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

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 N^ω 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.

Keywords: arginase; inducible nitric oxide synthase; inflammation; necrotizing enterocolitis.

Figure 1

LPS induced NO and urea production. rIEC-6 cells were treated with LPS or vehicle (control) for 24 h. (A) iNOS mRNA levels detected by qRT-PCR normalized to 18srRNA levels (n = 6 for each group). (B) Cell lysates were harvested and iNOS protein levels determined by Western blot. The densitometry data (n = 3 for each group) normalized to β-actin are shown relative to control. (C) Nitrites were analyzed by chemiluminescence for media harvested after a 24 h incubation with vehicle (control) or LPS (n = 9 for each group). Nitrite levels were measured in media and represented as a percent of control. (D) Cell lysates were examined for arginase II mRNA levels using qPCR normalized to 18s rRNA (n = 6 for each group). Densitometry results are shown as fold change from control (where control levels = 1). (E). Cell lysates were examined for arginase II protein levels by Western blot (n = 4 for each group). The densitometry data normalized to β-actin are shown relative to control. (F). Urea production was determined by colorimetric assay after harvesting medium from cells treated with vehicle (control) or LPS (n = 6 for each group). Urea levels were measured in media and are represented as a percent of control urea values. ^*LPS different from control, p < 0.05, ^**LPS different from control, p < 0.001.

Figure 2

LPS-induced NO production was augmented when urea production was inhibited. rIEC-6 cells were incubated for 24 h in media containing LPS and either vehicle or 100 μM N^ω-Hydroxy-nor-L-arginine acetate (NOHA). Urea (black bars) was measured using a colorimetric assay and the results are presented as percent of control (i.e., no LPS) urea values. Nitrites (gray bars) were measured in the media and are presented as a percent of control nitrite values. ^**LPS different from control; ^**LPS+NOHA different from control + NOHA, p < 0.001; multiple t-test and 2-way ANOVA.

Figure 3

Arginase inhibition resulted in increased 3-nitrotyrosine and decreased proliferation in LPS-treated rIEC-6 cells. rIEC-6 cells were treated with LPS or untreated (control), and either with the arginase inhibitor (NOHA) or its vehicle for 24 h. Cell lysate was examined for 3-NT, PCNA or β-actin by Western blot. (A). Densitometry data for 3-NT normalized to β-actin and presented as fold change from control (n = 6 for each group); ^**LPS+ NOHA-treated different from control + NOHA; LPS + NOHA different from LPS alone; LPS different from control; Control + NOHA different from control, p < 0.001. (B). Densitometry data for PCNA normalized to β-actin and presented as fold change from control (n = 9 for each group); #LPS different from LPS+NOHA control, p < 0.05. ^*LPS+NOHA different from control, p < 0.001.

Figure 4

Conditioned media up-regulates iNOS and arginase II protein levels. rIEC-6 cells were exposed to conditioned media (CM) for 24 h. CM media (DMEM with FBS) was collected from RAW 264.7 cells stimulated with 1 μg/ml LPS for 4 h. (A) Cell lysates (n = 6 for each group) were examined for protein levels of iNOS by western blotting. (B) The densitometry data normalized to β-actin are shown relative to control. (C) Cell lysates (n = 6 for each group) were examined for protein levels of arginase II by western blotting. (D) The densitometry data normalized to β-actin are shown relative to control. ^*CM different from control, p < 0.005.

Figure 5

Condtioned media increased apoptosis in rIEC-6 cells. Cells were exposed to conditioned media (CM) for 24 h. CM media (DMEM and FBS) was collected from RAW 264.7 cells stimulated with 1 μg/ml LPS for 4 h. Cell lysates were examined for levels of cleaved caspase-3 by western blotting. Typical western blots for cleaved caspase-3 and β-actin. The bar graph shows the densitometry data for cleaved caspase 3 normalized to β-actin (n = 6 for each group) are shown relative to control (i.e., control = 1). ^*CM different from control, P < 0.005.

Figure 6

Conditioned media results in more NO production and fewer viable cells and inhibition of arginase augments this response. rIEC-6 cells were incubated in either regular media (control) or conditioned media (CM), and with either vehicle or 100 μM NOHA added for 24 h. (A). Media was harvested for determination of NO production by assaying nitrites using chemiluminescence. Conditioned media resulted in a substantially greater NO production in vehicle treated cells (black bars) than did regular media (control). Inhibiting arginase with NOHA (gray bars) augmented the CM-induced production of NO. ^*CM different from control, p < 0.001. # NOHA different from vehicle, p < 0.005. (B). Equal numbers of rIEC-6 cells were seeded in each well of 6 well plates and treated as above. After 24 h viable cell numbers were determined using trypan blue exclusion. ^*CM different from control, p < 0.05. #CM + NOHA different from CM, p < 0.05.

Figure 7

Inhibition of arginase augments, while inhibition of iNOS attenuates, enterocyte apoptosis. rIEC-6 cells were grown ~80% confluence then incubated in either conditioned media (CM) alone or CM and the arginase inhibitor, NOHA (A), or CM alone or CM + the iNOS inhibitor, L-NAME (B), for 24 h. Cell lysates were collected and protein harvested to determine cleaved caspase-3 and β-actin protein levels. (A). Representative Western blots for cleaved caspase-3 and β-actin in CM and CM + NOHA-treated cells (n = 9 each group). The bar graph is the densitometry data for cleaved caspase-3 normalized to β-actin and shown relative to CM alone. ^*p < 0.05 CM + NOHA different than CM alone. (B). Representative Western blots for cleaved caspase-3 and β-actin in CM and CM + L-NAME-treated cells (n = 9 for each group). The bar graph is the densitometry data of cleaved caspase-3 normalized to β-actin as shown relative to CM alone. ^*p < 0.001 CM + L-NAME different than CM alone.