Immunology 1992 77 3 1-37
A clonal analysis of lung T cells derived by bronchoalveolar lavage of healthy individuals M. J. GARLEPP, A. H. ROSE, R. V. BOWMAN, N. MAVADDAT, J. DENCH, B. J. HOLT,* M. BARONHAY,* P. G. HOLT* & B. W. S. ROBINSON Department of Medicine, University of Western Australia, Queen Elizabeth II Medical Centre, Nedlands and *Division of Cell Biology, Western Australian Research Institute for Child Health, Princess Margaret Hospital, Subiaco, Western Australia
Acceptedfor publication 20 April 1992
SUMMARY The characteristics of the T-cell population in the healthy human lung have been investigated by analysing the properties of T-cell clones derived from bronchoalveolar lavage (BAL) samples and comparing them with T cells cloned from the blood ofthe same individuals. The proportions of CD4+ and CD8 + T cells in the starting populations from BAL and blood were similar although only 14% of BAL T cells were CD45RA+ compared to 70% of blood T cells. The precursor frequency of T-cell clones derived from BAL was less than from blood. The cytokine profiles [after phytohaemagglutinin (PHA) stimulation] of the clones derived from both sources were markedly different and these differences lay in the CD4+ population. BAL-derived CD4+ clones produced interferon-y (IFN-y) more frequently than did those from blood while blood-derived clones were more likely to produce interleukin-2 (IL-2) than those from BAL. IL-4 was produced by the majority of BAL- or bloodderived clones (93% and 88% respectively) either along with IFN-y (BAL) or IL-2 (blood). The cytokine profiles of BAL-derived T-cell clones are consistent with those derived from lung interstitium and suggest that the BAL T-cell populations reflect those in the lung wall. Whether the unique properties of lung T cells are acquired after leaving the blood or whether there is selective entry of T-cell subpopulations into the lung remains to be determined.
nosuppressive2 and more recently alveolar epithelial cells have been demonstrated to inhibit mitogen-stimulated lymphocyte proliferation in vitro.3 Recent work in man, and in other species, has shown that the lung is an important repository of T lymphocytes.4-8 At all times more lymphocytes are present in the lung interstitium than in the peripheral circulation.5 The tendency for lymphocytes to sequestrate in the lung has been known for some time9 and recent data suggest that some organ specificity is involved in this entrapment of T lymphocytes. Our recent studies of human interstitial T cells have demonstrated that these lung T cells differ from those derived from the blood in terms of their cloning efficiency and their cytokine-producing potential.5 Bronchoalveolar lavage (BAL) has become a useful technique with which to analyse the cellular content of the alveolar spaces.'0 The results of BAL are used as a diagnostic aid in interstitial lung diseases such as sarcoidosis and extrinsic allergic alveolitis'0"' and the T-cell content of such washings is thought to reflect that of the lung parenchyma. Some histochemical data are consistent with this assumption'2"3 although few data are available which have compared the functional capabilities of T lymphocytes from the two sources. Those studies which have examined T-lymphocyte populations in healthy human lungs
INTRODUCTION The alveoli of the lung provide an important interface between the environment and the body. The local defence mechanisms in the lower respiratory tract thus play a crucial role in the prevention of infection and in the clearance of non-infectious foreign material. The immune system in the lung is required to mount a rapid, specific active immune response as well as to ingest and degrade foreign material. The alveoli of the lung are, however, delicate structures, adapted for the rapid exchange of gases across a thin membrane and as such are highly susceptible to damage by excessive inflammatory processes. The immune response in the lung must therefore be under very tight control' and some candidate regulatory elements have been identified. For example, it has been clear for some time that the alveolar and interstitial macrophages of the lung are profoundly immuAbbreviations: BAL, bronchoalveolar lavage; IFN-y, interferon-y; IL, interleukin; PBL, peripheral blood lymphocytes; PHA, phytohaemagglutinin. Correspondence: Dr M. J. Garlepp, Dept. of Medicine, University of Western Australia, Queen Elizabeth II Medical Centre, Nedlands, Western Australia.
31
M. J. Garlepp et al.
32
have concentrated on phenotype analysis and responses to mitogen stimulation.4'-7 Although useful, such studies provide little information on the spectrum of functional activities possessed by lung T cells. Given the recently described functional heterogeneity of CD4+ cells, in particular,'8"9 it became clear that a more detailed analysis of the functional capabilities of lung T cells was necessary. We have, therefore, cloned T cells from BAL and blood samples from healthy individuals and have compared their cloning efficiencies, phenotypes and cytokineproducing potential. The data reported here confirm that T-cell clones derived from BAL are functionally similar to those derived from the lung interstitium and that their properties differ from those of T-cell clones from peripheral blood.
Table 1. Differential cell content (%) of bronchoalveolar lavage samples (n = 9)
Cell type Macrophages Lymphocytes Neutrophils Eosinophils
Cell preparation The recovered BAL fluid was immediately transported to the laboratory, where excess mucus was removed by straining through sterile gauze, the volume and appearance recorded, and a total cell count performed. Cell viability was determined using trypan blue exclusion. Cytospin preparations were made using 105 cells per slide and stained with May-Grunwald-Giemsa stain. A differential cell count was performed on 400 cells. Peripheral blood leucocyte (PBL) samples were prepared from blood taken from each subject at the time of the BAL, using Ficoll-Hypaque (Pharmacia, Uppsala, Sweden) centrifugation. Fresh lung tissue was obtained from three subjects undergoing lobectomy or pneumonectomy for localized pulmonary malignancy. The tissue studied was always obtained from an area of normal lung farthest from the malignancy. T cells were extracted as previously described using a combination of mechanical and enzymatic (DNAase and collagenase) disruptive techniques.5
T-cell cloning by limiting dilution Purified T cells were prepared from BAL, PBL or lung digests by two cycles of E rosetting as described elsewhere.4'5 T cells were seeded into wells of round-bottomed 96-well microtest plates (Nunc, Roskilde, Denmark) at densities of 16, 8, 4, 2, 1, 0 5 and 0 25 cells per well in a total volume of 200 p1 of RPMI-1640 (Flow Laboratories, McLean, VA) containing 15% human AB serum, I pg/ml highly purified phytohaemagglutinin (PHA)
Range
78-5 + 11-5 18 3 + 10 5 2-9+ 17 03 +04
61-95 5-35 0-8-3 0-1-3
1001 40-
MATERIALS AND METHODS
Study population Alveolar cells were obtained by BAL from nine healthy volunteers (age 54+ 10 years; eight men, one woman; one smoker, six non-smokers and two ex-smokers). None had clinical, radiological or physiological evidence of diffuse lung disease. This study was approved by the Human Rights Committee of the University ofWestern Australia and informed consent was obtained from each subject before the study. BAL was performed as previously described.'0 Briefly, the tip of a fibreoptic bronchoscope was positioned in a segmental bronchus and six 50 ml aliquots of warm (370) normal saline were instilled and immediately aspirated into a disposable sterile polycarbonate bottle. The fluid recovered from the first 50 ml aliquot was collected separately and the subsequent 250 ml (alveolar sample) was used as a source of alveolar T cells. Data presented in this study are from the alveolar specimen only.
Mean
\
CT
30-
0) 2
20 10 0 BAL
Blood
Figure 1. Cloning frequencies for T cells derived from BAL and blood
(paired data). 80
-
(a)
(b)
60-
40-
20
0
Original
Clones
Original
Clones
Figure 2. Phenotypes of starting T-cell populations and resultant clones from BAL (a) and blood (b). Graphs show mean + SEM CD4+ (-) or CD8+ (0) clones (n=9).
(Wellcome Laboratories, Beckenham, Kent, U.K.), 100 U/ml interleukin-2 (IL-2) (Cetus, Emeryville, CA) and 105 irradiated feeder cells. Feeder cells consisted of either irradiated (5000 rads) freshly prepared PBL or human spleen cells (3000 rads) which had been cryopreserved and cultured overnight before use. After 7 days, cultures were fed by replacement of 150 jl of medium with fresh medium containing 1 pg/ml PHA, 100 U/ml IL-2 and 105 irradiated feeder cells. Between Days 12 and 16 growing clones were scored under an inverted microscope and the precursor frequencies were determined according to the zero-order term of the Poisson probability distribution. Clones showing a probability of clonality >0 793 were selected for further expansion. Clones were maintained in RPMI with 15% human AB serum and 100 U/ml IL-2. Cells were split every 3-4 days and refed with fresh medium until sufficient cells were available for analysis.
33
Clonal analysis of lung T cells
-
80
80
-
60
60
-
40
40-
20
20-
0
0
32 80
(b)
60
60
>10
10
-
40-
20
20-
4-32
2-10
(d)
g 40
Surlface phenotipes Surface marker analysis of unfractionated blood and BAL T cells and subsequent clones was performed using fluorescence microscopy. The hybridoma lines OKT3 (CD3), OKT4 (CD4) and OKT8 (CD8) were obtained from American Type Culture Collection [(ATCC), Rockville, MD], grown in our laboratory and, after screening, the culture supernatants were used unpurified. Antibodies to the ;.(5 T-cell receptor (TcR) (TcR--,'-l ), the Y/J TcR (WT31) and CD45RA (Leu-18) were purchased from Becton Dickinson (San Jose, CA). Fluoresceinated sheep antimouse antibody (Silenus. Melbourne. Australia) was used as the detection system. Inidiction of (vtokines
Cloned T cells were seeded at a density of 106 cells ml in 200 Al of RPMI plus 10%' AB serum in 96-well microtitre trays (Nunc) and stimulated with I jig ml PHA. Supernatants were collected after 24 hr and stored frozen at -70 until assayed.
C(}tokine assai}s IL-2 was assayed as previously described." Proliferation of CTLL-2 (ATCC, TIB 214) cells was measured using [3H]thymidine uptake and units of IL-2 read from a standard curve generated with recombinant human IL-2 (Cetus). Interferon-; (IFN-- ) was measured using bioassay.> The reciprocal of the sample dilution which produced a 50%', inhibition of the cytopathic effect of encephalomyocarditis virus on human a
WISH cells (ATCC, CCL25) was determined and the concentration in units was calculated by comparison with the effect of a recombinant human IFN-; standard (Boehringer Ingelheim, Ingelheim, Germany). IFN activity was confirmed to be gamma in type by preincubating samples with antibody to IFN-; (Meloy Laboratories, Springfield, VA) for 1 hr at 37 prior to assay. IL-4 concentrations were measured using a commercially available ELISA assay (Genzyme, Boston, MA). The sensitivities of the assays for IFN-;', IL-2 and IL-4 were 2 U ml, 0 2 U ml and 0-1 ng, ml, respectively. Cytokine production was considered to be significant when the quantity measured was greater than 3 SD above the mean value for feeder cells alone, processed in the same manner as the clones. These cut-offs were 4 U ml (IFN--;) > 2 U ml (IL-2) and > 016 ng ml or 4 U ml (IL-4). Stal.stic.s
Statistical analyses UY-test.
were
carried out
using
the Mann-Whitney
RESULTS Cellular content of BAL and blood
The differential cell content of the BAL samples is shown in Table I. Of the lymphocytes in the BAL 86 + 8%°ao (mean + SEM) were CD3+ (cf. blood 76+9%V.), 3+2%, bore the ;'(i TcR (cf.
34
M. J. Garlepp et al. 80
60
any one subject were nine and 10 for BAL and blood respectively and the mean number of CD8+ clones was six for both BAL and blood.
(a)
-
IL-2 and IFN-y synthesis by CD4+ clones g 40-
20
0 32
Interferon (U/ml) 80
1
so
-
(b)
The cytokine-producing potentials of CD4 + and CD8 + cells are known to differ and furthermore there is heterogeneity even within the CD4+ population. We have therefore analysed CD4+ and CD8+ clones separately. CD4+ clones derived from BAL differed from those derived from blood in the frequency of IFN-j; versus IL-2-producing clones as well as in the quantity of each cytokine produced (Fig. 3a). The distributions of IFN-, and IL-2 concentrations were compared by the Mann-Whitney U-test. Clones derived from BAL were more likely to produce higher concentrations of IFN, than those from blood (P< 0 0001) while the frequency of IL-2 producers, and the quantity of IL-2 produced, was greater amongst blood clones (P
A clonal analysis of lung T cells derived by bronchoalveolar lavage of healthy individuals M. J. GARLEPP, A. H. ROSE, R. V. BOWMAN, N. MAVADDAT, J. DENCH, B. J. HOLT,* M. BARONHAY,* P. G. HOLT* & B. W. S. ROBINSON Department of Medicine, University of Western Australia, Queen Elizabeth II Medical Centre, Nedlands and *Division of Cell Biology, Western Australian Research Institute for Child Health, Princess Margaret Hospital, Subiaco, Western Australia
Acceptedfor publication 20 April 1992
SUMMARY The characteristics of the T-cell population in the healthy human lung have been investigated by analysing the properties of T-cell clones derived from bronchoalveolar lavage (BAL) samples and comparing them with T cells cloned from the blood ofthe same individuals. The proportions of CD4+ and CD8 + T cells in the starting populations from BAL and blood were similar although only 14% of BAL T cells were CD45RA+ compared to 70% of blood T cells. The precursor frequency of T-cell clones derived from BAL was less than from blood. The cytokine profiles [after phytohaemagglutinin (PHA) stimulation] of the clones derived from both sources were markedly different and these differences lay in the CD4+ population. BAL-derived CD4+ clones produced interferon-y (IFN-y) more frequently than did those from blood while blood-derived clones were more likely to produce interleukin-2 (IL-2) than those from BAL. IL-4 was produced by the majority of BAL- or bloodderived clones (93% and 88% respectively) either along with IFN-y (BAL) or IL-2 (blood). The cytokine profiles of BAL-derived T-cell clones are consistent with those derived from lung interstitium and suggest that the BAL T-cell populations reflect those in the lung wall. Whether the unique properties of lung T cells are acquired after leaving the blood or whether there is selective entry of T-cell subpopulations into the lung remains to be determined.
nosuppressive2 and more recently alveolar epithelial cells have been demonstrated to inhibit mitogen-stimulated lymphocyte proliferation in vitro.3 Recent work in man, and in other species, has shown that the lung is an important repository of T lymphocytes.4-8 At all times more lymphocytes are present in the lung interstitium than in the peripheral circulation.5 The tendency for lymphocytes to sequestrate in the lung has been known for some time9 and recent data suggest that some organ specificity is involved in this entrapment of T lymphocytes. Our recent studies of human interstitial T cells have demonstrated that these lung T cells differ from those derived from the blood in terms of their cloning efficiency and their cytokine-producing potential.5 Bronchoalveolar lavage (BAL) has become a useful technique with which to analyse the cellular content of the alveolar spaces.'0 The results of BAL are used as a diagnostic aid in interstitial lung diseases such as sarcoidosis and extrinsic allergic alveolitis'0"' and the T-cell content of such washings is thought to reflect that of the lung parenchyma. Some histochemical data are consistent with this assumption'2"3 although few data are available which have compared the functional capabilities of T lymphocytes from the two sources. Those studies which have examined T-lymphocyte populations in healthy human lungs
INTRODUCTION The alveoli of the lung provide an important interface between the environment and the body. The local defence mechanisms in the lower respiratory tract thus play a crucial role in the prevention of infection and in the clearance of non-infectious foreign material. The immune system in the lung is required to mount a rapid, specific active immune response as well as to ingest and degrade foreign material. The alveoli of the lung are, however, delicate structures, adapted for the rapid exchange of gases across a thin membrane and as such are highly susceptible to damage by excessive inflammatory processes. The immune response in the lung must therefore be under very tight control' and some candidate regulatory elements have been identified. For example, it has been clear for some time that the alveolar and interstitial macrophages of the lung are profoundly immuAbbreviations: BAL, bronchoalveolar lavage; IFN-y, interferon-y; IL, interleukin; PBL, peripheral blood lymphocytes; PHA, phytohaemagglutinin. Correspondence: Dr M. J. Garlepp, Dept. of Medicine, University of Western Australia, Queen Elizabeth II Medical Centre, Nedlands, Western Australia.
31
M. J. Garlepp et al.
32
have concentrated on phenotype analysis and responses to mitogen stimulation.4'-7 Although useful, such studies provide little information on the spectrum of functional activities possessed by lung T cells. Given the recently described functional heterogeneity of CD4+ cells, in particular,'8"9 it became clear that a more detailed analysis of the functional capabilities of lung T cells was necessary. We have, therefore, cloned T cells from BAL and blood samples from healthy individuals and have compared their cloning efficiencies, phenotypes and cytokineproducing potential. The data reported here confirm that T-cell clones derived from BAL are functionally similar to those derived from the lung interstitium and that their properties differ from those of T-cell clones from peripheral blood.
Table 1. Differential cell content (%) of bronchoalveolar lavage samples (n = 9)
Cell type Macrophages Lymphocytes Neutrophils Eosinophils
Cell preparation The recovered BAL fluid was immediately transported to the laboratory, where excess mucus was removed by straining through sterile gauze, the volume and appearance recorded, and a total cell count performed. Cell viability was determined using trypan blue exclusion. Cytospin preparations were made using 105 cells per slide and stained with May-Grunwald-Giemsa stain. A differential cell count was performed on 400 cells. Peripheral blood leucocyte (PBL) samples were prepared from blood taken from each subject at the time of the BAL, using Ficoll-Hypaque (Pharmacia, Uppsala, Sweden) centrifugation. Fresh lung tissue was obtained from three subjects undergoing lobectomy or pneumonectomy for localized pulmonary malignancy. The tissue studied was always obtained from an area of normal lung farthest from the malignancy. T cells were extracted as previously described using a combination of mechanical and enzymatic (DNAase and collagenase) disruptive techniques.5
T-cell cloning by limiting dilution Purified T cells were prepared from BAL, PBL or lung digests by two cycles of E rosetting as described elsewhere.4'5 T cells were seeded into wells of round-bottomed 96-well microtest plates (Nunc, Roskilde, Denmark) at densities of 16, 8, 4, 2, 1, 0 5 and 0 25 cells per well in a total volume of 200 p1 of RPMI-1640 (Flow Laboratories, McLean, VA) containing 15% human AB serum, I pg/ml highly purified phytohaemagglutinin (PHA)
Range
78-5 + 11-5 18 3 + 10 5 2-9+ 17 03 +04
61-95 5-35 0-8-3 0-1-3
1001 40-
MATERIALS AND METHODS
Study population Alveolar cells were obtained by BAL from nine healthy volunteers (age 54+ 10 years; eight men, one woman; one smoker, six non-smokers and two ex-smokers). None had clinical, radiological or physiological evidence of diffuse lung disease. This study was approved by the Human Rights Committee of the University ofWestern Australia and informed consent was obtained from each subject before the study. BAL was performed as previously described.'0 Briefly, the tip of a fibreoptic bronchoscope was positioned in a segmental bronchus and six 50 ml aliquots of warm (370) normal saline were instilled and immediately aspirated into a disposable sterile polycarbonate bottle. The fluid recovered from the first 50 ml aliquot was collected separately and the subsequent 250 ml (alveolar sample) was used as a source of alveolar T cells. Data presented in this study are from the alveolar specimen only.
Mean
\
CT
30-
0) 2
20 10 0 BAL
Blood
Figure 1. Cloning frequencies for T cells derived from BAL and blood
(paired data). 80
-
(a)
(b)
60-
40-
20
0
Original
Clones
Original
Clones
Figure 2. Phenotypes of starting T-cell populations and resultant clones from BAL (a) and blood (b). Graphs show mean + SEM CD4+ (-) or CD8+ (0) clones (n=9).
(Wellcome Laboratories, Beckenham, Kent, U.K.), 100 U/ml interleukin-2 (IL-2) (Cetus, Emeryville, CA) and 105 irradiated feeder cells. Feeder cells consisted of either irradiated (5000 rads) freshly prepared PBL or human spleen cells (3000 rads) which had been cryopreserved and cultured overnight before use. After 7 days, cultures were fed by replacement of 150 jl of medium with fresh medium containing 1 pg/ml PHA, 100 U/ml IL-2 and 105 irradiated feeder cells. Between Days 12 and 16 growing clones were scored under an inverted microscope and the precursor frequencies were determined according to the zero-order term of the Poisson probability distribution. Clones showing a probability of clonality >0 793 were selected for further expansion. Clones were maintained in RPMI with 15% human AB serum and 100 U/ml IL-2. Cells were split every 3-4 days and refed with fresh medium until sufficient cells were available for analysis.
33
Clonal analysis of lung T cells
-
80
80
-
60
60
-
40
40-
20
20-
0
0
32 80
(b)
60
60
>10
10
-
40-
20
20-
4-32
2-10
(d)
g 40
Surlface phenotipes Surface marker analysis of unfractionated blood and BAL T cells and subsequent clones was performed using fluorescence microscopy. The hybridoma lines OKT3 (CD3), OKT4 (CD4) and OKT8 (CD8) were obtained from American Type Culture Collection [(ATCC), Rockville, MD], grown in our laboratory and, after screening, the culture supernatants were used unpurified. Antibodies to the ;.(5 T-cell receptor (TcR) (TcR--,'-l ), the Y/J TcR (WT31) and CD45RA (Leu-18) were purchased from Becton Dickinson (San Jose, CA). Fluoresceinated sheep antimouse antibody (Silenus. Melbourne. Australia) was used as the detection system. Inidiction of (vtokines
Cloned T cells were seeded at a density of 106 cells ml in 200 Al of RPMI plus 10%' AB serum in 96-well microtitre trays (Nunc) and stimulated with I jig ml PHA. Supernatants were collected after 24 hr and stored frozen at -70 until assayed.
C(}tokine assai}s IL-2 was assayed as previously described." Proliferation of CTLL-2 (ATCC, TIB 214) cells was measured using [3H]thymidine uptake and units of IL-2 read from a standard curve generated with recombinant human IL-2 (Cetus). Interferon-; (IFN-- ) was measured using bioassay.> The reciprocal of the sample dilution which produced a 50%', inhibition of the cytopathic effect of encephalomyocarditis virus on human a
WISH cells (ATCC, CCL25) was determined and the concentration in units was calculated by comparison with the effect of a recombinant human IFN-; standard (Boehringer Ingelheim, Ingelheim, Germany). IFN activity was confirmed to be gamma in type by preincubating samples with antibody to IFN-; (Meloy Laboratories, Springfield, VA) for 1 hr at 37 prior to assay. IL-4 concentrations were measured using a commercially available ELISA assay (Genzyme, Boston, MA). The sensitivities of the assays for IFN-;', IL-2 and IL-4 were 2 U ml, 0 2 U ml and 0-1 ng, ml, respectively. Cytokine production was considered to be significant when the quantity measured was greater than 3 SD above the mean value for feeder cells alone, processed in the same manner as the clones. These cut-offs were 4 U ml (IFN--;) > 2 U ml (IL-2) and > 016 ng ml or 4 U ml (IL-4). Stal.stic.s
Statistical analyses UY-test.
were
carried out
using
the Mann-Whitney
RESULTS Cellular content of BAL and blood
The differential cell content of the BAL samples is shown in Table I. Of the lymphocytes in the BAL 86 + 8%°ao (mean + SEM) were CD3+ (cf. blood 76+9%V.), 3+2%, bore the ;'(i TcR (cf.
34
M. J. Garlepp et al. 80
60
any one subject were nine and 10 for BAL and blood respectively and the mean number of CD8+ clones was six for both BAL and blood.
(a)
-
IL-2 and IFN-y synthesis by CD4+ clones g 40-
20
0 32
Interferon (U/ml) 80
1
so
-
(b)
The cytokine-producing potentials of CD4 + and CD8 + cells are known to differ and furthermore there is heterogeneity even within the CD4+ population. We have therefore analysed CD4+ and CD8+ clones separately. CD4+ clones derived from BAL differed from those derived from blood in the frequency of IFN-j; versus IL-2-producing clones as well as in the quantity of each cytokine produced (Fig. 3a). The distributions of IFN-, and IL-2 concentrations were compared by the Mann-Whitney U-test. Clones derived from BAL were more likely to produce higher concentrations of IFN, than those from blood (P< 0 0001) while the frequency of IL-2 producers, and the quantity of IL-2 produced, was greater amongst blood clones (P
