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- MATERIALS AND METHODS
- 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.
- Top of page
- MATERIALS AND METHODS
- 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.