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Keywords:

  • pulmonary immune system;
  • lung;
  • lymphocytes;
  • bronchoalveolar space;
  • migration;
  • bronchial lymph node;
  • rat

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

Lymphocytes in the bronchoalveolar space are routinely obtained and examined in lung diseases such as asthma or sarcoidosis. In a pig model, labeled lymphocytes were found in regional lymph nodes after intrabronchial instillation, indicating that reentry of lymphocytes from the bronchoalveaolar space into the body is possible. In the present study, the route and kinetics of the reentry of bronchoalveolar lymphocytes were investigated in a congenic rat model using immunohistochemistry on cryostat and semithin sections and confocal laser scanning microscopy. As early as 15 min after intratracheal instillation lymphocytes were found to leave the bronchoalveolar space by transmigration through alveolar but not bronchial epithelium and were observed in interstitial alveolar tissue. At 6 hr after intratracheal instillation, T and B lymphocytes appeared in the draining lymph nodes of the lung with an increase after 24 and 48 hr. The kinetic pattern clearly differed in nondraining lymph nodes and other organs. After 6 hr, only single cells were found in nondraining lymph nodes, spleen, and blood with a slight increase after 24 hr, and only occasionally were single cells seen in the liver, thymus, or Peyer's patches 24 and 48 hr after instillation. In conclusion, T and B lymphocytes can leave the alveolar space by reentry into the lung tissue through alveolar epithelium. They reach regional lymph nodes by means of lymphatic vessels and are then distributed all over the body to rejoin the systemic immune system. Coming into contact with environmental antigens, these lymphocytes could perform an important function in the lung immune system and might be a target for inhalative therapy. Anat Rec 264:229–236, 2001. © 2001 Wiley-Liss, Inc.

The lungs are permanently in contact with the outside world and confronted with a large number of antigens and potentially harmful infectious agents. The lymphocytes of the pulmonary immune system are located in different lung compartments, e.g., the intravascular pool, the interstitial pool and the bronchoalveolar pool. They recognize foreign antigens specifically and activate the effector mechanisms that are required for host defense. These cells and their ability to migrate between the circulation, sites of antigen exposure, and lymphoid tissue are of critical importance for the generation of the immune response (Pabst and Tschernig, 1995). The complex system of lymphocyte recruitment and recirculation in the lung is still incompletely understood. The function and fate of the lymphocytes in the bronchoalveolar space is of particular interest, because they are routinely obtained by bronchoalveolar lavage (BAL) fluid for clinical and study purposes. The dynamics of these bronchoalveolar lymphocytes are dependent on their entry, proliferation, apoptosis, and exit from this pool (Pabst and Tschernig, 1997). The immigration of lymphocytes into the bronchoalveolar space has been shown in animals, e.g., by chimerism after lung transplantation (Tschernig et al., 1997; Schuster et al., 2000). The bronchoalveolar space, however, seems not to be the graveyard for these cells. Intrabronchially instilled, labeled autologous blood lymphocytes were found 1 day later in the regional lymph nodes in a pig model (Pabst and Binns, 1995). Moreover, Tournoy and Pauwels (2000) recently showed that, in a severe combined immune-deficient (SCID) mouse model, intratracheally (i.t.) instilled human peripheral blood mononuclear cells could later be found in the lung tissue. Therefore, the aims of this study were to detect the anatomic route of lymphocyte reentry from bronchoalveaolar space into lung tissue and to investigate the kinetics and location of lymphocyte distribution over the body.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

Animals

Adult male Lewis rats (RT7a) (n = 23) with a mean weight of 237 ± 16 g were used as recipients. Lewis rats (RT7b) served as lymphocyte donors, in each case one donor for one or two recipient animals. Lymphocytes from a congenic rat strain that only differs in the RT7 allogenic system were used as inoculum to avoid the manipulation of donor cells by in vitro labeling. In this congenic combination, no rejection phenomena occur (Tschernig et al., 1997). The animals were kept under specified pathogen free conditions and had free access to water and rat chow.

Lymphocyte Separation

The RT7b animals were exsanguinated under ether anaesthesia. Several lymph nodes, e.g., the mesenteric, axillary, and cervical lymph nodes, were excised, and a suspension was prepared by gentle disruption of the lymph nodes on a steel mesh in 0.9% NaCl. After centrifugation and lysis of red blood cells by osmotic shock, the lymphocytes were suspended in RPMI 1640. Samples were taken to determine the absolute number of lymphocytes. To characterize the lymphocyte subsets within the inoculum cytocentrifuge preparations of the inoculum were immunocytologically stained using monoclonal mouse anti-rat antibodies against surface markers [B cells (His 14), T cells (R 73), CD4+ T cells (W 3/25), CD8+ T cells (Ox 8), macrophages and dendritic cells (ED1), and NK cells (3.2.3), all from Serotec, Wiesbaden, Germany; the antibodies and the dilutions have been described previously (Westermann et al., 1993)]. To determine the percentage for each subset, 500–1,000 cells were counted in the cytocentrifuge preparations.

Lymphocyte Instillation

For i.t. instillation, the RT7a rats were anesthesized with ether and 0.5 ml of inoculum (at a concentration of 150 × 106 cells/ml to study the site of lymphocyte reentry and 200 × 106 cells/ml to investigate the kinetics of lymphocyte distribution) was injected by means of a thin cannula into the trachea followed by 0.5 ml of air. The cannula was removed immediately and the animals recovered consciousness a few seconds later, so that the inoculum could disperse quickly throughout the lung. As a control, three of the RT7a rats received 0.5 ml of inoculum with 200 × 106 cells/ml not i.t. but intravenously (i.v.), injected into the lateral tail vein by using a venous cannula.

Localization of Lymphocyte Reentry Into the Lung Tissue

The location of lymphocyte entry into the lung interstitium was examined in five animals. Two of them were given cells labeled with fluorescein isothiocyanate (FITC) (Isomer 1, Sigma, Munich, Germany) or carboxyfluorescein diacetate (CFSE) (Molecular Probes, Eugene, OR) before instillation to follow the migration in semithin, Epon-embedded sections. In vitro labeled cells had to be used, because the immunohistological detection of the congenic RT7b cells in plastic-embedded sections produced inconstant results. For labeling, the donor cells were incubated with FITC (Willführ et al., 1989) for 15 min at 37°C in RPMI 1640 at a final concentration of 50 μg FITC/ml and 2% fetal calf serum or with CFSE for 30 min at 37°C (Schuster et al., 2000). The lungs of the other three animals were investigated by confocal laser scanning microscopy (CLSM) so that unlabeled RT7b cells could be used. Fifteen minutes after cell instillation the animals were killed by exsanguination by means of the abdominal aorta under ether anesthesia. The lungs were excised en bloc from the thoracic cage and were infused with 8 ml of a 30% solution of optimum cutting temperature compound (OCT) (Sakura, Tokyo, Japan) in phosphate-buffered saline (PBS). The lungs of the two animals which had received FITC/CFSE-labeled cells were prepared for Epon embedding. First, the lungs were cut into small slices, fixed with formalin (2%) and glutaraldehyde (0.05%) in PBS, and fixed for 4–6 hr at 7°C. The fixed slices were dehydrated in graded concentrations of ethanol and embedded in Epon (Serva, Heidelberg, Germany). Sections (1 μm) were cut and mounted on poly-L-lysin–covered slides. The lungs of the three animals that had received unlabeled cells were frozen in liquid nitrogen and stored at −70°C. For CLSM, 12-μm sections were cut and placed on glass slides, air-dried, stored at −20°C, and stained by using immunohistochemistry (see below). CLSM was performed by using a laser scanning microscope (Bio-Rad MRC 1000, Hertfordshire, UK; amplification with crypton-argon laser, 488 and 568 nm) as described previously (Markus et al., 1998). About 8 sections over a distance of 1 μm were obtained (20× magnification) and overlayed.

Kinetics of Lymphocyte Distribution

Experiment with BAL lymphocytes.

In this experiment, the use of BAL lymphocytes for the observation of the hypothesized reentry was tested. A BAL with 5 × 5 ml of ice-cold saline per animal was performed in 26 Lewis rats. The BALs were centrifuged, and the cells were pooled. A total cell number of 20 × 106 was obtained. For an adherence step, the cells were incubated on an immunoglobulin-coated plate for 50 min at 37°C and 5% CO2. The nonadherent cells (3.3 × 106) were CSFE labeled as described above and instilled intratracheally into a Lewis rat (female, 170 g). After 24 hr, the lung-draining lymph nodes were obtained and immunohistology was performed as described above.

Experiments with congenic cells from mesenteric lymph nodes.

To investigate the migration route to the draining lymph nodes, the cell number for the intratracheal inoculum should be much higher than that in the previous experiment. In addition, it was not practicle to use hundreds of rats, therefore, the following kinetic experiments were performed by using mesenteric lymph node cell preparations as described above. The kinetics and location of lymphocyte distribution after i.t. instillation were studied in 18 recipient RT7a rats. The animals were killed by exsanguination 6, 24, and 48 hr after the administration of RT7b lymphocytes, forming three groups consisting of five animals with an i.t. and one with an i.v. injection of the inoculum for each period of time. The blood was collected in heparinized tubes. Several different thoracic (lung-draining) lymph nodes (posterior mediastinal, tracheal bifurcation, parathymic; Fig. 1) as well as cervical, axillary, and mesenteric (non–lung-draining) lymph nodes, thymus, spleen, liver, and Peyer's patches were excised. The lungs were taken out en bloc, and infused with OCT in PBS as described above. The hilar region was separated from both lungs to keep hilar lymph nodes untouched for cyrostat sections, because they were hardly visible macroscopically. The lungs were cut into longitudinal sections through the main bronchus. All organs were snap frozen in liquid nitrogen and stored at −70°C. For immunohistologic staining, serial cyrostat sections were cut at 6 μm, air-dried for 30 min, and stored at −70°C. The hilar region was examined for the presence of lymph nodes by serial sections stained with hematoxylin and eosin (H & E). Heparinized blood (0.5 ml) from each recipient animal was incubated in 10 ml of ammonium chloride solution (0.83% NH4Cl) supplemented with 0.1 g/l EDTA for 10 min at room temperature to remove red blood cells. After centrifugation (400× g, 15 min), the pellet was resuspended in 2 ml of PBS containing 1% bovine serum albumin (Serva, Heidelberg, Germany) and 0.1% NaN3. Cell numbers were determined by using a Coulter Counter (Coulter, Luton, UK), and cytospots for immunocytologic staining were prepared by centrifuging 1 × 105 cells for 8 min at 800 rpm in a Cytospin 3 cytocentrifuge (Shandon Scientific, Ltd., Runcorn, UK).

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Figure 1. Schematic drawing of the harvested draining (bold) and nondraining lymph nodes in the upper half of the rat chest.

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Immunohistologic staining.

To localize the donor lymphocytes in the different organs and blood of the recipient animals, the monoclonal mouse antibody His 41 (Dianova, Hamburg, Germany), recognizing leukocytes from the Lewis RT7b only, was used in the alkaline phosphatase–anti-alkaline phosphatase (APAAP) technique as described previously (Tschernig et al., 1997) by using Fast Blue (Sigma, Deisenhofen, Germany) as substrate for the alkaline phosphatase. In cryostat sections of lymph nodes, double staining with a biotinylated His 41 antibody combined with monoclonal mouse anti-rat antibodies against surface markers, which identify T cells (R73) and B cells (His14), was performed. Surface antibodies were revealed by the APAAP-technique as described above and the biotinylated His 41 antibody by a streptavidine-peroxidase complex (Dianova) and incubation with benzidine as a substrate. Controls produced the expected results. All cyrostat sections were counterstained with hemalaun, mounted, and examined by using light microscopy. At least four sections were analyzed in each organ. The donor cells appeared clearly as mononuclear cells and were counted by using a microscopic counting grid to determine the number of cells per square millimeter of tissue. By using blood cytocentrifuge preparations, 50,000–100,000 cells were counted to determine the number of His 41+ cells.

To detect FITC/CFSE-labeled cells in semithin sections, Epon was solved by using Na-methylate and the labeled cells were visualized by the APAAP technique as described above by using the FITC/CFSE recognizing monoclonal antibody DE1 (Boehringer, Mannheim, Germany). Cryostat sections for CLSM were first incubated with the His 41 antibody followed by a second step incubation with an anti-mouse IgG antibody conjugated with Cy5 (Dianova).

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

The inoculum of mesenteric lymph nodes of RT7b rats consisted of 34.6 ± 6.7% B cells, 44 ± 6.2% T cells, 35 ± 4.5% CD4+ T cells, 11 ± 2.6% CD8+ T cells, 1.5 ± 0.5% macrophages, and dendritic cells and 0.7 ± 0.5% NK cells.

Anatomic Sites of Lymphocyte Reentry Into the Lung Tissue

Two RT7a rats received FITC/CFSE-labeled lymphocytes by i.t. instillation and were killed 15 min later. In semithin sections, the FITC/CFSE-labeled cells could clearly be identified by immunohistochemistry with the monoclonal antibody DE1. The instilled donor cells were observed attached to the air side of the alveolar wall, and some appeared within the interalveolar interstitial tissue (Fig. 2A–D). Single cells were found in what seem to be lymphatic vessels. The donor cells were found neither near or in the bronchial epithelium, nor in bronchial interstitial tissue, nor in bronchus-associated lymphoid tissue (BALT).

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Figure 2. A–D: Semithin sections of the lung 15 min after intratracheally (i.t.) injection of fluorescein isothiocyanate carboxyfluorescein diacetate labeled cells [corresponding bright illumination (A,C) and phase contrast (B,D); alkaline phosphatase–anti-alkaline phosphatase using DE1 as primary antibody]. The labeled cells were lying attached to the air side of the alveolar epithelium but also within the interalveolar connective tissue (arrow). E,F: Confocal laser scanning microscopy of the lung 15 min after i.t. instillation of unlabeled RT7b cells (immunohistochemistry with His 41 as primary antibody and Cy5-conjugated secondary antibody). The red-labeled donor cells were seen adherent to the alveolar epithelium (arrowheads) and in a state of transmigration through the alveolar epithelium into the interstitial tissue (arrows). Scale bars = 250 μm in A (applies to A–D), 80 μm in E (applies to E,F).

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The lungs of three RT7a rats which had received unlabeled donor cells i.t. were processed for CLSM 15 min later. By using the RT7b-specific antibody His 41, the results found in semithin sections were confirmed. Occasionally deformed lymphocytes were seen, suggesting transmigration from the alveolar space through the alveolar epithelium into the interstitial alveolar tissue (Fig. 2E, F).

Kinetics of Lymphocyte Distribution

By using BAL-derived, CSFE-labeled lymphocytes, single cells were found in the marginal sinus and the T-cell area of draining lymph nodes 24 hr after the i.t. instillation (Fig. 3E). The kinetics of lymphocyte distribution throughout the body was studied in 15 RT7a rats killed 6, 24, and 48 hr after i.t. instillation of RT7b mesenteric lymph node leukocytes (n = 5 in each group). RT7b donor leukocytes were clearly detected in cryostat tissue sections by using the monoclonal antibody His 41.

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Figure 3. Cryostat sections of lung (A,C,F) and lung-draining mediastinal lymph node (B,D,E,G) 6, 24, and 48 hr after intratracheal instillation of donor lymphocytes (A–D,F,G) alkaline phosphatase–anti-alkaline phosphatase (APAAP) by using His 41 as primary antibody). The blue labelled lymphocytes were derived from mesenteric lymph nodes except the one in E, which was isolated from pooled BALs (APAAP, using DE1 as primary antibody). Numbers of the inoculated cells decreased in the lung tissue and increased in the draining lymph nodes over time. Scale bars = 500 μm in A (applies to A–D,F,G), 40 μm in E.

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At 6 hr after i.t. instillation, the lung contained a large number of His 41+ cells (Fig. 3A). The cells were spread throughout the whole lung and were found in the lumen of the great bronchi and the alveolar spaces. At 24 hr after instillation, the number of His 41+ cells in the lungs was reduced. They had accumulated near to the alveolar wall but were no longer seen in bronchi or bronchioli (Fig. 3C). At 48 hr after instillation, only a small number of His 41+ cells was found in the lung alveoli (Fig. 3F).

The draining lymph nodes of the lung contained approximately 2–4 His 41+ cells/mm2 at 6 hr after the i.t. instillation (Fig. 3B). In one animal, cells were located in the subcapsular sinus of the lymph node indicating entry by means of afferent lymphatic vessels. The number of lymphocytes in the draining lymph nodes increased on average 8 times higher per section at 24 and 48 hr after instillation compared with 6 hr, with up to 50–100 cells/mm2 (Fig. 3D,G). The highest numbers were reached in the posterior mediastinal lymph nodes in three animals, once in a lymph node of the tracheal bifurcation and once in a parathymic lymph node, possibly a result of the different drainage of different parts of the lung. Hilar lymph nodes were very small and only occasionally were single donor cells seen there. By using immunohistochemical double staining, it was possible to analyze subsets of the instilled lymphocytes. The donor cells found in the draining lymph nodes were mainly T cells, preferentially located in the T-cell area and only occasionally seen in B-cell areas. Remarkably, only few donor B cells were observed in the draining lymph nodes at all time points, although one-third of the cells in the inoculum were B cells.

The kinetic pattern was clearly different in the non–lung-draining lymph nodes, e.g., mesenteric lymph nodes. No His 41+ donor cells were found in these lymph nodes at 6 hr after instillation. At 24 and 48 hr after instillation, single His 41+ cells were found located near to high endothelial venules, implicating the arrival from the blood.

The appearance of His 41+ cells in the peripheral blood differed between the three time points. After counting at least 50,000–100,000 leukocytes/animal of the peripheral blood in cytocentrifuge preparations just one single His 41+ cell was found in one of the five recipient animals 6 hr after instillation. At 24 hr, the number of positive cells increased with a mean of 3 positive cells/100,000 in the peripheral blood of recipient animals. Only single cells were found 48 hr after instillation in two of the five recipients.

Examination of the spleen revealed approximately 1–2 His 41+ cells/cm2 at 6 hr after instillation, whereas 24 and 48 hr after instillation, the number of immigrant cells had increased to an average of 5 His 41+ cells/cm2. These cells were preferentially located in the periarteriolar lymphatic sheath of the white pulp. Some cells were found in the follicles, whereas only occasionally cells were located in the red pulp. Single His 41+ cells were present in the thymus, Peyer's patches, and the liver at 24 and 48 hr after instillation.

Three RT7a rats received an i.v. instead of i.t. injection of donor lymphocytes and were killed 6, 24, and 48 hr after injection. Huge numbers of His 41+ cells were present in all examined draining and nondraining lymph nodes and other organs such as spleen and liver regardless of the time after administration of donor cells. T cells and B cells were mainly found in the organs' specific T-cell and B-cell area, respectively. Thus, the kinetics of distribution of i.v. injected His 41+ cells was markedly different from that of the i.t. instilled cells.

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

The huge surface of the lung is permanently challenged by the outside environment, which is full of potentially harmful antigens. The antigens entering the respiratory tract are cleared by the mucociliar system or phagocytized by cells of the unspecific lung immune system, e.g., alveolar macrophages, dendritic cells, or neutrophils, which have all been shown to leave the alveolar space, reenter the lung tissue, and migrate to regional lymph nodes, where the primary immune response can be initiated (Corry et al., 1984; Harmsen et al., 1985, 1987; Havenith et al., 1993).

However, the bronchoalveolar space is not only the first site for antigen contact with the unspecific lung immune system but also with the specific lung immune system as about 10% of the cells obtained by BAL are lymphocytes (Berman et al., 1990). The fate and function of these bronchoalveolar lymphocytes are not well understood. Immigration of lymphocytes from blood into the bronchoalveolar space has been shown but seems to be slow in healthy rats with an increase during inflammation (Schuster et al., 2000). In addition, only 0.5–1% of the bronchoalveolar lymphocytes proliferate locally so that bronchoalveolar lymphocytes seem to form a stable cell population (Kracke et al., 2000). To our knowledge, no studies on the apoptosis of bronchoalveolar lymphocytes in the rat have been published; but, in healthy mice, remarkably approximately 18% of the bronchoalveolar lymphocytes have been found to be apoptotic (Milik et al., 1997) and the phagocytosis of apoptotic lymphocytes by alveolar macrophages was demonstrated to be defective in comparison to other organs (Hu et al., 2000).

Pabst and Binns (1995) reported that intrabronchially instilled autologous blood lymphocytes appeared after 24 hr in the draining lymph nodes of the lung in pigs, and Tournoy and Pauwels (2000) found i.t. instilled human mononuclear blood cells later in the lung tissue by using a SCID mouse model. These data indicate that reunion of bronchoalveolar lymphocytes with the systemic immune system is possible. In the present study using a congenic rat strain, these observations are extended as we were able to (1) confirm the results of Pabst and Binns (1995) in a different animal model, indicating that reentry of bronchoalveolar lymphocytes into the lung is probably a common phenomena not restricted to pigs, (2) to show the anatomic site of reentry into lung interstitium, and (3) to investigate the kinetics of distribution through different lymphoid organs after reentry into the body.

As early as 15 min after i.t. instillation, His 41+ donor lymphocytes were found closely attached to the alveolar epithelium and within the alveolar septa. In semithin sections, it was not possible to detect cells actually migrating through the alveolar epithelium, despite thorough inspection of lung sections in five animals. However, by using CLSM, “hand-mirror” shaped lymphocytes were observed, suggesting occasional transmigration from the alveolar space into lung tissue through the alveolar epithelium. Transmigration of instilled lymphocytes probably starts within a few minutes after instillation, the first step consisting of adhesion to the alveolar epithelium followed by rapid penetration through the epithelial layer, so that this second step of migration can only rarely be observed. Recently, Ichikawa et al. (1996) have demonstrated that intravascular lymphocytes could migrate to the perivascular space of the lung between endothelial cells after adhesion while changing their shape. Similarly, it is conceivable that the cells in this study pass the alveolar epithelium intercellularly. Interestingly, immigrated donor cells could be found neither in bronchial epithelium, nor in the epithelium covering BALT, nor in the BALT itself, not only 15 min after instillation but also at the later time points up to 48 hr after instillation. This finding is remarkable, because the BALT has been thought to play an important role in the integrated mucosal immune system and pulmonary immunity (Tschernig and Pabst, 2000), increasing in size and number during lung infections (Iwata and Sato, 1991; Delventhal et al., 1992).

To investigate the migration routes in the present experimental system, a high number of intrabronchially applied cells are necessary. Therefore, lymphocytes derived from mesenteric lymph nodes were used, which obviously has the disadvantage of a different composition and activation status. In the lung compartments, especially in the BAL, more than 90% of the lymphocyte fraction consists of activated, memory T cells (Tschernig et al., 1999), whereas in the mesenteric lymph nodes, the naive T cells are the major fraction. Despite technical difficulties, it was demonstrated in a “proof of principle experiment” that BAL-derived lymphocytes show similar behaviour. However, some minor differences in the migration pattern and numbers between these lymphocyte populations cannot be excluded.

In the present study, 6 hr after instillation—an early time point not investigated before (Corry et al., 1984; Harmsen et al., 1985, 1987; Havenith et al., 1993; Pabst and Binns, 1995)—donor lymphocytes were found in the draining lymph nodes, pointing to a rapid migratory capacity of bronchoalveolar lymphocytes once they have left the bronchoalveolar space with its suppressive effect on lymphocyte function (Kirby et al., 1985; Shimizu et al., 1988). Note that i.t. instillation of cells is an artifical intervention in the normal homeostatic situation and might influence the extent of motility as the intrinsic motility of intra-alveolar lymphocytes is not constant; Ohtsuka et al. (1994) demonstrated that BAL lymphocytes showed enhanced in vitro motility in patients with hypersensitivity pneumonitis and sarcoidosis.

At 24 hr, the number of lymphocytes in the draining lymph nodes had markedly increased and remained more or less constant 48 hr after i.t. instillation. Thus, lymphocyte immigration out of the lung into regional draining lymph nodes seems mainly to occur between 6 and 24 hr. The distribution of positive lymphocytes to the thoracic lymph nodes agrees with the results of Takahashi and Patrick (1987), who observed the drainage of i.t. instilled colloidal carbon in rats mainly to the posterior mediastinal lymph nodes. Double staining for T- and B-cell markers on the donor cells in the draining lymph nodes revealed that the immigrated cells were mainly T cells localized in T-cell areas, although only 40–50% of the cells in the inoculum had been T cells. Furthermore, detailed examination of subsets of immigrated donor cells in healthy animals and during lung inflammation will be important.

At 6 hr after instillation, hardly any His 41+ donor cells were detected in the blood, spleen, or several non–lung-draining lymph nodes. At 24 and 48 hr, donor cells occurred regularly in these compartments. In the other organs examined, e.g., Peyer's patches, thymus, and liver; single cells were only rarely found.

Concerning the physiological and immunological relevance, one could comment that a fraction of lymphocytes in the bronchoalveolar space reenters the lung tissue, then the draining lymph nodes, and finally the whole immune system. Taking into consideration that, in inflammation, these fractions may be increased, even a small number of lymphocytes together with macrophages and dendritic cells might be of great biological significance over time. However, the role in immune reactions and diseases of the lung has to be elucidated in further studies.

In conclusion, our studies indicate that, as early as 6 hr after i.t. instillation, the lung-draining lymph nodes were reached by the donor lymphocytes. Here in the regional lymph node of the lung, the whole cellular and humoral apparatus exists to start a specific immune reaction against intraalveolar antigens, including matured dendritic cells ready for antigen presentation, which have immigrated from the lung as immature antigen-laden dendritic cells (Holt and Stumbles, 2000). Therefore, an early specific immune response to pulmonary antigens seems possible. Further studies have to show whether dendritic cells in addition to T cells migrate so rapidly into draining lymph nodes or whether antigen presentation and activation of specific lymphocytes can take place even in the alveolar space or during the migratory process. At 24 hr after i.t. instillation, several non–lung-draining lymph nodes and spleen had been reached by the donor lymphocytes, so a rapid recirculation pathway exists between the bronchoalveolar space and the systemic immune system of the body.

The lung is probably the only organ where cells of the specific immune system can return to the systemic immune system of the body after being in the outside microbe- and antigen-laden environment. The lymphocyte migration from the alveolar space to the local immune system of the lung and reunion with the systemic immune system of the body is an important factor in understanding the relevance and function of bronchoalveolar lymphocytes. Furthermore, this finding may have clinical implications in the use of inhalation methods as a way to apply drugs and vaccinations.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

The authors thank K. Westermann, A. Weiss, and S. Fassbender for skillful technical assistance, S. Fryk for the correction of the English, as well as D. Stelte and M. Peter for help with the figure composition. The congenic rats were a gift of K. Wonigeit (Center of Surgery, Medical School of Hannover).

LITERATURE CITED

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED
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