Demonstration of Actin and Cytokeratin in the Nonciliated Bronchiolar Epithelial (Clara) Cell of Rat*

ACTA HISTOCHEM. CYTOCHEM. Vol. 22, No. 5, 1989

DEMONSTRATION OF ACTIN AND CYTOKERATIN IN THE NONCILIATED BRONCHIOLAR EPITHELIAL (CLARA) CELL OF RAT*

JUNZOSASAKI, SADAHIROWATANABE, SHIGETOKANDA, TAKAKONOMURA, YUKIKOTANAKA ANDNAGAYASU OTSUKA

Departmentof Anatomy, OkayamaUniversity Medical School, 2-5-1 Shikata-cho,Okayama 700

Received for publication June 23, 1989 and in revised form August 7, 1989

Previously, we perfused the lungs of several mammals (rat, mouse, guinea pig and hamster) with the detergent-containing medium and found that abundant filaments were present in apical caps of nonciliated bronchiolar epithelial (Clara) cells. These filaments were bound to the plasma membrane of the apical cap at one end and ran into the interior of the cytoplasm at the other end. In the present study, we used heavy meromyosin (HMM) and an immunohistochemical method to identify these filaments in rat Clara cell. Most filaments in apical caps bound HMM, but some filaments were not decorated with HMM. To examine the latter filaments, paraffin sections were stained immuno-histochemically utilizing the antibodies against intermediate filament proteins. Apical caps were positive for cytokeratin but were negative for other intermediate filament proteins, vimentin, desmin, neurofilament protein and glial fibrillary acidic protein. These results suggested that filaments in apical cap of nonciliated bronchiolar epithelial (Clara) cells were composed of actin and cytokeratin.

Nonciliated bronchiolar epithelial (Clara) cells of several mammals were defined by two morphologic characteristics: abundant agranular endoplasmic reticulum and membrane-bound ovoid granules, although the interspecies differences of cellular mor- phology in nonciliated cells were also emphasized (17). They are distributed mainly in the bronchioles (7, 27) and can be found in the upper airways of several mammals (7, 18). These cells contain large amounts of cytochrome P-450 and cytochrome P- 450 dependent oxidase (27) which indicates the metabolism of exogenous comp- ounds. The secretory function was demonstrated by the findings that several proteins purified from rat lavage surfactant were localized in Golgi apparatus, endoplasmic reticulum and granules of rat Clara cells (22, 26) and several proteins were released from isolated Clara cells (16). The molecular weights of these proteins were different. Recently, we have found abundant filaments in apical caps (protuberances) of these cells in several mammals by perfusing the lungs with Triton X-100 solution containing low concentrations of glutaraldehyde (19, 20) and we suggest that the presence of filaments is one of the morphologic characteristics of Clara cells and that the fila- * This work was supported by a Grant -in-Aid for scientific research (No. 62570007) from the Ministry of Education, Science and Culture, Japan.

491 492 Sasaki et al. ment may play an important role in the function of these cells which for the most part , is still unknown. The present study clarified the nature of the filaments by the use of heavy meromyosin and immunohistochemical methods .

MATERIALS AND METHODS For the experiments of HMM (Wako Co., Tokyo) decoration, tissues were prepared according to the procedures previously described for the observation of the cytoskeleton in nonciliated cells (19). As unfixed specimens were used , phenylmethyl-sulfonyl fluoride was added to all solutions. Lungs were perfused with buffered salt solution (NaC18 g, KC10.4 g, MgC126H2O 0.4 g, NaH2PO4 2H2O 0.078 g, Na2HPO412H2O 0.151 g, EGTA 0.76 g, NaHCO3 0.35 g, HEPES 2.38 g/l, pH 7.0) followed by 0.2% Triton X-100 in buffered salt solution. Omitting perfusion with glutaraldehyde-containing medium, the lungs were cut into small pieces (5 mm x 5 mm x 3 mm) and immersed in 20% sucrose solution in 0.1 M sodium phosphate buffer (pH 7.4). The pieces of lung were frozen in liquid nitrogen, and 20-25 pm thick sections were cut in a cryostat (Reichert Jung FRIGOCUT E 2800). These sections were placed on slide glasses coated with 0.1% poly L-lysine and air-dried. These slides with the sections were rinsed in salt solution, then the sections were covered with a few drops of 2-4 mg/ ml HMM solution (6) at 4°C for 2-4 hr and washed three times. They were fixed with 2.5% glutaraldehyde containing 0.1% tannic acid for 30 mm, then after thorough washing, they were fixed with 0.5% 0504. After dehydration with acetone, capsules with freshly prepared epoxy resin were placed on these sections and polymerized at 60°C for 48 hr. Thin sections were cut with an ultramicrotome (Sor- vall, MT-5000), stained with uranyl acetate and lead citrate and observed under an electron microscope (JEOL JEM100CX). For immunohistochemistry, untreated lungs were fixed by airway infusion of 3.8% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) at the pressure of 20 cm H2O. After 15 min of fixation, the inflated lungs were cut into small pieces, immersed and fixed in the same fixative for 24 hr at 4°C. Lung blocks were dehydrated and embedded in paraffin. Paraffin sections were attached onto glass slide, dewaxed and rinsed with distilled water. They were treated with 0.3% hydrogen peroxide methyl alcohol solution for 30 min, rinsed and immersed in 2% blocking serum for more than 1 hr at 37°C. Primary antibodies against intermediate filaments were placed on the sections and the sections were kept in a moist chamber overnight at 4°C. After rinsing, the sections were treated with biotinylated second antibody diluted 1 : 200 in PBS (154 mM NaCI, 10 mM sodium phosphate buffer, pH 7.4) for 1 hr at 37°C, rinsed and treated with avidin-biotinylated horseradish peroxidase complex diluted 1 : 100 in PBS for 1 hr at 37°C. After thorough rinsing, the sections were treated with 0.1% 3- 3'-diaminobenzidine solution containing 0.02% H2O2 at room temperature. After the reaction, they were stained with Mayer's hematoxylin. The monoclonal antibody to vimentin (15), monoclonal antibody to desmin (2) and monoclonal antibody to human neurofilament protein (10) were purchased from DAKO, and monoclonal antibody to cytokeratin 18 (11) from Amersham Int. Actin and Cytokeratin in Rat Clara Cell 493

RESULTS Figure la is an electron micrograph of the epithelium of rat bronchiole. As described in MATERIALS AND METHODS, the cells were treated with detergent to eliminate soluble cytoplasmic materials and to facilitate the penetration of heavy meromyosin into the cell. The epithelium is cut horizontally to the luminal surface. Nonciliated (Clara) cells with an indented nucleus and apical cap (protruded cytoplasmic region) of another Clara cell are seen. Cilia and ciliated cells surround Clara cells. Many filaments are present in the apical cap and in the cytoplasm around the nucleus. Figure lb is a higher magnification of part of Figure la. Cytoplasms of Clara cells were filled with filaments except the remnant of membrane organelles, pro- bably mitochondria. In the apical cap, most filaments are decorated with HMM and exhibit clear arrowhead labeling. In the perinuclear cytoplasm, some of these filaments were not decorated with HMM. The example of these filaments is shown by arrows. Figure 2 shows apical cap cut perpendicular to the luminal surface and the filaments extending from the luminal surface to the interior of the nuclear region. In some filaments, the arrowheads of HMM point away from the lumen toward the interior of the cell (arrows) although in other filaments its direction was unclear. Figures 3a-d show the results of immunohistochemistry. Reaction product was found in Clara cells of immunohistochemical preparations using antibody against cytokeratin 18 suggesting that cytokeratin 18 was present in Clara cells (Fig. 3a). It was observed in the perinuclear region and in the apical cap of Clara cell. Reaction product was found in the cells of connective tissue or in the smooth muscle of bronchiolar wall when antibodies against vimentin (Fig. 3b) or desmin were used (Fig. 3c), but neither of them was found in Clara cell. No reaction product was observed in immunohistochemical preparations using antibody against neurofilament protein (Fig. 3d) or filial fibrillary acidic protein (data not shown). Immunohistochemical study suggested that Clara cell was positive for cytokeratin but was negative for vimentin, desmin, neurofilament and filial fibrillary acidic proteins.

DISCUSSION Clara cells are cuboidal with an indented nucleus and often have an apical pro- tuberance (apical cap) at the center of the luminal surface. They are distinguished from the other cells by the presence of abundant agranular endoplasmic reticulum and electron-dense granules (17). The apical cap is covered with microvilli like projec- tions and has often been described as being concerned with apocrine secretion (7, 27). In the previous papers, we reported that apical cap including its cortical area was filled with abundant filaments in rat (19) and in other species (20). The presence of filaments leads to the idea that the apical cap is not an apocrine droplet but a stable

FIGS. la, b. a. The bronchiolar epithelium cut horizontally to the luminal surface. Cilia was cut transversely. The cells were treated with detergent-containing medium to observe the cytoplasmic filaments which were decorated with heavy meromyosin. N: Clara cell, C: Ciliated cell, Nac: Apical cap of Clara cell. Bar= 1 rim, X 17,000 b. High magnification of asterisk region in Figure la. Most filaments are decorated with heavy meromyosin. Intermediate sized filaments in the perinuclear region are not decorated with heavy meromyosin (arrow). Nac: Apical cap of Clara cell. Bar= 1 mm, X 34,000 494 Sasaki et al. Actin and Cytokeratin in Rat Clara Cell 495

Fin. 2. High magnification of apical cap cut perpendicular to the epilelium. The arrowheads of HMM point toward the interior of the cell in some cases (arrow). Bar =1 mm, X 45,000

structure. The presence of filaments, as well as the granules and smooth-surfaced endoplasmic reticulum, can be considered as a common feature of Clara cells and can be utilized to identify and distinguish Clara cells from other morphologically similar cell types such as serous cells which are distributed in the airway and have electron dense granules. Actin is one of the major components of microfilament and has been associated with amoeboid movement of the cells, protoplasmic movement, cleavage and cytokinesis, phagocytosis and secretory function (1). Actin in the cortical cytoplasm just underneath the plasma membrane was reported in many cells and virtual exclu- sion of granules and other organelles gives this region a homogenous appearance 496 Sasaki et a1. Actin and Cytokeratin in Rat Clara Cell 497

(24). Also in Clara cells, a peripheral fibrillar layer was previously reported (17, 23). In our previous paper (20), we observed the cortical homogeneous matrix where organelles were absent and which seemed to be composed of a fine filament network . In some cases, a granule crossed this cortical layer and was in close contact with the plasma membrane. By the detergent treatment we showed that abundant filaments were associated with plasma membrane in the cortical area and ran into the interior of the cell (19, 20). The present study showed that most filaments in apical cap of Clara cells consisted of actin by the use of heavy meromyosin. The precise role of abundant actin in Clara cell remains unknown, however, by the association of actin to plasma membrane, one of the functions of filaments is postulated to be the release of granules. Other filaments which did not bind HMM and were present principally around the nucleus of Clara cells were shown to contain cytokeratin (Fig. 3a). Other intermediate filament proteins, vimentin (Fig. 3b), desmin (Fig. 3c), neurofilament protein (Fig. 3d) and glial fibrillary acidic protein (data not shown) were not found in Clara cells. The expression of cytokeratin, vimentin, desmin, neurofilament and glial fibrillary acidic protein was mainly observed in epithelia, mesenchymal tissue, myogenic cells, neurons and astrocytes, respectively. Cytokeratins are a multigene family of polypeptides expressed in different sets in different epithelia, and cytokeratin 18 is one of the polypeptides expressed in simple epithelia (13). The demonstration of cytokeratin 18 in Clara cells is consistent with these previous reports. The expression of intermediate proteins is used as markers of histogenetic origin both of normal (3) and tumor cells (14, 21) in spite of the fact that coexpression of different class of intermediate filaments is reported in transformed cells (4). The presence of cytokeratin 18 can be utilized to investigate the origin of lung carcinoma which resembles Clara cells (8, 9). Whereas a great deal is known of the function of other cytoplasmic proteins, microfilaments and microtubules, the function of intermediate filaments remain the least understood except the evidence for site-specific, phosphorylation-dependent disassembly of vimentin filament is reported (5). Cytokeratin in Clara cell may function as mechanical integrators of cellular space, the cytoskeleton (12). Clara cell is one of the major components in the epithelium of bronchioles. Its function is not fully understood as compared with the ciliated cells. Although the evidence of granular release is demonstrated in Clara cells, the nature of granule con- tent is still unknown. Agranular endoplasmic reticulum in the cytoplasm is considered to be the site of oxidative metabolism of xenobiotics. Recent study shows the possibility that Clara cells play a role in absorbing liquid from the airway surface by ion transport activity in rabbits (25). The role of filaments in the latter functions is still unclear.

FIGS 3a-d. Paraffin sections of the lung are stained immunohistochemically using the antibodies to the intermediate filament proteins. N: Clara cell. a. When antibody against cytokeratin 18 is used, reaction product is found in the cytoplasm of Clara cells. X 1,500 b. When antibody against vimentin is used, reaction product is found in the cells of connective tissue (arrowhead) but is not in Clara cell. X 1,500 c. When antibody against desmin is used, reaction product is found in the smooth muscle cell (arrowhead) underlining the bronchiolar epithelium. X 1,500 d. When antibody against neurofilament protein is used, reaction product is not found in Clara cell. X 1,500 498 Sasaki et al.

ACKNOWLEDGMENTS

We wish to express our gratitude to Dr. Chao Liang Hsueh, Department of Pathology, Okayama University Medical School, for his suggestions regarding the immunohistochemical method.

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