Vol. 275, Issue 5, R1584-R1592, November 1998
Endotoxemia in mice stimulates production of complement C3 and
serum amyloid A in mucosa of small intestine
Quan
Wang,
Tory A.
Meyer,
Steven T.
Boyce,
Jing Jing
Wang,
Xiaoyan
Sun,
Greg
Tiao,
Josef E.
Fischer, and
Per-Olof
Hasselgren
Department of Surgery, University of Cincinnati, and the Shriners
Hospital for Children, Cincinnati, Ohio 45267-0558
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ABSTRACT |
We examined the effect of endotoxemia in
mice on protein and mRNA levels for the acute phase proteins complement
C3 and serum amyloid A (SAA) in jejunal mucosa. Endotoxemia was induced
in mice by the subcutaneous injection of 250 µg lipopolysaccharide per mouse. Control mice were injected with saline. C3 and SAA were
measured by ELISA. Messenger RNA levels were determined by Northern
blot analysis or competitive PCR. Immunohistochemistry was performed to
determine in which cell type(s) C3 and SAA were present. Mucosal C3 and
SAA protein and mRNA levels were increased in endotoxemic mice.
Immunohistochemistry showed that C3 was present in both enterocytes and
cells of the lamina propria, whereas SAA was seen mainly in lamina
propria cells. Results suggest that endotoxemia stimulates production
of C3 and SAA in small intestinal mucosa. The response may be regulated
at the transcriptional level and probably reflects increased synthesis
of the acute phase proteins in both enterocytes and cells of the lamina
propria.
gut; enterocyte
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INTRODUCTION |
IN PREVIOUS STUDIES from our laboratory (16, 30, 31),
protein synthesis was increased in intestinal mucosa during sepsis and
endotoxemia. Part of this response reflected increased cell turnover
caused by loss of cells from the tips of the villi and stimulated
enterocyte proliferation (28). In addition, the accelerated mucosal
protein synthesis reflected increased biosynthesis in the individual
enterocyte, as demonstrated by high protein synthesis rates in isolated
enterocytes from septic rats (14).
The intestinal mucosa is the production site of a number of
biologically important peptides and proteins, e.g., gut hormones. In
recent studies, we found that the production by the enterocyte of the
gut hormones vasoactive intestinal peptide and peptide YY was increased
during sepsis (15, 33). Another group of proteins that participates
significantly in the metabolic response to endotoxemia and sepsis is
the acute phase proteins. Although the majority of the acute phase
proteins are typically produced in the liver, there is recent evidence
that some of the acute phase proteins may be synthesized at
extrahepatic sites as well, including the intestinal mucosa. For
example, inflammatory bowel disease has been shown to be associated
with enhanced intestinal production of complements (1). Other studies
have provided evidence that the enterocyte expresses several of the
acute phase proteins (23).
The influence of sepsis and endotoxemia on mucosal acute phase proteins
is not known. In the present study, we tested the hypothesis that
endotoxemia in mice stimulates mucosal production of the acute phase
proteins complement C3 and serum amyloid A (SAA). Complement C3 is an
important acute phase protein involved in the local defense against
invading bacteria (8), and its expression in the enterocyte was
demonstrated recently (2, 3). SAA is one of the most abundant acute
phase proteins in mice, being increased up to 1,000-fold in serum after
stimulus (29). The role of SAA in the acute phase response is not fully understood, but there is evidence that it may play a role in enhancing the clearance of high-density lipoproteins (HDLs) (17). In a recent
study, the expression of SAA mRNA was upregulated in intestine of
endotoxemic mice but mucosal SAA protein levels were not
measured in that report (6).
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MATERIALS AND METHODS |
Animals and experimental design.
Male A/J mice (20-27 g) were purchased from Jackson Laboratory
(Bar Harbor, ME) and housed at a temperature of 22°C in a room with
a 12:12-h light-dark cycle for 1 wk before experiments. Endotoxemia was
induced by the subcutaneous injection of 250 µg/mouse of
lipopolysaccharide (Escherichia
coli 0111:B4; Calbiochem, LaJolla,
CA). Control mice were injected with corresponding volumes of sterile
saline. Food was withheld, but drinking water was provided after the
injection of endotoxin or saline. The dose of endotoxin used here was
based on a recent report in which we found that the subcutaneous
injection of the same amount of endotoxin in mice gave rise to a
maximal increase in total mucosal protein synthesis in the jejunum
(31). The endotoxin was injected subcutaneously rather than
intraperitoneally to avoid the influence of local metabolic changes
when intestinal mucosa was studied. Groups of mice were studied at
intervals up to 24 h after injection of endotoxin or saline. With mice
under pentobarbital sodium anesthesia (40 mg/kg ip), blood was
collected by heart puncture in heparinized tubes for determination of
plasma concentrations of C3 and SAA. We harvested the mucosa from a
10-cm segment of the jejunum by opening the intestine along the
antimesenteric border and by scraping the luminal side with a
microscope slide. The mucosa was immediately frozen in liquid nitrogen
and stored at 
70°C until used for determination of mRNA
levels and concentrations of complement C3 and SAA. The left liver lobe
was excised, frozen in liquid nitrogen, and stored at 70°C
until analysis. In a separate series of experiments, the superior
mesenteric artery was catheterized and slowly perfused with 0.5-1
ml of saline before harvesting of the intestinal mucosa to minimize the
contribution of acute phase proteins from the blood to the acute phase
proteins that were measured in the tissue. The intestinal wall was
noted to blanch before mucosa was harvested. All experiments were
performed, and the animals were cared for according to the National
Research Council's "Guide for the Care and Use of Laboratory
Animals." The experimental protocol was approved by the University
of Cincinnati Institutional Animal Care and Use Committee.
Measurement of complement C3 and SAA.
Plasma and tissue levels of complement C3 and SAA were measured by
ELISA. For determination of complement C3 and SAA in mucosa and liver,
tissue was ultrasonicated two times for 10 s in 1 ml of PBS containing
2 µg/ml each of the protease inhibitors leupeptin, aprotinin,
pepstatin A, and antipain (Sigma, St. Louis, MO) and 2 mM
phenylmethylsulfonyl fluoride (Sigma) and then centrifuged at 12,000 g at 4°C for 45 min. The
supernatants were used for determination of complement C3 and SAA.
Complement C3 was determined as described previously (13) using a goat
anti-mouse C3 antibody (IgG fraction 55463; Cappel Research Products,
Durham, NC). SAA was measured with a commercially available ELISA kit
using a rat anti-human SAA antibody (Biosource International,
Camarillo, CA). This antibody has a 100% cross-reactivity to mouse SAA
according to the manufacturer. The lower limits of detection were 10 ng/ml for complement C3 and 0.23 µg/ml for SAA. One concern when
measurements of C3 levels are performed is the specificity of the
anti-C3 antibody, i.e., whether the antibody recognizes the cleavage
products of C3 as well. In a control experiment using purified mouse C3
and purified human C3, C3a, C3b, and C3bi (purified mouse C3a, C3b, and
C3bi are not commercially available), we found evidence that the
anti-mouse C3 antibody used here recognizes C3, C3b, and C3bi but not
C3a. Thus the results obtained with this antibody may reflect the
presence both of C3 and of some of its cleavage products.
mRNA for complement C3 and SAA.
Northern blot analysis and competitive PCR were used to determine the
expression of mucosal and hepatic C3 and SAA mRNA. Mucosal samples were
harvested as described in Measurement
of
complement C3
and
SAA, and samples from two or three
mice were pooled for each time point. Total RNA was extracted by the
guanidinium thiocyanate-phenol-chloroform method (5) using a Stat-60
kit (Tel-Test B, Friendswood, TX).
For Northern blot analysis, RNA was denatured and separated by
electrophoresis on a 1% agarose gel containing formaldehyde. The RNA
was transferred from the gel to nylon membranes (Micron Separations,
Westboro, MA) by capillary action in 2× saline sodium citrate
(SSC) (1× SSC = 0.15 M NaCl and 15 mM sodium citrate) overnight.
RNA was immobilized either by baking at 80°C for 2 h or by
ultraviolet cross-linking. The blots were hybridized at 42°C for 4 h in 50% formaldehyde and 6× sodium chloride-sodium phosphate-EDTA (SSPE) (1× SSPE = 0.15 M NaCl, 10 mM
NaH2PO4
and 1 M EDTA), 5× Denhardt's solution, 0.5% SDS, and 100 µg/ml salmon sperm DNA. cDNA probes for C3 and SAA were labeled by
random priming with
[32P]dATP or
[32P]dCTP (Stratagene,
LaJolla, CA). The blots were hybridized with the
32P-labeled cDNA probes at
42°C overnight. The blots were then washed two times in 1×
SSC and 0.1% SDS and one time in 0.1× SSC and 0.1% SDS at room
temperature and autoradiographed at 70°C. The blots were
stripped and rehybridized with an 18S oligonucleotide probe to control
for equal loading of RNA.
Competitive PCR was performed as described in detail previously (26).
In short, total RNA was extracted from mucosa as described in
mRNA
for
complement
C3
and
SAA. Dissolved total RNA samples were
reverse transcribed to cDNA using a Moloney murine leukemia virus
reverse transcriptase (M-MLV RT) (Promega, Madison, WI). Briefly, 5 µl of total RNA samples were added to 1 µM 3'-primer in 50 mM
Tris · HCl (pH 7.3) and heated to 90°C followed
by cooling to 37°C over 15 min. Reaction buffer, 75 mM KCl, 3 mM
MgCl2, 20 mM dithiothreitol, 50 U
RNasin, 35 U DNase, 250 µM dNTP, and 250 U M-MLV RT were then added,
and the samples were incubated at 37°C for 40 min, followed by
freezing at 70°C for at least 15 min to inactivate the
enzyme. The resulting cDNA samples were stored at 70°C until
used. Aliquots of the cDNA samples were then added to a cocktail
containing PCR buffer, gelatin, KCl, MgCl2, dNTP, C3 primers, and
Taq polymerase (Promega). A sequence of 385 nt in mouse complement C3 mRNA
(exons
4-7)
(7) was used to construct the C3 primers: 5'-sense 5'-CAC
CGC CAA GAA TGC CTA C-3' (nt 2684-2703) and
3'-antisense 5'-GAT CAG GTG TTT CAG CCG C-3' (nt
3049-3068). PCR was performed using a Perkin-Elmer DNA thermocycler by heating the samples to 95°C for 2 min, followed by
25 cycles of 95°C for 1 min, 62°C for 1 min, and 72°C for 2 min. The samples were kept at 60°C for 7 min and cooled to 4°C. Ten microliters of the PCR products were then separated by
electrophoresis at 100 V in a 1.5% agarose gel stained with ethidium
bromide. To quantify mRNA expression, a PCR MIMIC construction kit
(Clontech, Palo Alto, CA) was used to construct nonhomologous DNA
fragments using the mouse C3 gene-specific primers and composite
primers. An initial PCR amplification using the composite primers was
performed on the neutral DNA fragment that incorporated the C3
gene-specific primer sequence onto the neutral DNA. The yield of this
PCR MIMIC was calculated as the ratio between the optical densities at
260 and 280 nm and diluted to 100 attomol/µl. Competitive PCR was then performed by adding 2 µl of the cDNA samples to 2 µl of
different 10-fold dilutions of PCR MIMIC
(100-10
6 attomol), PCR
buffer, MgCl2, nucleotide
triphosphates (Gibco-BRL, Grand Island, NY), 5'- and 3'-C3
gene-specific primers, and Taq polymerase (Promega). The mixture was heated to 94°C for 45 s, to
50°C for 45 s, and to 72°C for 90 s. The samples were kept at
72°C for 7 min and cooled to 4°C. Five microliters of the PCR produces were separated by electrophoresis at 100 V in a 1.5% agarose
gel stained with ethidium bromide. Comparison between PCR MIMIC bands
(known concentrations) and C3 bands was made to determine the
concentration of C3 mRNA present using a video image system (BioMax ID
Image Analytic System; Eastman Kodak, Rochester, NY).
Immunohistochemistry.
Immunohistochemistry was performed to determine in which cell type(s)
of the mucosa complement C3 and SAA were present. With mice under
pentobarbital anesthesia, the lumen of the jejunum was flushed with
saline and a 0.5-cm segment was excised from the midportion of the
jejunum. The intestinal segment was immersed in OCT
embedding medium (Miles, Elkhart, IN) and then placed on a metal board
frozen in liquid nitrogen. The samples were stored at 70°C
until analysis.
Five-micrometer sections were cut and embedded on
poly-L-lysine-coated slides. The
staining was performed as described previously (9). The sections were
dried at 45°C for 10 min and fixed in 100% methanol at
20°C for 10 min. The sections were then blocked with 0.01%
avidin (no. A-9275, Sigma) and 0.1%
D-biotin (no. B-4501) for 15 min
each and further blocked with 5% BSA for 30 min at room temperature.
The primary antibodies, which were the same as used for the ELISA
assays described but diluted 1:100, were added to the sections, which
were then incubated for 2 h at 37°C. Biotin conjugated rabbit
anti-goat IgG (no. 39559, Cappel Research Products) and fluorescence
conjugated streptavidin (no. 1055097; Boehringer, Mannheim, Germany)
were diluted 1:2,000 and used consecutively for further detection of
C3. Goat anti-rat IgG conjugated with biotin (no. 55790, Cappel
Research Products) was used as secondary antibody for detection of SAA.
During staining, Tris-buffered saline (0.05 M, pH 7.6) was
used as the buffering system throughout and each step was followed by
washes. Negative controls were performed by omitting the primary
antibody. The sections were examined with a Nikon microphot-FXA (Nikon,
Melville, NY) equipped with an epifluorescence illumination system, and
photographs were observed on Ektachrome 400 ASA professional daylight
film. Note that when the anti-mouse C3 antibody was used,
immunohistochemistry may reflect the presence of C3 as well as its
cleavage products C3b and C3bi, as described in
Measurement
of
complement
C3
and
SAA.
Statistical analysis.
Results are expressed as means ± SE. The Mann-Whitney rank
sum was used for statistical analysis.
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RESULTS |
Complement C3 levels in jejunal mucosa were increased by 50 and 86% 16 and 24 h after injection of endotoxin, respectively (Fig.
1A).
Complement C3 concentrations in liver were five to six times higher
than those in mucosa and were increased in endotoxemic mice at 8 h and
through the rest of the experimental period (Fig. 1B). Plasma levels of C3 were
increased 16 and 24 h after injection of endotoxin (Fig.
1C). Because the anti-mouse C3
antibody used in these experiments may recognize C3b and C3bi in
addition to C3, the results may reflect the sum of C3 and its cleavage
products C3b and C3bi. For clarity, results are described as C3
throughout the rest of the paper, but it is important to recognize this
limitation of the study.
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Fig. 1.
Concentration of complement C3 in jejunal mucosa
(A), liver
(B), and plasma
(C) of endotoxemic
(lipopolysaccharide, LPS) and saline-injected control mice. Groups of
mice were studied 4, 8, 16, and 24 h after subcutaneous injection of
250 µg/mouse of endotoxin or corresponding volume of saline;
n = 7 per group at each time point.
gww, Grams wet weight. * P < 0.05 vs. saline at corresponding time point.
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Endotoxemia resulted in an approximately fourfold increase in mucosal
SAA levels at 16 and 24 h (Fig.
2A). SAA
concentrations were higher in liver than in mucosa and responded to
endotoxemia in a similar way, with increased levels noted 16 and 24 h
after injection of endotoxin (Fig.
2B). As expected (29), plasma
concentrations of SAA were markedly elevated in endotoxemic mice, with
a more than 20-fold increase noted at 16 h (Fig.
2C). Interestingly, plasma levels of
SAA were increased already 4 and 8 h after injection of endotoxin,
i.e., before the increase in mucosal and liver concentrations took
place.
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Fig. 2.
Concentration of serum amyloid A (SAA) in jejunal mucosa
(A), liver
(B), and plasma
(C) of endotoxemic (LPS) and
saline-injected control mice. Experimental conditions were identical to
those in Fig 1. * P < 0.05 vs.
saline at corresponding time point.
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Because it is possible that blood-borne acute phase proteins, deposited
in the tissue, could have caused or at least contributed to the
increased mucosal levels noted at 16 and 24 h in endotoxemic mice, a
control experiment was performed in which the intestinal vasculature
was perfused with saline before the mucosa was harvested. In this
experiment, the basal C3 levels in the mucosa were somewhat lower than
in mucosa of intestine that had not been perfused, probably reflecting
in part removal of blood-borne C3 (Fig.
3A; compare with Fig. 1A). The
difference in mucosal C3 levels between control and endotoxemic mice,
however, persisted even after perfusion of the jejunal vasculature, and
the relative increase in endotoxemic mice was even more pronounced in
this experiment, being ~2.5- and 2-fold at 16 and 24 h, respectively
(Fig. 3A). The endotoxin-stimulated levels of SAA were somewhat lower in mucosa of perfused intestine than
in mucosa of intestine in which the vasculature had not been perfused
with saline, whereas the basal levels of SAA were not reduced by the
perfusion (Fig. 3B; compare with Fig.
2A).
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Fig. 3.
Concentrations of complement C3 (A)
and SAA (B) in jejunal mucosa of
endotoxemic (LPS) and saline-injected mice. Intestinal vasculature was
perfused with saline before mucosa was harvested to minimize
contribution of blood-borne C3 and SAA to tissue levels of proteins;
n = 7 per group at each time point.
* P < 0.05 vs. saline at
corresponding time point.
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To examine whether the increased mucosal complement C3 and SAA levels
during endotoxemia reflected an increased local production of the
proteins, possibly regulated at the transcriptional level, we next
measured mucosal levels of mRNA for C3 and SAA. When Northern blot
analysis was performed, no signals for C3 or SAA mRNA were noticed in
samples from saline-injected control mice. C3 mRNA was not detected on
Northern blots in mucosal samples of endotoxemic mice but was present
in liver tissue, with a faint band noticed at 2 h and a maximal
response noticed 16 h after injection of endotoxin (Fig.
4). SAA mRNA was induced in jejunal mucosa
of endotoxemic mice, with a weak signal noticed at 4 h after injection of endotoxin and a maximal response seen at 16 h (Fig.
5). In liver, the expression of SAA mRNA
was induced already 2 h after injection of endotoxin and was sustained
during the rest of the 24-h experimental period (Fig.
6). Note that in liver, two bands were
present on Northern blots, similar to a recent report by de Beer et al.
(6). The two mRNA bands reflect the fact that SAA is a
multigene family; the heavier band (between 18S and 28S) may correspond
to SAA5, and the lighter mRNA
(below 18S) may correspond to SAA2
(6). In jejunal mucosa, only one band for SAA mRNA was seen (Fig. 5)
and, on the basis of previous reports (6, 18), this band may represent
mRNAs for SAA1-3.
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Fig. 4.
Northern blot analysis of RNA extracted from livers of endotoxemic
(LPS) and saline-injected control mice. Blots were hybridized with a
cDNA probe for C3, stripped, and rehybridized with an 18S
oligonucleotide probe to control for equal loading of RNA.
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Fig. 5.
Northern blot analysis of RNA extracted from jejunal mucosa of
endotoxemic (LPS) and saline-injected control mice. Blots were
hybridized with a cDNA probe for SAA, stripped, and rehybridized with
an 18S oligonucleotide probe to control for equal loading of RNA.
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Fig. 6.
SAA mRNA levels in livers of endotoxemic and saline-injected mice
determined by Northern blot analysis. Two bands noted for SAA mRNA are
consistent with SAA multigene family and probably represent
SAA2
(bottom band) and
SAA5
(top band). See text for details.
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Because the absence of a clear mucosal signal for C3 mRNA on the
Northern blots could be due to insufficient amounts of C3 mRNA in the
mucosa, we performed PCR on RNA extracted from jejunal mucosa. Results
from that experiment showed that mRNA for complement C3 was detectable
in jejunal mucosa of both control and endotoxemic mice. To test whether
endotoxemia resulted in increased C3 mRNA levels, competitive PCR was
performed. Results from that experiment revealed an ~100% increase
in mucosal C3 mRNA levels 4 h after induction of endotoxemia and a
five- to sixfold increase at later time points (Fig.
7A).
Because of the high sensitivity of PCR, one concern was that the signal
for C3 mRNA noticed in mucosa may reflect "contamination" by
nonmucosal cells. A control experiment was therefore performed in which
mucosa was harvested after perfusion of the intestinal vasculature at
different time points after saline or endotoxin injection. Competitive
PCR showed an almost identical response to endotoxin as the one seen in
the experiment in which the vasculature was not perfused (Fig.
7B; compare with Fig.
7A). Also, the basal levels of C3
mRNA were similar in the two experiments, suggesting that the C3 mRNA
levels reflected local tissue mRNA rather than mRNA in circulating
cells.
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Fig. 7.
A: C3 mRNA in jejunal mucosa of
endotoxemic and saline-injected control mice determined by competitive
PCR. Groups of mice were studied 1, 4, 8, and 16 h after subcutaneous
injection of 250 µg/mouse of endotoxin or a corresponding volume of
saline; n = 3 per group at each time
point. B: C3 mRNA in jejunal mucosa
after perfusion of intestinal vasculature. Experimental conditions were
identical to those in A, except that
intestinal vasculature was perfused with saline before mucosa was
harvested. * P < 0.05 vs.
saline at corresponding timepoint.
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Although the results described suggest that mucosal levels of C3 and
SAA are increased during endotoxemia and that the increased protein
levels may reflect transcriptional upregulation of the protein, the
cellular origin of the proteins is not known. To test in which cell
type(s) of the mucosa complement C3 and SAA were present, we performed
immunohistochemical studies. C3 was present in both enterocytes and
cells of the lamina propria (Fig. 8),
whereas SAA was expressed mainly in cells of the lamina propria (Fig.
9). In endotoxemic mice, SAA was detected
in the intestinal lumen (Fig. 9C).
Evidence of mucosal injury was seen in the endotoxemic mice, with
disruption of the epithelial layer and desquamation of cells into the
intestinal lumen (Fig. 8, C and
D and Fig. 9, C and
D).
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Fig. 8.
Immunohistochemistry of jejunal mucosa from saline-injected
(A) and endotoxemic
(C) mice showing presence of C3 in
enterocytes and cells of lamina propria. Negative controls
(B and
D) were performed by omitting the
primary C3 antibody. Light green areas in
A and
C represent signal for C3. Mucosa was
harvested 24 h after injection of endotoxin or saline. Bar in
B, 100 µm.
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Fig. 9.
Immunohistochemistry of jejunal mucosa from saline-injected
(A) and endotoxemic
(C) mice showing presence of SAA
mainly in cells of lamina propria. Negative controls were performed by
omitting primary SAA antibody (B and
D). Light green areas in
A and
C represent signal for SAA. Mucosa was
harvested 24 h after injection of endotoxin or saline. Bar in
B, 100 µm.
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DISCUSSION |
In the present study, endotoxemia in mice was associated with increased
concentrations of the acute phase proteins complement C3 and SAA in
jejunal mucosa. Because C3 and SAA mRNAs were upregulated by endotoxin
and immunohistochemistry indicated that the proteins were expressed in
both enterocytes and nonepithelial mucosal cells, results are
consistent with the concept that the local production of C3 and SAA may
be increased in mucosa of small intestine during endotoxemia. The
results are important because they support the role of the intestinal
mucosa in the metabolic response to sepsis and endotoxemia and provide
further evidence that the gut is an active participant in the
inflammatory response to these conditions.
One major concern when the present results were interpreted was whether
the mucosal levels of C3 and SAA represented local production of the
proteins or merely a deposition or contamination of blood-borne
proteins. Although circulating C3 and SAA probably contributed to the
mucosal levels, several lines of evidence suggest that local production
of the proteins took place in the mucosa. 1) When the vasculature of the
jejunum was perfused with saline, the difference in mucosal C3 and SAA
levels between control and endotoxemic mice persisted;
2) the increased mRNA levels for C3 and SAA, in particular the persistent increase in C3 mRNA after perfusion of the intestinal vasculature, strongly support a local production of the proteins; 3) when
immunohistochemistry was applied, results showed that C3 was present in
enterocytes and other mucosal cells and SAA was present in
nonepithelial mucosal cells; and 4)
there is evidence that enterocytes can synthesize several acute phase
proteins, including C3 and SAA (2, 3, 23, 24, 27). Thus the increased
mucosal levels of complement C3 and SAA may at least in part reflect
increased local production of the proteins during endotoxemia.
To our knowledge, this is the first report of increased mucosal
production of C3 and SAA during endotoxemia. In a recent report, increased SAA mRNA levels were noticed in intestine of endotoxemic mice
but SAA protein levels were not measured (6). In other studies, mucosal
production of acute phase proteins was increased in inflammatory bowel
disease (1, 11, 12). In previous experiments, Molmenti et al. (23)
found evidence for an acute phase response in human intestinal
epithelial cells. In recent experiments in our laboratory, complement
C3 protein and mRNA were expressed in cultured Caco-2 cells, a human
intestinal epithelial cell line (23). Thus the intestinal mucosa may be
the production site of acute phase proteins, both in response to local
inflammation, such as inflammatory bowel disease (1, 11, 12), and in response to systemic inflammation, such as endotoxemia (present results).
Although the present report and other studies (23, 27) suggest that
acute phase proteins may be produced at extrahepatic sites, the liver
is probably the major site of acute phase protein synthesis, at least
from a quantitative standpoint. In the present study, the
concentrations of C3 and SAA were four to five times higher in liver
tissue than in mucosa, supporting the central role of the liver in the
acute phase response (29). The present finding of elevated circulating
levels of SAA in endotoxemic mice before liver and mucosal levels of
SAA were increased was surprising and may suggest that other tissues as
well are involved in the acute phase response. Alternatively, the
breakdown, excretion, and/or tissue distribution of circulating
SAA was influenced by endotoxin before changes in liver and mucosal
levels occurred.
The influence of endotoxemia on mucosal complement C3 levels was
examined in the present study because C3 may be particularly important
in the mucosa. Complement C3 converges the classical and alternative
pathways into a final common pathway in the complement cascade which
participates in the local defense against invading microorganisms and
may cause lysis of bacteria (8). SAA was studied because it is the
major acute phase protein in mice (4, 10, 29). Although the exact
biological role of SAA in the acute phase response is unidentified, SAA
is an apoprotein of HDL and may influence the formation and clearance
of HDL (17). It should be noted that murine SAA is encoded by multiple
genes and that at least five members of the SAA family have been
identified and labeled SAA1
through SAA5 (6, 18, 20). The SAA
proteins are highly homologous proteins (32), and the antibody used in the present study for ELISA and immunohistochemistry did not
discriminate between the different members of the SAA family. In
consideration of previous reports of the expression of different
members of the SAA family in various tissues (6, 18), our results most likely represented upregulated production of
SAA1-3 in the intestinal
mucosa and increased synthesis of
SSA2 and
SAA5 in liver.
It is not known from the present study if the increased mRNA levels for
C3 and SAA in liver and jejunal mucosa during endotoxemia reflected
increased mRNA stability or upregulated gene transcription. In previous
reports, increased SAA mRNA levels in liver tissue of endotoxemic mice
were associated with increased SAA gene transcription, although
posttranscriptional stabilization probably contributed to the increased
mRNA levels (18).
The mediators and cellular mechanisms of mucosal C3 and SAA production
during endotoxemia are not known from the present study. In other
reports, several proinflammatory cytokines stimulated acute phase
protein synthesis in human intestinal epithelial cells (23). In recent
experiments in our laboratory, tumor necrosis factor (TNF) and
interleukin (IL)-1 increased the production of C3 in cultured Caco-2
cells (24). In the same studies, endotoxin added in vitro to the Caco-2
cells did not influence C3 production. Thus it is possible that the
increased mucosal levels of complement C3 and SAA noted here in
endotoxemic mice were regulated by cytokines rather than by a direct
effect of endotoxin, although further experiments are needed to test
that notion. Increased mucosal levels of TNF, IL-1, and IL-6 during
endotoxemia, as reported elsewhere (21, 22, 25), suggest that these
cytokines may regulate acute phase protein synthesis in cells of the
intestinal mucosa in a paracrine or autocrine manner.
Perspectives
The present results support the concept that the intestinal mucosa is
an active participant, rather than only a passive bystander, in the
metabolic and inflammatory response to sepsis and endotoxemia. Although
the majority of the acute phase proteins are probably produced in the
liver, the observations in this study are important because they
suggest that the intestinal mucosa may contribute to the acute phase
response. It should be an important focus for future studies to
determine the biological role of mucosal acute phase
proteins. It is likely that they are most important for the local metabolic response in the intestinal mucosa. Because mucosal
acute phase proteins may be secreted into the portal vein, it is also
possible that they can regulate the hepatic response to sepsis and
endotoxemia.
| |
ACKNOWLEDGEMENTS |
This study was supported in part by Grants 8510 and 8450 from
Shriners of North America.
| |
FOOTNOTES |
Address for reprint requests: P.-O. Hasselgren, Dept. of Surgery, Univ.
of Cincinnati, 231 Bethesda Ave., M. L. 558, Cincinnati, OH 45267-0558.
Received 19 November 1997; accepted in final form 10 August 1998.
| |
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