In response to LPS, peritoneal macrophages produce IL-1, but, for the most part, newly synthesized cytokine molecules remain cell associated. Externalization and proteolytic processing of pro-IL-1β can be initiated by extracellular ATP. In this study, kinetics and inhibitor sensitivity of the stimulus-coupled mechanism were investigated with [35S]methionine-labeled macrophages. Optimal ATP concentrations required to promote cytokine post-translational processing suggest the involvement of a P2Z type of receptor. Proteolysis of pro-IL-1β initiates within 7.5 min of ATP addition; 17-kDa mature IL-1β is observed first intracellularly and subsequently extracellularly. In contrast, ATP-treated cells do not contain 17-kDa IL-1α. Macrophages exposed to ATP continuously or only for a 15-min pulse release IL-1α, IL-1β, and lactate dehydrogenase (LDH). Proteolytic maturation of IL-1β exceeds that of IL-1α in both formats, but pulsed cells process the externalized cytokines more efficiently. Ethacrynic acid and DIDS (4,4′-diisothiocyanato-stilbene-2,2′-disulfonic acid) block ATP-induced proteolysis of pro-IL-1β and prevent release of pro-IL-1α/β and LDH; they do not inhibit ATP-induced K+ (86Rb+) efflux. Ethacrynic acid inhibits release of both forms of IL-1 with a similar concentration dependence; within the arrested cells, procytokines accumulate in a Triton-insoluble fraction. An IL-1β-converting enzyme inhibitor blocks proteolysis of IL-1β, but it does not prevent release of pro-IL-1α, pro-IL-1β, or LDH. These results indicate that ATP stimulates externalization of both IL-1α and IL-1β. The ATP-induced cytokine release mechanism is accompanied by cell death and requires activity of an anion transport inhibitor-sensitive component, but this pathway operates independently of cytokine proteolytic processing.

Interleukin-1 is an important cytokine mediator produced in abundance by activated monocytes and macrophages (1). When administered to animals, IL-1 can initiate an inflammatory response, produce fever, and promote tissue degradation (2, 3, 4). Moreover, in some animal models, blockade of IL-1 production and/or function decreases the extent and/or severity of the condition, suggesting that the presence of this cytokine promotes the diseased state (5, 6). Elevated levels of IL-1 have been detected in patients suffering from a number of chronic disorders, including rheumatoid arthritis, Alzheimer’s disease, and acute myelocytic leukemia (7, 8, 9). Based on these observations, IL-1 appears to act as a master cytokine whose misregulation may underlie many chronic disease states. Understanding checkpoints that cells use to regulate IL-1 production, therefore, may provide novel targets for therapeutic approaches aimed at controlling inflammatory processes.

IL-1 biologic activity is derived from two distinct but related gene products, IL-1α and IL-1β (1). The primary translation products of the two mouse genes encode propolypeptides with apparent molecular masses of 31 to 35 kDa (10, 11). Pro-IL-1α can bind to receptors on target cells and elicit a biologic response (12). However, under some circumstances, pro-IL-1α is cleaved to yield a 17-kDa mature cytokine species that retains biologic activity; a calpain-like protease has been implicated in this cleavage (13, 14). In contrast, pro-IL-1β is a weak agonist for IL-1R, and this procytokine must be proteolytically processed to its mature 17-kDa form to demonstrate biologic activity (15). The protease responsible for cleavage of pro-IL-1β, IL-1-converting enzyme (ICE2 or caspase I), is the founder member of a family of cysteine proteases that cleave their substrates at an aspartic acid residue (16, 17). Activation of some members of this protease family occurs when cells are induced to undergo apoptosis (18, 19). Pro-IL-1α and pro-IL-1β do not contain signal sequences that direct them into the secretory apparatus of the cell (20). Rather, the newly synthesized cytokine products accumulate within the cytosol of activated monocytes/macrophages, where they appear to coexist with inactive forms of ICE (21, 22). In vitro studies indicate that the efficient proteolytic processing and release of mature IL-1 require a secondary signal in addition to the primary stimulus (e.g., LPS) that promotes transcription and translation (23, 24, 25). The need for a second stimulus provides an additional checkpoint by which cells may regulate biogenesis of this important cytokine product.

Efficient IL-1β post-translational processing has been achieved in vitro by treating LPS-activated monocytes and macrophages with ATP (23, 26), cytolytic T cells (23, 27), bacterial toxins (28), or potassium-selective ionophores (25, 26, 29). Likewise, peritoneal macrophages activated with LPS in vivo require a secondary stimulus to initiate efficient IL-1β maturation/release, and ATP can serve in this capacity (30). High concentrations of LPS can promote release of IL-1β from freshly isolated human monocytes (24), but this response is inefficient and is lost when these cells are maintained in culture (31). Fewer studies have examined the mechanism of IL-1α export; in some systems, this cytokine appears to remain associated with the plasma membrane (32). Mature forms of both IL-1α and IL-1β are externalized when thioglycolate-elicited/LPS-activated peritoneal macrophages are treated with ATP or cytolytic T cells (23). Likewise, both IL-1α and IL-1β are released from freshly isolated human monocytes in response to LPS activation (33). Differences in the rates at which the two cytokine species are externalized and in the extents to which the released cytokines are processed, however, are observed in both the mouse and human cell systems.

In this study, we characterize ATP’s ability to initiate post-translational processing of IL-1α and IL-1β from murine peritoneal macrophages in an effort to determine whether these two cytokine species are externalized via a similar stimulus-coupled maturation pathway. The data indicate that both cytokines are released in response to ATP stimulation to similar extents and at comparable rates, but that IL-1β is processed more efficiently and more rapidly to its mature form than is IL-1α. Moreover, release of both IL-1α and IL-1β is blocked by inhibitors of anion transport. ATP thus constitutes an efficient stimulus for promoting externalization of both cytokines; this stimulus-induced export pathway requires the activity of an anion transport inhibitor-sensitive component, but functions independently of the state of cytokine proteolytic maturation.

Mouse macrophages were isolated by peritoneal lavage from C3H/HeN mice (Taconic Laboratories, German Town, NY). The lavage medium consisted of RPMI 1640 containing 100 U/ml penicillin, 100 μg/ml streptomycin, 5% FBS, 2 mM glutamine, and 25 mM HEPES, pH 7.3 (culture medium). Cells from multiple animals were pooled and collected by centrifugation, washed once with culture medium, and then seeded at a density of 1 to 2 × 106 cells/well in Natrix-coated six-well plates (Collaborative Research, Bedford, MA). After 2 h at 37°C, adherent cells were washed twice with culture medium and then cultured overnight at 37°C in 2 ml of fresh culture medium in a 5% CO2 environment.

Macrophages were stimulated with 1 μg/ml Escherichia coli LPS (serotype 055:B5 obtained from Sigma Chemical Co., St. Louis, MO) for 75 min, then rinsed once with methionine-free αMEM medium containing 100 U/ml penicillin, 100 μg/ml streptomycin, 1% dialyzed FBS, 5 mM NaHCO3, 1 μg/ml LPS, and 20 mM HEPES, pH 7.3 (pulse medium). One milliliter of pulse medium containing 83 μCi/ml of [35S]methionine (Amersham Corp., Chicago, IL) was added to each well, and the macrophages were labeled at 37°C for 1 h. Labeled cells subsequently were rinsed twice with RPMI 1640 medium containing 100 U/ml penicillin, 100 μg/ml streptomycin, 1% FBS, 2 mM glutamine, 1 μg/ml LPS, and 25 mM HEPES, pH 7.3 (chase medium). One milliliter of chase medium was added to each well (with or without ATP), and the cells were chased at 37°C for the indicated times. Media then were harvested and clarified by centrifugation to remove cells and/or cell debris. Cellular monolayers were suspended in 1 ml of a lysis buffer composed of 1% Triton X-100, 150 mM NaCl, 25 mM HEPES, pH 7, 0.1 mM PMSF, 1 mg/ml OVA, 1 mM iodoacetic acid, 1 μg/ml pepstatin, and 1 μg/ml leupeptin. The clarified media samples were adjusted to the same Triton X-100 and protease inhibitor concentrations by addition of a concentrated stock of these reagents. After 30 min on ice, all samples were clarified by centrifugation at 45,000 rpm for 30 min in a Beckman (Palo Alto, CA) tabletop ultracentrifuge (TLA-45 rotor), and the resulting supernatants were recovered. Where indicated, the cell-associated Triton-insoluble pellets were suspended directly in SDS sample buffer. IL-1β was isolated from Triton-soluble supernatants by immunoprecipitation, as described previously (25). Goat anti-murine IL-1α (34) was obtained from Dr. Ivan Otterness (Pfizer Central Research, Groton, CT). This antiserum immunoprecipitated both a 31- and 17-kDa polypeptide from macrophage extracts and/or conditioned medium; addition of an excess of murine mature rIL-1α prevented recovery of both species, indicating that they are captured specifically by the Ab. The quantity of radioactivity associated with individual IL-1 species was determined by scanning dried gels with an Ambis Image Analysis System (San Diego, CA).

Macrophages (5 × 105 cells/well in Natrix-coated 24-well plates) simultaneously were incubated with 1 μg/ml LPS and 3 μCi/ml [86Rb+]Cl (Amersham Corp.) for 3 h. Monolayers then were rinsed twice with chase medium to remove non-cell-associated radioactivity. Fresh medium (0.5 ml) containing, where indicated, 4,4′-diisothiocyanato stilbene-2,2′-disulfonic acid (DIDS) or ethacrynic acid was added and the cells were preincubated for 15 min at 37°C. The preincubation media were removed and replaced with fresh media containing the same effector and, where indicated, an activating stimulus. Following an incubation at 37°C, plates were placed on ice and media immediately were harvested and clarified by centrifugation. Cells (including those recovered after clarification of the media samples) were solubilized in 0.5 ml of lysis buffer. Radioactivity associated with the media and cell extracts subsequently was determined by liquid scintillation counting, and the percentage of the total (cell + medium) released extracellularly was determined. DIDS, ethacrynic acid, ATP, UTP, and benzoyl-benzoic ATP were obtained from Sigma Chemical Co. Concentrated stock solutions of the nucleotide triphosphates were adjusted to pH 7 with NaOH before their addition to cells. YVAD-CHO was obtained from Bachem Bioscience (King of Prussia, PA).

Aliquots of media samples and cell extracts were assayed for LDH using pyruvate as substrate and a colorometric pyruvate detection assay (Sigma Chemical Co.); the percentage of total (sum of cell-associated and medium) LDH released extracellularly was determined.

To establish optimum conditions for IL-1β post-translational processing, LPS-activated, [35S]methionine-labeled macrophages were treated with several concentrations of ATP, after which the distribution and state of maturation of radiolabeled cytokine were determined. In the absence of ATP, no IL-1β was released to the medium following 30 or 60 min of incubation, and the cell-associated cytokine persisted as the 35-kDa procytokine species (Fig. 1). In the presence of ATP, however, proteolytic processing and release of IL-1β occurred. The cell-associated fractions contained a significant amount of 17-kDa IL-1β after 30 min of continuous ATP treatment, but the 35-kDa procytokine comprised the major intracellular species (Fig. 1,A). Media fractions recovered from ATP-treated cultures, on the other hand, contained predominantly the 17-kDa IL-1β species and smaller quantities of the procytokine and of a 28-kDa species (Fig. 1,B); this latter polypeptide is assumed to result from an alternate ICE cleavage event (16). Quantities of extracellular 17-kDa IL-1β were similar at ATP concentrations from 2 to 10 mM, but less mature cytokine was recovered at both lower (1 mM) and higher (100 mM) concentrations of the nucleotide triphosphate (Fig. 1 B). The decline in cytokine post-translational processing observed at the highest ATP concentration is most likely the result of an associated increase in osmolarity (ATP used as the disodium salt); hypertonic conditions previously were shown to inhibit the ATP response (35).

FIGURE 1.

ATP promotes IL-1β post-translational processing. LPS-activated/[35S]methionine-labeled macrophages were incubated for 30 or 60 min with the indicated concentration of ATP, after which IL-1β was recovered by immunoprecipitation from media and cell extracts. Immunoprecipitates were analyzed by SDS-PAGE, and autoradiograms of the cell (A) and media (B) samples are shown. Arrows indicate the migration positions of the 35- and 17-kDa forms of murine IL-1β.

FIGURE 1.

ATP promotes IL-1β post-translational processing. LPS-activated/[35S]methionine-labeled macrophages were incubated for 30 or 60 min with the indicated concentration of ATP, after which IL-1β was recovered by immunoprecipitation from media and cell extracts. Immunoprecipitates were analyzed by SDS-PAGE, and autoradiograms of the cell (A) and media (B) samples are shown. Arrows indicate the migration positions of the 35- and 17-kDa forms of murine IL-1β.

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Cells treated continuously for 60 min with ATP possessed less intracellular 17-kDa IL-1β and they released greater amounts of the 35-kDa procytokine than did their 30-min counterparts (Fig. 1). Likewise, at all ATP concentrations, release of the cytoplasmic marker LDH was increased at 60 min relative to 30 min, suggesting that the nucleotide triphosphate promoted cytolysis (data not shown). The absolute amount of extracellular 17-kDa IL-1β was not increased significantly by the longer treatment time, despite the persistence of pro-IL-1β intracellularly at lower (1 to 5 mM) ATP concentrations.

Activated macrophages were treated with a single concentration of ATP (10 mM), and the kinetics of cytokine post-translational processing were characterized. Within 7.5 min of ATP addition, 17-kDa IL-1β was observed, and at this early time, the mature cytokine was recovered exclusively intracellularly (Fig. 2). The absolute quantity of cell-associated 17-kDa IL-1β increased after 15 min of ATP exposure, and at this time, a small amount of mature cytokine also was observed extracellularly. Intracellular levels of 17-kDa IL-1β remained elevated at the 20- and 30-min harvests, after which quantities of this species declined (Fig. 2). In contrast, quantities of extracellular 17-kDa IL-1β increased progressively throughout the time course. Extracellular levels of pro-IL-1β remained low and constant throughout the initial 30 min of ATP exposure, but increased sharply at the 60-min harvest (Fig. 2). The appearance of extracellular procytokine coincided with the appearance of extracellular LDH (Fig. 2).

FIGURE 2.

Kinetics of ATP-induced IL-1β post-translational processing. LPS-activated, [35S]methionine-labeled macrophages were treated with 10 mM ATP. At the indicated times, individual cultures were harvested, cell- and medium-associated IL-1β were recovered by immunoprecipitation, and the immunoprecipitates were analyzed by SDS-PAGE. Radioactivity associated with individual IL-1β species was determined and is indicated for the 35-kDa intracellular (35i; □) and extracellular (35e; ▪) proforms, and the 17-kDa intracellular (17i; ○) and extracellular (17e; •) mature forms; all values were normalized to total culture-associated LDH content to correct for cell number differences. In addition, radioactivity associated with the 17-kDa species was multiplied by a factor of 2 to correct for loss of [35S]methionine that resulted from proteolytic processing. Pro-IL-1β contains eight methionines, but only four of these are located within the mature 17-kDa carboxyl-terminal fragment. Release of LDH into the medium (percentage of total) also is indicated. Each data point is an average of duplicate determinations.

FIGURE 2.

Kinetics of ATP-induced IL-1β post-translational processing. LPS-activated, [35S]methionine-labeled macrophages were treated with 10 mM ATP. At the indicated times, individual cultures were harvested, cell- and medium-associated IL-1β were recovered by immunoprecipitation, and the immunoprecipitates were analyzed by SDS-PAGE. Radioactivity associated with individual IL-1β species was determined and is indicated for the 35-kDa intracellular (35i; □) and extracellular (35e; ▪) proforms, and the 17-kDa intracellular (17i; ○) and extracellular (17e; •) mature forms; all values were normalized to total culture-associated LDH content to correct for cell number differences. In addition, radioactivity associated with the 17-kDa species was multiplied by a factor of 2 to correct for loss of [35S]methionine that resulted from proteolytic processing. Pro-IL-1β contains eight methionines, but only four of these are located within the mature 17-kDa carboxyl-terminal fragment. Release of LDH into the medium (percentage of total) also is indicated. Each data point is an average of duplicate determinations.

Close modal

LPS-activated, [35S]methionine-labeled cells were treated with 5 mM ATP, after which IL-1α and IL-1β were immunoprecipitated separately from the same cell and media extracts. IL-1α recovered after 20, 40, and 60 min of continuous ATP treatment migrated as the 31-kDa procytokine species; no mature IL-1α was detected intracellularly (data not shown) or extracellularly (Fig. 3,B). After the 20-min treatment, small quantities of pro-IL-1α were recovered in the medium, and extracellular levels of this species were increased at the 40- and 60-min harvests. In contrast, the same cell population produced 17-kDa IL-1β, and this species was released selectively during the initial 20 min of ATP exposure (Fig. 3,A). Both mature and precursor forms of IL-1β, however, were recovered extracellularly after 40 and 60 min of ATP treatment (Fig. 3,A). When radioactivity lost from IL-1β as a result of ICE cleavage was taken into account, a similar overall percentage of IL-1α, IL-1β, and LDH was found to be externalized after 60 min of continuous ATP treatment (Fig. 3 C). During the initial 20 min of ATP treatment, however, the percentage of radiolabeled IL-1β externalized was greater than that of IL-1α and LDH, reflecting the selective release of the 17-kDa species.

FIGURE 3.

ATP also promotes IL-1α externalization. LPS-activated, [35S]methionine-labeled macrophages were treated continuously with 5 mM ATP for the indicated times. After each harvest, IL-1β and IL-1α were recovered by immunoprecipitation from the same media samples, and the resulting immunoprecipitates were separated by SDS-PAGE; each condition was performed in duplicate. Autoradiograms of the IL-1β (A) and IL-1α (B) samples are shown. Arrows denote the migration positions of the 35- and 17-kDa forms of IL-1β and the 31-kDa pro-IL-1α species. Identity of the lower molecular mass species in the IL-1α immunoprecipitates is not known; these did not comigrate with authenic 17-kDa IL-1α. Total radioactivity recovered as all forms of the two individual cytokines were determined (and corrected for twofold loss of radioactivity from 17-kDa IL-1β) and the percentage of each recovered extracellularly is indicated in C as a function of time; each data point is an average of duplicate determinations. Medium LDH recovery also is indicated.

FIGURE 3.

ATP also promotes IL-1α externalization. LPS-activated, [35S]methionine-labeled macrophages were treated continuously with 5 mM ATP for the indicated times. After each harvest, IL-1β and IL-1α were recovered by immunoprecipitation from the same media samples, and the resulting immunoprecipitates were separated by SDS-PAGE; each condition was performed in duplicate. Autoradiograms of the IL-1β (A) and IL-1α (B) samples are shown. Arrows denote the migration positions of the 35- and 17-kDa forms of IL-1β and the 31-kDa pro-IL-1α species. Identity of the lower molecular mass species in the IL-1α immunoprecipitates is not known; these did not comigrate with authenic 17-kDa IL-1α. Total radioactivity recovered as all forms of the two individual cytokines were determined (and corrected for twofold loss of radioactivity from 17-kDa IL-1β) and the percentage of each recovered extracellularly is indicated in C as a function of time; each data point is an average of duplicate determinations. Medium LDH recovery also is indicated.

Close modal

In the original report describing ATP-induced IL-1 post-translational processing, thioglycolate-elicited peritoneal macrophages were exposed to ATP for 30 min and then incubated in ATP-free medium; cytokine release into the ATP-free medium was assessed over a subsequent 20-h period (23). To determine whether cells treated with a pulse of ATP differed with respect to the efficiency and/or extent of cytokine post-translational processing relative to cells maintained continuously in the presence of the nucleotide triphosphate, LPS-activated/[35S]methionine-labeled macrophages were treated with 2 mM ATP for 15 min and then chased in ATP-free medium. Processed IL-1β was detected in the medium of ATP-pulsed cells after only 15 min of chase (Fig. 4,A). Quantities of this 17-kDa extracellular species increased with an additional 15 min of chase, after which levels of extracellular 17-kDa IL-1β remained constant (Fig. 4,A). Extracellular 17-kDa IL-1β accounted for 55% of the total IL-1β recovered from the macrophage cultures at this time. Remarkably, ATP-pulsed macrophages released very little 35-kDa pro-IL-1β (Fig. 4 A).

FIGURE 4.

A pulse of ATP is sufficient to promote post-translational processing. LPS-activated, [35S]methionine-labeled macrophages were treated with 2 mM ATP for 15 min, new medium devoid of ATP was added, and cultures were chased for the indicated times. At each harvest, media were collected from duplicate cultures and IL-1β and IL-1α were recovered by immunoprecipitation. The immunoprecipitates were separated by SDS-PAGE, and autoradiograms of the gels are shown for the IL-1β (A) and IL-1α (B) samples. Arrows denote the migration positions of the 35- and 17-kDa forms of IL-1β and the 31- and 17-kDa forms of IL-1α.

FIGURE 4.

A pulse of ATP is sufficient to promote post-translational processing. LPS-activated, [35S]methionine-labeled macrophages were treated with 2 mM ATP for 15 min, new medium devoid of ATP was added, and cultures were chased for the indicated times. At each harvest, media were collected from duplicate cultures and IL-1β and IL-1α were recovered by immunoprecipitation. The immunoprecipitates were separated by SDS-PAGE, and autoradiograms of the gels are shown for the IL-1β (A) and IL-1α (B) samples. Arrows denote the migration positions of the 35- and 17-kDa forms of IL-1β and the 31- and 17-kDa forms of IL-1α.

Close modal

IL-1α also was released from ATP-pulsed macrophages. Pro-IL-1α was detected in the medium after 15 min of chase, and levels of this species increased with an additional 15 min of post-ATP exposure (Fig. 4,B). At longer chase times, several additional faster migrating species were detected within the extracellular IL-1α immunoprecipitates; one of these possessed an apparent molecular mass of 17 kDa and comigrated with murine mature rIL-1α. This 17-kDa species (assuming that two of six methionines were lost as a result of cleavage (11)) represented 31% of the recovered extracellular cytokine at the 3-h post-ATP harvest (Table I). Quantitation of the various cytokine species indicated that similar percentages of radiolabeled IL-1α and IL-1β were released from the ATP-pulsed macrophages (Table I). The overall efficiency at which the released cytokines were processed to the 17-kDa species, however, was much greater for IL-1β than IL-1α (Table I). Importantly, ATP-pulsed macrophages also released LDH in a time-dependent manner, and the extent of LDH release correlated with the extent of IL-1 release (Table I).

Table I.

Comparison of cytokine post-translational processing and LDH release induced by treatment of LPS-activated macrophages with a pulse of ATPa

Chase Time (min)IL-1αIL-1β
% ReleasedProcessing Efficiency% ReleasedProcessing EfficiencyLDH, % Released
15 19 1.2 32 93 25 
30 39 3.5 55 92 49 
45 51 4.1 65 91 54 
60 54 10.4 69 91 58 
120 51 20.2 75 93 61 
180 62 31.3 86 93 63 
Chase Time (min)IL-1αIL-1β
% ReleasedProcessing Efficiency% ReleasedProcessing EfficiencyLDH, % Released
15 19 1.2 32 93 25 
30 39 3.5 55 92 49 
45 51 4.1 65 91 54 
60 54 10.4 69 91 58 
120 51 20.2 75 93 61 
180 62 31.3 86 93 63 
a

LPS-activated [35S]methionine-labeled macrophages were treated with 2 mM ATP for 15 min after which the medium was replaced with fresh medium devoid of ATP, and the cultures were chased for the indicated times. Cell and media samples were harvested separately, each was divided into two equal fractions, and IL-1α and IL-1β were recovered by immunoprecipitation. The resulting immunoprecipitates were separated by SDS gel electrophoresis, and radioactivity associated with individual species was determined. The percentage of each cytokine released into the medium (after correcting for loss of radioactivity due to proteolytic processing to the mature 17-kDa species) and the efficiency at which the released cytokine was processed to the mature species are indicated. This percentage is based on radiolabeled cytokine recovered at each time point and does not account for losses due to cytokine turnover. In the case of IL-1β, a 2-fold loss of radioactivity (four of eight methionines) was assumed to occur when the procytokine was processed to the 17-kDa species; for IL-1α, a 33% loss (two of six methionines) was assumed. Aliquots of both the media and cell-associated extracts also were assayed for LDH content; the % of the total culture associated activity recovered in the medium is indicated.

The above experiment indicated that 17-kDa IL-1α appeared in the medium subsequent to the appearance of pro-IL-1α. Moreover, unlike the case with IL-1β, no 17-kDa IL-1α was detected intracellularly (data not shown). These observations suggested that pro-IL-1α cleavage occurred extracellularly. To test for this, LPS-activated/[35S]methionine-labeled macrophages were pulsed with 2 mM ATP and then chased for 30 min in ATP-deficient medium to allow release of pro-IL-1α (see Fig. 4). At this point, media were collected and these either were harvested immediately or incubated for an additional 2.5 h in the absence of cells. Other cultures were incubated for 2.5 h in the continued presence of macrophages. IL-1α ultimately was recovered by immunoprecipitation from all media samples and analyzed by SDS-PAGE.

Media recovered at the 30-min post-ATP harvest contained a predominance of 35-kDa pro-IL-1α and a small amount of the 17-kDa species (Fig. 5). Levels of extracellular 17-kDa cytokine were increased at the 3-h harvest (Fig. 5). Importantly, 30-min post-ATP harvest medium that was incubated for an additional 2.5 h in the absence of macrophages also yielded levels of 17-kDa IL-1α greater than those recovered from the 30-min harvest (Fig. 5). Moreover, the quantities of the 17-kDa species produced by the cell-free incubations were comparable with those recovered from cultures maintained in the presence of cells. Generation of the 17-kDa IL-1α species, therefore, did not require the continued presence of cells, and this behavior suggests that proteolytic conversion of 35-kDa IL-1α occurred post-externalization. Inclusion of 1 mM iodoacetic acid within the chase medium did not prevent appearance of the 17-kDa species (Fig. 5), indicating that a cysteine protease was not responsible for this conversion.

FIGURE 5.

Pro-IL-1α is cleaved extracellularly. LPS-activated, [35S]methionine-labeled macrophages were pulsed with 2 mM ATP for 15 min and then chased for 30 min in the absence of the nucleotide triphosphate. At this point, some cultures were harvested. Media recovered from some of the 30-min harvests were incubated for additional 2.5 h in the absence of cells (with or without 1 mM iodoacetic acid (IAA)). Other cultures were maintained for the 2.5-h incubation in the continued presence of macrophages (cells present). IL-1α subsequently was recovered by immunoprecipitation from all samples, and the immunoprecipitates were separated by SDS-PAGE; an autoradiogram of the dried gel is shown. Arrows indicate the migration positions of the 31- and 17-kDa species of IL-1α; each condition was performed in duplicate.

FIGURE 5.

Pro-IL-1α is cleaved extracellularly. LPS-activated, [35S]methionine-labeled macrophages were pulsed with 2 mM ATP for 15 min and then chased for 30 min in the absence of the nucleotide triphosphate. At this point, some cultures were harvested. Media recovered from some of the 30-min harvests were incubated for additional 2.5 h in the absence of cells (with or without 1 mM iodoacetic acid (IAA)). Other cultures were maintained for the 2.5-h incubation in the continued presence of macrophages (cells present). IL-1α subsequently was recovered by immunoprecipitation from all samples, and the immunoprecipitates were separated by SDS-PAGE; an autoradiogram of the dried gel is shown. Arrows indicate the migration positions of the 31- and 17-kDa species of IL-1α; each condition was performed in duplicate.

Close modal

Both anion transport and ICE inhibitors have been reported to affect IL-1β post-translational processing (17, 27, 36, 37, 38). To determine whether stimulated release of IL-1α was sensitive to these agents, ATP-pulsed macrophages were maintained in the absence or presence of various inhibitors. In the absence of an effector, ATP-pulsed cells released both IL-1α and IL-1β, and extracellular IL-1β was processed efficiently to the 17-kDa species (Fig. 6,A); IL-1α was recovered primarily as the 35-kDa species (Fig. 6,B). Two inhibitors of anion transport, ethacrynic acid and DIDS, inhibited post-translational processing of both IL-1α and IL-1β. In the presence of either of these agents, levels of extracellular IL-1β were reduced dramatically (Fig. 6,A). Importantly, no intracellular accumulation of 17-kDa IL-1β species was observed in the presence of the inhibitors (data not shown). Thus, anion transport inhibitors prevented proteolytic conversion of pro-IL-1β and cytokine release. Likewise, cells treated with ethacrynic acid or DIDS released less 35-kDa IL-1α than did their nontreated ATP-pulsed counterparts (Fig. 6,B); quantities of the 17-kDa IL-1α species also were reduced in the presence of the anion transport inhibitors (Fig. 6 B).

FIGURE 6.

Pharmacologic disruption of ATP-induced IL-1 post-translational processing. LPS-activated, [35S]methionine-labeled macrophages were pulsed with 2 mM ATP in the absence or presence of 10 μM ethacrynic acid, 100 μM DIDS, or 100 μM YVAD-CHO for 15 min and then chased for 3 h in medium lacking ATP, but containing the same effector. IL-1β and IL-1α individually were immunoprecipitated from harvested media, and the resulting immunoprecipitates were analyzed by SDS-PAGE. Autoradiograms of the IL-1β (A) and IL-1α (B) samples are shown. Arrows denote the migration of the 35- and 17-kDa species of IL-1β and the 31- and 17-kDa species of IL-1α; each condition was performed in duplicate.

FIGURE 6.

Pharmacologic disruption of ATP-induced IL-1 post-translational processing. LPS-activated, [35S]methionine-labeled macrophages were pulsed with 2 mM ATP in the absence or presence of 10 μM ethacrynic acid, 100 μM DIDS, or 100 μM YVAD-CHO for 15 min and then chased for 3 h in medium lacking ATP, but containing the same effector. IL-1β and IL-1α individually were immunoprecipitated from harvested media, and the resulting immunoprecipitates were analyzed by SDS-PAGE. Autoradiograms of the IL-1β (A) and IL-1α (B) samples are shown. Arrows denote the migration of the 35- and 17-kDa species of IL-1β and the 31- and 17-kDa species of IL-1α; each condition was performed in duplicate.

Close modal

The ICE inhibitor YVAD-CHO also inhibited ATP-induced cytokine post-translational processing. In the presence of this agent, much less 17-kDa IL-1β was released to the medium (Fig. 6,A), and no accumulation of 17-kDa IL-1β was observed within YVAD-CHO-treated cells (data not shown). Macrophages treated with the ICE inhibitor, however, continued to export pro-IL-1β (Fig. 6,A). Likewise, these cells released pro-IL-1α, and levels of the extracellular 17-kDa IL-1α species were greater than or equal to those recovered in the absence of the ICE inhibitor (Fig. 6 B).

In the absence of an effector, 56% of the culture-associated LDH activity was recovered in the medium collected at the 3-h post-ATP harvest. The anion transport inhibitors suppressed LDH release; extracellular LDH accounted for only 17 and 12% of the culture-associated LDH activity in the presence of 10 μM ethacrynic acid or 100 μM DIDS, respectively. YVAD-CHO-treated cells, however, released 68% of their LDH content.

Ethacrynic acid’s ability to inhibit externalization of IL-1α and IL-1β was dose dependent (Fig. 7). Release of mature IL-1β and pro-IL-1α displayed similar sensitivities to ethacrynic acid, suggesting that this agent impaired a common step in the export of both cytokines. Overall recovery of the radiolabeled cytokines in the presence of ethacrynic acid was reduced relative to the quantities recovered from ATP-treated cells maintained in the drug’s absence. To account for this loss, a pulse-chase experiment was performed. Compared with the quantities of pro-IL-1α and pro-IL-1β recovered immediately after a 1-h pulse labeling with [35S]methionine, total quantities of the two procytokines recovered after 3 h of chase in the absence of ATP were reduced; this loss is assumed to reflect normal cytokine turnover (Table II). The presence of ethacrynic acid did not alter this basal turnover (Table II). Macrophages treated with ATP, on the other hand, yielded quantities of the two cytokines (sum of all intracellular and extracellular species and corrected for loss of methionine due to proteolytic processing) comparable with those recovered immediately after the pulse, indicating that little cytokine turnover occurred when cytokine post-translational processing initiated. Macrophages treated with ATP in the presence of ethacrynic acid, on the other hand, yielded less of the radiolabeled cytokines than non-ATP-treated controls (Table II). A similar loss was observed in the presence of DIDS (data not shown). The reduction in extracellular cytokine observed in the presence of anion transport inhibitors raised the possibility that these agents did not block the post-translational processing mechanism, but, rather, enhanced the rate of cytokine turnover. This possibility seems unlikely, however, because the reduction in immunoprecipitable cytokine occurred in the absence of a change in total cell-associated radioactivity (data not shown). Moreover, loss of immunoprecipitable cytokine was accompanied by the appearance of two prominent radiolabeled polypeptides within the Triton-insoluble fraction of the cells that comigrated with the precursor forms of IL-1α and IL-1β (Fig. 8,B). The Triton-insoluble fraction isolated from macrophages treated with ethacrynic acid in the absence of ATP did not possess high levels of these two polypeptides (Fig. 8,A). Likewise, the Triton-insoluble fraction isolated from cells treated with ATP did not contain an abundance of the 31- and 35-kDa polypeptides, although changes in the overall pattern of radiolabeled polypeptides were observed relative to cells maintained in the absence of ATP (Fig. 8 A). Loss of immunoprecipitable IL-1α/β in the presence of a combination of ATP and ethacrynic acid, therefore, appears to result from partitioning of the procytokines into Triton-insoluble complexes.

FIGURE 7.

Ethacrynic acid’s effect on IL-1 post-translational processing is concentration dependent. LPS-activated, [35S]methionine-labeled macrophages were treated with 2 mM ATP in the absence or presence of ethacrynic acid for 15 min, after which new medium lacking ATP, but containing the same concentration of ethacrynic acid, was added, and the cultures were incubated for 3 h. IL-1β and IL-1α were recovered from the media fractions by immunoprecipitation, and the immunoprecipitates were analyzed by SDS-PAGE. Radioactivity recovered as extracellular 17-kDa IL-1β or 31-kDa IL-1α, normalized to LDH equivalents, is indicated as a function of ethacrynic acid concentration; each data point is an average of duplicate determinations.

FIGURE 7.

Ethacrynic acid’s effect on IL-1 post-translational processing is concentration dependent. LPS-activated, [35S]methionine-labeled macrophages were treated with 2 mM ATP in the absence or presence of ethacrynic acid for 15 min, after which new medium lacking ATP, but containing the same concentration of ethacrynic acid, was added, and the cultures were incubated for 3 h. IL-1β and IL-1α were recovered from the media fractions by immunoprecipitation, and the immunoprecipitates were analyzed by SDS-PAGE. Radioactivity recovered as extracellular 17-kDa IL-1β or 31-kDa IL-1α, normalized to LDH equivalents, is indicated as a function of ethacrynic acid concentration; each data point is an average of duplicate determinations.

Close modal
Table II.

Effect of ethacrynic acid and ATP on IL-1 recoverya

ConditionIL-1βIL-1α
35I28I35e28e17eTotal31I31e17eTotal
Pulse 6075 151 6226 3774 3774 
Chase 3812 82 3894 2871 2871 
Chase + ethacrynic acid 3311 95 3406 2560 2560 
Chase + ATP 759 100 249 180 5851 7139 1196 1902 134 3232 
Chase + ATP+ ethacrynic acid 954 116 92 20 953 2135 1713 338 2051 
ConditionIL-1βIL-1α
35I28I35e28e17eTotal31I31e17eTotal
Pulse 6075 151 6226 3774 3774 
Chase 3812 82 3894 2871 2871 
Chase + ethacrynic acid 3311 95 3406 2560 2560 
Chase + ATP 759 100 249 180 5851 7139 1196 1902 134 3232 
Chase + ATP+ ethacrynic acid 954 116 92 20 953 2135 1713 338 2051 
a

LPS-activated [35S]methionine-labeled macrophages were harvested immediately (Pulse) or treated with 2 mM ATP (where indicated) for 15 min and chased for 3 h in the absence or presence of 10 μM ethacrynic acid. Cell and media samples were harvested separately, each was divided into two equal fractions, and IL-1α and IL-1β were recovered by immunoprecipitation. The resulting immunoprecipitates were separated by SDS-PAGE, and radioactivity associated with individual species was determined. The quantity of radioactivity recovered intracellularly in association with the 35-kDa (35I) and 28-kDa (28I) forms and extracellularly in association with the 35-kDa (35e), 28-kDa (28e), and 17-kDa (17e) forms of IL-1β are indicated; counts recovered as the 17-kDa species of IL-1β were multiplied by 2 to correct for loss due to proteolytic processing. Likewise, the quantity of radioactivity recovered intracellularly in association with the 31-kDa (31I) form and extracellularly in association with the 31-kDa (31e) and 17-kDa (17e) forms of IL-1α are indicated; counts recovered as the 17-kDa IL-1α species were multiplied by 1.5 to correct for loss due to proteolytic processing. Total radioactivity (sum of all individual species) also is indicated. Each value is the average of duplicate determinations.

FIGURE 8.

Pro-IL-1 displays an altered intracellular distribution in the presence of ATP and ethacrynic acid. LPS-activated, [35S]methionine-labeled macrophages were harvested immediately (− chase) or pulsed for 15 min with 2 mM ATP in the absence or presence of 10 μM ethacrynic acid. ATP-treated macrophages then were chased for 3 h; ethacrynic acid was maintained in the medium of cultures that were treated with this agent during the ATP pulse. Post-harvest, Triton-insoluble fractions obtained after ultracentrifugation of cell extracts were solubilized and analyzed directly by SDS PAGE. A, An autoradiogram of the Triton-insoluble components is shown; each condition was analyzed in duplicate. Arrows denote migration positions of molecular mass standards of 46, 35, and 31 kDa. B, A sample of an SDS-disaggregated Triton-insoluble fraction obtained from ethacrynic acid-arrested/ATP-treated cells (lane 2) was run on an SDS gel side by side with samples of immunoprecipitated pro-IL-1β (lane 1) and pro-IL-1α (lane 3). Only the region of the autoradiogram containing the cytokines is shown.

FIGURE 8.

Pro-IL-1 displays an altered intracellular distribution in the presence of ATP and ethacrynic acid. LPS-activated, [35S]methionine-labeled macrophages were harvested immediately (− chase) or pulsed for 15 min with 2 mM ATP in the absence or presence of 10 μM ethacrynic acid. ATP-treated macrophages then were chased for 3 h; ethacrynic acid was maintained in the medium of cultures that were treated with this agent during the ATP pulse. Post-harvest, Triton-insoluble fractions obtained after ultracentrifugation of cell extracts were solubilized and analyzed directly by SDS PAGE. A, An autoradiogram of the Triton-insoluble components is shown; each condition was analyzed in duplicate. Arrows denote migration positions of molecular mass standards of 46, 35, and 31 kDa. B, A sample of an SDS-disaggregated Triton-insoluble fraction obtained from ethacrynic acid-arrested/ATP-treated cells (lane 2) was run on an SDS gel side by side with samples of immunoprecipitated pro-IL-1β (lane 1) and pro-IL-1α (lane 3). Only the region of the autoradiogram containing the cytokines is shown.

Close modal

DIDS previously was reported to block ATP binding to some types of P2 receptors (39). To rule out the possibility that DIDS and ethacrynic acid blocked IL-1 post-translational processing as a result of inhibiting ATP binding to cell surface receptors, peritoneal macrophages were loaded with the potassium analogue 86Rb+ and then treated with ATP in the absence or presence of these agents. ATP ligation of the P2Z receptor is known to promote rapid cell depolarization accompanied by loss of intracellular K+ (26). Release of 86Rb+ was greatly augmented by concentrations of ATP ≥1 mM or by benzoyl benzoic ATP (Table III); this latter agent is reported to act as a selective agonist of the P2Z receptor (40). In contrast, ATP concentrations ≤100 μM or 5 mM UTP only modestly enhanced 86Rb+ loss (Table III). The rapid extensive (>90%) loss of cell-associated 86Rb+, therefore, appears to proceed via the P2Z receptor based on: 1) the requirement for ATP concentrations in excess of 100 μM, the inactivity of UTP, and the agonist activity of benzoyl benzoic ATP. 86Rb+ loss from macrophages maintained in the absence of ATP remained low throughout 15 min of chase (Table IV). In contrast, cells maintained in the presence of 2 mM ATP released >93% of their radioactive cation content within 5 min of ATP addition (Table IV). Neither 10 μM ethacrynic acid nor 100 μM DIDS inhibited ATP-induced loss of 86Rb+ (Table IV).

Table III.

P2z receptor ligation promotes 86Rb+ release from macrophages

Stimulus86Rb+ (% Released)a
None 10b 
40 μM ATP 17.5 
100 μM ATP 21 
1 mM ATP 81 
5 mM ATP 94 
200 μM benzoyl benzoic-ATP 46 
1 mM benzoyl benzoic-ATP 95 
5 mM UTP 21 
Stimulus86Rb+ (% Released)a
None 10b 
40 μM ATP 17.5 
100 μM ATP 21 
1 mM ATP 81 
5 mM ATP 94 
200 μM benzoyl benzoic-ATP 46 
1 mM benzoyl benzoic-ATP 95 
5 mM UTP 21 
a

LPS-activated, 86Rb+-loaded macrophages were incubated for 5 min in the presence of the indicated stimulus. At the conclusion of this incubation, cells and media were separated, and the percentage of the radioactive isotope recovered in the medium was determined.

b

The value for the control (none) is a mean of eight individual wells; other values are averages of duplicate wells.

Table IV.

Ethacrynic acid and DIDS do not alter ATP-induced 86Rb+ efflux from LPS-activated macrophagesa

Time (min)% 86Rb+ Released
ControlATPATP + Ethacrynic AcidATP + DIDS
— — — 
13 93 92 92 
10 14 94 92 93 
15 20 95 94 95 
Time (min)% 86Rb+ Released
ControlATPATP + Ethacrynic AcidATP + DIDS
— — — 
13 93 92 92 
10 14 94 92 93 
15 20 95 94 95 
a

LPS-activated, 86Rb+-loaded macrophages were incubated in the absence (Control) or presence of 2 mM ATP; where indicated, 10 μM ethacrynic acid or 100 μM DIDS also were present. After incubation at 37°C, cells and media were separated, and the percentage of the radioactive isotope recovered in the medium was determined. Each value is the average of duplicate determinations.

Previous studies established that extracellular ATP can promote post-translational processing of IL-1α and IL-1β from monocytes and macrophages (23, 26). ATP’s activity as a secretion stimulus is not shared with other nucleotide triphosphates (26) and, as shown in this study, requires concentrations between 2 and 10 mM to achieve maximal processing of IL-1β. These properties indicate that ATP acts via a P2Z type of purinoreceptor (41, 42). A P2X7 receptor recently was cloned that possessed properties characteristic of the P2Z receptor (43); this novel member of the P2X superfamily, therefore, may be responsible for initiating IL-1 post-translational processing. New findings reported in this study indicate that ATP stimulation of LPS-activated murine peritoneal macrophages leads to the release of similar percentages of IL-1α, IL-1β, and LDH, and demonstrate that export of each of these three proteins follows a similar time course. Moreover, externalization of all three proteins is shown to be blocked by anion transport inhibitors. In contrast, an ICE inhibitor that blocks proteolytic processing of IL-1β is demonstrated not to suppress release of pro-IL-1α, pro-IL-1β, or LDH. ATP-induced cytokine externalization, therefore, does not require proteolytic maturation of the propolypeptides.

Macrophage IL-1β post-translational processing was initiated within 7.5 min of nucleotide triphosphate application. Importantly, at this time, 17-kDa IL-1β was observed intracellularly, but not extracellularly, indicating that proteolytic cleavage of pro-IL-1β occurred within the cell before externalization of the mature cytokine. Between 15 and 30 min of ATP treatment, levels of intracellular 17-kDa IL-1β remained constant, while extracellular levels progressively increased. Failure to accumulate large quantities of cell-associated 17-kDa IL-1β suggests that the mature cytokine is externalized soon after its formation. During this same 30-min time period, levels of extracellular pro-IL-1β remained low. Elevated levels of extracellular pro-IL-1β and LDH activity were observed after 60 min of continuous ATP exposure; at this time, the percentages of extracellular LDH and radiolabeled IL-1β were comparable. This similarity suggests that cells that initially selectively released 17-kDa IL-1β ultimately died and released their LDH. Likewise, the overall percentage of radiolabeled pro-IL-1α released in response to ATP was comparable with that observed for IL-1β and LDH. Export of pro-IL-1α, however, initially lagged behind the release of 17-kDa IL-1β.

Externalization of 17-kDa IL-1β from cells activated by a 15-min pulse of ATP occurred in parallel with the release of LDH and pro-IL-1α. Based on the rapid release of mature IL-1β following ATP addition noted above, it is likely that ATP-pulsed macrophages also released 17-kDa cytokine in the absence of LDH during the initial phase of the pulse protocol; the design of the experiment did not allow this to be assessed. Remarkably, ATP-pulsed macrophages did not release significant quantities of pro-IL-1β. In contrast, macrophages treated continuously with concentrations of ATP >2 mM released pro-IL-1β after prolonged (>30 min) treatment times. Release of pro-IL-1β from these cells may signify that continuous exposure to high levels of the nucleotide triphosphate leads to aberrant cytokine processing. In vivo, extracellular ATP is expected to exist only briefly due to ubiquitous ATPases; as such, a pulse of ATP may be more representative of a physiologically relevant stimulus. In both the pulse and continuous formats, ATP-treated macrophages ultimately released a similar percentage of their IL-1α, IL-1β, and LDH contents, suggesting that ATP-induced cytokine post-translational processing was closely associated with cell death. Previous studies also noted that cells subjected to prolonged activation of the P2Z receptor were destined to die (44). Cell death induced by ATP is characterized by both apoptotic- and necrotic-type changes (23, 45). Not all forms of cell death are sufficient to promote proteolytic maturation of pro-IL-1β (25); the mechanism of death initiated by ATP, therefore, must selectively activate IL-1 post-translational processing reactions.

IL-1α released from ATP-treated thioglycolate-elicited peritoneal macrophages is reported to be processed efficiently to the 17-kDa species (23). In contrast, pro-IL-1α was released from resident peritoneal macrophages in response to ATP both in vitro and in vivo (30). Perhaps elicited and resident macrophages differ with respect to their content of an IL-1α-processing protease. Interestingly, pro-IL-1α released to the medium as a result of the ATP pulse subsequently was cleaved to yield a 17-kDa species. The small quantities of this species that were produced precluded determination of the actual cleavage site. Previous studies indicated that calpain, a thiol protease, is responsible for proteolytic processing of pro-IL-1α (13, 14). Extracellular processing of the procytokine released from ATP-treated murine macrophages, however, was not blocked by the thiol protease inhibitor iodoacetic acid nor by an ICE inhibitor. Thus, a nonthiol protease appears to be responsible in this system for extracellular cleavage of pro-IL-1α. In contrast to IL-1β, processed 17-kDa IL-1α was never observed intracellularly. At present, we do not know whether the IL-1α protease was released from cells or was derived from serum in the medium.

In the presence of the ICE inhibitor YVAD-CHO proteolytic maturation of IL-1β induced by ATP was suppressed; a similar inhibition was observed previously (38). Macrophages treated with this ICE inhibitor, however, released quantities of pro-IL-1β that were comparable with the quantities of 17-kDa cytokine released in the inhibitor’s absence. Thus, the ICE inhibitor blocked proteolytic processing, but did not inhibit cytokine release. Likewise, quantities of IL-1α and LDH released by ATP-treated cells were not affected by YVAD-CHO. The inability of YVAD-CHO to prevent LDH release is consistent with previous suggestion that ICE is not required for ATP-induced cell death (38). Pro-IL-1β was shown previously to be externalized by heat-killed Staphylococcus aureus-activated human blood in the presence of an ICE inhibitor (17). Taken together, these observations indicate that IL-1β release from activated monocytes and/or macrophages is not dependent on its state of proteolytic processing.

Two inhibitors of anion transport, on the other hand, blocked release of IL-1α, IL-1β, and LDH. Moreover, in the presence of these agents, no intracellular accumulation of processed IL-1β was observed. When LPS-activated, 86Rb+-loaded peritoneal macrophages were treated with ATP in the absence and presence of ethacrynic acid or DIDS, rapid and complete release of the radioactive cation was observed. These agents, therefore, did not prevent binding of ATP to the P2Z receptor nor subsequent opening of the receptor-linked ion conductance (46). We demonstrated previously that ATP initiates a volume response in human monocytes that is sensitive to inhibitors of anion transport (35); identity of the affected transporter remains unknown. Ethacrynic acid and DIDS, therefore, may block ATP-induced macrophage activation by inhibiting a change to the intracellular ionic environment that is required for cytokine processing/release. Remarkably, within ATP-treated/ethacrynic acid-arrested macrophages, pro-IL-1α/β redistributed to a Triton-insoluble fraction. The significance of this localization is unclear, but previous studies have observed associations between IL-1 and cytoskeletal (Triton-insoluble) elements (47).

The mechanism(s) used by polypeptides lacking signal sequences to gain their release from cells remains largely unknown. In contrast to IL-1, leaderless polypeptides, such as basic fibroblast growth factor and galectin, are reported to be exported in the absence of cell death (48, 49), but specific components of the transport mechanisms remain to be identified. It seems reasonable to assume that IL-1α and IL-1β are released by monocytes/macrophages via a common pathway, rather than hypothesizing that each cytokine evolved a distinct and novel export mechanism. A previous study noted, however, that IL-1α release occurred independently of IL-1β; LPS-activated human monocytes externalized significant quantities of both the precursor and mature forms of IL-1β before releasing pro-IL-1α (33). The experimental system used in this previous study, however, made comparison difficult. First, human monocytes produce low levels of IL-1α relative to IL-1β (50); the low abundance of IL-1α hampers quantitative comparisons. Second, LPS was used as the sole stimulus to promote cytokine production; LPS is an efficient activator of IL-1 synthesis, but not of IL-1 post-translational processing (51). Murine peritoneal macrophages, on the other hand, produce comparable quantities of radiolabeled IL-1α and IL-1β, and when treated with ATP, they release a high percentage of both cytokines (23); comparison under these conditions, therefore, is less likely to be biased by differences in cytokine quantities and/or heterogeneity of the producing cells. In the murine peritoneal macrophage system, ATP promoted release of a similar percentage of IL-1α, IL-1β, and LDH. This similarity is consistent with the notion that a cell activated by ATP responds by releasing its entire content of cytokines and LDH. Externalization under these conditions did not require proteolytic maturation of IL-1α or IL-1β, although export of 17-kDa IL-1β preceded that of the procytokines; selective transport of mature IL-1 species has been observed in other systems (52).

The unusual post-translational processing requirements of IL-1 offer several potential points of therapeutic intervention aimed at limiting cytokine activity and, in turn, suppressing inflammation. Release of IL-1α/β from ATP-treated cells was not dependent on cytokine proteolytic maturation. If the ATP-coupled response is representative of a mechanism used in vivo, then agents that block proteolysis (such as ICE inhibitors) are not expected to prevent cytokine externalization. In view of pro-IL-1α’s biologic activity (12, 53) and the possibility that proteolytic processing of both pro-IL-1α and pro-IL-1β can occur extracellularly (54, 55), release of IL-1 proforms is expected to promote an inflammatory response. On the other hand, anion transport inhibitors prevent release of both mature and proforms of IL-1α/β; these agents may yield more profound anti-inflammatory effects than ICE inhibitors as a result of a larger reduction in extracellular cytokine. Therefore, therapeutic strategies that seek to achieve an ethacrynic acid- or DIDS-like response are expected to provide effective control of IL-1 activity in vivo.

2

Abbreviations used in this paper: ICE, interleukin-1β-converting enzyme; DIDS, 4,4′-diisothiocyanato-stilbene-2,2′-disulfonic acid; LDH, lactate dehydrogenase; YVAD-CHO, the tetrapeptide acetyl-tyrosine-valine-alanine-aspartic acid aldehyde.

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