Only Effector T Cells Accumulate and Proliferate in the Lamina Propria of the Bronchi |
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Abstract |
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Abstract
Introduction
Material and Methods
Results
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Conclusions
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T cells reach the lung via the pulmonary and bronchial arteries that supply the alveolar and bronchial regions. Although these regions are differentially affected by T cell-mediated diseases, the migration of T-cell subsets in these two regions has not been studied. Naive, memory, and effector T cells were injected into congenic rats and traced in sections of normal lung. All three T-cell subsets were found in large numbers in the alveolar region and exited again quickly. Only effector T cells accumulated in the lamina propria of the bronchi. Further, 72 h after injection 6% of the effector T cells still proliferated in the lung, whereas apoptotic effector T cells were only observed 1 h after injection (0.2%). Thus, not only effector and memory but also naive T cells continuously migrated through the lung. The preferential accumulation of effector T cells in the bronchial lamina propria may explain why some diseases preferentially affect the bronchial region.
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Introduction |
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Abstract
Introduction
Material and Methods
Results
Discussion
Conclusions
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The lung is constantly exposed to microbes and foreign particles (or "antigens") and is therefore an immunologically important organ. Although it is a nonlymphoid organ, the lung contains many lymphocytes (1, 2). For example, in humans the total number of lymphocytes found in the lung interstitium is similar to the total number in blood (about 1010) (3). Further, lymphocytes are also localized in the epithelium and the lamina propria of the bronchi, although the exact number is not known. Lymphocytes show unique migration kinetics through the lung. After intravenous injection, radioactively labeled thoracic duct lymphocytes were detected quickly and in high numbers in the lung, their numbers subsequently decreasing rapidly (4, 5). This migration pattern was later confirmed for B and T cells, for CD8+ and CD4+ T cells, for naive and memory CD4+ T cells, and for effector T cells (6). It is very likely that the situation in humans is comparable to that in animal models. Even the normal human lung contains significant numbers of T cells (1). In addition, after lung transplantation, donor-derived lymphocytes were found in the blood and in lymphoid and nonlymphoid organs of the recipient, which showed that lymphocytes are able to leave the lung (9). For a long time it was assumed that the high number of lymphocytes in the lung early after intravenous injection merely represented a passive retention of damaged cells in the first capillary bed. However, by injecting labeled lymphocytes intra-arterially (ascending aorta) it was shown that the labeled lymphocytes accumulated in the lung early after injection, although this time the lung vasculature was the second capillary bed that the vast majority of these lymphocytes had to pass (2). This clearly showed that the high number of lymphocytes in the lung immediately after injection represents an active process that retains living cells.
However, the experiments described earlier did not
consider that lymphocytes enter the lung by two different
routes: either via the pulmonary arteries (vasa publica),
which preferentially supply the alveolar region; or via the
bronchial arteries (vasa privata), which mainly supply the
bronchial region, i.e., connective tissue around the bronchi, arteries, and lymphatic vessels, and the lamina propria
of the bronchi. Moreover, several lung diseases which are
thought to be T cell-mediated (10, 11) initiate and manifest preferentially in one of these regions. For example, sarcoidosis mainly affects the alveolar region (12), whereas asthma mainly affects the bronchial region (11). Thus, in
addition to differences
e.g., in antigen exposure and antigen-presenting cell numbersa different migration pattern of T cells through the alveolar and bronchial regions
might explain this observation in part. And indeed, recently we found in the rat that injected T-cell subsets accumulated in the periportal field of the liver, which is often
affected by immunologically mediated liver diseases (13).
So far, it is not known whether, even under nonpathological conditions, T cells migrate in comparable numbers and
with similar kinetics through the two compartments of the
lung, the alveolar and bronchial regions. In addition, it is
unclear whether naive, memory, and effector T cells are
different in this respect. This would be important to know
inasmuch as they differ in their activation requirements
and the cytokines they secrete into the tissue. Understanding the migration of T-cell subsets under physiologic conditions through the two regions of the lung could help to
elucidate why lung diseases, in which T cells are involved
(12), affect either the alveolar or the bronchial region.
Therefore, in the present study, naive, memory, and effector T lymphocytes were separated, labeled, and injected
intravenously into rats. Using quantitative immunohistology (14, 15) the migration of these T-cell subsets through
the alveolar and bronchial regions of the lung was investigated. Further, the present study analyzes both the rate of
proliferation (5-bromo-2-deoxyuridine [BrdU] incorporation) and apoptosis (terminal deoxyribonucleotidyl transferase [TDT]-mediated deoxyuridine triphosphate-biotin
nick-end labeling [TUNEL] technique) among effector T
cells that have migrated into either of the two compartments.
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Material and Methods |
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Abstract
Introduction
Material and Methods
Results
Discussion
Conclusions
References
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Experimental Design
T cells were prepared by two methods, depending on whether naive and memory T cells or effector T cells were studied. To obtain naive and memory T cells, thoracic duct CD4+ T cells were separated into naive and memory phenotypes according to the high and low molecular-weight isoforms of CD45R, respectively. Effector T cells were generated in vitro by stimulating lymphocytes from peripheral and mesenteric lymph nodes (pLN and mLN, respectively) via the T-cell receptor and CD28. One type of donor T cell was then injected and the alveolar and bronchial regions of the host lung were investigated at different time points.
Naive (CD45RC+) and Memory (CD45RC
) T Cells
Congenic rats from the inbred PVG.7A (RT7a) and PVG.7B
(RT7b) strains were bred and maintained under barrier conditions in the Animal Unit at the University of Manchester (UK)
Medical School. Details of the purification procedure were described previously (7). Briefly, thoracic duct lymphocytes from
PVG.7B donors (6 to 13 wk old) were depleted of B cells, CD8+
T cells, and CD90+ recent thymic emigrants using a cocktail of
specific mouse monoclonal antibodies (mAbs) and antimouse
immunoglobulin-conjugated immunomagnetic particles. The resulting population (> 99% CD4+) was 80% CD45RC+. The
CD45RC subset was prepared in the same manner but with the
additional depletion of CD45RC+ cells. Cell purity was routinely > 97%. After intravenous injection of naive (CD45RC+) or
memory (CD45RC) T cells (20 × 106 cells), the lungs of recipients were removed at various time intervals.
In our study the injected naive T-cell suspension contained a
proportion of memory T cells (20%); we opted to tolerate this contamination to avoid purification by positive selectiona procedure that would coat the naive CD45RC+ subset with mAb. It
was unlikely, however, that the cells found in the lung after naive
cell injection all belong to the contaminating memory T-cell population, because if this were true the absolute number of cells we
found would have been only one-fifth of the number of memory
T cells. This was clearly not the case, because we found no difference between the numbers of naive and memory CD4+ T cells
migrating through the lung (Figure 2).
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It is known that memory cells (CD45RC) may revert back to
the CD45RC+ phenotype and re-express the high molecular-weight isoform of CD45R. Nevertheless, linking CD45RC+ and
CD45RC with naive and memory is a useful division; it distinguishes T cells waiting to encounter antigen (naive) from those
that have recently seen antigen (memory) (7).
In Vitro-Generated Effector T Cells
Rats from the standard inbred strain LEW/Ztm (RT.7a) and the
congenic strain LEW.7B/Won (RT.7b) were bred and maintained
at the central animal laboratory of the Hannover (Germany)
Medical School. As described by Luettig and colleagues (13), the
LEW.7B strain is identical to the congenic strain originally designated LEW.Ly1.2. The RT system is a diallelic polymorphism of
the CD45 molecular system. Cell suspensions were prepared
from LEW.7B rat pLNs (pooled axillary, brachial, and cervical
lymph nodes) and mLNs. The cells were stimulated in vitro via
the 
T-cell receptor (mAb R73) and CD28 (mAb JJ319) for 72 h as described (16). To follow only the T cells in vivo that had
gone through the cell cycle at least once, the cells were activated
in the presence of 5 µM BrdU. BrdU is incorporated into the
DNA during the S-phase of the cell cycle and can be detected in
cytologic and histologic preparations with specific antibodies (summarized in Ref. 17). Cytopreparations were made from pLN
and mLN cells after stimulation and before injection, and the incorporated BrdU was detected in T cells (antibody R73) using
the alkaline phosphatase antialkaline phosphatase (APAAP) and
peroxidase antiperoxidase techniques (17), showing that 79 ± 5% of the cells had incorporated BrdU and that 81 ± 3% of them
were T cells (n = 10). Although the inoculum contained some activated B cells, effector T cells always comprised the vast majority of cells found after injection in the lung (data not shown).
Among the T cells of the inoculum, 49 ± 6% were CD8+ and 53 ± 9% were CD4+. A mean of 60 × 106 BrdU+ T cells were injected
over 2 min intravenously into RT7a Lewis rats (5 to 6 wk old).
Detection of Donor Cells in the Recipient Organs
At various times after injection the rats were anesthetized with
ether and exsanguinated. To obtain standardized histologic sections, 2 ml of OCT compound Tissue-Tek (Sakura, Zoeterwoude, The Netherlands) was instilled via the bronchial system. In addition, the lung was always removed in the same way, frozen in liquid nitrogen, and stored at 
80°C. Cryostat sections of the middle of the right lung (thickness = 5 µm) were prepared. Care was
taken to obtain transverse sections of the whole organ containing
both the central and peripheral parts of the lung. These sections
were air-dried, wrapped in aluminum foil, and stored at 20°C.
To localize naive and memory cells in the organ, the sections
were fixed for 10 min in methanol and acetone (1:1) at 20°C and incubated with a mAb directed against the injected cells
(RT7b phenotype; mAb HIS 41 [16]). After washing with Tris-buffered saline containing 0.05% Tween 20 (TBS/Tween) the
bound antibody was revealed using a second antibody (rabbit antimouse; Dako, Hamburg, Germany) and a mouse antibody complex
(APAAP; Dako) for 30 min. Each of the last two steps was repeated
for 15 min. To visualize the antibodies, a mixture of APAAP substrate (Dako) and Fast Blue (Sigma, Deisenhofen, Germany) in Tris
Buffer, pH 8.2, was used. The naive and memory cells appeared blue.
Effector cells were identified by revealing their incorporated
BrdU. In addition, their phenotype was determined with one of
the following mAbs described by Luettig and associates (13). CD8+ cells, OX8; T cells, R73; B cells, HIS 14; major histocompatibility complex (MHC) class II-expressing cells, OX6; interleukin-2 receptor (IL2R )-expressing cells, OX39 (13). In
brief, the slides were fixed as described earlier, washed in TBS/
Tween, and incubated for 30 min at room temperature in a moist
chamber with the respective primary mAbs. Then the slides were
incubated with the second antibody (rabbit antimouse; Dako)
and the mouse antibody complex (APAAP; Dako) for 30 min.
Each of the last two steps was repeated for 15 min. To visualize
the antibodies, a mixture of APAAP substrate (Dako) and Fast
Blue (Sigma) in Tris Buffer, pH 8.2, was used. The positive cells
appeared blue. Next, the slides were washed in TBS/Tween, incubated in 70% ethanol for 30 min, and then air-dried for at least 30 min. To detect incorporated BrdU in activated lymphocytes,
DNA was denatured with formamide (Sigma) and NaOH (14).
Formamide (190 ml) was warmed to 70°C and 1 N NaOH (10 ml)
was added. The solutions were then mixed for 8 min. The slides were immersed in this solution for 30 s. After washing with TBS/ Tween, the slides were immersed in formamide containing 7.5 mM trisodium citrate for 15 min. These steps were done at 70°C.
Subsequently, the cells were washed in ice-cold TBS/Tween and
fixed in 1% formaldehyde (30 min) and 0.2% glutaraldehyde (10 min). Slides were incubated overnight with the mAb anti-BrdU
(Becton Dickinson, Mountain View, CA) dissolved in TBS/Tween.
After washing with TBS/Tween, the bound antibody was revealed
using rabbit antimouse (Dako) and the mouse antibody complex
(APAAP; Dako) for 30 min. Each of the last two steps was repeated for 15 min. Then the organ sections were incubated with a
mixture of APAAP substrate (Dako) and Fast Red for 25 min,
resulting in red staining of the BrdU+ cells (17). The slides were
counterstained with hematoxylin and mounted in glycergel
(Dako). Because both the applied antibodies and the activity of
the alkaline phosphatase were destroyed by the denaturation
procedure, the incorporated BrdU could be revealed using the
same system without unwanted cross-reactions.
Preparation and Injection of Effector T Cells to Test for Their Further Proliferation In Vivo
To study the local proliferation of effector T cells in the lung compartments, allotype-marked donor cells from LEW.7B (RT7b) rats were activated as described but in the absence of BrdU. At 3 d after injection of the effector T cells, LEW.7A rats (RT7a) received 5 mg BrdU/100 g body weight intravenously and 1 h later the lung, small intestine, and bone marrow were removed. Thus, only those lymphocytes that were in the S-phase of the cell cycle within the respective microenvironment were labeled. To test for proliferation of injected cells in the organs, the injected cells were first identified by using a mAb directed against the congenic phenotype (RT7b; mAb HIS 41) and visualized in blue as described earlier. BrdU incorporation was demonstrated in red. Proliferating donor cells appeared blue and red.
Identification of Apoptotic Cells in the Lung
Cryostat sections were fixed with 4% paraformaldehyde (pH 7.4 in phosphate-buffered saline) for 20 min. To localize donor cells in the lung, the sections were incubated with mAb directed against the congenic phenotype (RT7b; mAb HIS 41) and visualized in blue by the APAAP technique as described (14). To detect apoptotic cells by the TUNEL method (16), the sections were incubated with 70% ethanol for 30 min at room temperature. After washing in TBS/Tween, the sections were incubated with digoxigenin-labeled uridine triphosphate (UTP) (1.7 nM) and TdT (12.5 U) in TdT buffer (200 mM potassium cacodylate, 25 mM Tris-HCl, 1.25 mg/ml bovine serum albumin [pH 6.6], and 5 mM cobalt chloride solution) for 60 min at 37°C. After washing, the incorporated UTP was revealed by a peroxidase-conjugated antidigoxigenin Fab fragment antibody (Boehringer Mannheim, Mannheim, Germany) and subsequent immunohistology. Diaminobenzidine was used as a substrate. Thus, injected cells appeared blue and apoptotic cells appeared brown.
Evaluation
The lung sections were evaluated using a light microscope and an ocular grid divided into 100 squares. First, the alveolar region was analyzed by determining the area that was occupied by the alveolar septa, omitting the air-filled spaces. Then the area of the bronchial region was determined. This included the connective tissue surrounding the bronchi, arteries, and lymphatic vessels, including the lamina propria of the bronchi. The area comprised by the lumina of all structures was disregarded. In addition, bronchus-associated lymphatic tissue, which was present in less than 10% of the lungs analyzed, was not included in the present study. Next, the numbers of naive, memory, and effector T cells per tissue area were determined and expressed as the number of cells per area and per 20 × 106 injected cells. Means ± standard deviation (SD) were determined using SPSS for Windows. Differences between time points, tissues, or organs were analyzed using the Mann-Whitney U-test (P < 0.05 was considered significant).
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Results |
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Abstract
Introduction
Material and Methods
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Discussion
Conclusions
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Identification of Injected T Cells in the Alveolar and Bronchial Regions of the Lung
The different compartments of the lung, the alveolar and bronchial regions, could be clearly identified (Figure 1A). The bronchial region contained bronchi, pulmonary and bronchial arteries, lymph vessels, and the connective tissue surrounding these structures (Figure 1A, broken line). The alveolar region comprised the remaining area. Within the different compartments, individual injected cells could be determined (Figures 1B and 1C). The specificity of the staining was confirmed by the observation that no staining was seen in lungs of animals not having received donor T cells. Further, it was possible to determine whether the injected T cells were in the S-phase of the cell cycle (Figure 1D) or were dying by apoptosis (Figure 1E).
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CD4+ T Cells Migrate with Different Kinetics through the Alveolar and Bronchial Regions
CD4+ T cells showed different migration kinetics through the alveolar and bronchial regions of the lung. Early after injection (0.5 h), nearly 10 times as many CD4+ T cells entered the alveolar region compared with the bronchial region (Figure 2). By 2 h, donor T-cell numbers in the alveolar region had decreased significantly. In contrast, the number of CD4+ T cells in the bronchial region showed little variation over time (Figure 2).
Naive and Memory CD4+ T Cells Migrate in Comparable Numbers Through the Lung
Surprisingly, not only memory CD4+ T cells but also naive CD4+ T cells migrated into the lung. The lung is a nonlymphoid organ and thought to receive only memory T cells. However, the present study showed that naive CD4+ T cells migrated in comparable numbers and with comparable kinetics through the alveolar and bronchial regions as observed for memory CD4+ T cells (Figure 2).
Accumulation of Effector T Cells in the Bronchial Region of the Lung
Effector T cells generated in vitro by stimulating the T-cell receptor and CD28 of mLN or pLN lymphocytes were injected intravenously into congenic recipients. As already observed for naive and memory T cells, effector T cells localized in high numbers in the alveolar region early after intravenous injection and departed rapidly (Figure 3A). In contrast to naive and memory T cells, effector T cells showed a steady and significant accumulation in the bronchial region after 9 h (Figure 3A). Effector T cells generated either from mLN or pLN showed a similar migration pattern and the data were pooled. The kinetics of effector T cells in the blood were different from those observed in either the alveolar or bronchial region (Figure 3B).
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At 1 d after injection no difference was found in the number of naive, memory, and effector T cells in the alveolar region (Table 1). However, in the bronchial region the number of effector T cells was significantly higher than that of naive or memory T cells.
CD4+ and CD8+ Effector T Cells Migrate with Comparable Kinetics through the Lung
We investigated whether CD4+ and CD8+ effector T cells differed in their migration behavior through the lung. By analyzing the CD4+/CD8+ ratio among effector T cells before injection and by comparing it with the donor T-cell ratio in the alveolar and bronchial regions after injection, it became apparent that CD4+ and CD8+ effector T cells entered each compartment with comparable efficiency (no difference in CD4+/CD8+ ratio between inoculum and 1 h values; Figure 4). Further, neither subset showed any difference in migratory behavior into either the alveolar or bronchial region; the CD4+/CD8+ ratio remained constant between 1 and 96 h (Figure 4).
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The area of the lung tissue is dependent on the degree of inflation with air. To validate the determination of injected T cells per area of tissue, the ratio of effector to endogenous CD4+ T cells was analyzed over time. The number of endogenous cells was constant in rats of the same age and weight. Therefore, it was possible to assess the migration pattern in the alveolar or bronchial region independently of the size of the lung tissue at the time of removal. The results showed that in the alveolar region the number of CD4+ effector T cells decreased, whereas their number increased significantly in the bronchial region (Table 2). These results confirmed the data obtained on the migration kinetics of effector T cells as assessed by analysis on the basis of numbers of cells per area.
Effector T Cells Preferentially Accumulate in the Lamina Propria of the Bronchi
Next, we analyzed the migration kinetics of naive, memory, and effector T cells within the bronchial region, and determined the proportion of cells localized in the lamina propria of the bronchi at each time point after injection. Although the proportion of both naive and memory T cells declined over time (from 15% to 5%; Figure 5), the proportion of effector T cells significantly increased in the lamina propria of the bronchi, resulting in a 4-fold increase in number of effector T cells between 1 and 96 h after injection (Figure 5).
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Effector T Cells Proliferate to the Same Extent in the Alveolar and Bronchial Regions
To investigate whether the accumulation of effector T cells
in the bronchial region was due to local proliferation, effector T cells were injected. At 3 d later, the animals received
BrdU and the number of BrdU-positive effector T cells in
the lung was determined (Figure 1D). The effector T cells
generated from mLN proliferated to the same extent
(about 6%) in both regions (Figure 6). This correlates with
the percentage of effector T cells that were positive for
MHC class II (alveolar, 17 ± 2%; bronchial, 19 ± 5%) and
IL2R (alveolar, 16 ± 3%; bronchial, 15 ± 6%) in the alveolar and bronchial regions. Interestingly, the proliferation rate of effector T cells was about three times higher
than that of the endogenous T cells (Figure 6). This feature was observed only in the lung. In the small intestine,
both injected mLN effector T cells and endogenous T cells
proliferated at a similar rate (Figure 7). In bone marrow,
the mLN effector T cells proliferated at a rate that was significantly less than that of the endogenous bone marrow T
cells (Figure 7), but comparable to that of mLN effector T
cells in the blood (0.5%; 22/4,918). After injection, naive
and memory CD4+ T cells revealed a much lower proliferation rate than effector T cells both after migration into
the lung (0.06%; 2/3,080) and after migration into the lamina propria of the gut (0.2%; 1/421).
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Early After Injection, Effector T Cells Undergo Apoptosis in the Lung
It is known that effector T cells die by apoptosis in vivo. Therefore, we asked whether this also occurs in the lung. The number of apoptotic cells (TUNEL+) among injected effector T cells was determined in the lung (Figure 1E). About 0.2% of the effector T cells found in the lung 1 h after injection were apoptotic (Table 3). Due to the low number of apoptotic cells it was not possible to determine whether effector T cells demonstrated a different rate of apoptosis after migration into either the alveolar or bronchial region. Interestingly, apoptotic effector T cells were found in the lung only at 1 h after injection; no apoptotic cells were seen at later time points. Compared with injected effector T cells, endogenous T cells revealed an apoptosis frequency that was about 10 times lower.
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Discussion |
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Abstract
Introduction
Material and Methods
Results
Discussion
Conclusions
References
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In most studies investigating the migration of lymphocytes through the lung, radioactively labeled lymphocytes were injected and the radioactivity of the whole organ was determined so as to analyze lymphocyte migration patterns (2, 5, 8, 18). Recently, the migration of lymphocytes through the lung was investigated in more detail by identifying labeled lymphocytes in the marginal pool of the lung blood vessels, in the alveolar space, and in the tissue, and showed that the migration pattern of lymphocytes differed considerably, depending on the compartment being studied (21, 22). It is important to recall that lymphocytes reach the lung via two different arterial systems. The alveolar region is supplied with blood mainly by the pulmonary arteries (vasa publica) whereas the bronchial region is supplied with blood mainly by the bronchial arteries (vasa privata). In addition, the alveolar and bronchial regions are preferentially affected by different diseases (e.g., sarcoidosis and asthma, respectively) in which T cells play a major pathophysiologic role (10). In the present study, the migration of naive, memory, and effector T cells was investigated separately in the alveolar and bronchial regions of the normal rat lung. Although of considerable interest, the analysis of bronchus-associated lymphoid tissue (BALT) was not included in the present investigation. Only three out of 223 lung sections from 40 Lewis rats contained BALT. This shows that, as in other species, it is not a constitutive structure of the healthy rat lung (23). In addition, the migration kinetics of T cells through lymph nodes draining the lung was also not analyzed. Compared with the number of T cells continuously entering the lymph node via high endothelial venules, the number of T cells reaching the draining lymph nodes from the healthy lung via afferent lymph vessels is so small that it cannot be detected. The migration of naive, memory, and effector T cells through other lymph nodes and Peyer's patches has recently been described (15, 16).
T-Cell Subsets Migrate with Different Kinetics through the Alveolar and the Bronchial Regions of the Lung
Previous investigations reported that T cells recirculate through the lung (5, 7, 18, 20). Using quantitative immunohistology, the present report demonstrated that all T-cell subsets were found both in the alveolar and bronchial regions. Although it is known that damaged cells are sequestered in lung tissue, the donor cells in the lung were clearly intact, as seen by immunohistology. This supported earlier data showing that lymphocyte accumulation in the lung was part of an active rather than a passive process. Intact lymphocytes were temporarily retained in the environment of the lung; entrapment was not due to nonspecific binding of damaged lymphocytes (2).
The present study shows that early after injection, all T-cell subsets were found in high numbers in the lung alveolar region and departed again quickly. In contrast, the number of injected cells in the bronchial region remained constant or increased over time. Thus, the migration kinetics based on localization of labeled lymphocytes in the whole lung (2, 5, 7, 8, 18) primarily reflected the passage through the alveolar region. Because in the present experiments the vasculature of the lung was not perfused before analysis, it is not possible to definitely determine whether the injected T cells found in the alveolar and bronchial regions were located within the tissue or whether they were still within capillaries and small venules. The observation that the kinetics of effector T cells in the lung regions differed from those in the blood (Figure 3), and the fact that they showed a 12-fold higher proliferation rate in the lung compared with that in the blood (6% versus 0.5%), suggest that the majority of injected T cells analyzed in the lung was located within the tissue of the alveolar and bronchial regions.
Naive and Memory CD4+ T Cells Migrate with Comparable Kinetics through the Alveolar and Bronchial Regions
We found that naive and memory CD4+ T cells migrated equally through the lung of the rat. This was in agreement with reports that naive and memory T cells are present in the human lung (24), and clearly demonstrated that naive T cells continuously migrated through nonlymphoid organs such as the lung. Tietz and Hamann (20) reported that memory CD4+ T cells had a preference for the lung similar to that of activated lymphocytes. However, in their study activated T cells were not excluded from their memory-cell preparation. When followed directly, we found no evidence to indicate that naive and memory T cells migrated by different pathways through the alveolar and bronchial regions. This does not exclude preferential migration of a minority of memory T cells into the bronchoalveolar space as a cause for the enrichment of memory T cells in this compartment (25, 26). However, comparable migration routes for naive or memory CD4+ T cells have recently also been demonstrated in various lymph nodes (7, 15), Peyer's patches (15), thymus (14), and liver (13). This underlines the importance of directly tracing labeled lymphocyte subsets to define their migration routes in vivo (27). The idea that naive T cells migrate preferentially through lymphoid tissues whereas memory T cells traffic via nonlymphoid routes was not supported by the present investigation (20). Further studies are needed to determine whether similar kinetics apply to naive and memory CD8+ T cells.
Effector T Cells Accumulate in the Lamina Propria of the Bronchi
Similar to naive and memory T cells, effector T cells were
initially found in high numbers in the alveolar region and
quickly exited (Figure 3). This pattern was observed for
both CD4+ and CD8+ effector T cells, and is consistent
with data from the lungs of humans and mice that showed
a comparable endogenous CD4+/CD8+ ratio (1, 31). However, in contrast to naive and memory T cells, effector T
cells continued to accumulate in the bronchial region over
time. Our study showed that the high number of effector T
cells in the bronchial region is primarily due to their ability to accumulate in the lamina propria of the bronchi (Figure
5). The gradual decrease of naive and memory CD4+ T
cells and the increase of effector T cells in the lamina propria suggest that all three T-cell subsets enter this compartment. However, although naive and memory CD4+ T
cells were released, effector T cells were retained. This
correlates well with the high expression of intercellular adhesion molecule-1 and 4-integrins among the effector T
cells (32). The accumulation in the bronchial region was
observed for effector T cells generated from either pLN or
mLN, and for CD4+ and CD8+ T cells alike. Apparently,
the ability to accumulate was related more to the effector
status than to a specific cell type. An accumulation of effector T cells was recently reported for the periportal field of the liver (13), another nonlymphoid compartment.
In the Lung, Effector T Cells Proliferate and Die at a High Rate
Lung sections from rats injected with donor cells were histologically indistinguishable from normal lungs and free
from readily detectable cell infiltrates. By analyzing BrdU
incorporation at the single-cell level we showed that effector T cells were able to proliferate in situ after migration
into the lung. The proliferation rate of effector T cells was
the same in the alveolar and bronchial regions, thus excluding the possibility that the accumulation of effector T
cells was due to differential cell division at this site. The
proliferation rate of the effector T cells (6%) was significantly higher than that of the endogenous T cells (2%; Figure 6 [33]). In this respect the lung microenvironment differed from that of the lamina propria of the small intestine
(proliferation of effector and endogenous populations was
the same) and that of the bone marrow (endogenous T
cells had a significantly higher proliferation rate), and was
comparable to that of the periportal field of the liver (13).
This suggests that effector T cells maintained their ability
to proliferate, and that the amount of proliferation was determined by the local microenvironment. Interestingly, effector T cells generated in mLN had proliferation rates in
the lung (about 6%), in other nonlymphoid organs such as
the lamina propria of the small intestine (about 5%), and
in the periportal field of the liver (about 12%) (13) which
was equal or higher than those observed in the T cell areas
of mLN (about 3%) (32) and Peyer's patches (about 5%)
(32). Effector T cells generated from pLN had significantly lower proliferation rates in the organs listed earlier
(present study) (16). In addition, effector T cells generated
from mLN showed a significantly lower proliferation rate
after migration into pLN compared with that after migration into mLN (16). The increased proliferation in mLN
was recently found to be regulated by local production of
transforming growth factor-1 and IL-4 in this node (36).
There are clear similarities between the response of effector T cells in the microenvironments of the lung and the
gut-associated lymphoid tissues. Further work is needed to
elucidate the underlying mechanisms and to establish which cytokines, chemokines, and surface molecules are
driving the proliferation of effector T cells in the lung.
The present study shows that a small number of effector T cells died in the lung by apoptosis. Due to the low number of apoptotic cells in the lung it was not possible to ascertain whether differential cell death contributed to the unequal distribution of effector T cells between the alveolar and the bronchial regions. However, apoptosis was observed only within the first hour after cell injection and occurred at a rate of about 0.2%, i.e., about one-fifth of the rate of apoptosis in the cortex of the thymus. The high percentage of apoptotic T cells found in the bronchoalveolar lavage of healthy mice (more than 10% [34]) might indicate that at some point apoptotic T cells of the alveolar region are finally expelled into the bronchoalveolar space. Thus, the microenvironment of the lung not only supported the proliferation of effector T cells but also was the site where many effector T cells were removed from the circulation, thereby preventing the long-term circulation of potentially dangerous effector T cells in the blood.
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Conclusions |
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Top
Abstract
Introduction
Material and Methods
Results
Discussion
Conclusions
References
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Naive, memory, and effector T cells continuously migrate through the alveolar and bronchial regions of the lung. All three subsets localized in large numbers to the alveolar region but were not retained at this site. In contrast to naive and memory T cells, effector T cells accumulated in the lamina propria of the bronchi and proliferated in situ at a high rate. Future studies are needed to identify the as-yet-unknown factors involved in regulating these distinct migration and proliferation patterns. It will also be important to analyze how these T-cell subsets migrate through the two lung regions during various diseases (35). Modifying migration and proliferation of the different T-cell subsets may represent a way to increase or decrease their numbers in the lung, and could have clinical relevance for the treatment of immunologically mediated diseases of the lung.
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Footnotes |
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Address correspondence to: Jürgen Westermann, Institute of Anatomy, Medical University of Luebeck, Ratzeburger Allee 160, 23538 Luebeck, Germany.
(Received in original form October 21, 2000 and in revised form February 21, 2001).
Abbreviations: alkaline phosphatase anti-alkaline phosphatase; APAAP; 5-bromo-2-deoxyuridine, BrdU; interleukin-2 receptor, IL2R; monoclonal antibody, mAb; mesenteric lymph nodes, mLN; peripheral lymph
nodes, pLN; standard deviation, SD; Tris-buffered saline containing
0.05% Tween 20, TBS/Tween; terminal deoxyribonucleotidyl transferase,
TdT; TdT-mediated deoxyuridine triphosphate-biotin nick-end labeling, TUNEL.
Acknowledgments:
The authors thank Drs. J. Hohlfeld, N. Krug, R. Pabst, and
T. Tschernig (Hannover, Germany) for helpful critical comments; and K. Bankes, I. Dressendörfer, S. Lopez-Kostka, and F. Weidner for excellent technical assistance. This study was supported by grants to one author (J.W.) from
the Deutsche Forschungsgemeinschaft (We1175/4-3) and to one author (E.B.B.)
from The British Medical Research Council and The Arthritis and Rheumatism
Council (B0556).
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Top
Abstract
Introduction
Material and Methods
Results
Discussion
Conclusions
References
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