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Biochemical, Molecular, and Genetic Mechanisms

Glutamine Pretreatment Reduces IL-8 Production in Human Intestinal Epithelial Cells by Limiting IB Ubiquitination

Aurélie Hubert-Buron^*, Jonathan Leblond^*, Arnaud Jacquot^*, Philippe Ducrotté^*,, Pierre Déchelotte^*,,1 and Moïse Coëffier^*,

^* Appareil Digestif Environnement et Nutrition (ADEN EA-3234), Institut Fédératif de Recherches Multidisciplinaires sur les Peptides, Medical Faculty, 76183 Rouen, France and Rouen University Hospital, Rouen, France

¹ To whom correspondence should be addressed. E-mail: pierre.dechelotte{at}chu-rouen.fr.

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Glutamine, the most abundant amino acid in the human body, plays several important roles in the intestine. Recent studies showed that glutamine regulates protein metabolism and intestinal inflammation among other mechanisms by reducing proinflammatory cytokine release. Because regulation of the inflammatory response was shown to be linked to proteolysis regulation, we hypothesized that glutamine pretreatment could act on IL-8 production in human intestinal epithelial cells through the regulation of inhibitor B (IB) ubiquitination. The HCT-8 cells were pretreated for 24 h with 0.6, 2, or 10 mmol/L glutamine. IL-8 concentration and IB (free and ubiquitinated) expressions were assessed by ELISA and immunoblotting, respectively. A pretreatment with 10 mmol/L glutamine decreased IL-8 production under both basal and proinflammatory conditions (both P < 0.05). In the presence of a proteasome inhibitor (MG132), the ubiquitin-IB complex expression was not significantly modified by glutamine under basal conditions but decreased significantly under proinflammatory conditions (P < 0.05). After the addition of 10 mmol/L of glutamine, the free IB expression increased under basal and stimulated conditions (both P < 0.05). A glutamine pretreatment of 10 mmol/L did not affect ubiquitin expression or proteasome activity. This study indicates that glutamine pretreatment may reduce the intestinal inflammatory response by limiting the proteolysis of IB.

KEY WORDS: • glutamine • cytokine • IB • ubiquitin • intestine

Glutamine, the preferred substrate for both enterocytes and other rapidly dividing cells, such as immune cells, was proposed to have beneficial effects on clinical outcomes in critically ill patients (1,2). These effects could be explained by the influence of glutamine on intestinal functions as described in animal models (3). Indeed, glutamine improved intestinal integrity by increasing cell proliferation (4,5), reducing intestinal epithelial cell apoptosis (6), or improving gut protein metabolism (7,8).

Glutamine also modulates the inflammatory response. For example, glutamine influenced cytokine production by various cell types in vitro (9,10). Plasma IL-8 concentration was decreased by parenterally administered glutamine in septic rats (11) and in patients with severe pancreatitis (12). Intestinal cytokine production was also modulated by glutamine in different models. In rats, glutamine decreased tumor necrosis factor- (TNF-)² and IL-8 production and improved 2,4,6-trinitrobenzene sulfonic acid–induced tissue lesions (13), whereas it restored IL-4 and IL-10 intestinal production during total parenteral nutrition (14,15). In addition, we reported previously that glutamine decreased basal and IL-1ß–induced productions of IL-6 and IL-8 and increased IL-10 production by human duodenal mucosa (16–18). It was also reported that glutamine prevented nuclear factor (NF)-B activation in intestinal epithelial cells (19). However, pathways by which glutamine modulates cytokine production have yet to be fully clarified.

In fact, glutamine may reduce inflammatory response by increasing IL-10 production, but also inducing heat shock protein (hsp) expression (20,21) or improving antioxidant status (6). In addition, glutamine may regulate proteolysis because we reported that enteral glutamine reduced the ubiquitin mRNA level in human duodenal mucosa (8). Moreover, the ATP-ubiquitin-dependent proteolytic pathway contributes to the regulation of the inflammatory response. Indeed, inhibitor of B (IB) ubiquitination allows the translocation of NF-B in the nucleus and consequently the transcription of proinflammatory genes, including IL-8 (22). Liboni et al. (23) showed recently that the effect of induction of IL-8 production by glutamine depletion involved an increase in the IB-ubiquitin complex in Caco-2 cells. Nevertheless the effects of glutamine supplementation on IB degradation have not yet been examined.

Thus, the aim of our study was to evaluate the effects of glutamine pretreatment on IB ubiquitination in human intestinal epithelial cells.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
    Materials. The medium and products used for cell culture were obtained from Eurobio. Recombinant human IL-1ß and MG132, an inhibitor of proteasome activity, were supplied by Sigma-Aldrich; recombinant human TNF and recombinant interferon- (IFN) were from Tebu. Protein G-agarose, antibodies for IB (native form), and ubiquitin were obtained from Calbiochem. ELISA kits were from R&D Systems and Hyperfilm MP from Amersham Biosciences. HCT-8 cells, derived from human ileocecal epithelial adenocarcinoma, were obtained from the European Collection of Animal Cell Cultures.

    Cell culture. The cells were seeded at a density of 12 x 10³ cells/cm² into 21-cm² tissue culture dishes; they were grown in DMEM containing 2 mmol/L glutamine and supplemented with 10% heat-inactivated fetal bovine serum, 1% nonessential amino acid, and 1% antibiotics (0.1 U/L penicillin, 0.1 U/L streptomycin) as standard medium at 37°C in a water-saturated atmosphere with 5% CO₂. To study the influence of glutamine, glutamine-free and serum-free DMEM media were used.

Cells between passages 42 and 61 were used at confluence after 3 d of growth. Glutamine was added at various concentrations (0.6, 2, and 10 mmol/L) for 24 h and then cells were stimulated or not with a mixture of proinflammatory cytokines called cytomix (1 µg/L IL-1ß, 20 µg/L TNF, 10 µg/L IFN) as previously described (24–26). To study free and ubiquitinated IB, cells were treated 1 h before cytomix stimulation with the proteasome inhibitor MG132 at 20 µmol/L and then stimulated with or without cytomix for 30 min. At the end of the experiment, culture media were collected, filtered, immediately frozen in liquid nitrogen, and stored at –80°C until cytokine measurement. The cells were washed with PBS, then centrifuged at 1000 x g for 10 min, and the cell pellets stored at –80°C for protein measurement.

    IL-8 assay. After a pretreatment with increasing concentrations of glutamine for 24 h, cells were stimulated or not with cytomix for 18 h. IL-8 concentration was measured with ELISA kits using a 96-well plate reader (960 photometer, Metertech) at a wavelength of 450 nm (each sample was quantified in duplicate). The sensitivity was 13.9 ng/L for IL-8. Values were then normalized to total cellular protein assessed with the method of Lowry (27) using bovine serum albumin as a standard.

    Immunoprecipitation of IB. After pretreatment with increasing concentrations of glutamine for 24 h, cells were stimulated or not with cytomix for 30 min. Then, cells were washed with ice-cold PBS and Buffer A (50 mmol/L HEPES, 150 mmol/L NaCl, 10 mmol/L EDTA, 10 mmol/L ß-glycerophosphate, 100 mmol/L NaF, 2 mmol/L ortho-vanadate, pH 7.5). They were then lysed in lysis buffer (Buffer A supplemented with 1% Triton X-100, 1 mmol/L phenylmethyl sulfoxide, 20 µmol/L leupeptin, 2 nmol/L aprotinin) for 15 min at 4°C and centrifuged at 12,000 x g for 15 min. IB was immunoprecipitated in the supernatant by the addition of 1 µg of specific rabbit antibody preadsorbed on protein-A agarose for 2 h (28). Protein concentrations of cell extracts were measured by the method of Bradford (29).

    Immunoblotting for IB and ubiquitin. Total proteins (25 µg) and immunoprecipitated proteins (60 µg) were separated on 4–12% Tris-Glycine resolving gels (Invitrogen) and transferred to a polyvinylidene fluoride membrane. The membrane was blocked for 1 h at room temperature with 5% (wt:v) nonfat dry milk in Tris-buffered saline (TBS; 10 mmol/L Tris, pH 8; 150 mmol/L NaCl) plus 0.05% (wt:v) Tween 20 followed by an overnight incubation at 4°C with anti-IB (1:1000) or with anti-ubiquitin (1:500) antibodies. After 3 washes in a blocking solution of 5% (wt:v) nonfat dry milk in TBS:0.05% Tween 20, a 1-h incubation with peroxidase-conjugated goat anti-rabbit IgG (1:3000) was performed. After 3 additional washes, immunocomplexes were revealed using the enhanced chemiluminescence detection system (Amersham Biosciences). Protein bands were quantified by densitometry using IRIS 5.10 software.

    Proteasome activity. After being washed with cold PBS, cells were scraped into 200 µL of ice-cold lysis buffer containing 30 mmol/L Tris-HCl (pH 7.2), 1 mmol/L dithiothreitol, and 1% Triton X-100. Cells were placed on ice for 15 min and then centrifuged at 11,000 x g for 15 min at 4°C. Protein (20 µg) was incubated with 20 mmol/L Tris-HCl (pH 7.2), 0.5 mmol/L EDTA, 0.035% SDS, and 70 µmol/L of the fluorogenic proteasome substrate Suc-LLVY-MCA (Calbiochem) at 37°C for 1 h, as previously described (30). For each sample, incubation was also performed in the presence of a specific inhibitor, clasto-lactacystin ß-lactone (10 µmol/L). Measurements of proteolysis (unquenched MCA peptide) were realized in a microtiter plate fluorometer Mithras LB 940 (Berthold Technologies) (excitation, 355 nm; emission, 460 nm). The activity values shown were derived by subtracting the fluorescence obtained in the presence of the specific inhibitor from the value obtained in its absence and expressed in relative fluorescent units/60 min.

    Statistical analysis. Results (absolute values) are expressed as means ± SEM for the indicated number of independent experiments. Statistical analysis was performed using StatView 5.0 software (SAS Institute). When 2 conditions were compared, the statistical difference was calculated using a t test if comparable variances were observed. In other cases, the nonparametric Mann-Whitney test was used. When >2 conditions were compared, statistical analysis was performed using 1-way or 2-way ANOVA if comparable variances were observed. In others cases, a nonparametric test was used. Pairwise multiple comparisons were conducted using Student's t test as a post hoc test. The level of statistical significance was fixed at P < 0.05.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
    Effect of cytomix on IL-8 production and on free and ubiquitinated IB expression. The production of IL-8 was greater in the presence of cytomix compared with control cells (Fig. 1A). Indeed, IL-8 production increased 24-fold (P < 0.05). We then determined, using time courses, that the greatest degradation of IB occurred in HCT-8 cells at 30 min (data not shown) as was reported recently in Caco-2 cells (23). Free IB expression decreased (P < 0.05, Fig. 1B) and the ubiquitinated IB expression increased under proinflammatory conditions compared with control conditions (P < 0.05, Fig. 1C). Thus, we tested the effect of increasing concentrations of glutamine on these variables.

View larger version (9K): [in this window] [in a new window]   FIGURE 1  Effect of cytomix on IL-8 production, free IB, and ubiquitinated IB expression in HCT-8 cells. HCT-8 cells were cultured for 24 h with 0.6 mmol/L glutamine and then stimulated (+) or not (–) with cytomix (1 µg/L IL-1ß, 20 µg/L TNF, and 10 µg/L IFN). After 18 h of stimulation, IL-8 production (A) was measured in culture media by an ELISA assay. After 30 min of stimulation, the expression of free (B) and ubiquitinated (C) IB was evaluated by immunoprecipitation and immunoblotting. Values are means ± SEM, n = 4. *Different from unstimulated, P < 0.05.

View larger version (7K): [in this window] [in a new window]   FIGURE 2  Effect of glutamine on IL-8 production in HCT-8 cells under basal and stimulated conditions. HCT-8 cells were pretreated with 0.6, 2, or 10 mmol/L glutamine for 24 h. Then, cells were cultured for 18 h in the presence of 0.6, 2, or 10 mmol/L glutamine, without (A) or with (B) cytomix (1 µg/L IL-1ß, 20 µg/L TNF, and 10 µg/L IFN). Values are means ± SEM, n = 4. Means without a common letter differ, P < 0.05.

    Effect of glutamine on free and ubiquitinated IB expression.

View larger version (19K): [in this window] [in a new window]   FIGURE 3  Effect of glutamine on free and ubiquitinated IB expression in HCT-8 cells. HCT-8 cells were pretreated with 0.6, 2, or 10 mmol/L glutamine for 24 h. One hour before stimulation, 20 µmol/L MG132 was added to the culture media. Then, cells were cultured for 30 min in the presence of 0.6, 2, or 10 mmol/L glutamine and without (open bars; –) or with (black bars; +) cytomix (1 µg/L IL-1ß, 20 µg/L TNF, and 10 µg/L IFN). As illustrated (A), ubiquitinated IB (B) and free IB (C) expression was evaluated in the cytosolic fraction using immunoprecipitation of IB followed by immunoblot with anti-ubiquitin or anti-IB antibodies. Values are means ± SEM, n = 4. Means without a common letter differ, P < 0.05.

    Effect of glutamine pretreatment on ubiquitin protein expression and proteasome activity. Because ubiquitinated IB was studied 30 min after cytomix, a time that was likely too short to influence protein synthesis, ubiquitin protein expression and proteasome activity were assessed under basal conditions in response to glutamine pretreatment (0.6 and 10 mmol/L). Supplementation with 10 mmol/L glutamine did not modify ubiquitin protein expression or proteasome activity (data not shown).

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
In the present study, we showed that glutamine pretreatment reduced IL-8 production under both basal and proinflammatory conditions in human intestinal epithelial HCT-8 cells. Our data also indicated that the ubiquitination process of IB was involved in this effect.

The endogenous production of IL-8 by HCT-8 cells appeared minimal in the presence of 0.6 mmol/L glutamine, which is a physiologic plasma concentration (31). Indeed, recent evidence showed that glutamine deprivation enhanced IL-8 production after lipopolysaccharide (LPS) stimulation in Caco-2 cells (23,32,33). Under basal conditions, glutamine deprivation alone or associated with an inhibitor of glutamine synthetase, blocking de novo glutamine synthesis, did not increase IL-8 production (32). Under stimulated conditions, glutamine deprivation exacerbated the LPS response (23,32). On the other hand, glutamine supplementation was associated with a decrease in IL-8 intestinal production in experimental models in rats (13,34). We also showed previously that glutamine reduced proinflammatory cytokine production (IL-6, IL-8) by human duodenal mucosa (16–18) and others (32). In contrast, in previous studies, glutamine together with stimulation with cytomix did not affect IL-8 production in human intestinal epithelial cells (25,32). This discrepancy could be explained by different experimental procedures such as the dose of glutamine tested and the timing of supplementation of glutamine. Indeed, in our previous work (25), glutamine was not added 24 h before cytomix but at the same time as cytomix. Thus, pretreatment in the present study may be critical in eliciting a response to glutamine. In the present study, we compared 2 and 10 mmol/L glutamine to the physiologic plasma concentration (0.6 mmol/L), which was not examined in the earlier study (25). Thus, this wider range of concentrations allows a greater ability to demonstrate the effect of glutamine. These concentrations are relevant to in vivo studies because they mimic the concentrations that may be achieved locally during an enteral infusion of glutamine-supplemented diets.

The mechanisms by which glutamine modulates inflammatory cytokine production are still under debate. Indeed, glutamine may affect IL-8 production by the upregulation of anti-inflammatory and protective processes such as IL-10 production (18,35) or hsp expression (21,36–38). On the other hand, glutamine also affected the NF-B pathway (19,23). Liboni et al. (23) showed that enhanced IL-8 production by glutamine deprivation was mediated by an increase in IB ubiquitination and then its degradation, whereas the influence of glutamine supplementation on this pathway remains unknown. In the present study, ubiquitinated IB and free IB expressions were increased and decreased, respectively, by cytomix as previously described (22,23). Glutamine pretreatment partially restored these expressions. In fact, ubiquitinated IB expression was reduced by 10 mmol/L glutamine; consequently, free IB expression was increased. In addition, glutamine reduced IL-8 production 31%, whereas it increased ubiquitinated IB expression 28%. These results suggest that glutamine may limit IL-8 production through the degradation pathway of IB in HCT-8 cells.

Moreover, we demonstrated that the ubiquitin protein and the proteasome activity do not seem to be involved in this effect because glutamine did not affect these variables. In Caco-2 cells, the phosphorylation mechanisms were also not involved in the effects of glutamine on IB degradation (23). Thus, glutamine may influence IB ubiquitination by the regulation of specific ubiquitin-conjugating enzymes. IB is ubiquitinated by the SCF-ßTrCP ubiquitin-ligase complex and subsequently degradated by the proteasome (39). The influence of glutamine on these processes remains unknown. In addition, there are some differences between the effects of glutamine on cytokine-treated and -untreated cells; this may reflect the differential use of glutamine for metabolic or regulatory pathways according to the level of experimental stress. Further investigations are warranted to explore these mechanisms.

In conclusion, glutamine pretreatment decreases IL-8 production under both basal and proinflammatory conditions in human intestinal epithelial HCT-8 cells, through the regulation of IB ubiquitination.

	FOOTNOTES

 
² Abbreviations used: hsp, heat shock protein; IFN-, interferon-, IB, inhibitor of B; LPS, lipopolysaccharide; NF-B, nuclear factor-B; TNF-, tumor necrosis factor-.

Manuscript received 25 November 2005. Initial review completed 13 January 2006. Revision accepted 6 March 2006.

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TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

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