Journal of Histochemistry and Cytochemistry, Vol. 45, 551-558, Copyright © 1997 by The Histochemical Society, Inc.


ARTICLE

Phosphatidylinositol Transfer Protein in Lung: Cellular and Subcellular Localization

Abdul-Manaf A. Ibrahima and Jane D. Funkhousera
a Department of Biochemistry and Molecular Biology, University of South Alabama, Mobile, Alabama

Correspondence to: Jane D. Funkhouser, Dept. of Biochemistry and Molecular Biology, Univ. of South Alabama, 307 University Blvd., Mobile, AL 36688.


  Summary
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

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


  Introduction
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

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 Wirtz 1991 ). The protein of interest in this study transfers both phosphatidylinositol and phosphatidylcholine. However, it is designated as a phosphatidylinositol transfer protein (PtdIns-TP) based on transfer of phosphatidylinositol to a greater extent than phosphatidylcholine. The physiological functions of PtdIns-TPs have not been defined. Identification of similar proteins in yeasts and analysis by genetic methods suggested functions related to protein secretion and phosphatidylinositol metabolism (Cleves et al. 1991 ; Bankaitis et al. 1989 , Bankaitis et al. 1990 ). These possibilities are now being examined in mammalian cell lines. In the PC12 neuroendocrine cell line, PtdIns-TPs have been shown to participate in the formation of secretory vesicles (Ohashi et al. 1995 ) and in the fusion of vesicles with the plasma membrane (Hay et al. 1995 ; Hay and Martin 1993 ). In other cell lines, they participate in signal transduction mechanisms involving phosphorylated products of phosphatidylinositol (Kauffmann-Zeh et al. 1995 ; Thomas et al. 1993 ).

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 (Funkhouser and Read 1985 ; Funkhouser and Hughes 1983 ; Lumb et al. 1980 ; Tsao 1980 ; Engle et al. 1978 ). These organelles represent intracellular stores of pulmonary surfactant and are highly enriched in phosphatidylcholine, particularly dipalmitoyl phosphatidylcholine (van Golde et al. 1988 ). The bulk of surfactant phosphatidylcholine is synthesized in the endoplasmic reticulum (van Golde et al. 1988 ), and autoradiographic experiments suggest that it is transferred via the Golgi system to lamellar bodies, perhaps by a process that involves small lamellar vesicles as intermediates (Chevalier and Collet 1972 ).

The PtdIns-TP protein is the major phospholipid transfer protein transferring phosphatidylcholine in lung (Read and Funkhouser 1983 ) and has been considered a candidate for participation in the phospholipid sorting and vesicular trafficking associated with formation and secretion of lamellar bodies. A significant correlation is observed between the amount of phosphatidylinositol choline transfer activity in lungs of vertebrate species and the alveolar surface area and amount of surface active material (Lumb et al. 1980 ). PtdIns-TP is present in the alveolar Type II cells that produce pulmonary surfactant, and levels of the protein increase about twofold during prenatal lung development. However, the alveolar Type II cells are not enriched in PtdIns-TP compared to whole lung tissue (Batenburg et al. 1994 ). This argues against a prominent role in lamellar body formation or secretion, as these processes represent major functions of the alveolar Type II cell (van Golde et al. 1988 ). This communication addresses the cellular localization of the PtdIns-TP in lung and identifies the Clara cell, a nonciliated secretory cell lining the bronchioles, as the pulmonary cell most enriched in PtdIns-TP.


  Materials and Methods
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

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 (Singh et al. 1985 ; Singh and Katyal 1984 ).

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 {alpha}-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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Figure 1. Western blot analysis of a lung protein extract with anti-PtdIns-TP IgG fraction. A rat lung protein extract (50 µg protein per lane) was resolved on a 10-15% gradient SDS polyacrylamide gel and diffusion-transferred to PVDF membrane. Lane 1 was analyzed with the anti-PtdIns-TP IgG fraction at 1:3000 titer. Lane 2 was analyzed with the same IgG fraction, except that it was preabsorbed using 175 µM immunizing peptide. The antibody binding was detected using the ECL Western blot analysis system. Molecular weight standards are indicated in kD.



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Figure 2. Immunohistochemical localization of phosphatidylinositol transfer protein in rat lung tissue sections. A purified IgG fraction from anti-PtdIns-TP serum was used for immunohistochemical staining of rat lung sections prepared and processed as described in Materials and Methods. The staining reaction produces a brown peroxidase product. (A) Low-power micrograph showing staining in the context of the structural features of the portion of the lung examined. Bar = 76 µm. (C) Higher magnification of the region indicated by the arrow in (A), illustrating an area of stained bronchiolar epithelium. Bar = 19 µm. (B) A field of alveolar cells from the same section, showing little or no staining. Bar = 15 µm. (D) Control section treated the same as the experimental sections except that the anti-PtdIns-PT IgG fraction was preabsorbed by 1.0 mM immunizing peptide. Bar = 15 µm.

Figure 3. Serial sections of rat lung stained with anti-PtdIns-TP IgG fraction and antiserum to Clara cell protein. Lung sections prepared as in Figure 2 were processed with anti-PtdIns-TP IgG fraction (A) or with antibody to Clara cell protein (B). The bronchiolar epithelium shows the most intense staining with both antibodies. The cellular pattern of staining in the bronchiolar epithelium is almost identical for both proteins, indicating that the cells staining intensely for PtdIns-TP are Clara cells. Bars = 19 µm.

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.


  Results
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

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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Figure 4. Ultrastructural localization of PtdIns-TP in bronchiolalr epithelium. (A) Clara cells and ciliated bronchial epithelial cells in the same field. Bar = 2 µm. Electron-dense immunoreactivity is restricted to the apical region of the Clara cells (closed arrows) and is not present in the ciliated cells (open arrows). (B) A nonimmune IgG control exhibiting no immunoreactivity in the apical regions of Clara cells (closed arrows). Bar = 2 µm. (C) A higher magnification showing the interface between an immunoreactive Clara cell and a nonlabeled ciliated bronchial epithelial cell. Bar = 0.3 µm. The dark staining reaction at the plasma membrane (closed arrow) distinguishes the Clara cell surface from the adjacent nonlabeled ciliated cell surface (open arrow). Note the electron-dense reaction product associated with the membrane enclosing the granules (large arrowheads) and background staining in the area surrounding granules (small arrowheads). (D) Nonimmune IgG control. Note absence of staining at the plasma membrane of the Clara cell (closed arrow) and ciliated cell (open arrow) and the lack of electron dense material associated with granules (arrowheads) or cytoplasm surrounding the granules. Bar = 0.3 µm.

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).


  Discussion
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

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 (Funkhouser and Hughes 1983 ; Lumb et al. 1980 ; Spalding and Hook 1979 ; Engle et al. 1978 ). If this were the case, more intense PtdIns-TP immunoreactivity would be expected in the alveolar Type II cells, which have a major role in biosynthesis and selective sorting of phospholipid into lamellar bodies. The apparent enrichment of the protein in Clara cells suggests that the PtdIns-TP participates in a process that is more prominent in the Clara cells than in other lung cells.

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 (Massaro et al. 1979 ). The mechanisms regulating CCSP secretion are not yet completely defined. Secretion is stimulated in whole-animal and isolated perfused lung models by isoproterenol, a ß-adrenergic agonist. In isolated perfused lung, secretion is stimulated by dibutyryl cAMP and by an increased ventilation volume. The dibutyryl cAMP stimulation is consistent with a ß-adrenergic receptor-mediated response, and the effect of ventilation volume is believed to be mediated by prostaglandins (Massaro 1989 ).

In Clara cells, two different general types of secretion are supported by ultrastructural data (Peao et al. 1993 , and references cited therein). One involves apocrine secretion by extrusion of the dome-shaped apical projections of cell cytoplasm containing secretory granules, and the other is a merocrine process involving exocytosis by fusion of granules with the plasma membrane. In the merocrine secretion, the general secretory pathway is likely to be that described for other eukaryotic cells. This involves budding of secretory vesicles from a source membrane and vectorial transport of the vesicles to the target membrane, where docking and fusion occur. An abundance of experimental evidence indicates that phosphoinositides are important in regulating this type of vesicular traffic (reviewed in De Camilli et al. 1996 ), and that PtdIns-TP is involved in phosphoinositide metabolism (Liscovitch and Cantley 1995 ).

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 (Ohashi et al. 1995 ) and in Ca2+-activated norepinephrine secretion (Hay et al. 1995 ; Hay and Martin 1993 ). The consistent observation of accumulation of PtdIns-TP at the outer membrane of secretory vesicles and at the plasma membrane (Figure 4) represents, to our knowledge, the first suggestive evidence of subcellular localization of PtdIns-TP in intact secretory cells, and is compatible with the proposed roles for PtdIns-TP in formation of vesicles and in exocytosis of secretory granules (De Camilli et al. 1996 ). However, more definitive studies must be undertaken to confirm this observation.

The mechanistic role played by the PtdIns-TP in vesicle trafficking is not yet clearly defined. A considerable amount of data (reviewed in Liscovitch and Cantley 1995 ) supports the participation of phosphatidylinositol kinases in regulating the steps involved in secretory processes, and PtdIns-TP may participate by presenting PtdIns directly to the different phosphoinositide kinases involved in trafficking or signal transduction (Liscovitch and Cantley 1995 ).

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 (Read and Funkhouser 1983 ; Helmkamp et al. 1974 ) and loss of as much as 85% of the protein from permeabilized cells (Hay et al. 1995 ). PtdIns-TP is known to form in vitro complexes with the membrane-associated phosphatidylinositol-4 kinase and with the epidermal growth factor (EGF) receptor from A431 human epidermoid carcinoma cells. In these cells, PtdIns-TP is required for EGF signaling involving phosphatidylinositol-4,5 bisphosphate. Clara cell membrane proteins have not been studied, and an association of PtdIns-TP with membrane-associated Clara cell proteins remains to be demonstrated.

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 (Dierynck et al. 1995 ; Mantile et al. 1993 ) make mechanisms regulating its secretion an important target for further research.


  Acknowledgments

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.


  Literature Cited
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

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