Intraepithelial lymphocyte-derived interferon-gamma evokes enterocyte apoptosis with parenteral nutrition in mice

Hua Yang, Yongyi Fan, and Daniel H. Teitelbaum

Section of Pediatric Surgery, Department of Surgery, University of Michigan Medical School and C. S. Mott Children's Hospital, Ann Arbor, Michigan 48109


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Total parenteral nutrition (TPN) results in an increase in intraepithelial lymphocyte (IEL)-derived interferon-gamma (IFN-gamma ) expression as well as an increase in epithelial cell (EC) apoptosis. This study examined the role that IEL-derived IFN-gamma 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-gamma and Fas ligand (FasL) expression. EC apoptosis in IFN-gamma 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-gamma 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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-gamma has been reported to regulate apoptosis in many cells. Several researchers have reported that IFN-gamma 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-gamma . In addition, we have previously demonstrated (20) that TPN results in a significant increase in IEL-derived IFN-gamma expression. On the basis of this evidence, we hypothesized that an increased IEL-derived IFN-gamma expression resulting from TPN administration may induce an increase in EC apoptosis. Additionally, we hypothesized that TPN-induced EC apoptosis is mediated by IFN-gamma through a Fas/FasL interaction.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-gamma knockout mice (C57BL/6-Ifngtm1ts), FasL-deficient mice (B6Smn.C3H-FasLgld, gld mice), and Fas-deficient mice (B6.MRL-Faslpr, lpr mice) were also used. During the administration of intravenous solutions, the mice were housed in metabolic cages.

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-gamma knockout (INFKO) mice, FasL-deficient mice, and Fas-deficient mice were also used. Each group contained seven mice. All TPN groups received the same intravenous TPN solution at 7 ml/24 h. The control group received the crystalloid solution at 7 ml/24 h and standard laboratory mouse chow and water ad libitum. The TPN solution, as described in detail previously (20), contained a balanced mixture of amino acids, lipids, and dextrose in addition to electrolytes, trace elements, and vitamins. Caloric delivery was on the basis of estimates of caloric intake by the control group, so that caloric delivery was essentially the same among groups. The TPN group, IFNKO TPN group, and lpr TPN as well as gld TPN groups received a standard TPN solution intravenously at 7 ml/24 h with no oral intake. All animals were killed at 7 days by using CO2.

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, CD8alpha , and CD3; PE conjugated anti-mouse Fas and FasL; and IFN-gamma . Isotype-matched, irrelevant antibodies (PE hamster antibody IgG, FITC anti-rat IgG2alpha and PE antibody anti-rat IgG2alpha ) were used as the negative controls.

To identify IEL IFN-gamma expression, IEL cells were suspended in PBS and 2% FCS buffer (FACS), and stained with FITC-conjugated appropriate antibody at 4°C for 30 min. The cells were then washed, resuspended with Cytofix/Cytoperm (PharMingen) for 20 min at 4°C, and washed in 1× Perm/Wash solution (PharMingen). Phycoerytherin-conjugated antibody to IFN-gamma was then added, and the cells were incubated at 4°C for 30 min. Cells were washed again and resuspended in staining buffer before flow cytometry.

Flow cytometry was performed by using standard techniques (36). Acquisition and analysis were performed on a Becton-Dickinson FASCalibur (Becton-Dickinson, Mountainview, CA) by using a Macintosh G3 computer and CellQuest (Becton-Dickinson) software. Cells were gated on the basis of forward and side scatter characteristics for IEL and EC populations. Data are presented as the percentage of gated cells positive for Fas, FasL, or IFN-gamma expression.

In select experiments, isolated IEL were further separated from EC by using magnetic beads bound to anti-CD45 antibody according to manufacturer's instructions (BioMag SelectaPure Anti-Mouse CD 45R Antibody Particles, Polyscience). Purified cells, either bound to magnets (IEL) or unbound (EC), were then cultured for 48 h in microtiter plates and the supernatant was analyzed for IFN-gamma production (see ELISA analysis).

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-gamma Expression

Methods utilized are similar to those previously described (9, 40). Harvested IEL were cultured in RPMI 1640, 2 mM glutamine, penicillin, streptomycin, 2-mercaptoethanol (5 × 10-5 M) and 10% heat-inactivated FCS (GIBCO-BRL, Gaithersburg, MD) for 48 h in 96-well U-bottom plates. To maximize IFN-gamma expression, concanavalin A (2 µg/ml) was added to each well. Supernatants were then extracted and IFN-gamma measured by ELISA using matched-pair antibodies (PharMingen) according to the manufacturer's directions. Cytokine levels were determined graphically by using standard curves generated with recombinant murine IFN-gamma (Genzyme, Cambridge, MA).

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 at -70°C until assayed.

Poly 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 beta -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 beta -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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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 1.   Representative histology of jejunal biopsies using hematoxylin and eosin staining from control (A) and total parenteral nutrition (TPN) (B) (×40 magnification). Note the marked change in villus height after 7 days of TPN compared with control mice. The TPN group received a standard TPN solution intravenously with no oral intake. Controls received physiological saline in addition to standard laboratory mouse chow and water ad libitum.


                              
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Table 1.   Jejunal mucosal measurements in each experimental group

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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Fig. 2.   Epithelial apoptosis is shown in the jejunal mucosa of wild-type, interferon-gamma knockout (IFNKO), lpr, and gld mice for both control and TPN groups. The apoptotic index is expressed as the percentage of apoptotic epithelial cells (EC) per 100 ECs, as determined by using terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining. TPN administration led to a significant increase in EC apoptosis compared with controls for all study groups (*P < 0.01). There was also a statistically significant difference in the rate of EC apoptosis between wild-type TPN mice and IFNKO, lpr, and gld TPN mice (dagger P < 0.05). n = 7 Mice per each group.

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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Fig. 3.   5-Bromo-2-deoxyuridine (BrdU)-positive cells in mouse jejunum with immunohistochemical staining. Mice were injected with BrdU, 50 mg/kg body wt, via intraperitoneal route 1 h before sacrificing. The BrdU-positive cells significantly increased after TPN. *P < 0.05 compared with the control. Data are expressed as ratio of the number of crypt cells incorporating BrdU to the total number of crypt cells.

TPN Increased IEL-Derived IFN-gamma Expression

Our laboratory has previously shown (20) that IEL-derived IFN-gamma mRNA expression increased by 53% when mice received TPN. This study examines IFN-gamma protein expression by using ELISA. In the TPN group, there was a 3.5-fold increase in IEL-derived IFN-gamma protein expression when compared with the controls (P < 0.01). IEL IFN-gamma expression rose from 190 ± 37 pg/ml in controls to 455 ± 65 pg/ml in TPN mice.

Because of the unique phenotype of the IEL, IFN-gamma 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-gamma . A mean of 32.6 ± 4.2% of small bowel IEL expressed IFN-gamma protein in the TPN group and 14 ± 2.1% of IEL in the control group expressed IFN-gamma (P < 0.05). Flow cytometry analysis showed that TPN significantly increased the CD8+ and CD3+ IEL-derived IFN-gamma protein expression but did not increase IFN-gamma in the CD4+ phenotype (Table 2).

                              
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Table 2.   Flow cytometry results for IEL-derived IFN-gamma protein expression after administration of TPN

IFN-gamma Mediates EC Apoptosis Through Fas/FasL Interaction

IFNKO mice were used to investigate the effect of IFN-gamma on the increase in EC apoptosis after TPN administration. We hypothesized that the increase in IFN-gamma would lead to an increase in EC apoptosis and that this would be mediated via a 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-gamma -dependent pathways that contribute to EC apoptosis. Thus IFN-gamma 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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Fig. 4.   Changes in Fas ligand (FasL) mRNA in intraepithelial lymphocyte (IEL) measured by RT-PCR from isolated IEL. Results are expressed as a ratio of FasL gene to beta -actin mRNA expression. Expression of FasL was markedly increased in TPN mice compared with control mice. However, FasL mRNA expression in the IFNKO TPN group was significantly lower than in the wild-type TPN mice. Statistical comparison of groups with ANOVA showed that the TPN group was significantly greater (P < 0.01) compared with the remaining groups.


                              
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Table 3.   Isolated epithelial cell Fas mRNA and protein expression

Lower IEL-derived FasL expression in IFNKO TPN mice. To investigate the role of IFN-gamma 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-gamma 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).

To determine whether Fas and FasL are involved in inducing EC apoptosis, the Fas and FasL protein expression was measured with flow cytometry. IEL expressed significantly (P < 0.05) higher levels of FasL (21.2 ± 4.1% cells stained positively for FasL) in the TPN group, compared with controls (11.4 ± 3.5%). Double staining of the IEL with FasL and either CD3, CD4, or CD8 allowed a better understanding of which IEL subpopulation expressed FasL. FasL expression in wild-type TPN mice was significantly higher (P < 0.05) than in IFNKO TPN mice (Table 4). There was no significant difference in FasL expression between the IFNKO control and IFNKO mice after TPN administration. This was the case for both the CD4+ and CD8+ IEL subpopulations. Fas expression was seen in 20.2 ± 4.1% of EC in controls and was not significantly different (P > 0.05) from the TPN group (24.9 ± 6.1%). We also found that 28.1 ± 7.3% of EC expressed Fas protein in the IFNKO TPN group, however, there was no significant difference in Fas expression in EC between the TPN and IFNKO TPN groups (Table 3).

                              
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Table 4.   IEL FasL protein expression

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-gamma treatment can induce human intestinal ECs into Fas-mediated apoptosis without changing Fas expression (26), we hypothesized that the increased expression of IFN-gamma 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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Fig. 5.   Apoptosis in small intestinal ECs in wild-type and IFNKO TPN mice after incubation with anti-CD95 (Fas) antibody. Annexin V-positive cells are ECs. Basal EC apoptosis rate is the percentage of apoptosis before incubation subtracted from the percentage of apoptosis after antibody incubation. Fas antibody induced EC apoptosis in wild-type TPN mice was significantly higher than EC apoptosis in IFNKO TPN mice and controls. Statistical comparison of groups with ANOVA showed that the TPN group was significantly greater (P < 0.01) compared with the remaining groups. There was no significant difference of EC apoptosis rates among the remaining groups (P > 0.05).

Fas/FasL interaction plays an important role in this TPN-induced apoptotic process. IFN-gamma was found to be responsible for a large part of EC apoptosis after TPN administration. This increase in IEL-derived IFN-gamma 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-gamma 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-gamma Expression on TPN-Induced Villus Atrophy

To understand if there is a potential relationship between our observed increase in EC apoptosis and TPN-induced villus atrophy, villus height was measured. As previously noted, TPN administration led to a significant decline in jejunal villus height. A number of additional mouse strains were used to detect a relationship between IFN-gamma expression and villus atrophy. There was an increase in both villus height and total height in the IFNKO TPN mice when compared with wild-type TPN mice (Table 1). The villus height in IFNKO TPN mice increased 19.5% when compared with wild-type TPN mice. Histological data also showed that villus height in both gld, and lpr TPN mice was significantly higher than wild-type TPN mice (Table 1). Villus height in the gld TPN mice increased 17.7% compared with wild-type TPN mice. Villus height in the lpr TPN mice increased 15.8% compared with wild-type TPN mice. Although a number of factors may influence the villus height in the TPN groups, including alterations in EC proliferation, the data suggest that the presence of IFN-gamma has a significant influence on these histological changes.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-gamma expression rose significantly after TPN. IEL FasL expression also significantly increased. To further investigate the role of IFN-gamma 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-gamma 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 CD8alpha beta + but not from CD8alpha alpha + IEL subset. Activated CD8alpha beta + 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-gamma 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-gamma 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-gamma 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-gamma (2, 21, 31). A study from Ruemmele, et al. (35) found that the EC apoptosis rate markedly increased after costimulation with IFN-gamma 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-gamma did not increase Fas expression. Rather, IFN-gamma 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-gamma 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-gamma 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-gamma , Fas/FasL dependent pathways that affects apoptosis. This other pathway would most likely be modulated via signaling through TNF-alpha 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-gamma , can markedly increase the susceptibility of enterocytes to apoptosis induced by Fas-FasL. How TPN-induces IEL expression of IFN-gamma 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-gamma 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.


    ACKNOWLEDGEMENTS

This research was supported by National Institute of Allergy and Infectious Diseases Grant AI-44076-01 and Abbott Laboratories, Hospital Division.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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