Pattern specification and immune response transcriptional signatures of pericardial and subcutaneous adipose tissue.

Cardiovascular disease (CVD) remains the leading cause of morbidity and mortality in the United States. Recent studies suggest that pericardial adipose tissue (PCAT) secretes inflammatory factors that contribute to the development of CVD. To better characterize the role of PCAT in the pathogenesis of disease, we performed a large-scale unbiased analysis of the transcriptional differences between PCAT and subcutaneous adipose tissue, analysing 53 microarrays across 19 individuals. As it was unknown whether PCAT-secreted factors are produced by adipocytes or cells in the supporting stromal fraction, we also sought to identify differentially expressed genes in isolated pericardial adipocytes vs. isolated subcutaneous adipocytes. Using microarray analysis, we found that: 1) pericardial adipose tissue and isolated pericardial adipocytes both overexpress atherosclerosis-promoting chemokines and 2) pericardial and subcutaneous fat depots, as well as isolated pericardial adipocytes and subcutaneous adipocytes, express specific patterns of homeobox genes. In contrast, a core set of lipid processing genes showed no significant overlap with differentially expressed transcripts. These depot-specific homeobox signatures and transcriptional profiles strongly suggest different functional roles for the pericardial and subcutaneous adipose depots. Further characterization of these inter-depot differences should be a research priority.

Autoimmune sensitization to cardiac myosin leads to acute rejection of cardiac allografts in miniature swine.

BACKGROUND: Recent studies in mice and patients suggest that posttransplantation induction of autoimmune responses to tissue-specific antigens contributes to the rejection of major histocompatibility complex mismatched allotransplants. The relevance of this phenomenon to the rejection of major and minor histocompatibility-mismatched allografts performed in large-animal models remains to be established. METHODS: Miniature swine were immunized with cardiac myosin (CM) in Freund's adjuvant and received heterotopic, minor antigen-mismatched heart transplants. T-cell (proliferation and delayed type hypersensitivity [DTH]) and B-cell (antibody) responses specific to CM were measured. The rejection of heart transplants was assessed histologically. RESULTS: Three of four swine that were immunized with CM before receiving a minor antigen-mismatched heart transplant exhibited potent DTH, T-cell proliferation and antibody responses to CM and rejected their grafts acutely. The fourth swine, which failed to mount a significant DTH response to CM and displayed low and transient anti-CM antibody titers, demonstrated long-term allograft survival. CONCLUSIONS: This large-animal study supports the relevance of autoimmunity to CM in the rejection of minor antigen disparate cardiac allotransplants.

Immunosuppression for lung transplantation.

As a result of advances in surgical techniques, immunosuppressive therapy, and postoperative management, lung transplantation has become an established therapeutic option for individuals with a variety of end-stage lung diseases. The current 1-year actuarial survival rate following lung transplantation is approaching 80%. However, the 5- year actuarial survival rate has remained virtually unchanged at approximately 50% over the last 15 years due to the processes of acute and chronic lung allograft rejection (1). Clinicians still rely on a vast array of immunosuppressive agents to suppress the process of graft rejection, but find themselves limited by an inescapable therapeutic paradox. Insufficient immunosuppression results in graft loss due to rejection, while excess immunosuppression results in increased morbidity and mortality from opportunistic infections and malignancies. Indeed, graft rejection, infection, and malignancy are the three principal causes of mortality for the lung transplant recipient. One should also keep in mind that graft loss in a lung transplant recipient is usually a fatal event, since there is no practical means of long-term mechanical support, and since the prospects of re-transplantation are low, given the shortage of acceptable donor grafts. This chapter reviews the current state of immunosuppressive therapy for lung transplantation and suggests alternative paradigms for the management of future lung transplant recipients.

Repetitive gastric aspiration leads to augmented indirect allorecognition after lung transplantation in miniature swine.

INTRODUCTION: Lung transplant recipients with documented gastroesophageal reflux disease (GERD) are at increased risk for graft dysfunction. Here, we present the first large-animal model of gastric aspiration after allogeneic lung transplantation and some preliminary data demonstrating the effect of chronic aspiration on the direct and indirect pathways of allorecognition. METHODS: Left orthotopic lung transplants (n=3) were performed in miniature swine across a major histocompatibility complex class I disparity, followed by 12 days of high-dose cyclosporine A. At the time of transplantation, a transtracheal catheter was placed at the carina, above the bronchial anastomosis. A gastrostomy tube was placed for daily aspiration of gastric contents. Subsequently, graft lungs were instilled with gastric aspirate daily (3 mL/hrX8 hr/day) for 50 days. Recipients were followed up with daily complete blood count, scheduled chest radiographs, and biopsies. In vitro studies, including cell-mediated lympholysis, mixed lymphocyte reactions, and peptide proliferation assays, were performed. Results from these three recipients were compared with those of historical controls (n=6) who were treated identically, except for the tracheal cannulation and simulated gastric aspiration. RESULTS: Two of the experimental animals were euthanized with nonviable lungs soon after the postoperative day 50 biopsy. In both cases the native lung was normal. The third animal survived over 180 days without the evidence of chronic rejection. After immunosuppressive treatment, all animals demonstrated donor-specific hyporesponsiveness by assays of direct alloresponse (cell-mediated lympholysis, mixed lymphocyte reaction). A significant response to synthetic donor-derived class I peptide, however, was seen in all animals. A more pronounced and diffuse response was seen in the animals rejecting their grafts. The historical controls showed medium-term graft survival with evidence of chronic rejection in the majority of animals, as previously reported. CONCLUSION: In a model of GERD after lung transplantation, a spectrum of clinical outcomes was observed. The in vitro data suggest that acid reflux enhances the indirect alloresponse to processed donor class I antigen, giving mechanistic insight into the manner in which GERD may be deleterious to the transplanted lung.

In primed allo-tolerance, TIM-3-Ig rapidly suppresses TGFbeta, but has no immediate effect on Foxp3.

T-cell immunoglobulin mucin-3 (TIM-3) is only expressed by differentiated TH1 cells following their proliferative response to antigen, functioning to terminate TH1-mediated immunity upon binding to the TIM-3 ligand, galectin-9. This critical regulatory process involves Treg cells via their stable expression of galectin-9. Soluble TIM-3-Ig blocks galectin-9 and prevents induction of peripheral tolerance. Here we have looked for evidence that TIM-3-Ig might also break established regulatory tolerance. Using allo-primed spleen cells cultured ex vivo and challenged with irradiated donor-type stimulator cells either alone or together with 20 microg/ml TIM-3-Ig, we measured daily cytokine release [IL2, inferon gamma (INFgamma), transforming growth factor beta (TGFbeta), IL6, IL10] and cellular Foxp3 protein. In allo-tolerance, a specific effect of TIM-3-Ig was some fourfold reduction in TGFbeta. Foxp3 was induced in the allo-tolerant response to donor and this was not altered by TIM-3-Ig over the 5-day culture period. No Foxp3 was detected in either rejected or donor stimulator cells at any time. Thus, in an ex vivo model of in vivo tolerance to heart allografts, TIM-3-Ig therapy appears to reduce the stable tolerogenic environment by a rapid and specific repression of TGFbeta release.

Mechanisms of chronic rejection in cardiothoracic transplantation.

Despite significant improvements in early post-transplantation survival rates, long-term patient and graft survival have remained poor, due in large part to the vexing problem of chronic allograft rejection. Attempts to combat this problem with intensification of immunosuppression have led to concomitant increases in the rates of fatal malignancies and infections. In cardiac transplantation, chronic rejection is manifested primarily by a disease entity known as cardiac allograft vasculopathy, an occlusive narrowing of the coronary vessels. In lung transplantation, chronic rejection is typified by obliterative bronchiolitis, an airflow limiting narrowing of the bronchioles. From an immunologic standpoint, chronic rejection is believed to be the end result of repeated immune and non-immune insults to the graft. This review examines the pathophysiology of heart and lung chronic, with emphasis on both immune and non-immune causes.

Regulatory transplantation tolerance and "stemness": evidence that Foxp3 may play a regulatory role in SOCS-3 gene transcription.

Immune self-tolerance is controlled by a subset of T lymphocytes that are regulatory (Treg) and epigenetically programmed to suppress autoreactive immune effector cells in vivo. Treg require expression of Foxp3, a transcription factor that not only represses the interleukin-2 gene promoter, but also sequesters key mediators of T-cell signal transduction by complexing with cytoplasmic NFAT and NFkappaB. We have discovered that expression of Foxp3 is linked to two stem cell-related factors, namely leukemia inhibitory factor (LIF) and axotrophin. Because both LIF and axotrophin each influence Foxp3, we now ask if reciprocal cross-talk occurs; for example, does Foxp3 in turn influence LIF and/or axotrophin? We compared the effect of wt-Foxp3 versus mutant DeltaE251-Foxp3, which lacks transcriptional activity, on transcript levels of axotrophin, LIF, and suppressor of cytokine signaling-3 (SOCS-3; a feedback inhibitor of LIF) in the Jurkat human T-cell line. Unexpectedly, a 50-fold increase in SOCS-3 transcripts occurred in the DeltaE251-Foxp3 cells, coincident with a dramatic decrease in LIF transcription. This implies that, either directly or indirectly, transcription of SOCS-3 is negatively regulated by wt-Foxp3. Suppression of SOCS-3 by Foxp3 would support a model wherein Foxp3 promotes LIF signaling in Treg and is further evidence of reciprocity between Foxp3, LIF, and axotrophin.

A systematic review of randomized trials comparing revascularization rate and graft patency of off-pump and conventional coronary surgery.

OBJECTIVE: Although many trials have been conducted to evaluate the benefits of off-pump coronary surgery, few have concentrated on graft patency. We sought to evaluate the impact of off-pump surgery on completeness of revascularization and graft patency compared with conventional surgery. METHODS: A systematic literature search was undertaken of all randomized trials of off-pump coronary surgery in MEDLINE, EMBASE, the Cochrane Library Controlled Trials Register, the National Research Register, and abstracts from major conferences. RESULTS: In total, 132 publications were identified. From this number, we excluded 43 without a conventional surgery arm and 80 that did not evaluate graft patency. One trial was excluded for selective angiography and one abstract was excluded because of insufficient information. A total of 7 trials were eligible for overview. On initial analysis, the relative risk of graft patency in off-pump coronary surgery compared with conventional surgery was 0.959 (95% confidence interval 0.936-0.983; P = .001). The analysis was repeated after excluding one specific trial because of clinical and statistical heterogeneity (chi2(6) = 27.78; P < .001), and a relative risk of 0.953 (95% confidence interval 0.927-0.980; P = .001) was obtained with no further evidence of heterogeneity (chi2(5) = 5.35; P = .374). In 5 trials that included the mean number of grafts performed per arm, the standardized mean difference in revascularization comparing off-pump with conventional surgery was -0.164 (-0.286 to -0.043; P = .008). CONCLUSION: In a meta-analysis of randomized trials, patients undergoing off-pump coronary surgery had a lower rate of revascularization and lower graft patency than did patients undergoing conventional coronary surgery.

Evidence for functional inter-relationships between FOXP3, leukaemia inhibitory factor, and axotrophin/MARCH-7 in transplantation tolerance.

In an ex vivo mouse model, regulatory transplantation tolerance is not only linked to Foxp3, but also to release of leukaemia inhibitory factor (LIF) and to expression of axotrophin (also known as MARCH-7), a putative ubiquitin E3 ligase associated with feedback control of T cell activation and of T cell-derived LIF. Given this coordinate correlation with tolerance, we now ask if Foxp3 expression is influenced by LIF or by axotrophin. In spleen cells from allo-rejected mice we found that exogenous LIF reduced interferon gamma release in response to donor antigen by 50%, but LIF had no direct effect on levels of Foxp3 protein in allo-primed cells that were either tolerant, or aggressive, for donor antigen. However, we did find an effect of axotrophin on Foxp3: in the axotrophin null mouse, thymic Foxp3 transcripts were reduced compared to axotrophin wildtype littermates. To test whether these findings in the mouse were of potential significance in man we measured transcript levels of axotrophin and LIF in peripheral blood cell samples collected for a recently published clinical study concerning haematopoietic stem cell recipients. In controls, human peripheral blood CD4+CD25+cells contained significantly more FOXP3 and axotrophin than CD4+CD25-cells. In bone marrow autograft recipients, where peripheral blood cell samples directly represent both the grafted tissue and the immune response, both FOXP3 and axotrophin negatively correlated with graft versus host disease (GVHD). These data suggest that (i) thymic Foxp3+T cell development is influenced by axotrophin; and (ii) clinical auto-GVHD inversely correlates with axotrophin transcript expression as has been previously reported for FOXP3.

Vascular endothelium does not activate CD4+ direct allorecognition in graft rejection.

Expression of MHC class II by donor-derived APCs has been shown to be important for allograft rejection. It remains controversial, however, whether nonhemopoietic cells, such as vascular endothelium, possess Ag-presenting capacity to activate alloreactive CD4(+) T lymphocytes. This issue is important in transplantation, because, unlike hemopoietic APCs, allogeneic vascular endothelium remains present for the life of the organ. In this study we report that cytokine-activated vascular endothelial cells are poor APCs for allogeneic CD4(+) T lymphocytes in vitro and in vivo despite surface expression of MHC class II. Our in vitro observations were extended to an in vivo model of allograft rejection. We have separated the allostimulatory capacity of endothelium from that of hemopoietic APCs by using bone marrow chimeras. Hearts that express MHC class II on hemopoietic APCs are acutely rejected in a mean of 7 days regardless of the expression of MHC class II on graft endothelium. Alternatively, hearts that lack MHC class II on hemopoietic APCs are acutely rejected at a significantly delayed tempo regardless of the expression of MHC class II on graft endothelium. Our data suggest that vascular endothelium does not play an important role in CD4(+) direct allorecognition and thus does not contribute to the vigor of acute rejection.

Fetal liver as a source of autologous progenitor cells for perinatal tissue engineering.

Mesenchymal progenitor cells, isolated from adult bone marrow, have been shown to have utility for autologous tissue engineering. The possibility of isolating from the fetal hematopoietic system a cell population with similar potential, which could be used for autologous reconstruction of prenatally diagnosed congenital anomalies, has not been explored to date. Liver stromal cells isolated from a portion of the right lateral hepatic lobe of midgestation fetal lambs were expanded in vitro. Passage 1 cells displayed a uniform fibroblast-like morphology but could be induced to differentiate into skeletal muscle, adipocytes, chondrocytes, and endothelial cells by selective medium supplementation. By manipulating the extracellular matrix in vitro, spontaneously contracting cardiac myocyte-like cells could be generated as well. Multilineage differentiation was confirmed by morphology, protein expression, and upregulation of lineage-specific mRNA. The potential for engineering myocardial tissue was then investigated by transplanting early-passage progenitor cells, organized on a three-dimensional matrix, into the ventricle of an immunocompromised rat utilizing a previously described model of left ventricular tissue engineering. Survival, incorporation into the host myocardium, and cardiomyocytic differentiation of the transplanted cells were confirmed. We have demonstrated that mesenchymal progenitor cells with multilineage potential can be isolated from the fetal liver and have potential utility for autologous tissue engineering.

Surrogate markers and risk factors for chronic lung allograft dysfunction.

Obliterative bronchiolitis (OB) is the histologic correlate of chronic allograft dysfunction in pulmonary transplantation. The histologic diagnosis of OB is challenging, therefore a physiologic definition, bronchiolitis obliterans syndrome (BOS) based on pulmonary function tests has been used as a surrogate marker for OB for the last decade. BOS has proven to be the best available surrogate marker for OB and is predictive of the ultimate endpoints of graft and patient survival. Multiple other clinical markers have been reported and proposed as alternates for or complements to BOS grade, but all need further evaluation and validation in large, prospective clinical trials. Lastly, given the early occurrence and high incidence of chronic allograft dysfunction, the easily measurable endpoint of BOS grade, and our lack of understanding of ways to prevent or alter the course of BOS, lung transplant recipients represent an ideal population for clinical trials targeting prevention and treatment of chronic allograft dysfunction.

Homeostatic proliferation is a barrier to transplantation tolerance.

Despite the ease of inhibiting immune responses by blockade of T-cell costimulation in naive rodent models, it is difficult to suppress those responses in animals with memory cells. Studies demonstrating the importance of alloreactive T-cell deletion during tolerance induction have promoted use of peritransplant T-cell-depleting therapies in clinical trials. But potentially complicating wide-scale, nonspecific T-cell depletion is the finding that extensive T-cell proliferation can occur under conditions of lymphopenia. This process, termed homeostatic proliferation, may induce acquisition of functional memory T cells. Here, using clinically relevant mouse models of peripheral T-cell depletion, we show that residual nondepleted T cells undergo substantial homeostatic expansion. In this setting, costimulatory blockade neither significantly suppresses homeostatic proliferation nor prevents allograft rejection. In addition, T cells that have completed homeostatic proliferation show dominant resistance to tolerance when adoptively transferred into wild-type recipients, consistent with known properties of memory cells in vivo. These findings establish the importance of homeostatic proliferation in clinically relevant settings, demonstrate the barrier that homeostatic proliferation can present to the induction of transplantation tolerance, and have important implications for transplantation protocols that use partial or complete peripheral T-cell depletion.

Clinical risk factors for primary graft failure following lung transplantation.

STUDY OBJECTIVE: s: Primary graft failure (PGF) is a devastating acute lung injury syndrome following lung transplantation. We sought to identify donor, recipient, and operative risk factors for its development. DESIGN: We conducted a cohort study of 255 consecutive lung transplant procedures performed between October 1991 and July 2000. We defined PGF as follows: (1) diffuse alveolar opacities exclusively involving allograft(s) and developing within 72 h of transplant, (2) a ratio of PaO(2) to fraction of inspired oxygen < 200 beyond 48 h postoperatively, and (3) no other secondary cause of graft dysfunction identified. Risk factors were assessed individually and adjusted for confounding using multivariable logistic regression models. SETTING: Tertiary-care academic medical center. RESULTS: The overall incidence was 11.8% (95% confidence interval [CI], 7.9 to 15.9). Following multivariable analysis, the risk factors independently associated with development of PGF were as follows: a recipient diagnosis of primary pulmonary hypertension (PPH; adjusted odds ratio [OR], 4.52; 95% CI, 1.29 to 15.9; p = 0.018), donor female gender (adjusted OR, 4.11; 95% CI, 1.17 to 14.4; p = 0.027), donor African-American race (adjusted OR, 5.56; 95% CI, 1.57 to 19.8; p = 0.008), and donor age < 21 years (adjusted OR, 4.06; 95% CI, 1.34 to 12.3; p = 0.013) and > 45 years (adjusted OR, 6.79; 95% CI, 1.61 to 28.5; p = 0.009). CONCLUSIONS: Recipient diagnosis of PPH, donor African-American race, donor female gender, and donor age are independently and strongly associated with development of PGF.

Temporal changes in left ventricular systolic function in heart donors: results of serial echocardiography.

BACKGROUND: Heart donor availability continues to limit cardiac transplantation rates and many donor hearts are not transplanted because of left ventricular dysfunction. The aim of this study was to determine whether intensive donor management results in improved left ventricular systolic function as measured by serial echocardiography. METHODS: Using the California Transplant Donor Network Database, all donors who underwent serial echocardiography during donor management (from 1996 to 2000) were identified. The study includes those donors with ejection fractions <50% or regional wall-motion abnormalities shown on the initial echocardiogram. The database provides clinical data describing donor characteristics, treatments, and recipient outcomes. The mean ejection fractions at the first and second echocardiograms were compared using the Wilcoxon signed rank test. RESULTS: In 13 of 16 subjects, initial ejection fractions were <50% and improved in 12 subjects after intensive donor management. Seventy-five percent of the donors received high-dose corticosteroids, 15 of 16 received dopamine, and none received thyroid hormone. In 12 subjects, the donor hearts were transplanted with a survival rate of 92% at an average follow-up of 16 months. CONCLUSIONS: This pilot study indicates that in some cases, intensive donor management is associated with improved donor left ventricular function. Prospective studies are indicated to determine the predictors of improved donor left ventricular dysfunction and of recipient survival when sub-optimal hearts are transplanted.

Hormonal resuscitation yields more transplanted hearts, with improved early function.

BACKGROUND: Brain death results in cardiovascular instability and poor organ perfusion in many brain-dead donors. Hormonal resuscitation stabilizes certain brain-dead donors and is associated with significant increases in the numbers of organs transplanted per donor. The goal of this study was to examine the quality of hearts recovered from donors treated with hormonal resuscitation. METHODS: A retrospective analysis of 4,543 recipients of hearts recovered from brain-dead donors, reported to the United Network for Organ Sharing/Organ Procurement and Transplantation Network database between November 1, 1999, and December 31, 2001, was conducted. Hormonal resuscitation consisted of a methylprednisolone bolus and infusions of vasopressin and either triiodothyronine or l-thyroxine. Univariate and multivariate analyses were used to evaluate the quality of hearts from donors who received three-drug hormonal resuscitation (3HR) treatment versus donors who did not receive all three drugs (non-3HR). Death within 30 days and early graft dysfunction were used as endpoints. RESULTS: Hearts from 3HR donors demonstrated a 1-month survival rate of 96.2%, compared with a 92.1% survival rate for non-3HR donor hearts (P<0.01). Early graft dysfunction occurred in 5.6% of 3HR donor hearts and 11.6% of non-3HR donor hearts (P<0.01). Multivariate results demonstrated a 46% reduced odds of death within 30 days and a 48% reduced odds of early graft dysfunction. Steroids alone and steroids plus triiodothyronine/l-thyroxine also significantly reduced prolonged graft dysfunction. CONCLUSIONS: This study suggests that 3HR treatment of brain-dead donors results in increased numbers of transplanted hearts, with improved short-term graft function.

Aggressive pharmacologic donor management results in more transplanted organs.

BACKGROUND: Brain death results in adverse pathophysiologic effects in many cadaveric donors, resulting in cardiovascular instability and poor organ perfusion. Hormonal resuscitation (HR) has been reported to stabilize and improve cardiac function in brain-dead donors. The goal of this study was to examine the effect of HR on the brain-dead donor on the number of organs transplanted per donor. METHODS: A retrospective analysis of all brain-dead donors recovered in the United States from January 1, 2000, to September 30, 2001, was conducted. HR consisted of a methylprednisolone bolus and infusions of vasopressin and either triiodothyronine or L-thyroxine. Univariate analyses and multivariate logistic regression analyses were used to detect differences between the HR group and those donors who did not receive HR. RESULTS: Of 10,292 consecutive brain-dead donors analyzed, 701 received three-drug HR. Univariate analysis showed the mean number of organs from HR donors (3.8) was 22.5% greater than that from nonhormonal resuscitation donors (3.1) (P <0.001). Multivariate analyses showed that HR was associated with the following statistically significant increased probabilities of an organ being transplanted from a donor: kidney 7.3%, heart 4.7%, liver 4.9%, lung 2.8%, and pancreas 6.0%. Extrapolation of these probabilities to the 5,921 brain-dead donors recovered in 2001 was calculated to yield a total increase of 2,053 organs. CONCLUSION: HR stabilizes certain brain-dead donors and is associated with significant increases in organs transplanted per donor.

Mouse vascular endothelium activates CD8+ T lymphocytes in a B7-dependent fashion.

Despite several studies examining the contribution of allorecognition pathways to acute and chronic rejection of vascularized murine allografts, little data describing activation of alloreactive T cells by mouse vascular endothelium exist. We have used primary cultures of resting or IFN-gamma-activated C57BL/6 (H-2(b)) vascular endothelial cells as stimulators and CD8(+) T lymphocytes isolated from CBA/J (H-2(k)) mice as responders. Resting endothelium expressed low levels of MHC class I, which was markedly up-regulated after activation with IFN-gamma. It also expressed moderate levels of CD80 at a resting state and after activation. Both resting and activated endothelium were able to induce proliferation of unprimed CD8(+) T lymphocytes, with proliferation noted at earlier time points after coculture with activated endothelium. Activated endothelium was also able to induce proliferation of CD44(low) naive CD8(+) T lymphocytes. Activated CD8(+) T lymphocytes had the ability to produce IFN-gamma and IL-2, acquired an effector phenotype, and showed up-regulation of the antiapoptotic protein Bcl-x(L). Treatment with CTLA4-Ig led to marked reduction of T cell proliferation and a decrease in expression of Bcl-x(L). Moreover, we demonstrate that nonhemopoietic cells such as vascular endothelium induce proliferation of CD8(+) T lymphocytes in a B7-dependent fashion in vivo. These results suggest that vascular endothelium can act as an APC for CD8(+) direct allorecognition and may, therefore, play an important role in regulating immune processes of allograft rejection.

Mechanism of T cell-mediated endothelial apoptosis.

BACKGROUND: Cytotoxic T lymphocyte (CTL)-mediated destruction of allogeneic vascular endothelium is important in the pathogenesis of both acute and chronic allograft rejection. Despite the importance of this phenomenon, the effector mechanisms responsible for endothelial cell killing are not well defined, and conflicting conclusions have been reached based on variation in experimental methodology. METHODS: We used a recently described method for isolating mouse vascular endothelium to evaluate endothelial cell lysis by CTLs. Endothelial cell destruction was assessed in vitro both by 51Cr release and DNA fragmentation using wild-type and lpr (Fas deficient) endothelium of C3H/HeJ (H2(k)) mice by MHC alloantigen-specific T cells of wild-type, gld (Fas ligand deficient), and perforin-deficient mice on a C57BL/6 (H2(b)) background. RESULTS: Although maximal lysis of 56.6+/-0.8% was seen when using wild-type targets and effectors, only a moderate decrease in apoptosis to 37.6+/-4.0% was detected when the Fas/Fas ligand death receptor pathway was eliminated. This decrease in cytotoxicity occurred despite the preserved functional capacity of this pathway. Alternatively, a significant decrease in cytotoxicity to 17.4+/-4.7% was seen when the perforin/granzyme exocytosis pathway was eliminated. CONCLUSIONS: These data indicate that CTLs destroy vascular endothelium primarily by the perforin/granzyme exocytosis pathway with only a minor contribution to apoptosis by the Fas/Fas ligand death receptor pathway. These data are critical for the proper interpretation of studies evaluating acute and chronic allograft rejection and for the design of rational strategies to ameliorate vascular injury concomitant to the rejection process.