Introduction

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Lung maturation is a complex process. Some 40 different cell types have been identified in the adult lung (1), yet little information exists concerning regulation of development of these cells. Several recent reports suggest that growth factors may have major roles in cellular development and airway branching within the primordial lung. Ligands or receptors for fibroblast growth factor (2), epidermal growth factor (3, 4), transforming growth factor  (5, 6), and insulin-like growth factor (7) have been either localized in developing pulmonary tissue or shown to influence fetal lung cell function. Recent evidence suggests that cytokine receptors also play an important role in lung development (8, 9).

Leptin, a newly recognized hormone produced by adipocytes, regulates body fat through a neural feedback mechanism (10). Receptors have been identified for leptin in the central nervous system and are likely involved in energy homeostasis. However, numerous other tissues also express leptin receptors or leptin mRNA (11). In studies that surveyed the distribution of leptin receptors in different tissues, it was demonstrated that the adult lung displays particularly high levels of the putative functional leptin receptor as well as its splice variants (12, 13). In addition, expression of leptin and leptin receptor mRNA was detected in fetal lung (14), suggesting a possible role in development of this tissue.

Given that leptin is intimately involved in regulation of lipid metabolism (15), the present study was undertaken to characterize further the presence of leptin receptors in lung and assess whether leptin may play a role in lung development. Furthermore, since one of the hallmarks of maturity in fetal lung is the ability to produce pulmonary surfactant, we examined the potential of leptin to regulate production of this lipoprotein material to determine whether leptin may have a role in regulating the activity of surfactant-producing fetal type II alveolar cells.

	Materials and Methods

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Materials

Timed pregnant rabbits were from Blue Farm Rabbitry (St. Andrew's, MB, Canada). Animals were allowed to breed at a designated time for a maximum of 1 h. This day was considered as time 0. Rabbits were killed on the 24th day of gestation. All animal work was approved through the Canadian Council on Animal Care. Leptin (mouse recombinant) was from R&D Systems (Minneapolis, MN) while newborn calf serum was from Sigma Chemical (St. Louis, MO). [¹²⁵I]leptin (2200 Ci/mmol) and [³H]choline (80 Ci/mmol) were from New England Nuclear (Boston, MA). Nuclear emulsion was from Amersham (Mississauga, ON, Canada). Culture materials were from GIBCO (Mississauga, ON, Canada). Antibodies to leptin receptor were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Two antibodies were used. M18 was raised in goat against the carboxy-terminal of mouse leptin receptor, while K20, also from goat, was against the amino-terminal sequence of mouse leptin receptor.

Ligand-Binding Assays

[¹²⁵I]leptin was used to identify leptin receptor sites in adult and fetal rabbit lung as described by Paterson and colleagues (16). Samples of lung from adult and 24^th gestational day fetal rabbit lung were snap frozen in isopentane cooled in dry ice, sectioned at 10 µm, and mounted on gel-coated slides. Tissue sections were preincubated in buffer (100 mM Hepes, pH 7.4, 120 mM NaCl, 1.2 mM MgSO₄, 2.5 mM KCl, 15 mM Na acetate, 10 mM dextrose) for 10 min at room temperature and transferred to fresh buffer with increasing concentrations of [¹²⁵I]leptin (0.1, 0.5, 1.0, 2.0 and 5.0 nM) with or without 1 µM-unlabeled leptin (Sigma) for 30 min. Sections were scraped into scintillation vials and binding measured by  counting. Identical sections were coated with nuclear emulsion and placed in the cold (20°C) for various lengths of time or applied to X-ray film (Eastman Kodak Co., Rochester, NY). The latter samples were removed after 1-week exposure and developed. X-ray images were captured using the analysis system from Labtronics (Mississauga, ON, Canada). Quantitation was done by viewing the X-ray image through a microscope and repetitively measuring the density within a standard area projected over the image of the section using the public domain NIH Image program (developed at the U.S. National Institutes of Health and available on the Internet at http://rsb.info.nih.gov/nih-image/ ) (17). Binding was determined by measuring the total counts of each section and dividing this by the area of that section to give mean counts per arbitrary unit area. The former samples were exposed for 4 wk, removed, fixed, developed, and stained as described previously (18). Control sections consisted of samples incubated with [¹²⁵I]leptin in the presence of 1 µM-unlabeled leptin. Samples were viewed under standard Kohler illumination.

Cell Culture

Fetal rabbit lung type II cells were isolated and grown as described by Batenburg and colleagues (19) and Scott (20). Briefly, lungs were dissected from fetal animals, chopped, digested with trypsin/ethylenediaminetetraacetic acid (0.05%/0.02%), and filtered through Nitex gauze. Cells were collected and plated into 75-cm² flasks and allowed to adhere for 1 h. Nonadherent cells were collected at the end of this period and replated for an additional hour. Again, nonadherent cells were collected and replated into 75-cm² flasks. After 18 h, the medium was changed to remove cellular debris and nonadherent cells. The cell monolayers were cultured for 3-4 d as described and routinely showed purities of 85-90% (20). Cells were grown to near confluence prior to use. Viability is regularly established by trypan blue exclusion and lactate dehydrogenase assay. The cells were used for immunoreceptor binding and to measure the effect of leptin on [³H]choline incorporation into disaturated phosphatidylcholine (DSPC), which is the major phospholipid component of surfactant and an established marker [19]. Cells were incubated with increasing concentrations of leptin (0.01, 0.1, 1.0 and 10 µg/ml) for 24 h in the presence of [³H]choline (1 UCi/ml). After this period, the cells were washed and scraped into a small volume of methanol. DSPC was isolated from the cells by chloroform/methanol extraction as described previously (21). The organic phase of the extract was reacted with OsO₄ and separated on thin layer chromatography plates (LK5D; Whatman, Fisher Scientific, Edmonton, AB, Canada) with authentic standards. Phospholipid spots were identified using iodine vapor, and radioactivity from the appropriate spots on the plates was measured by scraping the gel into scintillation vials and counted on a LS5801 scintillation counter. Quench was compensated using the method of H# based on the spectrum of ¹³⁷Cs (Beckman Instruments, Palo Alto, CA) (20).

Leptin Receptor

To characterize leptin receptor in lung, membranes of adult rabbit lung and membranes from isolated fetal rabbit type II cells were prepared. Whole lungs were homogenized in buffer using a Polytron homogenizer (Kinematica, Lucerne, Switzerland). In the case of the isolated cells, membranes were prepared by scraping the cells into ice-cold buffer with protease inhibitor (PMSF, 1 mM) and homogenizing using a hand-held Dounce homogenizer (Fisher Scientific). Debris was removed by centrifuging at 250 g. A membrane fraction was prepared by centrifuging the samples at 100,000 × g for 1 h. Protein levels were determined using the kit from Bio-Rad (Mississauga, ON, Canada). Tissue samples were suspended in sample buffer with or without 2-mercaptoethanol (4%). A total of 75 µg of protein was applied to 10% polyacrylamide gels (5 × 4 cm) with a stacking overlay of 4% acrylamide (22). Standards (Bio-Rad high-molecular weight) of known molecular mass were run on adjacent channels. Gels were run at 200 V for 1 h or until the bromophenol blue marker reached the lower end of the gel. Gels were removed, rinsed briefly in distilled water, and transferred to PVDF membranes (Mandel Scientific, Mississauga, ON, Canada) at 100 V for 1 h (23).

Blots were blocked with 5% skim milk powder for 1 h and reacted with antibody to K20 (1:250 dilution) and M18 (1:100 dilution) in PBS for 1 h. Strep-avidin-HRP-labeled secondary antibody was reacted with the blots and binding sites identified by enhanced chemiluminescence. Blots were rinsed with buffer and overlaid with X-ray film for 5 min. Films were developed and bands quantitated using Quantiscan (Biosoft, Cambridge, UK).

Statistical Analysis

Where appropriate, statistical analysis was made by post hoc application of Duncan's New Multiple Range Test (24) assuming a significant difference at P < 0.05.

	Results

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Figures 1A-1D show the general microscopic appearance of 24th gestational day fetal rabbit lung. For comparison, the morphological appearance of 25th gestational day fetal rabbit lung type II cells and isolated 24th gestational day fetal rabbit type II cells in culture are also shown. Glycogen was prevalent in the cytoplasm of lining cells on the 24th gestational day. Two different cell types could be distinguished. One was somewhat flattened while the other was of a cuboidal nature. Immature lamellar bodies, often closely associated with glycogen particles were detectable (Figures 1C and 1D). Glycogen was considerably reduced by the 25th gestational day (Figure 1E). Cultured fetal type II cells displayed numerous lamellar bodies (Figure 1F).

View larger version (118K): [in this window] [in a new window]   Figure 1.   (A-D) are from 24th gestational day fetal rabbit lung. (A) Light micrograph of two presumptive acini (Ac) within the fetal lung (×900). (B) Electron micrograph similar to the micrograph in (A) showing the lumen of an acinus (Ac) within the developing lung. Two major cell types are present within the epithelial lining. The first (small arrow) is somewhat flattened and displays an oblong nucleus while the second (large arrow) protrudes into the lumen, has a round nucleus, and has copious amounts of glycogen (Gl) within the cytoplasm (×1000). (C) Lamellae beginning to form around a core of electron dense particles within a lining cell of the acinus (×12,000). (D) A well-formed early lamellar body displaying concentric lamellae surrounding several electron-dense particles (×12,000). (E) A 25th gestational day fetal rabbit lung showing a single lamellar body (arrow). Note that glycogen is virtually absent from the cytoplasm (×4,500). (F) Fetal rabbit type II cell isolated from 24th gestational day lung and maintained in cell culture for 4-5 d. The base of the cell opposed to the plastic of the culture vessel is indicated by the large arrow. Numerous lamellar bodies (small arrows) are present (×3,500).

Figure 2 shows autoradiographic binding of [¹²⁵I]leptin to adult (Figure 2A) and fetal (Figure 2C) rabbit lung sections at ligand concentrations of 1.0 nM. Density of binding in adult rabbit lung was ~ 50% greater than that displayed by fetal lung. Corresponding nonspecific binding levels (Figures 2B and 2D) in the presence of 1.0 µM-unlabeled leptin showed that a majority of the label was associated with specific binding.

View larger version (54K): [in this window] [in a new window]   Figure 2.   X-ray images of [125I]leptin binding in adult and fetal rabbit lung. Binding of [125I]leptin to sections of adult (A and B) and fetal (C and D) rabbit lung (all magnifications at ×40). Sections were incubated with radioactive ligand (1.0 nM) for 30 min in the presence (B and D) or absence (A and C) of 1-µM-unlabeled leptin. Sections were applied to X-ray film for identical lengths of time and the film was developed. (E) Quantitation of density of [125I]leptin binding at 1.0 nM as displayed in the photograph. In each case, the total binding was significantly different from the nonspecific binding (P < 0.05). Binding in fetal lung was significantly less (P < 0.05) than that of adult lung.

Figure 3 shows the saturation curve for [¹²⁵I]leptin binding to adult rabbit lung sections at increasing leptin concentrations. Total binding increased over this range. Specific binding (total minus nonspecific), increased in a linear fashion to 2.0 nM.

View larger version (15K): [in this window] [in a new window]   Figure 3.   Specific and nonspecific leptin binding. Binding of [125I]leptin was quantified from sections identical to those in Figure 1. Sections were incubated with radioactive ligand (0-2.0 nM) for 30 min in the presence or absence of 1 µM-unlabeled leptin. Radioactivity was determined in representative sections by  counting. Cross-sectional areas were measure using image analysis of digital photographs of each section to standardize for relative areas.

Figure 4 shows the localization of [¹²⁵I]leptin at the microscopic level in adult (Figures 4A and 4B) and fetal (Figures 4C-4E) lung slices. Control lung sections of [¹²⁵I]leptin binding in the presence of 1.0 µM-unlabeled leptin showed only occasional random autoradiographic granules (data not shown). The majority of labeling in adult lung was detected over alveolar septal walls (Figure 4A), while certain cells in the walls appeared to bind leptin very avidly (Figure 4B), but the cell type could not be distinguished at this level. In fetal lung, label was particularly concentrated in developing prealveolar acini (Figure 4C). Conspicuously, lesser amounts of label were present in parenchymal tissue peripheral to the developing ducts and acini. The epithelial lining cells of the ducts in particular showed copious radioactive label binding (Figure 4D). Lining epithelium of small branching ducts also appeared to be sites of [¹²⁵I]leptin binding (Figure 4E).

View larger version (147K): [in this window] [in a new window]   Figure 4.   Tissue localization of [125I]leptin binding in fetal lung. [125I]leptin binding (as shown by black autoradiographic granules) in adult (A and B) and fetal (C-E) lung. Standard frozen unstained sections of lung were prepared, incubated with [125I]leptin, washed, and coated with Nuclear Trac emulsion. Slides were incubated in the dark for 4 wk prior to development. (A) Adult lung (×600). (B) Adult lung (×600): arrows, single unstained (frozen) cells of lung epithelium. (C) Developing fetal lung (×400): arrows, single cells in the developing epithelium of the fetal lung. (D) Developing fetal lung (×600): arrows, note localization of autoradiographic granules over epithelial cells. (E) Developing fetal lung (×600). D, duct, D1, small duct branching off main duct; E, epithelium of alveolar duct; P, parenchymal connective tissue underlying epithelium; Ad, alveolar duct.

Figure 5 shows Western blots of binding of leptin receptor antibodies to cell membrane proteins. Antibody to the K20 receptor epitope bound to four major bands of 225, 123, 68, and 47 kD apparent molecular mass in adult rabbit lung membranes (Figure 5A). In contrast to whole lung, isolated fetal type II cells displayed only a single band with an apparent molecular mass of 125 kD (Figure 5A).

View larger version (35K): [in this window] [in a new window]   Figure 5.   Receptor characterization in membranes of isolated fetal type II cells and adult lung. Antibodies against leptin receptors display binding in membranes of whole adult lung and isolated fetal rabbit type II alveolar cells. Antibodies were against the amino-terminal common to all leptin receptor types (K20) (A) or to the carboxy-terminal unique to the functional leptin receptor (M18) (B). Gels were scanned using the Quantiscan software program. (solid lines are scans of gels from isolated fetal type II alveolar cell membranes, while dashed lines are from adult rabbit lung membranes). The numbers to the side of each figure indicate the location of the molecular mass standards.

Binding of M18 leptin receptor antibody is shown in Figure 5B. In adult rabbit lung, four major binding sites of apparent molecular masses of 217, 123, 65, and 44 kD were detected in nonreduced samples. Once again, isolated fetal rabbit type II cells displayed a single binding site with an apparent molecular mass of 125 kD (Figure 5B).

Figure 6 shows the effect of leptin on the incorporation of [³H]choline into DSPC in isolated fetal rabbit type II cells. Leptin had a clear effect on radiolabeling of this phospholipid. Precursor incorporation into DSPC increased significantly (P < 0.05) at all ligand concentrations and peaked at 1.0 µg/ml.

View larger version (12K): [in this window] [in a new window]   Figure 6.   Leptin effect on DSPC synthesis in isolated fetal type II cells. Effect of leptin (0-10 µg/ml) on the incorporation of [3H]choline into DSPC in isolated fetal rabbit type II alveolar cells. Results are expressed as the mean ± SD of replicates of at least five samples. * indicates significantly different (P < 0.05) from the samples not exposed to leptin but only the vehicle controls (C).

	Discussion

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Leptin is an important factor in the regulation of energy balance that is mediated in part through a hypothalamic leptin receptor. Leptin receptors are also present in a variety of peripheral tissues, and there is increasing evidence that leptin may play a role in regulating other functions including the immune response (25), sympathetic activity (26, 27), angiogenesis (28), and reproductive function (29). Although leptin receptors are widely distributed in the periphery (11, 12), the functional significance of these receptors is largely unknown. The leptin receptor, Ob-R, is a member of the class I cytokine family of receptors of which there are at least six splice isoforms (Ob-Ra to Ob-Rf) of the receptor. The two most common isoforms of the receptor are Ob-Ra and Ob-Rb (11). These isoforms have identical extracellular and transmembrane domains and differ only in the length of the intracellular domain of the receptor. The Ob-Rb isoform, which has the longest intracellular domain of the six isoforms, is considered to be the fully functional receptor and is the most effective isoform with respect to activation of signal transduction pathways (11). Since the leptin isoforms have identical extracellular domains, binding to leptin is presumably similar among the various isoforms (28). The Ob-Ra isoform has a truncated intracellular domain (relative to the Ob-Rb receptor) and is much less effective in activating signal transduction pathways (30). In the adult, both isoforms have a wide distribution with the lung having the highest absolute levels of the Ob-Rb. Furthermore, in the fetus, lung is one of the few tissues that contains OB-R mRNA (9).

Results from our work show that ¹²⁵I-leptin binds in a specific manner to both adult and fetal rabbit lung although the former displays a much greater capacity for the protein. As noted previously, the lung contains some 40 different cell types, among the highest degree of cellular diversity of any tissue. Therefore we used fetal lung, which has fewer cell types, to provide initial evidence of functionality. Acini in developing lung displayed clear evidence of radiolabel binding along the epithelial margins. Furthermore the label was not uniformly distributed but appeared to be concentrated over certain cells within the acini, which may represent a different cell type.

Antibody binding to adult rabbit whole lung and isolated fetal rabbit lung type II cells displayed several binding sites. In adult lung, the K20 antibody, which recognizes the extracellular amino-terminal common to all the leptin (corresponding to the Ob-R, 12) receptor splice variants showed four major bands. However, the isolated fetal type II cells showed only a single band of 125 kD apparent mass, which probably corresponds to the 123 kD band detected in the adult tissue. Similarly, the M18 antibody, which recognizes the intracellular carboxy-terminal and therefore represents the functional leptin receptor, showed four bands in the adult lung tissue. Of these, bands at 65, 44, and 217 kD probably correspond to proteins detected by the K20 antibody of similar molecular masses, the largest of which may represent homo-oligomers of the receptor isoforms (32). The band at 123 kD would appear to represent the receptor in its functional (M18) configuration, as this protein is of a similar mass to the receptor identified in mouse placental tissue (14), as well as predicted from the DNA/mRNA sequence (31).

In contrast to the whole adult lung, isolated fetal rabbit type II cells expressed only a single immunoreactive band with either the K20 or M18 antibody. Both antibodies reacted with a protein of ~ 125 kD apparent molecular mass, which compares favorably with previously reported size of the leptin receptor (14, 31). Furthermore, the fact that both antibodies, raised against the extracellular and intracellular domains, displayed clear evidence of reaction with a similar protein suggests that these cells express functional leptin receptors. This supports findings of Lollman and colleagues (12) that lung shows high levels of functional OB-Rb receptor and may suggest that the apparent absence of Ob-Rb mRNA in fetal lung (14) may be species specific or related to the developmental stage of the organism.

In the lung, Tsuchiya and colleagues (33) have recently demonstrated that leptin stimulates cell proliferation in tracheal epithelial cells and in lung squamous cancer cells. O'Donnell and colleagues (34) conclude, based on shifts of myosin heavy chain types, that the absence of leptin results in changes in lung mechanical properties. Both groups have proposed a role for leptin as a growth factor. The response of isolated fetal type II cells to leptin in our study supports a role for this cytokine on lung development. The demonstration of specific [¹²⁵I]leptin binding at the level of the developing acinar epithelium and the dramatic increase in leptin-induced choline incorporation into DSPC by lung cells suggests that this peptide may act as a regulatory factor as the pulmonary epithelium differentiates.

In conclusion, we have demonstrated that lung as a whole and fetal type II cells in particular express functional leptin receptors and respond to leptin stimulation by increasing precursor incorporation into DSPC, a specific marker for pulmonary surfactant, suggesting synthesis of this phospholipid is increased. Elevation of DSPC synthesis, the hallmark of epithelial specialization in fetal lung, suggests leptin may have a role in pulmonary maturation.