ARTICLE |
Correspondence to: Jane D. Funkhouser, Dept. of Biochemistry and Molecular Biology, Univ. of South Alabama, 307 University Blvd., Mobile, AL 36688.
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Summary |
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Determination of the cellular distribution of phosphatidylinositol transfer protein in rat lung by immunocytochemistry revealed that the protein is more readily observed in the nonciliated bronchial epithelial cells (Clara cells) than in other lung cells. By light microscopy, the phosphatidylinositol transfer protein (PtdIns-TP) was localized to the dome-shaped apical region of Clara cells that were identified by staining with an antibody to Clara cell protein. Further investigation by electron microscopy revealed that the PtdIns-TP accumulated at the limiting membrane surrounding secretory granules and at the apical plasma membrane. This localization is compatible with the proposed roles for PtdIns-TP in formation of vesicles and exocytosis of secretory granules and, when considered in the context of the proposed role of PtdIns-TP in phosphatidylinositide metabolism, suggests that phosphatidylinositides may be involved in the mechanisms regulating Clara cell secretion. (J Histochem Cytochem 45:551-558, 1997)
Key Words: phosphatidylinositol transfer protein, phospholipid transfer protein, Clara cell, Clara cell protein, secretion
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Introduction |
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Phospholipid transfer proteins were identified and characterized on the basis of their ability to facilitate transfer of phospholipids between artificial and biological membranes in vitro. The proteins are found in a variety of cells and tissues and are classified on the basis of their specificity for the polar headgroup of the phospholipid transferred (reviewed in
To date, studies of phospholipid transfer proteins in lung have focused on a possible role in the phospholipid metabolism associated with formation of lamellar bodies in alveolar Type II epithelial cells (
The PtdIns-TP protein is the major phospholipid transfer protein transferring phosphatidylcholine in lung (
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Materials and Methods |
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Animals
Male Sprague-Dawley rats (100-125 g) were obtained from Charles River Laboratories (Wilmington, MA). They were maintained in animal quarters under the supervision of a veterinarian and were used within 4 weeks.
Antibodies
A 15-amino-acid peptide corresponding to the C-terminal of rat brain PtdIns-TP was synthesized by Immuno-Dynamics (La Jolla, CA). Immune serum was produced by Immuno-Dynamics by injecting the KLH-conjugated peptide into rabbit. Serum was tested at 2-week intervals by Western blot and ELISA. Serum samples taken before immunization were used as preimmune serum controls. A partially purified IgG fraction was obtained by ammonium sulfate precipitation. Protein from the 0-33% fraction was dissolved in Tris-buffered saline (TBS), pH 7.4, and dialyzed against three changes of TBS.
The antibody to the Clara cell secretory protein was a gift from G. Singh. The antibody is well characterized, specific for Clara cell proteins, and reacts exclusively with Clara cells in rat lung (
Western Blot
SDS-PAGE resolution of proteins and Western blot transfer was carried out using the PhastSystem (Pharmacia Biotech; Uppsala, Sweden). Lung protein extracts were resolved on 10-15% gradient precast SDS-PAGE gels for the PhastSystem (Pharmacia Biotech). The proteins were transferred by diffusion blotting to polyvinylidene fluoride (PVDF) membranes (Immobilon-P; Millipore, Bedford, MA). After transfer the membrane was cut into strips corresponding to each lane on the gel. The strips were incubated with 5% nonfat dry milk to block nonspecific binding sites. The strips were then washed and incubated with the indicated titers of primary antibody. Visualization of antibody binding was done using the ECL Western blotting analysis system with a horseradish peroxidase-linked anti-rabbit IgG (Amersham; Poole, UK).
Light Microscopic Immunohistochemistry
The Vectastain ABC staining kit (Vector Laboratories; Burlingame CA) was used except that the Vectastain Elite kit (approximately fivefold enhanced sensitivity) was used for the immunohistochemistry shown in Figure 3. Rat lungs were fixed with 4% paraformaldehyde in PBS for 2-3 hr. The lung was divided into lobes and embedded in paraffin. Five-µm sections were cut, placed on glass slides, dewaxed, and rehydrated through a series of ethanol solutions. The hydrated sections were rinsed in distilled water and incubated for 30 min in 0.3% hydrogen peroxide in methanol to quench any endogenous peroxidase activity. The sections were washed and blocked sequentially with normal goat serum diluted in PBS and 5% nonfat dry milk in PBS. The slides were then incubated with the indicated titers of primary antibody. For preadsorption controls, the anti-PtdIns-TP antibody was preincubated with the indicated amount of peptide for 4 hr at 4C before addition to the slides. After washing, the sections were incubated with biotinylated goat anti-rabbit IgG (H+L) for 2 hr at room temperature, followed by washing and incubation with the Vectastain reagent prepared in a buffer containing 0.2 M -methyl mannoside, which binds endogenous lectins that may produce nonspecific staining. After washing, antibody binding was visualized by incubating with the peroxidase substrate diaminobenzidine tetrahydrochloride (DAB). In a control experiment to demonstrate that all cells in the sections were accessible to the immunoglobulin, sections were stained with an antibody to tubulin using the same techniques. The results showed similar staining of all cell types present in the section (data not shown).
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Transmission Electron Microscopic Immunohistochemistry
Several fixation protocols commonly used for electron microscopy proved unsuitable for use with the PtdIns-TP antibody, although the reaction of antibody to the Clara cell protein could be readily detected in all protocols employed. Therefore, the fixation employed for light microscopy was used, and the immunohistochemistry was done before embedding to preserve reactivity of the PtdIns-TP with the antibody. Briefly, the tissue was fixed with formalin and the fixed tissue was glued onto a block and sectioned (100-µm sections) using a vibratome. The 100-µm sections were stained with Vectastain using the manufacturer's protocol. They were then osmicated (1% osmium tetroxide) and processed for electron microscopy using standard procedures for dehydration and infiltration with Spurr's plastic. The sections were mounted on glass slides coated with chromic potassium sulfate (chrome alum), a drop of 100% Spurr's plastic was placed on the section, and a second chrome alum-coated glass slide was used to cover the section. After drying overnight at 65C, the slides were separated with a blade and examined by light microscopy to locate an appropriate area for thin sections. An electron microscopy tissue peg was glued to the selected area and a blade used to section around the peg. The 100-µm section mounted on the tissue peg was used to cut 0.1-µm sections. The sections were stained with lead citrate and uranyl acetate and viewed with a Phillips 301 transmission electron microscope.
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Results |
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Characterization of the Antibody
Western blot hybridization was used to evaluate the specificity of the anti-peptide antibody. An IgG fraction from immune serum was used to probe a blot of resolved rat lung proteins (Figure 1). The antibody reacted with a 35-kD protein of the same Mr as PtdIns-TP (Figure 1, Lane 1). No other bands were seen on the gel, indicating that the antibody specifically binds a protein or proteins of 35 kD. Further evidence for the specificity of the reaction was obtained by preincubating the antibody with the peptide used for immunization. This completely eliminated the antibody reaction with the blot (Figure 1, Lane 2).
Cellular Pattern of Immunostaining
Rat lung sections stained with hematoxylin and eosin (H&E) demonstrated the characteristic architecture of lung tissue (data not shown). Most sections examined were in the regions of the bronchioles. Terminal and respiratory bronchioles, alveolar ducts, alveolar sacs, and alveoli could be identified.
The pattern of staining produced with the anti-PtdIns-TP antibody depended to some extent on the titer of antibody used. At a titer of 1~500, only cells in the bronchiolar epithelium were stained. The staining is shown in Figure 2. Figure 2A is a low-power micrograph illustrating the immunostaining in the context of the structural features of the portion of the lung examined. The arrow indicates the area shown at higher magnification in Figure 2C. Some cells in the bronchiolar epithelium stained more intensely than the other cells, and the staining was more intense in the luminal projections of these cells (Figure 2C). A field of alveolar cells from the same section that shows little or no staining at the 1~500 antibody titer is shown in Figure 2B. Figure 2D shows a control section in which the antibody was preabsorbed with the immunizing peptide at a concentration of 1.0 mM.
To identify the type of epithelial cell that stained intensely for PtdIns-TP, we obtained an antibody against Clara cell secretory protein (CCSP) to establish the identity of Clara cells in the tissue sections. Because both the CCSP antibody and the PtdIns-TP antibody were produced in rabbit, co-localization experiments could not be performed on the same sections. As an alternate approach, serial sections were used to compare the cell types containing CCSP protein and PtdIns-TP. For this experiment, a detection system more sensitive than that used for the experiments shown in Figure 2 was used (see Materials and Methods), and serial dilutions beginning at 1~2000 were used for both antibodies. Optimal staining was reached at 1~8000 for the PtdIns-TP antibody and at 1~16,000 for the CCSP antibody. The CCSP staining (Figure 3B) confirmed that the Clara cells are the dome-shaped cells with the apical projections. Comparison of the serial sections shown in Figure 3 indicated that these same cells immunostained with PtdIns-TP antibody (Figure 3A), although the intracellular staining for the PtdIns-TP was confined more to the apical regions of the cells.
Subcellular Localization of PtdIns-TP
Electron microscopy was used to further define the intracellular localization of PtdIns-TP in the Clara cells. The results are shown in Figure 4. Figure 4A shows Clara cells and ciliated bronchial epithelial cells in the same field. Dark staining representing the reaction of the antibody with the PtdIns-TP is observed in the apical region of the nonciliated Clara cells, which are readily recognized by their dome shape, with the dome projecting into the lumen of the airway. No staining is observed in the adjacent nonciliated epithelial cells.
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At higher magnification (Figure 4C), immunostaining for PtdIns-TP is observed at the plasma membrane of the nonciliated Clara cell (closed arrow) but not the ciliated cell (open arrow). The interface at the surface between the Clara cell and the ciliated epithelial cell is clearly demarcated by the staining reaction. Examination of the apical region containing secretory granules shows staining at the membrane surrounding the granules (Figure 4C, large arrowheads) and some background staining (small arrowheads) not seen in the control (Figure 4D).
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Discussion |
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The results of the localization of PtdIns-TP in the lung show that the protein is not evenly distributed over the lung cell population. PtdIns-TP immunoreactivity is more intense in the Clara cells than in the alveolar Type II epithelial cells or other lung cells. This suggests that its primary function in lung is not related to the sorting of phospholipids to intracellular membranes, as suggested from previous in vitro studies of phospholipid transfer activity (
The Clara cell is a nonmucous secretory cell containing abundant granules located in apical regions of the cell cytoplasm. The major secreted protein is the Clara cell secretory protein, a 16,000 MW homodimer that is abundantly expressed and secreted (
In Clara cells, two different general types of secretion are supported by ultrastructural data (
The greater density of PtdIns-TP staining in the apical region of Clara cells that contain many vesicles involved in protein secretion is consistent with the hypothesis that the PtdIns-TP is involved in membrane traffic and secretion, as has been demonstrated in the PC12 neuroendocrine cell line in which PtdIns-TP is known to participate in the formation of secretory vesicles (
The mechanistic role played by the PtdIns-TP in vesicle trafficking is not yet clearly defined. A considerable amount of data (reviewed in
The accumulation of PtdIns-TP at distinct membrane sites in the Clara cells presumably represents a transient interaction with the membrane, because PtdIns-TP is considered to be a cytosolic protein on the basis of its purification from soluble fractions of cell homogenates (
In summary, our observation regarding the distribution of PtdIns-TP in lung raises some interesting questions about its function in Clara cells. The molecular details regarding signaling and the mechanistic aspects of Clara cell secretion have not been explored. To date, polyphosphoinositides and PtdIns-TPs have not been implicated in mechanisms related to Clara cell secretion. However, when considered in the context of current data regarding PtdIns-TP function, the observation reported here concerning the cellular distribution and intracellular location of the PtdIns-TP in Clara cells suggests that this may be an important area for exploration in efforts to delineate mechanisms involved in secretion. Although the function of the Clara cell secretory protein has not been definitively established, suggestions that it may have anti-inflammatory and immunosuppressive properties (
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Acknowledgments |
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We thank Drs Phillip Fields and Leonard Aldes for helpful discussions and critically reading the manuscript, and Dr Shi Jun Zhang for assistance with the histology and electron microscopy. The antibody to Clara cell protein was a much appreciated gift from Dr G Singh at the VA Medical Center in Pittsburgh, PA.
Received for publication July 25, 1996; accepted November 26, 1996.
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Literature Cited |
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Bankaitis VA, Aitken JR, Cleves AE, Dowhan W (1990) An essential role for a phospholipid transfer protein in yeast Golgi function. Nature 347:561-562[Medline]
Bankaitis VA, Malehorn DE, Emr SD, Greene R (1989) The Sacharomyces cerevisiae SEC14 gene encodes a cytosolic factor that is required for transport of secretory proteins from the yeast Golgi complex. J Cell Biol 108:1271-1281[Abstract]
Batenburg JJ, Ossendorp BC, Snoek GT, Wirtz KWA, Houweling M, Elfring RH (1994) Phospholipid-transfer proteins and their mRNAs in developing rat lung and alveolar type-II cells. Biochem J 298:223-229[Medline]
Chevalier G, Collet AJ (1972) In vivo incorporation of choline-3H, leucine-3H and galactose-3H in alveolar type II pneumocytes in relation to surfactant synthesis. A quantitative radiographic study in mouse by electron microscopy. Anat Rec 174:289-310[Medline]
Cleves AE, McGee TP, Whitters EA, Champion KM, Aitken JR, Dowhan W, Goebl M, Bankaitis VA (1991) Mutations in the CDP-choline pathway for phospholipid biosynthesis bypass the requirement for an essential phospholipid transfer protein. Cell 64:789-800[Medline]
De Camilli P, Emr SD, McPherson PS, Novick P (1996) Phosphoinositides as regulators in membrane traffic. Science 271:1533-1539[Abstract]
Dierynck I, Bernard A, Roeis H, De-Ley M (1995) Potent inhibition of both human interferon-gamma production and biologic activity by the Clara cell protein CC16. Am J Respir Cell Mol Biol 12:205-210[Abstract]
Engle MJ, van Golde LMG, Wirtz KWA (1978) Transfer of phospholipids between subcellular fractions of the lung. FEBS Lett 86:277-281[Medline]
Funkhouser JD, Hughes ER (1983) The lung lamellar body as a functioning membrane in protein-catalyzed phosphatidylcholine transfer. Arch Biochem Biophys 221:449-506
Funkhouser JD, Read RJ (1985) Phospholipid transfer proteins from lung properties and possible physiological functions. Chem Phys Lipids 38:17-27[Medline]
Hay JC, Fisette PL, Jenkins GH, Fukami K, Takenawa T, Anderson RA, Martin TFJ (1995) ATP-dependent inositide phosphorylation required for Ca2+-activated secretion. Nature 374:173-177[Medline]
Hay JC, Martin TFJ (1993) Phosphatidylinositol transfer protein required for ATP-dependent priming of Ca2+-activated secretion. Nature 366:572-575[Medline]
Helmkamp GM, Jr, Harvey MS, Wirtz KWA, Van Deenen LLM (1974) Phospholipid exchange between membranes. Purification of bovine brain proteins that preferentially catalyze the transfer of phosphatidylinositol. J Biol Chem 249:6382-6389
Kauffmann-Zeh A, Thomas GMH, Ball A, Prosser S, Cunningham E, Cockcroft S, Hsuan JJ (1995) Requirement for phosphatidylinositol transfer protein in epidermal growth factor signaling. Science 268:1188-1190[Medline]
Liscovitch M, Cantley LC (1995) Signal transduction and membrane traffic: the PITP/phosphoinositide connection. Cell 81:659-662[Medline]
Lumb RH, Cottle DA, White LC, Hoyle SN, Pool GL, Brumley GW (1980) Lung phosphatidylcholine transfer in six vertebrate species correlations with surfactant parameters. Biochim Biophys Acta 620:317-321[Medline]
Mantile G, Miele L, Cordella-Miek E, Singh G, Katyal SL, Mukherjee AB (1993) Human Clara cell 10-kDa protein is the counterpart of rabbit uteroglobin. J Biol Chem 268:20343-20351
Massaro GD (1989) Nonciliated bronchial epithelial (Clara) cells. In Lung Cell Biology. Vol. 41. New York, Marcel Dekker, 81-114
Massaro GD, Paris M, Thet L (1979) In vivo regulation of secretion of bronchiolar Clara cells in rats. J Clin Invest 63:167-172[Medline]
Ohashi M, de Vries KJ, Frank R, Snoek G, Bankaitis V, Wirtz K, Huttner WB (1995) A role for phosphatidylinositol transfer protein in secretory vesicle formation. Nature 377:544-547[Medline]
Peao MND, Aguas AP, De Sa CM, Grande NR (1993) Anatomy of Clara cell secretion: surface changes observed by scanning electron microscopy. J Anat 183:377-388
Read RJ, Funkhouser JD (1983) Properties of a non-specific phospholipid-transfer protein purified from rat lung. Biochim Biophys Acta 752:118-126[Medline]
Singh G, Katyal SL (1984) An immunologic study of the secretory products of rat Clara cells. J Histochem Cytochem 32:49-54[Abstract]
Singh G, Katyal SL, Gottron SA (1985) Antigenic, molecular and functional heterogeneity of Clara cell secretory proteins in the rat. Biochim Biophys Acta 829:156-163[Medline]
Spalding JW, Hook GER (1979) Phospholipid exchange between subcellular organelles of rabbit lung. Lipids 14:606-613[Medline]
Thomas GMH, Cunningham E, Fensome A, Ball A, Totty NF, Truong O, Hsuan JJ, Cockcroft S (1993) An essential role for phosphatidylinositol transfer protein in phospholipase C-mediated inositol lipid signaling. Cell 74:919-928[Medline]
Tsao FHC (1980) Specific transfer of dipalmitoyl phosphatidylcholine in rabbit lung. Biochim Biophys Acta 601:415-426[Medline]
van Golde LMG, Batenburg JJ, Robertson B (1988) The pulmonarysurfactant system: biochemical aspects and functional significance. Physiol Rev 68:374-455
Wirtz KWA (1991) Phospholipid transfer proteins. Annu Rev Biochem 60:73-99[Medline]