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B/NF-B in Human Fetal and Adult Intestinal Epithelium1
Department of Pediatrics, College of Medicine, University of Florida, Gainesville, FL 32610-0296
2To whom correspondence should be addressed. E-mail: neuj{at}peds.ufl.edu.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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B (IB)/nuclear factor (NF)-B signaling pathway in fetal-derived (H4) and adult (Caco-2) enterocytes. In the absence of Gln with or without LPS, H4 cells expressed more interleukin (IL)-8) than Caco-2 cells. Gln supplementation partially prevented the LPS-induced elevation of IL-8 in both cell types. IB
was significantly decreased in both H4 and Caco-2 cells with Gln deprivation, and this was followed by an increase in NF-B p65 in the nucleus. DNA binding of NF-B was increased in both H4 and Caco-2 cells with Gln deprivation. IB phosphorylation was not altered by Gln status in either H4 or Caco-2 cells. Proteasomal inhibition after Gln depletion in Caco-2 cells was associated with an increase in the IB-ubiquitin complex, but a decrease in complex formation in H4 cells, indicating that Gln deprivation alters IB through a pathway that differs from Caco-2 cells. We speculate that a reduced capacity of the immature enterocyte (H4) to respond to Gln deprivation with increased synthesis of IB rather than increased proteolysis as seen in the Caco-2 cells is the underlying mechanism.
KEY WORDS: • glutamine • interleukin-8 • IB/NF- B • inflammation
The immature intestinal epithelium is exquisitely sensitive to mediators of inflammation compared with the adult epithelium (1). In premature infants, an exacerbated inflammatory response by the intestinal epithelium, after bacterial colonization, can lead to increased expression and secretion of proinflammatory mediators such as interleukin (IL)-8,3 a C-X-C chemokine (2), which stimulates migration of neutrophils from intravascular to interstitial and luminal sites. Expression of proinflammatory mediators must be controlled to prevent excessive tissue injury and damage to local and distal organs (3,4). At the molecular level, the extracellular stimuli of inflammatory molecules, such as lipopolysaccharide (LPS) and flagellin, acting through Toll-like receptors initiate a cascade of nuclear factor (NF)-B–dependent intracellular events that culminate with the transcription and translation of IL-8, which differs in magnitude of response in the immature and mature human enterocyte (1,5). A partial mechanism for developmental expression of inhibitor of B (IB)/NF-B transduction was described, in which increased levels of IB in the mature intestine dampen the brisk IL-8 response to bacteria found in immature enterocytes (5). This raises the question whether the availability of certain nutrients might alter levels of IB through mechanisms such as the prevention of degradation, thereby modulating IL-8 production in the intestinal epithelium.
Glutamine (Gln), the most abundant amino acid in the human body, performs multiple roles in the intestine and may even serve as a signaling molecule (6). Very little is known about the effect of Gln on IB/NFB transduction pathways, despite several recent studies suggesting that Gln can alter intestinal inflammation (7–11). In adult humans, Gln pretreatment significantly decreased production of proinflammatory cytokines (IL-6 and IL-8) by the intestinal mucosa (10,11). Low plasma concentrations of Gln have been associated with a higher incidence of necrotizing enterocolitis (NEC) (12), one of the major causes of mortality and morbidity in low-birth-weight infants. A recent trial of Gln supplementation in very-low-birth-weight infants showed a significant decrease in intraventricular hemorrhage and periventricular leukomalacia, disorders that are known to cause cerebral palsy, which is associated with high levels of proinflammatory cytokines (13). Recent evidence showed that in Caco-2 cells, Gln deprivation enhanced IL-8 production after LPS stimulation (8,9). Studies in infant rats demonstrated that Gln supplementation can decrease the production of intestinal inflammatory mediators (7). The relation between Gln status and the proinflammatory state of the intestine requires an understanding of the underlying mechanisms.
We hypothesized that Gln would downregulate IL-8 expression in both Caco-2 (a widely studied human adult intestinal epithelial cell model) and H4 (a recently developed human fetal nontransformed primary small intestinal epithelial cell model) cells, but the magnitude of the effect would differ between these models of adult and fetal intestinal epithelia. Furthermore, we hypothesized that the mechanism by which Gln interferes with IB/NFB-mediated IL-8 production is via altered synthesis or degradation of IB. The results of this study document a previously undescribed mechanism of IL-8 regulation mediated by Gln acting through IB, which differs between adult and fetal enterocytes.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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B (native and phosphorylated forms), IBß, NF-B p65, ubiquitin, ß-actin, and Protein A-agarose were obtained from Santa Cruz Biotechnology. Intestinal cell lines. H4 cells are a human fetal nontransformed primary small intestinal epithelial cell line that was developed from a 20-wk-old normal fetus and was well characterized by Sanderson et al. (14). Cell passages 5–10 were used. H4 cells were grown in a humidified incubator at 37°C under 5% CO2, 95% air in DMEM supplemented with 10% FBS, 2 mmol/L Gln, 100,000 U/L penicillin, 100 mg/L streptomycin, and 0.25 mg/L amphotericin B, MEM nonessential amino acid solution, 10mmol/L Hepes buffer, 1 mmol/L sodium pyruvate, and Human Recombinant Insulin (10 mg/L). Media were changed 3 times/wk. H4 cells were used to represent immature fetal enterocytes. The Caco-2 cell line is a well-established cell model (15) and was obtained from American Type Culture Collection. The cells were used (passages 17–25) as a model of mature differentiated enterocytes and collected by dissociation of a confluent stock culture with 0.25% trypsin and 1 mmol/L EDTA. Caco-2 cells were cultured as previously described (9). Experiments in H4 cells were initiated on d 7 after reaching confluence. In Caco-2 cells, experiments were initiated on d 14 after seeding, 3–7 d after confluence. Previous studies in our laboratory showed that this is a time at which Caco-2 cells begin to express alkaline phosphatase activity and are in an early stage of differentiation, corresponding to the upper crypt-lower villous stage of differentiation. Both cell lines were grown in triplicate.
ELISA. The 2 cell lines were seeded into 12-well plates (Costar®, Corning) at a density of 2–3 x 105 cells/cm2. Before treatment, monolayers were rinsed with PBS and then pretreated with serum-free medium containing 0 (Gln 0) or 0.5 mmol/L Gln (Gln 0.5), with or without 4 mmol/L MS (MS 4) for 24 h. LPS (1, 10, and 100 mg/L) was added to the culture media for another 24 h. Supernatants of culture media were collected and frozen at –20°C for determination of the concentrations of IL-8 by ELISA (OptEIATM, PharMingen).
High Binding Extra microtiter plates (96-well; IMMULON®4HBX, Dynex Technologies) were used to quantify the IL-8 production as described by the manufacturer. Plates were read at 450 nm using a kinetic microplate reader (Bio-Tek Instruments). The amount of cytokines was quantified within each supernatant in duplicate.
Nuclear protein extraction.
Cells (6–8 x 104 cells/cm2) in 100-mm diameter dishes were cultured for 7 (H4 cells) and 14 d (Caco-2 cells). After incubation with different concentrations of Gln in the presence or absence of MS for 24 h, cells were stimulated with or without 100 mg/L LPS for 2 h (H4 cells) and 15 min (Caco-2 cells). These times were chosen to ensure maximum IB degradation and NFB production; this dose of LPS was determined by preliminary studies. All nuclear extraction procedures were performed on ice with ice-cold reagents. Nuclear protein was extracted by the Nuclear Extraction Kit from Panomics. Protein concentrations of the nuclear extract and the cytosolic fraction were measured by using the BioRad Dc Protein Assay.
Immunoblotting.
After determination of the protein concentrations of the cleared lysates (nuclear and cytoplasm fractions), equal amounts of protein were separated by 12.5% SDS-PAGE. Electrophoresed proteins were transferred from the gel to a polyvinylidene difluoride membrane, blocked with 5% nonfat dry milk in Tris-buffered saline with Tween 20, incubated with the primary antibody (anti-IB, anti-IBß or anti-NF-B p65) and then with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibody. The blot was developed using enhanced chemiluminescence (ECL; Amersham Pharmacia Biotech) according to the manufacturer’s instructions. The membranes were then stripped and reprobed with antibody to ß-actin to confirm equal loading of lanes. Protein bands were quantified by densitometry using Adobe Photoshop software.
Immunoprecipitation of ubiquitinated-IB (Ub-IB).
After the cells were incubated with or without Gln and MS for 24 h, they were treated with 50 µmol/L MG-132 for 1 h (16) before stimulation with LPS (100 mg/L) for 15 min (Caco-2 cells) or 2 h (H4 cells). Cytoplasmic extracts (200 µg of protein), made up to 100 µL with buffer [20 mmol/L Tris, pH 7.5, 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 1% Triton X-100, 1 mmol/L sodium vanadate, and 1x complete protease inhibitor mixture (Roche Molecular Biochemicals) (17)], were incubated overnight at 4°C with anti-IB (1:100). The samples were then treated with 30 µL of protein A-agarose beads for 2 h at 4°C; then the samples were centrifuged and washed 5 times in buffer at 1000 x g for 30 sec. The beads were boiled in SDS-PAGE sample treatment buffer and electrophoresed on a 10% SDS-PAGE. The gel was blotted and incubated with a monoclonal antibody to ubiquitin (1:1000) overnight followed by incubation with the HRP-conjugated secondary antibody (1:1000). Antigen-antibody complexes were detected with ECL (Amersham Pharmacia Biotech).
Electrophoretic mobility shift assays. The binding tests for transcriptional factors were performed as described in the kit from Panomics, i.e, 10 µL of binding reaction mixtures containing 1 µg of poly (dI-dC) and 10 ng biotin-labeled transcription factor probe in binding buffer were incubated with 5 µg cell nuclear extracts at room temperature for 30 min, followed by fractionation on native 5% Tris borate EDTA polyacrylamide gels, transferred to a nylon membrane, and detected by Streptavidin HRP solution.
Statistical analysis. Results are presented as means ± SD of triplicate measurements. Pairwise multiple comparisons were conducted following significant 1-way ANOVA using the Bonferroni t test. All analyses were performed using Sigma Stat software (SPSS Science) and differences among means were considered significant at P < 0.05.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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B degradation.
The greatest degradation of IB occurred in H4 cells at 
120 min and in Caco-2 cells at 15 min (data not shown). Using these time courses, we were then able to evaluate the effects on the IB pathway in both H4 and Caco-2 cells using Western blots.
Gln alters LPS induced IkB expression in H4 cells.
To begin to investigate whether the mechanism of Gln-mediated alteration of IL-8 expression is via IB, H4 cells were incubated with or without Gln and MS and then stimulated by LPS as above. Fig. 2A and B depict the effect of extracellular (via the medium) and intracellular (via MS) Gln deprivation on LPS-stimulated H4 cells at 120 min (Fig. 2A) and 24 h (Fig. 2B). From the blots and bar graphs (n = 3 different determinations) it can be seen that the situation with the greatest Gln deprivation [no Gln in the medium (Gln 0) and inhibition of Gln synthetase with MS 4 (lane 3)] downregulates IB in the cytosol at 120 min (P < 0.05 vs. LPS/Gln 0.5/MS0; Fig. 2A) and at 24 h (P < 0.05 vs. LPS/Gln 0/MS 4; Fig. 2B), suggesting that Gln is necessary for the reappearance of normal levels of IB in the cell at 24 h. Replacing Gln in the medium reverses the effect of MS (lane 4), indicating that the MS effect was specific for Gln deprivation. This effect suggests that Gln is acting through maintenance of IB in the cytosol of these cells. No effect was seen when IBß was evaluated (data not shown).
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B expression in Caco-2 cells.
To determine whether H4 cells responded differently from cells representing adult epithelium, Caco-2 cells were treated similarly to the H4 cells and stimulated by LPS for 15 min. LPS had an effect at 15 min (Fig. 2Cand 2D), but there was no effect of Gln. However, Gln deprivation significantly decreased IB- at 24 h (Fig. 2D), indicating that Gln exhibited delayed effects on these cells, similarly to the H4 cells treated in the same way at 24 h (P < 0.05).
Effect of Gln and LPS on NFB p65 level in H4 cells and Caco-2 cells.
To evaluate the effect of Gln and LPS on NFB p65 level in the nucleus, a time course experiment was initially performed, which demonstrated that the maximum NFB p65 expression after LPS stimulation was at 120 min (P < 0.05) in H4 cells and at 15 min (P < 0.01) in Caco-2 cells (data not shown).
To evaluate the effect of Gln on NFB p65 level in H4 cells, cells were incubated with or without Gln and MS and stimulated by LPS for 120 min (Fig. 3A). At 120 min there was no Gln effect (Fig. 3A); however, at 24 h (Fig. 3B) Gln deprivation increased NFB p65 expression (P < 0.05).
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B p65 level in Caco-2 cells, cells were treated as above but stimulated by LPS for 15 min. No Gln effect was seen at 15 min (Fig. 3C); however, at 24 h, Gln deprivation tended to increase of NFB p65 expression (P = 0.08) (Fig. 3D).
Electrophoretic mobility shift assay (EMSA) for NFB in H4 and Caco-2 cells.
We then evaluated the effect of Gln on NF-B nuclear binding activity under different conditions of Gln and LPS. H4 cells were incubated with or without Gln and MS and then stimulated by LPS for 120 min (Fig. 4). The greatest binding occurred with the most stringent condition of Gln deprivation (no Gln in the medium and treatment with the Gln synthetase inhibitor), suggesting that Gln deprivation upregulates NF-B binding activity (lane 3) by 15% and the addition of Gln reversed this effect (lane 4). A similar increase of 15% in NF-B binding was seen in the most stringently Gln-deprived group in Caco-2 cells (data not shown).
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B phosphorylation and ubiquitination after Gln deprivation in H4 cells and Caco-2 cells.
To determine whether the increased IL-8 production occurred through altered phosphorylation or ubiquitin-proteasome–mediated proteolytic degradation of IB, H4 and Caco-2 were incubated with or without Gln and MS for 24 h, incubated with the proteasome inhibitor MG132 for 1 h, and stimulated by LPS for 120 min (H4 cells) or 15 min (Caco-2 cells) according to our previous time course experiments. The different Gln conditions did not alter phosphorylation in either cell type, but Gln deprivation decreased Ub-IB (lane 7) in H4 cells (Fig. 5) and increases Ub-I (Fig. 6) in Caco-2 cells; the addition of Gln in the media prevented this effect (lane 8) in both cell types.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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B/NFB transduction pathway of Gln-mediated modulation of the IL-8 response in these cells. The findings reported here document that in the presence of LPS, Gln deprivation decreases IB, which in turn allows greater nuclear translocation of NFB, a major factor regulating inflammatory and immune responses, including several cytokines and leukocyte chemoattractant chemokines such as IL-8 (18). In both cell types, deprivation of Gln had long-lasting effects on the IB/NFB pathway, possibly relating Gln status to sustained inflammation in the intestine during stress. The mechanism of the decrease in IB appears to differ between H4 and Caco-2 cells, i.e., in H4 cells, Gln deprivation in the presence of LPS resulted in decreased production of IB; in Caco-2 cells, it acted through increased proteolytic degradation.
The cells used for these studies were characterized previously (14,15). It was found that the exaggerated IL-8 developmental response to initiators of inflammation such as IL-1ß, LPS, and flagellin that occurs in H4 vs. Caco-2 cells also occurs in other immature and mature cell lines, in human intestinal explants derived from fetal vs. mature human intestine, and in primary cultures of rat infant and adult intestine (1,5). These are thus reasonable models for evaluation of maturational response of the inflammatory pathway to nutrients such as Gln. We do not know the reasons behind the large differences in time required for maximum IB degradation between cell types. It is possible that these differences stem from the fact that one cell line is colonic and transformed and the other is small intestinal and nontransformed. Additional future studies at different stages of Caco-2 cell differentiation and/or using primary rat intestinal epithelial cell cultures might validate and help explain these results.
There are several signaling pathways that could be involved in the transduction of the IL-8 response to initiators of inflammation. We recently showed a relation of Gln to downregulation of LPS-induced IL-8 production and the signal transducer and activator of transcription (STAT)-4 pathway in Caco-2 cells (9). This appears not to be the case in H4 cells because we did not find any EMSA binding of STAT-4 in the H4 cell line (data not shown), prompting a greater emphasis on comparing the NF-B pathway between the 2 cell types. NF-B regulates the expression of multiple immediate early genes involved in the immune, acute phase, and inflammatory responses (19). NF-B subunits are kept inactive in the cytoplasm by an endogenous inhibitor protein of the IB family (20). Under stimulation by IL-1, LPS, or bacteria, IB is phosphorylated, selectively ubiquitinated, and rapidly degraded. Once free of IB, NF-B translocates into the nucleus and induces target genes that have NF- B binding sites (20). In a previous study, we found that LPS induction of IL-8 in Caco-2 cells was independent of the NF-B p50 subunit (8). In this study, we showed that degradation of IB and nuclear translocation of the NF-B p65 subunit occur before induction of IL-8 production, and both IL-8 and IB/NF-B levels are modulated by Gln status.
The IB response to flagellin was shown recently to be developmentally regulated, with a greater response in the immature enterocyte compared with the adult due to decreased levels of IB proteins (5). Furthermore, flagellin-stimulated IL-8 production was decreased when immature enterocytes were transfected with IB. For both H4 cells and Caco-2 cells, there was a significant decrease of IB in Gln-depleted cells with subsequent elevation of the NF-B protein level and DNA binding. We attempted to determine whether the mechanism of decreased IB with Gln deprivation in LPS-stimulated enterocytes was due to increased proteolytic degradation via the ubiquitin-proteasome pathway. Our results in Caco-2 cells are consistent with this mechanism as evidenced by the marked increase in Ub-IB after proteasome inhibition with MG 132. However, in H4 cells, the Ub-IB decreased, suggesting utilization of a pathway that results in a decrease in IB secondary to Gln deprivation that is not due to increased uniquitin-proteasome–mediated degradation. This is a complex pathway that could have numerous explanations (18). One explanation for this response in immature cells might relate to an increased responsiveness to amino acid starvation caused by Gln deprivation. This response to amino acid deprivation leads to phosphorylation of eukaryotic initiation factor 2, inhibition of protein synthesis, and the activation of the endoplasmic reticulum stress response. Recent studies in a breast cancer cell line demonstrated that Gln deprivation significantly elevated both IL-8 and vascular endothelial growth factor; these were associated with altered gene expression via the unfolded protein response signaling pathway (21). Further studies are required to determine whether this pathway of cellular stress is activated in the H4 cells and whether this plays a role in Gln deprivation activation of IL-8 production.
Many of the major complications of prematurity, including chronic lung disease, periventricular leukomalacia, and NEC, are associated with elevations in proinflammatory mediators (22–24). In fact, IL-8 in bronchoalveolar lavage fluid from preterm infants ventilated for respiratory distress syndrome (24) in the first few days of life is high. IL-8 is also twice as high in extremely low-birth-weight infants as it is in term newborns (25). The migration of polymorphonuclear leukocytes (PMNs) into tissues is a hallmark of several inflammatory conditions. IL-8 is a C-X-C chemokine that recruits PMNs, which in turn mediate tissue destruction. This process has been implicated in the pathogenesis of various forms of tissue injury (26,27).
In the current study, the IL-8 response to LPS was exacerbated in both immature (H4) and adult (Caco-2) cells after Gln deprivation. However, the overall response was greatest and would be most meaningful in the immature enterocyte. In premature infants, there is a strong rationale for supplementation with Gln. When born prematurely, they are suddenly deprived of the abundant supply of Gln derived in utero from their mothers and the placenta. Furthermore, these infants are highly stressed, and have an increased utilization of Gln during their first several weeks of life (28). Low plasma concentrations of Gln have been associated with a high incidence of NEC (12). Although only a few trials of Gln supplementation have been performed in premature infants, the results have been mixed. One large trial of enteral Gln supplementation in very-low-birth-weight infants did not show differences in hospital-acquired sepsis, the primary outcome, but secondary analysis demonstrated a significant decrease in intraventricular hemorrhage and periventricular leukomalacia in survivors who were supplemented with Gln (13). This raises the question whether enterally administered Gln might have contributed to this protective response by decreasing the systemic load of IL-8 derived from the intestine.
In conclusion, the role of the injured intestine is becoming increasingly recognized as being a critical component of several pathologic entities, especially when it acts as a nidus for a proinflammatory response that can propagate to distal organs (liver, lung, and brain) and cause multiple organ dysfunctions. The current study points to a mechanism by which Gln deprivation can upregulate an important proinflammatory mediator (IL-8) via decreasing IB in both the immature and mature intestine, but with the immature intestine showing the greatest response. It also provides a further stimulus to evaluate the relation of immunonutrients such as Gln to the prevention of disease entities with a basis in gut-derived inflammation.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Abbreviations used: ECL, enhanced chemiluminescence; EMSA, electrophoretic mobility shift assay; Gln 0, glutamine 0 mmol/L; Gln 0.5, glutamine 0.5 mmol/L; HRP, horseradish peroxidase; IB, inhibitor of B; IL, interleukin; LPS, lipopolysaccharide; MS, methionine sulfoximine; MS 0, methionine sulfoximine 0 mmol/L; MS 4, methionine sulfoximine 4 mmol/L; NEC, necrotizing enterocolitis; NF-B, nuclear factor-B; PMN, polymorphonuclear leukocyte; STAT, signal transducer and activator of transcription; Ub, ubiquitinated.
Manuscript received 17 September 2004. Initial review completed 25 October 2004. Revision accepted 19 November 2004.
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LITERATURE CITED |
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A. Hubert-Buron, J. Leblond, A. Jacquot, P. Ducrotte, P. Dechelotte, and M. Coeffier Glutamine Pretreatment Reduces IL-8 Production in Human Intestinal Epithelial Cells by Limiting I{kappa}B{alpha} Ubiquitination J. Nutr., June 1, 2006; 136(6): 1461 - 1465. [Abstract] [Full Text] [PDF]
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L. Zhang, N. Li, R. Caicedo, and J. Neu Alive and Dead Lactobacillus rhamnosus GG Decrease Tumor Necrosis Factor-{alpha}-Induced Interleukin-8 Production in Caco-2 Cells J. Nutr., July 1, 2005; 135(7): 1752 - 1756. [Abstract] [Full Text] [PDF]
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