Identification of a novel antigen on the apical surface of rat alveolar epithelial type II and Clara cells

Gráinne M. Boylan1,2, James G. Pryde2, Leland G. Dobbs3, and Mary C. McElroy1,2

1 Department of Physiology, Trinity College, Dublin 2, Ireland; 2 Rayne Laboratory, Respiratory Medicine, University of Edinburgh, Edinburgh EH8 9AG, United Kingdom; and 3 Cardiovascular Research Institute, University of California, San Francisco, California 94118


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Here we describe a monoclonal antibody (MMC4) that recognizes a novel antigen on the apical surface of rat alveolar epithelial type II and Clara cells in the lung, proximal tubule epithelial cells in the kidney, and villus epithelial cells in the small intestine. Biochemical analysis showed that the MMC4 antigen was sensitive to heating and proteinase K digestion and that it is distributed in the detergent-rich phase after Triton X-114 phase separation. These data suggest that the MMC4 antigen is an integral membrane protein. Glycerol gradient sedimentation identified two forms of the MMC4 antigen: one with a sedimentation coefficient of 10.1 and one with a sedimentation coefficient of 1.66, suggesting that the antigen may be part of a multiprotein complex. During rat development (fetal day 16 to adult), the MMC4 antigen increased 12-fold in the lung and 200-fold in the kidney. In the intestine, the MMC4 antigen increased 150-fold by neonatal day 1 and then decreased to adult values. Our data demonstrate that the MMC4 antigen is unlike known type II cell- and Clara cell-associated proteins. The MMC4 monoclonal antibody will be useful as a marker of epithelial cell phenotype in development and injury studies.

kidney; intestine; development


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE ALVEOLAR AND BRONCHIOLAR REGIONS of the lung are composed of a number of morphologically distinct epithelial cell types. These cell types include type I and II cells in the alveolar region and Clara and ciliated cells in the terminal bronchiolar region. Alveolar epithelial type II and Clara cells are important for repair of the epithelium in response to injury (reviewed in Ref. 25). Alveolar epithelial type II cells proliferate and differentiate to form type I cells, whereas Clara cells proliferate and differentiate to form ciliated cells (25). However, it is also likely that other epithelial cell-repair relationships exist; for example, Clara cells may also be important for alveolar repair (9).

It has long been suspected that the degree of damage to a given lung epithelial cell type is dependent on the nature of the toxic insult. McElroy and colleagues (26, 27) have recently shown that alveolar epithelial type I cell injury can be quantified by measuring the level of RTI40 in bronchoalveolar lavage fluid. RTI40 is an integral membrane protein expressed on the apical surface of alveolar epithelial type I cells in rat lungs (10). In various rat models of acute lung injury, the amount of RTI40 recovered in bronchoalveolar lavage fluid was associated with the extent of morphological damage to alveolar epithelial type I cells (26, 27). However, the inability to detect and quantify damage to other epithelial cell types has hampered our understanding of how toxic agents damage the lung.

A number of integral membrane proteins are highly expressed on alveolar epithelial type II and Clara cells, including p172 (14), aminopeptidase N (13), alkaline phosphatase (12), and pneumocin (24). However, these proteins may not be suitable as biochemical markers of type II or Clara cell injury. Specifically, aminopeptidase N and alkaline phosphatase are also expressed on inflammatory cells (12, 15), whereas the potential of p172 and pneumocin to act as cell-specific markers of injury has not yet been evaluated in lung injury models. We have developed a monoclonal antibody (MAb) against the apical surface of rat alveolar epithelial type II and Clara cells, referred to as the MMC4 MAb.

As the first step toward developing a biochemical marker of type II and Clara cell injury, we characterized the MMC4 antigen. First, we determined the tissue distribution of the MMC4 antigen in adult rat tissues. Second, we determined whether the MMC4 antigen was a protein and, if so, whether it was a peripheral or integral membrane protein. Third, because we were unable to obtain a molecular weight by Western blotting, we determined the sedimentation coefficient (S20,w) of the MMC4 antigen by glycerol gradient centrifugation. Fourth, we determined whether the MMC4 antigen is regulated during development. Our data demonstrate that the MMC4 antigen is a novel integral membrane protein that is also expressed in the kidney and intestine. Our data suggest that the MMC4 antigen may be a useful marker of type II and Clara cell injury in models of lung injury.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Alveolar epithelial type II cell isolation. Type II cells were isolated from the lungs of male Sprague-Dawley specific pathogen-free rats (Charles River Laboratories, Irvine, CA) by previously described methods (10). Briefly, the lungs were digested by an intratracheal instillation of elastase (Boehringer Mannheim, Indianapolis, IN) followed by differential adherence of the cells on bacteriological plastic plates coated with rat immunoglobulin G (IgG; Sigma, St. Louis, MO). All animal procedures used in this study were approved by the University of California, San Francisco Animal Care Committee or under a license from the Department of Health (Ireland).

Immunization of mice. Female BALB/c mice (9 wk old) were obtained from Bantin Kingman (San Mateo, CA). Mice were anesthetized with pentobarbital sodium (45 mg/kg) and immunized by intrasplenic injection of 2 × 106 alveolar epithelial type II cells. Thirteen days later, mice were boosted with type II cells (1 × 106 cells) also injected directly into the spleen. Sera from immunized mice were tested 3 days later (day 15) on thin frozen lung sections for reactivity against alveolar type II cells; type II cells were identified as cuboidal cells containing inclusion bodies located in the corners of alveoli (8, 10). The spleen from the mouse showing the strongest staining against type II cells was used for the production of MAbs.

Production of MAbs. Spleen cells were fused with SP/0 cells (American Type Culture Collection) with polyethylene glycol 4000 following a standard technique (16). Supernatants from hybridomas were tested for reactivity against type II cells by indirect immunofluorescence on sections of frozen rat lung (2 µm thick). Positive hybridomas were recloned three times by serial dilution to ensure a single clone.

The isotype of MMC4 MAb was determined with an ISO-1 mouse MAb isotyping kit (Boehringer Mannheim).

Tissue fixation for thin frozen sections. Lungs from adult rats were fixed by intratracheal instillation of paraformaldehyde (4% wt/vol) prepared in phosphate-buffered saline (PBS). After 2 h of fixation, 2-mm3 pieces of lung were cryoprotected in 15% (wt/vol) sucrose for 18 h at 4°C. Blocks of lung tissue were frozen in liquid nitrogen-cooled Freon 22, and 2- or 5-µm lung sections were cut in a cryostat (Sorvall MT 6000).

Adult kidney and intestines were fixed with the same basic protocol as for lung tissue except for the following minor modifications. The kidneys were removed from the rats, sliced into thin sections, and then immersed in 4% (wt/vol) paraformaldehyde in PBS. For adult and neonatal intestines, the contents were removed and the lumen was filled with 4% (wt/vol) paraformaldehyde. The intestine was then immersed in 4% (wt/vol) paraformaldehyde and treated as for lung tissue. Fetal lungs and kidneys were removed and placed directly into 4% (wt/vol) paraformaldehyde before being processed as for adult lung tissue.

Indirect immunofluorescence. Tissue sections were thawed at 20°C, washed in PBS, and blocked for 30 min in PBS containing 10% (vol/vol) goat serum (GIBCO BRL, Life Technologies, Paisley, UK). Tissue sections were incubated with primary antibody (hybridoma supernatants or anti-RTI40 MAb) (10) for 30 min followed by FITC-conjugated goat anti-mouse Ig (Cappel, Organon Teknika, Durham, NC) for 30 min. In some experiments, FITC-conjugated anti-mouse IgG1 or rhodamine-conjugated anti-mouse IgG2a (Rockland Immunochemicals, Gilbertsville, PA) was used to specifically identify either the anti-RTI40 MAb or MMC4 MAb, respectively. Nuclei were stained with Hoechst DNA dye (100 ng/ml). Tissue sections were viewed with an Axiovert S100 fluorescence microscope. Images were either photographed or captured with Open Lab version 2.2 software.

Preparation of rat tissues for MMC4 screening. Adult rat tissues (lung, kidney, small and large intestines, spleen, serum, heart, skeletal muscle, stomach, testis, brain, eye, and liver) were analyzed for the presence of the MMC4 antigen. All tissues were homogenized in a Dounce homogenizer at a 10:1 (vol/wt) ratio in ice-cold 10 mM Tris · HCl (pH 8.2), containing 0.15 M NaCl, 2 mM EDTA, 2 mM EGTA, and 0.1 mM phenylmethylsulfonyl fluoride or Complete protease inhibitor cocktail (Boehringer Mannheim). Homogenized tissues were centrifuged at 10,000 average g (gav) for 1 min (Eppendorf Centrifuge), and the postnuclear supernatant (PNS) was frozen at -80°C for analysis at a later time point.

Protein assay. The protein concentration of all samples was determined with Bio-Rad reagent (Bio-Rad Laboratories). A standard curve was constructed with BSA (Pierce & Warriner, Chester, UK)

ELISA-based dot blot assay. The amount of the MMC4 antigen and RTI40 in different tissues was analyzed with an ELISA-based dot blot assay as previously described (27). Rat lung PNS was used to construct a standard curve (0.1-1.0 µg protein/well). Blots were incubated with either the MMC4 hybridoma supernatant or the anti-RTI40 MAb followed by peroxidase-conjugated anti-mouse IgG (Rockland Immunochemicals). Blots were developed by enhanced chemiluminescence (ECL reagents, Amersham Life Science) for 1-3 min. Test values are reported as relative densitometry units (RDU) per milligram of protein (27) or densitometry units per milligram of protein when all the test samples from one experiment were run on the same blot. The MMC4 antigen content of samples obtained after glycerol gradient sedimentation is expressed as densitometry units per milliliter.

Western blot analysis. Lung and kidney PNSs, membrane fractions, and plasma membrane fractions were solubilized in 62.5 mM Tris · HCl, pH 6.2, 10% (wt/vol) SDS, 20% (wt/vol) glycerol, and 0.02% (wt/vol) bromphenol blue, and the proteins were separated by SDS-PAGE gel electrophoresis (10-15%) (22) and electrophoretically transferred onto nitrocellulose membranes. RTI40 and the MMC4 antigen were probed with either the anti-RTI40 MAb or MMC4 MAb as described for the dot blot assay (27).

Proteinase K treatment. Rat lung and kidney PNSs were incubated with 10 mg/ml of proteinase K (Boehringer Mannheim) in 50 mM HEPES, pH 7.4, containing 0.15 M NaCl for 24 h at 56°C. Proteinase K solution was preincubated at 37°C to remove any residual glycosidase activity before tissue digestion. The extent of MMC4 MAb binding to proteinase K-digested lung and kidney PNSs was assessed by dot blot analysis. The sensitivity of rat lung RTI40 to proteinase K digestion was run as a positive control.

MMC4 antigen detergent solubilization studies. To determine whether the MMC4 antigen could be solubilized without loss of MMC4 MAb binding, lung and kidney PNSs were solubilized at 0°C for 30 min in either SDS, C12E8 (octaethyleneglycol mono-n-dodecyl ether; Calbiochem, CN Biosciences, Nottingham, UK), Triton X-114, or beta -octylglucoside (Calbiochem). Lung and kidney PNSs were solubilized at a 10:1, 5:1, or 1:1 detergent-to-protein ratio. Detergent-insoluble material was separated by centrifugation at 541,000 maximum g (gmax) for 10 min in a TL100.3 rotor (Beckman Optima ultracentrifuge). The extent of MMC4 MAb binding to the detergent-insoluble pellet and supernatant fractions was compared with that in nontreated PNSs.

Sodium carbonate wash. Lung and kidney membrane fractions were obtained by centrifugation of PNSs at 541,000 gmax for 10 min at 4°C. The membrane pellet was retained and washed three times with 0.1 M sodium carbonate solution, pH 11 (19), or 0.15 M NaCl. The washed membrane pellet was resuspended in 10 mM Tris · HCl, pH 8.2, with 0.15 M NaCl [Tris-buffered saline (TBS)], and the amount of MMC4 antigen recovered in the washed membranes was determined by dot blot analysis.

Triton X-114 phase separation. Triton X-114 was precondensed as described by Bordier (6) and used as a 10% (wt/vol) stock solution. Kidney PNS was solubilized in Triton X-114 (detergent-to-protein ratio of 10:1) for 30 min on ice. The Triton X-114-solubilized PNS was then centrifuged at 541,000 gmax for 10 min, and the supernatant was retained. The supernatant was warmed to 30°C for 5 min, and the detergent-rich phase, which forms at the cloud point of Triton X-114, was separated by centrifugation (28). The amount of MMC4 antigen was determined by dot blot analysis in each fraction, and the specific activity and the total amount of MMC4 recovered were calculated.

Preparation of rat kidney plasma membranes. Differential centrifugation through Dextran 6000 was used to obtain a kidney microsomal fraction (3). Adult rat kidney PNS (12 ml) was first centrifuged at 26,500 gav for 20 min to yield a postmitochondrial supernatant. The postmitochondrial supernatant was then centrifuged at 541,000 gmax for 10 min to yield a microsomal pellet. The microsomal pellet was resuspended in 10 mM Tris · HCl, pH 8.6, containing 5 mM magnesium sulfate and 0.1 mM phenylmethylsulfonyl fluoride with a glass Dounce homogenizer and dialyzed for 18 h against the Tris-Mg2+ buffer. The dialyzed microsomal fraction was layered onto a 20% (wt/vol) dextran step and centrifuged at 541,000 gmax for 15 min. The plasma membrane fraction at the Tris-Mg2+-dextran interface was collected and solubilized in TBS containing C12E8 (0.01% wt/vol).

Glycerol gradient estimation of MMC4 antigen sedimentation coefficient. Glycerol density gradient sedimentation was performed at 4°C on a 2.5-ml linear gradient of 8-35% (wt/vol) glycerol in TBS containing C12E8 (0.01% wt/vol) (1). Kidney plasma membrane [80 µg of protein in 0.05 ml of TBS containing 0.01% (wt/vol) C12E8] was layered onto the glycerol gradient and centrifuged for 12 h at 166,000 gav (Beckman type TLS 55 swinging bucket rotor).

The total amount of protein and the amount of MMC4 antigen were determined in fractions collected from the gradient. To determine the S20,w of the MMC4 antigen, proteins with known S20,w values (i.e., catalase 11.7, gamma -globulin 7.3, BSA 4.4, and carbonic anhydrase 3.3) (32) were used to calibrate the gradient.

Developmental expression of the MMC4 antigen. The amount of the MMC4 antigen in lung, kidney, and intestine was measured in fetal (days 16, 19, and 21) and neonatal (days 1, 2, 5, and 8) rats. Timed-pregnant, Sprague-Dawley rats (n = 3/time point) were obtained from University College (Dublin, Ireland) ~1 wk before giving birth. Fetal day 0 was defined as the day a vaginal plug was obtained. Pregnant dams and neonatal rats were anesthetized with pentobarbital sodium (45 mg/kg body wt). Fetuses were obtained by laparotomy. Fetal tissues from one mother were pooled. All tissues were stored at -80°C for analysis at a later point. Data are expressed as relative densitometry units per milligram of protein.

Statistics. Data are expressed as means ± SE. Comparison between samples was analyzed with one-way ANOVA, with Student-Newman-Keuls posttest analysis. P < 0.05 was considered to be significant. Tests were performed with GraphPad InsStat version 3.00 for Windows 96.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Immunofluorescence location of the MMC4 antigen in adult lung tissue. The MMC4 MAb (isotype IgG2a) bound the apical surface of epithelial cells in the corners of alveoli (Figs. 1A and 2A). These epithelial cells were judged to be type II cells by the presence of lamellar bodies (10) and from their location between alveolar epithelial type I cells (Figs. 1A and 2A) (8). The MMC4 MAb also bound bronchiolar epithelial cells in the airways (Figs. 1A and 2C). These airway epithelial cells were judged to be Clara cells because they had a protruding apical membrane and no cilia (Fig. 2, C and D) (31). The MMC4 MAb did not stain type I cells, macrophages, blood vessels, ciliated cells, or cells beneath the surface bronchiolar epithelial layer (Figs. 1C and 2C).


View larger version (110K):
[in this window]
[in a new window]
 
Fig. 1.   Immunofluorescence localization of the MMC4 monoclonal antibody (MAb) in adult rat lung. Lung tissues were incubated with both the MMC4 MAb (red) and the anti-RTI40 MAb (green) followed by isotype-specific secondary antibodies. Nuclei were stained with Hoechst DNA dye (blue). A: MMC4-positive cells were located in the corners of alveoli (red; arrows) between RTI40-positive cells (green; type I cells). MMC4-positive cells were also located on the surface of the bronchiolar epithelium (Br; red; arrows). Cells within the alveoli (*) and blood vessels (BV) did not bind the MMC4 MAb. A, airspaces. B: corresponding phase-contrast image. Original magnification, ×100.



View larger version (101K):
[in this window]
[in a new window]
 
Fig. 2.   High-magnification view of the immunofluorescence localization of the MMC4 MAb in adult rat lung. A: MMC4 MAb bound to the apical surface of cuboidal epithelial cells (red; arrow) located between RTI40-positive cells (green; type I cells). B: corresponding phase-contrast image. C: MMC4 MAb also bound to the apical surface of nonciliated bronchiolar epithelial cells (red; arrows). D: corresponding phase-contrast image demonstrating ciliated cells (*). These data are consistent with the localization of the MMC4 antigen on the apical surface of both alveolar epithelial type II and Clara cells. Original magnification, ×1,000.

The MMC4 antigen is present in the kidney and small intestine. Relative to lung, the MMC4 antigen was highly expressed in the kidney (14,079 ± 3,317 vs. 937 ± 271 RDU/mg protein for lung). Immunofluorescence microscopy on thin frozen sections showed that the MMC4 MAb bound the apical surface of selective tubules in the kidney cortex (Fig. 3, A and B). The MMC4-positive tubules were determined to be proximal tubules by the presence of a brush border at the apical surface of tubule epithelial cells (7). The MMC4 MAb did not stain distal or conducting tubules of the cortex or glomeruli (Fig. 3, A and B).


View larger version (106K):
[in this window]
[in a new window]
 
Fig. 3.   Immunofluorescence localization of the MMC4 MAb in adult rat kidney and small intestine. Kidney and small intestine sections were stained with the MMC4 MAb (red or green). Nuclei were stained with Hoechst DNA dye (blue). A: MMC4 MAb bound to selected tubules in rat kidney cortex (C; red; *) but did not bind tubules in the medulla region (M). B: higher-magnification view of rat kidney demonstrating that the MMC4 MAb bound to the apical surface of epithelial cells of proximal tubules in the kidney cortex (green; corresponding phase-contrast image not shown); the adjacent glomerulus (G) was negative. C: MMC4 MAb bound to epithelial cells along the tip and middle regions of villi in the small intestine (red; short arrows). Epithelial cells lining the crypts (long arrows) and other regions of the villi were negative. D: higher-magnification view of a villus demonstrating that the MMC4 MAb bound the apical surface of the epithelial cells (green; long arrows). MMC4 MAb fluorescence stopped abruptly at the tip of the villus (short arrows) at the location of a goblet cell (corresponding phase-contrast image not shown). Cells inside the villus [i.e., lamina propria (LP)] were negative. Original magnifications: ×100 in A and C; ×500 in B and D.

The MMC4 antigen was also highly expressed in the small intestine (1,628 ± 271 RDU/mg protein in the small intestine vs. 937 ± 271 RDU/mg protein in the lung). Immunofluorescence analysis showed that the MMC4 MAb stained the apical surface of intestinal villi epithelial cells (Fig. 3, C and D). The MMC4 MAb did not stain goblet cells, inside the lumen of the villi (i.e., lamina propria), or crypt cells (i.e., stem cells, undifferentiated epithelial cells, goblet cells, or Paneth cells; Fig. 3, C and D). Along different regions of the small intestine (i.e., ileum, jejunum, and duodenum), the MMC4 antigen was expressed uniformly (data not shown) and with the same immunofluorescence distribution (i.e., tip and middle epithelial cells of the villi; Fig. 3C).

Although by immunofluorescence analysis, no specific MMC4 staining was detectable in the brain or the eye, by dot blot analysis, the MMC4 antigen was present at very low levels in the brain, eye, and stomach (i.e., 20-45 RDU/mg protein). The MMC4 antigen was not detectable by dot blot analysis in the spleen, liver, heart, testis, trachea, or serum.

The MMC4 antigen is a protein. Heating lung and kidney tissue extracts (PNS) for 18 h at 56°C significantly decreased the extent of MMC4 MAb binding as assessed by dot blot analysis (Table 1). The addition of proteinase K reduced the MAb binding to almost zero (Table 1). The sensitivity of the MMC4 antigen to both heat denaturation and proteolysis suggests that it is a protein.

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Effect of temperature and proteinase K treatment on MMC4 MAb binding to lung and kidney

The MMC4 antigen was not detectable by Western blot analysis. It was not possible to obtain a molecular weight for the MMC4 antigen by SDS-PAGE and Western blotting under nonreducing or reducing conditions. Specifically, we did not detect a signal with lung, kidney, or intestine PNSs, kidney plasma membrane fractions, or dot blot-positive fractions obtained after glycerol gradient sedimentation (data not shown). These data suggest the MMC4 MAb does not recognize the SDS-solubilized antigen.

Detergent solubilization of the MMC4 antigen. Detergent solubilization was required to remove the MMC4 antigen from lung and kidney membrane fractions (541,000 gmax pellet from PNS). Nonionic detergents such as Triton X-114 and C12E8 solubilized the MMC4 antigen without loss of MMC4 MAb binding as assayed by dot blot analysis under nondenaturing conditions. However, exposure to anionic detergents such as SDS interfered with the binding of the MMC4 MAb (i.e., the amount of MMC4 binding in kidney PNS was reduced by 80% at a SDS detergent-to-protein ratio of 5:1).

MMC4 antigen is an integral membrane protein. Most of the MMC4 antigen (85-90%) was detected in the insoluble fraction after high-speed centrifugation (541,000 gmax) of lung and kidney PNSs. Moreover, when lung and kidney membrane fractions were subsequently washed with sodium carbonate to remove peripheral membrane proteins and membrane-adherent soluble proteins, the MMC4 antigen specific activity was not reduced. In addition, the MMC4 antigen partitioned in the detergent-rich phase after Triton X-114 phase separation (Table 2). Both the behavior of the MMC4 antigen after a sodium carbonate wash and phase separation into Triton X-114 suggests that the antigen is an integral membrane protein (6, 28).

                              
View this table:
[in this window]
[in a new window]
 
Table 2.   Distribution of MMC4 antigen after temperature-induced Triton X-114 phase separation

Sedimentation analysis of the MMC4 antigen. The S20,w of the C12E8-solubilized MMC4 antigen was determined by glycerol gradient sedimentation (1). Plasma membrane fractions from kidney and lung PNSs were isolated over a step gradient of Dextran 6000 (3). The MMC4 specific activity of plasma membrane fractions was typically four- to fivefold greater than the specific activity of PNSs.

Plasma membrane fractions were solubilized with C12E8 and layered onto glycerol gradients to separate the proteins. After centrifugation, the MMC4 antigen was detected by dot blot analysis in fractions 1-4 at the bottom of the gradient (Fig. 4A). When the gradients were calibrated with standard proteins, the MMC4 antigen had an S20,w value of 10.1. To determine whether the S20,w 10.1 form of the MMC4 antigen was associated noncovalently with other proteins, we treated fraction 2 with 4 M urea to disrupt any noncovalent bonds. Urea treatment did not reduce the extent of the MMC4 MAb binding compared with that in untreated control samples (data not shown). Moreover, when the urea-treated samples were recentrifuged over a glycerol gradient, the MMC4 antigen was recovered in fractions 18 and 19 at the top of the gradient (Fig. 4, B and C). The MMC4 antigen in fractions 18 and 19 had an S20,w value of 1.66. These data suggest that the MMC4 antigen exists as part of a protein complex that is held together by noncovalent bonds.


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 4.   Native molecular weight determination of the MMC4 antigen by glycerol gradient sedimentation. A: C12E8 (octaethyleneglycol mono-n-dodecyl ether)-solubilized MMC4 antigen (from kidney plasma membranes) sedimented predominantly in the denser fractions (i.e., fractions 1-4) after glycerol gradient sedimentation. Arrows, locations of standard proteins. C, catalase; GG, gamma -globulin; BSA, bovine serum albumin; CA, carbonic anhydrase. B: resedimentation of an MMC4-positive glycerol gradient fraction (fraction 2; Fig. 3A). The MMC4 antigen is predominantly located in the denser glycerol gradient fractions (i.e., fractions 1 and 2). C: resedimentation of an MMC4-positive glycerol gradient fraction after urea treatment (fraction 2). In contrast to Fig. 3B, the MMC4 antigen is now located in the least dense fractions of the gradient (i.e., fractions 18 and 19). DU, densitometry units.

Developmental expression of the MMC4 antigen. The amount of the MMC4 antigen per milligram of PNS protein increased 12-fold during lung development (Fig. 5A). Specifically, the amount of MMC4 antigen per milligram of protein increased 3.5-fold from fetal day 16 to postgestational day 1 (P < 0.05) and 3.3-fold from postgestational day 1 to adult values (P < 0.05; Fig. 5A). RTI40, a marker of alveolar epithelial maturation (33, 34), also increased 12-fold between gestational day 16 and adult values (P < 0.01; Fig. 5B). In fetal day 21 lung sections, MMC4 MAb bound the apical surface of single cells in tubules that were lined predominately with RTI40-positive epithelial cells (Fig. 6A). In larger tubules, MMC4-positive cells were flanked by both RTI40-positive and -negative cells (Fig. 6B). On fetal day 21, the MMC4 antigen and RTI40 did not to colocalize on the same epithelial cells.


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 5.   Expression of the MMC4 antigen during development of the lung, kidney, and intestine. A: lung MMC4. B: lung RTI40. C: kidney MMC4. D: intestine MMC4. The amount of RTI40 in lung tissue was measured as a marker of alveolar maturation. RDU, relative densitometry units; 16, 19, and 21, fetal days; 1, 5, and 8, postgestational days. Values are means ± SE; n = 3 experiments.



View larger version (120K):
[in this window]
[in a new window]
 
Fig. 6.   MMC4 MAb binding in fetal day 21 lung, kidney, and intestine. A: MMC4-positive cells (red; short arrow) in tubule with many RTI40-positive cells (green; long arrows). B: sets of MMC4-positive epithelial cells (red; short arrow) were located in larger tubules that also contain sets of RTI40-positive epithelial cells (green; long arrow). Some epithelial cells did not appear to express either the MMC4 antigen or RTI40 (*). As with the adult lung, MMC4 MAb was restricted to the apical surface of lung epithelial cells. C: MMC4 MAb bound to apical surface of epithelial cells in selected fetal kidney tubules (green; *). D: MMC4 bound to apical surface of fetal epithelial cells in intestinal villi (green; arrows). Original magnifications: ×630 in A and B; ×300 in C; ×500 in D.

During rat kidney development, there was an incremental accumulation of the MMC4 antigen per milligram of PNS protein (Fig. 5C). The amount of the MMC4 antigen increased 11-fold between fetal days 16 and 19 (P < 0.01), 1.7-fold between fetal day 19 and postgestational day 5, and 11-fold between postgestational day 5 and adult values (P < 0.05). Immunofluorescence localization of the MMC4 antigen on fetal day 21 revealed that the MMC4 antigen stained the apical surface of epithelial cells within selected tubules in the kidney cortex (Fig. 6C).

During development of the rat intestine, the amount of MMC4 antigen increased 20-fold between gestational days 16 and 19 (P < 0.01) and by a further 7.5-fold between fetal day 19 and postgestational day 1. However, the amount of the MMC4 antigen decreased fivefold (P < 0.05) between postgestational day 1 and adult values (Fig. 5D). As determined by immunofluorescence localization, the MMC4 antigen was located on the apical surface of intestinal epithelial cells (Fig. 6D). The pattern of MMC4 MAb binding in fetal day 21 intestine was very similar to staining in the adult small intestine (Fig. 3, C and D).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The ability to detect and quantify the extent of injury to distinct cells of the alveolar wall would greatly improve our understanding of lung damage induced by different toxic agents. McElroy and colleagues (26, 27) have previously demonstrated that an integral membrane protein, RTI40, can be used to measure type I cell damage. Membrane proteins associated with alveolar type II cells either have not yet been evaluated for their use as biochemical markers of epithelial damage or are unsuitable because they are also expressed on inflammatory cells (12, 15). We have developed a MAb (MMC4) against the apical surface of rat alveolar epithelial type II and Clara cells. The overall objective of our study was to characterize the MMC4 antigen to determine whether it might be a potential marker of cell-selective damage.

The MMC4 antigen is an integral membrane protein. The MMC4 antigen was determined to be a membrane protein by both immunofluorescence and biochemical criteria. The MMC4 MAb bound the apical surface of selective cells in the lung, kidney, and small intestine by immunofluorescence detection on frozen tissue sections (Figs. 1-3). The MMC4 antigen was recovered in the insoluble membrane fraction after a high-speed centrifugation of kidney and lung PNSs. In addition, the specific activity of the MMC4 antigen was concentrated in isolated plasma membranes. The MMC4 antigen was determined to be a protein by denaturation during heat treatment and by susceptibility to protease digestion (Table 1). The epitope recognized by the MMC4 antibody was solubilized in the presence of nonionic detergents such as C12E8 and Triton X-114. The integral membrane nature of the MMC4 antigen was shown by its resistance to removal from membrane fractions by washing with sodium carbonate (19) and by its partitioning into a detergent-rich phase after solubilization in Triton X-114 and phase separation (Table 2).

Several other proteins that are located on the apical plasma membrane of alveolar epithelial type II and Clara cells, for example, aminopeptidase N (13), p172 (14), alkaline phosphatase (12) and pneumocin (24), have been identified. However, the MMC4 antigen may be a novel protein based on its tissue distribution and its solubility in Triton X-114. The MMC4 antigen is not detectable in the adult rat liver, but both aminopeptidase N and pneumocin are present in the liver (24, 29). The MMC4 antigen is expressed in both the kidney and intestine, whereas p172 is not detectable in either of these tissues (14). Alkaline phosphatase, like the MMC4 antigen, is also located in the kidney and small intestine (reviewed in Ref. 15). However, after Triton X-114 solubilization, alkaline phosphatase is recovered in the insoluble pellet (18). In contrast, the MMC4 antigen partitions into a detergent-rich phase after Triton X-114 phase separation (Table 2). Insolubility in Triton X-114 is a common feature of glycosylphosphatidylinositol membrane-anchored proteins such as alkaline phosphatase (18). Therefore, our data also suggest that the MMC4 antigen is not a glycosylphosphatidylinositol-anchored membrane protein.

Unfortunately, we were not able to determine a molecular weight for the MMC4 antigen by SDS-PAGE and Western blotting. However, the MMC4 antigen was solubilized in a nonionic detergent as part of a protein complex (S20,w 10.1) when analyzed by glycerol gradient sedimentation (Fig. 4). Treatment of the MMC4 S20,w 10.1 complex with urea disrupted any noncovalent interactions and produced a smaller form of the MMC4 antigen that had an S20,w value of 1.66 (Fig. 4). Hydrolytic enzymes, located in the brush border of kidney and intestinal tissues, are mostly dimeric proteins held together by noncovalent bonds (21). Our sedimentation data suggest the MMC4 antigen is part of a protein complex, and, in common with other brush border integral membrane proteins (21), disulfide bonds are not required to maintain the complex. However, we do not know whether the MMC4 antigen exists as a multimeric or a heteromeric protein complex.

The MMC4 antigen is developmentally regulated. Many proteins associated with the apical surfaces of epithelial cells are developmentally regulated (2, 4, 14, 20, 30, 33-35). Our study demonstrates that the concentration of lung MMC4 antigen (in RDU/mg protein) increases during fetal and postnatal development (Fig. 5A). The MMC4 antigen was first detected in rat lung tissues on fetal day 16, which is before the morphological maturation of both alveolar epithelial type II and Clara cells (17). Other lung cell-selective proteins, such as RTI40 and aminopeptidase N, are also detected early in development (20, 35). In addition, a number of these cell-selective proteins are coexpressed on the same epithelial cell, suggesting that a common progenitor cell gives rise to different epithelial cell types (35). We did not investigate whether RTI40 and the MMC4 antigen were coexpressed in fetal day 16 lungs. However, by fetal day 21, a point at which type II and I cells are distinguishable from each other by electron-microscopic analysis (17), the MMC4 antigen and RTI40 are expressed on different epithelial cells (Fig. 6, A and B).

Most known type II- and Clara cell-associated proteins are developmentally regulated e.g., surfactant proteins A and B (30), aminopeptidase N (20), alkaline phosphatase (11), p172 (14), and pneumocin (20). The rate at which the MMC4 antigen accumulates during lung development is distinct from surfactant protein A, p172, and alkaline phosphatase (11, 14, 30). Both lung surfactant protein A and p172 are not strongly expressed until fetal day 19 when there is a dramatic increase in both proteins before fetal day 22 (birth) (30, 14). The increased expression of surfactant protein A and p172 is associated with the differentiation of alveolar epithelial type II cells (17, 30). Fetal lung alkaline phosphatase activity also increases rapidly before birth but then decreases during the neonatal period to adult values (11). The developmental expression of the MMC4 antigen in lung tissue most closely mirrors the developmental expression of RTI40 and aminopeptidase N (Fig. 5B) (20). The MMC4 antigen, RTI40, and aminopeptidase N are all detectable on or before fetal day 16 (20, 22, 35). In addition, the concentration of each of these proteins in lung tissue increases ~12-fold between fetal day 16 and adult values (Fig. 5, A and B) (20, 35).

During kidney development, the amount of MMC4 antigen increased dramatically at two different stages (Fig. 5C). The first stage was between fetal days 16 and 19, and the second stage was between postnatal day 5 and adult values. The first phase may be associated with a large (27-fold) expansion of the tubular compartment of the developing kidney (5). The second stage may be associated with an increase in the surface area of the brush border epithelial cells of the proximal tubules (23, 33). Like the MMC4 antigen, the specific activities of kidney brush border enzymes such as alkaline phosphatase and aminopeptidase N also increase rapidly after birth (33). However, unlike the MMC4 antigen, neither alkaline phosphatase nor aminopeptidase N are detected before fetal day 18 in the rat kidney (33).

In contrast to lung and kidney development, intestinal MMC4 antigen expression increased during fetal life, then decreased shortly after birth to adult values (Fig. 5D). Similar patterns of expression have been reported for known intestinal brush border enzymes (e.g., alkaline phosphatase and aminopeptidase N) (2, 4). The peak of MMC4 expression on neonatal day 1 (Fig. 5D) is consistent with morphological data showing maturation of rat intestinal villi on fetal day 21 (11). Specifically, the villi are long and cylindrical and the epithelial cells are columnar with microvilli (11). As in lung and kidney development, the MMC4 antigen was detected early in gestation before morphological maturation of the intestinal villus epithelial cells (2, 11).

In summary, we have described a new MAb that recognizes a novel integral membrane protein located on the apical surface of selective epithelial cells in the lung, kidney, and small intestine. We have also demonstrated that the MMC4 antigen is developmentally regulated and that expression of the MMC4 antigen is restricted to specific epithelial cell types on fetal day 21 in lung, kidney, and intestinal tissues. The MMC4 MAb will be a useful tool for analyzing epithelial cell phenotype in lung injury and developmental studies. Furthermore, like RTI40, we expect that the MMC4 antigen might be a useful biochemical marker of cell-selective damage in models of lung injury.


    ACKNOWLEDGEMENTS

We thank Profs. Christopher Haslett and Christopher Bell for support; Maria Maglio and Hadas Millo for excellent technical assistance; Mark Lawson for help with preparation of the immunofluorescent images; Ian Dransfield, Adriano Rossi, Graham Thomas, and Joseph Gray for comments on the manuscript; and Kirsty Tyrrell for proofreading the manuscript.


    FOOTNOTES

This work was funded by the California Lung Association, the Health Research Board (Ireland), and the Medical Research Council.

Address for reprint requests and other correspondence: M. C. McElroy, Rayne Laboratory, Univ. of Edinburgh, Teviot Place, Edinburgh EH8 9AG, UK (E-mail: mmcelroy{at}ed.ac.uk).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 16 October 2000; accepted in final form 8 January 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Apps, DK, Pryde JG, and Sutton R. Characterization of a detergent-solubilized adenosine triphosphatase of chromaffin granular membranes. Neuroscience 9: 687-700, 1983[ISI][Medline].

2.   Auricchio, D, Stellato A, and de Vizia B. Development of brush border peptidases in human and rat small intestine during fetal and neonatal life. Pediatr Res 15: 991-995, 1981[Abstract].

3.   Avruch, J, and Wallach DFH Preparation and properties of plasma membrane and endoplasmic reticulum fragments from isolated rat fat cells. Biochim Biophys Acta 233: 2334-2347, 1971.

4.   Bailey, DS, Cook A, McAllister G, Moss M, and Mian N. Structural and biochemical differentiation of the mammalian small intestine during foetal development. J Cell Sci 72: 195-212, 1984[Abstract].

5.   Bertram, JF, Young RJ, Spencer K, and Gordon I. Quantitative analysis of the developing rat kidney: absolute and relative volumes and growth curves. Anat Rec 258: 128-135, 2000[ISI][Medline].

6.   Bordier, C. Phase separation of integral membrane proteins in Triton X-114 solution. J Biol Chem 256: 1604-1607, 1981[Abstract/Free Full Text].

7.   Boylan, GM. Development and Characterization of Monoclonal Antibodies Against Rat and Human Alveolar Epithelial Cells (PhD thesis). Dublin, Ireland: Trinity College, 1999.

8.   Brooks, RE. Concerning the nomenclature of the cellular elements in respiratory tissue. Am Rev Respir Dis 94: 112-113, 1966[ISI].

9.   Daly, HE, Baecher-Allan CM, Paxhia AT, Ryan RM, Barth RK, and Finkelstein JN. Cell-specific gene expression reveals changes in epithelial cell populations after bleomycin treatment. Lab Invest 78: 393-400, 1998[ISI][Medline].

10.   Dobbs, LG, Williams MC, and Gonzalez R. Monoclonal antibodies specific to the apical surfaces of rat alveolar type I cells bind to surfaces of cultured, but not freshly isolated, type II cells. Biochim Biophys Acta 970: 146-156, 1988[ISI][Medline].

11.   Dunn, JS. The fine structure of the absorptive epithelial cells of the developing small intestine of the rat. J Anat 101: 57-68, 1967[ISI][Medline].

12.   Edelson, JD, Shannon JM, and Mason RJ. Alkaline phosphatase: a marker of alveolar type II cell differentiation. Am Rev Respir Dis 138: 1268-1275, 1988[ISI][Medline].

13.   Funkhouser, JD, Tangada SD, Jones M, O S-J, and Peterson RD. p146 Type II alveolar epithelial cell antigen is identical to aminopeptidase N. Am J Physiol Lung Cell Mol Physiol 260: L274-L279, 1991[Abstract/Free Full Text].

14.   Girod, CE, Shin DH, Hershenson MB, Solway J, Dahl R, and Miller YE. p172: an alveolar type II and Clara cell specific protein with late developmental expression and upregulation by hyperoxic lung injury. Am J Respir Cell Mol Biol 14: 538-547, 1996[Abstract].

15.   Goding, JW. Ecto-enzymes: physiology meets pathology. J Leukoc Biol 67: 285-311, 2000[Abstract].

16.   Harlow, E, and Lane D. Monoclonal antibodies. In: The Antibodies, edited by Harlow E, and Lane D.. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory, 1998, chapt. 6, p. 139-244.

17.   Hitchcock-O'Hare, KH, and Sheridan MN. Electron microscopic observations on the morphogenesis of the albino rat lung, with special reference to pulmonary epithelial cells. Am J Anat 127: 181-206, 1970[ISI][Medline].

18.   Hooper, MN, and Bashir A. Glycosyl-phosphatidylinositol-anchored membrane proteins can be distinguished from transmembrane polypeptide-anchored proteins by differential solubilization and temperature-induced phase separation in Triton X-114. Biochem J 280: 745-751, 1991[ISI][Medline].

19.   Howell, KE, and Palade GE. Heterogeneity of lipoprotein-particles in hepatic Golgi fractions. J Cell Biol 92: 833-845, 1982[Abstract].

20.   Jiang, X, Tangada S, Peterson RDA, and Funkhouser JD. Expression of aminopeptidase N in fetal rat lung during development. Am J Physiol Lung Cell Mol Physiol 263: L460-L465, 1992[Abstract/Free Full Text].

21.   Kenny, AJ, and Maroux S. Topology of microvillar membrane hydrolases of kidney and intestine. Physiol Rev 62: 91-127, 1982[Free Full Text].

22.   Laemmli, UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685, 1970[ISI][Medline].

23.   Larsson, L. Ultrastructure of the developing proximal tubule in the rat kidney. J Ultrastruct Res 51: 119-139, 1975[ISI][Medline].

24.   Lwebuga-Mukasa, JS. Isolation and partial characterization of pneumocin, a novel apical membrane surface glycoprotein marker of rat type II cells. Am J Respir Cell Mol Biol 4: 479-488, 1991[ISI][Medline].

25.   Mason, RJ, Williams MC, Moses HL, Mohla S, and Berberich MA. Stem cells in lung development, disease, and therapy. Am J Respir Cell Mol Biol 16: 355-363, 1997[ISI][Medline].

26.   McElroy, MC, Pittet JF, Allen L, Wiener-Kronish JP, and Dobbs LG. Biochemical detection of type I cell damage after nitrogen dioxide-induced lung injury in rats. Am J Physiol Lung Cell Mol Physiol 273: L1228-L1234, 1997[ISI][Medline].

27.   McElroy, MC, Pittet JF, Hashimoto S, Allen L, Wiener-Kronish JP, and Dobbs LG. A type I cell-specific protein is a biochemical marker of epithelial injury in a rat model of pneumonia. Am J Physiol Lung Cell Mol Physiol 268: L181-L186, 1995[Abstract/Free Full Text].

28.   Pryde, JG, and Phillips JH. Fractionation of membrane proteins by temperature-induced phase separation in Triton X-114. Biochem J 233: 525-533, 1986[ISI][Medline].

29.   Roman, LM, and Hubbard AL. A domain-specific marker for the hepatocyte plasma membrane: localization of leucine aminopeptidase to the bile canalicular domain. J Cell Biol 96: 1548-1558, 1983[Abstract].

30.   Schellhase, DE, Emrie PA, Fisher JH, and Shannon JM. Ontogeny of surfactant apoproteins in the rat. Pediatr Res 26: 167-174, 1989[Abstract].

31.   Smith, MN, Greenberg SD, and Spjut HJ. The Clara cell: a comparative ultrastructural study in mammals. Am J Anat 155: 15-30, 1979[ISI][Medline].

32.   Sober, HA. Selected data for molecular biology. In: Handbook of Biochemistry. Cleveland, OH: Chemical Rubber, 1968, p. C10-C11.

33.   Wachsmuth, ED, and Stoye JP. The differentiation of proximal and distal tubules in the male rat kidney: the appearance of aldolase isozymes, aminopeptidase and alkaline phosphatase during ontogeny. Histochemistry 47: 315-337, 1976[ISI][Medline].

34.   Williams, MC, and Dobbs LG. Expression of cell-specific markers for alveolar epithelium in fetal rat lung. Am J Respir Cell Mol Biol 2: 533-542, 1990[ISI][Medline].

35.   Williams, MC, Cao Y, Hinds A, Rishi AK, and Wetterwald A. TI protein is developmentally regulated and expressed by alveolar type I cells, choroid plexus, and ciliary epithelia of adult rats. Am J Respir Cell Mol Biol 14: 577-585, 1996[Abstract].


Am J Physiol Lung Cell Mol Physiol 280(6):L1318-L1326
1040-0605/01 $5.00 Copyright © 2001 the American Physiological Society