Section of Pediatric Surgery, Department of Surgery, University of Michigan Medical School and C. S. Mott Children's Hospital, Ann Arbor, Michigan 48109
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ABSTRACT |
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Total
parenteral nutrition (TPN) results in an increase in intraepithelial
lymphocyte (IEL)-derived interferon- (IFN-
) expression as well as
an increase in epithelial cell (EC) apoptosis. This study
examined the role that IEL-derived IFN-
has in the increase in EC
apoptosis. Mice received either TPN or oral feedings for 7 days. Small bowel EC apoptosis significantly rose in mice
receiving TPN. The administration of TPN also significantly increased
IEL-derived IFN-
and Fas ligand (FasL) expression. EC
apoptosis in IFN-
knockout (IFNKO) mice that received TPN
was significantly lower than in wild-type TPN mice. Sensitivity of EC
to Fas-mediated apoptosis in IFNKO mice was significantly lower
than in wild-type TPN mice. Apoptosis in Fas-deficient and
FasL-deficient mice that received TPN was significantly lower than in
wild-type mice that received TPN. The TPN-induced increase in IFN-
expression appears to result in an increase in Fas-L expression and EC
sensitivity to Fas, with a resultant increase in EC apoptosis.
This may well be one of the mediators of increased EC apoptosis
observed with TPN administration.
epithelial cells; cytokine; Fas; Fas ligand
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INTRODUCTION |
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INTESTINAL MUCOSAL HOMEOSTASIS depends on a balance between cell proliferation and cell death. A major outcome in which cell death exceeds cell proliferation is the development of villus atrophy (11). Atrophy is best exemplified both clinically and experimentally by the use of total parenteral nutrition (TPN) (5, 23). This atrophy may be seen as early as 1 wk in rats and 2 wk in humans and mice receiving TPN. The etiology of TPN-associated villus atrophy is unknown. However, recently, TPN-induced atrophy has been associated with a significant increase in epithelial cell (EC) apoptosis (6). The mechanism for the increase in TPN-associated EC apoptosis remains unknown.
Apoptosis is a cellular suicide program that is a regular, nonnecrotic form of cell death. Apoptosis in EC can be regulated by a variety of signals of which the Fas/Fas ligand (Fas/FasL) pathway is one of the best described (7, 19, 30). Fas is a transmembrane receptor in the TNF receptor family (19, 25) that transduces an apoptotic signal into the cell when cross-linked by FasL. Fas is expressed constitutively on the basolateral membrane of normal small intestinal epithelium (27). The ligand for Fas is expressed by intraepithelial lymphocytes (IEL) and lamina propria lymphocytes. Lymphocytes expressing FasL are not dependent on either classical or nonclassical major histocompatibility complex restriction but recognize cells expressing Fas (22, 29, 34). It has been shown that the small intestine expresses abundant levels of both Fas and FasL mRNA (39). Several lines of evidence suggest that Fas/FasL interactions are important in immune-epithelial communication in the intestine (24, 25). It has also been shown that IEL upregulates the expression of the FasL gene, inducing EC activation-induced apoptosis (12, 14, 17). There is increasing evidence that both cytokines and growth factors arising within the epithelium are potent regulators of enterocyte proliferation and apoptosis (3, 8, 15, 21, 35).
IFN- has been reported to regulate apoptosis in many cells.
Several researchers have reported that IFN-
can promote an
apoptotic process via an upregulation of Fas/FasL, as well as an
increased sensitivity of EC to Fas-mediated apoptosis (1,
24, 26). In a human colon cancer line, others (2)
have found that HT-29 cells rapidly undergo apoptosis in the
presence of IFN-
. In addition, we have previously demonstrated
(20) that TPN results in a significant increase in
IEL-derived IFN-
expression. On the basis of this evidence, we
hypothesized that an increased IEL-derived IFN-
expression resulting
from TPN administration may induce an increase in EC apoptosis.
Additionally, we hypothesized that TPN-induced EC apoptosis is
mediated by IFN-
through a Fas/FasL interaction.
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MATERIALS AND METHODS |
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Animals
All mice were obtained from Jackson Laboratory (Bar Harbor, ME). Male, specific, pathogen-free, adult C57BL/6J mice were maintained under temperature, humidity, and light-controlled conditions. In some experiments, IFN-TPN Model
The administration of TPN was performed as previously described (20). Mice were infused intravenously (via external jugular vein) with crystalloid solution (dextrose 5% in 0.45 normal saline with 20 meq KCl/l) at 4 ml/24 h. After 24 h, mice were randomized into either a TPN group or a control group. In some studies, IFN-Histology
A 5-mm segment of jejunum was removed after death and fixed in 10% formaldehyde for histological sectioning. Tissues were then dehydrated with ethanol and embedded in paraffin. Sections were cut and stained with hematoxylin-eosin. The villus height and total height for each specimen were measured by using a calibrated micrometer. Each measurement for villus height consisted of the mean of seven different fields.Terminal Deoxynucleotidyl Transferase-Mediated dUTP Nick-End Labeling Assay for EC Apoptosis Detection
An in situ cell death detection kit (Roche, Mannheim, Germany) was used for terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay according to the manufacturer's protocol. Briefly, jejunal tissues were fixed in 10% formalin embedded in paraffin and were cut transversely in 5-µm-thick sections. Sections were deparaffinized with xylene and taken through a graded ethanol (100 to 70%) rehydration. The sections were incubated with proteinase K (20 µg/ml) in 10 mM Tris · HCl (pH 8.0) for 30 min and washed with PBS. Slides were then incubated with a permeabilization solution (0.1% sodium citrate) on ice, washed and incubated with an endogenous peroxidase (3% H2O2 for 10 min), and washed again. The slides were then incubated with the TUNEL reaction mixture (enzyme and label solution) in a humidified chamber at 37°C for 30 min. After slides were washed with PBS, the sections were incubated with peroxidase at 37°C for 30 min and treated with diaminobenzidine staining solution for 5-10 min at room temperature. Finally, slides were counterstained with hematoxylin. The sections were examined by light microscopy at ×45 magnification. Positive apoptotic reaction cells were defined as cells with a brown-stained nuclei or as apoptotic bodies that are fragments of apoptotic cells engulfed by neighboring ECs. IELs were excluded by morphology. The ratio of TUNEL-positive cells to all ECs was recorded as apoptotic index (%).Mucosal Cell Isolation
IEL isolation. Isolation was performed after the protocol described by Mosley et al. (28). It has been proven that this isolation method does not penetrate into the lamina propria, avoiding the contamination of the IEL with lamina propria lymphocytes (28). Briefly, a section of the small intestine was removed and placed in tissue culture media (RPMI 1640, with 10% fetal calf serum). The section was cut into 5-mm pieces, washed in an extraction buffer, and incubated in the same buffer with continuous brisk stirring at 37°C for 25 min. The supernatant was then filtered rapidly through a glass wool column. After centrifugation, the pellets were purified in 40% isotonic Percoll (Upjohn & Pharmacia, Sweden) and reconstituted in RPMI tissue culture media. Viability of IELs exceeded 95% by using trypan blue exclusion staining.
EC isolation. EC were isolated by using a modified nonenzymatic technique (13, 28). Briefly, a section of the small intestine was removed and placed in tissue culture media (RPMI 1640, with 10% fetal calf serum). The intestine was opened longitudinally to expose the entire mucousal surface. Mucus and debris were removed. The section was cut into 5-mm pieces and incubated in PBS containing 1 mM EDTA plus 1 mM DTT with continuous brisk stirring at 37°C for 25 min. After this incubation, the cells suspended in isolation buffer were immediately passed through filtering cylinders prepared by nylon wool to a tissue culture bottle and then centrifuged at 4°C. Cells were then reconstituted in RPMI tissue culture media at 4°C.
For Fas/FasL gene expression study, EC were purified by using magnetic beads conjugated with antibody to CD45 (lymphocyte specific) to remove non-ECs (BioMag SelectaPure Anti-Mouse CD 45R Antibody Particles, Polyscience, Warrington, PA). Cells bound to beads were considered purified IEL, the supernatant containing EC was saved for RNA isolation.Flow Cytometry
Reagents and antibodies.
The following antibodies were also obtained from PharMingen (San Diego,
CA): FITC conjugated anti-mouse CD4, CD8, and CD3; PE conjugated
anti-mouse Fas and FasL; and IFN-
. Isotype-matched, irrelevant
antibodies (PE hamster antibody IgG, FITC anti-rat IgG2
and PE
antibody anti-rat IgG2
) were used as the negative controls.
Induction of Apoptosis Using Anti-Mouse Fas Antibody
Isolated EC from wild-type and IFNKO control and TPN mice were placed in 96-well plates at a concentration of 2×106 EC per well. Monoclonal antibody to Fas (Hamster anti-mouse) at 4 µg/ml (PharMingen) and 2 µg/ml Protein G (Sigma, St Louis, MO) were added to each well. Negative control wells contained medium alone without Fas antibody. Cells were incubated for 2 h at 37°C. EC apoptosis was identified by using an Apoptosis Kit (PharMingen) according to manufacturer's instructions. EC were double stained with fluorescein isothiocyanate (FITC)-conjugated AnnexinV (as a marker of apoptosis) and propidium iodide (as a marker of cell necrosis). Cells (10,000 per sample) were analyzed for apoptosis by using a FASCalibur (Beckman-Dickinson, Franklin Lake, NJ). The apoptotic index is expressed as percentage of apoptotic cells per 100 ECs determined by flow cytometry.IEL IFN- Expression
RT-PCR
A guanidine isothiocyanate/chloroform extraction method was used to isolate RNA. Cells were suspended at a concentration between 2 and 10 × 106 cells/ml in 50 µl of Hank's balanced salt solution. TRIzol (GIBCO-BRL) was used for total RNA extraction according to manufacturer's instruction. Specimens were frozen atPoly A tailed mRNA was reversed transcribed into cDNA by adding 50 µg/ml of total cellular RNA to the following reaction mixture: dATP, dCTP, dTTP, and dGTP nucleotides each at 1 mM (Boehringer-Mannheim, Indianapolis, IN), 8 U/ml Maloney Murine Leukemia virus (GIBCO-BRL), 2.5 mM oligo(dT) (New England Biolabs, Beverly, MA); 2 U/ml RNAase (Boehringer-Mannheim) and diethyl pyrocarbonate (DEPC)-treated H2O was added to adjust volume. Samples (50 µl) were incubated at 39°C for 1 h, and the reaction was terminated by heating to 95°C for 5 min.
A series of PCRs was performed with oligomers designed for Fas and
FasL. Oligomers were designed by using an optimization program (OLIGO
4.1; National Biosciences, Plymouth, MN). The sequences of the Fas
(GeneBank accession no. M83649) and FasL (GeneBank accession no.
U06948) oligonucleotide primers were as follows: Fas forward primer,
5'-AGG AGG GCA AGA TAG ATG AGA-'3; Fas reverse primer, 5'-CAA AGA GAA
CAC ACC AGG AGT-'3; FasL forward primer, 5'-CAA GGT CCA ACA GGT CAG
CTA-'3; FasL reverse primer, 5'-CGG CTC AGA AAA CAT TAG GTA-'3. For
beta actin (GeneBank accession no. M12481) forward primer was 5'-TCT
ACG AGG GCT ATG CTC TC-'3, and reverse primer was 5'-AAG AAG GAA GGC
TGG AAA AG-'3. The following protocol was used (final concentration): 2 µl of RT product, added to 3' and 5' specific oligomers (5 mM); PCR
buffer (with 10 mM Tris and 50 mM KCl), MgCl2 (2.5 mM) and
Taq polymerase (Perkin Elmer, Foster City, CA; 0.4 U/sample), with sufficient DEPC-treated H2O to allow for
appropriate concentrations. The following thermal cycler (PTC-100, MJ
Research, Watertown, MA) settings were used: 94°C for 2 min, followed
by 28-30 cycles of 94°C × 15 s; 55°C × 15 s and 72°C × 30 s; followed by 5 min at 72°C.
The number of thermocycles was adjusted to ensure that the DNA product
was at the exponential portion of the amplification curve. The PCR products were run out on a 2% agarose gel containing ethidium bromide,
for 1 h at 170 V and then visualized under ultraviolet light.
Quantification of cDNA product was performed by recording a digital
image (.tif file) with a video camera system, under ultraviolet light
(Alpha Imager, Alpha Innotech, San Diego, CA). The image was analyzed
and product bands quantified by using imaging software (ImageQuant V
4.2, Molecular Dynamics, Sunyvale, CA). Results are expressed according
to previously established methods (10, 38, 42, 44). For
each sample, the abundance of mRNA expression was normalized to the
expression of -actin. This allowed us to control for minor
variations in RNA loading, as well as qualitative differences in the
RNA between experimental groups. We thus expressed mRNA as a ratio of
target gene to
-actin mRNA expression. Once this correction was
performed, we then compared control groups to other experimental groups
by using ANOVA (see below).
EC Proliferation
Mice were injected intraperitoneally with 5-bromo-2-deoxyuridine (BrdU, 50 mg/kg; Roche Diagnostic, Indianapolis, IN) 1 h before mice were killed. Paraffin-embedded small bowel sections of 5-µm thickness were deparaffinized with xylene. Immunohisochemistry was done by using a BrdU in-site detection kit according manufacturer's guidelines (BD PharMingen). Briefly, endogenous peroxidase was quenched with 3% H2O2. Slides were then incubated with biotinylated anti-BrdU antibody in a 1:10 dilution, and then with streptavidin-horseradish peroxidase. Slides were then exposed to diaminobenzidine (DAB) substrate. Finally, slides were counterstained with hematoxylin. An index of the crypt cell proliferation rate was calculated by the ratio of the number of crypt cells incorporating BrdU to the total number of crypt cells. The total number of proliferating cells per crypt was calculated by counting proliferating cells in a total of 10 crypts per histological section (counted at ×45 magnification).Statistics
All data are expressed as means ± SD. Kruskal-Wallis one-factor ANOVA was used for statistical analysis. Differences were considered significant at P < 0.05. ![]() |
RESULTS |
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General Description
Body weights at the beginning and end of the study for the standard C57Bl/6 J mice were 25.6 ± 1.4 and 23.3 ± 1.3 g, respectively for the TPN group, and 25.0 ± 1.7 and 24.8 ± 1.4 g, respectively for the control group. There was no statistical difference between the two groups.Mice given 7 days of TPN experienced a significant loss of villus
height, compared with the controls (Fig.
1 and Table
1). There was a decrease in both villus
height and total height in the TPN group. Villus height in the TPN mice
decreased 32.3% compared with controls. Total height of jejunum in TPN
mice decreased 27.7% compared with the controls.
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TPN Leads to an Increase in EC Apoptosis and a Decrease in EC Proliferation
Based on previous investigator's results (6), we hypothesized that the administration of TPN would lead to an increase in EC apoptosis and a decline in EC proliferation. TUNEL positive cells were seen at the both crypt and villus in both control and TPN mice. The apoptotic rate was found to be significantly increased in small bowel ECs in mice receiving TPN. TPN administration led to a threefold increase in EC apoptosis as compare with control mice (P < 0.01) (Fig. 2).
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There was no difference in labeled cell position between groups of mice
injected with BrdU. BrdU-positive cells were all distributed in the
crypt of Lieberkuhn of the small intestine. TPN was associated with a
44.7% decrease in BrdU-positive cells when compared with controls
(P < 0.01) (Fig. 3). Our
results support our hypothesis of a TPN-induced increase in EC
apoptosis and a decline in EC proliferation. Although the
observed decline in villus height may be secondary to the increase in
EC apoptosis, other factors clearly have a role. Specifically,
the decline in EC proliferation may greatly influence changes in villus
height. Thus, to more directly observe TPN-induced changes, we
specifically examined EC apoptotic changes.
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TPN Increased IEL-Derived IFN- Expression
Because of the unique phenotype of the IEL, IFN- protein expression
was further analyzed by using intracellular staining followed by flow
cytometry. This allowed a detailed examination of the specific
subpopulations of the IEL that expressed or did not express IFN-
. A
mean of 32.6 ± 4.2% of small bowel IEL expressed IFN-
protein
in the TPN group and 14 ± 2.1% of IEL in the control group
expressed IFN-
(P < 0.05). Flow cytometry analysis
showed that TPN significantly increased the CD8+ and CD3+ IEL-derived IFN-
protein expression but did not increase IFN-
in the CD4+ phenotype (Table 2).
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IFN- Mediates EC Apoptosis Through Fas/FasL
Interaction
IFNKO TPN mice have lower rates of apoptosis than wild-type
TPN mice.
Analysis of TUNEL data showed that apoptosis in the IFNKO mice
that received TPN was significantly lower than in the wild-type (C57BL/6J) TPN group. The apoptotic rate in IFNKO TPN mice was nearly twofold lower than TPN wild-type mice (1.88 ± 0.17 vs. 3.71 ± 0.38) (Fig. 2). Despite this decline in apoptosis
in the IFNKO TPN group, levels of apoptosis remained
significantly higher than in control mice (Fig. 2). This suggests that
there are other non-IFN--dependent pathways that contribute to EC
apoptosis. Thus IFN-
has a significant role in the mediation
of TPN-associated EC apoptosis. To investigate the role of Fas
and FasL, we initially measured the expression of Fas and FasL in our
mouse model.
FasL expression significantly increased in mice receiving TPN.
To understand the changes in FasL gene expression after TPN
administration, RT-PCR was used to detect IEL-derived FasL mRNA expression with or without TPN. FasL mRNA expression in the TPN group
for standard C57BL/6 J mice was significantly higher than in controls
(Fig. 4; P < 0.01). EC-
derived Fas mRNA expression, however, did not significantly change
after TPN compared with controls (Table
3).
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Lower IEL-derived FasL expression in IFNKO TPN mice.
To investigate the role of IFN- in the increase in FasL mRNA
expression, IFNKO mice were used in the TPN model. FasL mRNA expression
in the IFNKO TPN group was significantly lower than in the wild-type
TPN group (P < 0.01). There was no significant difference in FasL mRNA expression between IFNKO TPN and INFKO control
groups (P > 0.05) (Fig. 4), suggesting that baseline
levels of FasL mRNA expression may not be influenced significantly by IFN-
in this model. Fas mRNA expression was also analyzed by RT-PCR.
There was no difference in Fas mRNA expression between the IFNKO and
wild-type groups (P > 0.05) (Table 3).
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TPN increased EC sensitivity to Fas-mediated apoptosis.
There was no significant difference in EC Fas expression between the
TPN and control groups. Despite this lack of change in Fas, EC
apoptosis was significantly higher in the TPN group. Because it
was recently shown that IFN- treatment can induce human intestinal ECs into Fas-mediated apoptosis without changing Fas expression (26), we hypothesized that the increased expression of
IFN-
with TPN administration would similarly increase EC sensitivity to Fas-mediated apoptosis. To address this, EC from wild-type and IFNKO mice were incubated with anti-CD95 (antibody to Fas). Fas
antibody induced EC apoptosis in TPN wild-type mice at a
significantly higher rate (45.3 ± 6.8%) than in control mice
(19.1 ± 4.9%). This level of apoptosis in TPN mice was
also significantly higher than in both IFNKO TPN and IFNKO control mice
(Fig. 5), P < 0.01.
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Fas/FasL interaction plays an important role in this TPN-induced
apoptotic process.
IFN- was found to be responsible for a large part of EC
apoptosis after TPN administration. This increase in
IEL-derived IFN-
appeared to mediate the increase in IEL FasL
expression and increase EC sensitivity to Fas-mediated
apoptosis. To further investigate the role of Fas and FasL in
the development of TPN-associated apoptosis, Fas-deficient
(lpr) and FasL-deficient (gld) mice were used. EC
apoptosis in lpr TPN mice was 1.86 ± 0.31%
and 2.05 ± 0.28% in gld TPN mice. Both lpr
and gld levels were significantly lower than in wild-type
TPN mice. However, there was no significant difference in EC
apoptosis between gld and lpr control
mice and wild-type control mice (Fig. 2) (P > 0.05).
The TPN-induced increase in IFN-
expression appears to result in an
increase in Fas-L expression and EC sensitivity to Fas, with a
resultant increase in EC apoptosis. This may well be one of the
mediators of increased EC apoptosis observed with TPN administration.
Influence of IFN- Expression on TPN-Induced Villus
Atrophy
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DISCUSSION |
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This study examined TPN-associated apoptotic changes in the
intestinal mucosa and the possible etiology of these changes. We found
that the removal of enteral nutrition, with the administration of TPN,
resulted in intestinal mucosal atrophy. Apoptosis was significantly increased, and proliferation was significantly decreased in small bowel EC after TPN administration. Along with these
apoptotic changes, IEL-derived IFN- expression rose
significantly after TPN. IEL FasL expression also significantly
increased. To further investigate the role of IFN-
in this
apoptotic process, IFNKO mice were used. EC apoptosis and
IEL FasL expression were found to be significantly lower in IFNKO TPN
mice compared with wild-type TPN mice. Additionally, the sensitivity of
EC to Fas-mediate apoptosis was found to be significantly
higher in TPN wild-type mice than in TPN IFNKO mice. EC
apoptosis in Fas and FasL-deficient TPN mice was also
significantly lower than in wild-type TPN mice. These results strongly
suggest that TPN-induced EC apoptosis is mediated to a large
extent by increased IFN-
expression via a Fas/FasL apoptosis signaling.
The small bowel epithelium is one of the most rapidly proliferating tissues in the body, renewing itself every 3-8 days (33). Homeostasis is achieved by the balance of cell proliferation located in the crypts of Lieberkühn and cell elimination by apoptosis occurring in both the crypts and villus compartments. Experiments in rats on TPN show extensive shortening of villus height occurring within 3-4 days after initiation of TPN (16). Numerous mechanisms responsible for this loss of villus height have been proposed (5, 32). Included among these are a loss of hormonal stimulation and a loss of nutritional substrate including a deficiency of glutamine (6, 32, 41). Recently, it has been observed that the process of villus atrophy with TPN administration is associated with a marked increase in EC apoptosis (6). This study similarly confirmed that TPN led to villus atrophy after 7 days of TPN. During this same time period, there was a threefold increase in epithelial apoptosis and a nearly twofold decrease in epithelial proliferation. Clearly, TPN administration affects the homeostatic balance between EC proliferation and cell death in these subjects.
Mechanisms of apoptosis vary among different cell types and may involve an array of signaling processes and regulators influencing multiple cellular functions. Apoptosis has been shown to be induced by IEL via two distinct pathways. First, by the secretion of perforin, a pore-forming protein is inserted into the plasma membrane of the EC. Perforin allows granzyme B to pass through it, where it activates the caspases. Second, IEL-mediated apoptosis may be through the ligation of IEL's FasL to the transmembrane Fas-receptor on EC. ECs constitutively express Fas in the basolateral membrane, whereas neighboring IEL cells, as well as lamina propia lymphocytes, express its cognate soluble ligand, FasL, in a more controlled manner (19, 29, 39). The ligation causes a trimerization of Fas molecules that form part of a death-inducing signaling complex.
Previous studies (39) have demonstrated that FasL
expressing cytotoxic lymphocytes kill Fas-expressing target cells via apoptosis. Additionally, Fas-FasL interactions have been
implicated in increased enterocyte apoptosis seen in
immune-mediated bowel injury. Two previous studies (12,
14) have addressed the role of FasL-mediated killing by IEL.
Both of these studies found that IEL cytotoxic activity was induced
from the CD8+ but not from CD8
+ IEL subset. Activated
CD8
+ IEL subsets were able to mediate Fas-based killing. To
further demonstrate the interaction between IEL FasL and Fas expression
on EC, Lin et al. (24) isolated IEL from week 3 graft-vs.-host disease mice that IEL display potent FasL-mediated
killing activity. These IEL were injected into lpr mice
(that have defective Fas expression) and wild-type mice (that have an
intact Fas expression). Although EC apoptosis was observed in
both cases, a significantly lower level of EC apoptosis was observed in lpr mice. This suggests that activated T cells
upregulate the expression of the Fas/FasL genes, and undergo
activation-induced apoptosis. Our findings of IEL-derived
IFN-
upregulating FasL, but not Fas is corroborated by a study from
Shiraishi et al. (37). These investigators found an
upregulation of FasL expression and an increased incidence of
apoptosis in hepatocytes of mice 5 days after a small
intestinal transplant. No change, however, was noted in Fas mRNA and
protein expression. Despite this lack of change in Fas expression,
functional changes mediated by IFN-
may still occur. Levels of
apoptosis remained elevated in lpr and control mice
above control levels. This suggests, that although Fas and FasL have a
role in TPN-mediated apoptosis, other mechanism mediating EC
apoptosis also have a role.
The role of IFN- in inducing EC apoptosis has been reported
by several studies (2, 18, 26, 35). EC apoptosis
via a Fas-FasL interaction was induced rapidly in HT-29 cells with prior exposure of the cell to IFN-
(2, 21, 31). A study from Ruemmele, et al. (35) found that the EC
apoptosis rate markedly increased after costimulation with
IFN-
in the presence of an anti-Fas agonist compared with anti-Fas
antibody alone. This suggests that in vivo the susceptibility of
enterocytes to Fas-induced apoptosis can be markedly enhanced
by locally secreted cytokines in zones of active inflammation.
Interestingly, Martin et al. (26) showed that IFN-
did
not increase Fas expression. Rather, IFN-
led to an increased
sensitivity of Fas when exposed to antibody to Fas. Likewise, our study
did not find any significant increase in EC Fas expression in the TPN
group. Despite this, apoptosis significantly increased after
TPN administration. This suggested that the rise in EC
apoptosis in our study may be mediated by both an increase in
FasL, as well as an increased susceptibility of EC to Fas-mediated
apoptosis. To confirm this, ECs were incubated with anti-Fas
antibody. EC apoptosis was found to be significantly greater in
cells isolated from TPN wild-type mice compared with wild-type
controls, as well as both IFNKO TPN and IFNKO control mice. These
results suggest that the sensitivity of ECs to Fas-mediated apoptosis is significantly increased after TPN. Because of the decline in apoptosis observed in the IFNKO mice, it appears
that this effect is predominately mediated by the increase in IFN-
expression. This study also found that EC apoptosis in both
Fas-deficient and FasL-deficient TPN mice was significantly lower than
in wild-type TPN mice. Data from the present study demonstrate that EC
undergo a marked increase in apoptosis through Fas/FasL
interaction, and is associated with an alteration in IFN-
protein
expression during TPN. Interestingly, although the apoptosis of
EC in IFNKO TPN mice was lower than wild-type TPN mice, the
apoptotic rate was still higher than the control. This suggest that
there are other non-IFN-
, Fas/FasL dependent pathways that affects
apoptosis. This other pathway would most likely be modulated
via signaling through TNF-
binding to TNF receptor
(38), because this mechanism has been well established in
intestinal ECs (4).
Our findings show that TPN induces an increase in apoptosis and
decrease in proliferation in EC. In addition, Fas/FasL interactions may
play an important role in TPN-induced apoptosis. The
costimulatory factors released locally, such as IFN-, can markedly
increase the susceptibility of enterocytes to apoptosis induced
by Fas-FasL. How TPN-induces IEL expression of IFN-
has not been
elucidated. Clearly, a number of factors can influence IEL to alter
expression of cytokines. More recently a number of reports (43,
45) have emphasized the relationship of crosstalk between EC and
IEL by their alteration in gene expression. Such factors as an
alteration in luminal nutrients, foreign antigens, as well as gut
peptides may have a role in mediating the increase in IFN-
expression. Our study demonstrates one mechanism by which TPN mediates
an increase in EC apoptosis. Future studies will hopefully
provide insight as to which additional mechanisms are involved in this process.
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ACKNOWLEDGEMENTS |
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This research was supported by National Institute of Allergy and Infectious Diseases Grant AI-44076-01 and Abbott Laboratories, Hospital Division.
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FOOTNOTES |
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Address for reprint requests and other correspondence: D. H. Teitelbaum, Section of Pediatric Surgery, Univ. of Michigan Hospitals, Mott F3970, Box 0245, Ann Arbor, Michigan 48109 (E-mail: dttlbm{at}umich.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published January 16, 2003;10.1152/ajpgi.00290.2002
Received 18 July 2002; accepted in final form 8 January 2003.
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