Cynthia R. Wagner

Cardiac Allograft Vasculopathy Association With Cell-Mediated but Not Humoral Alloimmunity to Donor-Specific Vascular Endothelium

BACKGROUND Cardiac allograft vasculopathy (CAV) is an accelerated form of coronary artery disease responsible for the majority of late deaths after cardiac transplantation. Although most consider this complication a manifestation of chronic allograft rejection, it has not been established whether this disease is a consequence of humoral or cell-mediated alloreactivity. METHODS AND RESULTS Human aortic endothelial cells (HAECs) were isolated from donor aortas obtained at the time of organ acquisition for 52 cardiac allograft recipients. Serum and peripheral blood mononuclear cells were obtained from these 52 allograft recipients at several time points during the first year after transplantation. Lymphocyte proliferation (LP) in response to donor-specific HAECs and alloantibody binding to interferon-gamma-treated donor-specific HAECs were performed and correlated with clinical parameters, including HLA matching, acute cellular rejection, and coronary artery disease on surveillance angiography. Ten of the 52 patients studied had angiographic or autopsy evidence of coronary artery disease in the first posttransplantation year (CAV+ group). The CAV+ group had higher LP responses to their donor HAECs at 1 week, 3 months, and 6 months after transplantation compared with the CAV- group (1 week: 1439 +/- 222 versus 824 +/- 141 counts per minute [cpm], P = .026; 3 months: 1282 +/- 388 versus 884 +/- 94 cpm, P = .07; 6 months: 2504 +/- 635 versus 1540 +/- 209 cpm, P = .036; CAV+ versus CAV-, respectively). Only 8 of the 52 patients had donor-specific alloantibodies, and there was no relation between antibody presence and CAV. Other clinical parameters that correlated with CAV included the level of HLA-DR mismatch and the presence of late acute rejection. CONCLUSIONS CAV is associated with donor-specific cell-mediated alloreactivity to vascular endothelium. Humoral immunity does not appear to have a major role in this disease.

The mechanism of antigen presentation by endothelial cells.

Endothelial cells line the vessels and lymphatics forming a barrier between circulating T cells and the extravascular tissue site of antigen. We have suggested that circulating T cells recognize antigen on the surface of endothelial cells, resulting in the activation of the endothelium such that the endothelial cells then release the key mediators of a cell-mediated immune response. To test this hypothesis, we have evaluated the extent to which endothelial cells can signal antigen-specific T cell activation. We have shown that cultured endothelial cells are as effective as macrophages in lymphocyte activation and that this activation is HLA-DR restricted. In additional experiments we have established that endothelial cells synthesize both Ia and IL-1 early in the signaling process. To eliminate any possible contribution of other cell types participating in the T cell-endothelial cell interaction, we have shown that cloned endothelial cells present antigen to cloned T cells. Moreover, there appeared to be a preference of selected T-cell populations for different types of antigen presenting cells. These experiments document that endothelial cells are independently competent antigen presenting cells.

Cytomegalovirus-induced regulation of major histocompatibility complex class I antigen expression in human aortic smooth muscle cells

Cardiac allograft vasculopathy (accelerated transplant atherosclerosis) is considered by most to involve a chronic allogeneic immune response to one or more constituents in the coronary vascular wall. Recent evidence suggests that there is an association between cytomegalovirus infection and the development of cardiac allograft vasculopathy (CAV). To determine whether CMV directly infects and/or potentially influences immunogenicity of vascular tissue, human umbilical vein (HU-VECs) or human aortic (HAECs) endothelial cells and human aortic smooth muscle cells (HASMCs) were isolated, cultured, and infected with CMV strain AD 169. Infection was detected using an immunoperoxidase-labeled monoclonal antibody to CMV immediate-early antigen (L-14). The presence and relative quantity of MHC class I and II antigens were determined flow cytometrically using monoclonal antibodies to monomorphic class I and class II HLA determinants. Gamma interferon was used as a positive control stimulant for the upregulation of MHC determinants. Both pooled HUVECs as well as 2 cell lines of HAECs served as targets for CMV infection though less than 10% of the cells were infected despite inocula of 10 pfu/cell. Infection of the pooled HUVECs resulted in no significant changes in the cell surface density of either MHC class I or II determinants. In contrast, HASMCs were excellent targets for CMV infection with virtually 100% of cells infected. CMV infection of 2 distinct HASMC cultures resulted in an increase of 254 +/- 158 relative fluorescence units (RFUs) in MHC class I antigen expression, as assessed by fluorescence intensity, in a variable portion of the HASMCs. A second population of cells exhibited a decrease of 73 +/- 16 RFUs in MHC class I antigen expression. No significant change in MHC class II antigen expression was noted. These results demonstrate that while HUVECs and HAECs are targets of CMV infection, human aortic smooth muscle cells can more readily be infected by CMV. Furthermore, CMV can regulate smooth muscle cell MHC class I expression, hence potentially altering immunogenicity. A pathophysiologic link between cardiac allograft vasculopathy and CMV disease can therefore be hypothesized.

Expression of I-region-associated antigen (Ia) and interleukin 1 by subcultured human endothelial cells☆

Activation of T cells requires three signals from an antigen-presenting cell: antigen, Ia determinants (HLA-D region determinants in man), and interleukin 1 (IL-1). Recent evidence has suggested that macrophages, dendritic cells, epidermal Langerhans cells, and endothelial cells can each function as antigen-presenting cells (APC). If these cell types can independently function as APC, they should synthesize Ia determinants and secrete IL-1. To determine if endothelial cells fulfill these requirements, we have propagated human umbilical vein endothelial cells by serial subculture for extended periods of time and assessed Ia expression and IL-1 secretion. The endothelial cells were subcultured for 8 months (approximately 20 subcultures) and were found to display classic morphology and immunofluorescent staining for the endothelial cell-specific marker Factor VIII-related antigen. In a separate paper we have shown that these subcultured endothelial cells can present antigen to T cells in a HLA-D region-restricted fashion (C. R. Wagner, R. M. Vetto, and D. R. Burger, Subcultured human endothelial cells can independently function as fully competent antigen-presenting cells, accepted for publication, Hum. Immunol.). In this paper we present evidence demonstrating that extensively subcultured endothelial cells biosynthesize both HLA-DR and HLA-DS molecules after exposure to T cells and antigen or to a supernatant from antigen-activated T cells. Evidence is also presented that when endothelial cells are cultured in the presence of lipopolysaccharide they secrete a molecule(s) with IL-1 activity as assayed by LBRM-33-IA5 cell line production of interleukin 2.

Subcultured human endothelial cells can function independently as fully competent antigen-presenting cells

Recent evidence has suggested that dendritic cells, epidermal Langerhans cells and endothelial cells (EC) as well as macrophages, fulfill the requirements of antigen-presenting cells. Despite a variety of controls, one weakness in the evidence that these latter cell types can independently serve as antigen-presenting cells is that the cell preparations may contain small numbers of contaminating macrophages or other cell types. The experiments described in this paper are directed towards providing firm evidence that human EC are independently capable of presenting antigen to T cells. EC were isolated from human umbilical veins and maintained continuously by serial subculture for periods of up to 8 months. The subcultured EC displayed classic EC morphology and uniform immunofluorescent staining for Factor VIII-related antigen. The subcultured EC (tested to the 18th subculture) presented both particulate and soluble antigens to macrophage-depleted T cells with an efficiency equivalent to freshly isolated cells. Monoclonal antibodies to HLA-DR and HLA-DS determinants inhibited antigen presentation by either autologous macrophages or EC. In addition, antigen presentation by the subcultured EC was not affected by the macrophage-specific monoclonal antibody Mac-120, which inhibited antigen presentation by autologous macrophages in the same experiments. These results are consistent with human EC being able to independently function as fully competent antigen-presenting cells.

Elimination of donor-specific alloreactivity prevents cytomegalovirus-accelerated chronic rejection in rat small bowel and heart transplants

BACKGROUND The primary cause for late failure of vascularized allografts is chronic rejection (CR) characterized by transplant vascular sclerosis (TVS). Cytomegalovirus (CMV) infection accelerates TVS and CR by unclear mechanisms involving direct effects of CMV, indirect effects of the recipients immune response to CMV, or interactions between CMV and the recipients alloreactivity. This study examined the role of CMV and the alloreactive response in the development of TVS using bone marrow chimerism (BMC) in rat small bowel (SB) and heart transplantation models. METHODS Fisher 344 (F344) rat heart or SB grafts were transplanted into F344/Lewis bone marrow chimera. F344 heart or SB grafts transplanted into Lewis recipients (low-dose cyclosporine) were positive controls for the development of TVS. Lewis heart or SB grafts transplanted into Lewis recipients (+/-cyclosporine) were transplantation controls. The effect of rat CMV (RCMV) (5x105 plaque-forming units) on TVS (neointimal index, NI) and graft survival was studied in these groups. RCMV infection was assessed by serologic analysis and quantitative polymerase chain reaction techniques (TaqMan). RESULTS RCMV infection accelerated the time to graft CR (SB 70-38 days; hearts 90-45 days) and increased the severity of TVS in both the SB allografts (day 38, NI=27 vs. 52) and the heart allografts (day 45, NI=43 vs. 83). Grafts from CMV-infected syngeneic recipients failed to develop TVS and CR. Donor-specific tolerance induced by BMC prevented allograft TVS and CR in both transplant models. In contrast to naïve Lewis recipients, RMCV infection failed to cause allograft TVS and CR in bone marrow (BM) chimeras. CONCLUSIONS The events in CMV-induced acceleration of TVS involve a crucial interplay between CMV infection and the recipients alloreactive immune response.

Cardiac allograft vasculopathy : Preferential regulation of endothelial cell-derived mesenchymal growth factors in response to a donor-specific cell-mediated allogeneic response

We have previously reported that cell-mediated immunity to vascular endothelium is associated with the development of cardiac allograft vasculopathy (CAV). The mechanism by which a cell-mediated immune response to the coronary vascular is translated into the development of CAV is, however unknown. Peripheral blood mononuclear cells (PBMCs) obtained serially following cardiac transplantation were cocultured with donor-specific human aortic endothelial cells (HAECs) in 47 allograft recipients, 9 of whom had CAV (CAV+) at 1 year by angiography. At 20 hr following coculture, HAEC poly (A+) RNA was isolated, reverse-transcribed, and the cDNA-amplified (PCR) for a panel of growth factors (GFs) known to alter smooth muscle cell proliferation or migration. Relative quantitation of PCR product was performed using high-pressure liquid chromatography (HPLC). Three patterns of GF regulation were observed depending on the GF, the time posttransplant, and whether the patient had CAV: (1) no regulation (TGF-beta, PDGF-A early post-tx); (2) upregulation irrespective of CAV (bFGF, PDGF-B, TGF-alpha early post-tx); and (3) preferential or exclusive upregulation by CAV+ patients (PDGF-A and TGF-alpha late post-tx, HB-EGF early and late post-tx). For example, using PBMCs as stimulators, obtained 6 months posttransplant from CAV+ patients, increases in HAEC-derived PDGF-A chain (31 +/- 7 to 69 +/- 11), TGF-alpha (97 +/- 27 to 201 +/- 23), and HB-EGF (78 +/- 16 to 173 +/- 27) mRNA were demonstrated (all P<0.05 or greater using HPLC peak area as units). These data demonstrate that cell-mediated activation of vascular endothelial cells in patients with CAV results in preferential upregulation of certain endothelial-derived mesenchymal growth factors capable of stimulating smooth muscle cell proliferation and migration.

Tolerance induced by bone marrow chimerism prevents transplant vascular sclerosis in a rat model of small bowel transplant chronic rejection.

BACKGROUND The major impediment to success in solid organ transplantation is chronic rejection (CR). The characteristic lesion of CR is transplant vascular sclerosis (TVS). Although the mechanism of TVS is thought to have an immunologic basis, in humans immunosuppression does not prevent or reverse it. One possible therapy to prevent TVS is induction of donor-specific tolerance. Bone marrow chimerism has been successful in inducing tolerance in acute and chronic rejection heart and kidney transplant models. The highly immunogenic small bowel (SB) allograft provides a rigorous test of the efficacy of this tolerance regimen. We examined whether induction of tolerance by bone marrow chimerism could prevent TVS in a model of Fisher 344 (F344) to Lewis (LEW) rat SB transplantation. METHODS Bone marrow chimeras (BMC) were created by transplantation of T-cell-depleted F344 bone marrow into irradiated LEW rats. Chimerism was assessed by flow cytometric method. F344 SB, heterotopically transplanted into the chimeras, was clinically and histologically assessed for CR. F344 SB grafts, transplanted into cyclosporine-A-treated LEW recipients, served as control grafts for CR. RESULTS Cyclosporine-A-treated LEW rats chronically rejected F344 SB grafts. By contrast, the BMC group demonstrated tolerance and had long-term SB graft survival (>120 days) without TVS. The BMC demonstrated immunocompetence by prompt rejection of third party ACI (RT1av1) SB allografts. CONCLUSIONS Bone marrow chimerism prevents chronic graft failure secondary to TVS in a model of chronic SB rejection. TVS fails to develop when tolerance is established, suggesting that the mechanisms involved in TVS are, in part, immunologically mediated.

A rat small bowel transplant model of chronic rejection: Histopathologic characteristics

BACKGROUND The major impediment to long-term success in solid organ transplantation is the development of chronic rejection (CR). The vascular lesion of CR, transplant vascular sclerosis (TVS) is characterized by neointimal smooth muscle cell proliferation, and is driven by both immune- and nonimmune-mediated mechanisms. Although the features of chronic heart and kidney allograft rejection have been well characterized, the more immunogenic small bowel allograft has not received similar study. METHODS F344 small bowel (SB) was transplanted heterotopically into Lewis recipients that were treated with low-dose Cyclosporine A for 15 days. Lewis recipients of F344 or Lewis SB grafts without immunosuppression, served as controls. Grafts were assessed histologically when recipients showed clinical signs of rejection or at predetermined time points. The immunological components involved in the chronic rejection process were evaluated by immunohistochemical staining. RESULTS All SB allografts (100%) developed histologic evidence of CR Cyclosporine A. TVS was seen in 36 of the 46 (78%) of these allografts. The median time to develop TVS was 45 days. Immunohistochemical staining of chronically rejected grafts showed infiltration predominantly by CD4+ cells and macrophages, uniform up-regulation of class II MHC molecule expression, moderate to intense ICAM-1 staining in grafts harvested at postoperative day 45, and uniform neointimal cell staining for smooth muscle cell alpha-actin in the TVS lesions. CONCLUSIONS This F344 to Lewis SB transplant model is a useful model that reproduces significant features of CR. The highly immunogenic nature of the SB allografts allows this model to serve as a stringent test for protocols designed to prevent CR.

Synthetic Peptide Dendrimers Block the Development and Expression of Experimental Allergic Encephalomyelitis

Multiple Ag peptides (MAPs) containing eight proteolipid protein (PLP)139–151 peptides arranged around a dendrimeric branched lysine core were used to influence the expression and development of relapsing experimental allergic encephalomyelitis (EAE) in SJL mice. The PLP139–151 MAPs were very efficient agents in preventing the development of clinical disease when administered after immunization with the PLP139–151 monomeric encephalitogenic peptide in CFA. The treatment effect with these MAPs was peptide specific; irrelevant multimeric peptides such as guinea pig myelin basic protein GPBP72–84 MAP (a dendrimeric octamer composed of the 72–84 peptide) and PLP178–191 MAP (a dendrimeric octamer composed of the PLP178–191 peptide) had no treatment effect on PLP139–151-induced EAE. PLP139–151 MAP treatment initiated after clinical signs of paralysis also altered the subsequent course of EAE; it limited developing signs of paralysis and effectively limited the severity and number of disease relapses in MAP-treated mice over a 60-day observation period. PLP139–151 MAP therapy initiated before disease onset acts to limit the numbers of Th17 and IFN-γ-producing cells that enter into the CNS. However, Foxp3+ cells entered the CNS in numbers equivalent for nontreated and PLP139–151 MAP-treated animals. The net effect of PLP139–151 MAP treatment dramatically increases the ratio of Foxp3+ cells to Th17 and IFN-γ-producing cells in the CNS of PLP139–151 MAP-treated animals.