Immunostimulated enterocytes activate extracellular arginase I which competes with enterocyte inducible nitric oxide synthase for arginine during inflammation

Introduction

We hypothesized that constitutively expressed arginase 1 (Arg-1) may be released from hepatocytes to the circulatory compartment to play the role of extinguisher at the front and to serve a protective function by modulating inflammation. We used a cell culture model of epithelial barrier dysfunction to determine whether liver cytosolic proteins could decrease NO^• production and preserve enterocyte paracellular barrier function on that basis.

Methods

We exposed immunostimulated Caco 2BBe enterocyte-like cells to human liver cytosol (LC). Cytomix (IFNγ, TNFα, and IL-1β) was used to stimulate the cells. Arginase activity in cell supernatants and murine serum was measured by following the generation of citrulline and urea from arginase. Tissue lysates and conditioned media were untreated or treated with the enzyme inhibitors (S)-(2-boronoethyl)-L-cysteine-HCl (BEC) and L-N(6)-(1-iminoethyl) lysine (L-NIL), which inhibit arginases and inducible nitric oxide synthase (iNOS), respectively.

Results

Cytomix increased paracellular permeability, and induced the expression of iNOS and release of NO^•. LC protein (400 μg/ml) applied to the basal but not apical compartment preserved barrier function and completely blocked the release of NO^• but only slightly decreased the magnitude of iNOS protein expression in a dose-dependent and time-dependent manner. Ultrafiltration and ultracentrifugation demonstrated that microsomal Arg-1 prepared from LC decreased iNOS-dependent NO^• production. BEC and anti-Arg-1 antibody inhibited the NO^• blocking ability of LC. Surprisingly, LC arginase activity required activation by a cell-derived factor and its release could be blocked by treating cells with L-NIL. Increased consumption of arginase by activated LC Arg-1 led to decreased iNOS dimerization, which decreased NO^• production. In the serum from endotoxemic mice, arginase activity was significantly increased. Furthermore, iNOS existed in ileal mucosa predominantly in the inactive monomeric form at 18 hours after lipopolysaccharide injection, consistent with decreased iNOS activity in the absence of arginase.

Conclusion

Arg-1 is one such liver-derived protein that increases in serum during endotoxemia and other inflammatory states. Modulation of mucosal iNOS activity following activation of circulating Arg-1 may be protective because it would be expected to decrease epithelial barrier dysfunction as a result of decreased NO^• production.