Elsevier

Cytokine

Volume 26, Issue 2, 21 April 2004, Pages 57-65
Cytokine

Mechanism of glutamine-mediated amelioration of lipopolysaccharide-induced IL-8 production in Caco-2 cells

https://doi.org/10.1016/j.cyto.2003.12.008Get rights and content

Abstract

The mechanism of glutamine (Gln)-mediated down-regulation of inflammation in the intestine is poorly understood. We hypothesize that Gln down-regulates lipopolysaccharide (LPS)-stimulated IL-8 production in intestinal epithelial cells via transcription factors that counteract the effect of LPS-mediated increase in IL-8. Caco-2 cells were incubated with different doses of Gln with or without methionine sulfoximine (MS), an inhibitor of glutamine synthetase for 24 h before stimulation by LPS (100 μg/ml for 24 h). Inhibitors of the mitogen activated protein kinase (MAPK) family were added to cells for 1.5 h following stimulation by LPS. The p38 inhibitor SB 203580 resulted in a significant decrease in IL-8 peptide production (p<0.01). However, p38 MAPK activity increased with Gln (p<0.05), suggesting that this was not involved with Gln-mediated down-regulation of IL-8. Screening of 54 transcription factors demonstrated that STAT-4 was the only inflammation-related transcription factor that was up-regulated by Gln depletion and down-regulated with Gln supplementation (2-fold increase), paralleling IL-8 production. EMSA analysis confirmed these findings (3.5-fold increase). These results indicate that Gln deprivation enhances IL-8 production by Caco-2 cells after LPS stimulation and that down-regulation of IL-8 production with Gln is associated with alterations in STAT-4 transcription factor binding.

Introduction

The intestinal epithelium comprises an important barrier between the intraluminal environment and the internal milieu of the host and serves as an early host-signaling system to the underlying intestinal mucosal immune cells [1]. In response to bacteria, toxins, and/or antigen attachment, the enterocyte, through signal transduction pathways, stimulates genes necessary to regulate inflammatory cytokine transcription and translation [2]. One of the pro-inflammatory cytokines/chemokines produced in the intestine, interleukin-8 (IL-8), has been demonstrated to be a particularly potent chemo-attractant for leukocytes and subsequent inflammation [3]. IL-8 initiates the acute inflammatory cascade and is an early marker of the inflammatory process and its expression must be controlled to prevent excessive tissue injury and damage to local and distal organs [4]. In patients dying of sepsis there is clear evidence of an imbalance in pro- and anti-inflammatory cytokine production and high levels of the activation of transcription factor, NF-κB [5]. The pathogenesis of nosocomial pneumonia in injured patients involves an increase in IL-8 levels in bronchoalveolar lavage fluid [6].

Glutamine (Gln), the most abundant amino acid in the human body, is thought to play several important roles in the intestine including fuel metabolism, serving as a precursor for nucleotides, hexosamines and other amino acids, and as a signaling molecule [7]. Gln has been shown to be beneficial to the prevention of infectious morbidity and mortality in seriously ill adult and neonatal patients [8], [9], [10]. Part of this effect may be secondary to decreased intestinal pro-inflammatory cytokine/chemokine production. Gln supplementation has been found to reduce pro-inflammatory cytokine release, decrease multiple organ system dysfunction and mortality in a rat model of endotoxemia [11]. Gln pretreatment significantly decreases production of pro-inflammatory cytokines (IL-6 and IL-8) by the human intestinal mucosa [12]. In a mouse model Gln preserved the anti-inflammatory cytokines (IL-4 and IL-10 mRNA expression) in LPS-stimulated intestinal lamina propria [13]. The beneficial effects of Gln may be a result of improved gut integrity, reduced bacterial translocation or immune function. But the exact mechanism of Gln's relationship to the inflammatory cascade in the intestine remains unclear.

The intracellular signal transduction pathways that regulate inflammation involve a complex interplay of cell surface receptors, intracellular activating enzymes, DNA-binding transcription proteins and post-transcriptional modifiers. LPS is thought to act via toll like receptors on the cell surface, which activates the dissociation of NF-κB from IκB and allows the translocation of the NF-κB to the nucleus where it acts as a transcriptional activator of pro-inflammatory chemokines such as IL-8 [14], [15], [16]. Other pathways are thought to act in conjunction with NF-κB, but their precise mechanisms are poorly understood. These include the MAPK and JAK/STAT pathways. Our previous LPS-induced IL-8 production studies support that Gln deprivation in Caco-2 cell cultures leads to exaggerated IL-8 production [17].

Here we find that Gln does not appear to act through the MAPK signaling pathway or NF-κB signal transduction pathway, but rather through the STAT-4 pathway, which is regulated through the JAK signaling cascade. The current studies investigate the mechanism of Gln-mediated regulation of IL-8. The strategy was to find a pathway already known to be involved with LPS-induced stimulation of IL-8, then to investigate other possibilities if the known pathway did not appear to be involved.

Section snippets

Effect of glutamine and signaling pathway inhibitors on LPS-mediated IL-8 production

Our previous studies demonstrated that Gln deprivation results in exaggerated LPS-induced IL-8 production in Caco-2 cells [17]. To evaluate the possible mechanism of Gln via inhibition of MAPK signaling pathways on IL-8 production, Caco-2 cells were incubated with and without 0.5 mM Gln in the presence or absence of 4 mM MS for 24 h, treated with the MAPK signaling pathway inhibitors SB 203580 (10 μM), PD 098059 (20 μM) and Curcumin (20 μM) for 1.5 h compared to DMSO used as a control and stimulated

Discussion

These results demonstrate: (1) Gln does not decrease LPS-induced IL-8 production by decreasing p38 MAPK; (2) p38 MAPK is up-regulated by LPS and Gln; (3) several functionally relevant transcription factors are affected by Gln, as determined by screening with protein/DNA arrays; and (4) STAT-4 was the only inflammation-related transcription factor that was up-regulated with Gln depletion and down-regulated with Gln supplementation. This parallels with Gln-mediated changes in IL-8 production.

The

Reagents

Minimum essential medium (MEM, l-glutamine free), Dulbecco's modified eagle media (DMEM, l-glutamine free), fetal bovine serum (FBS), antibiotic antimycotic solution and trypsin were purchased from Invitrogen Life Technologies (Carlsbad, CA). BD MITO+ Serum Extender (SE: a substitute of serum, including hormones, growth factors and defined metabolites, without Gln) was from Becton Dickinson Labware (Bedford, MA). LPS (E. coli O127:B8) was purchased from Sigma Chemical Co. (St. Louis, MO). DMSO

Acknowledgements

This study was supported by National Institutes of Health Grant RO1 HD 38954 to Dr. Josef Neu.

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