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Departments of
*
General Surgery and
Pulmonology, Maastricht University, Maastricht, The Netherlands
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, IL-6, and IL-1ß on the release of LBP and SAA by
intestinal epithelial cells (IEC). In addition, the induction of LBP
and SAA release by cell lines of intestinal epithelial cells and
hepatic cells was compared. The data obtained show that in addition to
liver cells, IEC also expressed LBP mRNA and released bioactive LBP and
SAA upon stimulation. Regulation of LBP and SAA release by IEC and
hepatocytes was typical for class 1 acute phase proteins, although
differences in regulation between the cell types were observed.
Endotoxin did not induce LBP and SAA release. Glucocorticoids were
demonstrated to strongly enhance the cytokine-induced release of LBP
and SAA by IEC, corresponding to hepatocytes. The data from this study,
which imply that human IEC can produce LBP and SAA, suggest a role for
these proteins in the local defense mechanism of the gut to endotoxin.
Furthermore, the results demonstrate that tissues other than the liver
are involved in the acute phase response. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The intestinal lumen contains high amounts of endotoxin, and the intestinal mucosa forms the interface between this potentially harmful material and the interior of the host. The intestinal epithelium contributes to the immunologic defense by secretion of proinflammatory cytokines (5) and microbicidal agents such as defensins (6). Although the liver has been held responsible for the production of circulating acute phase proteins, there is recent evidence for the synthesis of acute phase proteins in the intestinal mucosa as well (7, 8). This prompted us to study whether the release of LBP and the major acute phase protein serum amyloid A (SAA), which is involved in the routing and binding of high density lipoprotein (HDL) to inflammatory cells (9), is part of the epithelial response to inflammation. In the current study we examined the regulation of LBP and SAA secretion by a number of intestinal epithelial cell lines in detail and compared it to the regulation in hepatocytes. The data obtained show that intestinal epithelial cells can release both LBP and SAA, which strongly implicates a role for these proteins in the local inflammatory processes in the gut.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human recombinant TNF- was provided by BASF/Knoll
(Ludwigshafen, Germany); human recombinant IL-6 was provided by Prof.
W. Sebald (Physiologisch-Chemisches Institut de Universität
Wurzburg, Wurzburg, Germany); human rIL-1ß was a gift from Dr. S.
Gillis (Immunex, Seattle, WA); dexamethasone was a gift from Merck
Sharp, & Dohme (Haarlem, The Netherlands); MTT and LPS (from
Escherichia coli, serotype 055:B5) were purchased from Sigma
(St. Louis, MO). Human recombinant LBP was produced by transfected CHO
cells, provided by Dr. P. Tobias (Research Institute of Scripps Clinic,
La Jolla, CA). Polyclonal Abs to human rLBP were obtained by immunizing
rabbits with human rLBP. After protein A purification, IgG was
biotinylated. A specific anti-human LBP mAb HM14 was obtained by
immunizing mice with human rLBP following classical procedures. The
selection and properties of the mAb will be discussed in detail
elsewhere. In short, this mAb was selected on basis of its reactivity
with both free human LBP and human LBP that has formed a complex with
LPS. The SAA immunoassay was provided by Dr. P. C. Limburg
(Department of Rheumatology, University Groningen, Groningen, The
Netherlands).
Culture and stimulation of cells
The cell lines Caco-2, Colo-205, and T-84 were obtained from
American Type Culture Collection (Manassas, VA; ATCC HTB-37, CCL-222,
and CCL-248 respectively). Human colonic adenocarcinoma Caco-2 cells
undergo in vitro differentiation and express structural characteristics
and functional properties typical of small intestinal enterocytes. The
Colo-205 and T-84 cell lines are human colonic carcinoma cell lines.
The small intestinal epithelial cell line Int-407 was obtained from the
European Collection of Cell Cultures (Wiltshire, U.K.; ECACC 85051004).
Human hepatoma (HuH-7) cells were a gift of Dr. K. C. H.
Fearon, Department of Surgery, University of Edinburgh (Edinburgh,
U.K.). Cells were cultured at 37°C with 5%
CO2. HuH-7 and Colo-205 cells were cultured in
RPMI 1640 (Life Technologies, Paisley, U.K.) supplemented with 10% FCS
(HyClone, Logan, UT). Caco-2 cells were cultured in DMEM (Life
Technologies) supplemented with 10% FCS (HyClone), 1.0 mM sodium
pyruvate (Life Technologies), and 0.1 mM nonessential amino acids (Life
Technologies). Int-407 cells were cultured in MEM with Earle’s salts
(Life Technologies) supplemented with 10% FCS (HyClone) and 0.1 mM
nonessential amino acids (Life Technologies). All culture media were
supplemented with 100 IU of penicillin/ml and 100 µg of
streptomycin/ml (Life Technologies). Cell lines were maintained in
25-cm2 flasks (Costar, Cambridge, MA). Cultures
were split when they reached 80% confluence. For experiments designed
to investigate the effect of cytokines on the release of LBP and SAA,
cells were plated at a density of 1 x 104
cells/well on 96-well flat-bottom tissue culture plates (Costar). Cells
were cultured in complete medium as described above. The medium was
changed 48 h after plating and then every other day. After 2 wk,
Caco-2 monolayers display morphologic and functional properties similar
to those found in normal small intestinal enterocytes
(10). Therefore, all experiments with Caco-2 cells were
conducted between 14–17 days after seeding of the cells. Experiments
with Colo-205, T-84, Int-407, and HuH-7 cells were conducted after
confluence was established microscopically. At the start of each
experiment, culture medium was removed, and complete medium,
supplemented with IL-1ß, IL-6, TNF-, LPS, or dexamethasone, as
single agents or in combination as given in Results, was
added. Stimulation with LPS was performed in the presence of human
serum. At the end of the incubation period, the medium was removed and
centrifuged to remove cells and cell debris. Cell number and viability
were determined using the MTT method as described previously
(11). To determine whether the presence of serum affected
the cytokine-induced LBP and SAA release, Caco-2 cells were stimulated
under serum-free conditions using a protocol similar to stimulations
conducted in the presence of serum.
For experiments designed to determine whether endothelial cells also posses the capacity to release LBP and SAA, HUVEC were isolated from fresh umbilical cords by treatment with collagenase type I (Sigma). Cells were seeded into fibronectin-coated tissue culture flasks. For stimulation experiments cells were seeded in 96-well plates at a density of 1 x 104 cells/well and were cultured for 2–3 days to confluence. Cells were stimulated in a protocol similar to IEC and hepatocytes.
Detection of LBP mRNA
Total cellular RNA was isolated from quiescent and stimulated Caco-2 and HuH-7 cells using a Quick Prep Total RNA Extraction Kit (Pharmacia, Uppsala, Sweden). cDNA was obtained by RT of total RNA. PCR of ß2m was employed for standardization of the different RNA samples as described by Kloppenburg et al. (12). Human LBP-targeted PCR was performed in a 25-µl reaction mixture containing 100 µM of each dNTP, 200 nM of primers, and 0.5 U of Taq DNA polymerase (Perkin-Elmer/Cetus, Emeryville, CA). The primers were described by Su et al. (7): sense primer, 5'-AGG-GCC-TGA-GTC-TCA-GCA-TCT-3'; and antisense primer, 5'-CAG-GCT-GGC-CGT-GTT-GAA-GAC-3'. Thirty-five cycles were run under the following conditions: 95°C for 30 s, 57°C for 1 min, and 72°C for 1 min. The reaction product was analyzed on a 1.2% agarose gel containing ethidium bromide. A mock PCR (without cDNA) was included to exclude contamination.
Immunoassays
LBP and SAA release was evaluated using sandwich ELISAs. LBP was determined as described previously (13). In short, plates (Nunc-Immuno Plate Maxisorp, Roskilde, Denmark) were coated with polyclonal anti-human LBP Abs. Supernatants of the cells and a standard dilution series of rLBP were added to the plate. Detection was performed with a biotinylated polyclonal rabbit anti-human LBP IgG, followed by peroxidase-conjugated streptavidin (Zymed, San Francisco, CA) and tetramethylbenzidine (TMB) substrate (Kirkegaard & Perry Laboratories, Gaithersburg, MD). The detection limit was 200 pg/ml.
SAA was quantified as described previously (14). In short, SAA-specific mAb Reu 86.5 was used as capture reagent. The amount of SAA bound to the wells was quantified by incubation with a HRP-labeled monoclonal anti-SAA Ab (Reu 86.1) followed by TMB substrate. The detection limit for the SAA assay was 100 pg/ml.
Characterization of LBP released by Caco-2 cells
To confirm the identity of LBP released by Caco-2 cells, the protein was isolated from the medium by selective affinity immunosorption, followed by determination of the mr by Western immunoblotting. For this purpose the newly developed anti-LBP mAb HM14 was cross-linked to NHS-activated Sepharose (Pharmacia) according to the manufacturer’s instructions. Conditioned culture medium of Caco-2 cells was applied to the anti-LBP column, and the unbound proteins were washed out with PBS. Glycine-HCl buffer (pH 2.5) was used to elute bound LBP. For comparison, rLBP was isolated from the culture medium of transfected CHO cells by the same method. Gel electrophoresis of Caco-2-derived LBP, rLBP, and a prestained low mr marker was performed using 10–15% gradient polyacrylamide gels (Pharmacia) run in a Pharmacia Phast system followed by electrophoretic transfer onto an Immobilon-P membrane (Millipore, Bedford, MA). LBP bands were detected using a biotin-labeled polyclonal Ab to human LBP followed by peroxidase-labeled streptavidin and 3,3'-diaminobenzidine substrate.
The bioactivity of LBP produced by Caco-2 cells was determined by measuring the LPS binding capacity. For this purpose Immuno-Maxisorp plates were coated with the monoclonal anti-human LBP Ab HM14. Next, dilution series of LBP produced by Caco-2 cells and rLBP were added, followed by biotinylated LPS. For biotinylation of LPS a hydrazide-biotin reagent (Pierce, Rockford, IL) was used. Detection occurred with peroxidase-conjugated streptavidin and TMB substrate. The ODs obtained represent the LPS binding capacity of the LBP.
Statistical analysis
Data were analyzed using SPSS software (SPSS, Chicago, IL). All values in the figures and text are expressed as the mean ± SD of n observations. Groups were compared by Student’s t test. Results of experiments designed to study the effects of cytokines and dexamethasone on the secretion of LBP and SAA by Caco-2 and HuH-7 cells were hierarchically analyzed by four-way ANOVA, using dummy regression analysis after square root transformation of data. A p value of 0.05 or less was considered statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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First we investigated whether IEC express LBP mRNA. In vitro
differentiated human colonic adenocarcinoma (Caco-2) cells were used as
a model of normal human intestinal epithelial cells. The monolayers
were stimulated with a combination of IL-1ß (5 ng/ml), IL-6 (100
ng/ml), and TNF- (100 ng/ml). For comparison, human hepatoma (HuH-7)
cells were treated in a protocol similar to that used for Caco-2 cells.
LBP mRNA was expressed in quiescent Caco-2 and HuH-7 cells in minor
quantities. Subsequently, after culture of cells in the presence of
cytokines LBP mRNA expression was up-regulated substantially (Fig. 1
). The PCR product was of predicted size
(565 bp). These data are the first to demonstrate that intestinal
epithelial cells express LBP mRNA, which strongly suggests synthesis of
LBP by these cells.
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A).
IL-1ß and TNF- alone did not induce a marked up-regulation of the
LBP release, although both cytokines strongly enhanced the effect of
IL-6 (p < 0.01) and each other
(p < 0.001) on LBP release. The strongest
up-regulation was induced by the combination of IL-1ß, IL-6, and
TNF- (p < 0.01; Fig. 2A).
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B). The SAA release of the
cells was markedly up-regulated by IL-1ß (p
< 0.001), while treatment with either IL-6 or TNF- had no
significant effect (Fig. 2B). However, IL-6
(p < 0.01) enhanced IL-1ß-induced SAA
synthesis, while TNF further up-regulated the effect of the combination
of IL-1ß and IL-6 (p < 0.001). The necessity
of IL-1ß for apparent induction of SAA release by Caco-2 cells was
evident and is typical for class 1 acute phase proteins. In conclusion,
Caco-2 cells have the capability to release both LBP and SAA in
response to cytokines, while each protein has its own specific
induction pattern. To investigate whether the secretion of LBP and SAA is a general property of in vitro cultured cells, HUVEC were stimulated with proinflammatory cytokines. LBP and SAA was not detected in the supernatants (data not shown), which implicates that LBP and SAA secretion is not a common feature of cells in general or epithelial cells in particular.
Next, the concentration dependency of IL-6 and IL-1ß on the induction
of LBP and SAA release was studied (Fig. 3). A clear up-regulation of LBP release
is mediated in Caco-2 cells by 100 ng/ml Il-6. However, when cells were
treated with IL-6 plus IL-1ß simultaneously, the enhancement of LBP
release was evident at concentrations as low as 1 ng/ml IL-6 (Fig. 3A). The data demonstrate that 0.5 ng/ml IL-1ß is enough
to enhance SAA release. The synergistic effect of IL-6 on
IL-1ß-mediated SAA release was clear at 5 ng/ml (Fig. 3B).
Furthermore, synthesis of both proteins increased when concentrations
of IL-1ß and IL-6 were raised. The data from this experiment indicate
that cytokine concentrations that has been previously reported in
actively inflamed intestinal mucosa (16, 17) as well as in
the mesenteric blood (18) induce LBP and SAA secretion
by IEC.
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Time kinetics of LBP and SAA release by Caco-2 cells
Fig. 4 shows the time kinetics of
LBP and SAA release. After an incubation period of 24 h LBP and
SAA concentrations in the supernatants were modest. A substantial
increase was seen after 48 h, and at 72 and 96 h LBP and SAA
release was further increased. These data are in agreement with the
kinetics of LBP and SAA concentrations in the circulation during an
acute phase response in man (19, 20).
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The gastrointestinal tract contains high amounts of endotoxins that are important inducers of the acute phase response in vivo. To determine whether endotoxins can directly induce the release of LBP and SAA by IEC, endotoxin derived from Escherichia coli was added to Caco-2 cells cultured in the presence of human serum containing human LBP and soluble CD14, both necessary for nonimmune cells to respond to endotoxins. Concentrations up to 10 µg/ml endotoxin did not lead to enhanced release of LBP or SAA by Caco-2 cells (data not shown).
To elucidate whether endotoxins are able to enhance the cytokine-induced release of LBP and SAA, Caco-2 cells were incubated with a combination of different concentrations of endotoxin, IL-1ß, and IL-6. For all concentrations tested endotoxin did not affect the LBP and SAA release induced by IL-6 or IL-1ß (data not shown). In conclusion, these data indicate that LBP and SAA release by IEC is not influenced by endotoxins.
Regulation of LBP and SAA release in Caco-2 cells is different in HuH-7 cells
Acute phase proteins, such as LBP and SAA, are thought to be
predominantly liver derived. Therefore, the induction of LBP and SAA
release in liver cells was investigated for comparison. To this end,
HuH-7 cells were stimulated in a protocol similar to that used for
Caco-2 cells (Fig. 5). These experiments
revealed that with HuH-7 cells, as with Caco-2 cells, LBP release is
induced by IL-6 (p < 0.001), whereas SAA
release is induced by IL-1ß (p < 0.001).
Despite this conformity, several differences in the induction pattern
of both proteins were notable between both cell types. The data
demonstrate that IL-1ß (5 ng/ml) significantly
(p < 0.001) up-regulates the LBP release by
HuH-7 cells, whereas no effect on LBP release was observed in Caco-2
cells. Furthermore, TNF- potentiated the LBP and SAA synthesis
induced by the combination of IL-6 and IL-1ß in Caco-2 cells
(p < 0.01), whereas a down-regulation of this
induction was observed in HuH-7 cells (p <
0.01; Fig. 5). In addition, the amount of SAA released by Caco-2 cells
exceeded the LBP released by these cells, whereas the opposite was
found in HuH-7 cells. As in Caco-2 cells, endotoxin did not evoke
significant LBP and SAA release by HuH-7 cells (data not shown).
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An important feature of the systemic acute phase response is the
involvement of glucocorticoids. These glucocorticoids enhance the
IL-1-, TNF-, and IL-6-mediated induction of the acute phase response in
the liver. To elucidate the effect of glucocorticoids on
cytokine-mediated release of LBP and SAA by IEC, Caco-2 cells were
stimulated in the presence of 1 µM dexamethasone, a synthetic
glucocorticoid. Dexamethasone enhanced the LBP synthesis induced by
IL-6 (p < 0.05) and markedly enhanced the
induction by the combination of IL-6, IL-1ß, and TNF-
(p < 0.01; Fig. 6A). Furthermore, spontaneous
SAA release and SAA release induced by the combination of IL-1ß and
IL-6 were strongly enhanced by dexamethasone (Fig. 6B). The
enhancement of LBP and SAA release by glucocorticoids was concentration
dependent (data not shown). A negative interaction between
dexamethasone and the combination of IL-1ß, IL-6, and TNF- on SAA
release was found (p < 0.001). These data
indicate a role for glucocorticoids in the local acute phase response
by regulation of the cytokine-mediated LBP and SAA release in the
gut.
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To characterize LBP produced by Caco-2 cells the
Mr of the isolated protein was
determined by Western immunoblotting and compared with that of rLBP.
LBP produced by Caco-2 cells migrated as a single band with a
Mr of approximately 60 kDa, identical
with that of rLBP (Fig. 7).
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). These results ascertain the
functional capacity of LBP produced by intestinal epithelial cells.
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To elucidate whether the release of LBP and SAA is restricted to
Caco-2 cells or is a common feature of IEC, the capacities of three
other intestinal epithelial cell lines to release LBP and SAA were
evaluated. For this purpose Colo-205 and T-84 cells, both derived from
the human colon, and Int-407 cells, derived from human ileum/jejunum,
were cultured in the presence of IL-1ß (5 ng/ml), IL-6 (100 ng/ml),
TNF- (100 ng/ml), and 1 µM dexamethasone. LBP release was
inducible by cytokines in all tested cell lines. Nonstimulated cells
produced only minute amounts of LBP. SAA was only detectable in the
supernatant of stimulated Int-407 cells (Table I). These data confirm the capacity of
human intestinal epithelial cells to synthesize and release
LBP.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The results reveal that Caco-2, Colo-205, T-84, and Int-407 cells
produce LBP. To our knowledge, this is the first evidence for the
production of LBP by human IEC. Investigation of the induction of LBP
release revealed that IL-6 induced LBP release by Caco-2 cells, while
IL-1ß and TNF- showed a synergistic effect when used together with
IL-6. Similarly, LBP synthesis was induced in liver cells by IL-6,
whereas the effects of IL-1ß and TNF- on IL-6-induced LBP release
were additive. TNF- down-regulated the LBP synthesis induced by a
combination of IL-1ß and IL-6 in liver cells, whereas the opposite
effect was found in Caco-2 cells. The data concerning the release of
LBP by liver cells are supported by other reports (1, 21, 22).
SAA synthesis by Caco-2 cells was predominantly regulated by IL-1ß,
and this induction was enhanced by IL-6. This regulation is consistent
with the SAA synthesis in liver cells, which is also primarily
regulated by IL-1ß and shows strong synergism with IL-6
(23). The regulation of SAA synthesis by TNF- in Caco-2
cells was shown to differ substantially from the regulation of SAA
release by liver cells. TNF- down-regulated the IL-1ß-induced SAA
release by HuH-7 cells, while in Caco-2 cells IL-1ß and TNF-
showed an additive effect on SAA release. Subsequently, parallel to the
differences seen for LBP induction between these cell types, TNF-
suppressed the SAA release induced by the combination of IL-6 and
IL-1ß in liver cells, while a synergistic effect was seen in Caco-2
cells. In summary, both proteins behave like secretory class 1 acute
phase proteins in IEC, similar to their regulation in hepatocytes
(19, 21, 23, 24), although differences in the regulation
of LBP and SAA release between both cell types were discernible. These
differences possibly reflect a distinction between the regulation of
systemic levels, controlled by the liver, and the regulation of local
tissue levels in the intestine, for which epithelial cells are
responsible. The high levels of cytokines found in the intestine during
intestinal inflammation (25, 26) seem in line with our
hypothesis of a localized regulation of acute phase protein synthesis
in the gut. Furthermore, the ratio of SAA to LBP secreted by Caco-2
cells is high compared with that in HuH-7 cells. It is not possible to
conclude from the data that the absolute amount of SAA secreted by IEC
exceeds the secretion of SAA by liver cells, because the numbers of
Caco-2 and HuH-7 cells per well were not identical during experiments.
However, these data clearly show a dramatic increase in SAA synthesis
by Caco-2 cells in response to cytokines, which suggests that the
intestinal mucosa is an important extrahepatic production site of SAA
and which may reflect an immediate local requirement for this
apolipoprotein during inflammation.
In contrast to cytokines, endotoxin induced neither LBP nor SAA release by Caco-2 cells in the presence of soluble CD14. The lack of response to endotoxin in Caco-2 cells is described for other acute phase proteins as well (8). Hypothetically this absence of response protects the host from an ongoing stimulation by endotoxins present in the gut lumen. Our data indicate that an inflammatory reaction leading to cytokine release is a necessary step for the induction of LBP and SAA release by IEC. Endotoxin enters under pathological circumstances the lamina propria, where it causes inflammation and the release of cytokines such as IL-6, IL-1, and TNF. We presume that these cytokines induce the release of acute phase proteins by the neighboring epithelium. However, it has been described that IEC themselves can generate cytokines in response to an endotoxin or bacterial challenge (27), although no consensus has been achieved in the literature concerning this property of IEC (8).
Glucocorticoids were demonstrated to enhance the cytokine-mediated induction of LBP and SAA release by IEC. This result is consistent with the enhancing effect of glucocorticoids on cytokine-induced SAA (24) and LBP (19, 21) synthesis by liver cells, as reported previously and confirmed in this study (data not shown). The data indicate that the local synthesis of acute phase proteins in the gut is reinforced by glucocorticoids, which are synthesized by the adrenal glands in high amounts during a systemic acute phase response.
We hypothesize that the release of LBP by human intestinal epithelial cells is involved in the defense against endotoxins, which is pre-eminently important in the mucosa. Besides its role in neutralization and clearance of endotoxin, a basal concentration of LBP is known to enhance the sensitivity of the immune system to endotoxin by catalyzing the binding of endotoxin to macrophages. LBP produced by IEC may therefore also have a function in sensitizing the mucosal immune system to translocated endotoxins. However, high concentrations of LBP have been shown to decrease endotoxin activity (4). The ratio of LBP to endotoxin is reported to determine a number of essential processes: the monocytic response (4), the binding of LBP to HDL (28), and the inactivation of endotoxin by HDL (29). We suggest that local production of LBP by epithelial cells during inflammation in the intestine may result in high local concentrations of LBP and consequently to a reduced toxicity of endotoxin. The proposed importance of LBP in local defense against Gram-negative bacteria is supported by recent studies demonstrating that i.p. administration of bacteria is tolerated by wild-type mice, although it results in uncontrolled multiplication and spread of bacteria in LBP knockout mice (30). Moreover, i.p. injection of LBP can reduce mortality mediated by endotoxin and bacteria in mice (4).
Secretion of SAA by the intestinal epithelium refers to a role for SAA in the local inflammatory processes in the intestine. Although the primary function of SAA in the acute phase response is not fully understood, several immunological functions have been proposed. There is evidence that SAA enhances the binding of HDL, which possesses substantial endotoxin binding capacity (2), to macrophages (9). Furthermore, it was demonstrated recently that SAA has cytokine-like properties (31). Further studies are necessary to determine the biological role of SAA secretion by the intestinal mucosa.
In summary, we have demonstrated the release of LBP and SAA by IEC in response to specific sets of cytokines and glucocorticoids. Endotoxin is shown not to induce the release of LBP and SAA by IEC, indicating that cytokine release in the proximity of the epithelial cells is necessary for the induction of LBP and SAA release by these cells. The capability of IEC to release LBP and SAA suggests a role for the intestinal epithelium in the acute phase response and the local defense of the gut against bacteria and endotoxin.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. A. C. E. Vreugdenhil, Department of General Surgery, Maastricht University, P.O. Box 616, 6200 MD Maastricht, The Netherlands. E-mail address:
3 Abbreviations used in this paper: LBP, LPS binding protein; IEC, intestinal epithelial cells; SAA, serum amyloid A; HDL, high density lipoprotein; TMB, tetramethylbenzidine.
Received for publication March 11, 1999. Accepted for publication June 25, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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production linked to the toxicity of indomethacin for the rat small intestine. Br. J. Pharmacol. 124:1385.[Medline]
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 W. Khovidhunkit, M.-S. Kim, R. A. Memon, J. K. Shigenaga, A. H. Moser, K. R. Feingold, and C. Grunfeld Thematic review series: The Pathogenesis of Atherosclerosis. Effects of infection and inflammation on lipid and lipoprotein metabolism mechanisms and consequences to the host J. Lipid Res., July 1, 2004; 45(7): 1169 - 1196. [Abstract] [Full Text] [PDF]
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M. d. J. Arana, M. G. Vallespi, G. Chinea, G. V. Vallespi, I. Rodriguez-Alonso, H. E. Garay, W. A. Buurman, and O. Reyes Inhibition of LPS-responses by synthetic peptides derived from LBP associates with the ability of the peptides to block LBP-LPS interaction Innate Immunity, October 1, 2003; 9(5): 281 - 291. [Abstract] [PDF]
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D. D. Bannerman, M. J. Paape, W. R. Hare, and E. J. Sohn Increased Levels of LPS-Binding Protein in Bovine Blood and Milk Following Bacterial Lipopolysaccharide Challenge J Dairy Sci, October 1, 2003; 86(10): 3128 - 3137. [Abstract] [Full Text] [PDF]
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A. C. E. Vreugdenhil, C. H. Rousseau, T. Hartung, J. W. M. Greve, C. van 't Veer, and W. A. Buurman Lipopolysaccharide (LPS)-Binding Protein Mediates LPS Detoxification by Chylomicrons J. Immunol., February 1, 2003; 170(3): 1399 - 1405. [Abstract] [Full Text] [PDF]
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 R. Berner, B. Furll, F. Stelter, J. Drose, H.-P. Muller, and C. Schutt Elevated Levels of Lipopolysaccharide-Binding Protein and Soluble CD14 in Plasma in Neonatal Early-Onset Sepsis Clin. Vaccine Immunol., March 1, 2002; 9(2): 440 - 445. [Abstract] [Full Text] [PDF]
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J. Zweigner, H.-J. Gramm, O. C. Singer, K. Wegscheider, and R. R. Schumann High concentrations of lipopolysaccharide-binding protein in serum of patients with severe sepsis or septic shock inhibit the lipopolysaccharide response in human monocytes Blood, December 15, 2001; 98(13): 3800 - 3808. [Abstract] [Full Text] [PDF]
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 T. Gutsmann, M. Muller, S. F. Carroll, R. C. MacKenzie, A. Wiese, and U. Seydel Dual Role of Lipopolysaccharide (LPS)-Binding Protein in Neutralization of LPS and Enhancement of LPS-Induced Activation of Mononuclear Cells Infect. Immun., November 1, 2001; 69(11): 6942 - 6950. [Abstract] [Full Text] [PDF]
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 C. de Coupade, M. N. Ajuebor, F. Russo-Marie, M. Perretti, and E. Solito Cytokine Modulation of Liver Annexin 1 Expression during Experimental Endotoxemia Am. J. Pathol., October 1, 2001; 159(4): 1435 - 1443. [Abstract] [Full Text]
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A. C. E. Vreugdenhil, A. M. P. Snoek, J. W. M. Greve, and W. A. Buurman Lipopolysaccharide-Binding Protein Is Vectorially Secreted and Transported by Cultured Intestinal Epithelial Cells and Is Present in the Intestinal Mucus of Mice J. Immunol., October 15, 2000; 165(8): 4561 - 4566. [Abstract] [Full Text] [PDF]
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M. G. Scott, A. C. E. Vreugdenhil, W. A. Buurman, R. E. W. Hancock, and M. R. Gold Cutting Edge: Cationic Antimicrobial Peptides Block the Binding of Lipopolysaccharide (LPS) to LPS Binding Protein J. Immunol., January 15, 2000; 164(2): 549 - 553. [Abstract] [Full Text] [PDF]
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, significant negative interaction
between dexamethasone and cytokines (p <
0.001).
).
Concentration range of Caco-2-derived LBP and rLBP added to plates
coated with anti-LBP mAb HM14. Binding of biotinylated LPS to the
immobilized LBP was determined. Data are expressed as OD 450 nm.