ARTICLE |
Correspondence to: Leland G. Dobbs, U. of California San Francisco: Laurel Heights, 3333 California St., Suite 150, Box 1245, San Francisco, CA 94118.
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Summary |
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The alveolar epithelium is composed of two morphologically distinct types of cells, Type I and Type II cells. The thin cytoplasmic extensions of Type I cells cover more than 95% of the internal surface area of the lungs. Type I cells provide the very short diffusion pathway essential for gas exchange. Because there were no biochemical markers specific for human Type I cells, we developed a strategy to produce a monoclonal antibody (MAb) specific for human Type I cells. Isolated human lung cells were used as immunogens; >5000 clones from seven fusions were screened to identify an MAb specific for a 56-kD protein of Type I cells, HTI56. By Western blotting, HTI56 is unique to the lung. By immunoelectron microscopy, it is localized to the Type I cell apical plasma membrane. The pI of HTI56 is 2.53.5. HTI56 is glycosylated and has the biochemical characteristics of an integral membrane protein. HTI56 is detectable by Week 20 of gestation and its expression increases in fetal lung explant culture. HTI56 should be useful as a marker for human Type I cells both morphologically and biochemically. It may also be useful in studies of disease and as a marker for lung injury. (J Histochem Cytochem 47:129137, 1999)
Key Words: HTI56, type I cells, pulmonary, epithelium, lung, lung injury, integral membrane protein
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Introduction |
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IN MAMMALS, the development and maintenance of a normal alveolar epithelium are essential for life. The extremely large alveolar epithelial surface area of the human lung (100150 M2) is composed of only two morphologically distinct types of cells, Type I and Type II cells. These epithelial cells have been characterized morphologically by their location within the lung and by their unique ultrastructural characteristics. Type I cells are large, with calculated volumes of ~3000 µm3/cell and diameters of 50100 µm (
The lack of specific biochemical markers for Type I and Type II cells has impeded the study of alveolar epithelial development, differentiation, and the response to injury. Over the past several years, we have developed biochemical and molecular probes for integral membrane proteins specific to the apical plasma membrane of rat Type I or Type II cells. By producing monoclonal antibodies (MAbs) specific for rat Type I cells, we identified RTI40, a 4042-kD integral membrane protein specific in the lung for the apical plasma membrane of Type I cells (
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Materials and Methods |
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Isolation of Type I Cells and Screening of Hybridomas
Type I cells were partially purified from human lung tissue by the following method. First it was necessary to establish a protocol for identifying human Type I cells by techniques less time-consuming than transmission electron microscopy. We developed methods to identify freshly isolated Type I cells in cytocentrifuged preparations by light microscopy by comparing images of isolated Type I cells obtained by transmission electron microscopy, thick plastic sections at the light microscopic level, and cytocentrifuged preparations. After considerable cross-comparison of images, we were able to identify Type I cells in cytocentrifuged preparations.
We used human lung tissue removed during lobectomy or pneumonectomy. The protocol was approved by the Human Tissue Use Committee at the University of California, San Francisco. A distal airway was identified, cannulated, and the cannula sutured securely into place. The distal lung tissue was lavaged six times with Ca++,Mg++-free PBS containing 5 mM EDTA and 5 mM EGTA at 37C and then instilled with RPMI (Cell Culture Facility, UCSF) containing dextran 10% and elastase 80 U/ml (Worthington; Freehold, NJ). The use of dextran in this and in all subsequent solutions was important for the preservation of Type I cell integrity. We believe the use of dextran for this purpose to be innovative and new. The enzyme-instilled lungs were incubated at 37C for a total of 1 hr. Additional elastase solution was continuously instilled to keep the lung segment inflated. Lungs were minced to 1-mm3 fragments, shaken in a reciprocating water bath for 5 min, and then filtered sequentially through gauze (1 ply, 2 ply) and 150 µm nylon mesh (Tekto; Elmsford, NY). The resultant cell suspension was separated on a discontinuous Percoll (Pharmacia; Uppsala, Sweden) gradient (densities 1.020, 1.030, 1.035, and 1.040). By analyzing cytocentrifuged preparations of cells obtained from gradient fractions, we determined that the interface between densities of 1.035/1.040 contained the highest purity of Type I cells (>50%). Cells were centrifuged and the cell pellet (approximately 107 cells) was resuspended in Freund's adjuvant to immunize each mouse. Booster injections were administered at 3-week intervals. BALB/c mice were immunized according to the UCSF MAb protocol. Mice were boosted with cells on Days 14, 42, and 120. Spleens were removed on Day 123, and splenocytes were isolated and fused to NS-1 myeloma cells by conventional methods (
To screen rapidly propagating fusion clones, we developed a 96-well cytocentrifuge technique to screen multiple hybridoma supernatants simultaneously. Glass slides (3 x 4'') were precoated with a solution of 200 µg fibronectin/ml PBS, pH 7.4, to increase adherence of the cells. The slides were secured to a 96-well plexiglass manifold. Cells were diluted to a final concentration of 0.5 million cells/ml; 0.1 ml was placed in each well and the entire apparatus was centrifuged in an IEC centrifuge at 200 rpm at room temperature (RT) for 10 min. After this, the cytocentrifuged cells were allowed to partially air-dry and then were placed in 1% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) and stored at 4C for up to 3 weeks before use.
For screening hybridoma clones, 50 µl of supernatant liquid was placed on each dot of cytocentrifuged cells, washed, and FITCanti-mouse IgG (ICN Immunobiologicals; Costa Mesa, CA) was added to identify potential candidate hybridomas. These were then recloned three times to homogeneity, using the same screening methodology. Finally, hybridoma supernatants were tested with 2-µm-thick cryostat sections of human lung to verify that the supernatant was reactive only against Type I cells. Many clones were discarded because supernatants exhibited crossreactivity with other cell types in addition to Type I cells.
As a result of seven fusions and screening of approximately 5000 clones, one clone appeared to produce antibody positive only for Type I cells in the lung. MAb class and subclass were determined by Ouchterlony double-diffusion analysis (ICN).
Immunocytochemistry
Tissue for immunocytochemistry was fixed in a solution of freshly prepared 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4, cryoprotected by overnight submersion in 30% sucrose at 4C, then transferred to OCT (Tissue Tek; Miles, Elkhart, IN), and frozen in Freon 22 and liquid nitrogen. Cryosections 2 µm thick were picked up on Superfrost Plus slides (Fisher Scientific; Fair Lawn, NJ) and rings of rubber cement were applied around the sections to form wells to contain solutions. Tissue was sequentially immersed in the following solutions: 0.1% BSA/PBS + 0.3% Triton (BPT); 10% normal goat serum in BPT; primary MAbs in BPT + 10% goat serum overnight at 4C; BPT; goat anti-mouse IgG conjugated to FITC or rhodamine (affinity-purified; from Organon TeknikaCappel, Durham, NC) in BPT. Sections were briefly rinsed in distilled water and counterstained with 0.05% Pontamine Sky Blue (Sigma; St Louis, MO) in PBS with 10% DMSO for 40 min. Rubber cement wells were removed, tissue was briefly dried, and coverslips were mounted with DABCO (2.5% 1,4-diazobicyclo 2,2,2 octane, 10% PBS in glycerin, pH 8.6). Slides were examined and photographed using a Leitz Orthoplan microscope.
Cryoultramicrotomy
Ultrathin cryosections were cut at -100C from tissue blocks fixed in 4% paraformaldehyde in 0.1 M cacodylate buffer, pH 7.4, cryoprotected by overnight submersion in a 20% solution of polyvinylpyrrolidone in 2.3M sucrose (
Western Blotting
For one-dimensional Western blotting, organs were minced into 1-mm3 fragments and homogenized in nonreducing electrophoresis buffer (4% sodium dodecyl sulfate (SDS), 2 M urea, 20% glycerol in 5 mM Tris base, pH 8.0) with a polytron homogenizer (Brinkman; Lucerne, Switzerland) for 15 sec at Setting 6. The resulting homogenates were extracted for 1 hr and centrifuged at 20,000 x g for 30 min. The supernatant liquids were analyzed for protein (bicinchoninic acid method; Pierce, Rockford, IL). Aliquots containing 50 µg protein were electrophoresed on 10% polyacrylamide gels with 4% stacking gels containing SDS at 40 mA for 1 hr at 20C. Proteins were transferred onto nitrocellulose paper. HTI56 was detected according to the following protocol. MAb was biosynthetically labeled with [35S]-methionine according to the method of
Two-dimensional blots were used to analyze crude lung homogenate and purified HTI56. Lung tissue was processed by two different methods. For the blots with crude lung homogenate, tissue was frozen in liquid nitrogen, pulverized with a mortar and pestle, and then extracted at 90C for 15 min in 9 M urea, 2% NP-40, and 2% ampholines (310 pH; BioRad). The samples were microfuged for 10 min; 50 µl of the supernatant liquid was analyzed by 2D electrophoresis according to
For 2D analysis of purified protein, membranes were prepared from 100 g of lung tissue as follows. Tissue was homogenized in 5 mM Tris base, pH 8.0, containing 0.1% MEGA-8, 2 mM EDTA, 2 mM EGTA, 5 mM iodoacetamide, and 0.5 mM PMSF with a Polytron (Brinkman) at Setting 6 for 30 sec. The resultant lung homogenate was centrifuged at 1000 x g for 20 min at 4C. The supernatant liquid was pooled and centrifuged in a Beckman SW-28 rotor at 100,000 x g for 2 hr at 4C. Pellets were dissolved in an appropriate buffer containing 5% MEGA-8, 2% ampholines, 5% glycerol, and 9.5 M deionized urea and were loaded onto a preparative isoelectric focusing column (see below). Fractions containing HTI56 were pooled and dialyzed overnight against 2% SDS, 10% glycerol, and 50 mM Tris, pH 6.8, and then purified on a Sepharose-4Bwheat germ agglutinin (WGA) column (see below). Eluted protein was concentrated and dissolved in electrophoresis loading buffer (according to O'Farrell). Electrophoresis and Western blotting were performed as described above for the crude lung homogenate.
Liquid Phase Isoelectric Focusing
Approximately 25 g of human lung tissue was frozen in liquid nitrogen, pulverized, and extracted at 20C for 1 hr with 100 ml of a solution containing 9.5 M urea, 5% octylphenoxy ethanol (NP-40), 2 mM ethylenediaminetetraacetic acid (EDTA), 0.5 mM phenylmethysulfonyl fluoride (PMSF), 5 mM iodoacetamide, and 2 µg DNase I /ml (all reagents from Sigma). The lung homogenate was centrifuged at 100,000 x g for 2 hr. We initially performed isoelectric focusing with ampholines of pH 310. All of the antigenic activity was found very close to pH 3.0. To obtain a more precise pI, we used ampholines in the lower pH range. To 45 ml of supernatant liquid obtained from lung homogenate, we added 5 ml glycerol and ampholines (2.8 ml, pH 2.54.0, and 1.4 ml, pH 4.06.0; BioRad). The homogenate was loaded into a preparative isoelectric focusing chamber (Rotofor Cell; BioRad) and 12 W constant power was applied until the voltage stabilized (
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Organ Blots
Organs were obtained from autopsy material as approved by the Human Tissue Use Committee at UCSF and were immediately frozen in liquid nitrogen. Lung tissue was pulverized with a mortar and pestle and extracted in the same solution used for preparations of samples for 2D SDS-PAGE (see above). Protein was measured by the bicinchoninic acid method. In initial experiments, we determined that reduction of HTI56 caused loss of binding to MAb. For this reason, one-dimensional electrophoresis was performed in the following manner. Samples containing 20 µg protein were heated to 90C for 10 min in 5 mM Tris base, pH 8.0, containing 4% SDS, 2 M urea, and 20% glycerol, and were loaded into each lane. Proteins were separated by electrophoresis, transferred to nitrocellulose, and washed and treated with primary and secondary antibodies as described for 2D SDS-PAGE.
Glycosylation of HTI56
Glycosylation of HTI56 was assessed by neuraminidase treatment and binding of solubilized protein to immobilized WGA. For neuraminidase treatment, 1 mg of a membrane preparation of human lung tissue (see above) was dissolved in 50 mM acetic acid, pH 5.0, and incubated at 37C for 2 hr with or without neuraminidase (0.1 U) (Calbiochem; La Jolla, CA). After treatment, samples were analyzed by Western blotting as previously described.
HTI56 was partially purified on a lectin column with WGA. HTI56-containing fractions purified by liquid-phase isoelectric focusing were pooled and dialyzed overnight against 500 mM NaCl, 0.01% 4% octanoyl-N-methylglucamide (MEGA-8; Calbiochem), and 50 mM Tris, pH 7.2. Dialyzed protein was passed twice through a Sepharose 4B column (1 x 20 ml) and loaded onto a Sepharose 4BWGA column (1 x 5 ml) pre-equilibrated with dialysis buffer. Bound protein was washed with dialysis buffer until eluted protein absorbance A280 was <0.02. Bound protein was eluted with dialysis buffer containing 0.2 M N-acetylglucosamine. Eluted protein was concentrated and dissolved in electrophoresis loading buffer (
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Results |
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Localization of HTI56 to Lung
Figure 1 shows a photograph of a silver-stained SDS-PAGE gel of homogenates of various organs and an accompanying Western blot for HTI56. Only the lane containing homogenate of lung tissue exhibited immunoreactivity. No other organ tested was immunoreactive. These data demonstrate that, within the sensitivity of the Western blot assay, HTI56 is found only in lung tissue. By immunofluorescence performed with the same organs shown in the Western blot, only the lung contained specific immunofluorescence for HTI56 (data not shown). By Western blotting and immunofluorescence, rat lung and mouse lung were both negative for HTI56. Antigenicity of HTI56 is lost when the protein is reduced so that it is not possible to detect HTI56 in gels run under reducing conditions.
Localization of HTI56 to the Apical Plasma Membrane of Type I Cells
Indirect immunocytochemistry at the light microscopic level of lung tissue (Figure 2) demonstrates that neither airways nor blood vessels exhibited staining for HTI56. Alveolar staining was limited to the Type I cells; Type II cells and macrophages were negative (Figure 2 and Figure 3).
By EM immunocytochemistry with immunogold, the apical plasma membrane was immunoreactive (Figure 4). Cytoplasm and basolateral membranes were not decorated with immunogold.
Purification of HTI56 and 2D Gel and Western Blot of Lung Tissue
Two-dimensional gel electrophoresis of lung homogenate and an accompanying Western blot for HTI56 are shown in Figure 5. In the Western blot, HTI56 displayed a charge train spanning approximately 1 pH unit, suggesting post-translational modification.
Isoelectric Focusing
In initial isoelectric focusing using ampholines in the pH range 310, all of the antigenic activity was found close to pH 3.0, in fractions containing <3% of the loaded proteins. By liquid-phase isoelectric focusing (Figure 6), all of the antigenic activity was found in fractions 35. Accordingly, the pI of HTI56 is 2.53.5. Less than 2% of proteins have pIs in this range (
HTI56 Has the Biochemical Characteristics of an Integral Membrane Protein
HTI56 is poorly soluble in aqueous media or in buffers containing 2 M urea, 2 M NaCl, or 2% SDS, agents that are known to liberate peripheral membrane proteins (
Glycosylation of HTI56
Treatment with neuraminidase lowers the apparent MW (Figure 7) of HTI56 by approximately 5 kD but does not alter the antigenicity of the protein. This observation and the extent of the charge train observed in 2D electrophoresis supports the conclusion that HTI56 is glycosylated. The binding of HTI56 to WGA further suggests that HTI56 contains sialic acid and/or ß-D-N-acetylglucosamine (
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Expression of HTI56 occurs in the first trimester and increases when lung explants are maintained in culture. HTI56 was detectable by immunocytochemistry in a small number of cells lining potential airspaces in fetal tissue at approximately Weeks 18 and 20 of gestation. Culture of fetal lung explant tissue for 4 days induced diffuse expression of HTI56 in cells lining potential airspaces (Figure 8).
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Discussion |
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The lack of specific biochemical markers for Type I and Type II cells has impeded the study of alveolar epithelial development, differentiation, and the response to injury. Surfactant proteins, which are expressed in Type II cells but also in other cell types, have been used as markers for the Type II cell differentiated phenotype. There have been fewer markers for the Type I cell phenotype. Over the past several years, we and others have developed biochemical and molecular probes for plasma membrane markers of rat Type I cells (
To produce probes specific for human alveolar epithelial Type I cells, we employed a similar strategy to that previously successful for the production of cell-specific probes in rodent tissue, using isolated cells as immunogens to produce MAbs and then screening clones for cell and tissue specificity. After a screen of approximately 5000 clones (the result of seven immunizations and fusions), we were successful in identifying a single clone producing an MAb that reacts with a 56-kD lung-specific protein by Western blotting (Figure 1). By immunocytochemical evidence, this protein is localized to the alveolar region of the lung and is specific to the apical plasma membrane of Type I cells (Figure 2 Figure 3 Figure 4).
Although other markers for human Type I cells have been reported, none is as specific as HTI56 for Type I cells.
We partially purified HTI56 by affinity chromatography to WGA, suggesting that the protein is glycosylated and contains sialic acid and/or N-acetylglucosamine residues. Treatment with neuraminidase caused a decrease in apparent MW of approximately 5 kD, suggesting that the protein is sialylated.
Morphologically identifiable Type I and Type II cells develop late in gestation. In rodent lungs, Type II cells are recognizable at Day 18 (total gestation of 21 days) and Type I cells can be identified somewhat after this time. However, specific biochemical and molecular markers for Type I cells are detectable much earlier in gestation (
In this work, we have partially characterized HTI56, a glycosylated integral membrane protein of human alveolar epithelial Type I cells. It is likely that HTI56 will prove to be useful in studies of human lung development and in the characterization of clinical problems involving acute lung injury, in an analogous manner to RTI40 and rodent lung injury (
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Acknowledgments |
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Supported by NIH Grant HL 41958.
Received for publication October 5, 1998; accepted October 8, 1998.
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