Cytokine-stimulated nitric oxide production and inducible NO-synthase mRNA level in human intestinal cells: lack of modulation by glutamine

ARTICLE IN PRESS Clinical Nutrition (2003) 22(6): 523–528 r 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0261-5614(03)00054-2

ORIGINAL ARTICLE

Cytokine-stimulated nitric oxide production and inducible NO-synthase mRNA level in human intestinal cells: lack of modulation by glutamine R. MARION, M. COEºFFIER, A. LEPLINGARD, L. FAVENNEC, P. DUCROTTEŁ, P. DEŁCHELOTTE Appareil Digestif Environnement Nutrition (ADENEA 3234), Institut FeŁdeŁratif de Recherches Multidisciplinaires sur les Peptides (I.F.R.23), Rouen, France. (Correspondence to: P. De¤chelotte)

Abstract(Background & aims: Excess NO production has been reported during intestinal in£ammation. Modulation of the in£ammatory response with nutrients in critically ill patients has gained increasing interest. Glutamine has bene¢cial e¡ects on gut mucosa but its e¡ects on human intestinal NO production during an in£ammatory response are not known. Methods: Caco-2/TC7 and HCT-8 cells were stimulated with a cytokine mixture (IL-1b,TNFa, IFNg) and duodenal biopsies from human healthy volunteers in organ culture were stimulated with IL-1b. All cultures were performed in the presence of 2^10 mmol/l glutamine. NO release in culture supernatant and iNOS mRNA level in cultured cells or biopsies were assessed by nitrate reduction and Griess assay and RT-PCR, respectively. Results: In Caco-2, HCT-8 cells and duodenal biopsies, cytokine stimulation increased iNOS mRNA level 1.2-fold (ns), 3.8-fold (P=0.02), 4.7-fold (P=0.03) and NO production 1.4-fold (ns), 9.1 (P=0.01) and 1.7-fold (P=0.01), respectively. Increasing glutamine concentration had no signi¢cant e¡ect on NO production and iNOS mRNA in any type of culture, stimulated or not by cytokines. In various models of human intestinal cells, glutamine does not further increase NO production induced by pro-in£ammatory cytokines. r 2003 Elsevier Science Ltd. All rights reserved.

supplementation is also beneficial in critically ill patients (8, 9). In previous studies, we reported that glutamine has a specific inhibitory effect on the production of the pro-inflammatory cytokines IL-6 and IL-8 in human gut (10, 11). In vitro, glutamine protects rat intestinal epithelial cells against oxidant and lethal injuries (12). Accordingly, we also recently reported that enteral glutamine enhanced the expression of the heat-shock protein heme oxygenase-1, which has protective effects on the gut, in human duodenal mucosa (13). Several studies have emphasized the regulation of NO synthesis by nutrients (14), but data on the influence of glutamine on NO production are still limited. Given the importance of preserving gut function in critical illness, nutrients that modulate the induction or activity of iNOS in the intestine may have therapeutic potential. As glutamine may be a precursor for arginine and consequently NO production, assessing the effect of glutamine on NOS and NO production could be relevant as regards safety. In the context of the debate on potential harmfulness of arginine-enriched pharmaconutrition in critically ill patients (15), it seems important to assess if glutamine does not exacerbate the induction or activity of iNOS under basal or stimulated conditions. This study investigated the effects of glutamine on NO production and iNOS mRNA level production in

Key words: nitric oxide synthase; intestine; glutamine; human

Introduction Nitric oxide (NO) participates in the regulation of many intestinal functions such as absorption, secretion or motility (1). NO is a product of the enzymatic conversion of arginine to citrulline by a family of three distinct NO synthase (NOS) (2). Expression of the inducible isoform of NOS (iNOS) and concentration of NO produced could play a key role in the pathogenesis of inflammatory bowel disease (1). Intestinal overproduction of NO could also contribute to severe hemodynamic changes in critically ill patients, enhanced intestinal permeability and increased risk of multiple organ failure (3, 4). After exposure to stimuli such as pro-inflammatory cytokines, human intestinal epithelial cells up-regulate the expression of iNOS and NO production (1, 5, 6). Glutamine is the preferred substrate for both enterocytes and other rapidly dividing cells such as immune cells. Several studies have shown the protective effect of glutamine in the intestine (7). A recent meta-analysis demonstrated the beneficial effect of glutamine supplementation in post-operative patients (4); glutamine 523

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two types of human epithelial intestinal cell lines Caco-2 (TC7) and HCT-8 and in human duodenal mucosa in an organ culture model, both under basal conditions and after an experimentally induced inflammatory state.

Materials and methods Material All cytokines were purchased in lyophilized form and reconstituted according to manufacturer’s instructions. IFNg, TNFa were supplied by Tebu (Le Perray en Yvelines, France) and IL-1b was obtained from Sigma (Saint-Quentin Fallavier, France). NO kits were obtained from R&D systems (Abingdon, UK). Cell culture reageants (Dulbecco’s modified Eagle’s medium (DMEM), glutamine, non-essential amino acids and fetal calf serum (FCS) were supplied by Eurobio (Les Ulis, France). The tissue culture plates were obtained from ATGC (Noisy Le Grand, France). Thermoprime plus DNA was supplied by Abgene (Epsom, UK), Pd(N6) and a 33dATP were obtained from Amersham Pharmacia Biotech (Orsay, France) and all other reverse transcription-polymerase chain reaction (RT-PCR) reageants (RT-MMLV, RT buffer, dNTP, Rnasin) were supplied by Promega (Charbonnie`res, France). The oligonucleotide primers were supplied by Eurogentec (Seraing, Belgium). Cell culture and cytokine treatment Human epithelial intestinal adenocarcinoma cell lines Caco-2 clone TC7 and HCT-8 were used respectively between passage number 28–30 and 59–62 and were routinely grown at 371C with 5% CO2 in DMEM supplemented with 10% heat-inactivated FCS, 1% nonessential amino acid, antibiotics and 2 mmol/l lglutamine. Cells were re-fed every 2 days and passaged weekly. Culture media without FCS was used for stimulation. Caco-2/TC7 and HCT-8 were allowed to grow for 9 and 1 days, respectively, after confluence before use. Cytokines were added in medium at 1 ng/mL for IL-1b, 20 ng/mLfor TNFa and 10 ng/ml for IFNg. Each experiment was carried out independently four times in Caco-2/TC7 cells and three times each conducted in duplicate in HCT-8 cells. Protein concentration assessed by Lowry method was 0.36870.089 and 0.3677 0.027 mg /well of 6
wells-plate in Caco-2/TC7 and in HCT-8, respectively.

from the distal duodenum. Biopsies were immediately transferred in pre-weighed tubes containing DMEM supplemented with penicillin (100 U/ml) and streptomycin (100 mg/ml; Sigma, Germany) and processed within 1 h for organ culture as previously described (10). Whole biopsy specimens were used without cell separation procedures. Thus, cultured biopsies contained both enterocytes and other gut cells as immune cells, as verified by histological examination of preliminary experiments. Biopsies were placed in 24
wells plastic culture plates and bathed in 1 ml of DMEM supplemented with 10% FCS. Biopsies were cultured with varying concentrations of glutamine from 2–10 mmol/l under basal or IL-1b (2 ng/ml) stimulated conditions. The culture dishes were placed in chamber equilibrated with 5% CO2 and incubated at 37C. After 18 h, culture supernatant was removed and stored at 801C until NO determination and biopsies were transferred to vials containing guanidium solution and stored at 801C until mRNA analysis. RNA extraction—Reverse transcriptase-polymerase chain reaction RNA was extracted from biopsies or cells by a modifiedextraction method as previously described (16). The quality and quantity of total RNA were determined by spectrophotometer using the absorbency at A260/ A280 nm.The integrity was also controlled by visualization of 18S and 28S ribosomal bands. RNA expression of iNOS was studied by RT-PCR as previously (17). The number of amplification was 21, 22 and 24 cycles for GAPDH mRNA and 25, 25 and 24 for iNOS mRNA, respectively, in Caco-2/TC7, HCT-8 cells and in duodenal biopsies. The levels of amplified product were normalized to constant amounts of GAPDH mRNA. The entire RT-PCR procedure was performed in triplicate. Determination of NO production NO production was estimated from the sum of nitrites and nitrates (total NO) determined by using the total NO kit according to manufacturer’s instructions. Briefly, in a first step, nitrate is converted to nitrite by the enzyme nitrate reductase. The reaction was followed by a colorimetric detection of nitrite as an azo dye product of the Griess reaction. The detection limit is less than 1.35 mmol/l.

Biopsies culture

Cell viability measurement

Eight non-smoker healthy volunteers (aged 19–39 years) gave informed consent to participate in the study. This study was approved by the Ethical Committee of Rouen University Hospital. During upper endoscopy, multiple biopsy specimens (mean weight 8.171.9 mg) were taken

To control the viability of cells in culture, the release of lactate dehydrogenase (LDH) was measured in supernatant. Maximal LDH release was achieved by treatment of cells in the presence of the detergent triton X-100 (LDHo5%). Cell viability was not affected by

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incubation, for up to 24 h with cytokines. In previous experiments of organ culture, we also verified that the release of LDH in culture medium did not increase by more than 10%, indicating that intestinal tissue remained viable.

Table 1 Influence of glutamine on NO production and iNOS mRNA level in Caco-2 cells after 24 h exposure to cytokines Glutamine (mmol/l) NO (mmol/l) Control Stimulated

2

5

10

7.6 (1.4) 10.6 (0.6)

8.9 (0.7) 10.7 (1.3)

8.2 (1.9) 11.3 (2.4)

INOS mRNA (%/GAPDH) Control 84.5 (13.5) 79.5 (26.3) 70.1 (13.3) Stimulated 101.8 (22.5) 108.7 (19.4) 92.1 (33.6)

Statistical analysis Statistical comparison was performed using GraphPadInStat version 3.05 for win95/NT. Cell culture data are expressed as mean (SEM) from at least three independent experiments. For cultured biopsies, results are expressed as median (range). Statistical significance was assessed with Po0.05 by paired t-tests for cells and by non-parametric paired test (Wilcoxon or Friedman) for biopsies.

Results Unstimulated HCT-8 or Caco-2/TC7 cells (Fig. 1, Table 1) produced very low levels of NO (7.6 and 11.3 mM respectively in Caco-2 and HCT-8 cells). (A) Gapdh

iNOS

Gln (mmol/L)

2

5

10

2

5

10

Cytokines

-

-

-

+

+

+

150 * * 120

*

90

60

30

0 Gln(mmol/L)

2

5

10

2

5

10

Cytokines

-

-

-

+

+

+

Total NO production was determinated in the supernatant by nitrate reduction and Griess reaction. iNOS mRNA level was determinated by RT-PCR. iNOS mRNA level is expressed as the percentage of the internal standard GAPDH mRNA.

Effect of cytokines on NO production and iNOS mRNA level After exposure of the HCT-8 cells to the combination of three cytokines (IL-1b, TNFa and IFNg) during 18 h, a significant increase of NO production (11.3 vs 102.9 mmol/l, Po0.05) and iNOS mRNA level (3.8-fold, Po0.05) was observed (Fig. 1). In Caco-2 cells incubated with the same combination of cytokines over 24 h, a modest, unsignificant increase of NO production (7.6 vs 10.7 mmol/l) and iNOS mRNA level (1.3-fold) was observed (Table 1). Stimulation with IL-1b of duodenal biopsies resulted in an increase of NO production (Fig. 2) and an up-regulation of iNOS mRNA level (19.7 vs 93.0% GAPDH). Effect of increasing concentrations of glutamine on NO production and iNOS mRNA level

(B)

Total NO (µmol/L)

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Under basal conditions, increasing concentrations of glutamine from 2–10 mmol/l did not affect the NO production and iNOS mRNA level in any cell lines (Table 1, Fig. 1). In stimulated conditions, the addition of glutamine at varying concentrations from 2–10 mmol/l did not influence the cytokine-induced NO production and iNOS mRNA level in HCT-8 (Fig. 1) and Caco-2 cells (Table 1). In cultured duodenal biopsies (Fig. 2), NO production and iNOS mRNA level were not significantly affected by increasing concentrations of glutamine from 2–10 mmol/l, either under basal conditions or in IL-1bstimulated conditions.

Discussion

Fig. 1 Influence of glutamine on NO production and iNOS mRNA level in HCT-8 cells. HCT-8 cells were incubated during 18 h with increasing concentrations of glutamine (2–10 mmol/l) under basal or cytokines (IL-1b, TNFa, IFNg) stimulated conditions. (A) Representative autoradiogram (n=3) for mRNA expression of GAPDH and iNOS analysed by RT-PCR. (B): Total NO production in the supernatant determined by nitrate reduction and Griess reaction. Results are expressed as mean7SEM. *Po0.05 vs basal conditions.

Few studies have investigated the influence of glutamine on NO production. In the present study performed under conditions of stimulated inflammatory response, glutamine had no influence on NO production by two different types of enterocytic human cell lines and by cultured duodenal mucosa. We have chosen two different epithelial intestinal cell lines: Caco-2/TC7 and

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(A) 400

† †

Total NO (µmol/L/mgwet weight tissue)

350

† 300

250

200

150

100

50

0 Gln (mmol/L)

IL-1β

2

5

10

2

5

10

-

-

-

+

+

+

(B)

iNOS mRNA level (%/gapdh) 2

5

10

Control

19.7 (11.5-34.5)

24.7 (6.1-124.3)

19.4 (1.1-68.4)

IL-1β

93.0 * (27.5-197.8)

80.7 * (22.6-105.5)

78.0 * (19.9-168.8)

Gln (mmol/L)

Fig. 2 Influence of glutamine on NO production and iNOS mRNA level by cultured human duodenal biopsies of eight healthy volunteers. Biopsies were stimulated during 18 h with IL-1b (2ng/ml) in the presence of increasing concentrations of glutamine from 2 to 10 mmol/ l. (A): Total NO production was determinated in the supernatant by nitrate reduction and Griess reaction. yPo0.05 vs unstimulated conditions. (B) INOS mRNA level was determinated by RT-PCR. iNOS mRNA level is expressed as the percentage of the internal standard GAPDH mRNA. *Po0.05 vs unstimulated conditions.

HCT-8. Caco-2 cell line model has been previously used to study the effect of glutamine or other amino acids on absorption, transport, permeability (18–20), NO production (1, 3, 5, 6), as well as the effects of proinflammatory cytokines mimicking an inflammatory response (18). Thus, Caco-2 cells appear to be an appropriate model to study the effect of glutamine on NO production under conditions of stimulation with cytokines. As increasing concentrations of glutamine showed no effect on NO production and iNOS mRNA level in Caco-2 cells, we subsequently aimed to determine whether this lack of modulation of NO by glutamine was specific of the Caco-2 cell line. Thus, we also carried out experiments with an other human intestinal epithelial cell line HCT-8. This cell line has been previously used under inflammatory conditions by our group and others (21–23). Further on, since both in

vitro cell models demonstrated the same results, the duodenal explants culture has been used to assess the effect of glutamine on ex vivo human intestinal mucosa. Cultured biopsy contains other cells, especially immune cells, in addition to epithelial cells, that could also be reactive to glutamine. Results with cultured biopsies however confirmed the results obtained with epithelial cell lines. Thus, in these three models, glutamine has no effect on NO production and iNOS mRNA level in human intestinal cells. In agreement with other studies of intestinal epithelial cell lines (1, 3, 5, 6), we observed that NO and iNOS mRNA production in the cell lines Caco-2/TC7 and HCT-8 were low under basal conditions, and increased in response to a mixture of pro-inflammatory cytokines. The most striking effect of cytokines was observed in HCT-8, while Caco-2 cells did not respond significantly. Thus, the effect of glutamine could be evaluated in two different types of cell lines, with different pattern of response to cytokine stimulation, as far as NO production is concerned. Taking into account the level of stimulation is relevant, since excess stimulation of NO by arginine feeding has been related to the already high level of NO production before feeding (24). In duodenal biopsies, NO production and iNOS mRNA were sharply upregulated in response to IL-1b. This model has proven effective to study the modulating effects of nutrients or drugs on the production of cytokines or prostaglandins (10, 11, 25). IL-1b is able to induce a marked increase of pro-inflammatory cytokine production in human intestine (26), and we observed in preliminary experiments that addition of TNFa and IFNg to IL-1b did not further increase the inflammatory response of cultured biopsies. Thus, we used only IL-1b to stimulate duodenal biopsies. Using both types of cell lines and the organ culture model, we observed no effect of glutamine on NO production and iNOS mRNA level, suggesting that in human intestine the modulating effect of glutamine on inflammatory response previously observed (10, 11) is not mediated by a modulation of the NO pathway. In the present study, the lowest glutamine concentration used was 2 mM, i.e. the concentration required for optimal function in Caco-2 (27) and the most commonly used in HCT-8 cells. A lower concentration could be considered as a depletion of glutamine, and could trigger apoptosis, as reported in rat and human immune cells (28–31) and in rat intestinal epithelial cells (32). Similarly, glutamine deprivation from 2 to 0 mM depleted intracellular ATP in Caco-2 cells treated with TNFa and increased the translocation of bacteria (9). Thus, 2 mM is considered as a basal condition for optimal function of Caco-2 cells. For human duodenal explant culture, we observed in preliminary experiments (data not shown), that increasing glutamine concentration from 0.5 to 2 mM had no influence on NO production and iNOS mRNA level under basal or IL1 stimulated conditions. Thus, further experiments with

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cultured biopsies presented in this study were performed using 2–10 mM glutamine. Our present findings that cytokine-stimulated iNOS mRNA and NO production in stimulated conditions were not increased by glutamine at concentration as high as 10 mmol/l are in accordance with our previous results in unstimulated human biopsies (11), as well as with data in rats neutrophils incubated with up to 20 mmol/l glutamine (33). In rats, enteral infusion of glutamine even reduced systemic nitrate levels (34). In bovine endothelial cells, NO synthesis under both basal and stimulated conditions increased when glutamine was omitted in culture medium as compared to lower NO production in standard 2 mmol/l glutamine conditions (35). Thus, it appears from the available data that NO production in human gut is unlikely to be stimulated to a detrimental level even during provision to the gut of very high amounts of glutamine. This point is relevant to the debate on the safety of high amounts of ‘‘pharmaconutrients’’ administered in situations of exacerbated inflammatory response (15). Indeed, concern has arisen regarding the use of arginineenriched enteral formulas in patients with severe inflammatory response, since conversion of arginine by iNOS may generate excessive amounts of NO. Glutamine delivered via the enteral route may be partly converted to citrulline in the gut and later to arginine in the kidney, thus resulting in an increase in arginine plasma level (36). Although the main regulatory step in NO production is the activity of the NOS enzyme isoforms (14), some reports show that increasing arginine in vivo availability or in vitro concentration may increase NO production (14). In contrast, glutamine may act as a tonic inhibitor of in vitro NO release from arginine in other models (37). The available literature on glutamine supplementation in post-operative or severely injured patients failed to demonstrate any adverse event that could be attributed to excess NO production, as it was the case for high arginine supplementation (4, 24). Our present in vitro results do not support that high glutamine concentration promote intestinal NO production, even in stimulated conditions. However, this observation remains to be confirmed in the clinical situation.

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Submission date: 13 January 2003 Accepted: 31 March 2003

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