Interleukin-10 Inhibition of Nitric Oxide Biosynthesis Involves Suppression of CAT-2 Transcription Chun-Jen Huang,* , † , ‡ Bruce R. Stevens,§ R. Barton Nielsen,‡ Paul N. Slovin,‡ Xiaoying Fang,† David R. Nelson, ¶ and Jeffrey W. Skimming‡ ,  ,1 *Department of Anesthesiology, Mackay Memorial Hospital; †Graduate Institute of Pharmacology, National Yang-Ming University, Taipei, Taiwan; and ‡Department of Pediatrics, § Department of Physiology, ¶ Department of Medicine, and Department of Anesthesiology, University of Florida College of Medicine, Gainesville, Florida 32610 Received August 9, 2001; published online October 25, 2001 Interleukin-10 (IL-10) has been shown to attenuate li- popolysaccharide (LPS) stimulation of inducible nitric oxide synthase (iNOS) in various cell types. Guanosine triphosphate cyclohydrolase I (GTPCH) and type-2 cat- ionic amino acid transporter (CAT-2) are enzymes that regulate iNOS activity. We therefore sought to assess the effects of IL-10 on the expression of these regulatory enzymes in LPS-stimulated macrophages that are known to express iNOS. Five minutes after adding LPS to these macrophage cultures, various doses of recombi- nant human IL-10 were also added. The samples were harvested for analysis 18 h after exposure to both LPS and IL-10. In LPS-stimulated macrophages, IL-10 atten- uated the upregulation of nitric oxide and iNOS protein but not iNOS mRNA. IL-10 also attenuated the LPS- induced upregulation of CAT-2 mRNA. However, IL-10 and LPS had no effect on GTPCH mRNA expression. We therefore conclude that IL-10 inhibits nitric oxide for- mation in LPS-stimulated macrophages partly by de- creasing iNOS protein expression. Moreover, our data suggests that transcriptional control of CAT-2 plays a role in IL-10 mediated influences upon nitric oxide biosynthesis. © 2001 Elsevier Science (USA) Key Words: IL-10; nitric oxide; iNOS; GTPCH; CAT-2; LPS; macrophage. Exposure of macrophages to bacterial lipopolysac- charide (LPS) stimulates the formation of inducible nitric oxide synthase (iNOS) and results in nitric oxide overproduction (1). This overproduction of nitric oxide has been shown to play an important role in the in- flammatory response that results in tissue injury (2). One mechanism by which nitric oxide injures tissues is by reacting with superoxide to form highly reactive oxidants, such as peroxynitrite, which cause oxidative tissue damage (3). Another mechanism involves the induction of apoptosis (4). Previous studies have shown that efforts to decrease nitric oxide exposure, such as inhibiting iNOS activity (2, 5) or treating with a nitric oxide scavenger (6), reduce the pathological sequelae of inflammation. Several regulatory mechanisms involved in the for- mation of nitric oxide by iNOS have been identified. One of them involves the biosynthesis of tetrahydro- biopterin (BH 4 ), a necessary cofactor for iNOS (7, 8). Synthesis of BH 4 is rate-limited by guanosine triphos- phate cyclohydrolase I (GTPCH) (9), an enzyme that is reported to be coinduced with iNOS in cells after expo- sure to bacterial toxins (10 –12). A second mechanism involves cellular uptake of arginine, the sole substrate for iNOS, by the type-2 cationic amino acid transporter (CAT-2) (13). In macrophages stimulated to form iNOS, bacterial toxins have been shown to upregulate CAT-2 (14). Therefore, GTPCH as well as CAT-2 each represents a key regulatory site for nitric oxide forma- tion by iNOS. Interleukin 10 (IL-10) is an anti-inflammatory cyto- kine released by T helper-2 cells, some B cells, and activated monocytes (15, 16). The antiinflammatory activity of IL-10 involves suppression of IFN- produc- tion by T helper-1 cells and suppression of TNF-, IL-1, and IL-6 release by mononuclear cells (16 –18). IL-10 has also been shown to suppress the expression of iNOS and reduce the production of nitric oxide in var- ious cell types (19, 20). However, the effects of IL-10 on GTPCH and CAT-2 have not been previously investi- gated. We, therefore, designed this study to elucidate the effects of IL-10 on several key regulatory pathways affecting iNOS activity. Our hypothesis was that IL-10 inhibits transcription of both GTPCH and CAT-2. We surmised that these mechanisms could account for the 1 To whom correspondence and reprint requests should be ad- dressed. Fax: (352) 392-0547. E-mail: skimmjw@ufl.edu. NITRIC OXIDE: Biology and Chemistry Vol. 6, No. 1, pp. 79 – 84 (2002) doi:10.1006/niox.2001.0402, available online at http://www.idealibrary.com on 79 1089-8603/01 $35.00 © 2001 Elsevier Science (USA) All rights reserved.