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Am J Physiol Gastrointest Liver Physiol 292: G1411-G1419, 2007. First published February 15, 2007; doi:10.1152/ajpgi.00557.2006
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INFLAMMATION/IMMUNITY/MEDIATORS

Developmentally regulated tumor necrosis factor-{alpha} induced nuclear factor-{kappa}B activation in intestinal epithelium

Erika C. Claud,1 Xiaoqiong Zhang,1 Elaine O. Petrof,2 and Jun Sun3

Departments of 1Pediatrics, Section of Neonatology, 2Medicine, Section of Infectious Disease, and 3Pathology, The University of Chicago, Chicago, Illinois

Submitted 6 December 2006 ; accepted in final form 9 February 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 DISCLOSURES
 REFERENCES
 
Premature infants are susceptible to many conditions that are inflammatory in nature. For this patient population, which is expecting the intrauterine environment, pathways necessary for fetal life and development may not have completed the transitions necessary for extrauterine life. In this study, responses to tumor necrosis factor-{alpha} were compared in human fetal and adult intestinal epithelial cell lines along with preweaned and postweaned mouse intestinal sections to identify a potential developmental difference that may explain the heightened inflammatory response of preterm infants. The nuclear factor-{kappa}B (NF-{kappa}B) pathway regulates a wide variety of genes involved in immune and inflammatory processes. We report that, compared with adult intestinal epithelial cells, immature intestinal epithelial cells have increased NF-{kappa}B activity associated with increased NF-{kappa}B-DNA binding and transcriptional activity. This increased activity appears due to inadequate inhibition of signaling leading to NF-{kappa}B activation since there is also increased phosphorylation, ubiquitination, and degradation of the inhibitor of NF-{kappa}B in conjunction with decreased baseline expression and delayed resynthesis of this inhibitor. Thus we demonstrate a potential mechanism for the heightened inflammatory response of immature intestinal epithelial cells.

immature intestinal epithelium; inflammation; development; necrotizing enterocolitis

THE INTESTINAL MUCOSA IS a key interface with the external environment, with important roles in host defense, immune responses, nutrition, and fluid balance. Many of the functions of the intestine have been shown to be developmentally regulated and influenced by microbial flora (13). It is vital that the intestine defend against pathogenic insult yet appropriately tolerate commensal bacteria.

The production of proinflammatory cytokines is one means of host defense. However, the immature intestine, expecting a sterile intrauterine environment, may not appropriately control intestinal inflammatory responses. This has implications for preterm infants with an immature gut potentially ill prepared for the extrauterine environment and contact with bacteria and food substrate. Developmental downregulation of gut inflammatory responses is potentially a necessary process to avoid immune responses against normal intestinal flora.

The intestine is exposed to both endogenous and exogenous inflammatory mediators. Studies have shown that exogenous mediators such as bacteria interact with the intestinal epithelium via well-conserved pattern-recognition receptors such as the Toll-like receptors (8). These receptors trigger signaling pathways leading to nuclear factor-{kappa}B (NF-{kappa}B) activation and propagation of the inflammatory response. Similarly, endogenous inflammatory mediators such as tumor necrosis factor-{alpha} (TNF-{alpha}) and interleukin-1beta (IL-1beta) bind to their respective receptors and can also trigger further cytokine production via activation of the NF-{kappa}B pathway.

NF-{kappa}B proteins activate transcription of a wide variety of genes involved in immune and inflammatory responses including interleukin 8 (IL-8). In its resting state NF-{kappa}B dimers are bound in the cytoplasm to the inhibitory-{kappa}B (I{kappa}B) proteins (9). Cell stimulation can trigger signaling pathways leading to the activation of I{kappa}B kinase (IKK), which then phosphorylates I{kappa}B, targeting it for ubiquitination and degradation by the 26S proteasome. The NF-{kappa}B, thus liberated, moves to the nucleus, where it activates gene transcription.

Previously published work demonstrates exaggerated inflammatory cytokine production by immature intestinal epithelial cells (IEC) compared with adult IEC. Specifically, IL-8 secretion in response to bacteria, IL-1beta, and TNF-{alpha} has been shown to be greatly increased in human fetal small intestinal epithelial cells and fetal tissue organ culture samples compared with adult cells and older child biopsy samples (6, 18).

We further demonstrated that this increased IL-8 secretion is associated with decreased baseline expression of I{kappa}B isoforms in immature IEC compared with mature IEC (5). To further understand the mechanism behind exaggerated cytokine production in immature enterocytes, we hypothesized that this decreased I{kappa}B expression would correspond to increased NF-{kappa}B activity. This study demonstrates that immature human intestinal epithelial cells have increased NF-{kappa}B-DNA binding and transcription activity in response to the endogenous inflammatory mediator TNF-{alpha}. Increased activity is associated with not only decreased I{kappa}B{alpha} expression but also increased signaling for I{kappa}B{alpha} degradation and delayed resynthesis of I{kappa}B{alpha}, which collectively contribute to the exaggerated inflammatory response due to insufficient inhibition of the NF-{kappa}B pathway in immature enterocytes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 DISCLOSURES
 REFERENCES
 
Cell culture. H4 cells are a human fetal nontransformed primary intestinal epithelial cell line used as a model of immature IEC (21). They were cultured in DMEM with 10% heat-inactivated fetal calf serum, 1% glutamine, 1% sodium pyruvate, 1% amino acids, 1% HEPES, 50 units/ml penicillin, 50 µg/ml streptomycin, and 0.2 units/ml insulin. Cell passages 11–21 were used. T84 cells originate from adult IEC. T84 cells were grown in a 1:1 (vol/vol) mixture of DMEM and F12 medium (Invitrogen, Carlsbad, CA) with 10% heat-inactivated fetal calf serum, 1% glutamine, 50 units/ml penicillin, and 50 µg/ml streptomycin. Cell passages 50–64 were used. All cells were grown at 37°C in a 5% CO2 atmosphere.

Transfection and luciferase reporter assay. Transient transfection was accomplished by using Lipofectamine 2000 according to the manufacturer's instructions (Life Technologies, Rockville, MD) (7). Briefly, cells were grown to 75% confluence in antibiotic-free medium and incubated with Lipofectamine 2000 reagent plus an NF-{kappa}B promoter-dependent firefly luciferase reporter construct and a constitutively expressed Renilla luciferase reporter (Promega, Madison, WI) as an internal control for transfection efficiency. After 40 h, cells were stimulated with TNF-{alpha} 10 ng/ml for 6 h. Cells were then lysed and the Dual-Luciferase Reporter Assay (Promega, Madison, WI) was carried out by following the manufacturer's recommendations. The luminescence generated by the firefly luciferase was measured first, and then quenched, and a second measurement was obtained to determine Renilla luminescence. Results are expressed as firefly luminescence normalized to Renilla luminescence.

EMSA. Nuclear extracts were obtained by a method modified from Inan et al. (14). Briefly, cells were treated with 10 ng/ml of TNF-{alpha} at 0, 5, 15, 30, 90, and 120 min. The cells were washed and harvested in Tris-buffered saline and then incubated for 15 min in extract lysis buffer [10 mM HEPES, 10 mM KCl, 0.1 mM EDTA, 2 mM MgCl2, 0.5 mM sucrose, 0.05% Nonidet P-40, 0.5 mM PMSF, 1 mM DTT, 1 x Complete protease inhibitor (Roche, Indianapolis, IN)]. The cytosolic fraction was then removed and discarded. Nucleus-containing pellets were rinsed with extract lysis buffer without Nonidet P-40 and incubated for 40 min in 25 µl of high-salt buffer [20 mM HEPES pH 7.4, 1.5 mM MgCl2, 420 mM NaCl, 0.2 mM EDTA, 5% glycerol, 1 mM DTT, 0.5 mM PMSF, 1 x Complete protease inhibitor (Roche)]. The nuclear extract was removed and combined with 38 µl of low-salt buffer [20 mM HEPES pH 7.4, 50 mM KCl, 0.2 mM EDTA, 20% glycerol, 1 mM DTT, 0.5 mM PMSF, 1 x Complete protease inhibitor (Roche)]. Protein concentrations were then determined by the Bradford method.

At room temperature, nuclear extracts (5–10 µg) were incubated for 15 min with 5 mM Tris pH 7.5, 0.5 mM EDTA, 2% Ficoll, 0.5 mM DTT, 37.5 mM KCl, 1 µg poly(dI-dC) (Roche), and 50,000 cpm of a {gamma}32P-labeled probe encoding the NF-{kappa}B consensus sequence (5'-AGTTGAGGGGACTTTCCCAGGC-3') (Promega, Madison, WI). To determine oligonucleotide specificity, a 100-fold excess cold oligonucleotide was added to the reaction mixture before the 15-min incubation (data not shown). In supershift reactions, antibodies to NF-{kappa}B p50, NF-{kappa}B p65, NF-{kappa}B p52, NF-{kappa}B cRel, or NF-{kappa}B RelB (Santa Cruz, Santa Cruz, CA) were preincubated with the nuclear extract and reaction buffer in the absence of probe for 30 min. The probe was then added, and samples were incubated for an additional 15 min before electrophoresis on a native 5% polyacrylamide gel. The gel was then dried and exposed to film.

Immunoprecipitation. To concentrate TNF receptor levels, T84 and H4 cells were grown to confluence in 60-mm culture dishes and then lysed with 500 µl of cold immunoprecipitation (IP) buffer (1% Triton X-100, 150 mM NaCl, 10 mM Tris pH 7.4, 1 mM EDTA, 1 mM EGTA pH 8.0,). Protein concentration of the lysate supernatant was measured by Bio-Rad DC protein assay method (Bio-Rad, Hercules, CA). Immediately before IP, lysates were precleared with PANSORBIN (Calbiochem, San Diego, CA). Equal volume cell lysates were incubated with TNF receptor antibody with agitation, and then Protein A gel (Pierce, Rockford, IL) was added to the antigen-antibody complex. The resulting pellet was washed several times in IP buffer and then resuspended in 0.1 M glycine pH 2.5. Samples were then centrifuged, and 1 M Tris pH 8.0 was added to neutralize the pH of the supernatant. A 4 x sample buffer was added to each sample and boiled before immunoblotting again with TNF receptor antibody. To confirm equal protein loading, total cell lysates samples were taken after preclearing but before IP and loaded on the same gel with the IP samples, then probed with heat shock cognate 73 (Hsc 73) antibody in addition to TNF receptor 1 and 2 antibodies.

Immunoblotting. Following treatments as indicated, cells were washed in ice-cold phosphate-buffered saline (PBS) and then lysed in 1% Triton X-100 in 10 mM Tris pH 8, 150 mM NaCl, with 10 µg/ml of aprotinin and leupeptin and 2 mM phenylmethylsulfonyl fluoride. After the protein concentrations of the cleared lysates were estimated by using the Bio-Rad DC protein assay (Bio-Rad) per the manufacturer's protocol, equal amounts of total protein were separated by SDS-PAGE on 10% polyacrylamide gels. Electrophoresed proteins were transferred from the gel to a nitrocellulose membrane using a semi-dry transfer apparatus as described previously (1). The blot was blocked with 5% nonfat dry milk in Tris-buffered saline with Tween 20 and incubated with the appropriate primary antibody and then with the appropriate horseradish peroxidase-conjugated secondary antibody. The blot was developed by using the West Pico Super Signal chemiluminescence reagent (Pierce). The membranes were then stripped and reprobed with an antibody to glyceraldehyde phosphate dehydrogenase (GAPDH) (Research Diagnostics Institute, Flanders, NJ) or Hsc 73 (Stressgen, San Diego, CA) to confirm equal loading of lanes.

Preweaned/postweaned mouse intestinal tissue. Rag 1–/– knockout mouse strain 002216 (Jackson Laboratory, Bar Harbor, ME), which does not develop mature T cells or B cells, was used to allow investigation of inflammatory responses specifically by IEC, without contamination of inflammatory cells in tissue samples. Responses in immature intestine or preweaned pups 12–17 days old were compared with responses in postweaned mice 5–8 wk old. Animals were individually anesthetized with sevoflurane by inhalation in a bell jar in a fume hood and then weighed before injection with recombinant murine TNF-{alpha} (PeproTech, Rocky Hill, NJ) 0.4 mg/kg ip. Control animals were injected with sterile PBS alone. At the end of the specified time, as noted in RESULTS, mice were euthanized and full-length intestine was removed and fixed in 10% formalin before paraffin imbedding.

The protocol was approved by the University of Chicago Animal Care committee.

Immunofluorescence staining. Staining for NF-{kappa}B p65 was performed on paraffin-embedded sections (4 µm) of mouse small intestines from rag 1–/– mice treated with TNF-{alpha} as outlined above. Paraffin sections were baked in an oven at 56°C, and then the slides were deparaffinized in xylene and rehydrated by graded ethanol washes at room temperature. Antigen retrieval was achieved by boiling tissue sections in 10 mM sodium citrate buffer (pH 6.0) for 20 min. Slides were then incubated in peroxidase block solution (Dako, Carpinteria, CA) for 20 min at room temperature to block endogenous peroxidase activity. Samples were then permeabilized in 0.1% Triton X-100 in PBS for 20 min at room temperature followed by incubation for 20 min in 5% BSA in PBS to reduce nonspecific background. The slides were incubated with the primary antibody rabbit polyclonal p65 (Santa Cruz) diluted 1:50 in 1% BSA in PBS followed by incubation in the secondary antibody Alexa Fluor 488 goat anti-rabbit IgG (H+L) (Invitrogen) diluted 1:200 in 1% BSA. Nuclei were counterstained with DAPI (D21490 [GenBank] ) diluted 1:10,000 in 1% BSA. Slow Fade equilibration buffer (Invitrogen) was added before mounting. Slides were kept in the dark to avoid bleaching before visualization by use of a Leica SP2 AOBS spectral confocal microscope.

Immunohistochemistry. Staining for phospho-I{kappa}B{alpha} was performed on paraffin-embedded sections (4 µm) of mouse small intestines from Rag 1–/– mice treated with TNF-{alpha} as outlined above. Paraffin sections were baked in an oven at 56°C, and then the slides were deparaffinized in xylene and rehydrated by graded ethanol washes at room temperature. Antigen retrieval was achieved by boiling tissue sections in 10 mM sodium citrate buffer (pH 6.0) for 20 min. Slides were then incubated in Peroxidase block (Dako) for 20 min at room temperature to block endogenous peroxidase activity, followed by incubation for 20 min in 5% powdered milk in PBS to reduce nonspecific background. The slides were incubated with the primary antibody mouse monoclonal phospho-I{kappa}B{alpha} diluted 1:150 in 1% BSA for 1 h at room temperature followed by the secondary antibody horseradish peroxidase anti-mouse (Dako) for 30 min at room temperature. Antibody staining was visualized with 3,5'-diaminobenzidine (DAB) chromogen (Dako, 20 µl in 1,000 µl of DAB buffer) for 15 min at room temperature and counterstained with hematoxylin.

Reagents. TNF receptor 1 and 2 antibodies were obtained from Abcam (Cambridge, MA). I{kappa}B{alpha} antibody was obtained from Santa Cruz and Hsc 73 antibody was obtained from Stressgen. IKK, phospho-IKKbeta/{alpha}, and phospho-I{kappa}B{alpha} antibodies were obtained from Cell Signaling (Danvers, MA). GAPDH antibody was obtained from Research Diagnostics Institute. Ubiquitin antibody and MG262 were obtained from Active Motif (Carlsbad, CA). Murine TNF-{alpha} was obtained from PeproTech.

Statistical analysis. Results are presented as mean values ± SE. Statistical significance was determined by the t-test. P < 0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 DISCLOSURES
 REFERENCES
 
Receptor expression. To determine whether differences in cytokine production between immature and adult IEC could merely be explained by differences in receptor expression, TNF-{alpha} receptor expression was compared in the adult human intestinal epithelial cell line T84 and the immature human intestinal epithelial cell line H4 by IP and immunoblotting using specific antibodies. TNF-{alpha} signaling is mediated by binding of two receptors, TNFR1 (50 kDa) and TNFR2 (75 kDa). TNFR1 is believed to be the major signaling receptor, leading to activation of NF-{kappa}B (25). As shown in Fig. 1, receptor expression for both TNFR1 and TNFR2 was comparable for the two cell lines. Thus a difference in cytokine production cannot be explained by a difference in receptor expression leading to enhanced signaling.


Figure 1
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  		Fig. 1. Equivalent TNF receptor (TNFR) expression in immature and mature enterocytes. Cell lysates were obtained from the human adult intestinal epithelial cell (IEC) cell line T84 or the human fetal IEC cell line H4. The expression of TNF receptor 1 and 2 was revealed by immunoprecipitation and immunoblotting with specific antibodies with heat shock cognate 73 (Hsc 73) used as loading control from total cell lysates samples obtained after clearing but before immunoprecipitation. NIH Image J software was used for densitometry to determine relative TNF receptor band intensity. Values are presented below each band as fold difference compared with T84 cells. T84 band intensity was arbitrarily set to 1. The results shown are representative of 3 separate experiments.

 
Immature enterocytes have increased NF-{kappa}B transcriptional activity compared with mature enterocytes. TNF-{alpha} can induce cytokine production via activation of the NF-{kappa}B pathway. To determine whether the previously described increased inflammatory cytokine production in immature IEC (5, 6) was associated with increased NF-{kappa}B activity, H4 and T84 cells were transfected with an NF-{kappa}B promoter-driven firefly luciferase reporter construct, along with a Renilla luciferase reporter as an internal control before treatment with TNF-{alpha}. As shown in Fig. 2, the immature IEC line H4 had significantly greater firefly luciferase activity of 109 ± 8 luminescence units compared with the adult cells, which had 35 ± 3 luminescence units given the same inflammatory stimulus (P < 0.05), indicating increased transcription of the NF-{kappa}B promoter-dependent reporter. Thus increased cytokine production in immature IEC is associated with increased NF-{kappa}B transcriptional activity.


Figure 2
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  		Fig. 2. Immature enterocytes have increased NF-{kappa}B transcriptional activity at baseline and when stimulated by TNF-{alpha} compared with adult enterocytes. Adult T84 IEC and immature H4 IEC were transfected with an NF-{kappa}B promoter-driven firefly luciferase construct along with a Renilla luciferase reporter as an internal control for transfection efficiency. Cells were then treated for 6 h with TNF-{alpha} 10 ng/ml and luminescence was measured. Results are presented as mean ± SE firefly luminescence normalized to Renilla luminescence, n = 4. *P < 0.05 for H4 compared with T84 cells.

 
Immature enterocytes have increased NF-{kappa}B binding compared with mature enterocytes. To determine the point of developmentally regulated differential responsiveness between immature and mature enterocytes to inflammatory stimuli, we explored signaling associated with steps upstream of NF-{kappa}B activity, beginning with NF-{kappa}B binding to DNA. Cells were treated with TNF-{alpha} for 0, 5, 15, 30, 90, and 120 min, and nuclear extracts were obtained. NF-{kappa}B binding was determined by EMSA as described in MATERIALS AND METHODS. As shown in Fig. 3, NF-{kappa}B binding was more prominent and prolonged in the immature H4 cell line, with binding beginning at 5 min and significant binding present through 120 min. In the T84 cell line there is less binding, which is not as prolonged. Supershift reactions showed that these bands were primarily p50/p65 heterodimers (data not shown). There was no difference in baseline expression for the most common isoforms of NF-{kappa}B p65, p50, and p52 (data not shown). Thus increased NF-{kappa}B activity in immature enterocytes is associated with increased NF-{kappa}B binding compared with mature enterocytes.


Figure 3
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  		Fig. 3. Immature enterocytes have increased and prolonged NF-{kappa}B binding when stimulated by TNF-{alpha} compared with adult enterocytes. A: adult T84 IEC and immature H4 IEC were stimulated with TNF-{alpha} 10 ng/ml over time as indicated. Nuclear extracts were obtained and EMSA was performed using a {gamma}32P-labeled NF-{kappa}B consensus sequence probe. B: NIH Image J software was used for densitometry to quantify NF-{kappa}B binding. Data are presented as fold change compared with T84 cells at time 0 (set at 1.0). The results shown are representative of 4 separate experiments with similar results.

 
Immature enterocytes have more rapid I{kappa}B{alpha} degradation than mature enterocytes in response to TNF-{alpha}. NF-{kappa}B is a transcription factor retained in the cytosol by the inhibitor protein I{kappa}B. Signal-induced degradation of I{kappa}B allows NF-{kappa}B to move into the nucleus, bind to DNA, and activate the transcription of a variety of genes involved in inflammation and innate immunity. To determine whether differential regulation of NF-{kappa}B activity between immature and mature IEC is associated with differences in inhibition thus leading to the increased availability of nuclear NF-{kappa}B for DNA binding, I{kappa}B{alpha} degradation was examined. H4 and T84 cells were incubated with TNF-{alpha} for 0, 5, 15, 30, 60, or 90 min. I{kappa}B{alpha} levels were measured by Western blot, using an anti-I{kappa}B{alpha} antibody. Blots were then stripped and incubated with a GAPDH antibody to confirm equal protein loading. As shown in Fig. 4, consistent with previous observations immature H4 cells had less baseline expression of I{kappa}B{alpha} than the T84 adult cells (5). Furthermore, whereas T84 cells had evidence of decreased I{kappa}B{alpha} levels at 15 min and return to baseline levels by 60 min, in H4 cells I{kappa}B{alpha} decrease was evident by 5 min, the decrease was more complete, and there was a delay in return to baseline levels until 90 min. Decreased levels of I{kappa}B{alpha} due to lower baseline levels coupled with increased I{kappa}B{alpha} degradation and delayed return to baseline levels may correspond to increased NF-{kappa}B release and thus enhanced activation of the transcription of inflammatory cytokines in immature cells.


Figure 4
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  		Fig. 4. Stimulated immature enterocytes have more rapid inhibitory-{kappa}B{alpha} (I{kappa}B{alpha}) decrease and delayed return compared with mature enterocytes. T84 cells and H4 cells were treated with TNF-{alpha} 10 ng/ml over time as indicated. Cell lysates were obtained and the expression of I{kappa}B{alpha} was revealed by Western blot with GAPDH used as a control for equal protein loading. Immature H4 cells had lower baseline levels of I{kappa}B{alpha}, more rapid decrease of I{kappa}B{alpha}, and delayed return of I{kappa}B{alpha} protein levels following stimulus. Results shown are representative of n > 4 separate experiments with similar results.

 
Immature IEC have increased I{kappa}B{alpha} phosphorylation and ubiquitination compared with adult IEC. I{kappa}B{alpha} phosphorylation and subsequent ubiquitination targets I{kappa}B{alpha} for degradation by the proteasome. To determine the mechanism responsible for the TNF-{alpha} induced decreases in I{kappa}B{alpha} in immature IEC, we examined known steps upstream of I{kappa}B{alpha} degradation in the NF-{kappa}B pathway. Cells were treated with the proteasome inhibitor MG262 (1.5 µM) for 30 min before treatment with TNF-{alpha} at different time points as described above. MG262 preserves the phosphorylated and ubiquitinated forms of I{kappa}B{alpha} but concomitantly diminishes visible differences between individual time points. Cell lysates were then obtained and evaluated by immunoblotting. As shown in Fig. 5, top, by utilizing a specific phospho-I{kappa}B{alpha} antibody, H4 cells have increased I{kappa}B{alpha} phosphorylation compared with T84 cells. Similarly, H4 cells have increased I{kappa}B{alpha} ubiquitination compared with T84 cells (Fig. 5, middle). By utilizing an I{kappa}B{alpha} antibody that, because of MG262 proteasome inhibition, can now detect ubiquitinated forms of I{kappa}B{alpha}, the higher molecular weight bands of polyubiquitin not usually visible for regular I{kappa}B{alpha} immunoblotting can now be seen. IP studies with both I{kappa}B{alpha} and ubiquitin antibodies confirmed that these higher molecular bands are ubiquitin (data not shown). An increase in both I{kappa}B{alpha} phosphorylation and ubiquitination corresponds to enhanced I{kappa}B{alpha} degradation as the explanation for TNF-{alpha} induced decreases in I{kappa}B{alpha}.


Figure 5
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  		Fig. 5. Stimulated immature enterocytes have increased I{kappa}B{alpha} phosphorylation and ubiquitination compared with mature enterocytes. T84 and H4 cells were pretreated with the proteasome inhibitor MG262 for 30 min before treatment with TNF-{alpha} 10 ng/ml over time as indicated. Cell lysates were obtained and the expression of phosphorylated I{kappa}B{alpha} (pI{kappa}B{alpha}) and ubiquitinated I{kappa}B{alpha} and revealed by Western blot. GAPDH was used as a control for equal protein loading. Top: increased pI{kappa}B{alpha} in response to TNF-{alpha} in immature H4 cells compared with adult T84 cells. Middle: more polyubiquitin bands above I{kappa}B{alpha} in the H4 cells. Results shown are representative of 4 separate experiments with similar results.

 
Immature IEC have increased IKKbeta expression and phosphorylation compared with adult IEC. IKK is responsible for phosphorylating I{kappa}B{alpha}. IKK is composed of three subunits: IKK{alpha}, IKKbeta, and IKK{gamma} (NEMO). Only IKKbeta-deficient mice are defective in activation of IKK and NF-{kappa}B in response to TNF-{alpha} and IL-1beta, suggesting that IKKbeta plays the major role in IKK activation and induction of NF-{kappa}B activity (16). Cells were again treated with TNF-{alpha} at different time points as described above, then cell lysates were evaluated for IKKbeta and phospho-IKKbeta. The phospho-IKK antibody used was for both phospho-IKK{alpha} and phospho-IKKbeta. As demonstrated in Fig. 6, top, phospho-IKKbeta staining, visible as the upper bands on the immunoblot, was much stronger in the immature H4 cells than in the adult T84 cells. In immature cells phospho-IKKbeta increases by 5 min and then begins to diminish by 60 min and returns to baseline levels at 90 min. IKK phosphorylation is the signal for I{kappa}B{alpha} phosphorylation and degradation. This phospho-IKKbeta time course corresponds to the demonstrated time course of I{kappa}B{alpha} degradation in H4 cells, which begins at 5 min and returns to baseline by 90 min (Fig. 4). Phospho-IKK{alpha} bands, visible as the lower band of the immunoblot, were also greater in the H4 cells, but are not thought to play as important a role in NF-{kappa}B activation. In addition, as shown in Fig. 6 middle, there was increased baseline expression of IKKbeta in addition to increased IKKbeta phosphorylation in the immature H4 cells, potentially leading to the increased I{kappa}B{alpha} phosphorylation and downstream degradation of I{kappa}B{alpha}.


Figure 6
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  		Fig. 6. Stimulated immature enterocytes have increased I{kappa}B kinase-beta (IKKbeta) expression and phosphorylation compared with mature enterocytes. T84 and H4 cells were treated with TNF-{alpha} 10 ng/ml over time as indicated. Cell lysates were obtained and the expression of phospho-IKKbeta (pIKK, top) and IKKbeta (middle, upper band) revealed by Western blot. Hsc 73 was used as a control for equal protein loading. Results shown are representative of 4 separate experiments with similar results.

 
Immature IEC have delayed I{kappa}B{alpha} resynthesis. In addition to activating the transcription of inflammatory cytokines such as IL-8, NF-{kappa}B binding sites are also located on the I{kappa}B{alpha} promoter. Thus an elegant autoregulatory feedback loop is supposed to exist in which NF-{kappa}B activation leads to I{kappa}B{alpha} synthesis, which can subsequently terminate the NF-{kappa}B response (2, 23). The delay in return to baseline levels of I{kappa}B{alpha} previously described could be due to delayed resynthesis of I{kappa}B{alpha} following degradation or to prolonged signaling for degradation of the newly synthesized I{kappa}B{alpha}. I{kappa}B{alpha} is degraded by the proteasome; thus, to distinguish between these possibilities, cells were treated with the proteasome inhibitor MG262 15 min after treatment with TNF-{alpha} beginning with the 30-min time point of the time course as described above. This allowed initial signaling to be transmitted via the NF-{kappa}B pathway to induce I{kappa}B{alpha} resynthesis to occur yet block subsequent I{kappa}B{alpha} degradation. As shown in Fig. 7, even with inhibition of ongoing I{kappa}B{alpha} degradation in this fashion, delay in return of I{kappa}B{alpha} persists, suggesting that the delay in return is due not only to prolonged signaling leading to degradation but also to delayed resynthesis.


Figure 7
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  		Fig. 7. Inhibition of I{kappa}B{alpha} degradation by MG262 does not lead to more rapid return of I{kappa}B{alpha} after stimulation by TNF-{alpha} in immature enterocytes. T84 and H4 cells were treated with TNF-{alpha} 10 ng/ml over time as indicated with the proteasome inhibitor MG262 added 15 min after TNF-{alpha} addition beginning with the 30-min time point as indicated. This was done to block ongoing I{kappa}B{alpha} degradation but allow signaling for I{kappa}B{alpha} resynthesis. Cell lysates were obtained and the expression of I{kappa}B{alpha} was revealed by Western blot. GAPDH was used as a control for equal protein loading. Results shown are representative of 4 separate experiments with similar results.

 
Preweaned mice have increased p65 translocation compared with postweaned mice. To confirm that the phenomenon of increased NF-{kappa}B signaling was not merely a difference between the cell lines used, an in vivo model of immature and mature IEC was used. Preweaned (12- to 17-day-old) mice were used as a model of immature intestine and compared with postweaned (5- to 8-week-old) mice.

Mice were injected with TNF-{alpha} 0.4 mg/kg ip or sterile PBS as control. Small intestinal samples were harvested at time points ranging from 0 to 4.5 h. NF-{kappa}B activation begins with translocation of NF-{kappa}B proteins from the cytoplasm to the nucleus. The NF-{kappa}B protein p65 was visualized by immunofluorescence microscopy using rabbit polyclonal anti-p65 and Alexa Fluor 488 goat anti-rabbit IgG (H+L). By using NIH Image J v1.37 software color overlay, it is visible as green staining in Fig. 8. Nuclei were counterstained using DAPI and are visible as red staining in Fig. 8. Nuclear p65 appears as an overlap of red and green staining becoming yellow with increased green/p65 confluence. As shown in Fig. 8, postweaned mice had nuclear p65 translocation beginning ~3 h after TNF-{alpha} injection and fading by 4–4.5 h. In contrast, preweaned animals had evidence of much earlier nuclear p65 translocation at 1.5 h that persisted for several hours until beginning to fade at 4 h, consistent with cell culture findings of increased NF-{kappa}B activation in immature enterocytes.


Figure 8
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  		Fig. 8. TNF-{alpha} induces earlier and more prolonged nuclear translocation of NF-{kappa}B protein p65 in preweaned mice compared with postweaned mice. Preweaned mice (12–17 days old) and postweaned mice (5–8 wk old) were treated with TNF-{alpha} 0.4 mg/kg ip. After death at times postinjection as indicated, intestinal samples were harvested, and sections were obtained for immunofluorescence staining with the primary antibody rabbit polyclonal p65 conjugated to the secondary antibody Alexa Fluor 488 goat anti-rabbit IgG (H+L). Nuclei were counterstained with DAPI. Slides were visualized by using a Leica SP2 AOBS spectral confocal microscope with color overlay using NIH Image J software. To facilitate interpretation, images were pseudo-colored with NIH Image J software. Green indicates p65 staining, the blue color of DAPI was changed to red for nuclear staining, and, as indicated by arrows, nuclear translocation is visible as overlapping green-red staining appearing yellow-orange with increasing intensity. As shown, postweaned animals have overlapping green-red staining indicating p65 nuclear translocation beginning at 3 h, whereas preweaned animals had overlapping green-red staining of increased intensity appearing yellow beginning at 1.5 h and persisting for several hours. Results shown are representative of 3 separate experiments all with similar results.

 
Preweaned mice have increased I{kappa}B{alpha} phosphorylation compared with postweaned mice. To confirm that the phenomenon of increased I{kappa}B{alpha} degradation in immature enterocytes was not merely a difference between the cell lines used, intestinal samples from preweaned and postweaned mice treated with TNF-{alpha} as described were used for immunohistochemistry staining for phosphorylated I{kappa}B{alpha}. As described previously, I{kappa}B{alpha} phosphorylation is the signal for subsequent I{kappa}B{alpha} ubiquitination and degradation. As shown in Fig. 9, postweaned mice had phospho-I{kappa}B{alpha} staining beginning at 3 h whereas preweaned mice had much earlier evidence of phospho-I{kappa}B{alpha} staining beginning at 1 h post-TNF-{alpha} injection. This timing correlates well with the timing demonstrated for nuclear p65 translocation in these animals and is consistent with cell culture findings of more rapid I{kappa}B{alpha} degradation in immature enterocytes.


Figure 9
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  		Fig. 9. TNF-{alpha} induces earlier and more prolonged phosphorylation of I{kappa}B{alpha} in preweaned mice compared with postweaned mice. Preweaned mice (12–17 days old) and postweaned mice (5–8 wk old) were treated with TNF-{alpha} 0.4 mg/kg ip. After death at times postinjection as indicated, intestinal samples were harvested, and sections were obtained for immunohistochemistry staining with a mouse monoclonal phospho-I{kappa}B{alpha} antibody. Antibody staining was visualized with DAB chromogen and counterstained with hematoxylin. As shown and indicated by arrows, postweaned animals have evidence of phospho-I{kappa}B{alpha} staining beginning at 3 h, whereas preweaned animals had evidence of staining beginning at 1 h and persisting for several hours. Results shown are representative of 3 separate experiments all with similar results.

 

   DISCUSSION
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The NF-{kappa}B pathway is a key signaling pathway for immune and inflammatory responses. Tight regulation is supposed to ensure appropriate host defense mechanisms against pathogenic organisms but tolerance of beneficial commensal organisms. The findings reported here demonstrate insufficient inhibition of the NF-{kappa}B pathway in immature enterocytes, associated with accelerated signaling for degradation of as well as delayed resynthesis of I{kappa}B{alpha}, the primary inhibitor of NF-{kappa}B. Coupled with the decreased baseline expression of I{kappa}B{alpha} in immature enterocytes, our data suggest inadequate downregulation of inflammatory responses of the immature intestine corresponding to exaggerated IL-8 secretion in response to endogenous and exogenous inflammatory mediators as previously reported (5, 6).

Our data demonstrate increased levels of both IKK{alpha} and beta. Although both isoforms can phosphorylate I{kappa}B{alpha} in vitro, they appear to have distinct functions. IKK{alpha} itself has been shown to be important in development because IKK{alpha} knockout animals have abnormalities of skin and limb bud development (24). However, these animals do not seem to have abnormalities of NF-{kappa}B activation. In contrast, IKKbeta-deficient mice die at midgestation from extensive liver apoptosis very similar to what is seen in animals deficient in the NF-{kappa}B protein p65 (16). It is the IKKbeta isoform that appears to play the major role in IKK activation and induction of NF-{kappa}B activity. Thus our finding in immature IEC of increased IKKbeta baseline with more prolonged phosphorylation in response to TNF-{alpha} is consistent with our findings of prolonged I{kappa}B{alpha} phosphorylation and increased I{kappa}B{alpha} degradation, which may explain the demonstrated increased NF-{kappa}B activity.

It is interesting that our data suggest that there may be delayed synthesis of I{kappa}B{alpha} despite increased NF-{kappa}B activity in immature enterocytes. Studies in other cell lines have indicated specific NF-{kappa}B activity on individual promoters and different synthesis kinetics for NF-{kappa}B induced transcription of I{kappa}B{alpha} compared with inflammatory cytokines such as IL-6 and IL-8 (12, 20). Studies in macrophages have suggested that there is an elegant sequence of nuclear events regulating timing and duration of NF-{kappa}B binding to specific gene promoters leading to different waves of gene expression. Modification of the histone proteins associated with DNA is important in the regulation of gene expression. Histone-DNA contacts in individual nucleosomes and the folding of the nucleosomal chain limit the accessibility of specific genes. Rapid recruitment of NF-{kappa}B to a subset of target genes with constitutively heavily acetylated or rapidly phosphorylated promoters has been demonstrated (19). This group includes I{kappa}B{alpha}. In contrast, other target genes are not immediately accessible to NF-{kappa}B until 90–120 min after nuclear entry and require stimulus-dependent hyperacetylation or phosphorylation of the promoter to make them accessible to NF-{kappa}B (20). This includes inflammatory cytokines such as IL-6 and IL-8. Our data suggest the possibility that specific binding kinetics for different promoters may be developmentally regulated with increased transcription of inflammatory cytokines but decreased transcription of regulatory factors in immature enterocytes. Although beyond the scope of the present study, future studies should investigate developmental nuclear regulation of NF-{kappa}B in intestinal cells, which may account for differences in inflammatory and regulatory gene transcription in immature vs. mature enterocytes.

In addition, degradation of I{kappa}B{alpha} occurs in the 26S proteasome. It is known that this highly conserved entity is itself regulated. Others have demonstrated that the proteasome, which is comprised of multiple subunits, can change its degradation specificity on the basis of variations in activation of three of these subunits in response to modifications such as phosphorylation or glycosylation or in response to factors such as interferon-{gamma} (10, 15). It is conceivable that the signaling needs of fetal development warrant different degradation patterns by the proteasome of immature cells compared with that of mature cells. There may thus be developmental regulation of the proteasome itself. In addition to increased phosphorylation and ubiquitination of I{kappa}B{alpha} resulting in increased degradation, the proteasome itself may contribute to enhanced degradation. Our data demonstrate that the proteasome inhibitor MG262 did not lead to normalization of the return of I{kappa}B{alpha} after degradation, suggesting a delay in synthesis. An alternative possibility is that the immature proteasome responds differently to this inhibitor than the mature proteasome, allowing degradation of I{kappa}B{alpha} to continue. Proteasome activity was not evaluated in this study but is another potential point of developmental regulation.

Our studies utilized a cell culture model to systematically compare signaling events along the NF-{kappa}B pathway in immature and mature enterocytes. The comparison of H4 cells to the well-established adult IEC lines T84, Caco2, and HT29 has been used and validated in many other studies as a model of immature and mature IEC. Comparison of immature enterocytes (H4 cells) and mature enterocytes (confluent Caco2 cells) treated with lipopolysaccharide (LPS) or IL-1beta revealed that the immature enterocytes secreted more IL-8 (LPS, 8-fold; IL-1beta 20-fold) than mature enterocytes with a comparable increase in IL-8 mRNA in the immature enterocytes in response to both stimuli (18). These results were then confirmed in studies using small intestinal organ culture from fetuses (18–21 wk gestational age) compared with small intestinal biopsy samples from older infants and children. IL-8 secretion, along with IL-8 mRNA, was again increased in fetuses compared with older infants or children (LPS 2.5-fold, IL-1beta 200-fold) (18). Another study compared the response to cholera toxin in fetal H4 cells compared with adult T84 cells and documented a greater induction of cAMP in response to cholera toxin in the fetal cells. Likewise, organ culture of fetal intestine had a greater induction of cAMP than did biopsies from older infants and children (17). Thus findings in this cell culture system have been repeatedly validated by use of primary intestinal tissue in organ culture, thus justifying the use of the H4/adult enterocyte model utilized in our studies. However, there are inherent problems with a cell culture system including the possibility that the differences observed are unique to the utilized cell lines rather than representative of biological systems, as well as the limitation that interactions with other cell types cannot be studied. Thus NF-{kappa}B signaling was also investigated in an in vivo mouse model of immature and mature enterocytes. These studies confirmed increased phospho-I{kappa}B{alpha} and p65 translocation in preweaned mouse immature intestinal epithelium. Intestine from preweaned rodents less than 3 wk of age is considered to be developmentally comparable to third trimester human fetal intestine. After weaning at 3 wk of life, rat intestine undergoes developmental "closure," no longer allowing passage of macromolecules (4). Postweaned rat intestine is thus considered developmentally mature. Comparisons between preweaned and postweaned rat intestine have been used as a model to approximate differences in immature and mature human intestine in other studies such as those investigating developmental responses to cholera toxin (22).

Thus these studies demonstrate that the cell culture findings have physiological relevance and scientific merit. Because the in vitro and in vivo responses are identical, the more reductionist approach with cell cultures is highly appropriate and allowed development of important mechanistic insights into the NF-{kappa}B signaling in the immature intestinal epithelium. In combination, these findings suggest that immature IEC do have inadequate inhibition of the NF-{kappa}B pathway leading to earlier and prolonged activation of NF-{kappa}B.

These studies have focused specifically on IEC. Other immune cells in the gastrointestinal tract can also produce inflammatory cytokines that may contribute to the intestinal damage, and it is possible that lymphoid elements may have a role in facilitating the epithelial response. Future studies to investigate the interaction between immune and epithelial cells would further enhance our understanding of the development of gut inflammatory responses.

Necrotizing enterocolitis (NEC) is an inflammatory bowel disease seen only in premature infants and thought to be a consequence of inappropriate inflammatory responses to microbial interaction by the immature intestine. Studies in a rat model of NEC demonstrated increased NF-{kappa}B binding activity in the small intestine of animals 0–3 h after the induction of NEC, potentially suggesting that NF-{kappa}B activation is an early step in the intestinal injury (3). Our data demonstrate several points of developmental difference: increased NF-{kappa}B activity, increased NF-{kappa}B DNA binding, accelerated I{kappa}B{alpha} phosphorylation, ubiquitination and degradation, increased IKKbeta expression and phosphorylation along with decreased I{kappa}B{alpha} resynthesis. We speculate that failure to terminate NF-{kappa}B activation in immature IEC may partially explain the unique susceptibility of preterm infants to NEC.

The preterm intestine is essentially a fetal intestine not expecting the conditions of the extrauterine environment and not having completed the growth and maturation processes that normally continue until term. The NF-{kappa}B pathway activates transcription of many other genes in addition to inflammatory cytokines. Perhaps the immature intestine, expecting the sterile environment of the intrauterine environment, has increased NF-{kappa}B signaling not for an inflammatory response but rather for other effects important for fetal development.

Further understanding of the intestinal NF-{kappa}B pathway and the intricacies of NF-{kappa}B binding to promoters of specific genes has implications for NEC and other inflammatory bowel diseases. It may be an important developmental step for the immature intestine to downregulate inflammatory responses as it transitions from the sterile intrauterine environment to obtaining the microbial colonization of the extrauterine world. It is unclear whether this maturation is the result of a normal genetic pattern, is the effect of factors in amniotic fluid or breast milk, or is triggered by contact with the microflora itself. Ongoing investigation into the mechanisms responsible for developmental downregulation of NF-{kappa}B signaling is a logical next step.

As many of the long-term morbidities of prematurity are inflammatory in nature including chronic lung disease and periventricular leukomalacia in addition to NEC, understanding the signaling leading to exaggerated inflammatory responses will advance understanding of the developmental disease susceptibility of the premature infant. Therapy designed to not only limit disease progression but also hasten normal maturation of the intestine and modulate exaggerated inflammatory responses is important to alleviate not only the initial intestinal injury but the long-term morbidity associated with NEC (11).


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This study was supported by National Institutes of Health Grants K08 HD-043839 (to E. Claud), K08 DK-064840 (to E. Petrof), and K01 DK-075386 (to J. Sun). The DK-42086 grant supported pilot and feasibility awards (to E. Claud and J. Sun) and maintenance of core facilities used.


   DISCLOSURES
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 ABSTRACT
 MATERIALS AND METHODS
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REFERENCES
 
This study was partially supported by a GlaxoSmithKline Institute for Digestive Health research award (to E. Claud).


   ACKNOWLEDGMENTS
 
We thank Tonya Waypa for technical assistance with the EMSA assays.


   FOOTNOTES
 

Address for reprint requests and other correspondence: E. C. Claud, The Univ. of Chicago, Dept. of Pediatrics Section of Neonatology, 5841 S. Maryland Ave. MC6060, Chicago, IL 60637 (e-mail: eclaud{at}peds.bsd.uchicago.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.


   REFERENCES
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 REFERENCES
 

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