Rat intestinal alpha 1-antitrypsin secretion is regulated by triacylglycerol feeding

Qing-Mei Xie, Jian-Su Shao, and David H. Alpers

Division of Gastroenterology, Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri 63110-1010


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

alpha 1-Antitrypsin (AAT) is secreted by the enterocyte, but its regulation of expression, intramucosal distribution, and functional status are unclear. After corn oil gavage (plus Pluronic L-81 to block chylomicron release), rat intestine was examined for mRNA encoding AAT, immunoreactivity by light and electron microscopy, and protein content by Western blot. Species-specific antisera used were raised against both AAT and surfactant-like particle (SLP), a membrane secreted by the enterocyte in response to fat feeding. Purified luminal SLP was fractionated by Bio-Gel P-200 chromatography to assess its interaction with AAT. Triacylglycerol feeding maximally increased mucosal mRNA-encoding AAT and AAT intracellular protein content by 3 and 5 h, respectively. Immunocytochemistry revealed predominance of AAT in basolateral spaces around enterocytes and Pluronic-blocked extracellular accumulation of AAT, patterns nearly identical to those of secreted SLP. About 10% of AAT was reversibly associated with SLP. Luminal AAT was smaller (51 kDa) than mature AAT (55 kDa) and did not form a complex with pancreatic elastase. When the common bile duct was tied, excluding pancreatic proteases from the lumen, mature AAT that was cleaved by pancreatic elastase was secreted. The luminal secretion of AAT and its reversible association with SLP suggest an intracellular association and a possible role for AAT during lipid digestion and absorption.

immunocytochemistry; fat absorption; surfactant-like particle


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE PROTOTYPIC SERINE PROTEASE inhibitor is alpha 1-antitrypsin (AAT), whose major physiological function is to inhibit the activity of neutrophil elastase, cathepsin G, and proteinase 3 (23). It also inhibits a broad spectrum of other serine proteases, including protein C, thrombin, trypsin, and chymotrypsin, although the degree of inhibition for these proteases is less than that for neutrophil elastase (23). AAT is synthesized predominantly in hepatocytes, but extrahepatic synthesis occurs in blood monocytes, tissue macrophages, and pulmonary epithelial cell lines, the latter thought to be type II cells (24). In these cells expression of AAT is regulated by mediators of the inflammatory response (20). Human AAT is also expressed in intestinal epithelial cells, as are other hepatic proteins, such as liver fatty acid binding protein (27). In the human intestine the protein has been found to be concentrated in the crypt in Paneth cells (19) and in enterocytes mainly in the base of the villus and the upper crypt (10). The protein is produced by Caco-2 cells when they differentiate into a fetal ileal-like phenotype (25). In this cell line AAT is secreted both apically and basolaterally, with basolateral AAT predominating and preceding the appearance of AAT in apical medium (25). Although it seems likely that AAT is secreted into the intestinal lumen of intact animals, this has not been demonstrated, nor has functional luminal AAT been characterized.

The surfactant-like particle (SLP) is a phospholipid-rich membrane that is purified from the apical surface of the enterocyte and the colonocyte and shares some properties and proteins with pulmonary surfactant (6). Production and secretion of this membrane in the rat are increased after triacylglycerol feeding (30). A number of proteins produced in the enterocyte or colonocyte are present in SLP, including both membrane-bound (intestinal alkaline phosphatase) and surfactant (A and B) proteins (6, 7). In the lung the protein-phospholipid complex of pulmonary surfactant is converted from a larger to a smaller size by the action of an AAT-inhibitable protease (13-15). For this reason SLP freshly prepared from rat small intestine was examined by amino-terminal sequencing for the presence of AAT. After a positive identification in SLP, intestinal AAT expression and secretion were examined in the rat, and the relationship of AAT to SLP was clarified.


    MATERIALS AND METHODS
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INTRODUCTION
MATERIALS AND METHODS
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Materials. Vivonex Plus as a source of amino acids and glucose was the product of Sandoz Nutrition (Minneapolis, MN). Rabbit anti-rat AAT antiserum and the cDNA encoding rat AAT were the generous gifts of Dr. Julie Chao, University of South Carolina (Charleston, SC) (4). Rabbit anti-human AAT antiserum was purchased from Dako (Carpinteria, CA). Pluronic L-81 was provided by the generosity of Dr. Patrick Tso, Department of Physiology, University of Cincinnati Medical School (Cincinnati, OH). Porcine pancreatic elastase was purchased from Sigma Chemical (St. Louis, MO). Polyclonal antibody against porcine pancreatic elastase was purchased from Chemicon International (Temecula, CA). Immunohistochemistry was performed using Vectastain reagents from Vector Laboratories (Burlingame, CA). RNAzol B was the product of Biotecx Laboratories (Houston, TX). Gold-labeled secondary antibody was purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). Bio-Gel P-200 was obtained from Bio-Rad (Hercules, CA).

Preparation of tissues. Male Sprague-Dawley rats (Sasco, Omaha, NE) were gavage fed 2.0 ml of corn oil (1.84 g/2.28 mmol of triacylglycerol) to maximize the response of SLP (8) after an overnight fast. The rate of gastric emptying of this fat load is fairly rapid. After administration of 2.26 g of corn oil intragastrically to rats, 1.0 g empties from the stomach by 12 min after delivery (17). Other animals were fed a mixture of corn oil and the detergent Pluronic L-81 (0.014 ml of Pluronic/2 ml of corn oil) or an isocaloric amount of Vivonex Plus (4.5 g). At designated times animals were anesthetized with methoxyflurane and perfused through the left ventricle with normal saline (with the tip of the right atrium removed to provide run-off) until the liver was completely blanched, to minimize serum contamination of the intestinal samples. Under these conditions serum albumin cannot be detected in enterocyte preparations (26). The proximal half of the small intestine was removed and rinsed with saline. The ends of the proximal intestine were tied after distending the loop with citrate buffer (in mM: 1.5 KCl, 96 NaCl, 27 sodium citrate, and 8 KH2PO4, pH 7.3). The loop was incubated for 15 min at 37°C, the buffer was removed, and this luminal wash was used for NaBr gradient centrifugation to purify SLP (6). The fractions with a density of 1.07-1.08 g/ml were used for further analysis. For some experiments the luminal wash was collected from animals whose common bile duct had been ligated 18 h previously to produce a fall in luminal pancreatic protease activity of >95% (22). When enterocytes were needed, the loop rinsed with citrate buffer was again distended with EDTA-containing buffer (0.137 M NaCl, 2.69 mM KCl, 8.1 mM Na2HPO4, 1.47 mM KH2PO4, 1.5 mM EDTA, and 0.5 mM dithiothreitol, pH 7.41) and incubated for 40 min at 37°C, at which time most of the villus cells had been removed (1). This method is a modification of the method of Weiser (29), in that the pre-EDTA incubation buffer contains citrate, producing a better yield for recovery of SLP from the cell surface. The isolated cells were then centrifuged and homogenized as previously described (26). For RNA preparation the mucosa was removed by scraping with a glass slide. For immunocytochemistry one section of proximal intestine was removed, fixed in 10% buffered Formalin for 4 h, kept in 70% ethanol overnight, and embedded in paraffin blocks, after which 5-µm-thick sections were prepared.

Analysis of tissue samples. Total tissue RNA was isolated from freshly scraped mucosa by the addition of RNAzol according to the manufacturer's instructions. Samples were either used fresh or were frozen immediately in liquid nitrogen until further use. Samples were stable over time, as monitored by ethidium bromide staining for ribosomal RNA and by Northern blot analysis of housekeeping markers, such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Northern blot analysis was performed on total mucosal RNA separated by agarose-formaldehyde gel electrophoresis as described previously (19) and stained with ethidium bromide, followed by transfer to nylon membranes and hybridization with 32P-labeled rat AAT DNA. Loading was monitored by scanning of the ethidium bromide stain. All samples were found to contain total RNA within 10% of each other. The AAT hybridization detected is entirely or nearly entirely attributed to enterocyte AAT, as AAT is undetected in normal human small intestinal lamina propria by immunocytochemistry (10, 19) or by in situ hybridization (19) and is undetected in rat lamina propria (see Fig. 5). Western blotting was carried out on tissue samples using the enhanced chemiluminescence method (ECL, Amersham, Arlington Heights, IL) (31). Luminal SLP purified on NaBr gradients was separated by denaturing acrylamide electrophoresis, and the proteins were transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA) before microsequencing with an Applied Biosystems model 477A protein sequencer. Other samples of freshly purified small intestinal SLP were applied to a P-200 column (45 × 1.0 cm) at a flow rate of 2.5 ml/h. The gel was swollen overnight at room temperature in 10 mM Tris, pH 7.8, and the column was developed in that buffer. The samples eluted from the void, and included volumes were analyzed by Western blotting using antisera against rat AAT and rat small bowel SLP. Samples of purified SLP obtained from normal rats and rats with bile duct ligation were treated with porcine pancreatic elastase or trypsin at ratios of 0.2-20 µg protease/mg SLP protein for 30 min at 37°C before analysis by Western blotting. Northern blots and Western blots were performed four times each, with similar results each time.

Immunocytochemistry. Fixed tissues were embedded in paraffin and sectioned at 5-µm thickness. The slides were deparaffinized, rehydrated, and treated with 1% H2O2 in methanol. Slides were then blocked in PBS containing 5% BSA and 10% normal goat serum. The primary antibody, either rabbit anti-rat AAT antiserum or rabbit anti-rat SLP antiserum, was added at a dilution of 1:300 or 1:100, respectively, and the slides were incubated at 37°C for 60 min. The secondary antibody, goat anti-rabbit biotinylated IgG, was added at a dilution of 1:200 for 20 min at 37°C, after which the slides were washed in PBS and incubated with Vectastain ABC (reagents A and B, 20 µl/ml, for 30 min at 37°C). 3,3'-Diaminobenzidine tetrahydrochloride [Sigma Fast DAB tablet (0.7 mg/ml) + H2O2 (20 mg/ml)] was added to the slide for 3-5 min or until color was evident. Normal rabbit serum as primary antiserum was used as a control. When anti-human AAT antiserum was used, the signal was much less intense.

AAT was identified as a protein migrating at 55 kDa in some preparations of purified SLP (see Relation of AAT to SLP in RESULTS). Thus, to eliminate the possibility that antisera raised in against rat small bowel SLP would recognize AAT, a preparation of SLP was subjected to Bio-Gel P-200 chromatography as described above and to antiserum produced in New Zealand White rabbits. To test for potential cross-reactivity, the antisera raised against rat AAT and SLP were tested on Western blot against a recombinant fusion protein (glutathione-S-transferase/AAT). The source of rat AAT was a fusion protein produced by inserting the entire coding region of rat AAT in frame into the EcoR I site of pGEX-5X-1 (Pharmacia Biotech, Piscataway NJ). The resultant chimeric vector was expressed in DH5alpha Escherichia coli cells, induced with 1 mM isopropyl beta -D-thiogalactoside for 5 h at 37°C, and the cellular pellets that contained the fusion protein were harvested. As shown in Fig. 1, antiserum against rat SLP did not recognize purified human AAT when tested by Western blotting against this fusion protein. This result is due to the fact that stored SLP preparations were used to raise antiserum, and under these conditions AAT is no longer associated with SLP (see Fig. 8).


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Fig. 1.   Lack of cross-reactivity of antisera raised against rat alpha 1-antitrypsin (AAT) and surfactant-like particle (SLP). Antisera against rat AAT and rat SLP were used in Western blot against rat AAT expressed as a 67-kDa fusion protein, as described in MATERIALS AND METHODS. Major fragment identified by anti-AAT was 67 kDa. Neither this band nor faint 54-kDa band was positive using anti-SLP antiserum. Same amount of protein (~5 µg) was applied to each lane. Ab, antibody.

For immunofluorescence studies the same concentrations of first antisera were used (1:300 for AAT, 1:100 for SLP) as for histochemical analysis. Secondary antibodies were labeled with indocarbocyanine (Cy3) (Jackson) or FITC (Sigma) and used at 1:100 dilutions. After overnight incubation with the first primary antiserum at 4°C, secondary antibody labeled with Cy3 (red fluorescence) or FITC (green fluorescence) was added for 1 h at room temperature. For double-labeled studies the second primary and secondary antibodies were each added for 1 h at room temperature. Between applications of antisera, sections were extensively rinsed with PBS, the effectiveness of which was monitored by parallel incubation and washing of a section treated with normal rabbit serum. Sections were mounted with phosphate-buffered saline-glycerol (1:1) under coverslips and examined in a Zeiss Axioskop photomicroscope, using filters of appropriate wavelength.

Immunoelectron microscopy. Rat small bowel SLP freshly purified from NaBr gradients (6) was diluted to 10.5 ml in 10 mM Tris · HCl, pH 7.25, and centrifuged in an L7-55 ultracentrifuge (Beckman Instruments, Fullerton, CA) for 60 min at 4°C. The SLP pellet was fixed in buffered 4% paraformaldehyde. After a rinse with three changes (20 min each) of 10 mM PBS containing 3% sucrose, the pellet was incubated overnight at 4°C in the same buffer. Free aldehydes were quenched with 50 mM ammonium chloride in 10 mM PBS containing 3% sucrose for 1 h at 4°C. Dehydration in ethanol and embedding in Lowicryl K4M resin (a water-miscible matrix) were performed according to the manufacturer's instructions (Electron Microscopy Sciences, Fort Washington, PA). K4M-ethanol (1:1) was added to the pellet on a rolling mixer for 1 h at -35°C (all samples during infiltration steps were rocked at -35°C), followed by K4M-ethanol (3:1) for 1 h and two changes of 100% fresh K4M (1 h and overnight). Samples were embedded in fresh K4M resin in BEEM capsules and were polymerized at 366-nm wavelength (using an ultraviolet light source at 115 V, 60 Hz, 20 A, at a distance of 10 cm from the capsule) for 48 h at -35°C and for 66 h at -10°C. Specimen blocks were stored at room temperature until used.

All immunogold staining treatments were performed with a Pelco 3450 laboratory microwave processor oven. Pale gold thin sections were placed on 400-mesh nickel grids, which were immersed one or two times in 20 mM Tris · HCl-225 mM NaCl, pH 7.34, blotted on filter paper, placed on 30-µl drops of blocking solution (20 mM Tris · HCl-225 mM NaCl, 0.2% Tween 20, pH 7.34), and heated in the oven for 2.5 min at 100% power and 37°C. Primary antisera (rabbit anti-rat AAT and rabbit anti rat SLP) were added at 1:10 dilution on the same thin sections and were treated for 2.5 min at 37°C. Secondary antibodies labeled with electron microscopy-grade colloidal gold (Jackson) were added at 1:20 dilution, by using 12-nm particles when AAT was the primary antiserum and 18-nm particles when SLP was the primary antiserum. Between each application of antiserum, the grids were rinsed (3 times, 5 s each) with 20 mM Tris · HCl-225 mM NaCl. After immunostaining the sections were counterstained for 15 min in 2.5% uranyl acetate and 2 min in lead citrate. Sections were observed in a JEOL 100C transmission electron microscope at 60 kV. Transmission electron microscopy was performed after fixation in 4% p-formaldehyde-1% glutaraldehyde with 0.66% tannic acid added to preserve membranes, as described previously (5).


    RESULTS
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MATERIALS AND METHODS
RESULTS
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Expression of AAT after triacylglycerol feeding. RNA was isolated from scraped proximal intestinal mucosa before and after triacylglycerol feeding. Before fat feeding no mRNA encoding AAT was detectable using a labeled DNA probe (Fig. 2). However, at 3 h a mRNA of the appropriate size had increased markedly in content; by 5 h after feeding the AAT mRNA was again undetectable. Despite the large change in AAT-specific mRNA content at 3 h, comparable amounts of RNA were loaded in all lanes (Fig. 2). There was no reproducible response to Vivonex feeding (data not shown).


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Fig. 2.   Response of AAT mRNA to corn oil feeding in the rat. After fat feeding, intestinal mRNA was isolated and probed in Northern blots using cDNA encoding rat AAT, as described in MATERIALS AND METHODS. A: Northern blot shows detectable mRNA only at 3 h after corn oil feeding. B: gel shows ethidium bromide stain of RNA applied to the gels. RNA was intact, and pattern confirms that RNA loaded (15 µg/lane) was present in nearly equal amounts.

To determine whether the mRNA response was followed by protein production, Western blots were performed on the homogenates of enterocytes isolated from another series of rats also fed triacylglycerol. Figure 3 shows that AAT was present at 1 h in enterocytes and that the concentration rose sharply and peaked at 5 h after feeding. The protein bands migrating at 62 and 51 kDa at 3 and 5 h after feeding correspond to the AAT-neutrophil elastase complex and AAT minus its carboxy-terminal peptide, respectively (23). This result is consistent with the presence of small amounts of pancreatic proteases that are adsorbed to the enterocyte apical surface and may gain access to the AAT after tissue homogenization. By 7 h after feeding only bands corresponding with the intact AAT and the putative AAT-pancreatic elastase complex were seen, but these were quite faint. In the lumen, where elastase is much more abundant, only the 51-kDa product of AAT and pancreatic elastase was seen.


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Fig. 3.   Expression of AAT in intestinal tissues after corn oil feeding. After corn oil feeding, isolated cells (A) and luminal washes (B) were obtained as described in MATERIALS AND METHODS, from which equal amounts of protein (15 µg) were applied to denaturing polyacrylamide gels. Western blots were performed using antiserum against rat AAT (1:5,000).

AAT was noted in isolated enterocytes at 1 h, but not a time 0, whereas AAT was abundantly present in the lumen at all times, although the concentration was somewhat greater at 3-7 h than it was at 0 or 1 h after fat feeding (Fig. 3). The enterocyte AAT was unlikely to be due to adherent luminal AAT, as it was absent in the cells at time 0. It was theoretically possible that some of the enterocyte AAT could have been absorbed from the small amount of AAT-elastase complex present in the lumen (9). However, the proteolytically cleaved 51-kDa fragment of AAT, the dominant luminal form of AAT, does not compete for binding of AAT-elastase complexes for the serpin-enzyme complex (SEC) receptor (16).

Interactions between pancreatic elastase and luminal AAT. When purified SLP was obtained from the intestinal lumen of normal rats 5 h after fat feeding (Fig. 4A, lanes 4-6), some of the AAT detected on Western blots migrated at 51 kDa, consistent with the presumed loss of the 4-kDa carboxy-terminal fragment (Fig. 4A, lane 4). The majority of the AAT from the lumen of normal rats (Fig. 4A, lanes 4-6) migrated slightly faster with an apparent relative molecular mass of 48 kDa. Moreover, the addition of pancreatic elastase did not alter the apparent relative molecular mass of the 48- or 51-kDa form of AAT (Fig. 4A, lanes 5 and 6). On the other hand, when the source of luminal material was from rats with bile ducts ligated 18 h before being killed (Fig. 4A, lanes 1-3), most immunoreactive AAT migrated at 55 kDa when no elastase was added (Fig. 4A, lane 1). The addition of porcine pancreatic elastase at low concentrations produced an additional immunoreactive band at ~72 kDa (Fig. 4A, lane 2). This larger protein band identified with antibody against rat AAT contains porcine elastase, as demonstrated by the faint band at the same position as lane 2 in the Western blot in Fig. 4B, developed by using antibody against elastase. Most elastase is present as the free form (28 kDa in Fig. 4B), but a small amount migrates at 72 kDa, presumably because of formation of a complex between the two proteins. Addition of high concentrations of porcine elastase converted AAT to the 51-kDa form (Fig. 4A, lane 3), with no effect noted from trypsin alone (trypsin data not shown). Thus the luminal AAT in normal animals appears to have been already degraded, probably by elastase, which is the most abundant luminal protease in the rat.


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Fig. 4.   A: degradation of rat AAT by pancreatic elastase. SLPs as a source of rat AAT were isolated from the luminal washings of normal animals [without common bile duct ligated (-CD Tied); lanes 4-6] and of bile duct-ligated animals (+CD Tied; lanes 1-3), as described in MATERIALS AND METHODS. For each 20-µl incubation, 8 µg of SLP protein were added to various amounts of porcine elastase, and the mixture was incubated for 30 min at 37°C before applying to denaturing polyacrylamide electrophoresis gels. AAT was identified by Western blotting using 1:5,000 dilution of anti-rat AAT. B: a duplicate of the gel was probed with antibody against porcine pancreatic elastase. Lane 7 corresponds to lane 2 of A and demonstrates presence of elastase in the high-molecular-mass band.

Immunocytochemical localization of intestinal AAT. In fasting rats strong staining was found for this soluble secreted protein in the region just beneath the basal membrane of the enterocytes in the lamina propria (Fig. 5A, solid arrow), with weaker reaction detected intracellularly and almost no reaction in the lumen (Fig. 5A, open arrow). By 3 h after feeding the stain in the lamina propria was more extensive (Fig. 5B, straight arrow), and the intercellular (paracellular) space now showed obvious reaction (Fig. 5B, curved arrow). At 5 h membranous material in the lumen was much more reactive (Fig. 5C, open arrow), with still some staining between cells (curved arrow) and strong staining in the lamina propria (Fig. 5C, straight arrow). Individual enterocytes were now strongly stained (arrowhead), but no cells in the lamina propria appeared to be reactive. The location of AAT was followed after blocking chylomicron secretion by feeding the detergent, Pluronic L-81. One hour after feeding fat and Pluronic L-81 the lamina propria was positive (Fig. 5E, straight arrow), mostly in the outer one-third of the villus, but no intercellular or luminal debris (open arrow) staining was seen. Moreover, many cells at the villus tip were faintly stained, unlike the cells studied at 0 and 3 h after fat feeding without Pluronic L-81 (Fig. 5, A and B). By 3 h after fat plus Pluronic L-81 the apical cytosol of many villus-tip cells was still positive (Fig. 5F, curved arrow), and staining was stronger in the lamina propria (Fig. 5F, straight arrow). By 5 h after Pluronic L-81, cytoplasmic staining of villus-tip cells persisted (curved arrow), but the lamina propria showed only patchy staining (Fig. 5G, straight arrow) compared with 5 h after fat feeding alone (Fig. 5C). In addition, compared with Fig. 5C there was no clear staining of luminal debris (Fig. 5G, open arrow).


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Fig. 5.   Immunocytochemical localization of AAT in rat intestine after corn oil feeding. Corn oil was fed by gavage to rats with or without Pluronic L-81 to inhibit chylomicron secretion. Tissues were processed and analyzed for AAT location as described in MATERIALS AND METHODS. Antiserum against rat AAT was used at 1:300 dilution. A: fasting. Note stain largely at base of epithelial cells (solid arrow) but none in luminal debris (open arrow). B: 3 h after corn oil. Note presence of heavier staining at base of epithelial cells (straight arrow) and also between cells (curved arrow). C: 5 h after corn oil. Note presence of stain in some epithelial cells (arrowhead) and in luminal debris (open arrow) in addition to sites found at 3 h in B (straight and curved arrows). D: 3 h after corn oil, normal rabbit serum. Note absence of stain. E: 1 h after corn oil plus Pluronic L-81. There is faint staining in epithelial cells but none between cells. The base of the epithelial cells is stained heavily (solid arrow). No stain was found in luminal debris (open arrow). F: 3 h after corn oil plus Pluronic L-81. Compared with B there is cytosolic staining of many villous tip cells (curved arrow) and still none between cells. G: 5 h after corn oil plus Pluronic L-81. Compared with C (5 h after fat) there is much less stain in enterocytes (curved arrow) and at the base of epithelial cells (straight solid arrow) and none in luminal debris (open arrow). H: 3 h after corn oil plus Pluronic L-81, normal rabbit serum. No reaction was seen. Studies were performed on 3 rats at each time point, with similar results (×200).

No reactivity was seen in any of these tissue compartments when normal rabbit serum was used either for control (Fig. 5D) or Pluronic L-81-treated (Fig. 5H ) samples. To confirm the specificity of the reactivity using the antiserum against rat AAT, the antiserum was adsorbed with homogenates of DH5alpha E. coli cells transformed with rat AAT DNA, as the recombinant protein produced was largely insoluble. Figure 6B shows that, when adsorbed antiserum was used, no reactivity in the tissue was seen 3 h after fat feeding, compared with strong staining of the same section using standard antiserum (Fig. 6A).


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Fig. 6.   Immunocytochemical specificity for AAT antiserum. A: sample was processed and analyzed as in Fig. 5. B: one sample of antiserum was incubated with 5% vol/vol E. coli DH5alpha homogenate (transformed with rat AAT cDNA) for 1 h at room temperature and overnight at 4°C before centrifugation at 15,000 g for 10 min (B). Dilution was then adjusted to make it equivalent to 1:300 dilution used for A.

Relation of AAT to SLP. SLP was freshly purified from the proximal rat small intestine and separated by SDS-acrylamide electrophoresis, and the proteins transferred to PVDF membranes were sequenced. The 55-kDa band yielded a sequence of QSPTYRKISSNLADFAFSLYRELV-, identical with the amino terminus of mature rat AAT. Because AAT would not have been predicted to be associated with membranes, the nature of the association between AAT and SLP was explored, using Bio-Gel P-200 column chromatography to separate the SLP in the void volume from AAT in the included volume. AAT and SLP proteins were detected by Western blotting (Fig. 7). Pure human AAT eluted (fractions 14-17) within the first included column volume (fractions 9-18), consistent with its monomeric size (data not shown). Analysis of a freshly isolated SLP preparation showed the range of SLP-associated proteins eluting from the start of the void volume (fraction 9) to fraction 15, with a peak at fractions 10-12. AAT was found not only where expected by its monomeric size (fractions 14-17), but also asymmetrically skewed toward larger sizes (fractions 10-13). The overlap in the elution patterns between SLP-reactive proteins and AAT is demonstrated in densitometric scans of gels from two separate preparations (Fig. 8A). On the other hand, when SLP was frozen for several months before application to the column, no overlap was seen between the profiles of AAT and SLP proteins (Fig. 8B).


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Fig. 7.   Distribution of AAT and SLP proteins separated by Bio-Gel P-200 chromatography. Freshly prepared SLP [100 µl (200 µg of protein)] was applied to a Bio-Gel P-200 column, and 20 µl of the eluant in each fraction (1 ml) were assayed for presence of SLP proteins (A) and AAT (B) by using antibodies against SLP (A) and against AAT (B) for Western blots as described in MATERIALS AND METHODS. The void volume of the column was 9 ml. The peak of alkaline phosphatase activity (a marker component for SLP) was exactly coincident (fraction 12) with presence of SLP protein determined by Western blotting.



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Fig. 8.   Distribution of AAT and SLP proteins separated by Bio-Gel P-200 chromatography. Eluants of SLP separation were processed as described in Fig. 7, and the radiographs were analyzed by densitometry using an Eagle Eye II Still Video system (Stratagene, La Jolla, CA). A: small bowel SLP freshly isolated and processed within 24 h, as in Fig. 7 (mean data from 2 samples). B: small bowel SLP frozen at -20°C for 2 months (mean data from 2 samples). Note absence of any overlap in distribution of AAT and SLP proteins in the stored sample.

Double labeling with anti-AAT and anti-SLP. With the use of the two antisera that had no cross-reactivity on Western blotting, double labeling was performed using immunohistochemistry and immunoelectron microscopy. Figure 9, C and D, shows the double-labeled fluorescence with these two antisera. Figure 9, C and D, shows that most areas in the lamina propria (large straight arrows) and intercellular space (large curved arrow, Fig. 9D) and a few areas in the lumen (arrowheads) were double labeled. Occasional enteroendocrine cells, presumptively identified by their unique shape (small straight arrows), were double labeled. In contrast, in the lumen there were large areas that were positive for SLP (green) but not for AAT (red). Figure 9D demonstrates that both antigens also were localized to paracellular spaces between enterocytes, consistent with the interpretation that AAT is associated with SLP as it is secreted from the cell.


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Fig. 9.   Double-labeled (anti-AAT and anti-SLP) immunofluorescence of rat small intestine. Tissues were fixed and processed as described in MATERIALS AND METHODS. A: anti-AAT, using Cy3-labeled secondary antibody (×400). B: anti-SLP, using FITC-labeled secondary antibody (×400). C: double-labeled image (×400). D: double-labeled image (×600). Large straight arrows identify lamina propria reactivity just beneath basal membrane of enterocyte. Arrowheads identify luminal debris, small arrows identify enteroendocrine cells, and curved arrow identifies paracellular space between enterocytes. Note that much of the luminal debris contains SLP proteins alone but that all other tissue compartments show identical distribution of AAT and SLP proteins. These studies were performed on 2 separate animals killed 5 h after fat feeding.

The gel filtration study (Figs. 7 and 8) and the double-labeling experiment (Fig. 9) only establish that AAT and SLP occupy the same position in a gel elution pattern or in a tissue section, but they do not directly demonstrate physical association. To show that such association can occur and may account for many of the findings in Figs. 7-9, double labeling was done using immunoelectron microscopy. Figure 10A shows double labeling of purified luminal SLP with immunogold decoration by electron microscopy. The labeled antibodies appear in continuous linear folded patterns (corresponding with membrane structures seen in Fig. 10B) and are decorated with both 12- and 18-nm gold particles, representing antibodies against both antigens. Figure 10B shows a transmission electron microscopy of the SLP pellet, demonstrating the nature of the membranes that cannot be seen using Lowicryl embedding.


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Fig. 10.   Double-labeled (anti-AAT and anti-SLP) immunoelectron microscopy of isolated rat small intestinal SLP. A: immunoelectron microscopy, using 12 nm-labeled secondary antibody for anti-AAT (notched arrowhead) and 18 nm-labeled secondary antibody for anti-SLP (arrowhead). Note the association of both size gold particles with each other, and displayed in linear folded patterns (×10,000). B: transmission electron microscopy of isolated SLP preparation used for A (×10,000). Arrows show unilamellar linear folded membrane characteristic of SLP (6, 7).


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

There seems little doubt that human AAT is produced by epithelial cells of the small intestine. The protein has been found by immunofluorescence in normal human intestine (3, 10, 19) and in human intestine after treatment of celiac disease (21). Interestingly, only one of these four studies shows no reactivity in Paneth cells (10), and that same study shows reactive cells in the lamina propria. In the present study no lamina propria cells in the rat intestine reacted with antiserum, although the lamina propria just beneath the basal layer of the epithelium stained heavily. After fat feeding many enterocytes and intercellular spaces between enterocytes became very reactive, a finding that peaked 5 h after fat feeding, following the very large increase in mucosal mRNA content encoding AAT seen at 3 h. As the enterocytes in rat intestinal mucosa account for most of the cell content and no lamina propria cells appeared positive by immunohistochemistry, it seems reasonable to conclude that the enterocytes are the source of the new AAT synthesis after triacylglycerol feeding. The increased synthesis and secretion of AAT in Caco-2 cells during their maturation (25) and production of human AAT in enterocytes of mice expressing the human AAT transgene (3, 18) are consistent with this conclusion. In the transgenic animals both Paneth cells and villus enterocytes contained AAT by in situ hybridization (18) or by immunocytochemistry (3).

AAT was found abundantly in the lumen 0-7 h after fat feeding, whereas in the enterocytes it was not detected at 0 h, appearing first at 1 h, peaking at 5 h, and almost disappearing by 7 h. Northern blotting detected AAT-specific mRNA only at 3 h. Western blotting is often more sensitive than Northern blotting; thus it is likely that the AAT in cells appearing 1 h after fat feeding is produced in the cell. Because of the discrepancy between luminal and cellular AAT content, most especially at 0 and 7 h after fat feeding, it seems unlikely that the AAT is derived from the luminal AAT, which probably enters the lumen from both biliary and intestinal secretions. Most importantly, the form in which the luminal AAT is present, the 51-kDa fragment, does not bind to the SEC receptor that mediates internalization and intracellular catabolism of the macromolecular AAT-elastase complex (16). In situ hybridization studies have also located the mRNA encoding AAT to the enterocyte (19). Thus most, if not all, of the intracellular AAT at all times after fat feeding probably derives from the enterocyte.

The secretion of newly synthesized AAT is quite rapid, and most of the process is completed by 1 h in Caco-2 cells (25). Thus the peak of AAT content in cells 5 h after fat feeding (2 h past the peak of specific mRNA content) suggests that AAT secretion in vivo may be partially retarded. One possible reason for a delay in secretion would be association with SLP, as the peak concentration of SLP proteins in enterocytes after fat feeding occurs at 5 h (30).

Most of the secreted AAT in Caco-2 cells is released at the basolateral surface of cells (25), and immunohistochemistry in the rat after fat feeding suggests that the same basolateral predominance occurs in intact rat intestine. The localization in the extracellular compartments of the intestine could not, however, have been predicted for a mobile soluble protein, and its colocalization with SLP proteins suggested an association with that secreted membrane. The identification of AAT in a purified preparation of the lipid-rich SLP and the column chromatography of SLP in luminal contents on P-200 confirmed a weak interaction between AAT and SLP. Immunoelectron microscopy confirms a physical association on the same membrane. This type of interaction is reminiscent of the charge-related interaction reported earlier between pancreatic hydrolases and the brush-border membrane (2, 28). Such an interaction, however, might have a function. For example, a serine protease inhibited by AAT appears to be important in the metabolism of extracellular pulmonary surfactant during the conversion of tubular myelin to vesicular forms (13, 15). In addition, this conversion appears to occur in the lumen adjacent to the apical surface of the cells (alveoli) in the intact animal (14).

AAT appears to be secreted intact from the enterocyte, because in the absence of luminal pancreatic proteases the 55-kDa form is found in the lumen. Moreover, that form of AAT interacts with porcine pancreatic elastase (Fig. 4), as it also does with macrophage elastase (12). It would seem reasonable that AAT in the lumen could act as a protease inhibitor, although it would not be a potent one. Such inhibition might take place close to the apical surface of the enterocyte, where it may be localized because of a reversible interaction with the SLP segregated there (6). This location is suggested by finding immunoreactive AAT in the debris overlying the enterocytes but not on the brush border itself. In that location AAT might act to protect the SLP from degradation or to preserve the proteins in the glycocalyx or mucus layer.

In the lamina propria the dual localization is much more prominent. This colocalization raises the possibility that AAT in the lamina propria may play some role in protecting extracellular proteins by inhibiting extracellular proteases that might gain entry from plasma ultrafiltrate or from the lumen. Some (or much) of the AAT in the lamina propria could derive from the plasma, particularly in the fasting animal, although another soluble plasma protein of a size similar to AAT, i.e., albumin, does not accumulate in the lamina propria (11). Thus it is possible that, when AAT is secreted basolaterally, it associates with SLP during transit through the lamina propria. In this location, where it is transiently immobilized, it might protect the SLP (and other associated) proteins from degradation.


    ACKNOWLEDGEMENTS

This study was supported in part by National Institutes of Health Grant AM-14038.


    FOOTNOTES

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: D. H. Alpers, Dept. of Internal Medicine, Box 8124, Washington Univ. School of Medicine, 660 S. Euclid Ave., St. Louis, MO 63110-1010 (E-mail: dalpers{at}imgate.wustl.edu).

Received 2 July 1998; accepted in final form 22 February 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Gastroint Liver Physiol 276(6):G1452-G1460
0002-9513/99 $5.00 Copyright © 1999 the American Physiological Society




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