Abstract
In lung transplantation, a substantial number of donor leukocytes are transferred from the donor to the recipient by the graft. Using a rat model, it was analyzed in this study to what extent leukocytes leave the lung, to which phenotype they belong, and to which organs they migrate. The model used was the orthotopic transplantation of the left lung of LEW.7B(RT7b) rats into LEW(RT7a) recipients. Lung allografts are not rejected in this strain combination, which differs only in the RT7 system, a genetic polymorphism of CD45. Using the RT7b marker (monoclonal antibody His41), the distribution of donor leukocytes passively transferred with the graft was studied by immunohistology 2 wk after transplantation. At this time, 2.9 ± 0.1% (n = 6) of the peripheral blood leukocytes in the recipients were derived from the donor lung. The donor cell population detected in the blood consisted of T cells (59 ± 4%), B cells (5.1 ± 0.2%) and a surprisingly high fraction of natural killer (NK) cells (36 + 3%). No monocytes or granulocytes were found. In lymph nodes, spleen and thymus donor-derived T- and B-cells could be shown in typical T- and B-areas, respectively. Donor-derived leukocytes were found in the liver and the skin. In the tissue and the bronchoalveolar lavage (BAL) of the host lung, predominantly T cells were found. Furthermore, in the donor tissue and BAL more than 70% of T- and B-cells were host type, demonstrating that the donor lung had been repopulated to a great extent by host lymphocytes. This supports the relevance of BAL as a diagnostic tool in lung diseases. Thus, the lung is an immunologically important site, releasing lymphocytes which migrate to other organs and also attracting many lymphocytes from the circulation.
After clinical lung transplantation, leukocytes have been shown to leave the lung transplant and to circulate in the blood of the recipient (1, 2). This is not surprising because large numbers of leukocytes residing in different compartments of the lung are transferred with the transplant (3). In the bronchoalveolar space of the human lung about 5 × 108 lymphocytes are found (4) and the lymphocyte number of the interstitial tissue has been estimated to be 1010 which is approximately the same number as in the entire circulating pool (5). The marginal pool of the human lung is similar in size to the interstitial pool or even larger (3, 6). An unknown number of lymphocytes is also present in the bronchial lamina propria and in the epithelium. The mechanisms underlying the compartmentalization of the lymphocytes in the lung and regulating known differences in subset composition are not clear.
By extracorporal perfusion of the graft routinely performed before transplantation, it is not possible to remove the cells in all lung compartments. This only reduces the free cells in the lung vessels and part of the marginal pool. The emigration of cells from the graft after experimental lung transplantation has previously been studied in an allogeneic rat model by radioactive labeling (7, 8). In the immediate postoperative period the migration of cells from the donor lung to the recipient was clearly demonstrated.
The aim of the present investigation was to study donor lymphocytes released from a single transplanted lung without the influence of rejection. In addition, the migration of lymphocytes into the lung should be studied. To identify all donor strain leukocytes a congenic rat strain was used that only differs in the RT7 allogeneic system, representing a genetic polymorphism of CD45 (leukocyte common antigen). The RT7 difference does not induce cellular immune responses or graft rejection (9, 10). So-called “genetically” marked RT7b+ leukocytes from the donor strain could be identified and distinguished by immunohistology without the need for separation or in vitro labeling techniques. In combination with cell surface staining, it was possible to characterize T-lymphocytes and T-cell subsets, natural killer (NK) cells and monocytes. In the blood, the donor leukocytes were quantified and phenotyped in several subsets. The thymus, lymph nodes, and spleen were examined as representative of the lymphatic system, while specimens of the liver and skin were analyzed as examples of nonlymphatic organs. Furthermore, the tissue and bronchoalveolar lavage (BAL) of the host lung were studied. The tissue and BAL of the donor lung were examined for immigrated lymphocytes derived from the host.
In six single lung transplantations performed in this study, male Lew(RT7a) rats, 10 to 12 wk old with a weight of 308 ± 55 g (mean ± SD), were used as recipients and six Lew.7B(RT7b) rats served as lung donors (four female, 238 ± 18 g, two male, 422 ± 2 g). The animals were bred under SPF conditions.
Transplantation of the left lung of Lew.7B rats into Lew recipients was performed as described previously (11). Briefly, the left lung of the donor rat was prepared under intubation anesthesia. The left main bronchus and the left hilus were stripped of lymph nodes and the left lung was removed. The explanted lung was perfused with 10 ml ice-cold Ringer solution via the pulmonary artery and stored on ice. Then the left lung of the recipient was removed. The donor lung was implanted; vascular anastomoses were performed first and the bronchial anastomosis last. The success of the anastomoses was proven and the operation lasted under 3 h. The ischemia time for the explanted lung did not exceed 30 min. Clinical monitoring and chest X-rays were performed on day 1 and day 7 after the transplantation. BAL was performed 14 days after transplantation separately for the left and the right lung to check for any possible influence of the surgical treatment (as described below in detail).
For the immunohistological analysis, the grafted rats were killed after 2 wk. The animals were narcotized and exsanguinated via the abdominal aorta. The blood was collected in heparinized tubes and red blood cells were removed by incubating each 0.5 ml in 10 ml ammonium chloride solution (0.83% NH4Cl) supplemented with 0.1 g/liter ethylenediaminetetraacetic acid (EDTA) for 10 min at room temperature. After centrifugation (400 × g, 15 min), the pellet was resuspended in 2 ml phosphate-buffered saline (PBS) (Biochrom KG, Berlin, Germany) containing 1% bovine serum albumine (BSA) (Serva, Heidelberg, Germany) and 0.1% NaN3. Cell numbers were determined using a Coulter Counter (Coulter, Luton, UK) and cytospots were prepared by centrifuging 1 × 105 cells for 8 min at 800 rpm in a Cytospin 3 cytocentrifuge (Shandon Scientific Ltd, Runcorn, UK). For the transplanted lung and for the host lung, BAL was performed separately (5 × 2 ml of 0.9% sodium chloride solution, 4°C) (12). Spillover of BAL fluid was prevented with particular care. BAL cytospots were stained with May-Grünwald and with monoclonal antibodies (mAbs) for T (R73) and B (His14) lymphocytes (see below). The total number of alveolar macrophages was determined by morphologic analysis. Specimens of thymus, spleen, axillary lymph nodes, liver, skin, and host lung were obtained, frozen in liquid nitrogen, and stored at −70°C. For further processing, the frozen tissue samples were immersed in O.C.T. embedding medium (Miles Inc., Elkhart, IN), and 8-μm thick sections were cut and placed on glass slides. Cytospots and tissue slides were air dried and stored at −20°C. The question whether the transplantation technique used is associated with cotransplantation of bronchial lymph nodes was examined in two additional donor animals. The lungs were prepared as for transplantation and then fixed in formaldehyde. Subsequently, they were examined for the presence of lymph nodes by serial sections stained with hematoxylin and eosin (H&E).
The monoclonal mouse antibody His41 (PharMingen, Hamburg, Germany), recognizing leukocytes from the Lew.7B strain only (11), was used to identify donor cells from the transplanted lung. Analysis of leukocyte subset markers was performed with mAb characterizing T-lymphocytes (R73), CD4+ cells (W3/25) and CD8+ cells (Ox8), B-lymphocytes (His14), NK cells (3.2.3), monocytes/macrophages (ED1), and granulocytes (RP1). The characterization of these mAbs has previously been summarized (13, 14). RP1 (15) was a generous donation of Dr. F. Sendo, Department of Immunology, Yamagata University, Japan. All other mAbs were purchased from Camon, Wiesbaden, Germany. To identify the different subsets of the leukocytes, a double staining technique combining alkaline phosphatase and peroxidase reactions was employed. Cytospots and sections were fixed in methanol and acetone for 10 min (−20°C) and thereafter incubated with the primary antibody for 30 min at room temperature, washed with TBS-Tween (0.05% Tween 20; Serva, Heidelberg, Germany), and then incubated for 30 min with a polyclonal rabbit antimouse immunoglobulin (Dako, Hamburg, Germany) functioning as bridging antibody. Then the APAAP-complex (30 min; Dako) was applied. In order to increase the staining intensity, the incubation with the bridging antibody and the addition of the APAAP-complex were repeated once. Then, biotinylated His41 (containing 20% mouse serum) identifying the donor leukocytes was added and incubated for 30 min, followed by streptavidin-peroxidase for 15 min. Diaminobenzidine (Sigma, St. Louis, MO) served as chromogen (detection of donor lymphocytes) and fast blue (Sigma) as the substrate for alkaline phosphatase (detection of the phenotype of the donor lymphocytes). Positive and negative controls produced the expected results and cross reactivities were excluded. To determine the total fraction of donor cells of peripheral blood leukocytes (PBL), at least 200 cells per animal were counted. The subset composition of this fraction in the recipient blood was examined by analyzing at least 200 donor cells per animal for each subset. In the BAL and tissue of the host lung and the donor lung, the fraction of donor and host leukocytes was determined by analyzing 200 cells per animal. The term “immigrated cells” refers not only to donor cells (His41+) in the host lung but also to host leukocytes (His41−) in the donor lung.
Results are presented as means ± SEM. The differences between group means were analyzed using the Wilcoxon test for paired data (SPSS-Windows 6.0.1.; SPSS Inc., Chicago, IL). Statistical significances were indicated for P < 0.05.
All animals recovered quickly after the operation, and clinical features such as breathing, body temperature, and weight were normal. By chest X-ray examination, it could be shown that all transplanted lungs were normally ventilated on days 1 and 7 after transplantation. Macroscopic inspection and histological examination of all transplanted lungs showed no signs of rejection or infection. Fourteen days after lung transplantation, the same total cell number was found in the BAL of the donor and the recipient lung (Table 1). The percentages of T- and B-lymphocytes in the BAL were likewise not significantly different (Table 1). Since there was no difference between male and female rats, the data were pooled.
| BAL | Transplanted Lung | Host Lung | ||
|---|---|---|---|---|
| Total cell count of nucleated cells (× 106/ml) | 1.1 ± 0.1 | 1.1 ± 0.1 | ||
| T-lymphocytes (%) | 3.4 ± 0.6 | 3.1 ± 0.4 | ||
| B-lymphocytes (%) | 2.1 ± 0.4 | 1.4 ± 0.5 | ||
| Alveolar macrophages (%) | 93 ± 2 | 93 ± 2 |
The His41 antibody allowed a clear differentiation of donor and recipient cells in the blood (Figure 1). From all PBL (5,100 ± 350 cells/μl) 2.9 ± 0.1% were identified as being derived from the donor lung. The subset composition of the donor-derived cell population 2 wk after transplantation has been analyzed by two-color staining (Figures 1 and 2). The fraction of T cells was of similar relative size to that in host PBLs. By contrast, the B-cell population of the donor cells was only about 20% of that found in recipient PBL (Figure 2). The fraction of monocytes within the donor cells was clearly also reduced in comparison with PBL. The fraction of the donor NK cells, however, was 4-fold higher than that found in recipient PBL (Figure 2). No granulocytes of the donor type were found. Thus, the overall cellular composition of the donor-derived cell population was markedly different from the subset composition of normal PBL.
Fig. 1. PBL on a cytospot are demonstrated in the double staining technique as described in Materials and Methods. The blue cells are NK cells (arrow), the yellow cells are donor type (arrowhead) and the greyish cells are NK cells derived from the donor lung (thick arrow; A, bright-field illumination). The phase-contrast microscopy (a) of the same region also shows several cells which are neither NK cells nor donor-derived cells. Similar to A, a donor T cell is demonstrated on a cryostat section of the spleen (arrowhead; B, bright-field illumination, red pulp). All bars represent 50 μm.
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Fig. 2. The composition of host PBL 14 days after transplantation is compared with the subset distribution in the donor PBL. No differences were observed for T cells, but fewer B-lymphocytes and monocytes (< 1%) and considerably more NK cells were present in the donor PBL than in the host PBL (*P < 0.05).
[More] [Minimize]In the lungs (n = 2) which were prepared as for transplantation and analyzed by serial sections and H&E staining, no bronchial lymph nodes were found in the hilar area. This indicated that the donor lymphocytes detected in the host tissues were derived from the transplanted lung tissue and not from transplanted lymph nodes.
Lung-derived donor cells were present in lymphoid organs such as the thymus, spleen, and axillary lymph nodes (Figures 3a through 3c). These cells were found in the spleen at a frequency of 2% in the periarterial lymphatic sheath (PALS) and less than 0.5% in the thymic medulla. In the thymus, the immigrated cells were located only in the medulla but not in the cortex (Figure 3a). These were T cells, mainly positive for CD4. In the spleen, lung-derived donor cells were preferentially found in the PALS (Figure 3b). Again, nearly all of these were CD4+ T-cells, whereas in the follicle and corona only B cells occurred. Cells in the axillary lymph nodes (Figure 3c) were identified as T cells in the paracortex and as B cells in the follicle and medulla. Thus, the lung-derived donor lymphocytes showed a compartment-specific distribution within the lymphoid organs. Leukocytes from the transplanted lung were also detected in the liver (Figure 4a), where mainly T cells occurred and only isolated B cells. In the skin, the morphology of donor cells was similar to that of dendritic cells (Figure 4b). Identification of these cells by the double staining technique described above is not possible. All these findings were constant in all animals studied.
Fig. 3. Leukocytes derived from the lung are demonstrated in cryostat sections of the thymus, (A, a), the spleen (B, b) and the axillary lymph node (C, c). In the thymus, immigrated cells are only seen in the medulla but not in the cortex. In the spleen these cells are preferentially detected in the periarterial lymphatic sheath (PALS) and in the lymph node in the paracortex. Bars on the left side represent 200 μm, bars on the right side represent 50 μm.
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Fig. 4. Stained cells in cryostat sections of the liver (A, a) and the skin (B, b), as nonlymphatic organs, as well as in the host lung (C, c) are donor- derived immigrated cells. Bars on the left side represent 200 μm, bars on the right side represent 50 μm.
[More] [Minimize]Donor cells were also present in lung tissue of the host (Figure 4c) and the BAL (Figure 5). In the BAL of the host lung, 0.2 ± 0.1% of all nucleated cells were donor cells. These immigrated cells were exclusively T lymphocytes (Figure 6). No donor-derived B cells, NK cells, or macrophages were found in the BAL and tissue of the host lung. The vast majority of the T- and B-cells in the donor lung were not donor-type and therefore must have immigrated from the host. The fraction of immigrated T- and B-cells was comparable in the tissue and in the BAL of the donor lung (Figure 6). Furthermore, the B cells seemed to be exchanged more quickly (Figure 6).
Fig. 5. A donor leukocyte is shown on a cytospot of the BAL from the host lung in bright-field illumination (A) and with surrounding unstained macrophages in phase-contrast microscopy (a). Bars represent 50 μm.
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Fig. 6. The immigrated fraction of the T- and B-lymphocytes in the BAL is shown for the host and the donor lung. In the tissue and BAL of the host lung all immigrated cells were T-lymphocytes. In the tissue and BAL of the donor lung, the majority of T-and B-lymphocytes were host type and therefore exchanged after transplantation. B cells were exchanged significantly faster than T cells (*P < 0.05; for evaluation of the BAL cytology at least 50 T- and B-lymphocytes and in the lung tissue at least 200 T- and B-cells per animal were determined for the host/donor type to obtain the percentage of the immigrants).
[More] [Minimize]In clinical lung transplantation it has been shown that substantial numbers of donor-derived T cells, B cells, NK cells, and macrophages are transferred with the transplanted lung and can be found in the circulation during the early postoperative period (1). This observation and the finding that most patients with a successful graft develop a form of long-term hematopoietic microchimerism have raised significant interest in the fate of bone marrow-derived cells cotransplanted with organ grafts (2). In histocompatible allograft models without immunosuppression, the fate of these cells is strongly affected by rejection processes. If immunosuppression is applied, it is also likely to interfere with cell trafficking and the potential to home in recipient tissues. An animal model avoiding these difficulties is allotransplantation in congenic rat strains differing only in the RT7 system. This is a genetic polymorphism of the CD45 molecular family expressed on all nucleated bone marrow-derived cells. It permits a detailed analysis of donor-derived cells in various compartments of the recipients' organism by immunohistology and immunocytology. This is a prerequisite for the analysis of migratory routes and survival time of the donor cell population.
In concordance with the known failure of the RT7 allogenetic difference to induce allograft rejection, there was no sign of a rejection response in the lung transplanted animals studied here (10). This is supported by the good clinical condition of the animals and grossly normal X-ray findings after the transplantation. Furthermore, in the total and the differential cell count there were no differences between the BAL of the host and transplanted lung. In addition, macroscopical and histological findings of the transplanted lung were normal.
Although only one time point after transplantation was investigated in the present analysis, the data allow conclusions regarding the migratory routes of leukocytes. Nearly 3% of the circulating PBL were of donor origin. In this context, it is of interest that after intravenous injection of 108 fluorescein isothiocyanate (FITC)-labeled isogenic PBL (representing the number of the whole blood pool) into a rat, a comparable number of cells of the inoculum was found after 24 h (16). Therefore, a substantial number of cells must have left the donor lung for such a quantity to be found in the blood. B cells and monocytes were found less frequently and cells expressing the NK cell marker NKR-P1 much more frequently in the donor population than in the blood. It is not clear whether these cells represent an expanded NK cell population, or if T cells coexpressing the NKR-P1 molecule probably represent a distinct T-cell population (17). This high percentage was not just from the blood which had not been washed out by perfusion, but came from lung compartments, presumably from the larger compartments such as the marginal vascular pool and the interstitial pool. More time points for kinetics and longer postoperative periods should be used in further experiments to compare the migration pattern of lung- derived lymphocytes with that of the commonly used thoracic duct lymphocytes (18, 19).
The fact that a substantial number of donor-derived lymphocytes is still circulating two weeks after transplantation is most likely explained by entering the recirculating cell pool. This is in agreement with the finding of donor lymphocytes in all secondary lymphatic organs studied. The donor T and B cells were distributed in the T- and B-dependent areas of the spleen and lymph nodes in a compartment-specific manner. The release of various leukocyte subsets from the lung and their occurrence in the lymphatic organs might explain systemic immune responses after local vaccination or challenge of the lung (20).
Surprisingly, donor-derived cells immigrated into the medulla of the recipient thymus. This shows that the thymus not only exports lymphocytes but also receives lymphocytes from a nonlymphoid organ such as the lung. Furthermore, a recent study demonstrated that the spleen and other peripheral tissues also provide cells for the thymic medulla (21). Donor-derived lymphocytes in the thymic medulla after allogenic transplantation may influence graft acceptance (22).
Donor cells also may contribute to immune functions in the liver. For example, intrahepatic modulation of donor antigen-presenting cells influenced T-cell responsiveness (23, 24). Lung-derived cells within the skin may be relevant to triggering atopic dermatitis by inhalative allergens (25). However, it has to be cleared up whether the cells found in the skin after lung transplantation are dendritic T cells or antigen-presenting dendritic cells. Lung-derived T-lymphocytes also reached the tissue and BAL of the host lung. This is different from the results of other migration experiments in which hardly any cells were found in the BAL after a single intravenous injection of labeled cells (26) demonstrating a turnover of lymphocytes in the bronchoalveolar space after persistence of cells in the blood stream. Cellular traffic between the lungs has also been shown indirectly after autotransplantation of the lung in the dog (27). These observations could be of interest with regard to cellular mechanisms in “transplanted asthma” (28). Donor B lymphocytes were not detectable in the host lung in our experiments which might be due to the low number. However, we cannot exclude that B cells of one lung migrate to the other. Another remarkable aspect was the high proportion of host T- and B-lymphocytes in the tissue and BAL of the donor lung two weeks after transplantation. This confirms a substantial physiologic turnover of lymphocytes in the lung. At present, it is not clear to what extent local proliferation and apoptosis contribute to the number of cells in the BAL. Surprisingly, B cells seem to be exchanged faster than T cells. The difference in donor cells between lung tissue and BAL cells was low, indicating a short transit time for T- and B-cells from the tissue to the bronchoalveolar space. This underlines the relevance of BAL as a diagnostic tool in lung diseases.
In conclusion, it has been shown that a combination of experimental lung transplantation with the RT7 marker system is a useful tool to study the migration of lymphocytes from and into the lung. The lung not only contributes lymphocytes to lymphatic organs including the thymus, but also receives many lymphocytes from the body. This may be relevant to the development of further diagnostic and therapeutic approaches.
This research was funded by the German Research Foundation (DFG Pa 240/7-1 and We 1175/4-1). Special thanks go to Karin Westermann and Annette Weiß for excellent technical help and Sheila Fryk for correction of the language.
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