Human Lung Transplant Recipients Research Articles

Induction of IL-10 Suppressors in Lung Transplant Patients by CD4+25+ Regulatory T Cells through CTLA-4 Signaling

T cell-mediated autoimmunity to collagen V (col-V), a sequestered yet immunogenic self-protein, can induce chronic lung allograft rejection in rodent models. In this study we characterized the role of CD4+ CD25+ regulatory T cells (Tregs) in regulating col-V autoimmunity in human lung transplant (LT) recipients. LT recipients revealed a high frequency of col-V-reactive, IL-10-producing CD4+ T cells (T IL-10 cells) with low IL-2-, IFN-gamma-, IL-5-, and no IL-4-producing T cells. These T(IL-10) cells were distinct from Tregs because they lacked constitutive expression of both CD25 and Foxp3. Expansion of T IL-10 cells during col-V stimulation in vitro involved CTLA-4 on Tregs, because both depleting and blocking Tregs with anti-CTLA4 F(ab')2 mAbs resulted in loss of T IL-10 cells with a concomitant increase in IFN-gamma producing Th1 cells (TIFN-gamma cells). A Transwell culture of col-V-specific T IL-10 cells with Th1 cells (those generated in absence of Tregs) from the same patient resulted in marked inhibition of IFN-gamma and proliferation of T(IFN-gamma) cells, which was reversed by neutralizing IL-10. Furthermore, the T IL-10 cells were HLA class II restricted because blocking HLA class II on APCs resulted in the loss of IL-10 production. Chronic lung allograft rejection was associated with the loss of Tregs with a concomitant decrease in T IL-10 cells and an increase in T IFN-gamma cells. We conclude that LT patients have col-V-specific T cells that can be detected in the peripheral blood. The predominant col-V-specific T cells produce IL-10 that suppresses autoreactive Th1 cells independently of direct cellular contact. Tregs are pivotal for the induction of these "suppressor" T IL-10 cells.

Immune Mechanisms of Lung Allograft Rejection

Extended survival after lung transplantation is primarily limited by progressive airflow obstruction and fibrotic obliteration of the small airways, termed bronchiolitis obliterans syndrome (BOS) and bronchiolitis obliterans (BO), respectively. BO is thought to represent the pulmonary-specific manifestation of chronic allograft rejection and the end result of a spectrum of different immunological insults to the allograft. Historically, research has focused on the adaptive immune system and its cellular-based rejection as the driving factor in the development of BO. Recent research in animal lung transplant models and human lung transplant recipients has identified that chemokines, humoral immunity, autoimmunity, and innate immunity also contribute to lung allograft rejection and BO. This review explores the complex immunological mechanisms that promote the high rate of pulmonary allograft failure and significantly impair survival after lung transplantation. We also identify areas for further research critical to improving transplant outcomes.

244: Identification of a unique airway epithelial cell antigen defined by post-transplant sera from human lung transplant recipients with brochiolitis obliterans syndrome capable of protein kinase C activation and fibrotic growth factor production

244: Identification of a unique airway epithelial cell antigen defined by post-transplant sera from human lung transplant recipients with brochiolitis obliterans syndrome capable of protein kinase C activation and fibrotic growth factor production

Pirfenidone Inhibits Obliterative Airway Disease in Mouse Tracheal Allografts

Obliterative bronchiolitis (OB) is the histologic correlate of chronic airway rejection, which remains the most significant cause of death in long-term survivors of lung transplantation. Using an established murine heterotopic tracheal transplant model of chronic airway rejection, the effects of the oral anti-fibrotic agent pirfenidone on development of the OB-like lesion were evaluated. Tracheas from BALB/c mice were implanted into the sub-cutaneous tissue of C57BL/6 mice, and the allografts were evaluated morphologically for airway rejection changes and immunohistochemically for transforming growth factor (TGF)-beta at 16 or 28 days after transplantation. In addition, the potential additive effects of pirfenidone in combination with 2 immunosuppressive agents, cyclosporine or rapamycin, was evaluated. Compared with untreated controls, pirfenidone-fed mice showed less epithelial cell injury and luminal granulation tissue and fibrosis. Plasma TGF-beta levels and local TGF-beta expression based on immunohistochemistry were decreased in the pirfenidone-treated animals. Pirfenidone given on Day 9 or 16 post-transplant through Day 28 resulted in no significant improvement compared with controls. There was no significant additive effect of pirfenidone in combination with cyclosporine, whereas pirfenidone plus rapamycin demonstrated additive protection against the development of the obstructive airway lesion. In aggregate, these results show that the anti-fibrotic agent pirfenidone inhibits the development of the OB-like lesion in this mouse model of human chronic airway rejection, and that these effects may be mediated by TGF-beta. The results also suggest that pirfenidone may be worthy of investigation in human lung transplant recipients at high risk of developing OB.

Innate immune response is activated by TLR 4 signaling in human lung transplant recipients

Innate immune response is activated by TLR 4 signaling in human lung transplant recipients

Increased expression of CD40, B7–2, interleukin-15 and granzyme B in bronchoalveolar lavage cells from human lung transplant recipients

Purpose: Expression of co-stimulatory molecules (CD40 and B7–2), interleukin-15 (IL-15), and granzyme B have been shown to be up-regulated during allograft rejection of several organs. Thus, the goal of this study was to determine whether the expression of these costimulatory molecules and inflammatory mediators is also up-regulated during lung allograft rejection.

Expression of CD40 and granzyme B in bronchoalveolar lavage cells from human lung transplant recipients

Expression of CD40 and granzyme B in bronchoalveolar lavage cells from human lung transplant recipients

MODELING CHRONIC LUNG ALLOGRAFT REJECTION IN MINIATURE SWINE1

The success of lung transplantation has been limited by the perplexing problem of chronic rejection. The development of a large-animal model for the systematic study of the mechanisms underlying chronic lung rejection has been problematic. We have developed a new preclinical model of chronic lung rejection using MHC-inbred miniature swine. Using standard operative techniques, four orthotopic left lung allografts were performed using MHC-matched, minor-antigen-mismatched donors. Recipient animals received a 12-day course of postoperative cyclosporine. Grafts were followed with open biopsies and high-resolution computed tomography. Cellular immune responses were monitored by mixed lymphocyte reaction, cytometric analysis of graft-infiltrating lymphocytes, and skin grafting. All grafts survived > or = 5 months and developed manifestations of chronic rejection, including obliterative bronchiolitis, interstitial fibrosis, and occlusive vasculopathy. A mononuclear infiltrate was also present in all grafts by the fourth posttransplant month. High-resolution computed tomography demonstrated several cardinal radiographic findings known to correlate with chronic rejection. Cytometric analysis of graft-infiltrating lymphocytes showed a predominance of CD8+ cells. The development of alloreactivity in the host was confirmed by mixed lymphocyte reaction and skin grafting. We report a reproducible, whole-lung, large-animal model of chronic lung rejection. In this immunogenetically defined construct, we have observed a full spectrum of histopathologic lesions that reproduce with fidelity those lesions observed in human lung transplant recipients suffering from chronic rejection. We anticipate that this preclinical model will facilitate further study of the pathogenesis and therapy of chronic lung rejection.

Prolonged inhibition of obliterative airway disease in murine tracheal allografts by brief treatment with anti-leukocyte function-associated antigen-1 (CD11a) monoclonal antibody.

We have previously shown that anti-leukocyte function-associated antigen (LFA)-1 (CD11a) monoclonal antibody (mAb) prevents acute rejection and produces donor-specific unresponsiveness in murine recipients of heterotopic heart allografts. Here, we investigate the ability of this mAb to prevent the development of obliterative airway disease (OAD) in murine recipients of tracheal allografts. BALB/c tracheae were heterotopically transplanted into C3H mice. OAD developed by day 28 after transplantation and was characterized histologically by a loss of epithelial cell coverage and luminal obliteration of the tracheal allograft with a proliferation of fibrogenic mesenchymal cells, which is a lesion comparable to obliterative bronchiolitis in human lung transplant recipients. Monotherapy with anti-LFA-1 mAb preserved graft epithelium, prevented the development of OAD, and maintained unresponsiveness to donor antigen for more than 42 days after the final mAb administration. These findings suggest the potential for anti-LFA-1 mAb therapy to suppress both acute and chronic rejection in clinical lung transplantation.

Increased endothelin-1 associated with bacterial infection in lung transplant recipients.

Endothelin-1 (ET-1) has fibrogenic and inflammatory properties. Its pathogenic role in pulmonary fibrosis and certain inflammatory airway diseases is now well known. Its production is, in part, triggered by infectious processes. Episodes of infection are suspected to be involved in the development of bronchiolitis obliterans syndrome (BOS), which is the main feature of chronic lung rejection and the major factor limiting the long-term survival of transplanted patients. We postulated that ET-1 is upregulated during infectious complications arising from the graft and that this could partly explain the remodeling of airway structures observed in BOS. We, therefore, set up this study to assess ET-1 expression in relation to complications of the graft in human lung transplant recipients. ET-1 mRNA was quantified by reverse transcription-competitive polymerase chain reaction in cells from 119 samples of bronchoalveolar lavage (BAL) fluid from 17 lung transplant recipients. ET-1 and big ET-1 proteins were assessed in BAL cell culture supernatants by enzyme immunoassay. Transbronchial biopsies (n=21) were stained immunohistochemically for ET-1 receptors. Episodes of bacterial infection strongly correlated with increased ET-1 mRNA and protein expression. ET-1 receptors were also upregulated during these episodes, especially on endothelial and smooth muscle cells. Five of the seven patients with the highest ET-1 levels subsequently developed BOS. These results raise the possibility that ET-1, part of whose production is triggered by infectious postgraft complications, might play a role in the development of BOS through its potential effects on airway remodeling.

Development of bronchiolitis obliterans syndrome despite blood chimerism in human lung transplant recipients.

Bronchiolitis Obliterans Syndrome (BOS) remains the overwhelming obstacle to the success of lung transplantations (LTx). The presence of donor-specific microchimerism (DSM) and its association with lung allograft function is not well defined. To investigate the relationship between chimerism and BOS, blood was obtained from 21 LTx recipients. Genomic DNA was isolated from patient blood, and PCR-based techniques were used to identify recipient and donor HLA-DR. Fifty percent of the LTx recipients with BOS exhibited DSM at "T1" time post transplant, and 40% at one year follow-up (T2). However, 54% exhibited DSM in the BOS-free group at T1, and 44% at T2. Of the BOS-free, DSM-positive patients at T1, 29% developed BOS by T2. In contrast, 50% of BOS-free DSM-negative patients 50% developed BOS (P > 0.05). Double LTx had a higher prevalence of DSM (73%) and a lower prevalence of BOS (46%) than single LTx (50% and 80% respectively, P > 0.05). One-HLA-DR-antigen-matched LTx recipients show a low prevalence of DSM compared to non-matched (P < 0.05). This study demonstrates that the development of BOS in LTx recipients could also occur in the presence of blood chimerism.

Development of bronchiolitis obliterans syndrome despite blood chimerism in human lung transplant recipients

Development of bronchiolitis obliterans syndrome despite blood chimerism in human lung transplant recipients

Distribution of endothelin-1 in transplanted human lungs.

This study was designed to assess which cells in transplanted lungs express endothelin-1 (ET-1) and if expression of the peptide can be used to discriminate between rejection and infection in transplanted lungs. Transbronchial biopsies (n=104) from 29 human lung transplant recipients were stained immunohistochemically for ET-1. Cells expressing ET-1 (pneumocytes, endothelial cells, airway epithelial cells, lymphocytes, and macrophages) were quantified and correlated with clinical histopathology findings. ET-1 was expressed in airway epithelial cells (93% of the biopsies), infiltrating macrophages (86%), and lymphocytes (19%) but not in endothelial cells or pneumocytes. ET-1 expression did not vary with rejection, obliterative bronchiolitis, or infection. ET-1 expression did not correlate with age, grade of rejection, pulmonary function, or time after transplantation. In transplanted human lungs, ET-1 is expressed in airway epithelial cells and infiltrating macrophages, and expression does not vary with pathological processes. Therefore, immunostaining for ET-1 probably cannot be used to discriminate between rejection and infection in transplanted lungs.

Obliterative airway disease after heterotopic tracheal xenotransplantation: pathogenesis and prevention using new immunosuppressive agents.

The purpose of this study was to investigate whether obliterative bronchiolitis might occur after xenogenic pulmonary transplantation. A model for obliterative airway disease (OAD) after tracheal allograft transplantation in the rat undergoes tracheal obliteration with histologic features characteristic of obliterative bronchiolitis in human lung transplant recipients. Using this model, the pathogenesis of OAD and its prevention with immunosuppressive drugs was studied in rat recipients of hamster tracheal grafts. Tracheae from 30 hamsters (xenografts) or 23 Brown-Norway rats (allografts) were implanted and wrapped in the greater omentum of untreated Lewis rats. The grafts were removed on day 1, 3, 7, 14, 21, or 28 after transplantation and stained with hematoxylin and eosin and Masson's trichrome and by immunohistochemistry and immunofluorescence (IFL) techniques. In addition, 25 recipients were treated with cyclosporine (CsA, 10 mg/kg p.o.), leflunomide (LFM, 20 mg/kg p.o.), or rapamycin (RPM, 6 mg/kg i.p.) for 14 or 21 days (5 animals per treatment group). Visual and morphometric analyses were used to evaluate the extent of airway obliteration, luminal coverage by respiratory or flattened cuboidal epithelium, and extent and density of peritracheal cellular inflammation. In all xenografts, a neutrophilic infiltration of the mucosa and submucosa was observed from day 1 until day 14 and was associated with complete loss of tracheal epithelium by day 14. A marked peritracheal mononuclear cellular infiltrate mixed with plasma cells and eosinophils was seen on days 7 and 14. Both the extent of peritracheal inflammation and the density of the mononuclear cell infiltrate were significantly increased in xenograft tracheae when compared with the allografts. Tracheal obliteration began on day 14 and reached a maximum of 43% on day 21 with evidence of intraluminal fibrosis. In contrast to IFL of allografts, IFL of xenografts demonstrated marked deposition of rat immunoglobulin in the peritracheal tissue on days 7 and 14. The effects of treatment with immunosuppressive drugs on tracheal graft narrowing and protection of respiratory epithelium were as follows: After 14 days of treatment, the percentage of tracheal graft narrowing was 12%, 23%, and 19% in the no treatment, CsA, and LFM groups, respectively; the percentage of respiratory epithelium at 14 days was 0%, 21%, and 95%. After 21 days of treatment, the percentage of tracheal graft narrowing was 43%, 49%, 12%, and 5% for the no treatment, CsA, LFM, and RPM groups, respectively; the percentage of respiratory epithelium at 21 days was 0%, 39%, 86%, and 0%. Using computerized morphometry, the extent and densities of the peritracheal cellular infiltrates were significantly reduced in LFM- and CsA-treated groups when compared with untreated xenograft controls. LFM and RPM, but not CsA, significantly reduced the degree of luminal obliteration compared with no treatment (P<0.05). LFM and, to a lesser extent, CsA were able to prevent the loss of normal respiratory epithelium. Analysis by IFL revealed a marked decrease in rat immunoglobulin deposition in xenografts from LFM- and RPM-treated groups compared with xenografts from CsA-treated or untreated rats. (1) OAD occurs not only after tracheal allotransplantation but also after xenotransplantation. (2) Subepithelial infiltration of neutrophils and the appearance of plasma cells and eosinophils in the peritracheal infiltrates distinguished the histology of rejected xenografts from allografts. (3) Antibody deposition was detected by IFL only in xenografts. (4) Treatment with LFM or RPM significantly decreased the severity of luminal obliteration. Importantly, LFM also prevented the loss of respiratory epithelium.

Bronchoalveolar lavage fluid endotoxin elevation in human single lung transplant recipients during rejection

Endotoxin has been implicated as an aetiological factor in liver transplant rejection and acute graft-versus-host disease. To investigate the role of endotoxin in human single lung transplant rejection we measured the level of endotoxin in bronchoalveolar lavage (BAL) fluid from six subjects at baseline and during rejection, which was defined histologically from transbronchial biopsy. Differential cell counts of BAL fluid cells, the levels of protein and albumin in BAL fluid, and serum albumin levels were also examined. BAL fluid albumin to serum ratio was calculated to evaluate alveolar-capillary leakage. A significant elevation of BAL fluid endotoxin with rejection compared with baseline was observed. Standardized endotoxin levels to BAL fluid volume, protein, or albumin were all of similar significance. Examination of BAL fluid cell population revealed a significant elevation in the percentage of lymphocytes with rejection. No significance between BAL fluid protein levles, BAL fluid albumin levels, BAL fluid albumin to protein ratio, serum albumin levels, or BAL fluid albumin to serum albumin ratio was seen with rejection. We conclude that BAL fluid endotoxin levels increase during lung rejection, endotoxin levels can be accurately standardized to millilitres of BAL fluid, and abnormal alveolar-capillary leakage does not appear to account for the increased BAL fluid endotoxin.

Analysis of bronchoalveolar lavage from human lung transplant recipients by flow cytometry

Bronchoalveolar lavage (BAL) cells and peripheral blood leucocytes (PBL) from 24 lung transplant recipients were analysed for leucocyte subsets and expression of cell surface antigens. Total and differential white cell counts were performed on BAL, and lymphocyte subsets were evaluated in both BAL and peripheral blood. Measurement of immunofluorescence by flow cytometry was used to assess the percentage of: T cells (CD3+); T-helper cells (CD4+); T-cytotoxic/suppressor cells (CD8+); and activated lymphocytes (HLA − DR+). Lymphocyte subsets in BAL demonstrated marked differences to those in blood. A lower percentage of CD3+ and CD4+ lymphocytes were found in BAL, whereas CD8+ cells were more prevalent in BAL than in PBL. The mean CD4:CD8 ratio was significantly lower in BAL (1:1) than in blood (2·1:1). In the absence of pulmonary infection, there was a trend for a lower CD4:CD8 ratio in BAL associated with acute rejection (1·1:1) and obliterative bronchiolitis (1:1), when compared to the group with no evidence of rejection (1·4:1). In the absence of pulmonary rejection, pulmonary infection was associated with a marginally lower CD4:CD8 ratio in BAL (0·7:1), than when infection was absent (1·4:1). This difference was more evident in cases of cytomegalovirus (CMV) infection with a mean CD4:CD8 ratio of 0·3:1, compared to 1·5:1 in the absence of CMV disease ( P<0·05).

Plasma Endothelin-1 Levels in Human Lung Transplant Recipients

Plasma Endothelin-1 Levels in Human Lung Transplant Recipients

Plasma Endothelin-1 Levels in Human Lung Transplant Recipients

Endothelin-1 (ET-1) is a potent vasoconstrictor peptide produced by vascular endothelial cells and is believed to play an important role in the pathophysiology of the pulmonary vasculature. We have investigated the early changes in ET-1 plasma levels in lung transplant recipients at various intervals before and after isolated lung transplantation. The mean plasma ET-1 level immediately before transplantation was 0.8 +/- 0.1 pg/ml. This was significantly increased to 2.6 +/- 0.5 pg/ml shortly after unclamping and vascular reperfusion of the allograft, and remained high 4 h (2.3 +/- 0.5 pg/ml), 6 h (2.4 +/- 0.6 pg/ml), and 12 h (1.7 +/- 0.3 pg/ml) after transplantation. Thereafter, the level of ET-1 started to decline, reaching 1.3 +/- 0.3 pg/ml 24 h and 0.9 +/- 0.3 pg/ml 1 week after transplantation. This early increase in ET-1 plasma level is consistent with our observation of alterations in pulmonary vascular resistance, pulmonary artery pressure and cardiac output early after transplantation. These findings demonstrate that an increase in plasma ET-1 levels shortly after lung transplantation may have a detrimental effect during the postoperative course early after transplantation.

Cytokine gene expression in human lung transplant recipients.

The polymerase chain reaction was used to evaluate cytokine gene expression in bronchoalveolar lavage (BAL) cells and peripheral blood leukocytes in 31 human lung transplant recipients. All patients were maintained on a triple immunosuppression regimen consisting of CsA, AZA, and prednisone. Posttransplant survival ranged from 0.5 to 100.5 months (mean = 16.3 months). Cytokines IL-1 alpha, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, TNF-beta, and IFN-gamma were studied. In BAL, transcripts for IL-1 alpha, IL-7, IL-8, and TNF-beta were found in over 60% of samples and those for IL-5, IL-6, and IFN-gamma in 40-50%, while IL-2 and IL-4 mRNA were rarely found (< 20%). Considerable variation in the frequency of cytokine gene expression between BAL and peripheral blood was observed. When analyzed for the presence of acute pulmonary allograft rejection (without infection), transcripts for IL-4 and IL-6 in BAL demonstrated the greatest increase in frequency compared with nil rejection (P = 0.07 and P = 0.17, respectively). Pulmonary infection (without rejection) was associated with a modest increase in the expression of genes for IL-1 alpha and IFN-gamma (> 10%). Transcripts for IL-4 were not found in association with pulmonary infection, suggesting that this cytokine may be useful as a discriminatory rejection marker.

Photofiberoptic bronchoscopic findings during lung transplant rejection.

The role of flexible bronchoscopy in detecting rejection following lung transplantation was studied at four- to 10-day intervals in 47 dogs, including 31 allograft recipients, ten autograft recipients, and six controls without operations. Anastomotic narrowing inevitably occurred within the first postoperative week. After allografting without immunosuppression, anastomotic narrowing was progressive. Despite rejection, segmental and subsegmental bronchi were remarkably normal. During rejection delayed by immunosuppression, there was a 33% reversibility rate for anastomotic narrowing. Bronchial biopsies were not helpful. Mucosal exudate and pus were regularly seen when pneumonia was the cause for graft failure. We conclude that progressive bronchial anastomotic narrowing is a manifestation of rejection. Bronchoscopy will be helpful in management of human lung transplant recipients by identifying anastomotic stenosis and early endobronchial signs of pneumonia.