Hormones and the lung II. Immunohistochemical localization of thyroid hormone binding in type II pulmonary epithelial cells clonally-derived from adult rat lung

Hormones and the Lung II. IMMUNOHISTOCHEMICAL LOCALIZATION OF THYROID HORMONE

BINDING IN TYPE I1 PULMONARY EPITHELIAL CELLS CLONALLY-DERIVED FROM ADULT RAT LUNG '

MARJORIE WILSON,*' KAREN R. HITCHCOCK,'5 WILLIAM H. J. DOUGLAS * AND RONALD A. DELELLIS Departments of 2Anatomy and Pathology, Tufts University School of Medicine, Boston, Massachusetts 02111

ABSTRACT The type I1 pulmonary epithelial cell is the recognized site of surfactant synthesis and storage. Results of recent studies indicate that the thyroid hormones, triiodothyronine (T,) and thyroxine (TJ, may be important regulators of surfactant production and/or release. Direct and indirect immunofluorescence techniques were used in an attempt to demonstrate binding of T3 and T, in monolayer cultures of isolated type I1 cells. These cultured epithelial cells are clonally-derived from adult rat lung, retain a diploid karyotype through 35 population doublings in vitro, contain granular inclusions (lamellar bodies) in the perinuclear cytoplasm, and synthesize phosphatidylcholine via the CDP-choline pathway.

In isolated type I1 cells, either of two fluorescent patterns was observed: (a) nuclear fluorescence accompanied by a reticular perinuclear network; or (b) diffuse cytoplasmic accumulations with concentrations around perinuclear cytoplasmic inclusions. Ultrastructurally these inclusions had the typical appearance of lamellar bodies. Histochemical studies demonstrated that these inclusions contained surfactant-associated nonspecific esterases and stained with Nile blue hydrochloride. The positive reactions with these two recognized markers for pulmonary surfactant indicate that these inclusions are indeed lamellar bodies, the putative sites of surfactant synthesis and/or storage. These findings suggest that the type I1 pulmonary epithelial cell contains specific binding sites for thyroid hormones, and support the hypothesis that thyroid hormones are regulators of surfactant metabolism.

Hormonal factors which may affect lung development and regulate the synthesis andlor release of pulmonary surfactant have been the subject of intensive investigation in recent years. While most work to date has focused on the glucocorticoids (for reviews, see Avery, '75; Ballard and Ballard, '76; Ballard et al., '77; Farrell, '77), recent work suggests that the thyroid hormones may affect surfactant metabolism. Redding et al. ('72) found that adult rats treated with thyroxine (T,) ex- hibited selective hypertrophy of type I1 pulmonary epithelial cells. These cells showed increased numbers of intracellular lamellar bodies, and lung washings from treated ani- mals showed increased surfactant levels. Wu et al. ('73) showed that injection of T, into rab-

ANAT. REC. (1979) 195: 611-620

bit fetuses and their amniotic sacs caused accelerated lung maturation, characterized by the early appearance of both lamellar bodies and surface activity. Similar findings have been reported in the rat by Hitchcock ('79). Recently, Hitchcock and Reichlin ('78) showed that the adult rat lung concentrates triiodothyronine (TJ from the plasma. In

Received July 19. '78. Accepted June 11, '79. 'This study was supported by Grants HL15316, HL21008,

HL19513 from the National Heart, Lung and Blood Institute, N.1.H , and Grant R-274 from The United Cerebral Palsy Education and Re- search Foundation. Preliminary results were presented at the Ninetieth meeting of the American Association of Anatomists, De- troit, Michigan.

'Present address: Children's Hospital, 300 Longwood Avenue, Boston, Massachusetts 02115.

Correspondence: Dr. Karen Hitchcoek. Department of Anatomy, Tufts University School of Medicine, 136 Hamson Avenue, Boston, Massachusetts 02111.

611

612 WILSON, HITCHCOCK, DOUGLAS AND DELELLIS

addition, while low in late gestation, pulmonary tissue to plasma ratios of T, rise dra- matically in the early neonatal period (Hitch- cock et al., '78). Finally, it has been reported that adult rat lung nuclei (Morishige and Guernsey, '78) and fetal and adult rabbit lung nuclei (Lindenberg et al., '78) possess specific receptors for T,. Such findings suggest that the lung is a target organ for thyroid hormones and that these hormones may have a specific role in surfactant metabolism.

In the present study, immunofluorescence techniques using antibodies directed against T, or T, were used in an attempt to localize these hormones in cultures of cells derived from adult rat type I1 pulmonary epithelial cells, the cells implicated in surfactant metabolism (for review, see Hitchcock-O'Hare et al., '76). The particular cells utilized for this study are a well-characterized population of isolated type I1 cells. These cells exhibit an epithelial morphology in vitro and retain type 11 cell-specific functions for up to 35 population doublings in vitro (Douglas et al., '79). Perinuclear granular inclusions are present in the isolated type I1 cells, and a number of mor- phologic, histochemical and biochemical techniques have been used to establish the identity of these granules (Douglas and Kaighn, '74; Douglas et al., '76, '79; Douglas and Far- rell, '76; Douglas and Chapple, '77). Ultra structural studies (Douglas and Kaighn, '74; Douglas et al., '76; Douglas and Farrell, '76; Douglas and Chapple, '77) have demonstrated that these cytoplasmic inclusions are morpho- logically identical to the lamellar bodies present in type I1 cells of intact lung (Douglas et al., '75). Indeed, while this manuscript was in preparation, Lindenberg et al. ('78), utilizing competitive binding studies, reported the presence of specific nuclear T, receptors in this same cell line.

MATERIALS AND METHODS

Type I I cell monolayers Cultures of type I1 pulmonary epithelial

cells, clonally-derived from adult rat lung (Douglas and Kaighn, '74) and a t population doubling level 25, were grown as monolayers on glass slides. Cultures were examined either a t 24 hours or 48-72 hours after plating. Cells were grown in Ham's F12K growth medium (Flow Labs, Rockville, Maryland) supplemented with 10% fetal bovine serum. The sera used contained two different levels of thyroid hormone: (a) normal fetal bovine serum (T,,

1.596 ng/ml; T,, 41.05 ng/ml; Flow Labs, Rockville, Maryland); and (b) normal fetal bovine serum with lo-% sodium Levothyrox- ine added (Pharmex Corp., Hollywood, Flor- ida). The T, and T, concentrations in all sera utilized were determined in our laboratory using the radioimmunoassay methods of Ne- jad et al. ('75).

Monolayer cultures were prepared for fine structural analysis by fixation for one hour a t 4°C in 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.3. Cultures were rinsed for 5 minutes at 4°C in 0.2 M cacodylate buffer and then postfixed for 30 minutes a t 4°C in 1% osmium tetroxide in 0.1 M cacodylate buffer. Samples were dehydrated in a graded series of ethanol in water and embedded in Epon-Araldite.

Fibroblast cultures Monolayer cultures of fibroblasts from

adult rat lung (ARL) and adult rat prostate (ARP) served as the control cultures. ARL and ARP cells, a t population doubling levels 6 and 3, respectively, were plated onto glass surfaces in Ham's F12K medium supplemented with the same serum combinations detailed above for the type I1 cells. These cells were examined either a t 24 hours or 48-72 hours after plating under the same conditions described for the type I1 cells.

Immunofluorescence Cell monolayers were rinsed for 30-60 sec-

onds in Hanks Balanced Salt Solution con- taining 1.26 mM Ca++ and 0.89 mM Mg++ (HBSS, Grand Island Biological Co., Grand Is- land, New York). Monolayers were fixed at 4°C for 10 minutes in absolute methanol and rinsed for 10 minutes in 3 changes of phosphate-buffered saline (PBS, 0.01 M, pH 7.3). All subsequent steps were performed a t room temperature.

As in previous studies on the thyroid gland (Wilson et al., '78), 4 primary antisera raised in rabbit and specific for either T, or T, were used. These were anti-T, (Endocrine Sciences, Oxnard, California) ; and 3 different antisera to T, (designated as antisera A, B and C; Cap- pel Laboratories, Cochranville, Pennsylva- nia). Antiserum A binds only to T,. Anti- serum B binds significantly to both T, and the bovine serum albumin (BSA) carrier (anti-T,/ BSA), thus yielding a background fluorescence which can be removed by absorbing with BSA. Antiserum C is similar to A but is con- jugated to fluorescein isothiocyanate (FITC).

THYROID HORMONE BINDING IN TYPE I1 CELLS 613

The anti-T, serum was used a t a 1:20 dilution; all anti-T, sera were used a t a 1 : l O dilution. The FITC-anti-T, was used for direct immunofluorescence; all other antisera were used for indirect immunofluorescence techniques (Coons et al., '55). The secondary antiserum was the FITC conjugate of goat anti-rabbit immunoglobulin (IgG) (Cappel Laboratories, Cochranville, Pennsylvania) which was used a t a 1 : l O dilution. Undiluted antisera were stored a t - 70°C; dilutions with phosphate buffer (0.1 M, pH 7.3) were prepared immediately prior to use. FITC conjugates were cen- trifuged prior to use to remove fluorescein ag- gregates.

Using the technique of Coons et al. ('551, specimens were incubated with primary antisera for 30 minutes in a moist chamber and then rinsed for 15 minutes in 3 changes of PBS. Specimens for indirect immunofluorescence were incubated with secondary antiserum for 30 minutes and rinsed again for 15 minutes in 3 changes of PBS. Slides were mounted in glycerin: PBS (1: 11, coverslipped and examined with a fluorescence microscope using UG1 and BG12 excitation filters and Zeiss barrier filters 47 and 65. Photomi- crographs were taken using the same ex- posure times for experimental and matching control specimens.

Controls In order to assess specificity of the reaction,

absorbed antisera were utilized. These were prepared by addition of 100 mg/ml of T,, T, (Sigma, St. Louis, Missouri) or BSA (ICN, Cleveland, Ohio) to concentrated antiserum; incubation for 1 hour a t 4°C was followed by centrifugation. Cross-absorbed antisera were similarly prepared by adding T, (100 mg/ml) to anti-T, serum and T, (100 mg/ml) to anti-T, serum. Absorbed antisera were prepared and diluted ( 1 : l O ) in phosphate buffer (0.1 M, pH 7.3) immediately prior to use.

Methodologic controls consisted of replacing primary antisera with either normal (pre- immune) rabbit serum, normal goat serum (Cappel Laboratories, Cochranville, Pennsyl- vania), or PBS.

Finally, some cultures were maintained in Ham's F12K medium supplemented with 10% serum obtained from a thyroidectomized calf (Grand Island Biological Co., Grand Island, New York). The T, and T, levels in this serum were undetectable by radioimmunoassay (i.e., T, < 0.125 ng/ml; T, < 1.6 ng/ml).

Fig. 1 Phase contrast micrograph of a n isolated type I1 cell demonstrating the epithelial morphology of the cell and granular appearing perinuclear inclusions (lamellar bodies). x 600.

Cytochemical methods Esterase technique

Type I1 cells growing on glass slides were rinsed for 30-60 seconds in HBSS and proc- essed for cytochemistry according to the method developed by Vatter et al. ('68) for the localization of nonspecific esterases. The p-ni- trophenylthiol butyrate substrate (Poly- sciences, Warrington, Pennsylvania) was utilized. The cells were counterstained with 1% aqueous toluidine blue for 1 minute, rinsed in buffer, dehydrated in graded alcohols, coverslipped, and examined with a light microscope.

Nile blue hydrochloride The Nile blue hydrochloride technique of

Bracco and Curti ('74) was modified for use with the cultured cells. Type I1 cells growing on glass slides were rinsed for 10-30 seconds in HBSS. The cells were covered with 2 drops of a Nile blue hydrochloride solution, coverslipped and immediately examined using a fluorescence microscope with ultraviolet light or


Fig. 2 Electron micrograph of an isolated type 11 cell a t the same population doubling level (251 as those cells utilized for the immunohistochemical studies. Characteristic lamellar bodies Prc pwminpnt in the cytoplasm of these epithelioid cells. x 27,000

with substage white light illumination. For fluorescence microscopy, UG1 and BG12 excitation filters and Zeiss barrier filters 41 ancl 44 were used.

RESULTS

In culture, the isolated type I1 pneumonocytes grew as monolayers and these cells contained typical perinuclear granular inclusions (fig. 1). Ultrastructural studies confirmed that these cytoplasmic inclusions were mor- phologically identical to the lamellar bodies present in type I1 cells of intact lung (fig. 2). In the present study, histochemical studies were also utilized to confirm the identity of these granules in the same population of cells being utilized for the immunofluorescent studies. These granules are the major sites of nonspecific esterase activity (fig. 3), esterases which are established markers for type I1 cell lamellar bodies (Hitchcock-O’Hare et al., ‘76). Treatment of the isolated type I1 cells with Nile blue hydrochloride, another accepted marker of type I1 cell lamellar bodies (Bracco and Curti, ’741, produced similar results (fig. 4a) ; fluorescence was localized to the granular inclusions in the perinuclear zone. For orien- tation, figure 4b illustrates the same Nile blue hydrochloride-treated cells when viewed with substage white light illumination.

The immunofluorescence studies indicated

that monolayer cultures of type I1 cells grown in nmmal or IT,-supplemented sera displayed identical fluorescence patterns with no ap- parent differences in fluorescence intensity. The results of these two groups will, therefore, be described together. Two distinctly different fluorescent patterns were observed regardless of the antisera used. One pattern was present in freshly (within 24 hours of plating) subcultured cells, and the second in cells maintained in vitro for two or t,hree days following subculture.

In freshly subcultured cells, combined nuclear and cytoplasmic fluorescence was observed !fig. 5). The diffuse nuclear fluorescence was punctuated by intensely fluorescent nuclear structures (possibly nucleoli); nuclear envelopes were also fluorescent. Cyto- plasmic fluorescence appeared as a fine. reticular network with greatest density in the perinuclear zone. This cytoplasmic network contained circular, non-fluorescent areas similar in location and size to the perinuclear lamellar bodies.

In the cultures that were maintained two or three days in vitro, a second fluorescent pattern was observed with all four primary antisera. This consisted of a diffuse cytoplasmic fluorescence with dense accumulations around the perinuclear inclusions. Figure 7a, a photomicrograph of an isolated type I1 cell

TWiROID HORMONE R I N D i N C I& TYPE I1 CELLS 615

treated with anti-T,, demonstrates such perinuclear fluorescence. Accumulations of fluorescein were found around perinuclear, cytoplasmic inclusion bodies which are in the same location as the granular inclusions demonstrated in figures 1.4. Figure 6 illustrates an isolated type I1 cell treated with anti-T,. Again, the fluorescence is located diffusely in the perinuclear zone with dense accumulations around these intracellular inclusions. In this particular cell, an additional dense concentration of fluorescein is seen a t the cell pe- riphery located around another cluster of inclusion bodies (fig. 6). In most instances in which this diffuse cytoplasmic pattern is found, the nucleus is devoid of fluorescence.

Several controls were employed to assess specificity of the reaction. Figure 7b illustrates typical results of the negative controls. In this particular case, absorption of anti-T, serum with T, eliminated essentially all fluorescence. Absorption of anti-?', serum with T, produced identical results.

A further negative control consisted of maintaining the type I1 cells in the absence of thyroid hormone. Type I1 cells were plated in Ham's F12K medium with serum from a thyroidectomized calf and were examined at the same time intervals as those used for the experimental cultures. Cells placed in this medium were devoid of fluorescence when sub- jected to either direct or indirect immunofluorescence techniques.

In order to test for nonspecific binding due to culture conditions, monolayer cultures of adult rat lung and prostate fibroblasts were grown in the same conditions as the type I1 cells. In experiments identical to those for type 11 cells, specific fluorescence was not observed.

Further controls consisted of replacing the primary antiserum with normal rabbit serum, normal goat serum, and PBS. In all cases fluorescence was absent from the type I1 cells. As shown in studies of the thyroid gland (Wilson et al., '781, cross-absorption of anti-T, with T, did not cause any observable loss of fluorescence, suggesting that the observed fluorescence was due to T,. However, when anti-T, was absorbed with T, ,there was a significant decrease in the observed fluorescence in the type I1 cells; this suggests that most of the fluorescence observed with this antiserum was also due to T,. This was an unexpected finding since radioimmunoassay data indi-

cated less than 45 cross-reactivity of ant,i-T, for T, (DISCUSSION).

DISCUSSION

Inimunofluorescence provides a powerful tool for localizing antigens to their cellular binding sites. The results of this study indicate for the first time that T, and T, can be di- rectly localized in their target cells in vitro utilizing immunofluorescence techniques. The high degree of specificity possible with immunofluorescence is demonstrated in the present study by the results of the absorption studies which show that cells treated with pre-absorbed antisera are devoid of fluorescence (fig. 7b). As already demonstrated in the thyroid gland (Wilson et al., '78), the results of cross-absorption controls indicate that anti-T, serum binds specifically to tissue sites of T,. However, the anti-T, serum does bind sub- stantially to T, antigenic sites. Therefore, we cannot be certain how much of the observed fluorescence, if any, is due to the presence of T,. Indeed, maintenance of cells in T,-supplemented sera did not produce any observable changes in fluorescence intensity. These results corroborate and extend those of Hitch- cock and Reichlin ('78) and Hitchcock et al. ('78) who showed that the major thyroid hormone concentrated from the plasma by peri- natal and adult rat lung is T,.

The demonstration that the lung can con- centrate T, from the plasma (Hitchcock and Reichlin, '78; Hitchcock et al., '78) and that rat and rabbit lung nuclei possess receptors for T, (Morishige and Guernsey, '78; Linden- berg et al., '78), indicate that the lung is a target organ for thyroid hormone. The present study corroborates these findings and demonstrates, using immunofluorescence, that T, is bound by cells in culture which are derived from adult rat type I1 pulmonary epithelial cells. These results confirm and extend the studies of Lindenberg et al. ('78). Utilizing the same cells described in this study, these inves- tigators demonstrated specific nuclear binding sites for T,. Competitive binding studies demonstrated 2,280 nuclear sitedcell, a level of sites commensurate with other thyroid hormone target tissues (Oppenheimer et al., '74).

Two different fluorescent patterns were observed in the cells. One pattern was demonstrated by freshly subcultured cells and the second by cells that had been maintained in vitro for two to three days. Cells in the latter


culture were in a subconfluent state when examined. Freshly plated type I1 cells enter a rapid growth phase. Smith ('77) reported that production of saturated phospholipids by type I1 cells was inversely proportional to cellular growth rate. As the density of the cultures ap- proached confluency, growth rate declined and metabolic processes were directed toward synthesis of secretory product as evidenced by the significant increase in the percent incorporation of precursor into saturated phospholipids (Smith, '77). This difference in metabolic state may be responsible for the two major fluorescence patterns observed. A number of studies (DeGroot e t al., '76, '77; Martial e t al., '77; Oppenheimer et al., '72) indicate that thyroid hormone exerts control over cell metabolism by binding to the nuclear chro- matin and stimulating messenger RNA synthesis. In freshly subcultured cells, fluorescence was observed on nuclear envelopes, throughout the nucleoplasm, and in a reticular network in the perinuclear zone which might represent the rough endoplasmic retic- ulum. After two to three days in culture, the nucleus is usually devoid of fluorescence, and while the perinuclear zone contains diffuse fluorescence, the major accumulations occur in cytoplasmic areas adjacent to the lamellar bodies. Indeed, studies utilizing radiolabeled precursors of surfactant (Chevalier and Col- let, '72) have demonstrated that these same cytoplasmic areas are sites of surfactant synthesis. Such fluorescent patterns are, therefore, in keeping with proposed mechanisms of action of thyroid hormones. In addition, localization of thyroid hormones to cytoplasmic areas involved in surfactant synthesis sup- ports the results of studies which indicate that T, and T, may be involved in the regulation of surfactant synthesis by, or release from, the type I1 cell of the lung (Redding et al., '72; Wu e t al., '73; Hitchcock, '79).

In summary, the present study adds strong support to the accumulating body of evidence that thyroid hormones influence lung metabolism. Results indicate that cells derived from type I1 cells contain specific binding sites for thyroid hormones, especially for T,, and that these binding sites are located both in the nucleus and in specific locations within the cytoplasm. It is through these specific binding sites that T, may exert a regulatory effect on type I1 cell metabolism.

LITERATURE CITED

Avery, M. E. 1975 Pharmacologic approaches to the acceleration of fetal lung maturation. Br. Med. Bull., 31: 13-17.

Ballard, R. A., and P. L. Ballard 1976 Use of prenatal glucocorticoid therapy to prevent respiratory distress syndrome: A supporting view. Am. J. Dis. Child., 130: 982-987.

Ballard, P. L., B. J. Benson and A. Brehier 1977 Gluco- corticoid effects in the fetal lung. Am. Rev. Resp. Dis., 115: 29-36.

Bracco, V. M., and P. C. Curti 1974 Beschreibung einer histochemischen, fur die oberflachenaktive substanz der alveolen spezifischen methode. Arzneimittel-Forschung, 24: 845-847.

Chevalier, G., and A. J. Collet 1972 In uiuo incorporation of cholineJH, l e ~ c i n e - ~ H and galactose-3H in alveolar type I1 pneumocytes in relation to surfactant synthesis. A quantitative radioautographic study in mouse by electron microscopy. Anat. Rec., 174: 289-310.

Coons, A. H., E. H. Leduc and J. M. Connolly 1955 Antibody production. I. Method for the histochemical determina- tion of specific antibody and i ts application to a study of the hyperimmune rabbit. J. Exptl. Med., 102: 49-59.

DeCroot, L. J., L. Hill and P. Rue 1976 Binding of nuclear triiodothyronine (T,) binding protein-T, complex to chro- matin. Endocrinology, 99: 1605-1611.

DeGroot, L. J., P. Rue, M. Robertson, J. Bernal and N. Scherberg 1977 Triiodothyronine stimulates nuclear RNA synthesis. Endocrinology, 101: 1690-1700.

Douglas, W. H. J., and P. J. Chapple 1977 Characterization of monolayer cultures of type I1 alveolar pneumonocytes that produce pulmonary surfactant in uitro. In: Develop- ments in Biological Standardization. Vol. 37. Interna- tional Association of Biological Standardization, S. Kar- ger, ed. Basel, Switzerland, pp. 71-76.

Douglas, W. H. J., P. Del Vecchio, R. W. Teel, R. M. Jones and P. M. Farrell 1976 Culture of type I1 alveolar lung cells. In: Lung Cells in Disease. A. Bouhuys, ed. North- Holland Biomedical Press, New York, pp. 53-68.

Douglas, W. H. J., and P. M. Farrell 1976 Isolation of cells that retain differentiated functions in uitro: Properties of clonally isolated type I1 alveolar pneumonocytes. En- viron. Health Perspectives, 16: 83-88.

Douglas, W. H. J., and M. E. Kaighn 1974 Clonal isolation of differentiated rat lung cells. In Vitro, 10: 230-242.

Douglas, W. H. J., J. A. McAteer, J. R. Smith and W. R. Braunschweiger 1979 Type I1 alveolar pneumonocytes in uitro. International Review of Cytology, in press.

Douglas, W. H. J., R. A. Redding and M. Stein 1975 The lamellar substructure of osmiophilic inclusion bodies present in rat type I1 alveolar pneumonocytes. Tissue and Cell, 7: 137-142.

Farrell, P. M. 1977 Fetal lung development and the influence of glucocorticoids on pulmonary surfactant. J. Steroid Biochem., 8: 463-470.

Hitchcock, K. 1979 Hormones and the lung. I. Thyroid hormones and glucocorticoids in lung development. Anat. Rec., 194: 15-40.

Hitchcock, K. R., andS. Reichlin 1978 Thyroid hormones in the adult ra t lung. Am. Rev. Resp. Dis., 117: 807-810.

Hitchcock, K. R., S. Reichlin and J. Harney 1978 Thyroid hormones in lungs of fetal, newborn and adult rats. Am. Rev. Resp. Dis., 117: 349 (Abstract).

Hitchcock-O'Hare, K. R., E. Meymaris, J. Bonaccorso and S. B. Vanburen 1976 Separation and partial characterization of surface-active fractions from mouse and rat

THYROID HORMONE BINDING IN TYPE I1 CELLS 617

lung homogenates. Identification of a possible marker system. J. Histochem. Cytochem., 24: 487-507.

Lindenberg, J. A., A. Brehier and P. L. Ballard 1978 Tri- iodothyronine nuclear binding in fetal and adult rabbit lung and cultured lung cells. Endocrinology, 103: 1725-1731.

Martial, J. A., J. D. Baxter, H. M. Goodman and P. H. Seeburg 1977 Regulation of growth hormone messenger RNA by thyroid and glucocorticoid hormones. Proc. Natl. Acad. Sci. (U.S.A.), 74: 1816-1820.

Morishige, W. K., and D. L. Guernsey 1978 Triiodo- thyronine receptors in ra t lung. Endocrinology, 102: 1628-1632.

Nejad, I., J. Bollinger, M. A. Mitnick, P. Sullivan and S. Reichlin 1975 Measurement of plasma and tissue triiodothyronine concentration in the rat by radioimmunoassay. Endocrinology, 96: 773-780.

Oppenheimer, J. H., D. Koerner, H. L. Schwartz and M. I. Surks 1972 Specific nuclear triiodothyronine binding sites in ra t liver and kidney. J. Clin. Endocrinol. Metab.,

Oppenheimer, J. H., H. L. Schwartz and M. I. Surks 1974 35: 330-333.

Tissue differences in the concentration of triiodothyronine nuclear binding sites in the rat: liver, kidney, pitui- tary, heart, brain, spleen, and testis. Endocrinology, 95: 897-903.

Redding, R. A., W. H. J. Douglas and M. Stein 1972 Thyroid hormone influence upon lung surfactant metabolism. Sci- ence, 175: 994-996.

1977 Cell line A549: A model system for the study of alveolar type I1 cell function. Am. Rev. Resp. Dis., 115: 285-293.

Vatter, A. E., 0. K. Reiss, J. K. Newman, K. Lindquist and E. Groeneboer 1968 Enzymes of the lung. 1. Detection of esterase with a new cytochemical method. J. Cell. Biol., 38: 80-98.

Wilson, M., K. R. Hitchcock and R. A. DeLellis 1978 Immu- nohistochemical localization of thyroid hormone binding in ra t thyroid gland. J. Histochem. Cytochem., 26: 1121-1124.

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Smith, B. T.

PLATE 1

EXPLANATION OF FIGURES

3 Photomicrograph of the sites of surfactant-associated nonspecific esterases in an isolated type 11 cell. The association of reaction product with the perinuclear granular inclusions indicates that these structures are lamellar bodies, the major storage sites of pulmonary surfactant. Substrate, p-nitrophenyltbiol butyrate; stained with toluidine blue. x 1,400.

Photomicrograph of an isolated type I1 cell treated with Nile blue hydrochloride. The characteristic orange fluorescence occurs a t sites of intracellular lamellar bodies. x 1,240.

4b Photomicrograph of the same cell depicted in figure 4a when viewed with substage, white light illumination. The brilliant perinuclear granules correspond to the fluorescent lamellar bodies shown in figure 4a. X 1,240.

Fluorescence micrograph of freshly subcultured type I1 cells treated with FITC anti-T,. These cells demonstrate a diffuse nuclear fluorescence which is punctuated by intensely fluorescent nuclear structures (possibly nucleoli) ; fluorescence is also associated with the nuclear envelopes. Cytoplasmic fluorescence is confined to a reticular perinuclear network. x 1,200.

This fluorescence micrograph depicts an isolated type I1 cell after two days in vitro which has been treated with anti-T, and FITC goat anti-rabbit serum. As with anti-T, (fig. 7a), diffuse fluorescence is observed in the perinuclear cytoplasm with accumulations around intracellular lamellar bodies. In this cell, two groups of inclusions are depicted; a large cluster in the usual perinuclear location with a second large group producing a protrusion of the plasmalemma. x 1,600.

7a Fluorescence micrograph of an isolated type I1 cell maintained for two days in vitro and treated with anti-T, and FITC goat anti-rabbit serum. The cell demonstrates diffuse fluorescence in the perinuclear cytoplasm with accumulations around perinuclear lamellar bodies. x 1,200.

7b Absorption control of an isolated type I1 cell maintained under the same conditions as described for figure 7a. The cell was treated with absorbed T, antisera and FITC goat anti-rabbit serum. Absorption of the anti-T, serum with T, has eliminated virtually all fluorescence. x 1,200.

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THYROID HORMONE BINDING IN TYPE I1 CELLS Wilson, Hitchcock, Douglas and DeLellis

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Documents

Hormones and the lung II. Immunohistochemical localization of thyroid hormone binding in type II pulmonary epithelial cells clonally-derived from adult rat lung