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
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ABSTRACT |
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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
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INTRODUCTION |
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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.
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METHODS |
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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 -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,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.
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RESULTS |
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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).
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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).
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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.
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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).
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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.
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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.
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DISCUSSION |
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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 |
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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.
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FOOTNOTES |
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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.
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