Nitric Oxide Prevents IL-1β and IFN-γ-Inducing Factor (IL-18) Release from Macrophages by Inhibiting Caspase-1 (IL-1β-Converting Enzyme)

Young-Myeong Kim, Robert V. Talanian, Jianrong Li and Timothy R. Billiar
J Immunol October 15, 1998, 161 (8) 4122-4128;

Abstract

Procytokine processing by caspase-1 is required for the maturation and release of IL-1β and IFN-γ-inducing factor (IGIF) (or IL-18) from activated macrophages (Mφ). Nitric oxide (NO) has emerged as a potent inhibitor of cysteine proteases. Here, we tested the hypothesis that NO regulates cytokine release by inhibiting IL-1β-converting enzyme (ICE) or caspase-1 activity. Activated RAW264.7 cells released four to five times more IL-1β, but not TNF-α, in the presence of the NO synthase inhibitor NG-monomethyl-l-arginine. Stimulated peritoneal Mφ from wild-type mice (inducible NO synthase (iNOS)+/+) also released more IL-1β if exposed to NG-monomethyl-l-arginine, whereas Mφ from iNOS knockout mice (iNOS−/−) did not. Inhibition of NO synthesis in stimulated RAW264.7 cells also resulted in a threefold increase in intracellular caspase-1 activity. The NO donor S-nitroso-N-acetyl-dl-penicillamine inhibited caspase-1 activity in cells as well as the activity of purified recombinant caspase-1 and also prevented the cleavage of pro-IL-1β and pro-IGIF by recombinant caspase-1. The inhibition of caspase-1 by NO was reversible by the addition of DTT, which is consistent with S-nitrosylation as the mechanism of caspase-1 inhibition. An in vivo role for the regulation of caspase-1 by NO was established in iNOS knockout animals, which exhibited significantly higher plasma levels of IL-1β and IFN-γ than their wild-type counterparts at 10 h following LPS injection. Taken together, these data indicate that NO suppresses IL-1β and IGIF processing by inhibiting caspase-1 activity, providing evidence for a unique role for induced NO in regulating IL-1β and IGIF release.

Caspases are a family of at least 10 human cysteine proteases that specifically cleave protein after aspartate residues. Various lines of evidence suggest that caspases 2, 3, 6, 7, 8, 9, and 10 participate in apoptosis (1, 2). Caspase-1, or IL-1β-converting enzyme (ICE)3 (3, 4), and perhaps caspases 4 and 5 (5, 6), participate in cytokine maturation. Precursors of both IL-1β (3, 4) and IFN-γ-inducing factor (IGIF) (or IL-18) (6, 7, 8) undergo proteolytic cleavage by caspase-1, permitting the activated cytokine to exit the cell. Peptide inhibitors of caspase-1 block IL-1β and IGIF release from activated macrophages (Mφ) in vitro (8). Caspase-1 knockout (KO) mice fail to exhibit elevations in IL-1β, IL-1α, and IFN-γ following an LPS challenge and are resistant to LPS-induced death (9, 10). Thus, activated caspase-1 is essential for cytokine processing both in vitro and in vivo.

Caspases exist in cells as zymogens that must themselves undergo proteolytic activation. Nitric oxide (NO) has recently emerged as a potent inhibitor of activated caspases (11, 12, 13, 14) and, in hepatocytes, as an inhibitor of caspase activation (12). NO or an NO reaction product reversibly inhibits caspase activity by S-nitrosylation at the active site cysteine (11, 12, 13). S-nitrosylation of caspase-3-like proteases accounts in part for the capacity of NO to inhibit apoptosis both in vitro (12, 13, 14, 15) and in vivo (12). These profound influences of NO on cell viability mediated by NO-caspase interaction suggest that NO could also down-regulate cytokine processing through the inhibition of caspase-1. We report here that NO suppresses Mφ caspase-1 activity and inhibits the release of IL-1β protein and IGIF activity in vitro and in vivo. The key role of the inducible NO synthase (iNOS) (or NOS-2) in the regulation of cytokine processing is demonstrated in iNOS-deficient animals, which exhibit exaggerated IL-1β and IFN-γ levels in response to LPS. These results indicate a novel regulatory mechanism for the down-regulation of IL-1β and IGIF release during inflammation.

Materials and Methods

DMEM, penicillin, streptomycin, l-glutamine, and HEPES were purchased from Life Technologies (Gaithersburg, MD). ELISA kits for IL-1β, IFN-γ, and TNF-α were obtained from R&D Systems (Minneapolis, MN). N-acetyl-Tyr-Val-Ala-Asp-p-nitroanilide (Ac-YVAD-pNA) and N-acetyl-Try-Val-Ala-Asp-aldehyde (Ac-YVAD-cho) were obtained from Alexis (San Diego, CA). Active recombinant human caspase-1 (rhcaspase-1) was generated with an N-terminal polyhistadine tag in Escherichia coli and purified by nickel-chelating Sepharose and size-exclusion chromatography as described previously (16). S-nitroso-N-acetyl-dl-penicillamine (SNAP) was synthesized as described previously (17). Mouse RBCs were prepared as described previously (18). A Th1-type T cell clone was obtained from R. A. Hoffman (University of Pittsburgh). All other chemicals were purchased from Sigma (St. Louis, MO) unless indicated otherwise.

Isolation of mouse peritoneal Mφ and cell culture

Murine peritoneal Mφ were isolated from 6-wk-old iNOS KO (iNOS−/−) and wild-type (wt) (iNOS+/+) mice as described previously (19). Primary peritoneal Mφ and the murine Mφ-like cell line RAW264.7 were cultured in DMEM containing 5% low-endotoxin calf serum supplemented with 15 mM HEPES (pH 7.4), 2 mM l-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin. Some cells were treated with LPS (2 μg/ml E. coli 0111:B4; Sigma) and IFN-γ (10 U/ml) in the presence or absence of 1.5 mM of NG-monomethyl-l-arginine (NMA). Cells in culture were used for various assays, including Northern blot, Western blot, or caspase-1 activity, while media was obtained for nitrite and cytokine analyses. Th1-type murine T cell lines (cloned and provided by R. A. Hoffman) were maintained in culture with irradiated murine spleen cells in Con A-conditioned media.

Measurement of cytokines, caspase-1 activity, and nitrite

The levels of TNF-α, IFN-γ, and IL-1β in cell culture medium and in serum were measured by ELISA according to the manufacturer’s protocols. In brief, culture media were removed from the Mφ cultures and centrifuged at 12,000 × g for 5 min; the supernatant was subjected to ELISA or nitrite analysis. For assay of caspase-1 activity, the culture media were removed, and the cells were washed with PBS and collected by plastic scrapper and subsequently pelleted by centrifugation at 400 × g for 10 min at 4°C. The cell pellets were washed with ice-cold PBS and resuspended in 100 mM of HEPES buffer (pH 7.4) containing protease inhibitors (5 μg/ml aprotinin, 5 μg/ml pepstatin A, 10 μg/ml leupeptin, and 0.5 mM PMSF). The cell suspension was lysed by three freeze/thaw cycles, and the cytosolic fraction was obtained by centrifugation at 12,000 × g for 20 min at 4°C. Protein concentration was determined with the BCA protein assay reagent (Pierce, Rockford, IL). Cytosol containing 200 μg of protein was combined with 400 μM of the synthetic substrate Ac-YVAD-pNA in 150 μl of 100 mM HEPES (pH 7.4) containing 20% glycerol and protease inhibitors, and the reaction was conducted for 1 h at 37°C. Cytosolic caspase-1 activity was assayed by measuring the increased absorbance at 405 nm (12). Nitrite accumulation in culture medium was measured by the Griess reaction (17). rhcaspase-1 (8 μg) was treated with various concentrations of SNAP on ice for 1 h. The enzyme was then separated from the NO donor through a Sephadex G-25 column preequilibriated with 100 mM of HEPES buffer (pH 7.4). rhcaspase-1 (200 ng) was used to measure the catalytic activity using the colorimetric substrate Ac-YVAD-pNA (11).

In vitro cleavage of pro-IL-1β and pro-IGIF

[35S]methionine-labeled murine pro-IL-1β and pro-IGIF were synthesized using a TNT-coupled transcription and translation system (Promega, Madison, WI) (12). Aliquots (4 μl) of in vitro-translated, 35S-labeled proteins were incubated with rhcaspase-1 (8 ng), which was pretreated with or without 400 μM of SNAP in 10 μl of the total reaction volume in the presence or absence of 20 mM of DTT at 37°C for 1 h. The cleavage reaction was stopped by mixing with an equal volume of 2× SDS sample buffer and heating the mixture for 2 min. Cleavage profiles of pro-IL-1β and pro-IGIF were examined by electrophoresis on 15% SDS-PAGE and protein visualized by fluorography.

Northern and Western blot analysis

Total RNA was isolated from RAW264.7 cells with RNAzol B (17). Aliquots (20 μg) of RNA underwent electrophoresis on a 1% agarose gel and were blotted to GeneScreen (Dupont New England Nuclear, Boston MA). Membranes were hybridized to cDNA probes for murine iNOS, TNF-α, or IL-1β. Relative levels of 18S rRNA were measured using an 18S-specific probe to assess RNA loading. For Western blot analysis, the cytosolic extracts from Mφ were obtained by three freeze/thaw cycles and by centrifugation at 12,000 × g for 20 min at 4°C. Cytosolic proteins (45 μg) were mixed with an equal volume of 2× SDS-reducing sample buffer and boiled for 2 min. Samples were separated on a 12% SDS-PAGE. Proteins were transferred onto a nitrocellulose membrane, and the membrane was blotted by 5% nonfat milk in PBS containing 0.01% Tween 20 (PBS-Tween) for 1 h at room temperature and hybridized with anti-IL-1β Ab (R&D Systems). After three washes with PBS-Tween, the blot was hybridized with goat anti-mouse IgG linked to horseradish peroxidase. The membrane was developed with chemiluminescence reagent (DuPont New England Nuclear) and exposed to Kodak X-Omat film to visualize the protein bands (12). The intensity of the protein bands was measured with a Bio-Rad densitometer (Hercules, CA).

Maturation of pro-IGIF in caspase-1-transfected cells

COS cells (3.5 × 105 cells per 35-mm dish) were transfected with expression plasmids for murine pro-IGIF (3 μg) and/or human ICE (3 μg) (20) using lipofectamine as described previously (7). Cells were cultured with or without SNAP, Ac-YVAD-cho, or SNAP and RBCs for 24 h. Culture media were collected and centrifuged at 12,000 × g for 20 min at 4°C. After serial dilution, the supernatants were incubated with nonadherent Th1-type murine T cell lines (1.4 × 105 cells/150 μl/well of 96-well plates) for 18 h. IFN-γ-producing activity was assessed by measuring IFN-γ production in T cells by ELISA.

In vivo production of IFN-γ and IL-1β

iNOS KO and wt mice were injected i.p. with 10 mg/kg LPS. Some mice received aminoguanidine (0.7 mmol/kg/injection) at 0 and 5 h following LPS injection. Blood was collected via heart puncture at 10 h following LPS injection, and serum was obtained by centrifugation at 12,000 × g for 10 min at 4°C. IFN-γ and IL-1β levels were measured by ELISA.

Statistical analysis

Experiments were typically repeated a minimum of three times; data in the figures depict the results from either a representative experiment or combined data from all experiments as indicated in the figure legends. Data are presented as mean ± SD except for the Northern and Western blots. Significance was determined by the Student t test using the StatView statistics program (Abacus Concepts, Berkeley CA). Statistical significance was established at a p value of <0.05.

Results

iNOS expression inhibits IL-1β release from Mφ

We hypothesized that induced NO would inhibit the release of IL-1β from activated Mφ by direct inhibition of the proteolytic activity of caspase-1. First, we characterized the time course for iNOS and IL-1β expression by LPS plus IFN-γ-stimulated RAW264.7 cells in the presence and absence of the NOS inhibitor NMA (Fig. 1⇓A). TNF-α release, which should not be suppressed by NO, was also measured. iNOS, IL-1β, and TNF-α mRNA, which were not detectable before stimulation, were easily detected by Northern blot analysis at 6 h poststimulation. iNOS and IL-1β mRNA levels remained elevated to the 24 h timepoint, while TNF-α mRNA levels had returned to baseline. Inclusion of NMA resulted in higher mRNA levels for all three induced genes at 12 h and beyond. Accumulation in the media of nitrite, a product of NO metabolism, increased from 6 to 24 h following stimulation (Fig. 1⇓B). This increase was suppressed by NMA. IL-1β levels in the media began to increase at 9 h, with a much greater elevation in the NMA-treated cultures (Fig. 1⇓C). In contrast, TNF-α levels were increased already by 3 h, with additional increases to 12 h (Fig. 1⇓B); the addition of NMA resulted in only a small increase in TNF-α levels. The addition of NMA in the absence of LPS plus IFN-γ stimulation did not stimulate IL-1β release (data not shown). Similar effects of NMA on NO production and IL-1β release, but not on TNF-α production, were observed in RAW264.7 cells treated with LPS alone (data not shown). Figure 2⇓, A and B shows a similar NO-dependent suppression of IL-1β accumulation using peritoneal Mφ from either iNOS KO mice or their wt counterparts. As expected, stimulation of Mφ for 18 h with LPS plus IFN-γ resulted in nitrite release from iNOS+/+ cells but not from iNOS−/− cells. iNOS−/− cells released more IL-1β than did iNOS+/+ cells upon stimulation; however, this release was not statistically significant (p = 0.065). More importantly, the addition of NMA increased IL-1β accumulation in the culture medium of iNOS+/+ cells but had no effect on IL-1β release from iNOS−/− Mφ. Western blot analysis for IL-1β was performed on lysates from the peritoneal Mφ. iNOS−/− cells had lower levels of pro-IL-1β than iNOS+/+ Mφ following stimulation along with detectable levels of processed IL-1β (Fig. 2⇓C). NMA reduced the intracellular level of pro-IL-1β in iNOS+/+ cells but not in iNOS−/− cells. Taken together, these data indicate that induced NO prevents the processing of pro-IL-1β and IL-1β release without suppressing mRNA levels.

FIGURE 1.
FIGURE 1.

Production of NO, IL-1β, and TNF-α in RAW264.7 cells stimulated with LPS plus IFN-γ. A, RAW cells (80% confluence) were treated with LPS (2 μg/ml) plus IFN-γ (10 U/ml) with or without NMA (1.5 mM), and the total RNA was isolated for Northern blot analysis at various timepoints. Relative abundance of each mRNA was visualized by autoradiography. B, Cells were cultured with LPS (2 μg/ml) plus IFN-γ (10 U/ml) in the presence or absence of NMA (1.5 mM) and assayed for NO2− accumulation in the culture media by the Griess method. IL-1β (C) and TNF-α (D) release were analyzed in the culture media of the RAW264.7 cells by ELISA. Data presented in B, C, and D are from a representative experiment and depict the mean ± SD from quadruplicate cultures.

FIGURE 2.
FIGURE 2.

Production of IL-1β and NO in the peritoneal Mφ from iNOS KO and wt mice. A, The peritoneal Mφ isolated from iNOS KO mice and wt mice were cultured with LPS (2 μg/ml) plus IFN-γ (10 U/ml) with or without NMA (1.5 mM) for 18 h. NO2− production was measured in the culture media by the Griess method. Data represent the mean ± SD from three separate experiments performed in triplicate. B, IL-1β production was measured in the culture media by ELISA. Data depict one of three similar experiments and represent the mean ± SD of triplicate measurements. C, Cytosolic fractions were prepared from cultured peritoneal Mφ exposed to LPS plus IFN-γ in the presence or absence of NMA. Proteins were separated on 15% SDS-PAGE, and IL-1β protein was detected by Western blot analysis.

An NO donor and a caspase inhibitor prevent IL-1β and IGIF release

If NO prevents cytokine release by inhibition of caspase-1, then exposure to NO should mimic the effect of a known caspase-1 inhibitor. As shown in Figure 3⇓A, the addition of the NO donor SNAP to RAW264.7 cells stimulated with LPS plus IFN-γ in the presence of NMA almost completely blocked IL-1β release. This dose of SNAP (200 μM) did not reduce cell viability during the 18 h time course of this study (data not shown). A similar inhibition was seen when the caspase-1 inhibitor Ac-YVAD-cho was used. NO is known to stimulate cyclic GMP (cGMP) synthesis in cells (21); however, the addition of the cell membrane-permeable analogue 8-bromo-cGMP did not reverse the enhanced release of IL-1β seen in the presence of NMA (Fig. 3⇓A).

FIGURE 3.
FIGURE 3.

SNAP inhibits the production of IL-1β and IGIF. A, RAW cells were treated with LPS (2 μg/ml) plus IFN-γ (10 U/ml) in the presence or absence of NMA (1.5 mM), SNAP (200 μM), 8-bromo-cGMP (200 μM), or Ac-YVAD-cho (250 μM) for 18 h. IL-1β release into the culture media was measured by ELISA. B, COS cells were transfected with expression plasmids for murine pro-IGIF and/or human ICE in 6-well plates by the lipofectamine method. Cells were cultured with or without SNAP, Ac-YVAD-cho, or SNAP plus RBCs for 24 h. Culture media were collected and centrifuged at 12,000 × g for 20 min at 4°C in a microcentrifuge. Cloned Th1 cells (1.4 × 105 cells/150 μl/96-well plate) were cultured with the 10-fold dilution of the supernatant for 18 h. IFN-γ production by the T cell clone was assayed in culture media by ELISA. All data are presented as the mean ± SD of three experiments performed in duplicate.

Caspase-1 is also required for the release of mature IGIF (7, 8). To determine whether NO also inhibited IGIF maturation and release, COS cells were transfected with expression plasmids for murine pro-IGIF, human caspase-1, or both. These cells were incubated with Ac-YVAD-cho or SNAP for 24 h. The presence of mature IGIF was assessed by placing culture media from the COS cells (1/10 dilution) on cloned Th1 cells and measuring IFN-γ release after 18 h. Cells transfected with both pro-IGIF and caspase-1 released active IGIF (Fig. 3⇑B). The release of IGIF was suppressed if the COS cells were incubated with Ac-YVAD-cho or SNAP. RBCs, which scavenge NO, prevented the SNAP-induced suppression. Thus, NO mimics the effect of a caspase-1 inhibitor.

iNOS expression inhibits caspase-1 activity in RAW264.7 cells

Caspase-1 activity was monitored in cell lysates from RAW264.7 cells exposed to LPS plus IFN-γ. Compared with unstimulated controls, caspase-1 activity increased slightly at 9 h poststimulation (Fig. 4⇓A). The addition of NMA, however, resulted in a marked increase in activity between 9 and 12 h that persisted to 24 h. Recent studies (11, 12, 13) have shown that NO inhibits caspase activity by S-nitrosylation. S-nitrosylated caspases can be reactivated in the presence of the reducing agent DTT (12). When lysates from LPS plus IFN-γ-treated RAW264.7 cells were incubated with DTT for 20 min, caspase-1 activity increased by ∼68% (Fig. 4⇓B). Lysates from RAW264.7 cells stimulated with LPS plus IFN-γ in the presence of NMA exhibited higher levels of caspase-1 activity, and this activity was not increased further by treatment with DTT. Therefore, NO suppresses caspase-1 activity in cells, at least in part, by a mechanism consistent with S-nitrosylation.

FIGURE 4.
FIGURE 4.

NO inhibits caspase-1 activity. A, RAW264.7 cells were cultured with LPS (2 μg/ml) plus IFN-γ (10 U/ml). Cells were harvested and washed twice with ice-cold PBS. Cells were lysed in 100 mM HEPES (pH 7.4) by three freeze/thaw cycles. Cell lysates were collected by centrifugation at 12,000 × g for 20 min at 4°C and used for caspase-1 activity assay. The caspase activity was assayed with Ac-YVAD-pNA in a colorimetric assay. The data are presented as the mean ± SD of one of three similar experiments performed in triplicate. B, RAW264.7 cells were cultured with LPS (2 μg/ml) plus IFN-γ (10 U/ml) in the presence or absence of NMA (1.5 mM) for 14 h. Cells were harvested and lysed by three freeze/thaw cycles. After centrifugation, supernatants were incubated with or without 20 mM of DTT for 20 min at room temperature; caspase-1 activity was measured with Ac-YVAD-pNA by colorimetric assay. ∗, p < 0.05 by the Student t test vs lysates without DTT. Data represent the mean ± SD of two experiments performed in duplicate.

NO inhibits cleavage of pro-IL-1β and pro-IGIF by recombinant caspase-1

To determine whether NO would directly inhibit caspase-1 activity, rhcaspase-1, prepared as described previously (16), was incubated with various concentrations of SNAP for 1 h; activity was measured by a colorimetric assay. Caspase-1 activity was inhibited by NO generated from SNAP in a concentration-dependent manner (Fig. 5⇓A). If the SNAP-treated caspase-1 was subsequently exposed to 20 mM of DTT, the inhibition was reversed. SNAP (400 μM) also prevented the proteolytic cleavage of purified pro-IL-1β (Fig. 5⇓B) and pro-IGIF (Fig. 5⇓C) by caspase-1. Cytokine maturation was also inhibited by Ac-YVAD-cho and the thiol-modifying agents HgCl2 and N-ethylmaleimide. DTT reversed the inhibition seen with SNAP and HgCl2, which is consistent with reversible modification of the reactive site thiol by NO and HgCl2.

FIGURE 5.
FIGURE 5.

NO inhibits purified caspase-1 activity. A, Purified rhcaspase-1 was incubated with various concentrations of SNAP for 1 h at room temperature. The enzyme activity was measured with Ac-YVAD-pNA, and the rate of NO production was estimated using the Griess method, which detects NO2−. Data are presented as the mean ± SD of a representative experiment of three total experiments performed in triplicate (without SNAP) or quadruplicate (with SNAP). B, Purified rhcaspase-1 was treated with SNAP (400 μM), HgCl2 (200 μM), or N-ethylmaleimide (200 μM) for 1 h. The chemicals were then removed using a Sephadex G-25 column preequilibriated with 100 mM HEPES (pH 7.4). Some samples were incubated with 20 mM of DTT for 20 min. The caspase-1-containing samples were incubated with 35S-labeled pro-IL-1β at 37°C for 1 h. For some samples, Ac-YVAD-cho (250 μM) was directly added to this reaction mixture. The reaction was stopped by the addition of 2× SDS sample buffer and boiling for 2 min. Samples were electrophoresed on 15% SDS-polyacrylamide gel. Proteins were transferred to nitrocellulose membrane, and radioactive-labeled protein was visualized by fluorography. C, rhcaspase-1 that had been treated as described in B was incubated with 35S-labeled pro-IGIF at 37°C for 1 h. The cleavage activity of caspase-1 was analyzed as described in B.

iNOS expression inhibits IL-1β and IFN-γ release in vivo

Experiments were conducted to determine whether iNOS expression regulated cytokine release in vivo. IL-1β and IFN-γ levels were measured at 10 h after an i.p. LPS injection in wt (iNOS+/+) and KO (iNOS−/−) mice. This timepoint was chosen because iNOS expression peaks in several tissues in rodents between 8 and 12 h in vivo following LPS injection (22). KO mice exhibited 6- to 10-fold higher levels of both cytokines (Figs. 6⇓, A and B) compared with wt mice at this timepoint. Administration of the iNOS inhibitor aminoguanidine with the LPS resulted in higher IL-1β and IFN-γ levels in wt mice but not in iNOS-deficient animals.

FIGURE 6.
FIGURE 6.

Production of IL-1β and IFN-γ in LPS-injected mice. Mice were injected i.p. with LPS (10 mg/kg) in the presence or absence of aminoguanidine. Blood was collected after 10 h, and plasma was obtained by centrifugation. Plasma levels of IL-1β and IFN-γ were measured by ELISA. Each group consisted of four animals. ∗, p < 0.05 by the Student t test vs LPS plus aminoguanidine. Data represent the mean ± SD for four to five mice per group.

Discussion

We (11, 12) and others (13, 14) have recently provided conclusive evidence that NO is a potent inhibitor of caspase activity in vitro (11, 12, 13) and in vivo (12, 15). These previous studies demonstrated that NO suppressed apoptotic signaling in part through the inactivation of caspase-3-like proteases by S-nitrosylation (12). Here, we establish that NO suppresses proteolytic maturation of pro-IL-1β and pro-IGIF through the inhibition of caspase-1 activity. The reversal of the NO-induced inhibition by DTT suggests that the inactivation of caspase-1 also takes place through S-nitrosylation. The importance of iNOS in the down-regulation of cytokine processing is confirmed in vitro and in vivo using iNOS null mice. Thus, induced NO can suppress the inflammatory response by down-regulating proinflammatory cytokine release. Either IL-1β, IFN-γ, or both can up-regulate iNOS expression in a wide range of cell types, including Mφ (23, 24). Therefore, an added consequence of the decrease in IL-1β and IFN-γ levels may be a decrease in the signals needed for further iNOS expression.

The essential role of caspase-1 in cytokine processing was demonstrated in caspase-1 KO mice (9). Otherwise overtly normal, these mice had much lower IL-1β, IL-1α, and IFN-γ levels in response to LPS than their wt counterparts. The reductions in IL-1β levels were most likely due directly to impaired processing of these cytokines, while the inhibition of IFN-γ release could be accounted for by the failure of these animals to release mature IGIF. Caspase-1, like the other members of the caspase family, is produced in cells as an inactive zymogen that must be proteolytically cleaved to form the active protease (25). Endogenous regulators of caspase-1 activation have not been identified, but it is presumed that other caspases, or even caspase-1 itself, may serve this function. The pox virus protein CrmA (26) and the baculovirus product p35 (20) both inhibit caspase-1 activity and consequently may serve to suppress cytokine processing as a mechanism to suppress immune responses. Our results identify NO as the first endogenous regulator of caspase-1 activity. By suppressing caspase-1 activity, NO may act to down-regulate inflammatory responses and prevent tissue damage. Under certain circumstances, induced NO may exert direct tissue damage (27) or even promote other inflammatory pathways (28). Both IL-1β (29) and IFN-γ (30) participate in the up-regulation of iNOS in several cell types. Therefore, down-regulation of these cytokines might represent a feedback mechanism to inhibit further iNOS expression. It is interesting to note that the expression of iNOS parallels the release of IL-1β and the increases in caspase-1 activity in our in vitro experiments. In fact, the large increases in caspase-1 activity in the presence of NOS inhibition (Fig. 4⇑) occur at the timepoint (∼9 h) of maximal NO production, raising the possibility that one function of induced NO is to regulate caspase activity. We have shown that NO inhibits total protein synthesis in RAW264.7 cells through the phosphorylation of eukaryotic initiation factor-2α (31), raising the possibility that induced NO could also inhibit caspase-1 production. Lower levels of pro-caspase-1 protein could account for the failure of DTT treatment to increase caspase-1 activity in iNOS-expressing cells to levels seen in cells stimulated with LPS plus IFN-γ in the presence of NMA (Fig. 4⇑B).

The failure of DTT treatment to recover caspase-1 activity completely in Mφ lysates raises the possibility that NO may also suppress caspase-1 activation. We found a similar response for caspase-3-like activity in hepatocytes stimulated to undergo apoptosis, in which NO not only suppressed caspase activity by S-nitrosylation but prevented increases in caspase-3-like activity and presumably activation by a cGMP-dependent mechanism (12). The addition of 8-bromo-cGMP did not suppress cytokine release in RAW264.7 cells, suggesting that a similar mechanism does not apply to caspase-1. Further studies are required to determine whether proteolytic activation of caspase-1 is influenced by NO or whether NO suppresses pro-caspase-1 protein levels as suggested above.

NOS inhibition did not influence TNF-α release, which is consistent with the notion that the effect of NO on cytokine processing is specific for IL-1β and IGIF. This outcome is not surprising, since TNF-α release does not require caspase-1 for maturation but instead requires TNF-α convertase, a metalloprotease that should not be susceptible to NO inhibition (32). Furthermore, maximal TNF-α release occurs before the peak in NO production. NOS inhibition did result in higher steady-state mRNA levels for TNF-α, IL-1β, and even iNOS itself at 12 h and beyond, suggesting that NO has influences on either transcription rates or mRNA degradation. The uniformity of this response suggests a nonspecific mechanism and, again, may even relate to suppression of total protein synthesis by NO. The specificity for the down-regulation of cytokine processing by NO seems to lie in the susceptibility of caspase-1 to S-nitrosylation by NO. Both NO and other thiol-modifying agents completely inhibited caspase-1-mediated cleavage of pro-IL-1β and pro-IGIF. The reversal of the NO-mediated inhibition by DTT is consistent with S-nitrosylation as a mechanism, as described both for other caspases (11, 12) and for other S-nitrosylated proteins (33, 34). It is unlikely that NO itself S-nitrosylates caspase-1; rather, it is more likely that a reaction product with NO+ activity carries out this chemistry. Candidates include N2O3, the reaction product of NO+O2 (35), or even peroxynitrite (formed from NO+O2−) (36) in the presence of transition metals (37).

These results provide yet another role for induced NO in the direct regulation of the inflammatory response. In addition to our observations that induced NO can inhibit cytokine release, others have shown that iNOS inhibits neutrophil accumulation in endotoxemia (38). These observations are contrasted by findings in which induced NO clearly participates in proinflammatory signaling. We have shown that iNOS expression participates heavily in the activation of the transcriptional factors NF-κB and STAT3 as well as in the expression of IL-6 and granulocyte CSF in resuscitated hemorrhagic shock (39). Others have shown that iNOS expression is required for the up-regulation of the cytokines during Leishmania infection (40). Although iNOS expression has typically been viewed as a direct effector of tissue damage, it is now clear that induced NO also modulates specific inflammatory signaling pathways. Understanding which factors dictate a pro- vs antiinflammatory function for iNOS represents an important area for continued investigation.

Acknowledgments

We thank Drs. C. Nathan and J. MacMicking (Cornell University) and Dr. J. S. Mudgett (Merck Research Laboratory) for providing the iNOS knockout mice. We also thank D. L. Williams and A. Green for technical assistance.

Footnotes

  • ↵1 This work was supported by National Institutes of Health Grants GM-44100, GM-37753, and GM-53789 and by Korean Scientific Foundation Grant 981-0714-100-2.

  • ↵2 Address correspondence and reprint requests to Dr. Timothy R. Billiar, A1010 Presbyterian University Hospital, 200 Lothrop Street, Pittsburgh, PA 15213.

  • ↵3 Abbreviations used in this paper: ICE, IL-1β-converting enzyme; IGIF, IFN-γ-inducing factor; Mφ, macrophage(s); NO, nitric oxide; NOS, NO synthase; iNOS, inducible NOS; KO, knockout; rhcaspase, recombinant human caspase; SNAP, S-nitroso-N-acetyl-dl-penicillamine; wt, wild-type; NMA, NG-monomethyl-l-arginine; cGMP, cyclic GMP.

  • Received February 17, 1998.
  • Accepted June 8, 1998.

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The Journal of Immunology
Vol. 161, Issue 8
15 Oct 1998
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