Interleukin-1 production by mouse macrophages is regulated in a ...

Interleukin-1 production by mouse macrophages is regulated in a feedback fashion by nitric oxide Florian Obermeier, Volker Gross, Ju¨rgen Scho¨lmerich, and Werner Falk Department of Internal Medicine I, University of Regensburg, Germany

Abstract: The pleiotropic cytokine interleukin-1 (IL-1) is an inducer of the inducible nitric oxide synthase (iNOS). It was surprising to find that treatment of normal mice with an iNOS inhibitor resulted in detectable IL-1␤ mRNA in colon and spleen, suggesting feedback regulation. When mouse peritoneal exudate cells (PEC) or RAW 264.7 cells were stimulated with lipopolysaccharide (LPS), concomitant inhibition of iNOS resulted in an increase of IL-1␤ and IL-1␣ protein secretion. Conversely, after addition of the NO-generating compound NOC-18, IL-1␤ and IL-1␣ concentrations in supernatants were dose-dependently reduced. Costimulation with interferon-␥ (IFN-␥) reversed the NOC-18-mediated suppression of IL-1␣ protein concentration into an almost fivefold increase in RAW 264.7 cells. This effect was specific for IL-1␣ and was also seen in PEC. The mRNA expression for IL-1␤ and IL-1␣ in RAW 264.7 cells correlated with the protein levels, suggesting transcriptional regulation by NO. Dysregulated IL-1/NO crossregulation may play a role in inflammatory diseases. J. Leukoc. Biol. 66: 829–836; 1999. Key Words: interferon-␥ · interleukin-1␣ · interleukin-1␤ · peritoneal exudate cells · RAW 264.7 · lipopolysaccharide

models of disease like dextran sulfate sodium (DSS)-induced chronic colitis [10], viral and autoimmune encephalitis [4], MPTP-induced parkinsonism in mice [11] and baboons [12], as well as in several diseases occurring in humans such as ulcerative colitis [13–15], arthritis [4, 16], glomerulonephritis [4, 16], diabetes [17, 18], stroke [19], pertussis [20], chronic hepatitis [21], and toxic shock [4, 8, 9]. Consequently, NO synthesis needs to be tightly regulated to favor self-defense and avoid self-destruction. The cytokines interferon-␥ (IFN-␥), tumor necrosis factor (TNF), and interleukin-1␣ and -␤ (IL-1␣, IL-1␤) have been shown to be endogenous inducers of iNOS [22–27], whereas LPS is a potent exogenous inducer of iNOS [28]. Recent data, however, show that NO not only acts as an end-stage toxic effector molecule but also seems to work as an autocrine or paracrine mediator regulating cytokine production. Deakin et al. [29] found NO to down-regulate IL-6 production and to up-regulate tumor necrosis factor (TNF) release in IFN-␥- and LPS-costimulated J774 cells, a murine macrophage cell line. IL-12 release was reduced after iNOS blocking in the macrophage cell line IC 21 stimulated with IFN-␥ [30].We report here that under certain conditions NO can down-regulate IL-1␤ and IL-1␣ production. Because these cytokines themselves are able to induce iNOS, a feedback mechanism apparently controls NO levels, which can be reversed by IFN-␥ for IL-1␣.

MATERIALS AND METHODS INTRODUCTION Nitric oxide (NO) that is produced by macrophages [1] is known as an end-stage effector molecule of the immune system. Until today three enzymes generating NO were found. Two calciumdependent, constitutive forms, the endothelial NO synthase (ecNOS) and the neuronal NO synthase (ncNOS), generate comparatively small amounts of NO [2]. The third form is the calcium-independent inducible NO synthase (iNOS), which can generate large amounts of NO, exceeding those produced by ec/ncNOS more than 100 times during immune response to inflammation [3]. NO has been identified as an important antimicrobial, antiviral, and antiparasitic effector [4, 5]. Even tumor cell death can be induced by NO [6, 7]. iNOS-deficient mice have an altered immune response to Leishmania major infection and are also resistant to lipopolysaccharide (LPS)-induced shock [8, 9]. NO, however, proved to be a double-edged sword. Produced in inadequate amounts, the toxicity of the radical NO may turn from friend to foe because it has been shown in several animal

Materials Murine IFN-␥ was a kind gift from Dr. Gu¨nther Adolf, Bender, Vienna. LPS from Salmonella typhimurium, the iNOS blocker aminoguanidine hemisulfate (AG), nitrate reductase from aspergillus spp., NADPH⫹, and diaminonaphtalene were obtained from Sigma-Aldrich Chemie (Deisenhofen, Germany). The NO donor (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2diolate] (NOC-18) continuously releasing NO with a half-life of 56 h at 22°C and pH 7.4, the iNOS-specific NO synthase blocker s-methylisothiourea.sulfate (ITU) and 8-Br-cGMP were purchased from Alexis Corp. (Gru¨nwald, Germany). MTS for proliferation assays was a kind gift from Terence C. Owen (Tampa, FL).

Treatment of mice For inhibition of iNOS synthesis female BALB/c mice were treated with 10 mg AG intraperitoneally (i.p.) in a volume of 100 µL phosphate-buffered saline

Correspondence: Dr. Werner Falk, Department of Internal Medicine I, University of Regensburg, D-93042 Regensburg, Germany. E-mail: werner. [email protected] Received December 3, 1998; revised June 9, 1999; accepted June 10, 1999.

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(PBS) per mouse per day for 5 days. Control animals received 100 µL of PBS i.p. Mice were killed on day 5 and colons and spleens were used for RNA extraction. To determine the onset of IL-1␤ expression mice were treated as described above for 5 days and each day, 30 min after treatment, one mouse was killed by cervical dislocation and RNA was extracted from colons and spleens. For control, RNA was extracted from colons and spleens of untreated healthy female BALB/c mice.

Peritoneal exudate cells (PEC) PEC were isolated from male NMRI mice (Charles River, Germany). Mice were treated with 500 µL PBS i.p. After 16 h mice were killed and peritoneal lavage was performed using ice-cold culture medium. PEC were washed three times and adjusted to a density of 106/mL. PEC (5 ⫻ 105/well) were allowed to adhere to bottoms of wells of a 24-well plate for 3 h before nonadherent cells were removed by repeated washing with culture medium.

Cell cultures RAW 264.7 cells, a murine macrophage cell line, were cultured in RPMI-1640 containing 10% heat-inactivated fetal calf serum (FCS), 100 units/mL penicillin, and 100 µg/mL streptomycin from GIBCO-BRL (Eggenstein, Germany) in 75-cm2 flasks from Costar (Cambridge, UK). Cells were used for experiments on days 3–4 after splitting (1:10) of the cultures. All cell cultures were done at 37°C in humidified air with 5% CO2.

Stimulation experiments RAW 264.7 cells were washed and divided into aliquots of 5 ⫻ 106 cells. Cell pellets were suspended in 5 mL of pre-prepared medium containing stimulation agents alone (LPS or LPS ⫹ IFN-␥) or with iNOS-blocking agents or the NO-donor NOC-18 in different concentrations. Culture medium without LPS and IFN-␥ was used for controls. Cells were added to a 24-well plate with each well containing 106 cells in a volume of 1 mL and incubated for 4–20 h as indicated in Figures 1–6. Four to five wells per condition were used. After incubation, supernatants were removed for cytokine and nitrite/nitrate determination. PEC were treated identically. To determine the time course of NO production cells were incubated at the same density as described above but in a volume of 5 mL in six-well plates, and 100 µL was removed at the given times for nitrite/nitrate determination. Using the MTS assay, toxic effects of iNOS inhibitors, NOC-18 or 8-Br-cGMP in concentrations used for experiments on cellular mitochondrial activity (converting tetrazolium salt to formazan), were excluded [31]. There was a minor increase in MTS conversion in LPS-treated compared to untreated cells but the addition of IFN-␥ and iNOS modifiers alone or in combination had no effect (data not shown). Conversion of a tetrazolium salt to formazan has been previously used successfully to determine toxic effects of NO on macrophages [32].

IL-1␣ and IL-1␤ protein determination Supernatants were used for IL-1␤ and IL-1␣ determinations using enzymelinked immunosorbent assay (ELISA) kits from Amersham (Braunschweig, Germany; IL-1␤) and from Innogenetics (Zwijndrecht, Belgium; IL-1␣).

Nitrite/nitrate determinations Fourteen milliunits of nitrate reductase plus 10 µL of NADPH/H⫹ (40 µM) were added to 100 µL of the supernatants. The reduction of nitrate was stopped after 5 min with the addition of 50 µL of water and 10 µL diaminonaphtalene (0.05 mg/mL). After another 10 min of incubation in the dark at 20°C the reaction was stopped with 5 µL of 2.8 M NaOH [33]. Finally, the volume was adjusted to 400 µL with water and fluorescence was determined by a fluorescencespectrophotometer (F-2000, Hitachi, excitation 365 nm, emission 405 nm). Nitrite concentrations were calculated from a standard curve using NO2⫺ concentrations in a range from 1 to 200 µM.

Northern blot analysis RAW 264.7 cells (5 ⫻ 106) were incubated for 4 or 12 h as described above and lysed in the wells with 750 µL of 4 M guanidinium thiocyanate (GTC) solution

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according to the method of Chomczynski and Sacchi [34]. The amount of extracted RNA was determined by measuring the absorbance at 260 nm. RNA extraction from spleen or colon tissue was performed by homogenizing the tissue in 10 mL of ice-cold GTC solution in a blender. Further isolation was done as described above. Ten micrograms of total RNA from cells or 30 µg of RNA from spleen or colon was subjected to agarose gel electrophoresis in the presence of formaldehyde. RNA was blotted on a positively charged nylon membrane from Appligene (Illkirch, France) and exposed to UV light (Stratalinker, Stratagene Europe, Amsterdam). RNA was stained with methylene blue. Blots were probed with IL-1␤, IL-1␣, and ␤-actin cDNAs 32P-labeled by random priming with the Prime-It kit from Stratagene (Heidelberg, Germany) according to the manufacturer’s instructions. Hybridization was done as described [35]. Blots were exposed to X-ray films (Hyperfilm, Amersham) for 1 day (RAW 264.7 cells, IL-1␣ and IL-1␤ mRNA detection) or 3 h for ␤-actin detection and 3 days (mouse spleen and colon) at ⫺70°C with intensifying screens. Signal intensity was quantified by Image Quant software installed on a Personal densitometer from Molecular Dynamics and calculated in percent. Signal intensity of the LPS or LPS ⫹ IFN-␥-stimulated cells was set to 100%.

Polymerase chain reaction cDNAs were synthesized from 1 µg total RNA using 2.5 µM poly(dT), 12.5 U AMV reverse transcriptase, 0.5 mM of each dNTP, 5 mM MgCl2, and 32 U RNase inhibitor in a final volume of 20 µL (all reagents from Pharmacia, Freiburg, Germany). PCR was performed for 35 cycles (0.5 min at 94°C, 1 min at 56°C, 1 min at 72°C) in a volume of 40 µL. The reaction mixture contained 10 mM Tris, pH 8.3, 50 mM KCl, 2 mM MgCl2, 400 µM of each dNTP, 1 U Taq DNA polymerase (all from Pharmacia). Primer pairs for iNOS (kindly provided by Dr. Christian Bogdan, Erlangen, Germany) [36] and ␤-actin (Biometra, Go¨ttingen, Germany) were added to a final concentration of 250 nM. Plasmid L1 containing iNOS cDNA (generously provided by Dr. Christian Bogdan) and RNA from LPS (1 µg/mL)-stimulated RAW 264.7 cells served as positive controls, and RNA from unstimulated RAW 264.7 cells as negative control. PCR amplicons (iNOS, 472 bp; ␤-actin, 574 bp) were analyzed on a 1.5% agarose gel.

RESULTS We have shown in a model of acute DSS-induced colitis in BALB/c mice that therapies aimed at neutralization of IL-1 and TNF activities led to exacerbation of the disease [37]. Because IL-1 and TNF are known as potent inducers of iNOS, we tried to investigate the role of NO in this animal model. Mice were treated over 5 days with AG [10]. We were surprised to find that not only animals with acute colitis expressed IL-1␤ mRNA in colon and spleen (data not shown) but also control mice treated with AG only (Fig. 1, A and B), whereas PBS-treated control mice expressed no detectable IL-1␤ mRNA (Fig. 1, A and B) under the experimental conditions. To determine the onset of IL-1␤ mRNA expression, mice were treated daily with 10 mg AG and expression in spleen and colon was monitored. Figure 1, C and D shows that from day 2 on IL-1␤ mRNA was expressed in spleen and colon, respectively. Because Figure 1E shows that iNOS mRNA was detectable in colon and spleen of untreated mice one could argue that IL-1␤ transcription was initiated by iNOS inhibition. Because IL-1␤ is known as an inducer of iNOS we hypothesized that constitutive expression of IL-1␤ might be dependent on NO, suggesting a negative feedback mechanism. To verify this assumption, PEC were stimulated with 1 µg/mL LPS in the absence or presence of the specific iNOS inhibitor ITU, the NO-releasing compound NOC-18, or 8-Br-cGMP, a cGMP analog. cGMP is considered to be involved in mediating physiological effects of NO such as http://www.jleukbio.org

A

B

C

D

E

Fig. 1. Induction of IL-1␤ mRNA expression by AG in spleens and colons of mice. (A, B) BALB/c mice were treated for 5 days with 10 mg/day AG or 100 µL PBS. Mice were killed on day 5 and total RNA was extracted from spleens (A) and colons (B). IL-1␤ mRNA was detected by Northern blotting (top panel, A and B) and 28S RNA was stained with methylene-blue (bottom panel, A and B). (C, D) Each day one mouse was killed and total RNA was extracted from spleens (C) and colons (D). IL-1␤ mRNA was detected by Northern blotting (top panel, C and D) and 28S RNA was stained with methylene blue (bottom panel, C and D). The results are representative of two separate experiments. (E) RT-PCR was performed on RNA isolated from colons and spleens of untreated mice to detect iNOS mRNA (top panel). iNOS cDNA (lane 1) and RNA from LPS-stimulated RAW 264.7 cells (lane 2) were used as positive controls and RNA from unstimulated RAW 264.7 cells as negative control (lane 3). ␤-Actin was amplified as control confirming adequate RNA extraction and cDNA synthesis (bottom panel).

smooth muscle relaxation. After 6 h of incubation increased NO levels caused by the addition of NOC-18 led to a significant dose-dependent reduction of IL-1␤ (to 56%) and IL-1␣ (to 63%) protein concentrations compared with those from LPStreated cells (Fig. 2A). 8-Br-cGMP and iNOS inhibition by ITU had no effect (Fig. 2A). However, after 20 h of incubation (Fig. 2B) IL-1␤ secretion was significantly increased by ITU (1.9-

fold), and still decreased by NOC-18 in a dose-dependent fashion. IL-1␣ secretion was also increased by ITU and decreased by NOC-18, however, without reaching statistical significance (Fig. 2B). The cGMP agonist 8-Br-cGMP again showed no significant change in IL-1␤ and IL-1␣ secretion at two different concentrations (Fig. 2B). To verify that the lack of effect of 8-Br-cGMP was not due to insufficient concentrations,

Obermeier et al. IL-1 and NO cross-regulation in macrophages

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Fig. 2. Effect of NO concentration on IL-1␤ and IL-1␣ release by stimulated PEC. PEC from PBS-treated mice were stimulated with LPS (1 µg/mL) with and without NOC-18 [50 µg/mL (306 µM) or 10 µg/mL (61.2 µM)] or ITU (10 µg/mL) or 8-Br-cGMP (10 µM or 1 µM; B). Supernatants were removed after 6 (A) or 20 h (B) of incubation and IL-1␤ protein (A, B left panel) and IL-1␣ protein (A, B right panel) was measured by ELISA. The results are the means ⫾ SEM of four replicates per condition. The result is representative of three separate experiments. *Significantly different from LPS-treated cells and P values were derived from Student’s t test.

it was used at up to 1 mM without having any effect on IL-1 production (data not shown). In contrast to IL-1␣ and IL-1␤, TNF production was significantly and dose-dependently reduced by 8-Br-cGMP in both RAW 264.7 and PEC in the concentrations used for experiments shown in Figure 2 (data not shown). The mouse macrophage-like cell line RAW 264.7 was used for further investigations. In experiments not shown the most effective concentrations for the NO-modulating agents were determined. The addition of the agents in the concentrations used for the experiments did not lead to any IL-1␤ production

in unstimulated cells (data not shown). The NO-donating substance NOC-18 reduced IL-1␤ production in a dosedependent manner, with a maximal effect (81% reduction vs. LPS-stimulated cells) at a concentration of 50 µg/mL (306 µM) (Fig. 3A). The iNOS-blocking substances (AG, ITU) significantly increased IL-1␤ levels 2.5-fold (AG) and 2.8-fold (ITU; Fig. 3A). Qualitatively, the same results were found for IL-1␣ (Fig. 3B). The time course of NO production and the influence of NOC-18 or the iNOS inhibitors (AG, ITU) or 8-Br-cGMP in LPS or LPS ⫹ IFN-␥-stimulated RAW 264.7 cells was investigated.

Fig. 3. Effect of NO concentration on IL-1␤ and IL-1␣ release from LPSstimulated RAW 264.7. RAW 264.7 cells were stimulated with LPS (1 µg/mL) with or without NOC-18 [50 µg/mL (306 µM), 10 µg/mL (61.2 µM), and 2 µg/mL (12.24 µM)] or AG (100 µg/mL) or ITU (10 µg/mL). Supernatants were removed after 20 h of incubation and IL-1␤ (A) and IL-1␣ (B) were determined by ELISA. Results are means ⫾ SEM of five replicates per condition and are representative of five separate experiments. *P ⬍ 0.05 and **P ⬍ 0.0001 significantly different from LPS-treated cells. P values were derived from Student’s t test.

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Figure 4 shows that in LPS or LPS ⫹ IFN-␥-stimulated RAW 264.7 cells nitrite/nitrate levels started to increase after 8 h of incubation. iNOS inhibition by AG or ITU reduced NO production to the concentrations in unstimulated cultures. Comparable results were obtained using PEC, however, NO production started about 2 h later compared to RAW 264.7 cells (data not shown). Because NOC-18 decomposed during measurement, no time-dependence of NO release from NOC-18 was measurable. The presence of IFN-␥ did not lead to further iNOS activity, which can be explained by a finding from Xie et al. [38], who found synergism of LPS and IFN-␥ in iNOS induction using LPS concentrations of 1 up to 100 ng/mL. However, at 1 µg/mL of LPS, costimulation with 50 units/mL IFN-␥ did not result in a significant increase in iNOS promoter activity. To investigate whether the inhibiting effect of NO on IL-1␣ and IL-1␤ production was also demonstrable on the transcriptional level, RNA was extracted from RAW 264.7 cells and

A

Fig. 5. Effect of NO concentration on IL-1␤ and IL-1␣ mRNA in LPSstimulated RAW 264.7. RAW 264.7 cells were stimulated with LPS (1 µg/mL) with or without NOC-18 [50 µg/mL (306 µM), 10 µg/mL (61.2 µM) (A, B), and 2 µg/mL (12.24 µM) (A)] or AG 100 µg/mL (A, B), 20 µg/mL, and 4 µg/mL (B). Cells were lysed in the wells after 4 (A) or 12 h (B) and total RNA was extracted and analyzed by Northern blotting. Blots were stained with methylene blue (28S RNA, bottom panel; A) and probed with IL-1␤, IL-1␣ (A, B), or ␤-actin (B). Signal intensity was measured using Image Quant software and calculated as percent of the signal intensity of LPS-stimulated cells (A). For quantification in B signal intensities were normalized to ␤-actin and then calculated as percent of LPS-stimulated cells. The results are representative of three separate experiments.

Fig. 4. Time course of NO production in LPS or LPS ⫹ IFN-␥-stimulated RAW 264.7 cells. RAW 264.7 cells were stimulated with LPS (1 µg/mL; top panel) or LPS ⫹ IFN-␥ (5 U/mL; bottom panel) with or without NOC-18 [50 µg/mL (306 µM), 10 µg/mL (61.2 µM), 2 µg/mL (12.24 µM)], ITU (10 µg/mL), or AG (100 µg/mL). Unstimulated cells served as control. Between 4 and 20 h of incubation supernatant was removed at 4-h intervals for nitrite/nitrate determination as described. Nitrite/nitrate values in supernatants of NOC-18-treated cells were determined after 20 h of incubation and are displayed as bars. The results are the means ⫾ SEM of three replicates per condition. The results are representative of two separate experiments.

analyzed by Northern blotting. Figure 5A shows that expression of IL-1␤ and IL-1␣ mRNA at 4 h of incubation was decreased dose-dependently by the activity of NOC-18. iNOS inhibition by AG at this time point had no effect, a result in line with the detection of secreted protein from PEC after 6 h (Fig. 2A). After 12 h of stimulation, however, AG resulted in a dose-dependent increase in IL-1␣ and -␤ mRNA and NOC-18 was still able to suppress IL-1␣ and -␤ mRNA levels (Fig. 5B). When PEC were stimulated with the combination of LPS (1 µg/mL) and IFN-␥ (10 U/mL), IL-1␤ production was again inhibited by NOC-18 and increased by ITU (Fig. 6A). In contrast, IL-1␣ production into the supernatant increased 1.7-fold in the presence of NOC-18 and decreased in the presence of ITU (Fig. 6A). The same IFN-␥-induced reversion of the effect of NO was observed in RAW 264.7 cells. Although there was no qualitative change in the effect of NO concentration on IL-1␤ secretion, IL-1␣ secretion in the presence of IFN-␥ was increased by NO (Fig. 6B). This IL-1␣-specific reversion was also evident for IL-1␣ mRNA production by


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Fig. 6. Effect of IFN-␥ on NO-regulated IL-1␣ and IL-1␤ protein production by PEC and RAW 264.7 and mRNA production by RAW 264.7. (A) PEC were stimulated with LPS (1 µg/mL) and IFN-␥ (10 U/mL) with and without NOC-18 [50 µg/mL (306 µM) or 10 µg/mL (61.2 µM)] or ITU (10 µg/mL) or 8-Br-cGMP (10 µM). Supernatants were removed after 20 h of incubation and IL-1␣ (right panel) and IL-1␤ (left panel) protein was measured by ELISA. These data together with results presented in Figure 2B were taken from the same experiment. (B) RAW 264.7 cells were stimulated with LPS (1 µg/mL) and IFN-␥ (5 U/mL) with and without NOC-18 (50 µg/mL) or AG (100 µg/mL). Supernatants were treated as described in panel A. Results are means ⫾ SEM of four to five replicates per condition and are representative of three separate experiments. Results for IL-1␣ concentrations (pg/mL ⫾ SEM) of LPS-stimulated cells not shown are as follows: LPS, 93.8 ⫾ 7.1; LPS⫹AG (100 µg/mL), 145.3 ⫾ 18.5; LPS ⫹ NOC-18 (50 µg/mL), 34.5 ⫾ 3.9. *Significantly different from LPS ⫹ IFN␥-treated cells, P values are derived from Student’s t test. (C) RNA was extracted from LPS (1 µg/mL) and IFN-␥ (5 U/mL)stimulated RAW 264.7 cells after 4 h of stimulation and analyzed by Northern blot. Northern blots were stained with methylene blue (28S RNA, bottom panel) and probed with IL-1␣ cDNA (top panel). Signal intensity was measured as explained in the legend to Figure 5. Signal intensity of LPS ⫹ IFN-␥-stimulated cells was set at 100%. The results are representative of two separate experiments.

RAW 264.7 cells (Fig. 6C), again suggesting an effect of NO on gene transcription.

DISCUSSION Blocking iNOS in normal untreated mice by injection of AG led to activation of IL-1␤ transcription in the spleen and colon in vivo. Blocking NO generation in LPS-activated adherent mouse PEC led to an increase of IL-1␤ and IL-1␣ protein levels. Conversely, addition of a compound that released NO, i.e. stimulation in the presence of NO, resulted in a reduced IL-1␤ and IL-1␣ release. The findings obtained in vivo and with PEC were confirmed using the macrophage-like cell line RAW 264.7. IL-1␤ and IL-1␣ protein in supernatants was increased by the iNOS inhibitors AG and ITU and strongly decreased by NOC-18. 834


These observations in vivo, ex vivo, and in vitro are compatible with the view that IL-1 and NO production by macrophages is counter-regulated in autocrine or paracrine fashion. The fact that blocking NO generation by iNOS in normal mice led to increased IL-1␤ transcription also suggests a steady state at a low level regulating NO/IL-1␤ concentrations. This hypothesis is in line with our finding that iNOS mRNA was detected by RT-PCR in colons and spleens of untreated mice and is also supported by others demonstrating that chronic abrogation of NO production in guinea-pigs caused ileitis and leukocytosis [39]. In contrast to iNOS inhibition over 20 h of stimulation, iNOS inhibitors did not result in an increase of IL-1␣ and IL-1␤ protein secretion at 6 h in PEC and IL-1␣ and -␤ mRNA levels in RAW 264.7 cells at 4 h of stimulation. This is explained by the late onset of NO release at about 8 h in RAW 264.7 and 10 h http://www.jleukbio.org

in PEC after stimulation. Therefore at 4 and 6 h after incubation there was no iNOS activity to be suppressed. Recently multiple immunoregulatory roles of NO became evident. NO was shown to favor a T-helper 2 type immune response [40] by suppressing IFN-␥ and IL-2 [27, 41] and increasing IL-4 secretion of lymphocytes [42], leading to elevated IgE serum levels [43]. Conversely, iNOS-deficient mice showed a bias toward a Th1 immune response after infection with Staphylococcus aureus [44]. Information concerning a possible regulatory role of NO on cytokine production by macrophages is limited and partly controversial. Inhibition of NO led to decreased IL-12 levels in the macrophage cell line IC 21 after activation by IFN-␥ [30] and to decreased TNF levels in thioglycollate-induced PEC after activation by LPS ⫹ IFN-␥ [45]. In contrast, in LPSstimulated RAW 264.7 cells, TNF production was increased after inhibition of iNOS [46]. IL-6 production was shown to be suppressed by NO in J774 cells [29] and alveolar macrophages [47]. The effect of NO on IL-1 production is controversial. NO increased IL-1 bioactivity produced by resident macrophages from CD1 mice after activation with IFN-␥ and LPS [48]. In contrast, Persoons et al. [47] described an inhibitory role of NO on IL-1␤ production in LPS-stimulated alveolar macrophages. Our finding of regulation by NO on the transcriptional level could be explained by NO-induced inhibition of NF-␬B [49, 50] as both IL-1␣ and IL-1␤ transcription, is controlled by NF-␬B [51, 52]. Furthermore, our results show that cGMP, which is involved in mediating several physiological effects of NO, such as smooth muscle relaxation [27] or platelet function [53], does not seem to play a role in NO-regulated IL-1 production. It is interesting to note that we found IFN-␥ reversed the suppression of IL-1␣ release and mRNA expression by NO. In the presence of IFN-␥ and LPS NO significantly enhanced IL-1␣ release in both PEC and RAW 264.7 and increased mRNA expression. NO regulation of IL-1␤ release was not reversed by IFN-␥ in both cell types. Because in studies from Hill et al. [48] IL-1␣ contributed to 90% of IL-1 bioactivity, the observed NO-induced increase after stimulation of mouse macrophages with LPS and IFN-␥ is in line with our findings, as are the results from Persoons et al. [47]. The IFN-␥-induced reversion of the NO effect on IL-1␣ protein production might also help to explain why in LPS-stimulated PEC, NO suppression of IL-1␣ production after 20 h did not reach significance (in contrast to RAW 264.7 cells) because some contaminating lymphocytes might secret small amounts of IFN-␥ sufficient to counteract the effects of NO. Considering IL-1␣ as TH2cytokine [54], a compensatory up-regulation of IL-1␣ expression after costimulation with the TH1-cytokine IFN-␥ is consistent with data from others describing NO as a strong TH2-favoring mediator [27, 40–44]. In contrast, IL-1␤, which is down-regulated in LPS and IFN-␥-stimulated cells seems to be more related to a TH1 type of immune response [55]. Because the effects of NO were also observed on the transcriptional level, the different posttranslational processing requirements of the two cytokines do not suffice to explain this differential effect of IFN-␥. However, fundamental differences between the gene promoter regions of IL-1␣ and IL-1␤ have been found, suggesting the possibility of differential regulation

[52]. For example there are no TATA box motifs in the IL-1␣ gene promoter region in contrast to the IL-1␤ promoter region [52]. In fact, different regulation of IL-1␣ and IL-1␤ gene transcription was previously described [56]. However, to our knowledge, no IL-1␣-specific IFN-␥-dependent promoter region or a specific effect of IFN-␥ on IL-1␣ mRNA stability has been described that might overcome the suppressive effect of NO on IL-1␣ production in LPS-stimulated macrophages. The observation of differential transcriptional regulation of IL-1␣ and IL-1␤ production by NO in LPS- and IFN-␥costimulated cells is under further investigation. We could demonstrate that NO is not only able to suppress IL-1␣ and IL-1␤ production of LPS-stimulated macrophages, but also has the potential to up-regulate IL-1␣ production in the presence of IFN-␥. This indicates that the immunoregulatory role of NO can vary fundamentally depending on the surrounding microenvironment. Further understanding of the NO-cytokine cross-regulatory network may give some insight into the control of diseases such as inflammatory bowel disease or toxic shock.

ACKNOWLEDGMENTS We thank Krishna Mondal and Hans Herfarth for critical reading of the manuscript.

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