Chronic Cardiac Allograft Rejection Research Articles

Inhibition of endothelin-converting enzyme attenuates transplant vasculopathy and rejection in rat cardiac allografts.

Transplant vasculopathy in kidney and heart allografts is associated with marked elevation of endothelin-1 (ET-1), but a role for ET-1 in the pathogenesis of transplant vasculopathy and chronic rejection has not been established. We, therefore, tested whether inhibition of ET-1-converting enzyme by phosphoramidon (PA) would attenuate rejection in a rat model of chronic cardiac allograft rejection (Lewis [LEW] to F344). Donor LEW rats were pretreated 24 hr before transplantation with a bolus injection of vehicle (water) or PA. Twenty- four hour after transplantation, water or PA was continuously administered through an osmotic mini-pump. Plasma ET-1 levels in Fisher 344 (F344) recipients were 0.8+/-0.1 pg/ml in water-treated rats and 0.2+/-0.2 pg/ml (P<0.01) in PA-treated rats, demonstrating that the PA treatment protocol effectively lowered ET-1 biosynthesis. LEW cardiac allografts treated with water survived (i.e., palpable heart beat) for 16.0+/-0.5 days (n=6). Inhibition of ET-1 secretion by PA improved allograft survival to 28.8+/-3.3 days (P<0.01, n=8). An analysis of cardiac arteries demonstrated that PA treatment attenuated transplant vasculopathy. A morphometric scale of neointima formation (0-5) was 1.4+/-0.2 and 3.6+/-0.2 in PA- or water-treated rats, respectively (P<0.01). The percent of luminal occlusion, as measured by microscopic image analysis, was 19+/-6% in PA-treated animals and 38+/-6% (P<0.01) in animals treated with water. PA treatment also reduced infiltration of ED-1-positive monocytes/macrophages into the vascular neointima. We conclude that, even in the absence of concomitant immunosuppression, inhibition of ET-1 biosynthesis significantly attenuates transplant vasculopathy and improves survival of LEW to F344 cardiac allografts.

Immunomodulatory functions of hyaluronate in the LEW-to-F344 model of chronic cardiac allograft rejection.

CD44 is an important leukocyte cell surface glycoprotein with diverse functions including cell adhesion, homing, migration, and activation. Because administration of the principal ligand of CD44, hyaluronate (HA), in soluble form, can inhibit CD44-HA interaction, we tested the effects of HA in vivo in an established model of chronic allograft rejection. Control F344 recipients of LEW hearts received either no treatment or low-dose cyclosporine (CsA) for 30 days from the day of transplantation. Experimental animals received 30 days of CsA in combination with 30 or 90 days of low molecular weight HA (LMW-HA). CsA therapy alone resulted in approximately 40% long-term (>100 days) graft survival, whereas CsA + LMW-HA (30-day and 90-day protocols) significantly increased long-term graft survival to 60% and 92%, respectively. Light microscopy and immunohistology of CsA-treated and CsA + LMW-HA-treated grafts harvested at day 30 after transplantation demonstrated that LMW-HA + CsA therapy decreased mononuclear cell infiltration and afforded better preservation of myocardial architecture. In addition, LMW-HA + CsA-treated grafts exhibited decreased expression of interferon-gamma and the growth factors transforming growth factor-beta, platelet-derived growth factor, and fibrogenic growth factor-beta. Long-term surviving grafts were assessed for arteriosclerosis, the sine qua non of chronic rejection in this model. Using a standardized scoring system, significantly less arteriosclerosis was seen in grafts from LMW-HA + CsA-treated animals at 120 days after transplantation compared with CsA alone-treated grafts. This difference was not significant, however, in grafts harvested at >150 days. This is the first report indicating that CD44-HA interactions play an important role in chronic allograft rejection.

The detection of chronic heart graft rejection by 31P NMR spectroscopy.

The usefulness of phosphorus-31 nuclear magnetic resonance spectroscopy (31P NMRS) for detecting heart graft rejection after transplantation has been investigated by several researchers, and it has thus been demonstrated to be a valid technique for detecting acute myocardial rejection. In this study, we investigated the value of 31P NMRS to assess chronic cardiac allograft rejection. Lewis rat hearts were transplanted into the femoral region of F-344 rat recipients which were treated with cyclosporine, 5mg/kg body weight, by a daily intramuscular injection for 30 days beginning on the day of transplantation. The control isografts employed Lewis donors and recipients not given cyclosporine. The ratios of phosphocreatine (PCr) to inorganic phosphate (Pi), beta-adenosine triphosphate (beta-ATP) to Pi, and PCr to beta-ATP were monitored using surface coil 31P NMRS. 31P NMRS was performed 3, 30, and 60 days after transplantation, and the degree of the rejection and arteriosclerosis of the coronary arteries were then assessed histologically. The PCr:Pi and beta-ATP:Pi ratios for the allografts demonstrated a significant decrease on postoperative day (POD) 60 from that on POD 30 (PCr:Pi, P < 0.001; beta-ATP:Pi, P < 0.01). Although a significant difference existed between the isografts and allografts on POD 60 (PCr: Pi, P < 0.01; beta-ATP:P, P < 0.01), no significant difference was found in the PCr:beta-ATP ratio between the allografts and the isografts. On POD 60, the allografts showed significant graft rejection and arteriosclerotic changes in the coronary arteries. These findings therefore demonstrated the effectiveness of 31P NMRS for detecting chronic graft rejection in a rat model.

Gene therapy for attenuating chronic rejection of cardiac allografts

Gene therapy for attenuating chronic rejection of cardiac allografts

Antibody-induced transplant arteriosclerosis is prevented by graft expression of anti-oxidant and anti-apoptotic genes.

We investigated the pathogenesis of chronic allograft rejection in mouse cardiac allografts. Long-term survival occurred after administration of monoclonal antibody to CD4 or CD40-ligand (CD40L) plus donor cells. Both treatments induced permanent graft survival, but, in contrast to transplants in mice treated with CD4 monoclonal antibody, grafts in mice treated with CD40L monoclonal antibody lacked evidence of chronic rejection, including transplant arteriosclerosis. Freedom from chronic rejection in the group treated with CD40L monoclonal antibody correlated with vascular expression of the 'protective' genes heme oxygenase-1 (HO-1), Bcl-xL and A20. Moreover, arteriosclerosis was induced in allografts in immunoglobulin-deficient mice by antibody transfer only when the transfer was done before expression of protective genes. A direct role for protective gene expression in endothelial cells was demonstrated by in vitro experiments in which induction of HO-1 or Bcl-xL suppressed alloantibody-stimulated endothelial activation. Finally, induction of HO-1 in vivo protected allografts against chronic injury. These data show a role for protective genes in the prevention of chronic rejection, and indicate new approaches to protect grafts against development of transplant arteriosclerosis.

NOS2 Mediates Opposing Effects in Models of Acute and Chronic Cardiac Rejection: Insights from NOS2-Knockout Mice

To compare regulatory effects of NOS2 in acute and chronic cardiac allograft rejection, we used NOS2 knockout mice as recipients in a cardiac transplant model. To study acute and chronic rejection separately but within the same genetic strain combination, we compared allografts placed into recipients without or with immunosuppression (anti-CD4/8 for 28 days). NOS2 mRNA and protein expression were compared using 32P-RT-PCR and immunohistochemistry. In our acute rejection model, NOS2 was predominately localized to graft-infiltrating immune cells. At day 7, grafts in NOS2-deficient recipients (n = 7) showed reduced inflammatory infiltrates and myocyte damage resulting in significantly lower rejection scores (1.6 +/- 0.4) compared to wild-type controls (n = 18; 2.8 +/- 0.2, P = 0.002). In contrast, in our chronic rejection model, additional NOS2 expression was localized to graft-parenchymal cells. At day 55, grafts in NOS2-deficient recipients (n = 12) showed more parenchymal infiltration and parenchymal destruction (rejection score 3.8 +/- 0.1) than wild-type controls (n = 15; 1.6 +/- 0.2, P < 0.0001). This was associated with a significant decrease in ventricular contractility (palpation score 0.3 +/- 0.1 compared to 2.3 +/- 0.3 in wild-type, P < 0.0001). Hence, NOS2 promotes acute but prevents chronic rejection. These opposing effects during acute and chronic cardiac allograft rejection are dependent on the temporal and spatial expression pattern of NOS2 during both forms of rejection.

Evidence Against the Role of Th1 in the Development of Chronic Cardiac Allograft Rejection

384 Transient depletion of CD4+ T cells in mouse vascularized cardiac allograft recipients prolongs allograft survival, however grafts exhibit signs of chronic rejection characterized by collagen deposition and neointima development. It has been suggested that chronic rejection is associated with persistent low-level immune responses directed against the graft. While it is well established that Th1 cells promote acute allograft rejection, the role of these cells in chronic rejection remains unclear. The present study was designed to determine whether IFNγ producing Th1 cells contribute to the development of chronic cardiac allograft rejection. To this end, splenocytes obtained from C57BL/6 (B6) recipients bearing Balb/c hearts with signs of chronic rejection were adoptively transferred into B6 SCID cardiac allograft recipients. Transfer of these cells resulted in accelerated and exacerbated chronic allograft rejection in the majority of SCID recipients. Hence, this transfer system allowed us to explore the underlying mechanisms involved in chronic cardiac allograft rejection and the participation of Th1 in this process. Characterization of splenocytes prior to adoptive transfer into B6 SCID allograft recipients revealed a hyporesponsiveness in Th1 function. However, splenocyte proliferative responses to mitogens or alloantigens and precursor CTL and IL-2 producing T lymphocyte frequencies were comparable to naive splenocytes. Further, transferred splenocytes recovered from SCID allograft recipients with advanced signs of chronic rejection remained deficient in Th1 function, suggesting that Th1 are not involved in this disease process. Two additional observations indicated that Th1 are not associated with chronic rejection: 1) chronic cardiac rejection developed in allografts of IL-12 knockout recipients treated transiently with anti-CD4 mAb, and 2) transfection of grafts with TGFβ1 ablated Th1 responses, but resulted in accelerated chronic rejection. Finally, when splenocytes used for adoptive transfer retained Th1 function, transfer of these cells resulted in acute allograft rejection in SCID recipients. Hence, the absence rather than the presence of Th1 was associated with chronic rejection. These data suggest that Th1-independent effector mechanisms are responsible for chronic rejection in this model. Ongoing studies employing this transfer system are aimed at identifying the cellular and cytokine mediators in this disease process.

CHRONIC CARDIAC ALLOGRAFT REJECTION IS INDEPENDENT OF DONOR MHC CLASS II ANTIGENS

381 Donor MHC class II antigens are expressed on the vascular endothelium during chronic rejection. However, whether expression of donor MHC class II antigens is required for chronic rejection is not known. The purpose of this study was to determine whether allografted MHC II-/- hearts which do not reject acutely in the presence of only CD4 T cells would develop chronic vascular lesions. To test this proposition, heterotopic cardiac allografts from C57BL/6 (H-2b) MHC II +/+ or MHC II-/- mice were transplanted into immune-deficient C.B-17scid (H-2d, SCID) mice. SCID mice bearing established cardiac grafts were: (1) left unreconstituted, (2) reconstituted by adoptive transfer with unfractionated lymph node cells from BALB/c (H-2d) donors (10 ×106 cells), or (3) reconstituted with purified CD4 BALB/c T cells (10× 106 cells). Hearts were monitored for rejection by daily palpation. CD4 T cells isolated by antibody column purification (Biotex) and had undetectable numbers (<0.5%) of contaminating CD8 T cells or B220 B cells by flow cytometry.RESULTS: Unreconstituted SCID mice did not reject either cardiac MHC II +/+ or MHC -/- allografts (n=10, n=6, graft survival >60 days). Adoptive transfer of unfractionated cells to SCID recipients triggered acute rejection of both MHC II+/+ allografts (n=7, days 8,9,10, 10, 11, 19, 19) and MHC-/- allografts (n=6, days 8,10,10,10,10,11). MHC II+/+ allografts in SCID mice reconstituted with purified CD4 cells rejected in a time course similar to control mice (n=10, days 8, 9, 9, 10, 10, 10, 11, 11, 16, 22). MHC II-/- allografts in SCID mice reconstituted with purified CD4 cells did not reject acutely (n=7, 4/5 grafts >60 days, p<0.005), but all developed intimal thickening and evidence of chronic rejection. Unreconstituted long term (>60 days) allografts from either MHC class II+/+ or -/- donors did not develop evidence of chronic rejection. Acutely rejecting allografts demonstrated a typical lymphocytic infiltrate but did show evidence of chronic rejection. CONCLUSIONS: 1) Donor MHC class II antigens are not required for chronic rejection of cardiac allografts. 2) CD4 T cells are sufficient for chronic rejection of allografts, independently from either CD8 T cells or B cells. 3) Furthermore, we propose that CD4-dependent chronic graft rejection can be mediated through the indirect (host APC) dependent pathway.

Cytomegalovirus infection enhances the neointima formation in rat aortic allografts: effect of major histocompatibility complex class I and class II antigen differences.

The development of chronic rejection has emerged as a major cause of long-term graft failure. Previous studies have demonstrated that cytomegalovirus (CMV) infection is associated with an increased incidence of chronic allograft rejection in renal, cardiac, and aortic allografts. This study was designed to investigate the effects of the major histocompatibility complex (MHC) class I or class II mismatches on CMV-enhanced chronic rejection. Aortic transplantation was performed between different inbred rat strain combinations; the Lewis to RP combination was class I-mismatched and Wag/Rij to RP class II-mismatched. At 7, 28, and 90 days after transplantation, the intensity of chronic rejection in mismatched grafts with or without CMV infection was evaluated using histological and immunohistological analysis. The results of this study demonstrated that CMV infection led to an increased influx of monocytes/ macrophages in class I-mismatched grafts at 1 week after transplantation and enhanced infiltration of T lymphocytes in class II-mismatched grafts at 4 weeks. Although more vascular lesions were observed in the class II-mismatched combinations, an intensified neointima formation by CMV infection was observed only in the MHC class I-mismatched allografts. CMV infection may increase neointima formation of allografts when an MHC class I disparity between donor and recipient is present. This may be associated with the increased perivascular influx of monocytes/macrophages observed in CMV-infected animals early after transplantation.

Evidence Against the Role of Th1 in the Development of Chronic Cardiac Allograft Rejection

Evidence Against the Role of Th1 in the Development of Chronic Cardiac Allograft Rejection

Tumor necrosis factor in the heart.

The heart is a tumor necrosis factor (TNF)-producing organ. Both myocardial macrophages and cardiac myocytes themselves synthesize TNF. Accumulating evidence indicates that myocardial TNF is an autocrine contributor to myocardial dysfunction and cardiomyocyte death in ischemia-reperfusion injury, sepsis, chronic heart failure, viral myocarditis, and cardiac allograft rejection. Indeed, locally (vs. systemically) produced TNF contributes to postischemic myocardial dysfunction via direct depression of contractility and induction of myocyte apoptosis. Lipopolysaccharide or ischemia-reperfusion activates myocardial P38 mitogen-activated protein (MAP) kinase and nuclear factor kappa B, which lead to TNF production. TNF depresses myocardial function by nitric oxide (NO)-dependent and NO-independent (sphingosine dependent) mechanisms. TNF activation of TNF receptor 1 or Fas may induce cardiac myocyte apoptosis. MAP kinases and TNF transcription factors are feasible targets for anti-TNF (i.e., cardioprotective) strategies. Endogenous anti-inflammatory ligands, which trigger the gp130 signaling cascade, heat shock proteins, and TNF-binding proteins, also control TNF production and activity. Thus modulation of TNF in cardiovascular disease represents a realistic goal for clinical medicine.

Effects of pravastatin on chronic rejection of rat cardiac allografts.

Pravastatin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, inhibits coronary transplant vasculopathy in the clinical setting. To further delineate the immune modulatory effect of this agent, it was tested in a rat cardiac transplant model of chronic rejection. Rat heterotopic abdominal cardiac transplants were performed using a Lewis to Fischer 344 combination. Fischer 344 recipients received a brief course of cyclosporine to decrease the incidence of acute rejection. Experimental groups were treated with either high-dose (10 mg/kg) or low-dose (5 mg/kg) pravastatin for 120 days, while a control group did not receive pravastatin. The effect of pravastatin on chronic rejection of cardiac allografts was analyzed by histology, and the expression of laminin, fibronectin, macrophages, and T cells was assessed by immunohistochemistry. Coronary transplant vasculopathy was inhibited in both groups of pravastatin-treated animals, as compared with controls. Immunohistochemistry revealed that control animals had degraded laminin and fibronectin which paralleled the degree of tissue necrosis. In contrast, pravastatin-treated animals had modest amounts of extracellular matrix proteins retained within intermyocytes and endothelium, a pattern seen in native cardiac tissue. The pravastatin-treated groups also had fewer graft-infiltrating macrophages, specifically within the arterial intima and perivascular areas. Progressive chronic vascular rejection, a leading cause of allograft failure, can be inhibited by pravastatin in a well-defined rat cardiac transplant model. Pravastatin appears to inhibit the synthesis and subsequent degradation of extracellular matrix proteins and block the infiltration of macrophages to the graft, which emphasizes that this inflammatory cell plays a major role in the pathogenesis of transplant chronic rejection.

New approaches to the management of acute and chronic cardiac allograft rejection.

There are still many problems to be faced in the field of heart transplantation. Acute and chronic rejection are still the major medical obstacles. In this review, we describe recent research in this field undertaken in our laboratory. The induced intercellular adhesion molecule-1 (ICAM-1) and MHC class II antigen resulting from rejection can be visualized in vivo by radioimmunoscintigraphy. This non-invasive method is sensitive for detecting early rejection and allows quantitative assessment of rejection. Short-term administration of monoclonal antibodies to ICAM-1 and leukocyte function-associated antigen-1 (LFA-1) results in an indefinite acceptance of cardiac allografts by induction of antigen-specific tolerance, as evidenced by acceptance of the secondary skin allografts. The characteristics and possible mechanisms of this tolerance induction are discussed. Immunohistopathologic features of graft coronary arteriopathy are shown. Adhesion molecules, cytokines, and growth factors are associated with intimal hyperplasia and phenotypic transformation of smooth muscle cells in the allograft coronary arteries. Dramatic reduction in this intimal hyperplasia was demonstrated by antisense gene therapy targeting cyclin-dependent kinase 2 kinase. We hope that these investigations will contribute to the improvement of the management of patients who undergo heart transplantation.

Adenosine decreases post-ischaemic cardiac TNF-alpha production: anti-inflammatory implications for preconditioning and transplantation.

Tumour necrosis factor-alpha (TNF-alpha) is an autocrine contributor to myocardial dysfunction and cardiomyocyte death in ischaemia-reperfusion injury (I/R), sepsis, chronic heart failure and cardiac allograft rejection. Cardiac resident macrophages, infiltrating leucocytes, and cardiomyocytes themselves produce TNF-alpha. Although adenosine reduces macrophage TNF-alpha production and protects myocardium against I/R, it remains unknown whether I/R induces an increase in cardiac TNF-alpha in a crystalloid-perfused model (in the absence of blood), and, whether adenosine decreases cardiac TNF-alpha and protects function after I/R. To study this, isolated rat hearts were crystalloid-perfused using the Langendorff method and subjected to I/R, with or without adenosine pretreatment. Post-ischaemic cardiac TNF-alpha (enzyme-linked immunosorbent assay and bioassay) and function were determined (Langendorff). I/R increased cardiac TNF-alpha and impaired myocardial function. Adenosine decreased cardiac TNF-alpha and improved post-ischaemic functional recovery. This study demonstrates that: first, I/R induces an increase in cardiac tissue TNF-alpha in a crystalloid-perfused model: second, adenosine decreases cardiac TNF-alpha and improves post-ischaemic myocardial function; third, decreased cardiac TNF-alpha may represent a mechanism by which adenosine protects myocardium; and fourth, adenosine-induced suppression of cardiac TNF-alpha may provide an anti-inflammatory link to preconditioning and have implications for cardiac allograft preservation.

Morphometric analysis of neointimal formation in murine cardiac grafts: III. Dissociation of intestitial fibrosis from neointimal formation.

This study examined the relationship between transplant vascular sclerosis (TVS) and tissue fibrosis, features of chronic rejection that can develop rapidly in accepted heterotopic murine cardiac allografts. The rate of development of interstitial fibrosis or TVS development was determined by computerized analysis of tissue sections from DBA/2-->C57BL/6 heterotopic cardiac allografts after immunosuppression with gallium nitrate. In accepted cardiac allografts, neointimal fibrosis developed by 30 days after transplant, whereas TVS was minimal by day 30, and maximal by day 60. Variable levels of fibrosis were found throughout the allografts. DBA/2-->DBA/2 cardiac isografts never displayed TVS in this time period, but displayed allograft-like fibrosis within 60 days of transplantation. Interstitial fibrosis can be dissociated from the TVS development in this experimental model of chronic cardiac allograft rejection. Apparently, it is caused, at least in part, by alloantigen-independent factors other than TVS-related tissue ischemia.

Expression and localization of platelet-derived growth factor ligand and receptor protein during acute and chronic rejection of rat cardiac allografts.

The molecular mechanisms of cardiac allograft vasculopathy (CAV) remain largely unknown. Using rat cardiac allografts, we examined by immunohistochemistry the expression and localization of platelet-derived growth factor ligand (PDGF-AA and -BB) and receptor (R alpha and R beta) proteins during acute and chronic rejection. In acute rejection, a prominent induction of both PDGF ligand and receptor proteins occurred in the interstitial mononuclear inflammatory cells (P<.05), most of which were ED1-immunoreactive. PDGF-R beta was also induced in the capillary endothelium (P<.01). In cardiac allografts with severe intimal thickening, PDGF-AA expression was localized to the media and intima, whereas PDGF-BB expression was less prominent and was detected mainly in interstitial ED1-immunoreactive inflammatory cells. Double staining revealed that intimal cells expressing PDGF-AA were alpha-smooth muscle actin-positive but also alpha-smooth muscle actin-negative myofibroblast-like cells and to a lesser extent, ED1-immunoreactive cells. Both PDGF-R alpha and -R beta expression occurred in intimal, arterial endothelial, and interstitial mononuclear inflammatory cells. High-dose cyclosporin A (CsA) treatment significantly reduced both PDGF-AA and PDGF-R alpha expression in intimal cells. Furthermore, linear regression analysis revealed that PDGF-AA, PDGF-R alpha, and PDGF-R beta expression in intimal cells and PDGF-BB expression in interstitial mononuclear inflammatory cells correlated with intimal thickening. Alloimmune injury induces the expression of PDGF ligands, especially of PDGF-AA, in the graft vasculature and sufficient immunosuppression with CsA suppresses the expression of PDGF and inhibits the development of CAV. PDGF may have a substantial role in the regulation of smooth muscle cell migration and proliferation in an autocrine or paracrine manner during the development of CAV.

20 Pravastatin protects against development of chronic rejection in rat cardiac allografts and upregulates immunosuppressive TGF-B2

20 Pravastatin protects against development of chronic rejection in rat cardiac allografts and upregulates immunosuppressive TGF-B2

T-cell costimulatory blockade in experimental chronic cardiac allograft rejection: effects of cyclosporine and donor antigen.

Chronic rejection is a T cell-dependent process and blockade of the CD28-B7 T-cell costimulatory activation pathway by the fusion protein CTLA4Ig has been shown to prevent the development of accelerated graft arteriosclerosis in a rat model of chronic cardiac allograft rejection. The effectiveness of T-cell costimulatory blockade at preventing chronic allograft rejection in a clinically relevant model in combination with cyclosporine therapy has not been investigated. Using the well-established LEW into F334 heterotopic cardiac allograft model, we show that when cyclosporine is administered in combination with CTLA4Ig, it abrogates the previously demonstrated protective effect of CTLA4Ig in preventing chronic allograft rejection. Long-term surviving allografts from animals treated with a combination of cyclosporine and CTLA4Ig had a mean vascular luminal occlusion of 42.2%, affecting more than 90% of graft vessels due to accelerated arteriosclerosis. This was associated with up-regulation of intragraft expression of mRNA for CD4, the costimulatory molecule B7, the T-cell cytokine interferon-gamma, monocyte chemoattractant protein-1, and the fibrogenic growth factor transforming growth factor-beta; all have been previously shown to be associated with development of chronic rejection in this model. In comparison, the addition of donor splenocytes to the combination of CTLA4Ig and cyclosporine therapy protocol significantly reduced the amount of arteriosclerosis; mean vascular luminal occlusion was 11.3%, affecting approximately 50% of vessels. This was associated with decreased intragraft expression of CD4, B7, interferon-gamma, monocyte chemoattractant protein-1, and transforming growth factor-beta. These data indicate that the mechanism of action of CTLA4Ig in attenuating chronic rejection is cyclosporine sensitive, and that strategies implying combination of CTLA4Ig and cyclosporine may not be clinically desirable. Administration of donor antigen may be necessary if CTLA4Ig and cyclosporine are to be combined, to prevent the process of chronic rejection.

Triple drug immunosuppression significantly reduces aortic allograft arteriosclerosis in the rat.

We evaluated the effect of triple drug immunosuppression (cyclosporine A 10 mg . kg(-1) . d(-1), methylprednisolone 0.5 mg . kg(-1) . d(-1) on the development of allograft arteriosclerosis (chronic rejection). The recipients of rat aortic allografts from the DA (AG-B4,RT1a) to the WF (AG-B2,RT1u) strain were either treated with triple drug immunosuppression (n=23) or left untreated (n=23) and used as controls. The grafts were removed 7, 14, 30, 90, and 180 days after transplantation, and vascular wall changes were evaluated by quantitative histology, [3H]thymidine autoradiography, and immunohistochemistry. Nonimmunosuppressed aortic allografts developed progressive arteriosclerotic alterations 1 to 6 months after transplantation that were virtually identical to those observed during chronic rejection in human cardiac allografts. Linear regression analysis revealed that triple drug immunosuppression with clinically relevant dosages of drugs significantly reduced intimal thickening (r=.69 versus r=.88, P<.05). Concomitantly, there was a marked reduction in the number of inflammatory cells (P<.01) and their rate of proliferation (P<.025) in the allograft adventitia during the period of acute inflammation (30 days after transplantation). Immunohistochemistry revealed that the number of helper T cells (W3/25) and monocyte/macrophages (OX42) but not cytotoxic T cells (OX8) or natural killer cells (3.2.3) was significantly (P<.05) reduced. The number of adventitial cells expressing interleukin-2 receptor (CD25) (P<.05), MHC class II (OX6) (P<.05) and leukocyte function-associated antigen-1 alpha-chain (CD11a) (P<.025) was also significantly reduced at 30 days. Triple drug immunosuppression downregulated the induction of MHC class II and intercellular adhesion molecule-1 on the graft endothelium but had no significant effect on the number of subendothelial inflammatory cells. In addition, [3H]thymidine autoradiography demonstrated that triple drug immunosuppression significantly reduced the rate of cell proliferation in the media, composed of smooth-muscle cells, 30 and 90 days after transplantation. Thus, triple drug immunosuppression efficiently reduced the development of allograft arteriosclerosis by down-regulating the inflammatory response and the level of immune++ activation in the allograft adventitia during the acute rejection period, resulting in diminished intimal thickening of the graft in the long run. These results support the concept that allograft arteriosclerosis is due to or at least initiated by immune injury of the graft.

Cytokine and alloantibody networks in long term cardiac allografts in rat recipients treated with rapamycin.

Treatment with rapamycin (RPM) prevents accelerated rejection of (LEW x BN)F1 cardiac allografts in LEW rats presensitized with BN skin grafts. This study analyzed the influence of RPM on cytokine (IL-2, IL-4, IL-10, and IL-12) and alloantibody networks in this model. Accelerated (24-h) rejection was associated with strong expression of intragraft IL-2 and IL-12 (p40) mRNAs, which reached maximal levels 3 to 6 h post-transplantation. IL-4 and IL-10 mRNAs were readily detectable throughout the observation period. RPM therapy abrogated rejection at 24 h and prolonged cardiac allograft survival to about 50 days. This effect was correlated with a profound initial depression of IL-2 mRNA; delayed expression of IL-2 mRNA was detected in well functioning grafts at > 20 days. In RPM-treated hosts, expression of IL-12 (p40) mRNA was low at the early time points (6-24 h), but prominent in long term grafts. The expression of both IL-4 and IL-10 mRNAs was preserved in RPM-conditioned hosts. Immunohistologic analysis of long term allografts revealed an interstitial cellular infiltrate and areas of intimal proliferation within small arteries indicative of early transplant arteriosclerosis. Analysis of cytokine proteins showed dense labeling of mononuclear and some endothelial cells for IL-4 and IL-12 (p70), but not for IL-2 or IFN-gamma alloantibody in the early post-transplant period. However, an increase in circulating and intragraft IgM and, to a lesser extent, IgG, primarily of the IgG2b subclass, was evident in long term recipients. Thus, RPM treatment reduces, but does not completely inhibit, the expression of Th1-type and preserves the expression of Th2-type cytokines. The demonstration of IL-12 in long term allografts after RPM therapy may reflect late activation of macrophages that, coupled with the appearance of IgG2b, may contribute to the chronic rejection of cardiac allografts.