Coinduction of nitric-oxide synthase and arginase I in cultured rat peritoneal macrophages and rat tissues in vivo by lipopolysaccharide.

Nitric oxide is synthesized by nitric-oxide synthase from arginine, a common substrate of arginase. Rat peritoneal macrophages were cultured in the presence of bacterial lipopolysaccharide (LPS), and expression of the inducible isoform of nitric-oxide synthase (iNOS) and liver-type arginase (arginase I) was analyzed. mRNAs for iNOS and arginase I were induced by LPS in a dose-dependent manner. iNOS mRNA appeared 2 h after LPS treatment and increased to a near maximum at 8-12 h. On the other hand, arginase I mRNA that was undetectable prior to the treatment began to increase after 4 h with a lag time and reached a maximum at 12 h. Immunoblot analysis showed that iNOS and arginase I proteins were also induced. mRNA for arginase II, an arginase isozyme, was not detected in the LPS-activated peritoneal cells. mRNA for CCAAT/enhancer-binding protein β (C/EBPβ), a transactivator of the arginase I gene, was also induced, and the induction was more rapid than that of arginase I mRNA. Changes in iNOS and arginase I mRNAs were also examined in LPS-injected rats in vivo iNOS mRNA increased rapidly in the lung and spleen, reached a maximum 2-6 h after the LPS treatment, and decreased thereafter. Arginase I mRNA was induced markedly and more slowly in both tissues, reaching a maximum in 12 h. Thus, arginase I appears to have an important role in down-regulating nitric oxide synthesis in murine macrophages by decreasing the availability of arginine, and the induction of arginase I is mediated by C/EBPβ.

arginine synthesis in ureotelic animals are the liver, where arginine generated in the urea cycle (ornithine cycle) is rapidly converted to urea and ornithine by arginase, and the kidney, where arginine is synthesized from citrulline and released into the blood circulation (see Ref. 4 for a review). In other tissues and cell types, arginine can be generated from citrulline, which is produced as a coproduct of the NOS reaction, forming a cycle that is composed of NOS, argininosuccinate synthetase, and argininosuccinate lyase and that is termed the "citrulline-NO cycle" (5)(6)(7)(8)(9)(10). The inducible isoform of NOS (iNOS) and argininosuccinate synthetase are coinduced in activated murine macrophage-like RAW 264.7 cells (8), in cultured vascular smooth muscle cells (9), and in vivo (10,11). Argininosuccinate lyase is also induced in vivo (10,11).
On the other hand, arginine is utilized for both the arginase and NOS reactions. Thus, these two enzymes compete for arginine. At least two isoforms of arginase are present. Liver-type arginase (arginase I) is expressed almost exclusively in the liver and catalyzes the final step of urea synthesis. Arginase I activity (12) and mRNA (13) in rat liver increase markedly in the perinatal period, in coordination with other urea cycle enzymes. The enzyme is regulated by dietary protein (14) and hormones (15). Arginase I consists of three identical subunits of about 35,000 Da. cDNA clones were isolated from rat (16,17) and human (18,19) liver. The rat (20) and human (21) genes are 11.5-12 kb long and consist of 8 exons. Promoter and enhancer regions of the rat gene were characterized (22)(23)(24). In addition to arginase I, an isozyme (arginase II) is present in extrahepatic tissues, including kidney, small intestine, and lactating mammary gland (25)(26)(27). The coinduction of NOS and arginase II activities in RAW 264.7 cells activated by lipopolysaccharide (LPS) was reported (28). We isolated cDNA for arginase II and showed that it is 59% identical with arginase I on the amino acid level (29). We also found that the enzyme is mitochondrial and that its mRNA is induced by LPS, dexamethasone, and cyclic adenosine monophosphate and is reduced by interferon-␥ (29). To better understand the role of arginase isoforms in NO synthesis, we examined expression of the isoforms and iNOS in cultured rat peritoneal cells and in LPS-treated rats using RNA blots, immunoblots, and immunocytochemical analyses. We report here that iNOS and arginase I mRNAs and proteins are coinduced by LPS in cultured rat peritoneal macrophages and in the lung and spleen in vivo. The induction of mRNA for CCAAT/enhancer-binding protein ␤ (C/ EBP␤), a transactivator of the arginase I gene, is also described.

EXPERIMENTAL PROCEDURES
Materials-A monoclonal antibody against mouse iNOS was obtained from Transduction Laboratories (Lexington, KY). Antiserum * This work was supported in part by Grant-in-aid 08877035 from the Ministry of Education, Science, Sports and Culture of Japan (to M. M.) and a grant from Taisho Pharmaceutical Co., Tokyo, Japan (to M. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
against human arginase I was raised in a rabbit. Purified recombinant human arginase I (30) was provided by M. Ikemoto of Kyoto University, Kyoto, Japan. A monoclonal antibody RM4 against rat macrophages will be described elsewhere (41).
Preparation and Culture of Rat Peritoneal Cells-Specific pathogenfree male Wistar rats (7 weeks of age) were given 5 ml of 10% polypeptone (Difco) intraperitoneally, and peritoneal cells were harvested 3 days after the injection (31). The cells were seeded in 10-cm culture dishes at 5 ϫ 10 6 cells/dish in RPMI 1640 medium with 10% heatinactivated fetal calf serum. The cells were cultured in the absence or presence of LPS at 37°C under 5% CO 2 in air.
Animals and LPS Treatment-Male Wistar rats (5-6 weeks of age) were given Escherichia coli LPS (serotype 0127:B8, Sigma) intraperitoneally at 1.0 mg/kg of body weight and killed at the indicated times following anesthetization with ether.
Immunoblot Analysis-Rat tissues were excised and homogenized in 9 volumes of 20 mM potassium HEPES buffer, pH 7.4, containing 1 mM dithiothreitol, 50 M antipain, 50 M leupeptin, 50 M chymostatin, and 50 M pepstatin. Packed rat peritoneal cells were homogenized in 9 volumes of the same buffer containing 0.5% Triton X-100. The homogenates were centrifuged at 25,000 ϫ g for 30 min at 4°C and the supernatants were used as tissue or cell extracts. The extracts were subjected to SDS-polyacrylamide gel electrophoresis and proteins were electrotransferred to nitrocellulose membranes. Immunodetection was performed using an ECL kit (Amersham Corp.) according to the manufacturer's protocol. Protein was determined with protein assay reagent (Bio-Rad) using bovine serum albumin as a standard. Immunocytochemical Staining-Cultured peritoneal cells were prepared for immunocytochemical staining by cytocentrifuge. Briefly, 5 ϫ 10 4 cells were suspended in 100 l of RPMI 1640 medium containing 10% heat-inactivated fetal calf serum, and then the cell suspension was applied on cytospin slides by centrifugation at 600 rpm for 5 min (Cytospin 2, Shandon, Astmoor, United Kingdom). Immunocytochemical staining was done as described (10).

Induction by LPS of iNOS and Arginase I mRNAs in Rat
Peritoneal Cells-Rat peritoneal cells were cultured in the presence of LPS, and expression of iNOS and arginase I mRNAs was studied. Fig. 1 shows the effects of LPS concentration on iNOS and arginase I mRNAs. iNOS mRNA of about 4.5 kb, which was not detectable in the absence of LPS, was evident at 1 ng/ml and gradually increased with increasing concentrations of LPS up to 10 g/ml. mRNA for arginase I (liver-type arginase) of about 1.7 kb was also induced by LPS. However, dose dependence differed from that of iNOS mRNA. Arginase I mRNA was barely detectable with 1 ng/ml of LPS; it was detected with 10 ng/ml and the level increased with up to 10 g/ml. The maximal mRNA concentration seen with 10 g/ml of LPS was comparable to that in the liver. Fig. 2 shows the time course of induction of iNOS and arginase I mRNAs in the peritoneal cells treated with 10 g/ml of LPS. iNOS mRNA was detected at 2 h, increased with time, reached a near maximum at 8 h and a maximum at 12 h, and remained at the near maximal level at 24 h. On the other hand, arginase I mRNA began to increase after 4 h with a lag time and reached a maximum at 12-24 h. Thus, iNOS and arginase I mRNAs are coinduced by LPS in rat peritoneal cells, but arginase I mRNA is induced more slowly than iNOS mRNA. Arginase II mRNA, which is 57% identical with arginase I mRNA, was not detected in LPS-treated peritoneal cells under conditions where the mRNA of 1.8 kb was clearly detected in the rat intestine (data not shown).
Induction by LPS of C/EBP␤ mRNA in Peritoneal Cells-We reported that C/EBP family members bind to both the promoter and enhancer regions of the rat arginase I gene (22,23) and that the promoter is activated by C/EBP␤ and other members (24). We then asked whether C/EBP␤ mRNA would also be induced by LPS in rat peritoneal cells. mRNA was present at a very low level prior to the LPS treatment; it increased with time, reached a maximum at 12 h, and then decreased (Fig. 2,  A, c, and B, c). Thus, induction of C/EBP␤ mRNA was more rapid than that of arginase I mRNA. Fig. 3 shows the time course of induction of iNOS and arginase I proteins in cultured peritoneal cells after exposure to LPS. iNOS protein of about 150 kDa was induced 8 h after the treatment; it increased at 24 h, then decreased. On the other hand, two polypeptides of about 35 and 38 kDa immunoreacted with the arginase I antibody, comigrated with the two forms of arginase I in the liver, and were observed 8 h after the treatment, then increased to 48 h. These two polypeptides of arginase I apparently arose by alternative translation initiation from the two methionine residues located 30 base pairs apart (17,20,34). Concentration of arginase I protein in the LPS-treated peritoneal cells was comparable to that in the liver; these findings were in accord with the observation that mRNA concentrations in the activated peritoneal cells and in the liver were comparable (see above).

Induction of iNOS and Arginase I Proteins in LPS-treated Peritoneal Cells-
Immunocytochemical Detection of iNOS and Arginase I in Peritoneal Cells-To identify cells positive for iNOS and arginase I in peritoneal cells, immunocytochemical analysis was performed (Fig. 4). iNOS immunoreactivity was absent prior to LPS treatment. However, most cells became strongly positive for iNOS immunoreactivity after the treatment. Arginase I was negative prior to the LPS treatment, but most cells became positive after the treatment. Distributions of the iNOS-positive cells, the arginase I-positive cells, and macrophages positive to the macrophage-specific antibody RM 4 were similar. However, cellular staining patterns of iNOS and arginase I differed from that with antibody RM4; iNOS and arginase I are cytosolic proteins, whereas the antibody RM4 is specific to lysosomes in macrophages. 2 Therefore, iNOS and arginase I are coinduced in practically all peritoneal macrophages.
Induction of mRNAs and Proteins for iNOS and Arginase I in the Lung and Spleen of LPS-treated Rats-We then examined expression of iNOS and arginase I in vivo. We previously reported that iNOS mRNA was induced in various tissues of rats injected intraperitoneally with LPS (10); the induction was the strongest in the lung and spleen. mRNAs for arginase I as well as for iNOS in the lung were measured at various times after LPS treatment (Fig. 5). iNOS mRNA in the lung increased to a near maximum 2 h after the treatment, reached a maximum at 6 h, decreased thereafter, and returned to a hardly detectable level at 24 h. Arginase I mRNA was also induced. mRNA was present at a very low level before treatment, began to increase after 2 h with a lag time, reached a maximum at 12 h on the average, and then decreased slowly. Concentration of the mRNA at 12 h was over 10-fold lower than that in the liver (data not shown).
iNOS mRNA in the spleen showed an increase similar to that in the lung, but the level decreased more slowly than seen in the lung (10). Arginase I mRNA was also induced in the spleen, but less markedly than in the lung, and the time course was similar to that in the lung and slower than that of iNOS mRNA in this organ (data not shown).
We reported that iNOS protein was markedly induced in the lung and spleen of LPS-treated rats (10). The induction of arginase I protein in the lung was also examined using immunoblot analysis. A polypeptide that comigrated with the larger form of arginase I was induced 24 h after LPS-treatment (data not shown). Concentration of arginase I in the lung of the LPS-treated rat was over 10-fold lower than that in the liver. DISCUSSION NO synthesis is regulated by depending on the availability of arginine, substrate of the NOS reaction, as well as by NOS activity and other factors. Arginine can be obtained via the blood circulation or by endogenous synthesis from citrulline. Arginine transport into cultured macrophages increases in response to LPS and to interferon-␥ (35,36). The cat-2 gene of the arginine transporter in cultured vascular smooth muscle cells is stimulated by interleukin-1␤ and by tumor necrosis factor-2 (37). Furthermore, argininosuccinate synthetase and argininosuccinate lyase, which together synthesize arginine from citrulline, are induced in stimulated murine macrophages (8,10) and aortic smooth muscle cells (9), and intracellular arginine synthesis is enhanced.
Activity of arginase that degrades arginine is also induced in activated macrophages (38,39). Arginase induced in activated RAW 264.7 cells, a mouse macrophage-like cell line, was shown to be arginase II (28,29). On the other hand, in the present work, we noted that arginase I, not arginase II, is induced by LPS in primary cultured rat peritoneal macrophages and in the lung and spleen in vivo. This was unexpected because arginase I has been thought to be expressed almost exclusively in the liver. Differential induction of the two arginase isoforms in the mouse macrophage-like cell line and in primary cultured rat macrophages may be due to differences in animal species or to differences between the established cell line and the primary cultured cells. Arginase I mRNA was induced more slowly than iNOS mRNA. The induced arginase I probably decreases arginine availability for the NOS reaction in activated macrophages and may down-regulate the overproduction of NO. All of these results suggest that there is a complex regulation of genes encoding enzymes and transporter proteins involved in arginine metabolism that together control NO production in cells.
Arginase I mRNA was induced slowly in cultured peritoneal macrophages and also in vivo with an apparent lag time of a few hours. Therefore, the induction of arginase I appears to be mediated by a transcription factor(s) that is synthesized de novo in response to LPS stimulation. C/EBP family members bind to the promoter and enhancer regions of the arginase I gene and activate the promoter (Refs. 22-24; see Ref. 40 for a review), C/EBP␤ being the most potent. The present study also shows that C/EBP␤ mRNA is induced in the LPS-stimulated peritoneal macrophages. The induction of C/EBP␤ mRNA is more rapid than that of arginase I mRNA. Thus, the induction of arginase I appears to be mediated, at least in part, by the induction of C/EBP␤.

FIG. 5. Time course of induction of iNOS (a) and arginase I (b) mRNAs in the lung after LPS treatment.
A, total RNA was isolated from the rat lung at the indicated times after LPS injection. Two rats at 0 and 24 h and three rats at 2, 6, and 12 h were used. RNAs (1.0 g) were subjected to blot analysis. c shows 28 S and 18 S rRNAs. B, the results in A were quantified and are represented by mean Ϯ S.D. (solid bar; n ϭ 3) or mean Ϯ range (dotted bar; n ϭ 2). Maximal values are set at 100. The results on iNOS mRNA (A, a and B, a) were already reported (10), but are shown here for comparison with arginase I mRNA.