0 1990 Wiley-Liss, Inc.
Cytometry 11:395-405 (1990)
Separation and Characterization of Basal and Secretory Cells From the Rat Trachea by Flow Cytometry N.F. Johnson', J.S. Wilson, R. Habbersett, D.G. Thomassen, G.M. Shopp, and D.M. Smith Inhalation Toxicology Research Institute, Lovelace Biomedical and Environmental Research Institute, Albuquerque, New Mexico 87185 (N.F.J., D.G.T.);Life Sciences Division, Los Alamos National Laboratory, Los Alamos, New Mexico 87545 (N.F.J., J.S.W., R.H., D.M.S.); Lovelace Medical Foundation, Albuquerque, New Mexico 87108 (G.M.S.) Received for publication May 8, 1989; accepted October 26, 1989
Basal and secretory cells have been separated as highly enriched viable populations from single-cell suspensions of rat tracheal epithelial cells. Isolation of the populations was achieved by preparation of a cell suspension and separation by flow cytometry using contour maps generated from 2"and 90"light scatter signals. Flow cytometric analysis of cells showed 10% of the whole preparation were cells in SG,M phase of the cell cycle. The secretory cells accounted for 86% of these cycling cells; the remainder were accounted for by the basal cells. Culture of sorted populations of basal and secretory cells in serum free defined medium showed that basal cells had a lower (0.6%) colony-forming efficiency
The lung is a complex organ composed of more than 40 cell types (3).While many of these cells can contribute to the development of primary lung cancer in humans, epithelial cells appear to be the major cell types with progenitorial capacity. Many human lung cancers occur centrally and are found mainly within the first five generations of the conducting airways (4). Predominant cell types in this region are basal, secretory, and ciliated cells (17). Ciliated cells are terminally differentiated and play a minor role in repair and replacement in the tracheobronchial lining (15). Basal and secretory cells are the potential progenitor cells and are thought to be the cells that undergo neoplastic transformation and give rise to lung carcinomas (15) in the upper respiratory tract. Until recently, basal cells of the airway have been viewed analogously to the basal keratinocytes of skin, and generally considered the major proliferative cell type in the upper respiratory tract (1)and are considered the critical cell a t risk from inhaled toxicants (7). In experimental studies to determine the role of
than secretory cells (3.4%). Significant differences in blue auto-fluorescence, Hoechst 33342 uptake, and lectin staining were apparent between basal and secretory cells. These results suggest that the secretory cell rather than the basal cell is primarily the cell type involved in maintenance of the normal tracheal epithelium. Secretory cells are greater in number, have a higher proliferative potential, and greater metabolic capability. Because of these traits they may be a critical cell at risk from damage by environmental agents. Key terms: Trachea, lectins, cycling cell fraction, potential target cells
basal and secretory cells, tracheas from rodents are frequently used a s the source of cells. While humans experience few tracheal tumors, the cell types present in the trachea are those found lining the first few divisions of the bronchial tree. Recent studies by McDowell and colleagues show that secretory cells in the hamster trachea, and not basal cells, play a major proliferative role in repair of denuding injuries to the tracheal epithelium (12). This approach to delineating the proliferative and progenitorial capacity involves a n artificially induced injury. During the response to this injury, cell populations involved can undergo phenotypic changes making identification of cell types difficult. In addition, i t is difficult to follow accurately the lineage of the various cell
'Address reprint requests to Dr. Neil F. Johnson, Lovelace Inhalation Toxicology Research Institute, P.O. Box 5890, Albuquerque, NM 87185.
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types. Morphological studies indicate that the basal cell may have a n important role in adhesion of columnar ciliated and secretory cells to the basement membrane (6). Two recent studies have shown that basal cells from the rabbit trachea are capable of reestablishing a complete tracheal epithelial lining following their inoculation into denuded heterotopic tracheal grafts (8,9).The basal cells in one case were isolated by centrifugal elutriation (8),a n approach used by Mahler et al., who showed that isolated basal cells from rat trachea had a low proliferative capacity in culture (14). In the second study of Inayama et al. basal cells were further purified by a n in vitro cloning step (9) to overcome some of the potential contamination problems associated with centrifugal elutriation. The present study was undertaken to develop a methodology for separating pure populations of viable basal and secretory cells from the normal tracheal epithelium, and to characterize these populations by flow cytometry with particular reference to their proliferative capacities. The separated cells were also characterized using ultrastructural analysis and lectin binding. With such cell separations, pure populations can be studied to determine progenitorial and other characteristics relating to neoplasia.
MATERIALS AND METHODS Female Fischer 3441N rats (6-12 wk of age) from the Inhalation Toxicology Research Institute (ITRI) breeding colony were sacrificed using CO, inhalation, and their tracheas were removed aseptically. The laryngeal end of the trachea was cannulated with polythene tubing (ID 1-14 mm, OD 1.57 mm) fitted with a n 18 gauge hypodermic needle, and the tracheal lumen was flushed with medium (RPMI with 25 mM HEPES buffer, 20 mgiL DNase and 20 mliL antibiotic solution (Sigma, St. Louis, MO). The distal end of the trachea was occluded with a n artery clamp and the lumen filled to distension with a solution of hyaluronidase (1.6 mgi ml) and cytochalasin B (1 kgiml) made up in aMEM (Sigma, St. Louis, MO) and incubated for 60 min a t 37°C. The lumen was then flushed with 10 ml medium (RPMI with 25 mM HEPES buffer, 20 mgil DNase, and 20 mliL antibiotic solution, Sigma, St. Louis, MO), which was collected and kept a t 4°C. The lumen was re-inflated with aMEM containing pronase (3.3mgiml) and incubated for 30 min a t 37°C. The lumen was then flushed with 20 ml medium (as above, but containing 10% fetal calf serum), which was collected with the previous sample. This pooled sample was then used to flush the tracheal lumen a n additional 5 times (5 x 10 ml). The resultant cell suspension was centrifuged (1,100 rpm, 15 min a t 19°C) and the supernatant was carefully removed and discarded. The cell pellet was resuspended gently in 4 ml of fresh RMPI medium (no fetal calf serum) and counted using a haemacytometer. Viability was assessed using trypan blue dye exclusion. Cell suspensions were adjusted to a concentration of approximately 0.5 x lo6 cellsiml and analyzed either
a t Los Alamos National Laboratory using a multiparameter flow cytometer (20) operated by the National Flow Cytometry Resource (NFCR) or a t the Lovelace Medical Foundation (LMF), Albuquerque, NM on a n EPICS V (Coulter Electronic, Hialeah, FL). At NFCR, cells were analyzed using 2" and 90" light scatter, axial light-loss, and blue, green, and red fluorescence. Commonly, two lasers were used, one operating in the UV and the other at a wavelength of 488 nm, a t powers of 300 and 400 mW, respectively. The UV enhanced laser provided excitation for blue and red fluorescence, while the 488 nm wavelength laser provided excitation for green fluorescence, forward angle light scatter, and axial light-loss signals. The data generated from 10 to 30,000 events per sample were stored in correlated list mode fashion. The data subsequently were processed a s individual frequency distribution histograms and two-parameter contour diagrams. The contour diagrams were used to delineate discrete cell populations. At LMF, the cells were examined using a single laser (488 nm wavelength, 0.4-0.5 mW power) to generate 2" and 90" light scatter signals. Two-parameter contour diagrams were used to separate defined cell populations. With both flow cytometers, two cell populations were sorted at the same time and 10-30,000 cells were collected from each sample for ultrastructural analysis or cytological preparations. In addition, 3,000 sorted basal or secretory cells were sorted directly in 60 mm culture dishes (Lux, Naperville, IL) containing 4 ml of serum-free defined medium. This medium has been previously described by Thomassen et al. (21). Cells were incubated in this medium a t 37°C in a humid, 5% CO, and 95% air atmosphere, fixed and stained after 10 days and colonies with 2 3 0 cells were scored. The sorted cells and the original cell suspension were processed for electron microscopy according to the method of Sebring et al. (18).In brief, the cells were fixed in buffered glutaraldehyde and centrifuged within a n agar-plugged, narrow-bore tube. Following secondary fixation with buffered osmium tetroxide, the cell pellet was encapsulated in agar. The agar containing the cells was removed from the tubing and processed conventionally for electron microscopy. These preparations were used to verify the morphology of the cells and the purity of the various cell types. The purity of the cell populations was assessed by identifying a t least 100 cell cross sections. Cells used for cytospin preparations were centrifuged at 2,500 rpm for 10 min and all but 200 pl of supernatant removed and discarded. The cells were resuspended in the remaining supernatant, cytology preparations were made using the Shandon Cytospin 2 (Pittsburgh, PA) and stained with Diff Quik stain (American Scientific Products, McGaw Park, IL). The cell surface sugar moieties were determined in the intact tracheal epithelial lining and in isolated cell preparations using lectins labeled with ferritin (for electron microscopy) o r fluorescein (for histology and
SEPARATION OF TRACHEAL BASAL AND SECRETORY CELLS
397
FIG. 1. Light micrograph of a cytospin preparation of isolated tracheal epithelial cells. The cell suspension is composed predominantly of single cells
flow cytometry). Samples of tracheal tissue were fixed in 10% buffered formalin. After dehydration, the tissues were embedded in paraffin and 5 pm sections were cut for histology. After deparaffnization in xylene, the sections were brought through decreasing concentrations of alcohol to water, rinsed in phosphate-buffered saline (PBS), and stained for 30 min with various concentrations of fluorescein-conjugated lectins (10-100 pg/ml) in PBS (pH 7.2) and then gently washed twice in 5 ml of PBS. The stained sections were mounted in 10% buffered glycerin solution and examined with a fluorescence microscope. The lectins used were Ulex europaeus (UEA I), Pisum sativum (PSA), Lens culinaris (LCA), Dolichos biflorus (DBA), Glycine max (SBA), Maclurapomifera (MPA),Arachis hypogea (PNA),Titicum vulgaris (WGA), Concanavalin A (CON A), Helix pomatia (HPA), Wisteria floribunda (WFA), Banderiraea simplicifolia (BSA I), Phaseolus limus (PHA L), and Limulus polyphemus (LPA). As a control, sections were either stained with PBS only or fluorescein-conjugated lectin with the appropriate inhibitory sugar (0.4 mM). The sugars used were L-fucose (UEA I), Dmannose (PSA, LCA, CON A), D-glucose (Con A), Nacetyl-D-galactosamine (DBA, MPA, SBA), D-galactose (BSA I, MPA), N-acetyl-1-D glucosamine (WGA, HPA) and sialic acid (LPA). The preparations involving UEA I employed PBS containing calcium and magnesium ions; all the other preparations used PBS with-
out magnesium or calcium ions. The lectins and inhibitory sugars were obtained from Sigma Chemical Co. (St. Louis, MO). For flow cytometry the cell suspensions were adjusted to a concentration of approximately 0.5 x lo6 celldm1 and stained with 10-100 pg lectin/ml or lectin plus inhibitory sugar for 30 min a t 0-1°C and then examined by flow cytometry for the presence of cell-associated green fluorescence. Cells used for flow cytometry were investigated unstained or stained singly or in combination with the following: Hoechst 33342 dye (5 pgiml for 30 min a t 37°C) for DNA content, pyronin Y (10 pg/ml, 5 min room temperature) for RNA content and propidium iodide (10 pgiml, 5 min room temperature) for viability determinations.
RESULTS The initial cell suspension contained 1.0-3.0 x lo6 cells with a viability in excess of 95%. Similar estimates of viability were obtained with trypan blue dye exclusion and flow cytometric evaluation using propidium iodide. The cell suspension was primarily composed of single cells (Fig. 1). The differential cell count was undertaken using electron microscopy. Estimates were obtained from three separate preparations in which 100 cell cross sections were viewed for each sample. The individual values were for basal cells, 20, 22, and 28% (average 23%);secretory cells, 59,61, and 67%
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A X I A L LIGHT LOSS
2'LIGHT SCATTER
FIG. 2. Two-parameter contour diagrams. A 90" light scatter vs. axial light loss (NFCR).B: 90" light scatter vs. 2" light scatter (LMF). Two populations, a and b, can be resolved. Population a represents large granular cells, while population b represents small agranular
cells. Ultrastructural features of these populations show a to be secretory cells and b to be basal cells. The events aligned along the y-axis (90"light scatter) with low 2" light scatter are composed of cell debris.
(average 62%); ciliated cells, 4, 14, and 20% (average 13%)and other cells, 1, 2, and 3% (average 2%). The latter cells were composed of macrophages, red blood cells, and unidentified cells. Flow cytometric analysis was conducted using a variety of intrinsic and extrinsic parameters. The intrinsic parameters, 2" light scatter and axial light-loss, reflect cell size; 90" light scatter relates to the granularity of the cell (20);and blue auto-fluorescence indicates the inherent levels of NADPH (22). Extrinsic parameters employed were Hoechst 33342 for DNA content, pyronin Y for RNA content, and lectins for cell-surface sugar moieties. Ungated flow cytometric analysis of the cell suspensions involving 90" light scatter vs. axial light-loss or 2" light scatter using either the custom made flow cytometer (NFCR) or the commercial instrument (LMF) produced similar two-dimensional contour maps (Fig. 2). The location of viable DNA-containing cells was established by dual staining with Hoechst 33342 and propidium iodide. Data was acquired from events that were Hoechst positive and propidium iodide negative. These events were displayed a s a 90" light scatter vs. 2" light scatter contour diagram, revealing two major cell populations (Fig. 2). Hoechst positive, propidium iodide negative events were regarded a s being related to viable cells, while the Hoechst negative, propidium iodide positive events were taken as either dead cells or debris. The two subpopulations contained within the total viable cell population had distinct morphological characteristics (Fig. 3); one was composed of small agranular cells and the other of larger granular cells. Ultrastructural analysis of the two cell types showed them to be either basal or secretory cells, respectively. Secre-
tory cells were of the serous type, with electron-dense, membrane-bound secretory granules; numerous mito-
B FIG. 3. Light microscopy of cytospin preparations of sorted basal cells (A) and sorted secretory cells (B). The populations were sorted according to their 2" and 90" light scatter characteristics using defined parameters shown in Figure 2B.
SEPARATION OF TRACHEAL BASAL AND SECRETORY CELLS
399
B
A
FIG. 4. Electron micrographs of cells from populations sorted on the basis of 90" light scatter and axial light loss (as defined in Fig. 2A). Population A contains cells possessing secretory granules, numerous mitochondria, and well-developed elements of rough endo-
plasmic reticulum. Population B represents basal cells with scant cytoplasm containing few mitochondria elements of rough endoplasmic reticulum. Prominent bundles of microfilaments are seen in the perinuclear region (bar represents 0.5 pm).
chondria and cisternae of rough endoplasmic reticulum; and a n eccentric, indented nucleus (Fig. 4A). The isolated basal cells had scant cytoplasm containing prominent perinuclear microfilaments, few mitochondria, and elements of rough endoplasmic reticulum; and the cell surface possessed numerous fine micro villi (Fig. 4B). The ultrastructural appearance of isolated cells was similar to that seen in the intact tracheal epithelium with the exception of the loss of cell shape. The distinctive ultrastructural morphologies of the isolated basal and secretory cells were used to quantify the purity of cell separations. With these parameters, the purity of the basal and secretory cells was 94 1% and 91 3%, respectively (average of 3 estimations -+ SEM). The remaining cells could not be identified a s they had no definitive cytological features. The colonyforming efficiency of the cells sorted directly in culture dishes was 0.6% -+ 0.15% for basal cells and 3.4% 0.42% for secretory cells. The colony-forming efficiency
of the unsorted parent population was 2.6% 0.15%. These values were obtained from 5 separate sorts on different occasions. Flow cytometric analysis (5) using Hoechst 33342 showed the number of cells within the suspension that were in S, G2, or M phase of the cell cycle to be 14.2% 0.23% (standard error of mean n = 5). This figure required correction to allow for the drop in the number of ciliated cells within the cell suspension compared to the intact trachea. The intact trachea contains approximately 37-40% ciliated cells (11)compared to 13%in the cell suspension. The corrected value of the dividing cells in the original tracheal epithelium was 9.1% and this showed that only a fraction of the cells are in S, G2, and M phases of the cell cycle a t any one time. Reanalysis of the cycling cells using 2" and 90" light scatter showed the majority (>86%) of cells to be relatively large, with granular cytoplasm (Fig. 51, which are characteristics of secretory cells. Similar results
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FIG. 5. Flow cytometric analysis of the tracheal cells suspension. A Two-parameter contour diagram of blue fluorescence (DNA) content vs. 2" light scatter: population A represents predominantly cells in S G, M phase of the cell cycle and population B represents cells in
G,G, phase of the cell cycle. Replotting the data from population A on the basis of 2" and 90" light scatter (C) shows these cells to be in the region of the secretory cell population (arrow) of the parent cell suspension (B).
were obtained when the two major populations of the original cell suspension were analyzed in terms of their DNA content; the secretory cell population had approximately six times more cells in the S, G2, and M phases of the cell cycle than the basal cell population. A satisfactory staining reaction was obtained with 5 pg Hoechst 333421m1 and a 30 min incubation period (coefficient of variation of G,/G, peak was < 6%); however, during the initial stages of dye uptake, there was a bimodal staining reaction (Fig. 6). Analysis of the single parameter histogram using data from the two major staining peaks and replotting it as a bivariate contour diagram (2' light scatter or axial light-loss vs. 90" light scatter) showed that the slowly staining cells were predominantly small agranular cells (basal cells), while the more rapidly staining cells were larger and more granular (secretory cells) (Fig. 6). The blue auto-fluorescence response showed a bimodal distribution, one population with low fluorescence, and another with a positive blue auto-fluorescent response. Analysis of these data on a bivariate contour diagram with axial light-loss showed two major populations. The small cells gave a small blue auto-fluorescence signal, while the larger cells were positive (Fig. 7). A similar response was seen with pyronin Y staining for RNA (data not shown). There was good correlation between negative blue auto-fluorescence and lowhegative pyronin Y fluorescence; 91% of the former cells were in the latter population. The fluorescein-conjugated lectins displayed a variable pattern of cell labeling. WFA, SBA, WGA, and
BSA all gave a positive response (Fig. 81, while the remainder elicited no fluorescent signal up to a lectin concentration of 100 pgiml (Table 1). All the lectins gave a negative response when they were combined with their inhibitory sugar. Reanalysis of the fluorescence positive cells on a bivariate contour diagram (90" light scatter vs. 2" light scatter) shows that WGA stains two cell populations (characteristic of basal and secretory cells); SBA, BSA-I, and WFA stained predominantly single populations (secretory-SBA and BSA-I and basal cells-WFA) (Fig. 9). The staining characteristics of the isolated cells were similar to those seen in the intact epithelium whether i t was stained with ferritin or fluorescein-conjugated lectins (Table 1).
DISCUSSION Pure, viable populations of either basal or secretory cells from the rat trachea were produced. The isolation procedure did not appear to affect cells adversely, as there were marked similarities in the ultrastructural appearance and the expression of surface sugar moieties between isolated cells and the cells of the intact epithelium. In addition, the cells have been successfully grown in culture in defined serum free medium. The separation of basal and secretory cells is based on intrinsic differences between the two cell types in terms of their ability to scatter light. The basal cells are relatively small and agranular and, consequently, have low 2" and 90" light scatter, while the secretory cells are larger, with prominent secretory granules that result in high 2" and 90" light scatter signals. Sort-
SEPARATION OF TRACHEAL BASAL AND SECRETORY CELLS
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with WGA (19). However, they did demonstrate that CON A and RCA (Ricinus communis agglutin) bound to the cells. CON A in the present study was negative. Lectin labeling of human bronchial glands showed that the secretory cells stain positively with a wide range of lectins, however, serous cells were negative with HPA (16). This result is similar to the present study where the majority of secretory cells in the rat trachea are of the serous type. Studies on the mouse trachea demonstrated that PNA and DBA did not label the epithelial lining cells (23). Ultrastructural and flow cytometric profiles of the cells show the secretory cells to be more active metabolically than the basal cells. This is seen in the numerous mitochondria, cisternae of rough endoplasmic reticulum, secretory granules, RNA content, and blue auto-fluorescence of the secretory cells. Blue auto-fluorescence is a n indicator of inherent NADPH content (22) and, therefore, of general biochemical synthetic activity. In contrast, the basal cells have few mitochondria, little RNA, no blue auto-fluorescence, and exiguous cytoplasm. Working a t a concentration of 5 bg Hoechst 333421m1 provides a n adequate DNA histogram (coefficient of variation of GIG, peak < 6%); however, during the initial phases of dye uptake, there is a bimodal response. The two major cell populations (basal and secretory cells) take up stain differently and may provide a parameter with which to sort the cells. The slowly staining cells have a flow cytometric profile (small agranular cells) compatible with basal cells, while the more rapidly staining cells have a profile (large granular cells) similar to secretory cells in other experiments. This confirms other studies that showed that different cell types take up Hoechst 33342 stain a t different rates (13).Differential staining patterns must be taken into account when assaying the DNA content of heterogenous cell populations. The results showed that the secretory cell population contains the majority (> 85%) of cycling cells in the normal trachea. These results agree with the work of McDowell and Trump (15), indicating that secretory cells play a major role in repair of the tracheal lining following a denuding injury. The results confirm previous findings that both basal and secretory cells are capable of division (2); however, here secretory cells play a more significant role than basal cells. The higher colony-forming efficiency of secretory cells compared to basal cells also indicates the greater proliferative potential of the former cells. These studies also show that sorting cells by flow cytometry does not appear to affect the growth of tracheal epithelial cells in culture. The value for colony-forming efficiency obtained in this study are comparable to those of Mahler et al., who found 0.1-1.0% CFE for a cell fraction obtained from the rat by elutriation containing 70% basaloid cells and a CFE of 5-12% for a fraction containing only 20-30% basaloid cells (14). Inayama et al. (8,9), using similar elutriation techniques a s Mahler et al.
L'L DNA/BLUE FLUORESCENCE
FIG. 6. Flow cytometric analysis of Hoechst staining. A is a twoparameter countour diagram of the tracheal cell suspension, using 90" light scatter and axial light loss signals. Two major populations are defined. Population A was shown to correspond to secretory cells and population B to basal cells. An adequate DNA histogram can be obtained with 5 kg Hoechstiml with a 30 min incubation period a t 37°C (C). During the initial stain uptake, a bimodal staining reaction is seen (B) = 5 kgiml, 5 min incubation). Analysis of the blue fluorescence data from population A at the time point (D) shows the majority of cells taking up stain rapidly. Similar analysis for population B (E) shows cells taking up stain more slowly than population A.
ing windows for the two cell types can be adjusted so there is no apparent overlap of cell types. This approach produces pure populations, but it leads to the collection of a proportion of each cell phenotype. The differential lectin staining of the two cell types may provide a means to sort pure populations from the entire spectrum of each cell phenotype. This approach is currently under investigation. The binding patterns of the various lectins show that the cell surfaces of isolated tracheal epithelial cells are similar to their counterparts in the intact epithelium. The positive staining of in situ epithelial cells and isolated cells with WGA is in contrast to the results of Shiba et al., who found no staining of rat tracheal cells
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basis of blue auto-fluorescence shows that these cells have a positive fluorescence response (C) compared to population B in which the cells have low auto-fluorescence (B).
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ml) staining showing presence of labeled cells. D: BSA-I (10 pglml) and E, SBA (10 pgiml) reveals positive labeled cells. F PNA (50 Fgiml) staining elicits no fluorescence response.
403
SEPARATION O F TRACHEAL BASAL AND SECRETORY CELLS
Table 1 Lectin Affinity of Tracheal Epithelial Cells I n Situ and in Single Cell Suspensions" In situ epithelia Lectin UEA I PNA BSA I BSA I1 WFA WGA MPA DBA SBA LCA CONA HPA
B
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PHA-L "This table shows the lectin affinity of tracheal epithelial cells in situ and in single cell suspensions. The former cells were investigated using fluorescent light microscopy (FITC labeled lectins) and electron microscopy (ferritin labeled lectins), while the latter were studied by flow cytometry (FITC labeled lectins). There was no detectable difference between the cells of the intact epithelium and those in suspension. UEA = Ulex europeans agglutinin; PNA = peanut agglutinin; BSA I and BSA I1 = Bandeiraea simplicifolia agglutinin; WGA = wheat germ agglutinin; MPA = Maclura pomifera agglutinin; SBA = soyabean agglutinin; LCA = Lens culinaris agglutinin; Con-A = concanavalin A; HPA = Helix pomatia agglutinin; WFA = Wisteria floribuinda agglutinin; DBA = Dolichos biflorus agglutinin; PHA-L = Phaseolus limus agglutinin. B = basal cell; S = secretory cell; C = ciliated cell; ND = not done; + = positive; + + = markedly positive.
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FIG. 9. Flow cytometric analysis of fluorescein labeled lectin binding. A is a contour diagram of the parent tracheal cell suspension using 90" light scatter and 2" light scatter indicating the locations of basal and secretory cells. B is a contour diagram (2" vs. 90" light scatter) using data from weakly stained WGA events; the stained cells correspond to basal cells of the parent cell suspension. C is a contour
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diagram (2" vs. 90" light scatter) using data from strongly positive stained WGA events; the stained cells correspond to the secretory cells of the parent cell population. Results similar to this occur with SBA and BSA-I stained cells. D is a contour diagram (2" vs. 90" light scatter) using data from WFA positive cells. The stained cells correspond to the basal cells of the parent population.
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(14), showed that cell suspension obtained from rabbits containing 83-94% basal cells, when inoculated into denuded tracheal grafts are capable of reestablishing a complete tracheal epithelium. The basal cells may have a low proliferative capacity but have the ability to act as progenitor cells. However, comparison of results between those experiments are complicated by the use of different species and isolation techniques. The choice of isolation or separation techniques may influence the composition of the initial cell suspension and the extent of contaminating cells. Minor modifications to the enzyme digest technique can affect the relative proportion of cell types in the initial cell suspension prior to any separation procedure. The method reported here of a combined hyaluronidase and cytochalasin treatment resulted in a cell suspension containing 13% ciliated cells; in a n earlier method (11) using separate hyaluronidase and cytochalasin, the proportion of ciliated cells was 32.6%. In this latter study and our present study, non-granulated secretory cells were not identified by electron microscopy. Jeffery and Reid (10) have shown by electron microscopy that these cells are infrequent and may show signs of ciliogenesis; however, by light microscopy non-granulated secretory cells can account for 13-27% of the total tracheal cell population. Flow cytometry, while producing a relatively low number of cells (up to 1 x lo6), produced higher-purity populations than can be obtained by elutriation. Flow cytometry produced a basal cell population of 95% purity. The contaminating cells could not be identified by electron microscopy. Elutriation techniques produced a n 85% pure basal cell population using electron microscopy to characterize the cells (8).This level of purity may be insufficient for in vitro or in vivo studies; if the 85% fraction of the entire population has a low proliferative capacity (as is the case with basal cells) it could easily become replaced by a highly proliferative cell type contained within the 15% impurity. To increase the purity of basal cell fractions obtained from elutriation, a postelutriation in vitro cloning step has been used (9). This in vitro step increases the effort needed to achieve a highly pure population of basal cells and allows the possibility of the selection or differentiation of a new population of cells. Flow cytometry provides a simple method of separation for basal and secretory cells from the rat trachea. This may also be true of other pulmonary cells such a s the alveolar type IT cells (24). Basal cells of the tracheobronchial lining are generally assumed to have a role analogous to basal cells of the stratum basale of the epidermis. As a result of this assumption, basal cells generally are considered to be the cells a t risk from the carcinogenic and toxic effects of xenobiotics. Many calculated dose-effect relationships, especially for radioactive materials, are based on this premise (7).The secretory cells, however, appear to be the critical cells at risk because they have a greater potential metabolic capability, they are more numerous and have a higher proliferative potential than the
basal cells and receive a higher dose from inhaled material because they are closer to the luminal surface. The overall biological risk for a specific exposure condition does not change if the secretory cells are considered the critical cells; however, the effective dose changes. The ability to sort and characterize pure cell populations will allow for investigations of differentiation pathways associated with these populations. In addition, the use of well defined mixtures of various cell types will help to delineate the role of cell-to-cell interactions in the repair and maintenance of the normal tracheobronchial lining. A further potential use of this system is in determination of the sensitivity of defined cell populations to toxicant damage.
ACKNOWLEDGMENTS Research was sponsored by the U S . Department of Energy’s Office of Health and Environmental Research under contract No, DE-AC04-76EV01013 and by Los Alamos National Flow Cytometry Resource funded by the Division of Research Resources of NIH (Grant p41rr01315) and the Department of Energy. Flow cytometry facilities were also kindly provided by Lovelace Medical Foundation. Technical support was provided by E. A. Margiotta and J. Milisa, whose help is gratefully acknowledged.
LITERATURE CITED 1. Blenkinsopp WK: Proliferation of respiratory tract epithelium in the rats. Exp Cell Res 46:144-154, 1967. 2. Boren HG, Paradise W: Cytokinetics of lung. In: Pathogenesis and Therapy of Lung Cancer, Volume X, Harns CC (ed).Dekker, New York, 1978, pp 369-418. 3. Breeze RG, Wheeldon EB: The cells of the pulmonary airways. Am Rev Respir Dis 116:705-777, 1977. 4. Dunnill MS: Pulmonary Pathology. Longman Group, London, 1982. 5. Dean PN, Jett JH: Mathematical analysis of DNA distributions derived from flow cytometry. J Cell Biol 60:253-261, 1976. 6. Evans MJ, Plopper C G The role of basal cells in adhesion of columnar epithelium to airway basement membrane. Am Rev Respir Dis 138:481-483, 1988. 7. Harley NH, Pasternack BS: Environmental radon daughter alpha factors in five lobed human lung. Health Phys 42:789-799, 1982. 8. Inayama Y, Hook GER, Brody AR, Cameron GS, Jetten AM, Gilmore LB, Gray T, Nettesheim P: The differentiation potential of tracheal basal cells. Lab Invest 58:706-717, 1988. 9. Inayama K, Hook GE, Brody AR, Jetten AM, Gray T, Mahler J, Nettesheim P: I n uitro and in uiuo growth and differentiation of clones of tracheal basal cells. Am J Pathol 134:539-549, 1989. 10. Jeffery PK, Reid L: New observations of rat airway epithelium: A quantitative and electron microscopic study. J Anat 120: 295-320, 1975. 11. Johnson NF, Margiotta EA, Wilson JS, Sebring R J , Smith DM: Preparation of viable single cell suspensions of tracheal epithelial cells. Br J Pathol 68:157-165, 1987. 12. Keenan KP, Wilson TS, McDowell EM: Regeneration of hamster tracheal epithelium after mechanical injury. V. Histochemical, immunocytochemical and ultrastructural studies. Virchows Arch [Cell Path011 43:213-240, 1983.
SEPARATION OF TRACHEAL BASAL AND SECRETORY CELLS 13. Lalande ME, Miller RG: Fluorescence flow analysis of lymphocyte activation using Hoechst 33342 dye. J Histochem Cytochem 27:394, 1979. 14. Mahler J , Hook GER, Gilmore L, Gray T, Nettesheim P: Proliferative activity of tracheal epithelial cell fractions isolated by centrifugal elutriation. Am Rev Respir Dis 137:322, 1988. 15. McDowell EM, Trump BF: Conceptual review: Histogenesis of preneoplastic and neoplastic lesions in tracheobronchial epithelium. Sur Syn Pathol Res 2:235-279, 1984. 16. Mazzuca M, Lhermitte M, Lafitte J J , Roussel P: Use of lectins for detection of glycoconjugates in the glandular cells of the human bronchial mucosa. J Histochem Cytochem 30:956-966, 1982. 17. Rhodin JAG: Ultrastructure and function of the human tracheal mucosa. Am Rev Respir Dis 93:l-15, 1966. 18. Sebring RJ, Johnson NF, Spa11 WD: Transmission electron microscopy of small numbers of sorted cells. Cytometry 9:88-92, 1988. 19. Shiba M, Ohiwa T, Klein-Szanto AJP: Lectin-binding sites in
20. 21.
22. 23.
24.
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preneoplastic and neoplastic lesions of human and rodent respiratory tracts. JNCI 72:43-51, 1984. Steinkamp JA: Flow cytometry. Rev Sci Instrum 55:1375-1400, 1984. Thomassen DG, Safiotti U, Kaighn ME: Clonal proliferation of rat tracheal epithelial cells in serum-free medium and their responses to hormones, growth factors and carcinogens. Carcinogenesis 2033-2039, 1986. Thorell B: Intracellular red-ox steady-states as basis for cell characterization by flow cytofluorometry. Blood Cells 6:745-751, 1980. Watanabe M, Muramatsu T, Shirane H, Vgai K: Discrete distribution of binding sites for dolichos biflorus agglutinin (DBA)and for peanut agglutinin (PNA)in mouse organ tissues. J Histochem Cytochem 29:779-790, 1981. Wilson JS, Steinkamp JA, Lehnert BE: Isolation of viable type I1 alveolar epithelial cells by flow cytometry. Cytometry 7:157-162, 1986.
Cytometry 11:395-405 (1990)
Separation and Characterization of Basal and Secretory Cells From the Rat Trachea by Flow Cytometry N.F. Johnson', J.S. Wilson, R. Habbersett, D.G. Thomassen, G.M. Shopp, and D.M. Smith Inhalation Toxicology Research Institute, Lovelace Biomedical and Environmental Research Institute, Albuquerque, New Mexico 87185 (N.F.J., D.G.T.);Life Sciences Division, Los Alamos National Laboratory, Los Alamos, New Mexico 87545 (N.F.J., J.S.W., R.H., D.M.S.); Lovelace Medical Foundation, Albuquerque, New Mexico 87108 (G.M.S.) Received for publication May 8, 1989; accepted October 26, 1989
Basal and secretory cells have been separated as highly enriched viable populations from single-cell suspensions of rat tracheal epithelial cells. Isolation of the populations was achieved by preparation of a cell suspension and separation by flow cytometry using contour maps generated from 2"and 90"light scatter signals. Flow cytometric analysis of cells showed 10% of the whole preparation were cells in SG,M phase of the cell cycle. The secretory cells accounted for 86% of these cycling cells; the remainder were accounted for by the basal cells. Culture of sorted populations of basal and secretory cells in serum free defined medium showed that basal cells had a lower (0.6%) colony-forming efficiency
The lung is a complex organ composed of more than 40 cell types (3).While many of these cells can contribute to the development of primary lung cancer in humans, epithelial cells appear to be the major cell types with progenitorial capacity. Many human lung cancers occur centrally and are found mainly within the first five generations of the conducting airways (4). Predominant cell types in this region are basal, secretory, and ciliated cells (17). Ciliated cells are terminally differentiated and play a minor role in repair and replacement in the tracheobronchial lining (15). Basal and secretory cells are the potential progenitor cells and are thought to be the cells that undergo neoplastic transformation and give rise to lung carcinomas (15) in the upper respiratory tract. Until recently, basal cells of the airway have been viewed analogously to the basal keratinocytes of skin, and generally considered the major proliferative cell type in the upper respiratory tract (1)and are considered the critical cell a t risk from inhaled toxicants (7). In experimental studies to determine the role of
than secretory cells (3.4%). Significant differences in blue auto-fluorescence, Hoechst 33342 uptake, and lectin staining were apparent between basal and secretory cells. These results suggest that the secretory cell rather than the basal cell is primarily the cell type involved in maintenance of the normal tracheal epithelium. Secretory cells are greater in number, have a higher proliferative potential, and greater metabolic capability. Because of these traits they may be a critical cell at risk from damage by environmental agents. Key terms: Trachea, lectins, cycling cell fraction, potential target cells
basal and secretory cells, tracheas from rodents are frequently used a s the source of cells. While humans experience few tracheal tumors, the cell types present in the trachea are those found lining the first few divisions of the bronchial tree. Recent studies by McDowell and colleagues show that secretory cells in the hamster trachea, and not basal cells, play a major proliferative role in repair of denuding injuries to the tracheal epithelium (12). This approach to delineating the proliferative and progenitorial capacity involves a n artificially induced injury. During the response to this injury, cell populations involved can undergo phenotypic changes making identification of cell types difficult. In addition, i t is difficult to follow accurately the lineage of the various cell
'Address reprint requests to Dr. Neil F. Johnson, Lovelace Inhalation Toxicology Research Institute, P.O. Box 5890, Albuquerque, NM 87185.
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types. Morphological studies indicate that the basal cell may have a n important role in adhesion of columnar ciliated and secretory cells to the basement membrane (6). Two recent studies have shown that basal cells from the rabbit trachea are capable of reestablishing a complete tracheal epithelial lining following their inoculation into denuded heterotopic tracheal grafts (8,9).The basal cells in one case were isolated by centrifugal elutriation (8),a n approach used by Mahler et al., who showed that isolated basal cells from rat trachea had a low proliferative capacity in culture (14). In the second study of Inayama et al. basal cells were further purified by a n in vitro cloning step (9) to overcome some of the potential contamination problems associated with centrifugal elutriation. The present study was undertaken to develop a methodology for separating pure populations of viable basal and secretory cells from the normal tracheal epithelium, and to characterize these populations by flow cytometry with particular reference to their proliferative capacities. The separated cells were also characterized using ultrastructural analysis and lectin binding. With such cell separations, pure populations can be studied to determine progenitorial and other characteristics relating to neoplasia.
MATERIALS AND METHODS Female Fischer 3441N rats (6-12 wk of age) from the Inhalation Toxicology Research Institute (ITRI) breeding colony were sacrificed using CO, inhalation, and their tracheas were removed aseptically. The laryngeal end of the trachea was cannulated with polythene tubing (ID 1-14 mm, OD 1.57 mm) fitted with a n 18 gauge hypodermic needle, and the tracheal lumen was flushed with medium (RPMI with 25 mM HEPES buffer, 20 mgiL DNase and 20 mliL antibiotic solution (Sigma, St. Louis, MO). The distal end of the trachea was occluded with a n artery clamp and the lumen filled to distension with a solution of hyaluronidase (1.6 mgi ml) and cytochalasin B (1 kgiml) made up in aMEM (Sigma, St. Louis, MO) and incubated for 60 min a t 37°C. The lumen was then flushed with 10 ml medium (RPMI with 25 mM HEPES buffer, 20 mgil DNase, and 20 mliL antibiotic solution, Sigma, St. Louis, MO), which was collected and kept a t 4°C. The lumen was re-inflated with aMEM containing pronase (3.3mgiml) and incubated for 30 min a t 37°C. The lumen was then flushed with 20 ml medium (as above, but containing 10% fetal calf serum), which was collected with the previous sample. This pooled sample was then used to flush the tracheal lumen a n additional 5 times (5 x 10 ml). The resultant cell suspension was centrifuged (1,100 rpm, 15 min a t 19°C) and the supernatant was carefully removed and discarded. The cell pellet was resuspended gently in 4 ml of fresh RMPI medium (no fetal calf serum) and counted using a haemacytometer. Viability was assessed using trypan blue dye exclusion. Cell suspensions were adjusted to a concentration of approximately 0.5 x lo6 cellsiml and analyzed either
a t Los Alamos National Laboratory using a multiparameter flow cytometer (20) operated by the National Flow Cytometry Resource (NFCR) or a t the Lovelace Medical Foundation (LMF), Albuquerque, NM on a n EPICS V (Coulter Electronic, Hialeah, FL). At NFCR, cells were analyzed using 2" and 90" light scatter, axial light-loss, and blue, green, and red fluorescence. Commonly, two lasers were used, one operating in the UV and the other at a wavelength of 488 nm, a t powers of 300 and 400 mW, respectively. The UV enhanced laser provided excitation for blue and red fluorescence, while the 488 nm wavelength laser provided excitation for green fluorescence, forward angle light scatter, and axial light-loss signals. The data generated from 10 to 30,000 events per sample were stored in correlated list mode fashion. The data subsequently were processed a s individual frequency distribution histograms and two-parameter contour diagrams. The contour diagrams were used to delineate discrete cell populations. At LMF, the cells were examined using a single laser (488 nm wavelength, 0.4-0.5 mW power) to generate 2" and 90" light scatter signals. Two-parameter contour diagrams were used to separate defined cell populations. With both flow cytometers, two cell populations were sorted at the same time and 10-30,000 cells were collected from each sample for ultrastructural analysis or cytological preparations. In addition, 3,000 sorted basal or secretory cells were sorted directly in 60 mm culture dishes (Lux, Naperville, IL) containing 4 ml of serum-free defined medium. This medium has been previously described by Thomassen et al. (21). Cells were incubated in this medium a t 37°C in a humid, 5% CO, and 95% air atmosphere, fixed and stained after 10 days and colonies with 2 3 0 cells were scored. The sorted cells and the original cell suspension were processed for electron microscopy according to the method of Sebring et al. (18).In brief, the cells were fixed in buffered glutaraldehyde and centrifuged within a n agar-plugged, narrow-bore tube. Following secondary fixation with buffered osmium tetroxide, the cell pellet was encapsulated in agar. The agar containing the cells was removed from the tubing and processed conventionally for electron microscopy. These preparations were used to verify the morphology of the cells and the purity of the various cell types. The purity of the cell populations was assessed by identifying a t least 100 cell cross sections. Cells used for cytospin preparations were centrifuged at 2,500 rpm for 10 min and all but 200 pl of supernatant removed and discarded. The cells were resuspended in the remaining supernatant, cytology preparations were made using the Shandon Cytospin 2 (Pittsburgh, PA) and stained with Diff Quik stain (American Scientific Products, McGaw Park, IL). The cell surface sugar moieties were determined in the intact tracheal epithelial lining and in isolated cell preparations using lectins labeled with ferritin (for electron microscopy) o r fluorescein (for histology and
SEPARATION OF TRACHEAL BASAL AND SECRETORY CELLS
397
FIG. 1. Light micrograph of a cytospin preparation of isolated tracheal epithelial cells. The cell suspension is composed predominantly of single cells
flow cytometry). Samples of tracheal tissue were fixed in 10% buffered formalin. After dehydration, the tissues were embedded in paraffin and 5 pm sections were cut for histology. After deparaffnization in xylene, the sections were brought through decreasing concentrations of alcohol to water, rinsed in phosphate-buffered saline (PBS), and stained for 30 min with various concentrations of fluorescein-conjugated lectins (10-100 pg/ml) in PBS (pH 7.2) and then gently washed twice in 5 ml of PBS. The stained sections were mounted in 10% buffered glycerin solution and examined with a fluorescence microscope. The lectins used were Ulex europaeus (UEA I), Pisum sativum (PSA), Lens culinaris (LCA), Dolichos biflorus (DBA), Glycine max (SBA), Maclurapomifera (MPA),Arachis hypogea (PNA),Titicum vulgaris (WGA), Concanavalin A (CON A), Helix pomatia (HPA), Wisteria floribunda (WFA), Banderiraea simplicifolia (BSA I), Phaseolus limus (PHA L), and Limulus polyphemus (LPA). As a control, sections were either stained with PBS only or fluorescein-conjugated lectin with the appropriate inhibitory sugar (0.4 mM). The sugars used were L-fucose (UEA I), Dmannose (PSA, LCA, CON A), D-glucose (Con A), Nacetyl-D-galactosamine (DBA, MPA, SBA), D-galactose (BSA I, MPA), N-acetyl-1-D glucosamine (WGA, HPA) and sialic acid (LPA). The preparations involving UEA I employed PBS containing calcium and magnesium ions; all the other preparations used PBS with-
out magnesium or calcium ions. The lectins and inhibitory sugars were obtained from Sigma Chemical Co. (St. Louis, MO). For flow cytometry the cell suspensions were adjusted to a concentration of approximately 0.5 x lo6 celldm1 and stained with 10-100 pg lectin/ml or lectin plus inhibitory sugar for 30 min a t 0-1°C and then examined by flow cytometry for the presence of cell-associated green fluorescence. Cells used for flow cytometry were investigated unstained or stained singly or in combination with the following: Hoechst 33342 dye (5 pgiml for 30 min a t 37°C) for DNA content, pyronin Y (10 pg/ml, 5 min room temperature) for RNA content and propidium iodide (10 pgiml, 5 min room temperature) for viability determinations.
RESULTS The initial cell suspension contained 1.0-3.0 x lo6 cells with a viability in excess of 95%. Similar estimates of viability were obtained with trypan blue dye exclusion and flow cytometric evaluation using propidium iodide. The cell suspension was primarily composed of single cells (Fig. 1). The differential cell count was undertaken using electron microscopy. Estimates were obtained from three separate preparations in which 100 cell cross sections were viewed for each sample. The individual values were for basal cells, 20, 22, and 28% (average 23%);secretory cells, 59,61, and 67%
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A X I A L LIGHT LOSS
2'LIGHT SCATTER
FIG. 2. Two-parameter contour diagrams. A 90" light scatter vs. axial light loss (NFCR).B: 90" light scatter vs. 2" light scatter (LMF). Two populations, a and b, can be resolved. Population a represents large granular cells, while population b represents small agranular
cells. Ultrastructural features of these populations show a to be secretory cells and b to be basal cells. The events aligned along the y-axis (90"light scatter) with low 2" light scatter are composed of cell debris.
(average 62%); ciliated cells, 4, 14, and 20% (average 13%)and other cells, 1, 2, and 3% (average 2%). The latter cells were composed of macrophages, red blood cells, and unidentified cells. Flow cytometric analysis was conducted using a variety of intrinsic and extrinsic parameters. The intrinsic parameters, 2" light scatter and axial light-loss, reflect cell size; 90" light scatter relates to the granularity of the cell (20);and blue auto-fluorescence indicates the inherent levels of NADPH (22). Extrinsic parameters employed were Hoechst 33342 for DNA content, pyronin Y for RNA content, and lectins for cell-surface sugar moieties. Ungated flow cytometric analysis of the cell suspensions involving 90" light scatter vs. axial light-loss or 2" light scatter using either the custom made flow cytometer (NFCR) or the commercial instrument (LMF) produced similar two-dimensional contour maps (Fig. 2). The location of viable DNA-containing cells was established by dual staining with Hoechst 33342 and propidium iodide. Data was acquired from events that were Hoechst positive and propidium iodide negative. These events were displayed a s a 90" light scatter vs. 2" light scatter contour diagram, revealing two major cell populations (Fig. 2). Hoechst positive, propidium iodide negative events were regarded a s being related to viable cells, while the Hoechst negative, propidium iodide positive events were taken as either dead cells or debris. The two subpopulations contained within the total viable cell population had distinct morphological characteristics (Fig. 3); one was composed of small agranular cells and the other of larger granular cells. Ultrastructural analysis of the two cell types showed them to be either basal or secretory cells, respectively. Secre-
tory cells were of the serous type, with electron-dense, membrane-bound secretory granules; numerous mito-
B FIG. 3. Light microscopy of cytospin preparations of sorted basal cells (A) and sorted secretory cells (B). The populations were sorted according to their 2" and 90" light scatter characteristics using defined parameters shown in Figure 2B.
SEPARATION OF TRACHEAL BASAL AND SECRETORY CELLS
399
B
A
FIG. 4. Electron micrographs of cells from populations sorted on the basis of 90" light scatter and axial light loss (as defined in Fig. 2A). Population A contains cells possessing secretory granules, numerous mitochondria, and well-developed elements of rough endo-
plasmic reticulum. Population B represents basal cells with scant cytoplasm containing few mitochondria elements of rough endoplasmic reticulum. Prominent bundles of microfilaments are seen in the perinuclear region (bar represents 0.5 pm).
chondria and cisternae of rough endoplasmic reticulum; and a n eccentric, indented nucleus (Fig. 4A). The isolated basal cells had scant cytoplasm containing prominent perinuclear microfilaments, few mitochondria, and elements of rough endoplasmic reticulum; and the cell surface possessed numerous fine micro villi (Fig. 4B). The ultrastructural appearance of isolated cells was similar to that seen in the intact tracheal epithelium with the exception of the loss of cell shape. The distinctive ultrastructural morphologies of the isolated basal and secretory cells were used to quantify the purity of cell separations. With these parameters, the purity of the basal and secretory cells was 94 1% and 91 3%, respectively (average of 3 estimations -+ SEM). The remaining cells could not be identified a s they had no definitive cytological features. The colonyforming efficiency of the cells sorted directly in culture dishes was 0.6% -+ 0.15% for basal cells and 3.4% 0.42% for secretory cells. The colony-forming efficiency
of the unsorted parent population was 2.6% 0.15%. These values were obtained from 5 separate sorts on different occasions. Flow cytometric analysis (5) using Hoechst 33342 showed the number of cells within the suspension that were in S, G2, or M phase of the cell cycle to be 14.2% 0.23% (standard error of mean n = 5). This figure required correction to allow for the drop in the number of ciliated cells within the cell suspension compared to the intact trachea. The intact trachea contains approximately 37-40% ciliated cells (11)compared to 13%in the cell suspension. The corrected value of the dividing cells in the original tracheal epithelium was 9.1% and this showed that only a fraction of the cells are in S, G2, and M phases of the cell cycle a t any one time. Reanalysis of the cycling cells using 2" and 90" light scatter showed the majority (>86%) of cells to be relatively large, with granular cytoplasm (Fig. 51, which are characteristics of secretory cells. Similar results
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FIG. 5. Flow cytometric analysis of the tracheal cells suspension. A Two-parameter contour diagram of blue fluorescence (DNA) content vs. 2" light scatter: population A represents predominantly cells in S G, M phase of the cell cycle and population B represents cells in
G,G, phase of the cell cycle. Replotting the data from population A on the basis of 2" and 90" light scatter (C) shows these cells to be in the region of the secretory cell population (arrow) of the parent cell suspension (B).
were obtained when the two major populations of the original cell suspension were analyzed in terms of their DNA content; the secretory cell population had approximately six times more cells in the S, G2, and M phases of the cell cycle than the basal cell population. A satisfactory staining reaction was obtained with 5 pg Hoechst 333421m1 and a 30 min incubation period (coefficient of variation of G,/G, peak was < 6%); however, during the initial stages of dye uptake, there was a bimodal staining reaction (Fig. 6). Analysis of the single parameter histogram using data from the two major staining peaks and replotting it as a bivariate contour diagram (2' light scatter or axial light-loss vs. 90" light scatter) showed that the slowly staining cells were predominantly small agranular cells (basal cells), while the more rapidly staining cells were larger and more granular (secretory cells) (Fig. 6). The blue auto-fluorescence response showed a bimodal distribution, one population with low fluorescence, and another with a positive blue auto-fluorescent response. Analysis of these data on a bivariate contour diagram with axial light-loss showed two major populations. The small cells gave a small blue auto-fluorescence signal, while the larger cells were positive (Fig. 7). A similar response was seen with pyronin Y staining for RNA (data not shown). There was good correlation between negative blue auto-fluorescence and lowhegative pyronin Y fluorescence; 91% of the former cells were in the latter population. The fluorescein-conjugated lectins displayed a variable pattern of cell labeling. WFA, SBA, WGA, and
BSA all gave a positive response (Fig. 81, while the remainder elicited no fluorescent signal up to a lectin concentration of 100 pgiml (Table 1). All the lectins gave a negative response when they were combined with their inhibitory sugar. Reanalysis of the fluorescence positive cells on a bivariate contour diagram (90" light scatter vs. 2" light scatter) shows that WGA stains two cell populations (characteristic of basal and secretory cells); SBA, BSA-I, and WFA stained predominantly single populations (secretory-SBA and BSA-I and basal cells-WFA) (Fig. 9). The staining characteristics of the isolated cells were similar to those seen in the intact epithelium whether i t was stained with ferritin or fluorescein-conjugated lectins (Table 1).
DISCUSSION Pure, viable populations of either basal or secretory cells from the rat trachea were produced. The isolation procedure did not appear to affect cells adversely, as there were marked similarities in the ultrastructural appearance and the expression of surface sugar moieties between isolated cells and the cells of the intact epithelium. In addition, the cells have been successfully grown in culture in defined serum free medium. The separation of basal and secretory cells is based on intrinsic differences between the two cell types in terms of their ability to scatter light. The basal cells are relatively small and agranular and, consequently, have low 2" and 90" light scatter, while the secretory cells are larger, with prominent secretory granules that result in high 2" and 90" light scatter signals. Sort-
SEPARATION OF TRACHEAL BASAL AND SECRETORY CELLS
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with WGA (19). However, they did demonstrate that CON A and RCA (Ricinus communis agglutin) bound to the cells. CON A in the present study was negative. Lectin labeling of human bronchial glands showed that the secretory cells stain positively with a wide range of lectins, however, serous cells were negative with HPA (16). This result is similar to the present study where the majority of secretory cells in the rat trachea are of the serous type. Studies on the mouse trachea demonstrated that PNA and DBA did not label the epithelial lining cells (23). Ultrastructural and flow cytometric profiles of the cells show the secretory cells to be more active metabolically than the basal cells. This is seen in the numerous mitochondria, cisternae of rough endoplasmic reticulum, secretory granules, RNA content, and blue auto-fluorescence of the secretory cells. Blue auto-fluorescence is a n indicator of inherent NADPH content (22) and, therefore, of general biochemical synthetic activity. In contrast, the basal cells have few mitochondria, little RNA, no blue auto-fluorescence, and exiguous cytoplasm. Working a t a concentration of 5 bg Hoechst 333421m1 provides a n adequate DNA histogram (coefficient of variation of GIG, peak < 6%); however, during the initial phases of dye uptake, there is a bimodal response. The two major cell populations (basal and secretory cells) take up stain differently and may provide a parameter with which to sort the cells. The slowly staining cells have a flow cytometric profile (small agranular cells) compatible with basal cells, while the more rapidly staining cells have a profile (large granular cells) similar to secretory cells in other experiments. This confirms other studies that showed that different cell types take up Hoechst 33342 stain a t different rates (13).Differential staining patterns must be taken into account when assaying the DNA content of heterogenous cell populations. The results showed that the secretory cell population contains the majority (> 85%) of cycling cells in the normal trachea. These results agree with the work of McDowell and Trump (15), indicating that secretory cells play a major role in repair of the tracheal lining following a denuding injury. The results confirm previous findings that both basal and secretory cells are capable of division (2); however, here secretory cells play a more significant role than basal cells. The higher colony-forming efficiency of secretory cells compared to basal cells also indicates the greater proliferative potential of the former cells. These studies also show that sorting cells by flow cytometry does not appear to affect the growth of tracheal epithelial cells in culture. The value for colony-forming efficiency obtained in this study are comparable to those of Mahler et al., who found 0.1-1.0% CFE for a cell fraction obtained from the rat by elutriation containing 70% basaloid cells and a CFE of 5-12% for a fraction containing only 20-30% basaloid cells (14). Inayama et al. (8,9), using similar elutriation techniques a s Mahler et al.
L'L DNA/BLUE FLUORESCENCE
FIG. 6. Flow cytometric analysis of Hoechst staining. A is a twoparameter countour diagram of the tracheal cell suspension, using 90" light scatter and axial light loss signals. Two major populations are defined. Population A was shown to correspond to secretory cells and population B to basal cells. An adequate DNA histogram can be obtained with 5 kg Hoechstiml with a 30 min incubation period a t 37°C (C). During the initial stain uptake, a bimodal staining reaction is seen (B) = 5 kgiml, 5 min incubation). Analysis of the blue fluorescence data from population A at the time point (D) shows the majority of cells taking up stain rapidly. Similar analysis for population B (E) shows cells taking up stain more slowly than population A.
ing windows for the two cell types can be adjusted so there is no apparent overlap of cell types. This approach produces pure populations, but it leads to the collection of a proportion of each cell phenotype. The differential lectin staining of the two cell types may provide a means to sort pure populations from the entire spectrum of each cell phenotype. This approach is currently under investigation. The binding patterns of the various lectins show that the cell surfaces of isolated tracheal epithelial cells are similar to their counterparts in the intact epithelium. The positive staining of in situ epithelial cells and isolated cells with WGA is in contrast to the results of Shiba et al., who found no staining of rat tracheal cells
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basis of blue auto-fluorescence shows that these cells have a positive fluorescence response (C) compared to population B in which the cells have low auto-fluorescence (B).
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Table 1 Lectin Affinity of Tracheal Epithelial Cells I n Situ and in Single Cell Suspensions" In situ epithelia Lectin UEA I PNA BSA I BSA I1 WFA WGA MPA DBA SBA LCA CONA HPA
B
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PHA-L "This table shows the lectin affinity of tracheal epithelial cells in situ and in single cell suspensions. The former cells were investigated using fluorescent light microscopy (FITC labeled lectins) and electron microscopy (ferritin labeled lectins), while the latter were studied by flow cytometry (FITC labeled lectins). There was no detectable difference between the cells of the intact epithelium and those in suspension. UEA = Ulex europeans agglutinin; PNA = peanut agglutinin; BSA I and BSA I1 = Bandeiraea simplicifolia agglutinin; WGA = wheat germ agglutinin; MPA = Maclura pomifera agglutinin; SBA = soyabean agglutinin; LCA = Lens culinaris agglutinin; Con-A = concanavalin A; HPA = Helix pomatia agglutinin; WFA = Wisteria floribuinda agglutinin; DBA = Dolichos biflorus agglutinin; PHA-L = Phaseolus limus agglutinin. B = basal cell; S = secretory cell; C = ciliated cell; ND = not done; + = positive; + + = markedly positive.
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FIG. 9. Flow cytometric analysis of fluorescein labeled lectin binding. A is a contour diagram of the parent tracheal cell suspension using 90" light scatter and 2" light scatter indicating the locations of basal and secretory cells. B is a contour diagram (2" vs. 90" light scatter) using data from weakly stained WGA events; the stained cells correspond to basal cells of the parent cell suspension. C is a contour
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2' L I G H T S C A T T E R
diagram (2" vs. 90" light scatter) using data from strongly positive stained WGA events; the stained cells correspond to the secretory cells of the parent cell population. Results similar to this occur with SBA and BSA-I stained cells. D is a contour diagram (2" vs. 90" light scatter) using data from WFA positive cells. The stained cells correspond to the basal cells of the parent population.
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JOHNSON ET AL.
(14), showed that cell suspension obtained from rabbits containing 83-94% basal cells, when inoculated into denuded tracheal grafts are capable of reestablishing a complete tracheal epithelium. The basal cells may have a low proliferative capacity but have the ability to act as progenitor cells. However, comparison of results between those experiments are complicated by the use of different species and isolation techniques. The choice of isolation or separation techniques may influence the composition of the initial cell suspension and the extent of contaminating cells. Minor modifications to the enzyme digest technique can affect the relative proportion of cell types in the initial cell suspension prior to any separation procedure. The method reported here of a combined hyaluronidase and cytochalasin treatment resulted in a cell suspension containing 13% ciliated cells; in a n earlier method (11) using separate hyaluronidase and cytochalasin, the proportion of ciliated cells was 32.6%. In this latter study and our present study, non-granulated secretory cells were not identified by electron microscopy. Jeffery and Reid (10) have shown by electron microscopy that these cells are infrequent and may show signs of ciliogenesis; however, by light microscopy non-granulated secretory cells can account for 13-27% of the total tracheal cell population. Flow cytometry, while producing a relatively low number of cells (up to 1 x lo6), produced higher-purity populations than can be obtained by elutriation. Flow cytometry produced a basal cell population of 95% purity. The contaminating cells could not be identified by electron microscopy. Elutriation techniques produced a n 85% pure basal cell population using electron microscopy to characterize the cells (8).This level of purity may be insufficient for in vitro or in vivo studies; if the 85% fraction of the entire population has a low proliferative capacity (as is the case with basal cells) it could easily become replaced by a highly proliferative cell type contained within the 15% impurity. To increase the purity of basal cell fractions obtained from elutriation, a postelutriation in vitro cloning step has been used (9). This in vitro step increases the effort needed to achieve a highly pure population of basal cells and allows the possibility of the selection or differentiation of a new population of cells. Flow cytometry provides a simple method of separation for basal and secretory cells from the rat trachea. This may also be true of other pulmonary cells such a s the alveolar type IT cells (24). Basal cells of the tracheobronchial lining are generally assumed to have a role analogous to basal cells of the stratum basale of the epidermis. As a result of this assumption, basal cells generally are considered to be the cells a t risk from the carcinogenic and toxic effects of xenobiotics. Many calculated dose-effect relationships, especially for radioactive materials, are based on this premise (7).The secretory cells, however, appear to be the critical cells at risk because they have a greater potential metabolic capability, they are more numerous and have a higher proliferative potential than the
basal cells and receive a higher dose from inhaled material because they are closer to the luminal surface. The overall biological risk for a specific exposure condition does not change if the secretory cells are considered the critical cells; however, the effective dose changes. The ability to sort and characterize pure cell populations will allow for investigations of differentiation pathways associated with these populations. In addition, the use of well defined mixtures of various cell types will help to delineate the role of cell-to-cell interactions in the repair and maintenance of the normal tracheobronchial lining. A further potential use of this system is in determination of the sensitivity of defined cell populations to toxicant damage.
ACKNOWLEDGMENTS Research was sponsored by the U S . Department of Energy’s Office of Health and Environmental Research under contract No, DE-AC04-76EV01013 and by Los Alamos National Flow Cytometry Resource funded by the Division of Research Resources of NIH (Grant p41rr01315) and the Department of Energy. Flow cytometry facilities were also kindly provided by Lovelace Medical Foundation. Technical support was provided by E. A. Margiotta and J. Milisa, whose help is gratefully acknowledged.
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