microarray analysis defined different entities in the immune responses of CMV infection and acute rejection. The differences and similarities of the gene expression profiles related to those in CMV infection and rejection may help to understand the intragraft immunologic events.     

Inducible nitric oxide synthase inhibitors prolonged the survival of skin xenografts through selective down-regulation of pro-inflammatory cytokine and CC-chemokine expressions

To elucidate the possible immunoregulatory role of nitric oxide (NO) in cellular xenograft rejection we performed rat-to-mouse skin xenotransplantation. The rat skin engrafted mice were treated with the inducible NO synthase (iNOS) inhibitors, aminoguanidine (AMG, 200 mg/kg) and NG-nitro-L-arginine methyl ester (L-NAME, 60 mg/kg) every other day until rejection. Skin xenograft survival was monitored and immune cell infiltration and intragraft cytokine and chemokine mRNA expressions were analyzed 7 days after grafting. Compared with the control mice, the AMG- and L-NAME treated mice showed delayed xenograft rejection by approximately 3 days (8.9 +/- 0.7 days vs. 11.7 +/- 1.2 and 12.0 +/- 0.9 days, respectively). Infiltrations of CD11b+, MOMA-2+ cells and neutrophils were significantly reduced in both AMG- and L-NAME treated graft but CD4+ and CD8+ cells were not. The expression of cytokines such as IL-1beta, IL-2, IL-6, IL-12 and IFN-gamma in AMG- and L-NAME treated grafts were significantly decreased (P<0.01), whereas IL-10, TNF-alpha and TGF-beta1 were unchanged or enhanced. Additionally, the expressions of CC-chemokines, such as RANTES and MIP-1alpha, were significantly reduced (P<0.01) whereas the expressions of CXC-chemokines, such as IP-10 and MIG, were unchanged. These results imply that prolonged rat-to-mouse skin xenograft survival by iNOS inhibitors may be due to the selective inhibition of pro-inflammatory cytokines and chemokines and suggest the possible regulatory role of NO in cytokine and chemokine expressions during xenotransplant rejection.     

细胞因子对异种脱细胞真皮基质免疫调节作用的临床研究

目的　探讨烧伤患者接受异种 /异体脱细胞真皮基质 (xeno /allo ADM )移植后全身和局部多种细胞因子与移植物近期转归的关系。　方法　在大面积烧伤患者的四肢切痂创面上 ,移植xeno ADM (12例 ,19块 )或allo ADM(15例 ,18块 ) ,其上覆盖自体超薄断层皮片 ,并以 6例单纯移植自体中厚断层皮片 (auto TTS)的烧伤患者为对照。移植物成活后 4～ 8周 ,收集局部组织标本、血清和xeno ADM排斥后的创面渗出液 ,采用免疫组织化学染色与酶联免疫吸附法 (ELISA) ,检测白细胞介素 (IL) 1β、IL 4、IL 6、肿瘤坏死因子α(TNF α)和γ型干扰素 (IFN γ)的含量。　结果　免疫组织化学染色结果显示 ,移植物内IL 1β、IL 4、IL 6、TNF α和IFN γ的阳性细胞密度或着色强度相比较 ,xeno ADM >allo ADM >auto TTS (P <0 .0 5 )。ELISA检测结果显示 ,xeno ADM被排斥后创面渗出液中IL 4、IL 6、TNF α和IFN γ水平明显高于自体血清 ,但其血清IL 4与IFN γ水平分别低于和高于未排斥时。xeno ADM移植后血清中IL 4、IFN γ水平明显高于allo ADM和auto TTS(P <0 .0 5～ 0 .0 1)。　结论　xeno ADM移植后可在局部检测到高水平的IL 1β、IL 4、IL 6、IFN γ ,可能与细胞杀伤和细胞因子介导的免疫放大作用有关。这些细胞因子的动态变     

Portal donor-specific blood transfusion and mycophenolate mofetil allow steroid avoidance and tacrolimus dose reduction with sustained levels of chimerism in a pig model of intestinal transplantation

BACKGROUND: In a pig model of intestinal transplantation, we previously showed that hepatic conditioning through portal donor-specific blood transfusion (pDSBT), high-dose tacrolimus (TAC), and steroids prevented rejection and increased survival Our current study tests a protocol of pDSBT, short-term mycophenolate mofetil (MMF), and low-dose TAC to eliminate the use of steroids, reduce TAC dosage, and increase the level of chimerism in the peripheral blood. MATERIALS AND METHODS: Four groups of outbred, mixed lymphocyte culture (MLC)-reactive pigs underwent bowel transplants and pDSBT. Immunosuppression (group 1, high-dose TAC and steroids; group 2, low-dose TAC and MMF; group 3, low-dose TAC, MMF, and aminoguanidine; group 4, low-dose TAC, MMF, and arginine) was discontinued after 28 days. RNA was extracted from intestinal graft and native liver biopsies for cytokine measurements. Chimerism levels were determined using a Q-PCR analysis. RESULTS: Pig survival and death rates due to rejection did not significantly differ between the four groups. Chimerism levels determined by Q-PCR analysis were not different until day 28. After discontinuation of immunosuppression, we noted a trend (P = 0.15) toward higher mean chimerism levels on day 60 for groups 2, 3, and 4 (9%) vs. group 1 (0.5%). Tissue cytokine and serum nitrate levels did not significantly differ between the four groups. Attempts to modify nitric oxide synthase activity offered no added benefit. CONCLUSIONS: The combination of pDSBT, MMF, and low-dose TAC (vs. high-dose TAC and steroids) allowed sustained levels of mixed chimerism to develop after discontinuation of immunosuppression.     

猪节段性小肠移植免疫耐受的研究

目的 探讨免疫耐受在移植领域的应用前景,建立猪节段性小肠移植免疫耐受模型。方法 １２只幼猪为受者,随机分为实验组及对照组,另选体重相近的异性猪为供者,实验组用供者骨髓细胞预处理,２周后行节段性小肠移植,术后不使用免疫抑制剂;对照组不作预处理。结果 实验组部分移植小肠获长期存活,Ｔ细胞亚群明显下降,外周血白细胞介素（ＩＬ－６均无明显变化,外周血白细胞发现嵌合现象,病理表现为轻度急性排异和慢性排异或无     

Alpha beta TCR+ T cells play a nonredundant role in the rejection of heart allografts in mice.

BACKGROUND: Although the transplantation of solid organs and cellular grafts is a clinical routine, the morbidity and mortality associated with immunosuppression is significant. This could be avoided by the induction of donor-specific tolerance. To develop targeted antirejection strategies and regimens to induce donor-specific tolerance, cell populations in the recipient-mediating rejection of solid organ and cellular grafts must be defined. In this study we examined the role of alpha beta-TCR+ cells in the rejection of allogeneic heart grafts, by use of knockout (KO) mice deficient in the production of alpha beta-TCR+ T cells. METHODS: C57BL/6-TcrbtmlMom (alpha beta-KO) and C57BL6/J (B6) recipient mice were transplanted with B10.BR/SgSnJ (B10.BR) or BALB/c heart allografts. Animals also received bone marrow from normal B10.BR donors, followed by donor-specific or third-party heart transplants. RESULTS: Naive B6 control mice rejected B10.BR and BALB/c grafts within 16 days. In striking contrast, B10.BR and BALB/c heart allografts were indefinitely accepted in unmanipulated alpha beta-KO mice. The immune responsiveness was restored after bone marrow transplantation from normal donors. After bone marrow transplantation major histocompatibility-disparate BALB/c third-party heart grafts were rejected, whereas donor-specific grafts were still accepted. CONCLUSIONS: alpha beta-TCR+ T cells play a nonredundant role in the rejection of heart allografts in mice. Bone marrow chimerism is associated with donor-specific transplantation tolerance.     

Long-term results of a controlled prospective study with transfusion of donor-specific bone marrow in 57 cadaveric renal allograft recipients

Experimental studies have shown that antilymphocyte globulin combined with transfusion of donor-specific bone marrow cells can induce partial tolerance to allograft tissue. We have adapted these protocols to clinical use and present the results of 57 cadaveric renal allograft recipients who received Minnesota ALG followed by the transfusion of cryopreserved donor-specific bone marrow. A group of 54 patients received the contralateral kidney and similar immunosuppression without the marrow transfusion and serve as controls. Both groups received quadruple immunosuppression with MALG, cyclosporin, azathioprine, and prednisone. In the bone marrow group, after a 10-14 day induction course of ALG, cryopreserved marrow was transfused on the seventh day after the last dose of ALG. The median follow-up in both groups is 16 months, (range 2.5-33 months). Six grafts have been lost in the bone marrow group, (three rejections, 2 deaths [Cr 2.0, 2.3], 1 recurrent disease). In the control group 16 grafts have been lost (13 rejections, 3 deaths [Cr 1.7, 2.5, 3.0]). Five patients in the control group have biopsy-proved chronic rejection compared to one in the bone marrow group. 17 patients in the bone marrow group have been tapered off the prednisone, and three of these patients have had mild late rejection episodes without graft loss. The two groups were compared for differences in the number of rejection episodes, estimated renal plasma flow, glomerular filtration rate, and urine protein. No differences were found. The allograft survival of the bone marrow group was significantly greater (P less than .01) than the control group. The graft survival rates for the bone marrow group at 12 and 18 months were 90% (confidence limits [CL] 85-94) and 85% (CL 78-90), respectively. In the the control group the 12 and 18 month allograft survival rates were 71% (CL 63-78) and 67% (CL 58-74), respectively. The survival in the control group was similar to our overall transplant experience with quadruple therapy. Mixed lymphocyte culture analysis shows a trend to diminished donor-specific responsiveness in the bone marrow group. The use of cryopreserved donor-specific bone marrow is associated with improved allograft survival in cadaveric kidney allograft recipients. However, a more effective induction protocol is needed to reduce the overall number of rejection episodes.     

Selectin inhibitor bimosiamose prolongs survival of kidney allografts by reduction in intragraft production of cytokines and chemokines

Binding of the P-, L-, and E-selectins to sialyl Lewis(x) (sLe(x)) retards circulating leukocytes, thereby facilitating their attachment to the blood vessels of allografts. Whether the selectin inhibitor bimosiamose (BIMO; C(46)H(54)O(16) . 0.25 H(2)O [867.4 molecular weight]) inhibits the rejection process of kidney allografts in a rat model was examined. Rat recipients acutely rejected kidney allografts at a mean survival time of 8.8 +/- 0.75 d. An intravenous 7-d infusion by osmotic pump of 2.5, 5, 10, or 20 mg/kg BIMO extended kidney allograft survival to 11.5 +/- 2.2 d (P < 0.03), 25.4 +/- 11.4 d (P < 0.006), 37.4 +/- 13.6 d (P < 0.001), and 39.8 +/- 34.5 d (P < 0.01), respectively. Combination of BIMO with cyclosporine produced synergistic interactions, as documented by the combination index (CI) values of 0.34 to 0.43 (CI <1 is synergistic; CI = 1 is additive; and CI >1 is antagonistic). Similarly, BIMO interacted synergistically with sirolimus (CI = 0.64) and FTY720 (CI = 0.22). While the mechanism of immunosuppression was being analyzed, decreased infiltration of CD4(+), CD8(+), and macrophages on day 7 after grafting was observed. Multiple cytokines were also expressed, including IL-1alpha, IL-1beta, IL-2, IL-4, IL-6, IL-10, IL-12, IL-18, TNF-alpha, and IFN-gamma in kidney allografts on days 3, 5, and 7 after grafting, as measured by a ribonuclease protection assay. Furthermore, at similar time points, BIMO treatment reduced intragraft expression of P-selectin glycoprotein ligand-1, CX(3)CL1, CCL19, CCL20, and CCL2. Thus, BIMO blocks allograft rejection by reduction of intragraft expression of cytokines and chemokines.     

Chronic rejection: insights from a novel immunosuppressive-free model of kidney transplantation

The use of immunosuppressive drugs in models of chronic rejection may limit their usefulness for mechanistic studies. We have developed a new minor histocompatibility-mismatched rat kidney transplant model without the need for immunosuppression. Kidneys from LEW (RT1(l)) donors were transplanted to congenic WF.1L (RT1(l)) recipients and compared with the reversed strain combination and isogenic controls. Urinary protein excretion was measured serially in all recipients; kidneys were harvested 90, 120, and 180 d after transplantation for morphologic analysis and cytokine gene expression. In vitro lymphocytic reactivity and cytokine analysis of mixed lymphocyte reaction (MLR) culture supernatants by ELISA was also carried out. LEW into WF.1L kidney grafts developed proteinuria starting 120 d after transplantation and were associated with morphologic changes of focal segmental glomerulosclerosis together with interstitial cell infiltrates, upregulated gene expression of IL-1beta, IL-2, and TNF-alpha/-beta, as well as IL-2, IFN-gamma, and TNF-alpha production by lymphocytes in MLR culture supernatants. WF.1L kidneys transplanted into LEW recipients did not develop chronic rejection and had upregulation of Th2 cytokines, both within the allograft and in MLR supernatant of recipient lymphocytes cultured with WF.1L cells. Furthermore, these lymphocytes produced both Th1 and Th2 cytokines when cultured with WF cells, unlike lymphocytes from the LEW isografts, which produced Th1 cytokines when challenged with WF cells. These studies show that indirect allorecognition can cause strain-dependent chronic rejection associated with Th1-like cytokine production, whereas production of Th2 cytokines is associated with protection from the development of chronic rejection.