Báo cáo khoa học: A pathway through interferon-c is the main pathway for induction of nitric oxide upon stimulation with bacterial lipopolysaccharide in mouse peritoneal cells pot

A pathway through interferon-c is the main pathway for induction of nitric oxide upon stimulation with bacterial lipopolysaccharide in mouse peritoneal cells Motohiro Matsuura 1 , Shinji Saito 1 , Yoshikazu Hirai 1 and Haruki Okamura 2 1 Department of Microbiology, Jichi Medical School, Tochigi, Japan; 2 Institute for Advanced Medical Sciences, Hyogo College of Medicine, Nishinomiya, Hyogo, Japan Production of nitric oxide (NO) in response to bacterial lipopolysaccharide (LPS) was investigated using cultures of mouse peritoneal exudate cells (PEC) and the macrophage cell line RAW264.7. In the presence of anti-(interferon-c) (IFN-c), NO production was markedly suppressed in the PEC culture but not in the RAW264.7 culture. In the PEC culture, LPS induced both IFN-c production and activation of IFN response factor-1, which leads to the gene expression of inducible NO synthase, but neither was induced in the culture of RAW264.7 cells. In addition to anti-(IFN-c), antibodies against interleukin (IL)-12 and IL-18 showed a suppressive effect on LPS-induced NO production in the PEC culture, and these antibodies in synergy showed strong suppression. Stimulation of the PEC culture with IL-12 or IL-18 induced production of IFN-c and NO, and these cytokines, in combination, exhibited marked synergism. Stimulation of the culture with IFN-c induced production of NO, but not IL-12. The macrophage population in the PEC, prepared as adherent cells, responded well to LPS for IL-12 production, but weakly for production of IFN-c and NO. The macrophages also responded well to IFN-c for NO production. For production of IFN-c by stimulation with LPS or IL-12 + IL-18, nonadherent cells were required in the PEC culture. Considering these results overall, the indi- rect pathway, through the production of intermediates (such as IFN-c-inducing cytokines and IFN-c) by the cooperation of macrophages with nonadherent cells, was revealed to play the main role in the LPS-induced NO production pathway, as opposed to the direct pathway requiring only a macro- phage population. Keywords: lipopolysaccharide; nitric oxide; interferon-c; interleukin-12; interleukin-18. The ability of bacterial lipopolysaccharide (LPS) to stimu- late the mammalian immune system is mediated by the action of LPS-induced mediators such as cytokines, chemo- kines and lipid mediators [1,2]. Nitric oxide (NO) is also regarded as an important mediator, with its unique characteristic as a liquid-soluble gas. Multiple physiological functions of this molecule, in relation to LPS activity, have been reported, including enhancement of resistance against microbial infections and tumor cells as well as induction of tissue damage, hypotension and shock [3–6]. Macrophages have been shown to be the primary cellular source to recognize LPS by CD14 [7] and transduce its signals through the Toll-like receptor 4 (TLR4)–MD-2 complex [8] for the production of mediators, including NO. Production of NO is governed by the activity of three NO synthase (NOS) isoforms in which neuronal NOS and endothelial NOS are constitutively expressed, while another macro- phage-inducible NOS (iNOS or mac-NOS), is transcrip- tionally induced in response to immune stimuli such as LPS and produces large amounts of NO [9,10]. Expression of the iNOS gene is therefore an intracellular event that must occur before NO production in macrophages. In the promoter of this gene, two important regions, region 1 containing nuclear factor-jB(NF-jB)-binding sequence [11,12] and region 2 containing interferon response factor-1 (IRF-1)-binding sequence [13–15], have been defined. Acti- vation of region 1 is essential for expression of the iNOS gene in murine macrophages stimulated with LPS. This indicates that LPS can directly induce NO production by macrophages through a pathway activating NF-jBandits binding to region 1 without the help of cofactors such as cytokines. In the case of region 2 activation, stimulation of the macrophages with interferon-c (IFN-c), but not with LPS, was found to be effective for inducing the binding of IRF-1toregion2,leadingtoiNOS gene expression. The important role of IFN-c in the pathogenesis of LPS- induced shock was confirmed using mice deficient for the IFN-c receptor [16,17]. IFN-c is produced primarily by natural killer (NK) cells and a certain subpopulation of T lymphocytes (T helper 1 cells) [18] upon stimulation with Correspondence to M. Matsuura, Department of Microbiology, Jichi Medical School, 3311-1, Yakushiji, Minamikawachi-machi, Tochigi, 329-0498, Japan. Fax: + 81 285 44 1175, Tel.: + 81 285 58 7332, E-mail: mmatsuur@jichi.ac.jp Abbreviations:IFN-c, interferon-c; IL, interleukin; iNOS, inducible nitric oxide synthase; IRF-1, interferon response factor-1; LPS, lipopolysaccharide; NF-jB, nuclear factor-jB; NK, natural killer; NO, nitric oxide; PEC, peritoneal exudate cells; TLR4, Toll-like receptor 4; TNF-a, tumor necrosis factor-a. (Received 27 June 2003, revised 31 July 2003, accepted 14 August 2003) Eur. J. Biochem. 270, 4016–4025 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03792.x interleukin (IL)-12 [19], IL-18 [20,21] and some others, but not with LPS. A single report described the detection of IFN-c mRNA, without secreted protein, in murine macro- phages upon LPS stimulation [22]. This mechanism is, however, hard to consider as the main underlying mechan- ism of LPS-induced IFN-c production in vivo,inviewofthe large amount of IFN-c (detectable as protein level) secreted in the serum of mice after LPS challenge. Recently, LPS- induced production of IFN-c in large amounts in in vitro systems was reported by our group using murine peritoneal cells [23] and by other groups using murine spleen cells [24,25]. These results indicate that additional cellular populations, besides the macrophage population, are required for effective production of IFN-c in response to LPS, unlike the production of the usual LPS mediators such as IL-1, IL-6 and tumor necrosis factor-a (TNF-a), for which the macrophage population alone is sufficient. In the course of our study concerning the mechanism of LPS-induced NO production, participation of IFN-c was not suggested when murine macrophage cell lines such as RAW264.7 [26] and J774.1 [27] were used. However, in a study using murine peritoneal cells [23], participation of endogenously produced IFN-c in LPS-induced NO pro- duction was thought to occur. In the present study, we aimedtoclarifytheroleofIFN-c in LPS-induced NO production and revealed the important role of IFN-c as a key mediator in the main pathway for LPS-induced NO production in murine peritoneal cell culture. The underlying mechanisms in the IFN-c-mediated pathway were also elucidated. Materials and methods Animals and cells Mice of the C3H/HeN and C3H/HeJ strains (mutants with a defect in the function of TLR4) [28], were obtained from Japan Charles River (Tokyo, Japan) and Clea Japan, Inc. (Tokyo, Japan), respectively, and used at the age of 7–10 weeks. All animal experiments in the present study were conducted according to the guidelines of the Labor- atory Animal Center, Jichi Medical School. Peritoneal exudate cells (PEC) were obtained from mice that had been injected intraperitoneally with 2 mL of thioglycollate broth (Difco Laboratories, Detroit, MI, USA) 4 days previously. The PEC usually contained 87–92% macrophages and 6–9% lymphocytes, as determined by morphological criteria. The murine macrophage cell line RAW264.7, originally from the American Type Culture Collection (Manassas, VA, USA) and maintained in our laboratory, was also used. For cell culture, RPMI-1640 (Dainippon Pharmaceutical Co. Ltd, Osaka, Japan) supplemented with 10 m M Hepes, 2 m ML -glutamine, 100 UÆmL )1 penicillin, 100 lgÆmL )1 streptomycin, 0.2% NaHCO 3 , and 5% heat- inactivated fetal bovine serum (Flow Laboratories Inc., Rockville, MD, USA) was used. All cells were suspended in the culture medium, plated onto a 24-well culture plate (Corning Inc., Corning, NY, USA) at a dose of 6 · 10 5 cells per well (final volume, 0.6 mL) and cultured in a humidified chamber at 37 °Cwith5%CO 2 and 95% air. Cells were cultured for 5 h before stimulation with LPS and cytokines. In some experiments, the 5 h cultures were washed three times with Hanks’ balanced salt solution (Gibco-BRL, Gaithersburg, MD, USA) to remove nonadherent cells and the adherent cells only were used as the macrophage culture. Reagents The LPS used was a kind gift from C. Galanos (MIP fu ¨ r Immunbiologie, Freiburg, Germany), prepared from Sal- monella enterica serovar Abortus-equi, as described previ- ously [29]. The antibodies used were anti-(mouse IL-1b) (R&D Systems, Minneapolis, MN, USA), anti-(mouse IL-6) and anti-(mouse IL-12) (C17.8) (Genzyme Inc., Cambridge, MA, USA), anti-(mouse IL-15) (Torrey Pines Biolabs Inc., Houston, TX, USA), anti-(mouse IFN-b) (MB-7) (Seikagaku Co., Tokyo, Japan), anti-(mouse IFN-c) (R4-6A2) (Endogen, Woburn, MA, USA), anti- (mouse TNF-a) (a kind gift from Suntory Co. Ltd, Osaka, Japan) [26], anti-(mouse CD14) (4C1) (kindly donated by Y. Adachi, Tokyo University of Pharmacy and Life Science, Tokyo, Japan) [30] and anti-(mouse IL-18), prepared as described previously [20]. The cytokines used were recom- binant murine IFN-c (Pepro Tech EC, London, UK), recombinant mouse IL-12 (Genzyme Inc.) and recombinant mouse IL-18, prepared as described previously [20]. Assay of NO and cytokines Production of NO was determined as the amount of nitrite, a stable end-product of NO, in the culture supernatant obtained 48 h after stimulation with LPS or cytokine. Nitrite was measured by a colorimetric assay using the Griess reagent (1% sulfanilamide and 0.1% N-1-naphtyl- ethylendiamine dihydrochloride in 2.5% H 3 PO 4 solution) [31]. The absorbance at 540 nm was measured by the Biomek 1000 spectrophotometer and the nitrite concentra- tion was quantified (in l M ) using sodium nitrite as the standard in each assay. Production of IFN-c and IL-12p70 (active form) was determined as the amount in the culture supernatant obtained 24 h after stimulation. The concentration of each cytokine was measured using a specific sandwich ELISA, according to the manufacturer’s instructions (Endogen) using matched antibody pairs. A 96-well EIA/RIA plate (Corning Inc.), coated with a coating antibody and masked with skim milk, was incubated with test samples and standard solutions. A biotin-labeled detecting antibody was then added to the plate followed by reaction with a streptavidin–horseradish peroxidase conjugate. Enzyme reaction of the peroxidase was performed with a substrate solution (0.01% 3,3¢,5,5¢-tetramethylbenzidine and 0.03% H 2 O 2 in 0.11- M acetate buffer, pH 5.5). The color reaction was stopped by adding 0.18 M H 2 SO 4 and the absorbance was measured at 450 nm. Quantification of each cytokine (in ngÆmL )1 for IFN-c andinpgÆmL )1 for IL-12p70) was performed based on the standard curve in each assay. Preparation of nuclear extract The peritoneal cells and RAW264.7 cells were stimulated with LPS or IFN-c, and nuclear proteins of the cells were prepared as described previously [27]. Briefly, the cells were washed with NaCl/Pi and resuspended by vortexing for 10 s Ó FEBS 2003 LPS-induced NO production through IFN-c (Eur. J. Biochem. 270) 4017 in hypotonic buffer A [10 m M Hepes (pH 7.8), 10 m M KCl, 0.1 m M EDTA, 0.5% Nonidet P-40 (NP-40), 1 m M dithiothreitol, 0.5 m M phenylmethanesulfonyl fluoride, 5 lgÆmL )1 aprotinin, 5 lgÆmL )1 pepstatin and 5 lgÆmL )1 leupeptin). Nuclei were separated from the cytosol by centrifugation at 1600 g for 1 min, resuspended in buffer C (50 m M Hepes pH 7.8, 0.42 M KCl, 0.1 m M EDTA, 5 m M MgCl 2 , 20% glycerol, 1 m M dithiothreitol, 0.5 m M phenyl- methanesulfonyl fluoride, 5 lgÆmL )1 aprotinin, 5 lgÆmL )1 pepstatin and 5 lgÆmL )1 leupeptin), and incubated on ice for 30 min with occasional vortexing. Nuclear extracts were obtained from the suspension by centrifugation at 15 000 g for 15 min and stored in small aliquots at )80 °C. EMSA Oligonucleotide containing the downstream NF-jB-binding site (nts )85 to )76) of the mouse iNOS promoter plus the downstream 47 base pairs, designated NF-jBd (5¢-CAT GGG GAC TCT CCC TTT GGG AAC AGT TAT GCA AAA TAG CTC TGC AGA GCC TGG AGG GGT CGA-3¢) [12] and the IRF-1 consensus sequence oligo- nucleotide (5¢-GGA AGC GAA AAT GAA ATT GAC T-3¢) were constructed as probes for EMSA. The oligonucleotides were annealed and labeled with [ 32 P]dCTP[aP]. Binding reactions were performed (20 lL of the total volume) by incubating these probes ( 20 000 counts per minute) with nuclear extracts (5 lg of protein content) at room tempera- ture for 30 min in binding buffer. The binding buffer consists of 10 m M Hepes (pH 7.8), 50 m M KCl, 1 m M EDTA, 5 m M MgCl 2 , 10% glycerol, 5 m M dithiothreitol, 0.7 m M phenylmethanesulfonyl fluoride and 2 lgÆmL )1 poly(dI-dC). Reaction products were electrophoresed on 5% polyacrylamide with 0.25 · TBE (Tris/borate/EDTA; 22.5 m M /22.2 m M /0.5 m M ) for analysis by autoradiogra- phy. Before starting the binding reaction, nuclear extracts were incubated for 20 min on ice in the presence or absence of competitors and antibodies. As competitors, a 100-fold excess of unlabelled oligonucleotide probes was used for analysis of binding specificities. As antibodies, anti-IRF-1, anti-p50, anti-p65 and anti-(c-Rel) Igs (1 lg; all from Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) were used for supershift assays. Results Suppression, by anti-IFN-c Ig, of LPS-induced NO production in murine PEC but not in the RAW264.7 murine macrophage cell line Cells of murine PEC and of the RAW264.7 murine macrophage cell line were cultured and stimulated with LPS in the presence or absence of anti-(IFN-c) Ig. In both cultures, a large amount of NO was produced upon LPS stimulation at doses of > 1 ngÆmL )1 , as shown in Fig. 1. In the presence of anti-(IFN-c) Ig, production of NO was suppressed markedly in the PEC culture but not at all in the RAW264.7 culture. Another murine macrophage cell line, J774.1, was also used for the experiments and results similar to those for RAW264.7 were obtained. Namely, no suppression of LPS-induced NO production by anti-IFN- c Ig was observed (data not shown). These results indicate that LPS-induced NO production in PEC depends largely on IFN-c, which is probably produced upon LPS stimula- tion, but that IFN-c does not participate at all in LPS- induced NO production in macrophage cell lines such as RAW264.7 and J774.1. Production of IFN-c and IL-12 by murine PEC upon LPS stimulation We then investigated the production of IFN-c and related cytokines upon LPS stimulation of PEC and RAW264.7 cells. As shown in Fig. 2A, IFN-c was produced dose dependently upon stimulation with LPS in PEC, but not in RAW264.7cells,atdosesofLPSupto10ngÆmL )1 .Asfor the production of IL-12p70, results similar to those for IFN- c production were obtained; namely, a dose-dependent production of IL-12p70 in PEC and no production in RAW264.7 cells (Fig. 2B). An attempt to determine the LPS-induced production of IL-18 was also made as IL-18 is known to be a representative IFN-c-inducing cytokine, in addition to IL-12. Production of IL-18 in PEC culture was detected, although at a very low level (<70 pgÆmL )1 at 1ngmL )1 of LPS); IL-18 was also detected in RAW264.7 cells, but at a somewhat higher level (data not shown). Fig. 1. Effect of anti-(IFN-c)IgonLPS-inducedNOproductionby peritoneal cells of C3H/HeN mice (A) and by RAW264.7 mouse macrophage line cells (B). Peritoneal exudate cells (PEC) were obtained from C3H/HeN mice which had been injected with thioglycollate broth intraperitoneally 4 days previously. The PEC and RAW264.7 cells were cultured with 5% fetal bovine serum–RPMI in the presence (d)orabsence(s)of10lgÆmL )1 anti-(IFN-c).LPSwasaddedtothe cultures at the indicated concentrations  1 h later. Culture super- natant obtained 48 h after LPS stimulation was assayed to determine the concentration of NO. Data represent mean ± SEM of triplicate samples. A representative result from three independent experiments is shown. 4018 M. Matsuura et al. (Eur. J. Biochem. 270) Ó FEBS 2003 Improvement of the ELISA sensitivity may help to deter- mine more precisely the level of IL-18 produced in response to LPS stimulation. Effect of cytokine antibodies on LPS-induced production of NO and IFN-c in PEC Various cytokines other than IFN-c, IL-12 and IL-18 are produced by PEC upon LPS stimulation. To estimate the participation of those cytokines, known as LPS media- tors, in LPS-induced NO production, PEC were cultured in the presence of antibodies to those cytokines and stimulated with LPS. Even at the highest concentration of antibodies tested (10 lgÆmL )1 ) most showed no significant suppressive effect of NO production (Fig. 3). Only anti-(IL-12) and anti-(IL-18) Igs exhibited a signi- ficant suppressive effect, although the effect was weaker than that of anti-(IFN-c) Ig. When anti-(IL-12) and anti- (IL-18) Igs were combined at the same concentration (5 lgÆmL )1 each, giving a total protein concentration of 10 lgÆmL )1 ), the NO production was strongly suppressed to a level similar to that achieved using the anti-(IFN-c) Ig, and more effective than the suppression achived using either anti-(IL-12) or anti-(IL-18) alone at 10 lgÆmL )1 . Neither of these antibodies showed cross-reactivity to neutralize the antigenicity of murine IFN-c (data not shown). These results, together with those presented in Fig. 2, indicate that IFN-c is produced by PEC upon stimulation with LPS, and that both IL-12 and IL-18 participate in the process of the LPS-induced IFN-c production. Besides these cytokine antibodies, anti-CD14 Ig (4C1) was also used in this experiment. Strong suppression of the LPS-induced production of NO and IFN-c indicates that the signals of LPS for the produc- tion of these mediators are transduced through CD14. Stimulatory effect of IFN-c and IL-12 + IL-18 on NO production by PEC of C3H/HeN and C3H/HeJ mice TheroleofIFN-c,aswellasofIL-12andIL-18,as intermediates in the pathway of LPS-induced NO produc- tion, was indicated from the results obtained above. Preparations of recombinant IFN-c, IL-12 and IL-18 were added to PEC cultures of HeN and HeJ mice, and production of NO in the culture supernatant was measured. As shown in Fig. 4A, a significant effect of IFN-c on NO production was observed at concentrations of > 1 ngÆmL )1 in the cultures of both mouse strains. Concerning IL-12p70, no significant production was observed in response to IFN-c in both cultures (data not shown), unlike the case of LPS stimulation. No response of the HeJ culture to LPS was observed for the production of NO, IFN-c and IL-12p70 (data not shown). We also examined the effect of IL-12 and IL-18 on NO production and found that the combined use of these cytokines was more effective than when either was used alone. Both PEC cultures of HeN and HeJ mice responded to these cytokines for production of NO (Fig. 4B). At the same time, the production of IFN-c was also determined. As shown in Fig. 4C, both cultures responded to a Fig. 2. Production of IFN-c (A) and IL-12 (B) in response to stimulation with LPS by peritoneal cells of C3H/HeN mice and by RAW264.7 cells. Peritoneal exudate cells (PEC) of C3H/HeN mice (d) and RAW264.7 cells (s) were cultured in the presence of LPS at the indicated con- centrations. Culture supernatant obtained 24 h after LPS stimulation was assayed, by ELISA, to determine the concentrations of interferon- c (IFN-c) and IL-12p70 (active form). Data represent mean ± SEM of triplicate samples. A representative result from three independent experimentsisshown. Fig. 3. Effect of cytokine antibodies on LPS-induced production of NO and IFN-c by mouse peritoneal cells (PEC). PEC of C3H/HeN mice were cultured in the presence or absence of cytokine antibodies and anti-CD14. The cultures were stimulated with 1 ngÆmL )1 LPS  1h later. Culture supernatants obtained at 24 h and 48 h time-points were assayed to determine the concentration of IFN-c and NO, respectively. A representative result with the highest concentration of antibodies tested (10 lgÆmL )1 ) is shown. In the case of anti-(IL-12) + anti-(IL-18) Igs, the antibodies were mixed at the same concentration, i.e. 5 lgÆmL )1 of each antibody was included. Data represent mean ± SEM of triplicate samples. Similar results were obtained in two other independent experiments. Ó FEBS 2003 LPS-induced NO production through IFN-c (Eur. J. Biochem. 270) 4019 combination of these cytokines for the production of IFN-c, similarly to NO production. The responses, of the HeN culture, were not suppressed by anti-CD14 Ig (data not shown). These results suggest that NO production by IL-12 + IL-18 is mediated by IFN-c via signaling path- ways that do not depend on either CD14 or TLR4, unlike LPS-induced signals. This is further supported by the observation that production of NO upon stimulation with IL-12 + IL-18 was suppressed by anti-(IFN-c)Ig, but that upon IFN-c stimulation was not suppressed by anti-(IL-12) + anti-(IL-18) (data not shown). Effect of nonadherent cells in the PEC of HeN mice on the production of NO and cytokines in response to LPS and its mediators In the preparation of the PEC used in this study, non- adherent cells were usually present at  10%, the remaining 90% of cells (almost all of which were macrophages) being adherent. To investigate the role of nonadherent cells, PEC were cultured for 5 h to permit adhesion, then the non- adherent cells were washed off. The adherent cells were cultured as macrophage culture and stimulated with LPS, IFN-c and IL-12 + IL-18. Production of NO, IFN-c and IL-12p70 by the adherent cell culture was compared with that of the whole-cell PEC culture containing nonadherent cells. The production of NO in response to LPS and IL-12 + IL-18 was markedly reduced in the adherent cell culture compared with that of the whole cell culture, while the production in response to IFN-c was only slightly reduced (Fig. 5A). The adherent cell culture showed a marked reduction in the production of IFN-c in response to LPS and IL-12 + IL-18 (Fig. 5B), similarly to that found for NO production. Production of IL-12p70 in response to LPS was reduced only slightly in the adherent cell culture compared with that in the whole-cell culture (Fig. 5C), and the rate of the reduction was far less than that observed in the LPS-induced production of NO and IFN-c. Represen- tative cytokines produced from macrophages upon LPS stimulation, e.g. IL-6 and TNF-a, were also measured in the culture supernatant. Similarly to the production of IL-12p70, the production of these cytokines in response to LPS was only marginally reduced in the adherent cell culture compared with that in the whole cell culture (data not shown). The IFN-c response of the adherent cells and RAW264.7 cells to produce NO was strong, but the response to produce IL-6 and TNF-a was not observed (data not shown), similarly to the case of IL-12p70. These results indicate that nonadherent cells participate largely in the production of NO and IFN-c in response to LPS and IL-12 + IL-18, but very little in the production of NO in response to IFN-c and weakly in the production of IL-12p70 in response to LPS. In another experiment, nonadherent cells recovered from the PEC culture of C3H/HeJ mice were added to the adherent cell culture of HeN-PEC and the mixed population culture was stimulated with LPS. Production of both NO and IFN-c in the mixed population culture was remarkably enhanced compared with that in the adherent cell culture alone and reached the level observed in the whole cell culture of HeN-PEC (data not shown). This indicates that the production of IFN-c by nonadherent cells occurs independently of LPS, as neither adherent cells nor non- adherent cells of HeJ mice respond to LPS. Effect of LPS stimulation on the activation of IRF-1 and NF-jB in PEC and RAW264.7 cells Activation of transcription factors relating to iNOS gene expression, such as IRF-1 and NF-jB, was then investi- gated by EMSA using PEC and RAW264.7 cells stimulated with LPS. Activation of IRF-1 in the PEC of HeN mice was observed 18 h after LPS stimulation, but not at 2 or 6 h, while activation after IFN-c stimulation was already evident Fig. 4. Stimulation effect IFN-c and IL-12 + IL-18 on peritoneal cells of C3H/HeN and C3H/HeJ mice for production of NO. Peritoneal exudate cells (PEC) of HeN mice (d)andHeJmice(s), which have a defect in the function of Toll-like receptor 4 (TLR4), were stimulated with the indicated concentrations of IFN-c (A) and IL-12 + IL-18 (mixed at the equal concentrations indicated) and the culture super- natant was assayed to determine the concentrations of NO (B) and IFN-c (C). Data represent mean ± SEM of triplicate samples. A representative result from three independent experiments is shown. 4020 M. Matsuura et al. (Eur. J. Biochem. 270) Ó FEBS 2003 at the 2-h time-point and maintained up to the 18-h time- point (Fig. 6A). Specificity of IRF-1 binding to the DNA probe was confirmed from the result that the strong bands observed at 6 h as a result of stimulation with IFN-c were remarkably reduced in intensity by the addition of excess nonradiolabeled cold probe (lane cp in competition), but not by the addition of a mutant probe (lane mp), which lacks the binding ability owing to substitution of two AA residues in the IRF-1 binding motif to GG, and also because the bands were supershifted out of the range by preincu- bation of the nuclear extract with anti-(IRF-1) Ig (lane Ab). Production of IFN-c in the PEC upon LPS stimulation was first detectable at the 8-h time-point, becoming maximal at  20 h (data not shown). These results support the idea that IRF-1 is activated by IFN-c, but not by LPS, and that the IFN-c induced by LPS is the direct activator of IRF-1 in the LPS-stimulated PEC. In RAW264.7 cells, activation of IRF-1 was observed (to some extent) without stimulation, and enhancement from that level of activation was obscure by LPS stimulation up to 18 h while that by IFN-c stimulationwasclearfrom2hupto18h(Fig.6B).This result is understandable considering the above result that IFN-c was not induced by LPS during the culture of RAW264.7 cells (Fig. 2A). When the NF-jB consensus sequence oligonucleotide was constructed as a probe for EMSA, activation of NF-jBwas clearly observed in both the PEC and RAW264.7 cells in response to LPS and also to IFN-c (data not shown). We determined that the specificity of this probe was too low for our experiments to detect activation of NF-jB leading to expression of the iNOS gene and selected a probe designated NF-jBd with higher specificity (as described in the Materials and methods) for further experiments. In the PEC of HeN mice, activation of NF-jB upon LPS stimulation was weak and that upon IFN-c stimulation was no higher than observed in the negative control (none) without stimulation (Fig. 7A). In RAW264.7 cells, the activation was clearly observed at 1 and 2 h after LPS stimulation but no more clearly at 4 h, and the activation after IFN-c stimulation was not observed at any of the time-points examined (Fig. 7B). Specificity of NF-jB binding to the DNA probe was confirmed from the result that the appearance of the strong bands at 1 h with LPS stimulation was completely sup- pressed by the addition of excess nonradiolabeled cold probe (Fig. 7B, lane cold probe). These results support the idea that activation of NF-jB participates in the direct induction of NO by LPS (as seen in RAW264.7 cells), but not in the induction by IFN-c (as seen in both types of cells) or in the indirect induction by LPS via IFN-c (as seen in the PEC). Attempts were made to characterize, by supershift assay, the components of the activated NF-jB members. Nuclear extracts obtained from RAW264.7 cells at 1 h after LPS stimulation were incubated with antibodies against p50, p65 and c-Rel subunits of NF-jB followed by addition of the NF-jBd probe for binding to NF-jB. Both the upper and Fig. 5. Removal of nonadherent cells from peritoneal exudate cells and the subsequent effect on the production of NO (A), IFN-c (B) and IL-12 (C) in response to stimulation with LPS, IFN-c and IL-12 + IL-18. PEC of C3H/HeN mice were cultured for 5 h and the nonadherent cells washed off to obtain macrophages as adherent cells. The whole-cell culture (grey column) without washing and the adherent cell culture (white column) were stimulated with the indicated stimulants. Culture supernatant obtained at 24 h was assayed to determine the concentrations of IFN-c and IL-12p70, and culture supernatant obtained at 48 h was assayed to determine the concentration of NO. Data represent mean ± SEM of triplicate samples. Similar results were obtained in two other independent experiments. N.D., not determined. Fig. 6. Effect of LPS stimulation on the activation of IRF-1 in peritoneal cells of HeN mice and in RAW264.7 cells. Peritoneal exudate cells of HeN mice (A) and RAW264.7 cells (B) were stimulated with LPS (1 ngÆmL )1 )orIFN-c (10 ngÆmL )1 ) for the indicated times. Nuclear extracts of the cells were prepared and EMSA performed using an IRF-1 probe. Part of the nuclear extract from PEC, prepared 6 h after IFN-c stimulation, was incubated with a 100-fold excess of unlabeled cold probe (lane cp) or mutant probe (lane mp), or with 1 lgofanti- (IRF-1) Ig (lane Ab) before the EMSA. Lanes labeled as ÔprobeÕ and ÔnoneÕ indicate free probe without nuclear extract and nuclear extract prepared from unstimulated cells, respectively. Similar results were obtained from three independent experiments. Ó FEBS 2003 LPS-induced NO production through IFN-c (Eur. J. Biochem. 270) 4021 the lower bands, clearly observed without antibody (Fig. 7B, lane 1 in LPS), supershifted dramatically with anti-p50 Ig (lane p50 in Ab) and less extensively with anti- p65 Ig. Supershift of the upper band alone was clearly observed with anti-c-Rel Ig, and the resulting weak upper band seemed to supershift completely by the addition of anti-p65 Ig (lane p65 + c-Rel in Ab). It is assumed, from these results, that the lower band consists predominantly of p50 homodimer and that the upper band consists of the mixed heterodimers of p50/c-Rel and p50/p65. Discussion Most of the LPS mediators studied, to date, have been shown to be produced directly from macrophages upon stimulation with LPS. NO is thought to be one such mediator. It is known that there is a direct pathway for the production of NO in macrophages stimulated with LPS, via activation of NF-jB and its binding to region 1 in the promoter of the iNOS gene [11,12]. In the present study, LPS-induced NO production in the RAW264.7 macro- phage cell line was not suppressed by anti-(IFN-c)Ig (Fig. 1) and activation of NF-jB was clearly observed in the LPS-stimulated RAW264.7 cells (Fig. 7), indicating NO production through this direct pathway. On the other hand, the NO production in murine PEC was strongly suppressed by anti-(IFN-c)Ig.IFN-c is known to be an inducing factor of NO from macrophages through the activation of IRF-1 and its binding to region 2 in the promoter of iNOS gene [13–15]. This indicates that NO is produced from LPS not only through a direct pathway but also through an indirect pathway via IFN-c. Activation of IRF-1 in relation to production of IFN-c in the PEC upon LPS stimulation was observed (Fig. 6A), and the amount of NO was reduced to < 10% by the anti-(IFN-c), indicating that the indirect pathway is mainly used for production of NO in the PEC culture, which contains varied cellular populations and is closer to in vivo situations than cultures with a single cellular population of macrophage. This is the first report demon- strating that the indirect pathway through IFN-c partici- pates in LPS-induced NO production and, moreover, that it playsamajorrole. The role of IFN-c as a mediator of LPS has been indi- cated in in vivo experiments [32], but in vitro investigations to elucidate the role have found little. The present study is one such in vitro study and clarifies the role of IFN-c as a mediator of LPS in NO production. A synergistic effect of exogenous IFN-c + LPS on the induction of LPS medi- ators in macrophage cultures is well known. The effect is, however, influenced profoundly by the sequence of stimu- lation with IFN-c and LPS, as reported previously [33]. It was indicated that exposure of macrophages to IFN-c, before or simultaneously with LPS stimulation, induced significant levels of NO release, but that exposure to LPS prior to IFN-c resulted in poor induction of NO release. We examined the effect of the sequence of stimulation with IFN-c and LPS on the production of NO using the adherent cell culture of HeN-PEC prepared under our experimental conditions. We found that addition of IFN-c to the culture, before or simultaneously with LPS stimulation, induced a synergistic effect on the production of NO, addition of IFN-c 3 h later still showed some synergistic effect, but addition of IFN-c 8 h later showed no synergistic effect (data not shown). In the whole-cell culture of HeN-PEC, IFN-c was detectable only 8 h after LPS stimulation. These results indicate that the endogenously induced IFN-c contributes little to enhance LPS-induced NO production but largely to the induction of NO by itself under the experimental conditions of the present study. In addition to these activities, IFN-c has diverse activities participating in both innate and acquired immunity [34], and such activities should also be taken into consideration in further investi- gations of the role of this cytokine as an LPS mediator. The present PEC culture also showed the advantage of using mixed-cell cultures for the investigation of LPS mediators induced indirectly after interaction with different cellular populations. Using such mixed-cell cultures, new LPS mediators are expected to be found. IL-12 and IL-18 have been shown to play important roles as IFN-c-inducing cytokines and exhibit marked synergism in combination [35,36]. Production of IL-12 by PEC in response to LPS was clearly detected (Fig. 2B) but that of IL-18 was not so clear. Production of IL-18 was detected; however, the amount was too small to determine accurately using our current ELISA system. Anti-(IL-18) Ig showed a suppressive effect, similar to that of anti-(IL-12) Ig, against the production of IFN-c and NO, and combined treatment using these two antibodies exhibited a synergistic effect (Fig. 3), suggesting the participation of IL-18 in concert with IL-12 in the indirect pathway for NO production. We Fig. 7. Effect of LPS stimulation on the activation of nuclear factor (NF)-jB. Nuclear extracts prepared from peritoneal exudate cells of HeN mice (A) and RAW264.7 cells (B), stimulated as indicated in the legend to Fig. 6, were analysed by EMSA using an NF-jBd probe. Part of the nuclear extract from RAW264.7 cells, prepared 1 h after LPS stimulation, was incubated with a 100-fold excess of unlabeled cold probe (lane cold probe) before the EMSA. Another part of this nuclear extract was also used for supershift experiments by incubating with 1 lg of antibodies against NF-jB subunits, such as anti-p50 Ig (lane p50), anti-p65 Ig (lane p65), anti-(c-Rel) Ig, or a combination of anti-p65 and anti-(c-Rel) Igs (lane p65 + c-Rel) before the EMSA. Lanes of probe and none are as indicated in the legend to Fig. 6. Similar results were obtained from three independent experiments. 4022 M. Matsuura et al. (Eur. J. Biochem. 270) Ó FEBS 2003 confirmed that the anti-(IL-18) Ig used in the present study does not inhibit the antigenicity of IL-12 by ELISA and that the same results as shown in the present study are obtained even when another anti-mouse IL-18 mAb (clone 93-10C; MBL Co. Ltd, Nagoya, Japan) is used instead of the present antibody (data not shown). It has been reported that the active form of IL-18 is secreted from cells after processing the precursor form of IL-18 by caspase-1 [37,38]. In preliminary experiments, we observed the suppressive effect of caspase-1 inhibitor on LPS-induced NO production by the PEC, supporting further the requirement for active IL-18. It is possible that only a small quantity of this cytokine is produced, as detected using our ELISA, or it may be rapidly degraded under the culture conditions; further investigations are therefore required to elucidate more fully how IL-18 contributes in the production of IFN-c and NO. Besides IL-12 and IL-18, IL-15 has also been reported to play a role as an LPS-induced mediator for IFN-c production and participate in the exhibition of an LPS- induced general Shwartzman reaction [39]. The suppressive effect of anti-(IL-15) Ig on the LPS-induced production of IFN-c and NO was not clear in the present study (Fig. 3). The effect of recombinant IL-15 on the induction of IFN-c in the PEC was not evident, even in combination with IL-12 or IL-18 (data not shown). These results indicate that IL-15 contributes far less to the indirect pathway of NO produc- tion than IL-12 and IL-18. Induction of IL-12 by LPS was not observed in the PEC of HeJ mice (Fig. 2) and the strong induction observed in the PEC of HeN mice was suppressed by anti-CD14 (Fig. 3). These results indicate that LPS activates macrophages initially through CD14 and the TLR4–MD-2 complex on their cellular surface to trans- duce the signals inside the cells for production of IL-12, in a manner recently revealed as the usual way for LPS to induce a macrophage response [2,8]. The cellular populations contributing to a synergistic effect of IL-12 and IL-18 on IFN-c production have been shown to be T helper 1 cells [40,41] and NK cells [42], and these populations are known to be nonadherent cells. The response of HeJ cells to IL-12 + IL-18 for the production of IFN-c (Fig. 4), and no suppressive effect of anti-CD14 in this response of HeN cells, indicates that CD14 and TLR4–MD-2 are not required in this step. For the final step to produce NO in response to IFN-c, macrophages obtained as adherent cells were sufficient (Fig. 5) without the help of nonadherent cells. The response of HeJ (Fig. 4), and the effect of anti-CD14 on HeN, indicates that this final step is also independent of CD14 and TLR4–MD-2, although the macrophage population is the target. These processes, participating in the pathways of LPS- induced NO production, are schematically summarized in Fig. 8. In the present study, the important role of the indirect pathway through IFN-c in LPS-induced NO production in murine peritoneal cells was clarified, for the first time, by in vitro experiments. Acknowledgement This work was supported, in part, by Grant 13670280 (to M.M.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. References 1. Ulevitch, R.J. & Tobias, P.S. (1995) Receptor-dependent mechanisms of cell stimulation by bacterial endotoxin. Annu. Rev. Immunol. 13, 437–457. 2. Alexander, C. & Rietschel, E.T. (2001) Bacterial lipopoly- saccharide and innate immunity. J. Endotoxin Res. 7, 167–202. 3. Culotta, E. & Koshland, D.E.J. (1992) NO news is good news. Science 258, 1862–1865. 4. Stamler, J.S., Single, D.J. & Loscalzo, J. (1992) Biochemistry of nitric oxide and its redox-activated forms. Science 258, 1898–1902. 5. Schumidt, H.H.H.W. & Walter, U. (1994) NO at work. Cell 78, 919–925. Fig. 8. Pathways for LPS-induced NO production in mouse peritoneal cells. The direct pathway through activation of nuclear factor (NF)-jBis considered to be the sole pathway for LPS-induced NO production in murine macrophage cell line culture, but a minor pathway in peritoneal cell culture. The indirect pathway, through IFN-c, was indicated to play the major role in the peritoneal cell culture in the present study. Ó FEBS 2003 LPS-induced NO production through IFN-c (Eur. J. Biochem. 270) 4023 6. MacMicking, J.D., Nathan, C., Hom, G., Chartrain, N., Fletcher, D.S.,Trumbauer,M.,Stevens,K.,Xie,Q W.,Sokol,K., Hutchinson, N., Chen, H. & Mudgett, J.S. (1995) Altered response to bacterial infection and endotoxin shock in mice lacking inducible nitric oxide synthase. Cell 81, 641–650. 7. Wright, S.D., Romos, R.A., Tobias, P.S., Ulevitch, R.J. & Mathison, J.C. (1990) CD14, a receptor for complexes of lipo- polysaccharide (LPS) and LPS binding protein. Science 249, 1431–1433. 8. Akashi, S., Shimazu, R., Ogata, H., Nagai, Y., Takeda, K., Kimoto, M. & Miyake, K. (2000) Cutting edge: cell surface expression and lipopolysaccharide signaling via the Toll-like receptor 4–MD-2 complex on mouse peritoneal macrophages. J. Immunol. 164, 3471–3475. 9.Lowenstein,C.J.,Glatt,C.S.,Bredt,D.S.&Snyder,S.H. (1992) Cloned and expressed macrophage nitric oxide synthase contrasts with the brain enzyme. Proc. Natl Acad. Sci. USA 89, 6711–6715. 10. Xie, Q W., Cho, H.J., Calaycay, J., Mumford, R.A., Swiderek, K.M., Lee, T.D., Ding, A., Troso, T. & Nathan, C. (1992) Cloning and characterization of inducible nitric oxide synthase from mouse macrophages. Science 256, 225–228. 11. Lowenstein, C.J., Alley, E.W., Raval, P., Snowman, A.M., Snyder, S.H., Russell, S.W. & Murphy, W.J. (1993) Macrophage nitric oxide synthase gene: two upstream regions mediate induc- tion by interferon c and lipopolysaccharide. Proc. Natl Acad. Sci. USA 90, 9730–9734. 12. Xie, Q W., Kashiwabara, Y. & Nathan, C. (1994) Role of tran- scription factor NF-jB/Rel in induction of nitric oxide synthase. J. Biol. Chem. 269, 4705–4708. 13. Kamijo, R., Harada, H., Matsuyama, T., Bosland, M., Gerecit- ano, J., Shapiro, D., Le, J., Koh, S.I., Kimura, T., Green, S.J., Mak,T.W.,Taniguchi,T.&Vilcek,J.(1994)Requirementfor transcription factor IRF-1 in NO synthase induction in macro- phages. Science 263, 1612–1615. 14. Martin, E., Nathan, C. & Xie, Q W. (1994) Role of interferon regulatory factor 1 in induction of nitric oxide synthase. J. Exp. Med. 180, 977–984. 15. Weisz, A., Cicatiello, L. & Esumi, H. (1996) Regulation of the mouse inducible-type nitric oxide synthase gene promoter by interferon-c, bacterial lipopolysaccharide and N G -monomethyl- L-arginine. Biochem. J. 316, 209–215. 16. Kamijo, R., Le, J., Shapiro, D., Havell, E.A., Huang, S., Aguet, M., Bosland, M. & Vilcek, J. (1993) Mice that lack the interferon-c receptor have profoundly altered responses to infection with bacillus Calmette–Gue ´ rin and subsequent challenge with lipo- polysaccharide. J. Exp. Med. 178, 1435–1440. 17.Car,B.D.,Eng,V.M.,Schnyder,B.,Ozmen,L.,Huang,S., Gallay, P., Heumann, D., Aguet, M. & Ryffel, B. (1994) Interferon c receptor deficient mice are resistant to endotoxic shock. J. Exp. Med. 179, 1437–1444. 18. Billiau, A. (1996) Interferon-c: biology and role in pathogenesis. Adv. Immunol. 62, 61–130. 19. Trinchieri, G. (1995) Interleukin-12: a proinflammatory cytokine with immunoregulatory functions that bridge innate resistance and antigen-specific adaptive immunity. Annu. Rev. Immunol. 13, 251–276. 20. Okamura, H., Tsutsui, H., Komatsu, T., Yutsudo, M., Hakura, A.,Tanimoto,T.,Torigoe,K.,Okura,T.,Nukada,Y.,Hattori, K., Akita, K., Namba, M., Tanabe, F., Konishi, K., Fukuda, S. & Kurimoto, M. (1995) Cloning of a new cytokine that induces IFN- c production by T cells. Nature 378, 88–91. 21. Dinarello, C., Novick, D., Puren, A.J., Fantuzzi, G., Shapiro, L., Mu ¨ hl, H., Yoon, D Y., Rezinikov, L.L., Kim, S H. & Rubin- stein, M. (1998) Overview of interleukin-18: more than an inter- feron-c inducing factor. J. Leukoc. Biol. 63, 658–664. 22. Fultz, M.J., Barber, S.A., Dieffenbach, C.W. & Vogel, S.N. (1993) Induction of IFN-c in macrophages by lipopolysaccharide. Int. Immunol. 5, 1383–1392. 23. Tominaga, K., Saito, S., Matsuura, M., Funatogawa, K., Matsumura, H. & Nakano, M. (1999) Role of IFN-c on dissoci- ation between nitric oxide and TNF/IL-6 production by murine pertoneal cells after restimulation with bacterial lipopolysaccha- ride. J. Leukoc. Biol. 66, 974–980. 24. Balkhy, H.H. & Heinzel, F.P. (1999) Endotoxin fails to induce IFN-c in endotoxin-tolerant mice: deficiencies in both IL-12 het- erodimer production and IL-12 responsiveness. J. Immunol. 162, 3633–3638. 25. Varma, T.K., Lin, C.Y., Toliver-Kinsky, T.E. & Sherwood, E.R. (2002) Endotoxin-induced c-interferon production: contributing cell types and key regulatory factors. Clin. Diagn. Lab. Immunol. 9, 530–543. 26. Funatogawa, K., Matsuura, M., Nakano, M., Kiso, M. & Hase- gawa, A. (1998) Relationship of structure and biological activity of monosaccharide lipid A analogues to induction of nitric oxide production by murine macrophage RAW264.7 cells. Infect. Immun. 66, 5792–6798. 27. Saito, S., Matsuura, M., Tominaga, K., Kirikae, T. & Nakano, M. (2000) Important role of membrane-associated CD14 in the induction of IFN-b and subsequent nitric oxide production by murine macrophages to bacterial lipopolysaccharide. Eur. J. Biochem. 267, 37–45. 28. Poltorak, A., He, X., Smilnova, I., Liu, M Y., Van Huffel, C.X., Birdwell, D., Alejos, E., Silva, M., Galanos, C., Freudenberg, M., Ricciardi-Castagnoli, P., Layton, B. & Beutler, B. (1998) Defective LPS signaling in C3H/HeJ and C57BL/ScCr mice: mutation in the Tlr4 gene. Science 282, 2085–2088. 29. Galanos, C., Lu ¨ deritz, O. & Westphal, O. (1979) Preparation and properties of a standardized lipopolysaccharide from Salmonella abortus equi. Zentbl. Bakteriol. Mikrobiol. Hyg. Abt. 1 Orig. A 243, 226–244. 30. Adachi, Y., Satokawa, C., Ohno, N., Tamura, H., Tanaka, S. & Yadomae, T. (1999) Inhibition by a CD14 monoclonal antibody of lipopolysaccharide binding to murine macrophages. J. Endo- toxin Res. 5, 139–146. 31. Green, L.C., Wagner, D.A., Glogowski, J., Skipper, P.L., Wish- nok, J.S. & Tannenbaum, S.R. (1982) Analysis of nitrate, nitrite, and [ 15 N]nitrate in biological fluids. Anal. Biochem. 126, 131–138. 32. Heinzel, F.P. (1990) The role of IFN-c in the pathology of experimental endotoxemia. J. Immunol. 145, 2920–2924. 33. Lorsbach, R.B. & Russell, S.W. (1992) A specific sequence of stimulation is required to induce synthesis of the antimicrobial molecule nitric oxide by mouse macrophages. Infect. Immun. 60, 2133–2135. 34. Bochem, U., Klamp, T., Groot, M. & Howard, J.C. (1997) Cellular responses to interferon-c. Annu. Rev. Immunol. 15, 749–795. 35. Okamura, H., Kashiwamura, S., Tsutsui, H., Yoshimoto, T. & Nakanishi, K. (1998) Regulation of interferon-c production by IL-12 and IL-18. Curr. Opin. Immunol. 10, 259–264. 36. Nakahira,M.,Ahn,H J.,Park,W R.,Gao,P.,Tomura,M., Park,C S.,Hamaoka,T.,Ohta,T.,Kurimoto,M.&Fujiwara, H. (2002) Synergy of IL-12 and IL-18 for IFN-c gene expression: IL-12-induced STAT4 contributes to IFN-c promoter activation by up-regulating the binding activity of IL-18-induced activator protein 1. J. Immunol. 168, 1146–1153. 37. Gu, Y., Kuida, K., Tsutsui, H., Ku, G., Hsiao, K., Fleming, M.A., Hayashi, N., Higashino, K., Okamura, H., Nakanishi, K., Kuri- moto, M., Tanimoto, T., Flavell, R.A., Sato, V., Harding, M.W., Livingston, D.J. & Su, M.S S. (1997) Activation of interferon-c inducing factor mediated by interleukin-1b converting enzyme. Science 275, 206–209. 4024 M. Matsuura et al. (Eur. J. Biochem. 270) Ó FEBS 2003 38. Ghayur, T., Banerjee, S., Hugunin, M., Butler, D., Herzog, L., Carter, A., Quintal, L., Sekut, L., Talanian, R., Paskind, M., Wong, W., Kamen, R., Tracey, D. & Allen, H. (1997) Caspase-1 processes IFN-c-inducing factor and regulates LPS-induced IFN-c production. Nature 386, 619–623. 39. Fehninger, T.A., Yu, H., Cooper, A., Suzuki, K., Shah, M.H. & Caligiuri, M.A. (2000) Cutting edge: IL-15 costimulates the gen- eralized Shwartzman reaction and innate immune IFN-c pro- duction in vivo. J. Immunol. 164, 1643–1647. 40. Robinson, D., Shibuya, K., Mui, A., Zonin, F., Murphy, E., Sanna,T.,Hartley,S.B.,Menon,S.,Kasteline,R.,Bazan,F.& O’Garra, A. (1997) IGIF does not drive Th1 development but synergizes with IL-12 for interferon-c production and activates IRAK and NFjB. Immunity 7, 571–581. 41. Ahn, H J., Maruo, S., Tomura, M., Mu, J., Hamaoka, T., Nakanishi, K., Clark, S., Kurimoto, M., Okamura, H. & Fuji- wara, H. (1997) A mechanism underlying synergy between IL-12 and IFN-c-inducing factor in enhanced production of IFN-c. J. Immunol. 159, 2125–2131. 42. Tomura,M.,Zhou,X.Y.,Maruo,S.,Ahn,H J.,Hamaoka,T., Okamura, H., Nakanishi, K., Tanimoto, T., Kurimoto, M. & Fujiwara, H. (1998) A critical role for IL-18 in the proliferation and activation of NK1.1 + CD3 – cells. J. Immunol. 160, 4738– 4746. Ó FEBS 2003 LPS-induced NO production through IFN-c (Eur. J. Biochem. 270) 4025 . A pathway through interferon-c is the main pathway for induction of nitric oxide upon stimulation with bacterial lipopolysaccharide in mouse peritoneal. by macrophages through a pathway activating NF-jBandits binding to region 1 without the help of cofactors such as cytokines. In the case of region 2 activation,