Rat intestinal
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
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ABSTRACT |
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
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INTRODUCTION |
THE PROTOTYPIC SERINE PROTEASE inhibitor is
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.
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MATERIALS AND METHODS |
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
DH5
Escherichia coli cells, induced with 1 mM isopropyl
-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
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.
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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).
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RESULTS |
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.
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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).
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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.
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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).
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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 DH5
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 DH5
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.
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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.
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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 |
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 |
1.
Alpers, D. H.,
D. K. Lock,
N. Lancaster,
K. Poksay,
and
G. Schonfeld.
Distribution of apolipoprotein A-1 and B among intestinal lipoproteins.
J. Lipid. Res.
26:
1-10,
1985[Abstract].
2.
Alpers, D. H.,
and
M. Solin.
Characterization of rat intestinal amylase.
Gastroenterology
58:
833-842,
1970[Medline].
3.
Carlson, J. A.,
B. B. Rogers,
R. N. Sifers,
H. K. Hawkins,
M. J. Finegold,
and
S. L. C. Woo.
Multiple tissues express
1-antitrypsin in transgenic mice and man.
J. Clin. Invest.
82:
26-36,
1988[Medline].
4.
Chao, S.,
K. X. Chai,
L. Chao,
and
J. Chao.
Molecular cloning and primary structure of rat
1-antitrypsin.
Biochemistry
29:
323-329,
1990[Medline].
5.
DeSchryver-Kecskemeti, K.,
R. Eliakim,
S. Carroll,
W. F. Stenson,
M. A. Moxley,
and
D. H. Alpers.
Intestinal surfactant-like material: a novel secretory product of the rat enterocyte.
J. Clin. Invest.
84:
1355-1361,
1989[Medline].
6.
Eliakim, R.,
K. DeSchryver-Kecskemeti,
L. Nogee,
W. F. Stenson,
and
D. H. Alpers.
Isolation and characterization of a small intestinal surfactant-like particle containing alkaline phosphatase and other digestive enzymes.
J. Biol. Chem.
264:
20614-20619,
1989[Abstract/Free Full Text].
7.
Eliakim, R.,
G. S. Goetz,
S. Rubio,
B. Chailley-Heu,
J.-S. Shao,
R. Ducroc,
and
D. H. Alpers.
Isolation and characterization of surfactant-like particles in rat and human colon.
Am. J. Physiol.
272 (Gastrointest. Liver Physiol. 35):
G425-G434,
1997[Abstract/Free Full Text].
8.
Eliakim, R.,
A. Mahmood,
and
D. H. Alpers.
Rat intestinal alkaline phosphatase secretion into lumen and serum is coordinately regulated.
Biochim. Biophys. Acta
1091:
1-8,
1991[Medline].
9.
Gardner, M. L. G.
Gastrointestinal absorption of intact proteins.
Annu. Rev. Nutr.
8:
329-350,
1988[Medline].
10.
Geboes, K.,
M. B. Ray,
P. Rutgeerts,
F. Callea,
V. J. Desmet,
and
G. Vantrappen.
Morphological identification of
1-antitrypsin in the human small intestine.
Histopathology
6:
55-60,
1982[Medline].
11.
Granger, D. N.,
B. H. Cook,
and
E. A. Taylor.
Structural locus of transmucosal albumin efflux in canine ileum.
Gastroenterology
71:
1023-1027,
1976[Medline].
12.
Gronski, T. J.,
R. L. Martin,
D. K. Kobayashi,
B. C. Walsh,
M. C. Holman,
M. Huber,
H. E. Van Wart,
and
S. D. Shapiro.
Hydrolysis of a broad spectrum of extracellular matrix proteins by human macrophage elastase.
J. Biol. Chem.
272:
12189-12194,
1997[Abstract/Free Full Text].
13.
Gross, N. J.
Extracellular metabolism of pulmonary surfactant: The role of a new serine protease.
Annu. Rev. Physiol.
57:
135-150,
1995[Medline].
14.
Gross, N. J.,
V. Bublys,
J. D'Anza,
and
C. I. Brown.
The role of
1-antitrypsin in the control of extracellular surfactant metabolism.
Am. J. Physiol.
268 (Lung Cell Mol. Physiol. 12):
L438-L445,
1995[Abstract/Free Full Text].
15.
Gross, N. J.,
and
R. M. Schultz.
Serine proteinase requirement for the extra-cellular metabolism of pulmonary surfactant.
Biochim. Biophys. Acta
1044:
222-230,
1990[Medline].
16.
Joslin, G.,
A. Wittwer,
S. Adams,
D. M. Tollefsen,
A. August,
and
D. H. Perlmutter.
Cross-competition for binding of
1-antitrypsin (
1 AT)-elastase complexes to the serpin-enzyme complex receptor by other serpin-enzyme complexes and by proteolytically modified
1 AT.
J. Biol. Chem.
268:
1886-1893,
1993[Abstract/Free Full Text].
17.
Kaplan, J. M.,
W. Siemers,
and
H. J. Grill.
Effect of oral versus gastric delivery on gastric emptying of corn oil emulsions.
Am. J. Physiol.
273 (Regulatory Integrative Comp. Physiol. 42):
R1263-R1270,
1997[Abstract/Free Full Text].
18.
Koopman, P.,
S. Povey,
and
R. H. Lovell-Badge.
Widespread expression of human
1-antitrypsin in transgenic mice revealed by in situ hybridization.
Genes Dev.
3:
16-25,
1989[Abstract].
19.
Molmenti, E. P.,
D. H. Perlmutter,
and
D. G. Rubin.
Cell-specific expression of
1-antitrypsin in human intestinal epithelium.
J. Clin. Invest.
92:
2022-2034,
1993[Medline].
20.
Molmenti, E. P.,
T. Ziambarsas,
and
D. H. Perlmutter.
Evidence for an acute phase response in human intestinal epithelial cells.
J. Biol. Chem.
268:
14116-14124,
1993[Abstract/Free Full Text].
21.
Nielsen, K.
Coeliac disease:
1-antitrypsin contents in jejunal mucosa before and after gluten-free diet.
Histopathology
8:
759-764,
1984[Medline].
22.
Pelot, D.,
and
M. I. Grossman.
Distribution and fate of pancreatic enzymes in the small intestine of the rat.
Am. J. Physiol.
202:
285-288,
1962.
23.
Perlmutter, D. H.
1-Antitrypsin: structure, function, physiology,
In: Acute Phase Proteins, edited by A. Mackiewicz,
I. Kushner,
and H. Baumann. Boca Raton, FL: CRC, 1993, p. 149-167.
24.
Perlmutter, D. H.,
F. S. Cole,
P. Kilbridge,
T. H. Rossing,
and
H. R. Colten.
Expression of
1-proteinase inhibitor gene in human monocytes and macrophages.
Proc. Natl. Acad. Sci. USA
82:
795-799,
1985[Abstract].
25.
Perlmutter, D. H.,
J. D. Daniels,
H. S. Auerbach,
K. DeSchryver-Kecskemeti,
H. S. Winter,
and
D. H. Alpers.
The
1-antitrypsin gene is expressed in a human intestinal epithelial cell line.
J. Biol. Chem.
264:
9485-9490,
1989[Abstract/Free Full Text].
26.
Schonfeld, G.,
N. Grimme,
and
D. H. Alpers.
Detection of apolipoprotein C in human and rat enterocytes.
J. Cell. Biol.
86:
562-567,
1980[Abstract].
27.
Shields, H. M.,
M. L. Bates,
N. M. Bass,
C. J. Best,
D. H. Alpers,
and
R. K. Ockner.
Light microscopic immunocytochemical localization of hepatic and intestinal types of fatty acid-binding proteins in rat small intestine.
J. Lipid. Res.
27:
549-557,
1986[Abstract].
28.
Ugolev, A. M.
Membrane (contact) digestion.
Physiol. Rev.
45:
555-595,
1965[Free Full Text].
29.
Weiser, M. M.
Intestinal epithelial cell surface membrane glycoprotein synthesis. I. An indicator of cellular differentiation.
J. Biol. Chem.
248:
2536-2541,
1973[Abstract/Free Full Text].
30.
Yamagishi, F.,
T. Komoda,
and
D. H. Alpers.
Secretion and distribution of rat intestinal surfactant-like particles following fat feeding.
Am. J. Physiol.
266 (Gastrointest. Liver Physiol. 29):
G596-G605,
1994[Abstract/Free Full Text].
31.
Yeh, K.-Y.,
M. Yeh,
P. R. Holt,
and
D. H. Alpers.
Developmental and hormonal modulation of postnatal expression of intestinal alkaline phosphatase mRNA species and their encoded isozymes.
Biochem. J.
301:
893-899,
1994[Medline].
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