Jochum Prop

LATE AIRWAY CHANGES CAUSED BY CHRONIC REJECTION IN RAT LUNG ALLOGRAFTS

Airway disease after lung or heart-lung transplantation is one of late major complications, affecting the prognosis of the transplants. Little is known about the causes of airway changes. We performed rat lung transplantation and investigated the late airway changes of the long-term surviving lung grafts: allografts, BN to Lewis; isografts, BN to BN rat. All recipients were treated with CsA. We found airway changes, i.e., mucosal ulceration, granulation, submucosal fibrosis, which was located in the large airways, in four of five allografted lungs. The lung isografts showed no pathological abnormalities. Immunopathological studies disclosed the localized expression of MHC class II antigens on the bronchial epithelium of the large airways where recipient type dendritic cells accumulated in the submucosa and CD4 positive predominant lymphocytes infiltrated. These findings support the idea that the late airway changes in lung transplants are caused by immunologically mediated chronic rejection.

RESPIRATORY VIRAL-INFECTIONS AGGRAVATE AIRWAY DAMAGE CAUSED BY CHRONIC REJECTION IN RAT LUNG ALLOGRAFTS

Airway damage resulting in bronchiolitis obliterans occurs frequently in patients after heart-lung and lung transplantation. Generally, chronic rejection is assumed to be the most important cause of bronchiolitis obliterans. However, viral infections might also be potential causes of airway damage after lung transplantation. In the present study, we investigated whether viral infections could induce airway damage in rat lung transplants in the absence or presence of chronic rejection. We compared the histopathology of the airways in 3 groups of rats: (1) nontransplanted LEW lungs, (2) LEW-to-LEW syngeneic lung transplants, and (3) BN-to-LEW allogeneic lung transplants. Nontransplanted and transplanted rats were treated with CsA to induce permanent graft acceptance of the allografts. Six months after transplantation, 4 noninfected rats of each group were killed for histological investigation (another 4 noninfected allografted rats were killed 56 days later). The remaining 16 rats in each group were infected with Sendai virus (parainfluenza type 1) intratracheally. These rats were killed for histological investigation 4, 7, 21, and 56 days after infection. In the lungs of the noninfected rats of the nontransplanted and syngeneically transplanted groups, airway changes were absent. After viral infection in these lungs, mild inflammation developed in the airways that was transient and completely resolved by day 56 after infection. In contrast, in the allogeneically transplanted lungs the viral infection caused severe and permanent damage of the airways. In the bronchioles and the large airways throughout the allogeneic lung transplants, inflammation with epithelial necrosis and formation of granulation tissue was present. On day 56 after infection, the bronchioles showed scarring in the submucosa and obliteration of the lumen, typical features of bronchiolitis obliterans. This study shows that a respiratory viral infection aggravates the airway damage in rat lung allografts with chronic rejection. The findings suggest that viral infections and chronic rejection play a synergistic role in the development of bronchiolitis obliterans after human heart-lung and lung transplantation: the virus infection may stimulate chronic rejection and rejection may hamper the local defense against the virus.

Lung allograft rejection in the rat. I. Accelerated rejection caused by graft lymphocytes

To find out to what extent rejection of lungs differs from that of other organs, functional rejection of lung allografts was studied in five combinations of inbred rat strains. Rejection could be monitored accurately by perfusion scintigraphy, and equally well by chest roentgenography. The rejection of lung grafts was found to proceed remarkably fast, when compared with heart grafts, in combinations with strong RT1-incompatibilities. This accelerated rejection pattern could be converted into rejection at a normal pace by pretreatment of the donor with 10 Gy roentgen irradiation one day before transplantation. Donor pretreatment depleted the lung grafts bronchus-associated lymphoid tissue (BALT) of lymphocytes. When grafts were depleted of all other passenger cells as well–by retransplantation from a cyclosporine-treated intermediate host–they showed an even more reduced immunogenicity, probably because of the loss of donor-type dendritic cells. These results indicate that lymphocytes from the BALT of lung grafts are capable of accelerating the rejection response.

EXPRESSION OF CLASS-II MAJOR HISTOCOMPATIBILITY COMPLEX ANTIGENS BY BRONCHIAL EPITHELIUM IN RAT LUNG ALLOGRAFTS

Variations in expression of class II major histocompatibility complex antigens on bronchial epithelial cells and vascular endothelium were investigated in normal rat lungs and allografted lungs during acute rejection and after cyclosporine (CsA) treatment. BN (RT1n) left lungs were transplanted into LEW (RT11) recipients. Lungs were excised during acute rejection in untreated rats on postoperative days 1 through 5, and after CsA treatment (25 mg/kg on days 2 and 3) on days 5 and 100. Cryostat sections were examined for class II antigen expression with an immunoperoxidase technique, using various monoclonal antibodies. In the normal lung, class II antigens were not expressed by epithelial or endothelial cells. In the allografts, induction of class II antigens closely correlated with the rejection process: on day 2, the ciliated bronchial epithelium was locally positive; it became uniformly positive with increasing cellular peribronchial infiltration on days 3 and 4. CsA treatment prevented class II antigen expression to a certain extent, leaving the bronchial epithelium weakly positive at 100 days. Endothelial cells were invariably negative for class II antigens in all allografted lungs. The class II antigens expressed on the bronchial epithelial cells were of graft origin, except for recipient-type class II molecules found on the ciliated surface in CsA-treated animals. We conclude that expression of class II antigens by bronchial epithelium is the result of a bronchus-directed rejection process, and hypothesize that such a rejection process may have caused bronchiolitis obliterans in several of the patients with combined heart-lung transplants. Important is the observation that class II molecules can be present on the membranes of cells that do not themselves produce these antigens.

Donor brain death aggravates chronic rejection after lung transplantation in rats

Background. Many recipients of lung transplants from brain-dead donors develop bronchiolitis obliterans, a manifestation of chronic rejection. It has been shown that brain death increases inflammatory mediators and accelerates acute rejection in kidney, liver, and heart transplants. In this study, the authors investigated the hypothesis that brain death increases inflammatory mediators in the donor lung and subsequently aggravates chronic rejection of the lungs after transplantation in rats. Methods. Brain death was induced in F344 rats by inflation of a subdurally placed balloon catheter. After 6 hr, donor lungs were assessed for influx of leukocytes, expression of cell adhesion molecules, and cytokine mRNA expression. For assessment of the lung after transplantation, lungs from brain-dead F344 rats were transplanted into WKY rats. Lung function after transplantation was monitored by chest radiographs during an observation period of 100 days. At the end of this period, the lungs were histologically examined; also, cytokine mRNA expression was measured. Lungs from ventilated living donors and living donors served as controls. Results. After 6 hr of brain death, influx of polymorphonuclear cells and macrophages and expression of vascular cell adhesion molecule-1 in the donor lungs was increased. After transplantation at postoperative day 100, the lung function was significantly decreased compared with allografts from living donors. In the lung allografts from brain-dead donors, histologic symptoms of chronic rejection were obvious, including severe intimal hyperplasia but without bronchiolitis obliterans. Interleukin-2 mRNA was significantly increased in allografts from brain-dead donors compared with living donors. Conclusions. This study shows that brain death induces an inflammatory response in the donor lung and subsequently aggravates chronic rejection after transplantation. This may explain the clinical difference in long-term function between lungs from cadaveric donors and living donors.

Lung Allograft Rejection In The Rat: Ii. Specific Immunological Properties Of Lung Grafts

The immunological mechanism of lung allograft rejection was studied in inbred rats, in order to explain the rapid progress of the rejection response against RT1-incompatible lung grafts. Histological appearances of the graft and of the recipients spleen were studied, migration patterns of graft and recipient lymphocytes were assessed, and titers of circulating alloantibodies were determined. Histologically, we discriminated four phases of the rejection response in lung grafts: sequentially the latent, vascular, alveolar, and destruction phases. Early in the vascular phase, recipient lymphocytes primarily infiltrated the bronchus-associated lymphoid tissue (BALT) of the graft, causing a local immune response. Concurrent with these local rejection phenomena in the graft, a strong systemic immune response developed in the recipients spleen, presumably induced by the great number of lymphocytes that migrated from the grafts BALT into the recipients lymphoid tissues. We conclude that BALT facilitates a fast and intensive interaction between lung graft and recipient that is likely to accelerate the induction of the rejection response both locally in the graft and systemically in the recipients lymphoid organs.

MUCOSAL IMMUNOADJUVANT ACTIVITY OF LIPOSOMES: ROLE OF ALVEOLAR MACROPHAGES

Previously, we have reported on a liposomal adjuvant system for stimulation of both systemic IgG and mucosal s‐IgA responses against viral antigens (influenza virus subunit antigen or whole inactivated measles virus) administered intranasally to mice. Immune stimulation is observed with negatively charged, but not with zwitterionic, liposomes and is independent of a physical association of the antigen with the liposomes. Furthermore, liposome‐mediated immune stimulation requires deposition of the liposomes and the antigen in the lower respiratory tract. In the present study, it is shown that alveolar macrophages (AM) are the main target cells for negatively charged liposomes administered to the lungs of mice. AM isolated from animals, to which negatively charged liposomes were administered beforehand, showed large intracellular vacuoles, suggestive of massive liposome uptake. Under ex vivo conditions, both AM and RAW 264 cells exhibited a high capacity to take up negatively charged liposomes. The deposition of negatively charged liposomes, but not zwitterionic, liposomes in the lung reduced the phagocytic and migratory behaviour of AM, as assessed on the basis of transport of carbon particles to the draining lymph nodes of the lungs. Depletion of AM in vivo with dichloromethylene diphosphonate, facilitated an enhanced systemic and local antibody response against influenza subunit antigen deposited subsequently to the lower respiratory tract. In conclusion, these data provide support for the hypothesis that uptake of negatively charged liposomes blocks the immunosuppressive activity of AM, thereby facilitating local and systemic antibody responses.

THE COMBI-EFFECT - REDUCED REJECTION OF THE HEART BY COMBINED TRANSPLANTATION WITH THE LUNG OR SPLEEN

The term combi-effect was introduced to describe the phenomenon of a reduction in rejection of heart grafts after combined transplantation with the lung. In this study in rats we investigated whether the combi-effect was an immunological process and whether it could also be induced by combined transplantation of the heart with the spleen or with a lymphocyte-depleted spleen. Heart and spleen grafts were transplanted into the abdomen; left lungs were transplanted into the thorax of recipient rats. To deplete spleens of their lymphocytes, prospective donor rats were irradiated. Cyclosporine was injected once, on day 2 after transplantation. All heart allografts transplanted alone and treated with cyclosporine were rejected acutely (median survival time [MST] of 14.5 days). In contrast, after combined transplantation of a donor lung or spleen with the heart, almost all heart grafts survived indefinitely. Transplantation of a syngeneic lung or third-party spleen had little effect on heart graft survival (MST of

Lung allograft rejection in the rat. III. Corresponding morphological rejection phases in various rat strain combinations.

Rejection of RT1-incompatible lung grafts has been found in the study reported in our accompanying article to result in four consecutive morphological rejection phases: the latent, the vascular, the alveolar, and the destruction phase. The most prominent signs of rejection, however, occur early in the vascular phase in the bronchus-associated lymphoid tissue (BALT) of these grafts. In this study we investigated whether these four phases and the early rejection signs in BALT are universal phenomena of lung allograft rejection. Therefore, various donor-recipient combinations of inbred rat strains, incompatible for the MHC or for minor loci, were compared with respect to histological rejection phenomena--both in the lung graft and in the recipients spleen--and alloantibody formation. The four rejection phases appeared sequentially in grafts of all combinations. Duration of the phases depended on the degree of histoincompatibility of the graft. Again, BALT was involved early in the rejection process. During the vascular phase a strong immune response developed in the spleen, and in the alveolar phase antibodies circulated in the blood. We conclude that these morphological rejection phases are universal phenomena of the rejection process against lung allografts in rats. Corresponding phenomena have been described for other species, even in immunosuppressed recipients. Based on these data, a new concept of the universal rejection process of lung allografts is postulated.

Decreased donor-specific cytotoxic T cell precursor frequencies one year after clinical lung transplantation do not reflect transplantation tolerance: a comparison of lung transplant recipients with or without bronchiolitis obliterans syndrome.

BACKGROUND Decreased in vitro T cell alloreactivity, demonstrated by decreased frequencies of peripheral blood donor-specific T cell precursors, may reflect a tolerant state after transplantation and lower the risk for development of chronic graft dysfunction. It is unknown whether a decrease in donor-specific T cell frequencies also occurs after clinical lung transplantation and if such a decrease lowers the risk for bronchiolitis obliterans syndrome (BOS), a hallmark of chronic graft dysfunction. Therefore, we compared changes in posttransplant donor-specific cytotoxic T lymphocyte (CTLp) and helper T lymphocyte precursor (HTLp) frequencies in lung allograft recipients with good graft function and in recipients with BOS. METHODS Donor and third party specific CTLp and HTLp frequencies were determined by limiting dilution assay in pre- and posttransplant (1 year) peripheral blood samples of lung allograft recipients with good graft function (n = 13) and BOS (n = 10). RESULTS In recipients with good graft function, mean donor-specific CTLp frequencies decreased after transplantation (183 vs. 16 precursors before and after transplantation, respectively). Additionally, HTLp frequencies decreased but this was not specific for donor alloantigens because third party-specific HTLp frequencies decreased also. Surprisingly, recipients with BOS also showed a decrease in mean donor-specific CTLp frequencies after transplantation (332 vs. 49 precursors before and after transplantation, respectively). Again, HTLp frequencies decreased nonspecifically. CONCLUSIONS We conclude that donor-specific CTLp frequencies decrease after lung transplantation, but that this does not result in transplantation tolerance protecting the lung against the development of chronic graft dysfunction.