Immunostimulated Arginase II Expression in Intestinal Epithelial Cells Reduces Nitric Oxide Production and Apoptosis

Maria M Talavera,Hongmei Cui,Leif D Nelin,Yi Jin,Sushma Nuthakki,Yusen Liu

doi:10.3389/fcell.2017.00015

Abstract

Increased production of nitric oxide (NO) and subsequent local cytotoxicity to mucosal epithelial cells has been proposed as a putative mechanism involved in the development of necrotizing enterocolitis (NEC). Intestinal epithelial cells (IECs) metabolize L-arginine to either nitric oxide (NO) by NO synthase (NOS) or to L-ornithine and urea by arginase. L-ornithine is the first step in polyamine synthesis important for cell proliferation, while NO production can lead to apoptosis. We hypothesized that in IECs immunostimulation increases both NOS and arginase expression, and that arginase activity mitigates NO production and apoptosis. Rat intestinal epithelial cells (rIEC-6) were immunostimulated by either incubation with lipopolysaccharide (LPS) alone for 24 h or by incubation with conditioned media (CM) for 24 h. CM was obtained from RAW 264.7 cells (a macrophage cell line) treated with LPS (E. coli 0127:B8; 1 μg/ml) for 4 h. The rIEC-6 stimulated with LPS or with CM had significantly higher levels of inducible NOS (iNOS) protein, NO production, and arginase II protein than did the control cells. Direct LPS stimulation of rIEC-6 produced a less robust increase in iNOS expression and NO (represented as nitrite percent of control) than did CM stimulation. Inhibition of arginase using Nω hydroxyl-L-arginine (NOHA) further increased stimulated NO production in rIEC-6. Viable cell numbers were significantly lower in CM stimulated cells after 24 h than in controls, and inhibition of arginase activity with NOHA resulted in a further significant decrease in viable cell numbers. We conclude that immunostimulated arginase expression of rIEC-6 cells tempers cytokine-induced iNOS-derived NO production and apoptosis.

Highlights

Necrotizing enterocolitis (NEC) is the most common gastrointestinal emergency in preterm infants and a leading cause of neonatal morbidity and mortality (Lin and Stoll, 2006; Neu and Walker, 2011)
We examined protein levels of proliferating cell nuclear antigen (PCNA), a marker of cell proliferation in each experimental group, hypothesizing that if nitric oxide (NO) production were greater in the LPS treated cells that PCNA levels would be lower and that arginase inhibition would further lower PCNA protein levels
The major findings of this study in rIEC-6 were that: (1) immunostimulation increased inducible NOS (iNOS) and arginase II protein levels, as well as NO and urea production; (2) inhibiting arginase resulted in greater immunostimulated NO production; (3) immunostimulation increased apoptosis; (4) inhibition of arginase resulted in further decreased viable cell number and increased apoptosis following immunostimulation; and (5) inhibition of NO synthase (NOS) production attenuated apoptosis following immunostimulation

Summary

Introduction

Necrotizing enterocolitis (NEC) is the most common gastrointestinal emergency in preterm infants and a leading cause of neonatal morbidity and mortality (Lin and Stoll, 2006; Neu and Walker, 2011). Of particular interest is the role of inflammation-induced vascular dysfunction and epithelial cytotoxicity in the development of NEC (MacKendrick et al, 1993; Ford et al, 1997; Nowicki et al, 2005). Increased production of NO and subsequent local cytotoxicity to mucosal epithelial cells has been proposed as one of the putative mechanisms in NEC development (Ford et al, 1997; Nadler et al, 2000; Chokshi et al, 2008). Of the three isoforms of NOS described, iNOS is not constitutively expressed, but induced at high levels during inflammation resulting in relatively high levels of NO production (Di Lorenzo et al, 1995; Chokshi et al, 2008). In experimental models of NEC, the inhibition of iNOS has been found to attenuate inflammatory intestinal injury (Ciftci et al, 2004; Giannone et al, 2006; Cintra et al, 2008)

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