The activity of inducible nitric oxide synthase in rejected skin xenografts is selectively inhibited by a factor produced by grafted cells

BACKGROUND: Production of nitric oxide (NO) by graft infiltrating macrophages has been suggested as an important effector mechanism of allograft rejection. Expression of the gene for the inducible NO synthase (iNOS) and the production of NO in rejected graft has been demonstrated in various models of allotransplantation. However, whether NO plays a role in rejection of skin xenografts has not been documented. METHODS: Explants of rejected skin allografts or xenografts (rat to mouse) were cultivated in vitro and the production of NO, interleukin (IL)-2, IL-4, IL-10 and interferon-gamma (IFN-gamma) by graft infiltrating cells was determined by the Griess reaction or ELISA. Effects of supernatants from cultures of xenograft explants on the expression of gene for iNOS, accumulation of iNOS protein and NO production were determined by RT-PCR or Western blots. Molecular mass of the factor with the suppressive activity was characterized by filtration on chromatography Sephacryl S-200 Superfine column. In addition, the effects of 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (AMT), a selective iNOS inhibitor, on survival of skin xenografts were tested. RESULTS: While explants of rejected mouse skin allografts produced substantial amounts of NO, undetectable or only very low levels of NO were found in supernatants from cultured rat skin xenografts. Cocultivation of bacterial lipopolysaccharide (LPS)-stimulated mouse macrophages which produce high quantities of NO, with pieces of rejected xenografts, but not of syngeneic grafts, allografts or normal rat skin, completely inhibited production of NO. Production of IL-6 and IL-10 by LPS-stimulated macrophages was not inhibited under the same conditions. The inhibition of NO production was mediated by a factor which was produced by rejected rat xenograft and which was eluted from chromatography Sephacryl S-200 Superfine column in a fraction representing a molecular mass of 67 kDa. The factor did not inhibit the expression of the gene for iNOS, reduce the level of iNOS protein in stimulated macrophages, or function as a scavenger of NO. Rather, the factor inhibited the function of iNOS. The finding that NO does not play an important role during rejection of skin xenografts is supported by the observation that treatment of graft recipients with AMT, a specific iNOS inhibitor, did not enhance xenograft survival, while the same treatment resulted in prolongation of survival of skin allografts. CONCLUSION: The results thus demonstrate that a 67-kDa molecule produced by rejected rat skin xenografts selectively inhibits iNOS activity in graft infiltrating macrophages. We suggest that NO does not play a significant role in rejection of skin xenografts as it does in the case of allograft rejection.     

Alloantigen-induced, T-cell-dependent production of nitric oxide by macrophages infiltrating skin allografts in mice

The immunological rejection reaction occurring after organ or tissue transplantation is characterized by a strong infiltration of the graft by T cells and macrophages. Since the rejection reaction is highly specific, we tested the role of T cells in the activation of macrophages and in the induction of nitric oxide (NO) production during graft rejection. The rejection of both MHC and non-MHC antigen-disparate skin allografts was associated with a significantly increased production of NO in the graft. The kinetics of NO production after transplantation correlated with the rejection reaction and with the fate of the allograft. A significant reduction in NO production was found in immunologically hyporeactive mice treated with cyclosporine, and no specific production of NO was found in tolerated skin allografts from neonatally tolerant mice. The production of NO was completely suppressed in graft explants from mice with depleted CD4(+) cells, but remained at a normal level in skin allografts from mice treated with anti-CD8 monoclonal antibody. The treatment of recipients of fully allogeneic skin grafts with 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (AMT), a specific inhibitor of the inducible NO synthase, resulted in a significant prolongation of graft survival. The results thus show CD4(+) T-cell-dependent, alloantigen-induced production of NO by graft-infiltrating macrophages and the role of NO in the rejection reaction. We suggest that this pathway may represent one of the local effector mechanisms of graft rejection.     

Corneal rat-to-mouse xenotransplantation and the effects of anti-CD4 or anti-CD8 treatment on cytokine and nitric oxide production

Corneal xenotransplantation may be an alternative approach to overcome shortage of allografts for clinical transplantation. Orthotopic corneal rat-to-mouse xenotransplantation and syngeneic transplantation was performed and the effects of anti-CD4 and anti-CD8 treatments on corneal xenograft survival and production of cytokines, interleukin (IL)-2, IL-4, IL-10, gamma-interferon (IFN-gamma) and nitric oxide (NO) were evaluated. RT-PCR was used to determine the expression of genes for cytokines and inducible nitric oxide synthase (iNOS) in the grafts. The presence of iNOS protein in grafts was detected by immunofluorescent staining. We found that corneal xenotransplantation was associated with a strong upregulation of genes for both Th1 and Th2 cytokines and with NO production in the graft. Treatment of xenograft recipients with mAb anti-CD4, but not anti-CD8, resulted in a profound inhibition of IL-2, IL-4 and IL-10 production, and in a significant prolongation of corneal xenograft survival. The results show that upregulation of Th2 cytokines after corneal xenotransplantation does not correlate with xenograft rejection. Rather, corneal graft rejection is associated with the expression of genes for IFN-gamma and iNOS and with NO production.     

Acute Xenograft Rejection Mediated by Antibodies Produced Independently of TH1/TH2 Cytokine Profiles

There is substantial support for the hypothesis that T(H)1 cytokine responses are critical for the normal elaboration of allograft rejection. Recent studies by Wang et al. (1) underscore the importance of T(H)2 responses in xenograft rejection and revealed that T(H)1 cytokines, IL-12 and interferon-gamma (IFN-gamma), can negatively regulate the development of humoral responses necessary for xenograft rejection. Their exceptional studies prompted us to test whether the ability of allografts to elicit cellular rejection and xenografts to induce humoral rejection also result from the differential ability to induce T(H)1 and T(H)2 responses. We compared the kinetics of antibody and cytokine (IFN-gamma and IL-4) production in C57BL/6 mice following allograft transplantation with BALB/c hearts and in C57BL/6 and BALB/c mice following transplantation with Lewis rat hearts. We also compared the ability of BALB/c mice, deficient in the ability to produce IL-4 or IFN-gamma, to reject xenografts and produce xenoantibodies. We observed that T(H)1/T(H)2 cytokine production minimally affected the kinetics of graft rejection but regulated the magnitude of IgG subclass production. Anti-graft IgM played a critical role in initiating acute antibody-mediated xenograft rejection, and the production antigraft IgM was unaffected by IL-4 or IFN-gamma deficiency. In contrast to the report by Wang et al. (1), we conclude that antibody-mediated xenograft rejection in the concordant Lewis rat heart-to-C57BL/6 mouse xenotransplantation model is dependent on anti-IgM production but independent of T(H) cytokine profiles.     

Non-depleting anti-CD4, but not anti-CD8, antibody induces long-term survival of xenogeneic and allogeneic hearts in alpha1,3-galactosyltransferase knockout (GT-Ko) mice

The anti-galactose-alpha1,3-galactose (Gal) antibody (Ab) response following pig-to-human transplantation is vigorous and largely resistant to currently available immunosuppression. The recent generation of GT-Ko mice provides a unique opportunity to study the immunological basis of xenograft-elicited anti-Gal Ab response in vivo, and to test the efficacy of various strategies at controlling this Ab response [1]. In this study, we compared the ability of non-depleting anti-CD4 and anti-CD8 to control rejection and antibody production in GT-Ko mice following xenograft and allograft transplantation. Hearts from baby Lewis rat or C3H mice were transplanted heterotopically into GT-Ko. Non-depleting anti-CD4 (YTS177) and anti-CD8 (YTS105) Abs were used at 1 mg/mouse, and given as four doses daily from day -2 to 1 then q.o.d. till day 21. Xenograft rejection occurred at 3 to 5 days post-transplantation in untreated GT-Ko recipients, and was histologically characterized as vascular rejection. Anti-CD4, but not anti-CD8, Ab treatment prolonged xenograft survival to 68 to 74 days and inhibited anti-Gal Ab as well as xeno-Ab production. In four of the five hearts from anti-CD4 mAbs-treated GT-Ko mice, we observed classic signs of chronic rejection, namely, thickened intima in the lumen of vessels, significant IgM deposition, fibrosis and modest mononuclear cell infiltrate of Mac-1+ macrophages and scattered T cells (CD8>CD4). Xenograft rejection in untreated, as well as anti-CD4- and anti-CD8-treated, recipients was associated with increased intragraft IL-6, IFN-gamma and IL-10 mRNA. C3H allografts were rejected in 7 to 9 days by untreated GT-Ko mice and were histologically characterized as cellular rejection. Treatment with anti-CD4 and anti-CD8 mAb resulted in graft survivals of >94.8 and 11.8 days, respectively. Anti-CD4 mAb treatment resulted in a transient inhibition of alloreactive and anti-Gal Ab production. The presence of circulating alloreactive and anti-Gal Abs at >50 days post-transplant was associated with significant IgM and IgG deposition in the graft. Yet, in the anti-CD4 mAb-treated group, the allografts showed no signs of rejection at the time of sacrifice (>100 days post-transplantation). All rejected allografts had elevated levels of intragraft IL-6, IFN-gamma and IL-10 mRNA, while the long-surviving anti-CD4-treated allografts had reduced mRNA levels of these cytokines. Collectively, our studies suggest that the elicited xeno-antibody production and anti-Gal Ab production in GT-Ko mice are CD4+ T-cell dependent. The majority of xenografts succumbed to chronic rejection, while allografts survived with minimal histological change, despite elevated levels of circulating alloAbs. Thus, immunosuppression with anti-CD4 mAb therapy induces long-term survival of allografts more effectively than to xenografts.     

冻干同种与异种骨移植免疫反应的比较研究

目的: 通过比较冻干同种与异种骨移植免疫反应, 探讨异种骨移植排斥机制。方法: 取C57BL/6小鼠 10只和新西兰兔 1只, 分别为同种和异种骨供体, 通过处理制成冻干骨。取 40只BALB/c小鼠为骨移植受体, 随机分为A、B两组 (每组 20只), 分别在其股后肌袋植入冻干同种和冻干异种骨。分别于术后 1、2、4、6周分批取材检测。通过观察术后受者的淋巴细胞刺激指数、淋巴细胞亚群分析、细胞因子产生及组织学表现, 比较冻干同种与异种骨移植免疫反应。结果: 冻干异种骨移植组的细胞刺激反应在各不同时期均比同种组强 (P<0. 05 ), 而且其CD4 和CD8 T细胞亚群及IL 2分泌均高于同种组 (P<0. 05)。组织学检查也表明其细胞浸润多、成骨少。结论: 冻干同种与异种骨移植排斥机制基本相同, 二者均以Th1反应为主, 但异种骨移植排斥反应较强。     

Late CD8+ T cell-dependent xenoantibody production in innate tolerant nude rats after hamster islet grafting but not after hamster heart grafting

BACKGROUND: Xenograft rejection can be provoked by both the innate and adaptive immune compartments and close reciprocal interactions exist between these two systems. We investigated the interdependent roles of T and B lymphocytes in vascularized (heart) and cellular (islet) xenograft rejection in a model with established xeno-nonreactivity of the innate immune system. METHODS: Specific innate xenotolerance was induced in nude rats bearing either a hamster heart or a hamster pancreatic islet graft by a tolerizing regimen consisting of donor antigen infusion, temporary natural killer cell depletion and a 4-week administration of leflunomide. One month after transplantation, syngeneic CD4 and CD8 T cells were adoptively transferred. RESULTS: Both vascular and cellular xenografts were rejected after CD4 T cell reconstitution, corresponding with production of high IgM and IgG xenoantibody titers. Deposition of xenoantibodies and complement was seen in the heart but not in the islet xenografts. After infusion of CD8 T cells, xenohearts underwent a delayed type of rejection without xenoantibody production and xenoislets were not rejected. In xenoislet recipients, CD8 dependent B cells were not tolerized, resulting in the production of IgG xenoantibodies belonging to Th2-dependent isotypes, known not to cause graft rejection, and deposited at the graft implantation site. CONCLUSIONS: We conclude that distinct mechanisms of immune activation underlie xenogeneic reactions against vascular and cellular grafts.     

Regulatory cell-mediated tolerance does not protect against chronic rejection

BACKGROUND: Regulatory cells prevent graft loss to acute rejection and induce tolerance, possibly by promoting Th2 deviation. Th2 cytokines stimulate B cells, which cause alloantibody-mediated chronic rejection. We searched to determine whether regulatory cell-mediated tolerance protects or not against chronic rejection. METHODS: Heart transplantation (Htx) was performed using RA (RT1P) and PVG (RT1c) rats as donor and recipients. Donor-specific blood transfusion (DSBT) was given on preTx day 12. Secondary grafts were implanted at day 100. Splenocytes were transferred from tolerant rats (and controls) into lightly irradiated (450 rad) naive PVG, which received RA Htx. Primary Htx were investigated for the development of vascular occlusion (VO), the production of Th1/Th2 intragraft cytokines, and for the nature of graft infiltrate as well as for endothelial deposition of immunoglobulin (Ig)G isotypes and complement (C3) binding. Results were compared with rejecting controls (no DSBT) and syngeneic Htx. RESULTS: RA Htx were rejected within 10 days (8, 9, 10x4). PreTx DSBT prolonged primary Htx survival indefinitely (>140 days) with acceptance of secondary donor-specific (but not third-party) grafts (P<0.001). Naive irradiated PVG rats given splenocytes from tolerant rats but not from controls accepted RA Htx, showing the existence of regulatory cells in allograft acceptors. Despite being tolerant, DSBT-treated rats displayed typical features of chronic rejection at day 90 (VO=77%; P<0.001 vs. VO=4% in syngeneic rats). An overt Th2 deviation, particularly intragraft production of interleukin (IL)-4, was observed at day 30. Simultaneously to this Th2 deviation, B cells emerged in the grafts and endothelial deposition of IgG1 (Th2 dependent) and C3 binding were observed. CONCLUSIONS: Regulatory cells that prevent graft loss to acute rejection in primary and secondary grafts do not protect against the development of chronic rejection.     

Chronic human skin graft rejection in severe combined immunodeficient mice engrafted with human PBL from an HLA-presensitized donor.

Mice with severe combined immunodeficiency (C.B-17 scid [SCID]) accepted xenografts of adult human peripheral blood leukocytes injected intraperitoneally as evidenced by production of human immunoglobulin (IgG and IgM), and circulation of human leukocytes in peripheral blood. SCID mice also accepted human split-thickness skin xenografts. Passenger leukocytes present in small numbers in such skin grafts could also recirculate in host peripheral blood and make detectable levels of human immunoglobulin. To test the immunocompetence of the transferred human PBL, SCID mice received a human skin xenograft from a second donor (HLA-mismatched with the PBL donor) either before (n = 6) or after (n = 23) xenografting of PBL. Skin was monitored daily for signs of rejection, and rejection was scored by histology 3-4 weeks after the second graft (PBL or skin) was placed. Of 19 SCID injected with PBL from an HLA presensitized patient (L.G.), 7/19 (37%) rejected a subsequent HLA-mismatched skin xenograft. Two of six SCID (33%) rejected a previously established skin xenograft when PBL were administered afterward. The rejection of the human skin was chronic, of relatively late onset (3-4 weeks), and was characterized grossly by contraction, glassy surface, and thickening. Histopathologic examination showed lymphocyte infiltration into the dermis with endothelial cell cuffing and destruction of capillaries, as well as lymphocyte tagging of the basal epidermis, hyperkeratosis, lymphocyte exocytosis and single epidermal cell necrosis. Immunostaining with monoclonal antibody to human CD2 or mouse CD3 revealed that human, but not mouse T lymphocytes were tagging the dermis/epidermis junction and infiltrating the epidermis of rejecting skin grafts. We conclude that a form of human skin graft rejection may be reproduced in an SCID mouse. The immune status of the transferred cells (sensitized vs. normal) and the lymphocytes ability to recirculate in SCID peripheral blood appear to be factors limiting the rejection process.     

Macrophages produce nitric oxide at allograft sites.

OBJECTIVE: The current study was designed to determine which cytokines produced during an alloimmune response stimulate macrophage nitric oxide (.N = O) production at allograft sites. SUMMARY BACKGROUND DATA: Previous work has demonstrated that rat sponge matrix allograft infiltrating cells produce more .N = O on stimulation with alloantigen than syngeneic graft-infiltrating cells. Addition of NG-monomethyl-L-arginine (NMA), an inhibitor of .N = O synthesis, promotes allospecific cytolytic T-lymphocyte effector function. METHODS: Polyurethane sponges were implanted subcutaneously in recipient Lewis rats and injected with 10 x 10(6) ACl splenocytes. On various days after grafting, graft-infiltrating cells were harvested for in vitro study. Adherent macrophages from the graft infiltrating cell population were obtained by a 2- to 3-hour incubation to plastic dishes with subsequent washing to remove nonadherent cells. RESULTS: Stimulation of unseparated graft-infiltrating cell populations with lipopolysaccharide or interferon-tau resulted in enhanced .N = O synthesis by allograft infiltrating cells compared with syngeneic graft-infiltrating cells, early after grafting. Macrophages recovered from an allograft site spontaneously produce more .N = O than macrophages recovered from syngeneic grafts (p < 0.001). Significantly enhanced levels of .N = O were produced by allograft macrophages compared with syngeneic graft macrophages on stimulation with lipopolysaccharide or interferon-tau (p < or = 0.025). CONCLUSIONS: Nitric oxide appears to be produced in response to the local cytokines secreted by an ongoing rejection reaction. Nitric oxide serves under these circumstances to modulate the alloimmune response.     

Requirement of MyD88 for macrophage-mediated islet xenograft rejection after adoptive transfer

BACKGROUND: Porcine antigen primed and CD4+ T-cell activated macrophages are able to migrate to and destroy porcine xenografts. However, the specific signaling mechanisms involved remain to be identified. METHODS: In this study macrophages which lack the universal toll-like receptor (TLR) adaptor MyD88 were used to investigate the role of TLR in the recognition and activation of macrophages in islet xenograft rejection. Macrophages were isolated from rejecting MyD88(-/-) and wild-type C57BL/6 mice that were recipients of neonatal porcine pancreatic cell cluster (NPCC) xenografts, and were transferred to NPCC recipient NOD-SCID mice. RESULTS: Both wild-type C57BL/6 and MyD88(-/-) mice rejected NPCC xenografts 8 and 10 days, respectively after transplantation, and the grafts were heavily infiltrated with CD4+ T cells and macrophages. However, graft infiltrating macrophages from rejecting MyD88(-/-) recipients demonstrated impaired up-regulation of TLR expression and impaired activation phenotype, when compared to those from rejecting C57BL/6 recipients. Transfer of NOD-SCID recipients with macrophages from rejecting C57BL/6 mice resulted in NPCC xenograft rejection along with massively infiltrated macrophages 8 days after transfer, whereas NPCC xenografts in NOD-SCID mice transferred with macrophages from rejecting MyD88(-/-) mice remained intact until the end of this study, 90 days after transfer, with insulin-positive islets and no infiltration by macrophages. CONCLUSION: This study demonstrates that deletion of MyD88 causes impaired macrophage activation after pig islet xenotransplantation. However, graft survival is not prolonged and xenografts are rejected rapidly by alternate mechanisms.     

Graft survival and cytokine production profile after limbal transplantation in the experimental mouse model

Limbal transplantation or limbal stem cell (LSC) transfer represents the only way to treat severe ocular surface damage or LSC deficiency. However, limbal allografts are promptly rejected in spite of extensive immunosuppressive therapy. To characterize immune response after limbal transplantation, we established an experimental model of limbal transplantation in the mouse. Syngeneic, allogeneic and xenogeneic (rat) limbal grafts were grafted orthotopically in BALB/c mice and graft survival was evaluated. The presence of graft donor cells and the expression of IL-2, IL-4, IL-10, IFN-γ and inducible nitric oxide synthase (iNOS) mRNA in the grafts were detected by real-time PCR. While syngeneic grafts survived permanently, allografts were rejected in 9.0±1.8 days and xenografts in 6.5±1.1 days. The manifestation of clinical symptoms of rejection correlated with the disappearance of donor cells in the graft and in the recipient cornea. Intragraft expression of iNOS mRNA and distinct expression patterns of Th1 (IL-2, IFN-γ) and Th2 (IL-4, IL-10) cytokines were detected during rejection of limbal allografts and xenografts. The limbal graft rejection was prevented with anti-CD4, but not anti-CD8 monoclonal antibody therapy. The results indicate that limbal grafts do not enjoy immune privilege of the eye and are promptly rejected by Th1 (allografts) or by a combined Th1 and Th2 (xenografts) type of immune response involving CD4+ cells and iNOS expression. Targeting this pathway may be an effective way to prevent and treat limbal graft rejection.     

Predominant expression of the Th2 response in chronic cardiac allograft rejection

Chronic rejection is the main cause of late allograft failure in patients. CD4+ T cells activated by indirect recognition of alloantigens are implicated in this rejection reaction. However, the type of T cell response (Th1 vs Th2) that contributes to chronic rejection has not been fully investigated. The purpose of this study is to examine whether chronic rejection is associated with a polarized T-cell response in a rat cardiac allograft model, where long-term graft survival is achieved by intrathymic immunomodulation with donor class I, RT1.Aa, allopeptides. All long-surviving allografts showed histological evidence of chronic rejection. Chronic rejection was associated with high levels of intragraft Th2 cytokines and the Th2-regulated alloantibodies. The Th2 response was systemic, since long-surviving allografts with chronic rejection had high levels of serum IL-10. The predominance of the Th2 cytokines demonstrates that the Th2 response was not sufficient for the prevention of chronic rejection in this model. The predominant expression of Th2 cytokines, together with the presence of Th2-regulated alloantibodies, suggests that the Th2 response may play a role in the development of chronic rejection.     

Roles of CD4+ and CD8+ T cells in discordant skin xenograft rejection

An essential role of murine CD4+ T cells in immune reactivity and skin graft rejection in discordant xenogeneic combinations have been reported. Our study was conducted to further clarify the roles of CD4+ and CD8+ T cells in discordant skin xenograft rejection, by using CD4 and CD8 knockout [C57BL/6 Cr Slc (B6; H-2b) background] mice. When human skins were grafted on CD8 knockout mice or B6 mice, both hosts rejected human skin grafts within 12 days after grafting. By contrast, survival of human skin grafts was significantly prolonged in CD4 knockout mice (mean survival times=19.3+/-(SD) 1.6 days; median 19 days). Fully allogeneic C3H/He Slc (H-2k) skin grafts were rejected within 14 days in CD4 knockout mice, suggesting that non-CD4+ T cells in CD4 knockout mice were immunocompetent for allograft rejection. In spleens of these recipient mice, CD8+ T cells seemed to be activated 10 days after human skin grafting. Immunohistological analysis revealed the infiltration of CD8+ T cells at the site of transplanted human skin on CD4 knockout mice. To further examine the role of CD8+ T cells in CD4 knockout mice, human skin grafting was performed on day 0 followed by administration of anti-CD8 monoclonal antibody on days 0, 5, and 14. The administration of anti-CD8 monoclonal antibodies caused the significant prolongation of human skin graft survival. These results indicate the following two conclusions: (1) CD4+ T cells have an essential role in rejecting discordant human skin xenografts rapidly and (2) however, CD8+ T cells also are capable of rejecting discordant human skin xenografts.     

Biodistribution of an anti-interleukin 2 receptor monoclonal antibody in rat recipients of a heart allograft, and its use as a rejection marker in gamma scintigraphy

Anti-interleukin-2 receptor monoclonal antibodies have been shown to prevent allograft rejection. This paper reports on the biodistribution of a mouse MoAb directed at the 55 Kd alpha chain of rat interleukin-2 receptor (IL2-R) during allograft rejection. Only a low percentage (approximately 1%) of intact 125I-labeled MoAb was detected in the rejected graft, and irrelevant control IgG1 was found at a similar level. This suggests that most of the injected intact MoAb bound to graft tissue via its monomorphic Fc segment. In contrast, OX39 F(ab')2 fragments showed a preferential localization in the rejected allograft and did not bind to the LEW-to-LEW syngeneic heart graft. Irrelevant F(ab')2 did not concentrate in the allogeneic graft. Accordingly, F(ab')2 fragments from OX39 or irrelevant MoAb were used for gamma-scintigraphy on allograft recipients together with biodistribution studies. Results show that scintigraphy was able to detect allograft accumulation of 131I OX39 F(ab')2, whereas no imaging was obtained when OX39 F(ab')2 was used in the syngeneic combination or when irrelevant 131-IgG1 F(ab')2 was given to allograft recipients. This method, applied to the clinical situation, could be of interest for detection of early graft rejection episodes by immunoscintigraphy using reagents specific for activation determinants on lymphocyte membranes, such as anti-interleukin-2 receptor MoAb.