Human Monocyte Interleukin-1β Posttranslational Processing

Interleukin (IL)-1β produced by monocytes and macrophages is not released via the normal secretory apparatus, and prior to its release, this cytokine must be proteolytically processed to generate a mature biologically active species. Biochemical mechanisms that regulate these posttranslational steps are not well understood. Lipopolysaccharide (LPS) is a poor activator of IL-1 posttranslational processing despite serving as a potent inducer of IL-1 synthesis. For example, freshly isolated human monocytes treated with LPS released <30% of their newly synthesized IL-1β as the mature 17-kDa cytokine species, and monocytes that were aged overnight in culture prior to LPS treatment released no 17-kDa cytokine. In contrast, addition of extracellular ATP promoted IL-1β posttranslational processing from both monocyte populations. Previous studies indicated that ATP, acting via surface P2Z-type receptors, promoted major intracellular ionic changes. To explore whether these ionic changes were required for cytokine posttranslational processing, LPS-stimulated human monocytes were maintained in ionically altered media. Hypotonic conditions promoted an efficient and selective release of mature 17-kDa IL-1β from LPS-activated monocytes in the absence of ATP. In contrast, hypertonic conditions blocked the ATP-induced posttranslational processing reactions. Both hypotonic stress- and ATP-induced processing were blocked when NaI was substituted for NaCl within the medium; substitution with NaSCN or NaNO3 also blocked the ATP response, but these salts were less inhibitory against the hypotonic stimulus. Sodium glucuronate substitution did not inhibit cytokine processing induced by either stimulus. Removal of divalent cations from the medium did not affect the ATP response, but pretreatment of monocytes with the phosphatase inhibitor okadaic acid dose-dependently suppressed ATP-induced IL-1β posttranslational processing. A volume-induced change to the intracellular ionic environment, therefore, may represent a key element of the mechanism by which IL-1β posttranslational processing is initiated. The strong dependence of this cytokine release mechanism on chloride anions suggests that selective anion transporters function as important components of this response.

Interleukin (IL)-1␤ produced by monocytes and macrophages is not released via the normal secretory apparatus, and prior to its release, this cytokine must be proteolytically processed to generate a mature biologically active species. Biochemical mechanisms that regulate these posttranslational steps are not well understood. Lipopolysaccharide (LPS) is a poor activator of IL-1 posttranslational processing despite serving as a potent inducer of IL-1 synthesis. For example, freshly isolated human monocytes treated with LPS released <30% of their newly synthesized IL-1␤ as the mature 17-kDa cytokine species, and monocytes that were aged overnight in culture prior to LPS treatment released no 17-kDa cytokine. In contrast, addition of extracellular ATP promoted IL-1␤ posttranslational processing from both monocyte populations. Previous studies indicated that ATP, acting via surface P 2Z -type receptors, promoted major intracellular ionic changes. To explore whether these ionic changes were required for cytokine posttranslational processing, LPS-stimulated human monocytes were maintained in ionically altered media. Hypotonic conditions promoted an efficient and selective release of mature 17-kDa IL-1␤ from LPS-activated monocytes in the absence of ATP. In contrast, hypertonic conditions blocked the ATP-induced posttranslational processing reactions. Both hypotonic stress-and ATP-induced processing were blocked when NaI was substituted for NaCl within the medium; substitution with NaSCN or NaNO 3 also blocked the ATP response, but these salts were less inhibitory against the hypotonic stimulus. Sodium glucuronate substitution did not inhibit cytokine processing induced by either stimulus. Removal of divalent cations from the medium did not affect the ATP response, but pretreatment of monocytes with the phosphatase inhibitor okadaic acid dose-dependently suppressed ATP-induced IL-1␤ posttranslational processing. A volume-induced change to the intracellular ionic environment, therefore, may represent a key element of the mechanism by which IL-1␤ posttranslational processing is initiated. The strong dependence of this cytokine release mechanism on chloride anions suggests that selective anion transporters function as important components of this response.
Interleukin (IL) 1 -1␤ is produced by many cells in response to inflammatory stimuli such as lipopolysaccharide (LPS) and tumor necrosis factor (1,2). When administered to target cells, IL-1␤ binds to cell-surface receptors and promotes various proinflammatory activities including enhanced production of matrix metalloproteinases, increased expression of adhesion molecules, and enhanced prostaglandin synthesis (3)(4)(5)(6). In view of its many pro-inflammatory activities, in vivo production of IL-1␤ is likely to be controlled at many levels. Indeed, recent in vitro studies indicate that IL-1␤ production is regulated via both transcriptional and posttranslational mechanisms (7)(8)(9)(10)(11)(12)(13)(14)(15)(16). Mouse peritoneal macrophages, for example, synthesize large quantities of IL-1␤ mRNA and protein in response to LPS stimulation. This translated product, however, corresponds to the inactive 35-kDa pro-IL-1␤ species which is neither proteolytically processed nor released into the medium (13,14). Externalization and proteolytic maturation of the pro-IL-1␤ polypeptide requires an additional stimulus. To date, identified agents that can serve as this secondary stimulus in vitro include ATP, cytolytic T-cells, and potassium ionophores such as nigericin (13,14,16). Likewise, production of mature biologically active IL-1␤ by LPS-stimulated freshly isolated human monocytes can be dissociated into two distinct steps. Low concentrations of LPS activate monocytes to produce pro-IL-1␤, but this newly synthesized cytokine is not proteolytically processed or released (15). On the other hand, high concentrations of LPS (Ͼ20 ng/ml) promote synthesis of comparable levels of pro-IL-1␤ and, in addition, stimulate its release and maturation (15).
The efficiency of IL-1 release from human monocytes treated only with LPS is variable and correlates with release of the cytoplasmic enzyme lactate dehydrogenase (LDH) (17). Moreover, agents like ATP and nigericin that promote efficient externalization of mature 17-kDa IL-1␤ from LPS-treated murine peritoneal macrophages cause cell death, possibly via an apoptotic mechanism (13). Interestingly, the enzyme responsible for the proteolytic cleavage of pro-IL-1␤ to its mature counterpart, interleukin convertase (ICE), is a homolog of the Caenorhabditis elegans cell death gene ced-3 (18), and overexpression of murine ICE in rat fibroblasts leads to apoptotic-like features (19). These observations suggest that the posttranslational processing and release of pro-IL-1␤ may be linked to cell death.
In a previous study we compared the processes by which nigericin and ATP promoted the posttranslational maturation of IL-1␤ from LPS-stimulated murine peritoneal macrophages (20). Both of these agents promoted efflux of potassium from the cell, and this efflux appeared necessary for the posttranslational processing reactions. This requirement for K ϩ export was confirmed recently, and two non-selective inhibitors of K ϩ channels, triethanolamine and 4-aminopyridine, were reported to block cytokine maturation (21). In a related study, release of mature IL-1␤ from LPS-stimulated human monocytes and mouse peritoneal macrophages was shown to be impaired by agents that disrupted anion transport processes, including DIDS, ethacrynic acid, and tenidap, a new anti-inflammatory/ anti-arthritic agent (16). Changes to the intracellular ionic environment, therefore, appear to be required for activation of the posttranslational maturation of pro-IL-1␤. In the present study we have continued to explore ionic conditions required for this unusual cytokine processing event. ATP and hypotonic stress are employed as alternative signals to activate human monocyte IL-1␤ posttranslational processing. Sensitivity of these cellular activation processes to changes of the ionic environment and to pharmacological effectors suggests that an anion transporter is a necessary component of the mechanism by which both hypotonic stress and ATP promote IL-1␤ posttranslational processing.

EXPERIMENTAL PROCEDURES
Human Monocytes-Mononuclear cells were isolated from blood (100 ml) drawn from normal volunteers as described previously (16). In an average experiment, 1 ϫ 10 7 mononuclear cells were added to each well of 6-well dishes in a total volume of 2 ml of RPMI 1640 containing 5% FBS, 25 mM Hepes, pH 7.2, and 1% penicillin/streptomycin (Maintenance Medium). Monocytes were allowed to adhere for 2 h, after which the supernatants were discarded and the attached cells were rinsed once with 2 ml of Maintenance Medium.
When freshly isolated monocytes were employed, the adherent cells immediately were exposed to 10 ng/ml LPS (Escherichia coli serotype 055:B5, Sigma) for 2 h. Alternatively, the adherent monocytes were incubated overnight at 37°C in a 5% CO 2 environment in Maintenance Medium prior to LPS activation; where indicated, 5 ng/ml recombinant human granulocyte macrophage colony stimulating factor (R & D Systems, Minneapolis, MN) was added to the culture medium. In all cases LPS-stimulated cells were labeled for 60 min in 1 ml of Pulse Medium (methionine-free RPMI 1640, 1% dialyzed FBS, 25 mM Hepes, pH 7.2, 83 Ci/ml [ 35 S]methionine (Amersham Corp., 1000 Ci/mmol)). The Pulse Medium subsequently was discarded; the radiolabeled cells were washed once with 2 ml of Chase Medium (RPMI 1640, 1% FBS, 20 mM Hepes, 5 mM NaHCO 3 , pH 6.9), and 1 ml of Chase Medium that contained various effector molecules was added to each well. Where indicated, ATP was added (from a 100 mM stock solution previously adjusted to pH 7 with NaOH) to a final concentration of 1-5 mM. Radiolabeled monocytes were chased at 37°C for various times after which the medium was recovered and clarified by centrifugation to remove cells that detached from the plate during the chase and cell debris; the resulting supernatants were harvested and adjusted to 1% in Triton X-100, 0.1 mM phenylmethylsulfonyl fluoride, 1 mM iodoacetic acid, 1 g/ml pepstatin, and 1 g/ml leupeptin by addition of concentrated stock solutions of these reagents. Cells were solubilized by addition of 1 ml of an extraction buffer composed of 25 mM Hepes, pH 7, 1% Triton X-100, 150 mM NaCl, 0.1 mM phenylmethylsulfonyl fluoride, 1 mM iodoacetic acid, 1 g/ml pepstatin, 1 g/ml leupeptin, and 1 mg/ml ovalbumin. After a 30-min incubation on ice, both the media and cell extracts were clarified by centrifugation at 45,000 rpm for 30 min in a Beckman tabletop ultracentrifuge using a TLA 45 rotor (Beckman Instruments, Palo Alto, CA).
Immunoprecipitation of IL-1␤ and Analysis of Radiolabeled Cytokine Product-IL-1␤ was immunoprecipitated from detergent extracts of cell and media samples as detailed previously (16). Disaggregated immunoprecipitates were analyzed by SDS-gel electrophoresis and autoradiography (22); gels were soaked in Amplify (Amersham Corp.) prior to drying. Quantitation of the amount of radioactivity associated with the various species of IL-1␤ was determined with the use of an Ambis Image Analysis System (San Diego, CA).
Lactate Dehydrogenase (LDH) Assays-Aliquots of media samples and cell extracts were assayed for LDH using pyruvate as substrate and a colorimetric pyruvate detection assay (Sigma).

Monocyte Populations Differ in Their Release of IL-1␤
Freshly isolated human monocytes are heterogeneous with respect to their size and functional properties (23,24). Moreover, when maintained in culture these cells may change their biochemical properties (25,26), including their ability to respond to extracellular ATP (27)(28)(29). Based on this known heterogeneity, a comparison of the IL-1␤ posttranslational processing capacity of freshly isolated monocytes and monocytes that were aged overnight in culture was performed. Each population was stimulated for 2 h with LPS, labeled for 60 min with [ 35 S]methionine, and chased in the absence or presence of ATP. In the absence of ATP, freshly isolated monocytes released proteolytically processed radiolabeled 17-kDa IL-1␤ (Table I); based on recovery of total radiolabeled IL-1␤ from these cultures, however, the 17-kDa species generally accounted for Ͻ30% of the newly synthesized cytokine. High concentrations of LPS are known to favor mature IL-1␤ release (15), but even at a concentration of 1 g/ml only 26% of the total radiolabeled cytokine was recovered as the extracellular mature species (experiment 2; Table I). Aged monocytes, on the other hand, did not release significant mature IL-1␤ in the presence of LPS (Table I). In contrast, both monocyte populations responded to ATP and released proteolytically processed 17-kDa IL-1␤; the nucleotide triphosphate, however, did not affect both populations equally. Thus, freshly isolated monocytes consistently released Ͼ90% of their IL-1␤ as the 17-kDa mature species but only 50 -60% of the radiolabeled cytokine was released as the mature species from aged monocytes (Table I).
In the presence of ATP an apparent over-recovery of radiolabeled IL-1␤ consistently was observed. For example, a total of 3970 counts were recovered as IL-1␤ from LPS-treated fresh monocytes in the absence of ATP (experiment 1; Table I). Following ATP treatment, however, total radioactivity recovered as immunoprecipitable IL-1␤ from an identical culture increased Ͼ4-fold (Table I). Since radiolabeled methionine was removed from the culture medium prior to the addition of ATP, new synthesis of IL-1␤ cannot account for this over-recovery.

release by fresh and aged human monocytes
Human monocytes were isolated by adherence and treated with LPS immediately (Fresh) or after overnight culture (Aged). After 2 h of LPS treatment, cells were labeled with [ 35 S]methionine for 60 min and then chased in the absence or presence of ATP; once added, LPS remained in all subsequent solutions. The amount of IL-1␤ recovered as the 17-kDa species (expressed as a % of the total radiolabeled IL-1␤ recovered) and the total radioactivity immunoprecipitated as IL-1␤ (sum of 17-, 28-, and 31-kDa species recovered intracellularly and extracellularly and corrected for the 2-fold loss of [ 35 S]methionine that occurred due to formation of the 17-kDa species) are indicated. Each value is the average of duplicate determinations. Fresh monocytes were treated with ATP at an extracellular pH of 7.3, whereas aged monocytes were treated with ATP in medium at pH 6.9; this pH difference affords maximal proteolytic processing (16). Fresh monocytes were chased for either 30 min (experiment 1) or for 3 h (experiment 2) and aged cells for 3 h; the LPS concentration in experiments 1, 3, and 4 was 10 ng/ml and was increased to 1 g/ml in experiment 2. Moreover, the antibody employed to capture radiolabeled IL-1␤ was not limiting. Treatment of monocyte extracts (derived from cells not exposed to ATP) with increasing polyclonal antibody did not yield additional radiolabeled IL-1␤. Rather, it appeared that conditions which led to an increase in IL-1␤ posttranslational maturation also led to an increase in cytokine recovery. This over-recovery may result from a decrease in the intracellular turnover of newly synthesized cytokine and/or a change in IL-1␤ that allows recognition by the antibody. Similar differences were observed in the quantity of radiolabeled IL-1␤ recovered in the absence or presence of ATP from aged monocyte cultures (Table I). Although the cause of this over-recovery remains to be determined, the results indicate that ATP is a potent inducer of IL-1␤ release and maturation and that the extent of these posttranslational processing reactions varies between different monocyte populations. These differences suggest that the ATP response is not passive but, rather, requires active participation of the IL-1␤ producing cell.

Hypotonic Stress Promotes Release of Mature IL-1␤ from LPS-activated Human Monocytes
Based on previous observations suggesting that anion and potassium fluxes are necessary for the posttranslational maturation of IL-1␤ (14,16,20,21), LPS-stimulated monocytes were subjected to hypotonic stress, a treatment known to promote KCl export in a wide variety of cells (30). Freshly isolated human monocytes were stimulated with LPS for 2 h, labeled with [ 35 S]methionine for 60 min, and then chased for 3 h in media of differing ionic strength. The base medium in this experiment consisted of a standard Dulbecco's formulation (Table II). Hypotonic media were prepared by varying the concentrations of NaCl, KCl, and KH 2 PO 4 ; concentrations of other media components, including Ca 2ϩ , Mg 2ϩ , NaHCO 3 , glucose, and Hepes, were maintained at constant values (Table II). The chase media varied in NaCl concentration from 132 to 27 mM.
In normal Dulbecco's medium (Formulation A, Table II), LPS-stimulated cells released little mature IL-1␤ and the cellassociated cytokine persisted as the 35-kDa species (Fig. 1). Addition of 2 mM ATP to this medium, however, promoted complete release of the radiolabeled cytokine, and externalized IL-1␤ was processed efficiently to the 17-kDa species (Fig. 1). Importantly, as the concentration of NaCl within the chase medium decreased (in the absence of exogenous ATP), mature 17-kDa IL-1␤ increased extracellularly. Thus, in medium containing 55 (Formulation D) and 27 (Formulation E) mM NaCl, 85 and 97%, respectively, of the total radiolabeled IL-1␤ was recovered extracellularly as the 17-kDa species (Fig. 1).
To define the kinetics of this posttranslational processing, LPS-stimulated [ 35 S]methionine-labeled monocytes were chased in hypotonic medium for various times. After a 10-min chase, only a small quantity of radiolabeled IL-1␤ was recov-ered in the medium, and the externalized polypeptides were not proteolytically processed (Fig. 2). Extracellular 17-kDa IL-1␤, however, was readily detected after 20 min of hypotonic treatment, and the quantity of this species increased during longer chase times (Fig. 2). Importantly, beyond the 20 min time point extracellular IL-1 was recovered almost exclusively as the 17-kDa species (Fig. 2). In contrast, at all times intracellular cytokine was composed primarily of the 31-kDa species with only minor amounts of the 17-kDa mature form being present (data not shown). Externalization of the mature cytokine species, therefore, occurred more efficiently than release of the pro-IL-1␤ polypeptide species. Moreover, only 20% of the cytoplasmic constituent lactate dehydrogenase (LDH) was displaced to the medium after 3-h of hypotonic treatment (Fig. 2). Thus, hypotonic stress promoted formation of 17-kDa IL-1␤ and selective release of this species from LPS-activated human monocytes. Hypotonically stressed monocytes (in medium containing Յ55 mM NaCl) demonstrated the same ballooned appearance as previously observed after ATP or nigericin treatment (data not shown). Although viability was not assessed, cells demonstrating this dramatically altered appearance are assumed to be non-vital.
The sum of all radioactivity recovered as IL-1␤ (corrected for the 2-fold loss of 35 S-labeled methionines that accompany formation of the 17-kDa IL-1␤ species) again indicated that a 4-fold over-recovery occurred during the 3-h posttranslational maturation period (Fig. 2). Since quantities of radiolabeled polypeptides are expected to decrease during the chase as a result of protein turnover, this over-recovery (relative to the amount recovered from cells in the absence of a chase) cannot S]methionine-labeled fresh human monocytes were placed in media that contained the indicated concentration of NaCl; where indicated, 2 mM ATP also was present. These cultures were incubated for 3 h after which cells and media were separated, and IL-1␤ was recovered from each by immunoprecipitation. The resulting immunoprecipitates were analyzed by SDS-gel electrophoresis; an autoradiogram is shown for the cell-associated (A) and media (B) samples. Each condition was performed in duplicate. Arrows on the right indicate the migration position of the 31-kDa pro-IL-1␤ and 17-kDa mature IL-1␤ species. reflect differences in protein degradation. Rather, the marked increase in immunoprecipitable radioactivity suggests that pulse-labeled cytokine molecules initially exist within monocyte detergent extracts as forms that are inaccessible to and/or not recognized by the antibody and are converted during hypotonic stress to forms that are recognized immunologically.

Medium Composition Affects the ATP and Hypotonic Stress Responses
To determine whether the ATP-activated IL-1␤ posttranslational processing mechanism also involved a volume-sensitive response, monocytes were treated with ATP in a hypertonic medium. Normal RPMI medium was made hypertonic by the addition of 0.2 M NaCl. LPS-activated [ 35 S]methionine-labeled cells treated with ATP in this medium did not efficiently process IL-1␤ to the 17-kDa species nor did they release significant cytokine into their medium (Fig. 3). In contrast, cells maintained in normal RPMI demonstrated an efficient ATP response (Fig. 3). ATP-treated control cultures released 26% of their LDH into the medium, but monocytes treated with ATP in the hypertonic medium released only 15% of this cytoplasmic marker.
To explore the role of anions in the ATP-induced response, LPS-activated [ 35 S]methionine-labeled monocytes were incubated in isotonic media that contained various sodium salts in place of NaCl; chaotropic anions (I Ϫ , NO 3 Ϫ , and SCN Ϫ ) and the membrane impermeant glucuronate anion were employed. In medium containing NaCl, monocytes demonstrated an efficient ATP response (Fig. 4); 17-kDa IL-1␤ was produced and externalized. In contrast, when monocytes were maintained in a NaI-or NaSCN-based medium, a marked decrease in formation and release of 17-kDa IL-1␤ was observed (Fig. 4). In the presence of either of these salts, production of 17-kDa IL-1␤ was reduced by 97% relative to cells maintained in NaClcontaining medium. Likewise, cells maintained in a NaNO 3based medium demonstrated an impaired response; 56% less 17-kDa IL-1␤ was produced by cells maintained in NaNO 3 relative to cells maintained in NaCl (Fig. 4). When the medium contained 137 mM sodium glucuronate, on the other hand, production of 17-kDa IL-1␤ was not impaired; rather, cells maintained in this medium consistently produced slightly greater amounts of the mature cytokine species (Fig. 4). The percentage of LDH released by ATP was 25, 10, 9, 16, and 31%, respectively, in media that contained 137 mM NaCl, NaI, NaSCN, NaNO 3 , or sodium glucuronate. Thus, chaotropic an- ions inhibited ATP-induced processing and release of IL-1␤ and the release of LDH.
To address the possibility that chaotropic anions impaired the ATP-induced cytokine response by blocking binding of ATP to its cell surface receptor, monocytes were treated with ATP at different times relative to the time of substitution of NaI for NaCl within the medium. When monocytes were placed in NaI either before or at the time of ATP addition, 17-kDa IL-1␤ production was blocked efficiently (data not shown). Likewise, when the medium substitution occurred within 15 min of ATP addition the cytokine response was inhibited by 87% (Fig. 5). When the NaI for NaCl substitution was delayed beyond 25 min of ATP addition, however, production of 17-kDa IL-1␤ was observed (Fig. 5). Thus, NaI blocked the ATP-induced cytokine response independently of an effect on the initial binding of ATP to its surface receptor. It should be noted that the anti-IL-1␤ antiserum immunocaptured radiolabeled IL-1␤ as efficiently from the NaI medium as from a NaCl medium; failure to recover 17-kDa IL-1␤ from the NaI medium, therefore, did not result from impaired immunoprecipitation.
Additional evidence that the anion effect on IL-1␤ processing was not due to inhibition of ATP binding to its receptor was obtained by asking if chaotropic anions altered cytokine processing promoted by hypotonic stress. LPS-activated [ 35 S]methionine-labeled monocytes were treated with hypotonic medium that contained 55 mM concentrations of the individual sodium salts. As expected, cells incubated in hypotonic NaCl produced and externalized 17-kDa IL-1␤ (Fig. 6). In contrast, cells maintained in hypotonic NaI produced only 5% as much 17-kDa IL-1␤ (Fig. 6). Production of 17-kDa IL-1␤ also was reduced in the presence of hypotonic NaSCN, but the extent of inhibition (Ͻ30%) was not equivalent to that obtained in the NaI-based medium. Monocytes maintained in hypotonic NaNO 3 or sodium glucuronate produced quantities of 17-kDa IL-1␤ Ն to that recovered from cells maintained in hypotonic NaCl (Fig. 6).

Pharmacological Effectors
Hypotonic stress promotes a regulated volume decrease (RVD) response in many cell types that is mediated by a net loss of KCl from the cytosol (30 -32); the mechanism of KCl loss is cell type-dependent and may occur by the concerted action of separate K ϩ and Cl Ϫ conducting channels or by an electroneutral KCl cotransporter (30 -32). In an attempt to link IL-1␤ posttranslational processing events to a specific KCl release mechanism, known effectors of the RVD response were profiled in the human monocyte cytokine release assay.
Okadaic Acid Inhibits ATP-induced IL-1␤ Posttranslational Processing-The protein phosphatase inhibitor okadaic acid impairs swelling activated KCl cotransport in rabbit red blood cells (33). To determine whether okadaic acid affected IL-1␤ posttranslational processing, LPS-activated [ 35  Each condition was performed in duplicate, and the migration positions of 31-and 17-kDa IL-1␤ species are indicated. Radioactivity associated with the 17-kDa IL-1␤ species was determined and is indicated in B as a function of the employed salt. Each bar is the average of duplicate determinations; numbers within (or above) the bars indicate the percentage that the radioactivity represents relative to that recovered from the NaCl-treated control.

FIG. 5. NaI substitution post-ATP addition inhibits IL-1␤ processing. LPS-stimulated [ 35 S
]methionine-labeled aged monocytes were placed in a modified Dulbecco's medium containing 137 mM NaCl, 1% FBS, and 2 mM ATP. At the times indicated, the medium was removed from duplicate cultures and replaced with a comparable medium except that 137 mM NaI was substituted for NaCl. All cultures were incubated with ATP for a total of 3 h. At the end of the 3-h treatment, cells and media were separated, and IL-1␤ was recovered by immunoprecipitation. Following the initial time of ATP treatment the NaCl-containing media were clarified by centrifugation (to remove cells that may have detached) and pooled with the final media samples prior to the immunoprecipitation step to allow recovery of any IL-1 that may have been released prior to the NaI medium switch. The media immunoprecipitates were analyzed by SDS-gel electrophoresis, and an autoradiogram of the gel is shown in A. The quantity of radioactivity recovered as extracellular 17-kDa IL-␤ is indicated in B as a function of the time of the medium substitution.
acid produced a dose-dependent inhibition in the formation of 17-kDa IL-1␤ (Fig. 7); the IC 50 for this agent was 150 nM.
Quinine Blocks Cytokine Posttranslational Processing-Quinine sensitivity is a criterion that often is employed to distinguish between the functioning of separate conductive channels or cotransporters in the RVD response (34,35). This agent is reported to block hypotonic stress-induced K ϩ channels, and quinine inhibition is reversed when gramicidin, a potassium conductive ionophore, is added to the system to provide an alternate K ϩ efflux mechanism (35). We previously reported that potassium selective ionophores such as nigericin and lasalocid can on their own promote maturation and release of IL-1␤ from monocytes and macrophages (20). Likewise, gramicidin promoted mature IL-1␤ release from LPS-activated human monocytes (Fig. 8). At a concentration of 2 M, monocytes treated with this channel forming ionophore (36) efficiently externalized their newly synthesized IL-1␤ in the form of the 17-kDa species (Fig. 8). At a concentration of 1 mM, comparable with concentrations reported to inhibit RVD (34), quinine partially blocked both the gramicidin (39%) and ATP (58%) responses (Fig. 8). Quinine-treated monocytes did not release additional procytokine nor did they accumulate processed IL-1␤ intracellularly (data not shown). Quinine's ability to impair both the ATP and gramicidin responses suggests that it is acting independently of a specific K ϩ channel. Monocytes activated by hypotonic stress also were treated with quinine; within the hypotonic medium, however, 1 mM quinine promoted release of LDH and 31-kDa pro-IL-1␤ suggesting that it was lytic under these conditions (data not shown).
Ca ϩ2 -free Media Do Not Affect the ATP Response-An RVD response mediated by separate conductive K ϩ and Cl Ϫ channels often is inhibited by removal of Ca 2ϩ from the medium (34). LPS-stimulated [ 35 S]methionine-labeled human monocytes treated with ATP in normal medium and in medium containing 10 mM EGTA yielded identical amounts of 17-kDa IL-1␤ (data not shown). Bumetanide and Furosemide Do Not Impair the ATP Response-Agents that impair anion transport processes effectively suppress ATP-induced maturation and release of IL-1␤; such agents include the stilbene derivatives DIDS and SITS and the antiinflammatory agent tenidap (16). In addition, we noted previously that ethacrynic acid is a potent inhibitor of the ATP response of human monocytes; the IC 50 against this response is 3 M (16). Ethacrynic acid is used as a diuretic, and this effect, in part, is attributed to inhibition of kidney cotransporters (37). Two other diuretic agents, bumetanide and furosemide, are reported to be selective inhibitors of the Na ϩ /K ϩ / 2Cl Ϫ cotransporter (38). At 100 M, a concentration sufficient to impair Na ϩ /K ϩ /2Cl Ϫ cotransport (39,40), neither of these agents inhibited ATP-induced cytokine posttranslational processing (Table III). DISCUSSION Posttranslational maturation of IL-1␤ promoted by ATP is not observed with similar concentrations of AMP, UTP, or GTP, and ADP is less efficient than ATP (20). This selectivity suggests that ligation of specific purinoreceptors (P 2 ) activates a signaling mechanism leading to the posttranslational processing of IL-1␤ (41,42). Further evidence that the monocyte ATP response involves a signal transduction mechanism is indi- ]methionine-labeled aged monocytes were placed in RPMI, pH 6.9, medium that contained the indicated concentration of okadaic acid. After a 20-min incubation, 1 mM ATP was introduced, and the cells were incubated for an additional 3 h. Cells and media were harvested, and IL-1␤ was recovered from each by immunoprecipitation. Media immunoprecipitates were analyzed by SDS-gel electrophoresis and autoradiography (A); the region of the autoradiogram that contained 31-and 17-kDa IL-1␤ is indicated as a function of the okadaic acid concentration. Each condition was performed in duplicate. The amount of radioactivity associated with the extracellular 17-kDa species is indicated in B; each bar is the average of the duplicate determinations. Numbers within the bars indicate the percentage relative to the control (O nM okadaic acid). cated by the differential sensitivity of various monocyte populations to ATP. Freshly isolated LPS-activated monocytes were highly sensitive to purinoreceptor stimulation, and these cells released Ͼ80% of their newly synthesized IL-1␤ as the 17-kDa cytokine species in the presence of extracellular ATP. In contrast, monocytes aged overnight prior to LPS activation were less sensitive to ATP and released 50 -60% of their IL-1␤ as processed 17-kDa IL-1␤. Loss of responsiveness may result from down-regulation of P 2 receptors and/or a decrease in an important post-receptor component of the signal transduction machinery. Indeed, expression of functional P 2Z receptors on human monocytes previously was observed to be affected by cell culture conditions (27)(28)(29)43).
Based on the ability of okadaic acid to inhibit the ATP response, alterations in the phosphorylation state of mechanistic elements of the signal transduction pathway also may contribute to changes in monocyte ATP responsiveness. Likewise, monocytes and macrophages are known to alter their surface channel properties when maintained in culture (44 -46), and these changes may affect ATP responsiveness. ATP promotes major changes in the intracellular ionic environment as a result of its binding to surface purinoreceptor family members (42,(47)(48)(49)(50)(51)(52). The identity (i.e. P 2X , P 2Y , P 2U , or P 2Z ) of the purinoreceptor subtypes that are present on monocytes and macrophages is not known; murine macrophage-like cells and human monocytes appear to express more than one type of P 2 receptor subtype (29,48,49). Likewise, mouse microglial cells appear to express at least two purinergic receptor subtypes, P 2Y and P 2Z , and ligation of the latter has been associated with IL-1 release (53). Thus, a monocyte's ATP responsiveness is likely to be governed not only by the presence of one or more members of the purinoreceptor family but also by the array of specific ion transporters that are components of the signal transduction pathway.
Data presented previously suggested that potassium and anion fluxes were necessary components of the mechanism by which ATP promoted IL-1␤ posttranslational processing (14,16,20,21). A remarkable feature of the release process induced by ATP is the dramatic morphological change that accompanies cytokine externalization. In the continuous presence of this nucleotide triphosphate LPS-stimulated monocytes and macrophages swell extensively (14,16,20). Mammalian cells possess a remarkable ability to regulate their cytoplasmic volume (30,31), and this regulation is easily visualized when cells are placed within an osmotically altered media. For example, Ehrlich ascites tumor cells initially swell after suspension in a hypotonic medium due to an influx of water. This swelling soon subsides, however, and within minutes a cell's volume normalizes despite its persistence within the hypotonic medium (30). This RVD response often occurs as a result of activation of K ϩ and Cl Ϫ channels or K ϩ /Cl Ϫ cotransporters within the plasma membrane. These ion transporters facilitate net loss of KCl followed by the passive movement of H 2 0 and a near restoration of the cell's original volume (30,31).
LPS-stimulated monocytes subjected to hypotonic stress responded by activating both the proteolytic maturation of pro-IL-1␤ and the release of the mature 17-kDa cytokine. This activation was not instantaneous, and monocytes suspended in 55 mM NaCl-containing buffer required Ͼ10 min of treatment to initiate the processing reactions. This time dependence is consistent with the notion that the hypotonic conditions triggered changes in the intracellular ionic environment which, in turn, activated the posttranslational processing reactions. Importantly, the employed hypotonic conditions did not simply lyse the monocytes. When mouse peritoneal macrophages are suspended in an extreme hypotonic medium, for example, they lyse and release their content of both LDH and IL-1␤; the released cytokine, however, is not processed to the mature 17-kDa species (14). Lysis, therefore, is not sufficient to promote correct posttranslational processing. In contrast, after 30 min of moderate hypotonic exposure, virtually all of the recovered IL-1␤ released by human monocytes was processed to the 17-kDa mature species. Cell-associated cytokine, on the other hand, remained predominantly as higher molecular mass species. Moreover, Ͻ10% of the total LDH associated with the  8. Gramicidin promotes IL-1␤ posttranslational processing. LPS-stimulated [ 35 S]methionine-labeled aged monocytes (previously cultured in the presence of 5 ng/ml granulocyte macrophage colony-stimulating factor) were placed in RPMI medium and treated for 3 h with either 2 mM ATP or 2 M gramicidin in the presence or absence of 1 mM quinine; cultures treated with quinine were pretreated with this agent for 15 min prior to the addition of the release stimulus. Media IL-1␤ immunoprecipitates were analyzed by SDS-gel electrophoresis and autoradiography (A); the region of the autoradiogram that contained 31-and 17-kDa IL-1␤ is shown. The amount of radioactivity associated with extracellular 17-kDa IL-1␤ is indicated in B; each bar is the average of the duplicate determinations. Numbers within the bars indicate the percentage relative to the ATP or gramicidin-treated controls. monocyte cultures was recovered in the medium after 30 min of hypotonic treatment. This selective externalization of the 17-kDa cytokine species, therefore, suggests that the majority of the monocytes possessed intact membranes during the initial period of hypotonic treatment. With prolonged treatment times, however, the amount of extracellular LDH increased indicating that membrane integrity was compromised. The ability of hypotonic stress to promote IL-1␤ posttranslational processing also has been reported by an independent group (21).
As with ATP-treated cells, monocytes subjected to hypotonic stress developed a swollen appearance that correlated temporally with the release of mature IL-1␤. Therefore, if hypotonic stress activated an RVD-like response in monocytes, this response apparently failed as the volume did not normalize. Swelling induced by ATP treatment of human monocytes was suppressed when these cells were maintained in the presence of a hypertonic medium; likewise, the hypertonic medium blocked ATP-induced posttranslational processing of IL-1␤. Volume-activated changes, therefore, appear to be important elements of both the ATP-and hypotonic-induced cytokine responses.
Substitution of chaotropic anions for Cl Ϫ ions in the medium also impaired the ATP and hypotonic responses. Iodide anions, for example, completely suppressed both responses. The thiocyanate anion was an effective inhibitor of the ATP response but was less effective against the hypotonic stress response, and nitrate anions partially suppressed the ATP response without affecting the hypotonic response. In contrast, glucuronate anions did not inhibit the ATP or hypotonic stress-induced posttranslational processing of IL-1␤. The employed chaotropic anions are, to varying degrees, permeant to biological membranes, and they are expected to enter the cytosol (54,55); the glucuronate anion, on the other hand, is membraneimpermeant. Cotransporters possess a very high selectivity for Cl Ϫ ions, and chaotropic anions often inhibit cotransport function (54 -56). Likewise, some Cl Ϫ channels are inhibited by I Ϫ anions (57) although others are less discriminating and will facilitate transport of divergent anions such as I Ϫ and SCN Ϫ (30,58). Based on the remarkable inhibition observed in the presence of the chaotropic anions, therefore, function of a selective chloride transporter is suggested. If ATP and hypotonic stress were initiating IL-1␤ posttranslational processing simply by opening non-selective pores in the membrane, then one would not expect to observe this strong anion dependence. The degree of inhibition obtained in the presence of chaotropic anions is likely to reflect their ability to penetrate the monocyte membrane and access the anion transporter under the different experimental situations.
Removal of extracellular Ca 2ϩ by addition of the calcium chelator EGTA did not impair the ATP response; entry of extracellular Ca 2ϩ , therefore, is not necessary, and this divalent cation independence suggests that a Ca 2ϩ -activated K ϩ channel is not operative in the cytokine response. These data, however, do not eliminate the possibility that release of Ca 2ϩ from intracellular stores is sufficient for channel activation. Quinine inhibited ATP-induced posttranslational processing of IL-1␤, but this inhibitory effect also was observed when gramicidin served as the stimulus. Since gramicidin is expected to facilitate K ϩ loss independently of a K ϩ channel, quinine's inhibitory effect does not appear to result from inhibition of a specific K ϩ channel; high concentrations of quinine required to block cytokine posttranslational processing probably affected multiple cellular components. Finally, anion transport inhibitors such as DIDS, 5-nitro-2-(3-phenylpropylamino)benzoic acid, and ethacrynic acid suppress ATP-induced IL-1␤ post-translational processing (16); these agents are not selective, and they are reported to impair cotransporters as well as anion channels (59,60). Two specific blockers of the Na ϩ /K ϩ /2Cl Ϫ cotransporter, bumetanide and furosemide, did not inhibit the ATP response at concentrations that were sufficient to inhibit cotransport functions.
Taken together, these data suggest a role for a specific anion transporter in the IL-1␤ response, but the nature of this transporter (channel versus cotransporter) remains to be determined. A number of different Cl Ϫ channels have been cloned (57), but the monocyte repertoire of such channels is unknown; these cells are reported to possess a SITS-inhibitable chloride channel (45). A K ϩ /Cl Ϫ cotransporter was cloned recently, but it is not known whether monocytes express this protein (61); moreover, this cloned cotransporter was inhibited by bumetanide suggesting that it is not involved in the bumetanideinsensitive monocyte cytokine response. The apparent diversity in the nature of agents that promote posttranslational maturation of IL-1␤ may be explained based on a shared ability of these agents to activate chloride and potassium transport. Ionophores such as gramicidin and nigericin are expected to depolarize monocytes. Likewise, ATP stimulation of murine macrophages causes depolarization as a result of the opening of a large non-selective pore in the membrane, and hypotonic stress also may elicit depolarization (34,47). Thus, membrane depolarization may serve as the common trigger that activates cytokine posttranslational processing. The exact nature of the ionic events that underlie subsequent steps culminating in cytokine posttranslational processing remains unclear, but lowering intracellular concentrations of KCl as a result of activation of KCl efflux and/or an increase in cell volume may facilitate ICE activation and conversion of pro-IL-1␤ to its mature counterpart. Catalytic activity of ICE is inhibited by ionic conditions in excess of 50 mM (62), and within LPSactivated cells this enzyme is inactive (63). After initiation of IL-1␤ posttranslational processing with nigericin, however, active ICE is observed (64). In addition, ionic signals may regulate IL-1 export (65).