T H E JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 269, No. 21, Issue of May 27, pp. 15195-15203, 1994 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

Interleukin-lP Maturation and Release in Response to ATP and Nigericin EVIDENCE THAT POTASSIUM DEPLETION MEDIATED BY THESE AGENTS IS A NECESSARY AND COMMON FEATURE O F THEIR ACTMTY*

(Received for publication, December 2, 1993, and in revised form, February 25, 1994)

David Perregaux and Christopher A. GabelS From the Department of Immunology and Infectious Diseases, Pfizer Znc., Central Research Division, Groton, Connecticut 06340

Lipopolysaccharide (LPS)-stimulated mouse perito- neal macrophages produce large quantities of interleu- kin (IL)-lp but in the absence of a secondary stimulus little of this cytokine is proteolytically processed to its mature biologically active state and externalized. The potassium-proton ionophore nigericin and ATP are known to promote the maturation and release of IL-1p from LPS-stimulated cells. We investigated the mecha- nisms by which these agents act in an attempt to under- stand requirements of the post-translational processing. Like nigericin, the ionophores A204 and lasalocid in- duced the release and maturation of IL-1p. The electro- genic potassium ionophore valinomycin, however, did not stimulate these post-translational events. Addition of nigericin or lasalocid to LPS-stimulated cells pro- duced a rapid intracellular acidification; A204, however, did not alter pH, indicating that an acidification was not necessary for activation of IL-lP maturation. Macro- phages treated with ATP became rounded and swollen, and after 30 min of treatment their appearance was com- parable with cells treated with nigericin. Post-transla- tional maturation and release of IL-1p began immedi- ately after ATP addition. The majority of the 17-kDa mature IL-lP produced within the first 30 min of treat- ment was recovered extracellularly; in contrast, during this same time period the 35-kDa IL-lp precursor and the cytoplasmic marker enzyme lactate dehydrogenase and the lysosomal enzyme P-N-acetylglucosaminidase remained cell-associated. ATP, therefore, promoted both the proteolytic maturation of IL-lp and the release of the biologically active species in the absence of cell lysis. Longer incubations with ATP caused cytolysis as judged by the release of the cytoplasmic enzymes, ADP was less active than ATP at initiating the post-translational maturation and release of IL-lp and AMP, GTP, and UTP were totally inactive. ATP, nigericin, A204, and lasalocid promoted a rapid and complete loss of the potassium analog 88Rb+ from cells that were preloaded with this cation; valinomycin-treated cells released only a portion of the radiolabeled cation. Agents that promoted the maturation and release of IL-lp from LPS-stimulated macrophages, therefore, shared an ability to mobilize intracellular potassium. Macrophages treated with ATP or nigericin in medium that contained KC1 rather than NaCl failed to proteolytically activate and to release IL-

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “uduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

and Infectious Diseases, Pfizer Central Research, Eastern Point Rd., t To whom correspondence should be addressed: Dept. of Immunology

Groton, CT 06340. Tel.: 203-441-5483; Fax: 203-441-5719.

lp. These data suggest that ATP and nigericin induce a net decrease in intracellular levels of K+ which is neces- sary for activation of the post-translational maturation of IL-1p.

Interleukin (IL)’--lp is an important cytokine produced by a variety of cell types in response to inflammatory stimuli such as lipopolysaccharide (LPS) (1, 2). Mouse peritoneal macro- phages, for example, produce large amounts of IL-1p when exposed to LPS in vitro (3, 4). Cytokine produced by these macrophages, however, is not released efficiently into the me- dium and only the biologically inactive 35-kDa molecular mass form of IL-1p is detected intracellularly (5). This precursor must be cleaved to generate a mature 17-kDa biologically ac- tive species which binds to specific receptors on target cells to elicit an inflammatory response (6-8). Likewise, the production of pro-IL-10 and the release of its mature counterpart by hu- man monocytes recently were demonstrated to represent two distinct events (9). Biochemical mechanisms that control the proteolytic activation and the release of IL-lp remain unclear.

The precursor form of IL-1p is cleaved by a variety of extra- cellular proteases to generate an active cytokine (i.e. a form that will bind to the IL-1 receptor and elicit a biologic re- sponse). Both trypsin and elastase, for example, generate an active IL-lp species in vitro (10-12). Moreover, a specific pro- tease termed interleukin-1/3 convertase has been identified that cleaves the human 35-kDa IL-1p precursor between as- partic acid 116 and alanine 117 to generate the mature 17-kDa cytokine (13, 14). This enzyme recently was cloned, and al- though it appears to be a cysteine protease, its sequence bears no similarity to known thiol proteases (15, 16). Pro-IL-lp is the only known substrate for interleukin-lp convertase, but the mRNA encoding this protease was detected in cell types that do not produce IL-1p. In view of this, interleukin-lp convertase may function to cleave other proteins in addition to pro-IL-lp. The amino acid sequence of interleukin-lp convertase does not contain an obvious signal sequence nor membrane-spanning domain, suggesting that the enzyme is a cytoplasmic constitu- ent; its precise subcellular distribution is unknown (15, 16). Both interleukin-1p convertase and pro-IL-lp appear to co- exist within the cytoplasm of LPS-activated macrophages. Since the cytokine is not processed in the absence of a second- ary stimulus (17, 181, the proteolytic activity of interleukin-lp convertase must be regulated. Interestingly, in vitro studies suggest that interleukin-1p convertase is inhibited by ionic conditions expected to exist within resting cells (13, 14).

The abbreviations used are: IL, interleukin; LPS, lipopolysaccha- ride; FBS, fetal bovine serum; BCECF, 2’,7’-bis-(2-~arboxyethy1)-5-(and -6)carboxyfluorescein.

15195

15196

The initial translation product of pro-IL-lf3 also does not contain an obvious signal sequence that could direct the newly synthesized cytokine into the rough endoplasmic reticulum (19-22). Moreover, the human cytokine is not glycosylated de- spite the presence of a consensus N-linked g~ycosylation se- quence (23, 24). These two features, the lack of a signal se- quence and the lack of N-linked oligosaccharides, suggest that this cytokine is produced within the cytosol on free polysomes; immunolocalization studies have confirmed that the cell-asso- ciated cytokine resides within the cytoplasm (25). As a cytosolic component, the pathway for IL-lP release appears to require a mechanism distinct fmm the standard secretory pathway in- volving the rough endoplasmic reticulum and Golgi apparatus 126). Human whole blood monocytes stimulated with Staphy- lococcus aureus in the presence of an inhibitor of interleukin-lp convertase continue to externalize newly synthesized pro-IL- 18, suggesting that release of this cytokine is not dependent on proteolysis (16). Release of mature IL-lp, however, is more efficient than release of the procytokine from P388Dl cells transfected with the two individual cytokine species (27).

Recent studies have indicated that both proteolytic activa- tion of pro-IL-lf3, and release of the cytokine can be achieved by treating LPS-stimulated mouse peritoneal macrophages with agents that induce cytolysis. Hogquist et ai. (171, for example, showed that ATP and allo-specific cytolytic T-cells induced the release of mature 17-kDa IL-16 from mouse macrophages; in the presence of these stimuli, the macrophages underwent ap- optosis or programmed cell death. Likewise, Perregaux et al. (18) reported that nigericin treatment of LPS-stimulated mouse peritoneal macrophages induced the efficient release and cleavage by interleukin-lp convertase of newly synthe- sized IL-la. Like ATP and cytolytic T-cells, nigericin treatment caused cell death. Other cytolytic agents, such as the calcium ionophore A23187 or the detergent saponin, did not induce the proteolytic maturation but did cause externalization of pro- IL-1p (18). The potassium-proton ionophore nigericin, there- fore, stimulated macrophages in a selective manner that re- sulted in activation of in te r Ie~in- lp convertase. In the present study we compared the effects of nigericin and ATP on LPS- stimulated mouse macrophages in order to determine whether these diverse agents act via a common mechanism that may mimic the in vivo biological activation process. These results suggest that a net eBux of potassium from the cell i s an im- portant and n e c e s s ~ y component of the activation process.

MATERJALS AND METHODS Cells-Resident mouse peritoneal macrophages were isolated from

C3WHeN mice (Taconic Laboratories, German Town, N y ) by peritoneal lavage; each mouse was lavaged with 8 ml of RPMI 1640 medium containing 5% FBS, 2 mM glutamine, 25 m~ Hepes, pH 7.3,lOO unitdm1 penicitIin and 100 pg/ml streptomycin (culture medium). Cells recov- ered from multiple animals were pooled, collected by centrifugation, and washed once with culture medium. The washed cells were seeded at a density of 1.5 x lo6 celldwell in 6-well multidishes; the dishes were precoated with Natrix (Collaborative Research, Bedford, MA). Afher a 2-h incubation at 37 "C, nonadherent cells were removed, and the at- tached cells were rinsed twice with culture medium and then incubated overnight in 2 ml of culture medium at 37 "C in an en~ronment of 5%

M e ~ a b o ~ ~ c Labeling of Cells and Imm~nopreei~i ta~io~ of IL-l@" Macrophages were stimulated with 1 pg/ml of LPS (Escherichia coli; Serotype 055:B5 from Sigma) for 75 min then rinsed once with methi- onine-free a-minimal essential medium containing 1% dialyzed FBS, 20 mM Hepes, pH 7.3, 5 mM NaHCO,, 100 units/ml penicillin, and 100 pg/ml streptomycin (pulse medium). Each well then received 1 ml of pulse medium that contained 83 pCi of [35Slmethionine (Amersham Corp.), and the cells were labeled for 60 min at 37 "C in the continued presence of LPS. The radiolabeled cell monolayers were rinsed once with 2 ml of RPMI 1640 containing 25 mx Hepes, pH 7.3,Z m~ gluta- mine, 1% FBS, I00 unitdml penicillin, and 100 p g / d streptomycin

co,.

(chase medium) and then 1 ml of fresh chase medium was added to initiate the chase; where indicated this medium contained ionophores or ATP. The cultures were chased at 37 "C, after which the media were recovered and clarified by centrifugation (6000 x g for 10 min); cells that detached from the dishes and were recovered in the pelfets after cfari- fication of the chase media were added back to the cell monolayer fractions. The clarified media samples were adjusted to 1% in Triton X-100, 0.1 m~ in phenylmethylsulfonyl fluoride, 1 pg/ml in pepstatin, and 1 pg/ml in leupeptin by addition of small volumes of concentrated stock solutions of these reagents. The adherent macrophages were solu- bilized by the 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 phenylmethylsul- fonyl fluoride, 1 pgiml leupeptin, 1 pg/ml pepstatin, and 1 mg/ml ovalbumin. After a 30-min incubation on ice, both the media and cell extracts were clarified by centrifugation at 50,000 rpm for 30 min in a Beckman tabletop ultracentrifuge using a TLA 100.3 rotor (Beckman Instruments). IL-lp was recovered from the clarified extracts by immu- noprecipitation as detailed previously (18). The immunoprecipitates were analyzed by SDS-gel electrophoresis and autoradio~aphy (18). In some cases, regions of the dried gel corresponding to radiolabeled IL-lp species were excised, and the radioactivity was solubilized by digestion of the gel piece in 0.5 ml of 0.1 M M s , pH 8, 20 m~ CaCl, containing 5 mg/ml pronase. After a minimum incubation of 4 h at 56 "C, the digests were cooled and diluted with 5 ml of scintillation fluid; the samples subsequently were analyzed by liquid scintillation counting.

Ionophores were dissolved in ethanol and added to the cultures so that the final ethanol concentration did not exceed 0.5%; this concen- tration of ethanol did not effect release andlor maturation of IL-lp. ATP (disodium salt) was dissolved in Dulbecco's modified salt solution to achieve a final concentration of 100 mM. The resulting solution was adjusted to pH 7.2 by the addition of NaOH and stored at -20 "C. ATP, nigericin, and valinomycin were obtained from Sigma; lasalocid and A204 were obtained from the Pfizer compound file. The Nai-containing medium was composed of 137 mx NaCI, 20 m~ Hepes, pH 7.2, 5 mM NaHCO,, 0.5 mM MgCl,, 1.5 mM KH,PO,, 2.7 m~ KCl, 0.9 mm CaCI,, 5 mM glucose, and the Na+-depleted medium was composed of 140 rnlw KCI, 20 m~ Hepes, pH 7.2, 0.5 mM MgCl,, 1.5 mM KH,PO,, 0.9 mM CaCl,, 5 mM glucose.

Intracellular pH Measurements-Mouse macrophages isolated by peritoneal lavage were seeded into six-well multidishes (2 x lo6 cells/ well) that contained glass coverslips; these coverslips were designed to fit into a cuvette of an SLWAminco spectrofluorometer (DMX 1000). After an overnight incubation the cells were stimulated for 2 h with 1 pg/ml LPS and then loaded with the pH-sensitive fluorescent reporter 2',7'-bis-(2-~arboxye~y1)-5-(and -6~arbox~uorescein (BCECF Ref. 28). Individual coverslips were rinsed twice with 2 ml of a modified Dulbecco's buffer that contained 20 mM Hepes, pH 7.2,137 mM NaCI, 0.9 mx CaCI,, 2.7 mM KCI, 1.5 mM KH,PO,, 0.5 mM MgCl,, 5 mM NaHCO,, 5 mM glucose, and 1% FBS. The coverslip subsequently was immersed in 1.3 ml of modified Dulbecco's buffer containing 4 pl of a 1 mgiml B C E C F - ~ solution dissolved in Me,SO (Molecul~ Probes, Eugene, OR). The macrophages were incubated at 37 "C for 10 min, after which the coverslip was rinsed twice with modified Dulbecco's buffer to remove non-cell-associated probe. The coverslip then was positioned within a cuvette that contained 3.5 ml of modified Dulbecco's buffer; this solution was stirred constantly. Fluorescence was measured using an excitation wavelength of 500 nm and an emission wavelength of 525 nm. After a base-line fluorescence intensity was established, an ionophore was in- troduced into the cuvette, and the resulting change in fluorescence intensity was recorded. All fluorescence measurements were recorded at 20 "C.

86Rb+ Effzuz Measurements-Macrophages (5 x lo5) seeded into 24- well dishes (coated with Natrix) were incubated simultaneously with 1 pg/ml LPS and 5 pCi/mt *%6Rb' ( h e r s h a m Corp.) for 3 h in culture medium. These monolayers then were rinsed twice with culture me- dium to remove non-cell-associated rubidium and suspended in 1 ml of fresh culture medium to initiate the chase; where indicated, ionophores or ATP were present in the chase medium. After a 20-min incubation at 37 "C, the media were removed and clarified by centrifugation. Cell monolayers were solubilized in a Triton X-100-containing extraction buffer; radioactivity associated with the chase media and cell extracts was determined by liquid scintillation counting.

Enzyme Assays-The content of lactate dehydrogenase within the clarified cell extracts and chase media was determined using pyruvate as substrate and a colorimetric pyruvate detection assay (Sigma). The content of the lysosomal enzyme p-~-acetylglucosaminidase within these same samples was determined using p-NO,-phenyl-p-N-acetyl- ghcosamine as substrate (29). Microscopy was performed with a Nikon

IL-1 p Post-translational Processing 15197 Diaphot inverted phase microscope and a x 40 objective.

With the exception of Fig. 8 (which was not repeated in its entirety), each presented experiment was reproduced at least twice and compa- rable results were obtained; results shown are representative of all of the data. The ATP responsiveness within RPMI medium, which was examined more than 10 times, is very reproducible in terms of the efficiency of IL-1p processing and the timing of the post-translational events.

RESULTS Ionophores That Mediate a n Electroneutral Potassium Efflux

Induce IL-lp Maturation and Release-In a previous study we showed that nigericin, but not the structurally related iono- phore monensin, mediated the release and processing of newly synthesized IL-lp produced by LPS-stimulated mouse perito- neal macrophages (18). Nigericin is expected to mediate an electroneutral exchange of intracellular K+ ions for extracellu- lar protons (30, 31). In contrast, monensin is expected to me- diate exchange of extracellular Na+ ions for intracellular pro- tons (30, 31). Based on these ionic selectivities, the differential activity observed between nigericin and monensin suggested that either an intracellular acidification and/or a n e M w of potassium was necessary for the maturation and release of IL-1p. To distinguish between these possibilities, several addi- tional ionophores were analyzed. Lasalocid, like nigericin, is expected to exchange intracellular K+ ions for extracellular pro- tons. In contrast, A204 has an equal affinity of K+ and Na+ ions and, as a result, is expected to mediate an electroneutral ex- change of intracellular K+ ions for extracellular Na' ions with- out an accompanying intracellular acidification. Valinomycin is an electrogenic potassium ionophore that mediates movement of K+ ions down a concentration gradient (30, 31).

A

B

a- 35 kDa

17 kDa

1 2 3 4 5 6 7 8 9 1 0 L a n e

35 kDa

, 4 1 7 kDa

and matura t ion of IL-lp. LPS-stimulated ["Slmethionine-labeled FIG. 1. Electroneutral potassium ionophores media te release

macrophages were treated with the indicated concentration of potas- sium ionophore. After a 30-min incubation, cells and media were sepa- rated, and I L l p was recovered by immunoprecipitation. The immuno- precipitates were fractionated by SDS-gel electrophoresis, and autoradiograms of the gels are shown: A, cell-associated immunopre- cipitates and B, media immunoprecipitates. The arrows on the right indicate the migration positions for the 35-kDa proform and 17-kDa mature form of IL-10. Each condition was performed in duplicate: 20 J ~ M nigericin (lanes 1 and 2) , 10 PM A204 (lanes 3 and 4 ) , 25 PM A204 (lanes 5 and 6), 10 PM lasalocid (lanes 7 and 8), 25 PM lasalocid (lanes 9 and 10).

35 kDa

c

B

q 0 N a E =I 0 -

FIG. 2. Ionophores that induce matura t ion and release of ILlp also cause cell lysis. LPS-stimulated [35S]methionine-labeled mac- rophages were treated with the various potassium ionophores for 30 min, after which the cells and media were separated. A, IL-1p was recovered by immunoprecipitation (see Fig. 1). Regions of the dried gel corresponding to the various species of I L l p were excised, and the radioactivity was solubilized by Pronase digestion. The quantity of ra- dioactivity recovered with the various species (as a percentage of the total countdmin recovered as IL-1p) is indicated as a function of iono- phore treatment; to correct for the 2-fold loss of radioactivity that oc- curred during proteolytic cleavage, counts/min recovered as 17-kDa IL-lp were multiplied by a factor of 2 prior to this calculation. B, extracts of the cells and media were analyzed for their content of lactate

these activities recovered in the medium (as a percentage of the total dehydrogenase and p-N-acetylglucosaminidase. The amount of each of

recovered intra- and extracellularly) is indicated as a function of iono- phore treatment.

LPS-stimulated [35Slmethionine-labeled mouse peritoneal macrophages were treated with the ionophores, and the fate of IL-1p was assessed by SDS-gel electrophoresis and autoradiog- raphy after immunoprecipitation of this cytokine. Control mac- rophages released <2% of their newly synthesized IL-1p follow- ing a 30-min incubation in the absence of an ionophore (data not shown). Macrophages treated with either lasalocid orA204, on the other hand, efficiently released radiolabeled IL-1p into their medium (Fig. 1). The released [35S]methionine-labeled cytokine consisted primarily of the 17-kDa mature cytokine species, whereas the cell-associated cytokine was predomi- nantly the 35-kDa species (Fig. 1). The medium also contained a small amount of a 28-kDa species; this species was observed with nigericin (Fig. 1) and presumably resulted from cleavage of pro-IL-lp by interleukin-lp convertase at an alternate cleav- age site (32). Both 10 and 25 p~ A204 caused efficient release of IL-1p into the medium during a 30-min incubation. Lasalocid

15198 Post-trans~ationa~ Processing

1

- 7.100 A - 1.300 c

2 - a -

- 0.000 - 0.000 l . . . . l . . . . l . . . . l . . , l . . , . I _ . . I . . . . ( . .

<O t 00 200 300 400 500> <O 100 200 300 400 500>

SEC SEC

0.820 D

t

0.000 I . . . . l . . . . l . . . . i . . I . .

<O 100 200 300 400 500> <O 100 200 300 400 500>

SEC SEC

FIG. 3. Nigericin and lasalocid induce an intracellular acidification. Mouse peritoneal macrophages were seeded onto glass coverslips. After an overnight adherence, the cells were stimulated with LPS and then loaded with pH-sensitive reporter BCECF. The coverslips were placed

(C), or vehicle only (D) was introduced, and the resulting change in relative fluorescence intensity (RFU) was recorded; the n axis indicates the in a cuvette, and a base-line fluorescence intensity was established. At the 50-s time point, an ethanol solution of nigericin (A), lasalocid (B) , A204

time (s) of the run. The final ionophore concentration was 20 PM, and all measurements were recorded at 20 "C.

a t 10 PM did not release all of the newly synthesized IL-lP, but a t 25 gm this agent caused a near quantitative release (Fig. 1). The radioactive regions of the dried gel c o ~ e s p o n ~ n g to the 17-, 28-, and 35-kDa species of IL-lp were excised, the radio- activity was solubilized by pronase digestion, and the released radioactivity was quantitated by liquid scintillation counting. In the presence of 10 and 25 1" A204, >75% of the radioactivity recovered as IL-1 was associated with the 17-kDa extracellular species (Fig. 2A 1. When the 35-kDa pro-IL-lp species i s cleaved to its mature 17-kDa counterpart, half of the methionines in- corporated into the protein are lost with the cleaved fragment (33). If this loss is taken into account, then the extracellular 17-kDa species produced in the presence of A204 accounts for >80% of the total newly synthesized cytokine, This efficiency is comparable with that observed with nigericin (Figs. 1 and 2A). Lasalocid was less efficient at promoting the production and release of 17-kDa IL-lp at 10 ~ I M (Fig. 2 4 ) ; at 25 pM the effi- ciency of cytokine release was comparable with that observed with A204 and nigericin, but the released cytokine was not processed as efficiently to the 17-kDa species (Fig. 2A). Valino- mycin (20 w) failed to induce the release or maturation of radiolabeled IL-lP (data not shown).

When LPS-stimulated mouse peritoneal macrophages are treated with nigericin, the release of IL-1p is accompanied by the liberation of other cytoplasmic constituents (18). Likewise, A204 and lasalocid induced the release of both the cytosolic enzyme lactate dehydrogenase and the lysosomal constituent P-~-acetylglucosaminidase (Fig. 2B). A204, at both 10 and 25 w, was as effective as nigericin at causing the release of these

two enzymes (Fig. ZB). Lasalocid at 10 p caused release of only 33% of the lactate dehydrogenase and 53% of the 8-N- acetylglucosaminidase activities (Fig. 2B), and at this concen- tration the ionophore caused release of only 57% of newly syn- thesized IL-la (Fig. 2 4 ) . At 25 PM, however, lasatocid released 280% of the lactate dehydrogenase activity and caused a greater externalization of IL-1p. Control cells or cells treated with 25 PM valinomycin released <5% of their lactate dehydro- genase and p-N-acetylglucosaminidase activities (data not shown). Release of IL-lp, therefore, closely paralleled the ex- tent to which the potassium ionophores induced cytolysis.

Cytoplasmic Acidification Is Not Necessary for the Release and Maturation of ZL-l@"As noted above, nigericin and lasalo- cid are expected to cause an intracellular acidification due to the exchange of extracellular protons for intracellular K+ ions. In contrast, A204 is expected to mediate the exchange of intra- cellular K+ ions with extracellular Na' ions rather than pro- tons. To verify this difference in selectivity, LPS-stimulated mouse peritoneal macrophages were loaded with the pH-sensi- tive fluorochrome BCECF, and the BCECF-loaded cells were treated with the ionophores. Both nigericin and lasalocid caused a rapid decrease in the fluorescence intensity indicative of an intracellular acidification (Fig. 3). In contrast, A204 did not change intracellular pH (Fig. 3 0 ; the slight decline in BCECF fluorescence intensity observed with A204 also was seen with the ethanol vehicle control (Fig. 30). This decrease most likely reflected loss of dye from the cells rather than an acidification. At concentrations of A204 that were sufficient to promote maturation and ex~rnalization of IL-1P, therefore,

IL-lp Post-translational Processing 15199

100 1

0 N

FIG. 4. ATP and the potassium ionophores stimulate =Rb+ ef- flux. LPS-treated mouse peritoneal macrophages were loaded with 86Rb+ and then chased for 20 min in the presence of the indicated effector molecules. After the chase, cells and media were separated, and distribution of “Rb+ was determined. The graph indicates the percent-

the total recovered in the cell + medium fractions) as a function of the age of the cation recovered in the medium (expressed as a percentage of

indicated concentration of ionophore or 5 mM ATP; each point is the average of duplicate determinations. The control culture contained only 0.1% ethanol in the chase medium, the vehicle used for solubilization of the ionophores.

the intracellular acidification was minimal relative to that ob- served with nigericin and lasalocid.

Mobilization of Intracellular 86Rb’ by the Zonophores-To demonstrate that the ionophores promoted potassium efflux from LPS-stimulated macrophages, cells were loaded with “Rb’ and release of this potassium analog was determined. After a 3-h co-incubation with 1 pg/ml of LPS and 5 pCi/ml of “jRb+, the macrophages contained 34,000 c p d 5 x lo5 cells. Following a subsequent 20-min incubation in ionophore-free medium, 32% of the cell-associated “Rb’ partitioned extracel- lularly (Fig. 4). Cells incubated in the presence of nigericin, lasalocid, and A204, on the other hand, released >95% of their “jRb’ into the medium (Fig. 4). Valinomycin-treated cells re- leased more “Rb’ than control cultures, but after 20 min of incubation these cells retained approximately 50% of their ra- diolabeled cation. Thus, valinomycin was not as effective as the other ionophores at promoting efflux of radiolabeled rubidium from LPS-treated macrophages.

ATP Znduces a Time-dependent Release and Maturation of ZL-lp from Mouse Peritoneal Macrophages-The release and maturation of IL-1p induced by ATP treatment of mouse peri- toneal macrophages is accompanied by apoptosis or cell death (17). LPS-stimulated [35S]methionine-labeled cells treated with 5 mM ATP rapidly changed their morphology. Within 7.5 min of ATP addition, the macrophages possessed a ruffled appearance that was distinct from the appearance of their non-ATP-treated counterparts (Fig. 5) . After 15 min of treatment the cells were rounded and contained cytoplasmic extensions; longer treat- ments led to continued swelling and an apparent clearing of the cytoplasm (Fig. 5) . After a 10-min treatment with 5 m~ ATP, little IL-lp was recovered extracellularly (Fig. 6). The cells, how- ever, were responding to ATP as evidenced by the appearance of 17-kDa IL-1p intracellularly. Between 20 and 30 min of treat- ment, the amount of 17-kDa IL-1p increased intracellularly, and the 17-kDa species also was observed extracellularly. Treatment

times >30 min led to the continued loss of IL-1p from the cell- associated fraction (Fig. 6); both the 35- and 17-kDa IL-1p spe- cies were detected extracellularly after the longer treatment times (Fig. 6). The ATP-induced release of IL-lP was accompa- nied by the release of cytoplasmic marker enzymes (Fig. 7). Both lactate dehydrogenase and p-N-acetylglucosaminidase were displaced from the cell-associated fraction into the medium dur- ing the ATP treatment (Fig. 7). The release of these cytoplasmic enzymes, however, initially lagged behind the release of 17-kDa IL-1p. Following the 30-min ATP treatment, for example, >55% of 17 kDa IL-lp was recovered extracellularly, whereas only 5 and 11% of lactate dehydrogenase and p-N-acetylglucosamini- dase, respectively, were recovered in the medium (Fig. 7). Dur- ing this same time period <4% of the 35-kDa pro-IL-lp species was recovered extracellularly. Only after longer ATP treatments when lactate dehydrogenase and p-N-acetylglucosaminidase fractionated into the medium did the 35-kDa pro-IL-lp species partition extracellularly (Fig. 7). ATP treatment, therefore, caused a selective release of 17-kDa IL-lp prior to the release of the other cytoplasmic constituents.

Release of ZL-Ip from Mouse Peritoneal Macrophages Is Not Induced by Other ATP Analogs-Mouse peritoneal macro- phages were reported previously to possess a receptor for ATP4-, which when bound to extracellular ATP, caused depo- larization of the cell (34). This receptor, the P,, receptor, is believed to be analogous to an ATP receptor found on mast cells (35). To confirm that the ATP effect on IL-1p was mediated via a specific receptor for this nucleotide triphosphate, mouse peri- toneal macrophages were treated with several analogs of ATP. LPS-stimulated [35Slmethionine-labeled cells treated with 5 mM UTP, GTP, or AMP released no more radiolabeled cytokine than control cells (Fig. 8). 5 mM ATP, on the other hand, led to the release of large quantities of 17-kDa IL-1p and 5 mM ADP also caused externalization of mature cytokine (Fig. 8). Quan- titation of the radioactivity recovered after pronase digestion of the radiolabeled regions of the dried gel indicated that the amount of extracellular 17-kDa IL-1p produced in the presence of ADP was 50% of that generated in the presence of ATP. The ability of ATP to induce the release and maturation of IL-1p from mouse peritoneal macrophages, therefore, was not shared by other nucleotide triphosphates. Moreover, ADP was less ef- fective than ATP and AMP was totally inactive. These data are consistent with the notion that the ATP response is initiated via a P,-type of receptor associated with the macrophage plasma membrane (36).

ATP Znduces the Release of 86Rb+ from Macrophages-ATP treatment of mouse 5774 macrophage-like cells causes a rapid membrane depolarization mediated, in part, by the efflux of potassium from the cells (37, 38). To determine whether ATP- treatment led to a similar efflux of K+ ions from LPS-stimulated mouse peritoneal macrophages, these cells were loaded with “Rb+ and exposed to 5 mM ATP. In the absence of ATP, the “jRb+-loaded macrophages retained the majority of the radiola- beled cation throughout a 20-min incubation (Fig. 4). In the presence of 5 mM ATP, however, the majority of the radioisotope was recovered extracellularly (Fig. 4). The extent of release was similar to that observed with nigericin, lasalocid, and A204.

Substitution of Potassium for Sodium within the Medium Inhibits the ATP- and Nigericin-induced Effects on ZL-l+The results presented above indicated that both ATP and potassium ionophores promoted the release of 17-kDa IL-1p from LPS- stimulated mouse peritoneal macrophages. Moreover, both ATP and the potassium ionophores caused release of intracellular potassium. If these two processes were related, then inhibition of the K+ efflux might impair the maturation and/or release of IL-1p. To examine this, LPS-stimulated [35S]methionine-la- beled macrophages were treated with ATP or nigericin while

15200 IL-lp Post-translational Processing

4

Q ,. I

'*

. "

FIG. 5. ATP induces a time-dependent change in macrophage morphology. LPS-stimulated macrophages (on noncoated plastic surfaces) were treated with 5 mM ATP, and the resulting morphological changes were recorded at 7.5 min (B), 15 min (C), 30 min (D), and 60 min ( E ) after addition of the nucleotide triphosphate. Cells in A were photographed prior to the addition of ATP.

IL-1 p Post-translational Processing 15201

A 10 20 30 60 120 180 (mln)

35 kDa

,4 17 kDa

B 1 2 3 4 5 6 7 8 9 10 11 12Lane

FIG. 6. ATP induces a t ime-dependent maturat ion and release of IL-lp. LPS-stimulated [''~5Slmethionine-labeled macrophages were treated with 5 mhl ATP and IL-1p subsequently was recovered by im- munoprecipitation from extracts of the cells and media; the immuno- precipitates were analyzed by SDS-gel electrophoresis and autoradiog- raphy. The autoradiograms show the IG lp species recovered within the cell (A) and media ( B ) extracts after the indicated treatment times. The arrows indicate the migration positions of the 35-kDa precursor and 17-kDa mature IL-1p species.

maintained in modified Dulbecco's medium that contained NaCl (Na+-containing Dulbecco's) or that contained KC1 in place of NaCl (Na+-depleted Dulbecco's). Cells maintained in Na+-containing Dulbecco's produced 17-kDa IL-1P and released the mature cytokine in response to both 5 mM ATP and 20 VM nigericin (Fig. 9). In contrast, cells maintained in Na+-depleted Dulbecco's did not generate 17-kDa IL-1P or externalize the radiolabeled cytokine in the presence of ATP or nigericin (Fig. 9). A small amount of extracellular 35-kDa IL-lP was recovered from these cells, but this quantity was much less than that observed from cells maintained in Na+-containing Dulbecco's. Cells treated with ATP in Na+-depleted Dulbecco's medium pos- sessed a small amount of a 20-kDa IL-1P species that was not detected in the other cultures (Fig. 9). Quantitation indicated that cells treated with ATP and nigericin in Na+-containing Dulbecco's released 31 and 80% of their IL-1P as the 17-kDa species, respectively. In contrast, cells treated with ATP and nigericin in Na+-depleted Dulbecco's released no detectable 17- kDa cytokine (Fig. 9).

DISCUSSION Mouse 5774 cells, a macrophage-like cell line, were shown

previously to alter their ionic content in the presence of extra- cellular ATP (34, 37-40). Application of ATP to these cells ac- tivated a Ca2+-dependent K+ channel (39,40) and caused a large nonselective conductance across the plasma membrane (34,37, 38). This nonselective conductance led to a rapid depolarization of the cells (34). Examination of the selectivity of this effect indicated that ATP- was the active species and that other ATP analogs were less effective (37, 38). Surprisingly, 5774 cells actually became permeable to small (<go0 kDa) organic mol- ecules in the presence of ATP, but the integrity of the plasma membrane was restored if the ATP was removed from the me- dium within a short time period (41). The available data sug- gested that both the ionic conductance and the movement of the small organic molecules occurred via the same ATP-induced pore; a recent study suggested that this pore may correspond to gap junction protein connexin-43 (42). Similar ATP receptors have been described on mast cells and lymphocytes (35). Based

*I

" I i

1 0 2 0 30 6 0 120 180

Time (min) FIG. 7. ATP induces a selective release of 17-kDa IL-lp. LPS-

stimulated [35Slmethionine-labeled macrophages were treated with 5 mM ATP, after which the cells and media were separated. A, I G l p was recovered by immunoprecipitation (see Fig. 6). Regions of the dried gel corresponding to the various species of I L l p were excised, and the radioactivity was solubilized by Pronase digestion. The quantity of ra- dioactivity recovered with the various species (as a percentage of the total countdmin recovered as IL-1p) is indicated as a function of the time of ATP treatment; to correct for the 2-fold loss of radioactivity that occurred during proteolytic cleavage, countdmin recovered as 17-kDa I G l p were multiplied by a factor of 2 prior to this calculation. B, extracts of the cells and media were analyzed for their content of lactate dehydrogenase and p-N-acetylglucosaminidase. The amount of each of these activities recovered in the medium (as a percentage of the total

ATP treatment time. recovered intra- and extracellularly) is indicated as a function of the

on the selectivity for ATP4 and the type of response elicited, the purinergic receptor that mediates this activity is distinguished as the P,, subtype (35,431.

LPS-stimulated mouse peritoneal macrophages, like 5774 cells, responded to extracellular ATP and mobilized their intra- cellular K+ stores. This was demonstrated by showing that macrophages preloaded with 86Rb+ lost the radioactive cation more rapidly in the presence of ATP than in its absence. The ionic imbalance created by ATP also led to striking changes in the morphology of the macrophages. Many of the LPS-treated cells were spread and tightly adherent to the tissue culture plates. Within minutes of ATP addition, however, the cells be- gan to ruffle and round up. After 15 min of ATP treatment many of the macrophages were ballooned in appearance and were completely detached from the dish. These ATP-induced morphological changes were very similar to changes induced by nigericin (18).

The extent of 86Rb+ loss induced by ATP from LPS-stimulated macrophages was matched by the potassium ionophores nigeri- cin, lasalocid, and A204, but not by valinomycin. The latter ionophore is electrogenic, and as a result of the valinomycin-

15202 IL-lp Post-translational Processing

4 1 7 kDa

B 1 2 3 4 5 6 7 a 9 1 0 1 1 1 2 L a n e

e 3 5 kDa

I

FIG. 8. The ATP-induced maturation and release of IGlp is not caused by other ATP analogs. LPS-stimulated [%]methionine-la- beled macrophages were incubated for 23 min in the presence of 5 mM concentrations of the indicated effectors. At the end of the chase, cells and media were separated, and IL-lP was recovered by immunoprecipi- tation. The immunoprecipitates were analyzed by SDS-gel electro- phoresis and autoradiograms corresponding to the cell-associated (A) and media ( B ) samples are shown. The arrows indicate the migration positions of the 35-kDa precursor and 17-kDa mature IGlp species.

induced efflux of K+ down its concentration gradient (between intracellular and extracellular compartments), the macrophage is expected to hyperpolarize (30,31). This hyperpolarization, in turn, limits the ability of valinomycin to dissipate the intracel- lular K+ ion pool. The "Rb+ efflux induced by nigericin and lasalocid was accompanied by an intracellular acidification. This change in pH, was expected based on an electroneutral exchange of intracellular K+ for extracellular H+ (30,31). A204, on the other hand, did not cause an intracellular acidification; this ionophore differs from nigericin and lasalocid in that it has an equal affinity for K+ and Na+ and, as such, is expected to mediate a neutral exchange of intracellular K+ for extracellular Na'. We did not ask if ATP affected pHi, because the ATP- induced pore was expected to facilitate release of BCECF from the cells (41); this loss would negate the experimental ap- proach.

Importantly, agents that induced a rapid and total 86Rb+ ef- flux also stimulated the release and maturation of IL-1p from LPS-stimulated macrophages. Thus, treatment with ATP, ni- gericin, lasalocid, or A204 led to the production and external- ization of 17-kDa IL-lp. In contrast, valinomycin did not induce an efficient 86Rb+ efflux and did not affect the release of IL-1p. Since A204 did not significantly alter pHi, yet was as effective as nigericin at stimulating the release and maturation of IL-lp, an intracellular acidification was not necessary for cytokine processing. Agents that induced the release and maturation of IL-1p also promoted cell lysis. We have shown previously, how- ever, that cell lysis is not sufficient to induce maturation of pro-IL-lp. Treatment of LPS-stimulated mouse peritoneal mac- rophages with agents such as saponin and the calcium iono- phore A23187, for example, induce the release of IL-lP from macrophages, but the liberated cytokine is not processed to its mature 17-kDa species (18). Moreover, the release of 17-kDa

A

B

' , 6 1 7 kDa

1 2 3 4 5 6 7 8 L a n e

1 4 1 7 kDa

ILlp is inhibited by substitution of NaCl with KC1 in the me- FIG. 9. ATP- and nigericin-induced release and maturation of

d i m . LPS-stimulated [""Slmethionine-labeled macrophages were in- cubated with 5 mM ATP or 20 PM nigericin in sodium-containing or sodium-depleted medium. m e r a 25-min incubation, cells and media were separated, and IL-1p was recovered by immunoprecipitation. The immunoprecipitates were analyzed by SDS-gel electrophoresis and au- toradiograms corresponding to the cell-associated (A) and media ( E ) samples are shown. The arrows indicate the migration positions of the 35-kDa precursor and 17-kDa mature IGlP species.

IL-1p from ATP-treated LPS-stimulated macrophages initially occurred in the absence of the release of other cytosolic compo- nents. Thus, after 30 min of incubation with 5 mM ATP, LPS- treated macrophages produced 17-kDa IL-1p and released 55- 60% of the mature cytokine into the medium. These same cells, however, released 4 % of the cytoplasmic marker enzyme lac- tate dehydrogenase and the lysosomal marker /3-N-acetylglu- cosaminidase; likewise, these cells released only 4% of the 35- kDa IL-1p species during this time period. Lysis of the plasma membrane, therefore, is neither necessary nor sufficient to cause the proteolytic maturation of pro-IL-lp by interleukin-lp convertase.

The ability of ATP to promote the release and maturation of IL-10 from macrophages was not shared by other nucleotide triphosphates nor by AMP ADP was less effective than ATP. Previous studies demonstrated that the pore opening induced by ATP within 5774 cells was elicited by nonhydrolyzable forms of ATP and to a lesser degree by ADP, but not by other nucle- otide triphosphates and AMP (37-39). Thus, the selectivity ob- served with respect to the effectiveness of the various ATP analogs to induce maturation of pro-IL-lp correlates with their ability to depolarize 5774 cells. This correspondence suggested that activation of IL-lp maturation is mediated via a P,,-type of receptor. The ATP-induced potassium emux and maturation of IL-1p were promoted equally well by nigericin, lasalocid, and A204. This similarity in activity between agents as structurally diverse as ATP and nigericin suggested that a potassium efflux and/or membrane depolarization was responsible for initiating maturation of pro-IL-lp. Medium substitution experiments

IL-1 Post-translational Processing 15203

supported the importance of a potassium efflux. Macrophages treated with ATP and nigericin in a sodium-containing medium responded to produce and externalize 17-kDa IL-1p. In con- trast, when these cells were treated with ATP and nigericin in medium that contained potassium chloride rather than sodium chloride, no 17-kDa IL-1p was produced. Since the high potas- sium concentration within the sodium-depleted medium is ex- pected to depolarize the membrane potential, membrane depo- larization alone does not appear sufficient to induce the release and maturation of IL-1p. Rather, inhibition of the IL-1 response observed in the presence of elevated concentrations of extracel- lular potassium suggests that a net movement of potassium out of the cell must occur in order to activate the maturation and release mechanisms. Elevated external K+ is expected to inhibit intracellular K+ depletion induced by ATP and nigericin. Meas- urements of total cell-associated K+ content were not per- formed, and direct proof of a net K+ depletion, therefore, re- mains to be established.

What processes could be activated by a depletion of intracel- lular K+? Studies of interleukin-1P convertase in vitro have indicated that its catalytic activity is inhibited by KC1 and NaCl concentrations in excess of 100 mM (13, 14). It is possible, therefore, that monovalent cations impair interleukin-lp con- vertase activity and that ATP and nigericin activate interleu- kin-lp convertase by depletion of intracellular K'. Alterna- tively, interleukin-lp convertase appears to be synthesized as a precursor that is proteolytically processed post-translationally (15, 16). Perhaps a K+ depletion stimulates conversion of inter- leukin-lp convertase from an inactive to an active state. Fi- nally, the ionic change may lead to the activation of pro-IL-lp maturation by destruction of a compartmental barrier that separates interleukin-lp convertase from its cytokine sub- strate within the resting cell. The ionic conductance promoted by ATP and nigericin is paralleled by dramatic changes in mac- rophage morphology. Changes that we observed at the light level undoubtedly are accompanied by changes of the intracel- lular architecture. Although interleukin-lp convertase and pro- IL-lp appear to co-exist within the cytosolic compartment, these two proteins may not be present within the same subcel- lular domains. K+ depletion may lead to architectural changes that liberate either interleukin-lp convertase or pro-IL-lp from a previously sequestered location. Interestingly, macro- phages are known to contain a cytoskeletal component, acu- mentin, whose association with actin is inhibited by high po- tassium concentrations (44). Moreover, IL-1p is reported to associate with microtubules in activated U937 cells (45). Acti- vation of IL-16 maturation, therefore, may require reorganiza- tion of cytoplasmic matrix components. Stimuli that induce maturation and release of IL-lp in vitro ultimately lead to cell lysis. When ATP was used as the stimulus, release of the 17- kDa IL-lp species initially was observed in the absence of re- lease of the 35-kDa species. This selectivity is consistent with the proposal that a secretory mechanism exists that prefers the mature cytokine species (27). Whether IL-lp maturation and release can be achieved in vivo in the absence of cell death remains to be determined.

AcknowledgmentsSpecial thanks to Ann Cunningham for provid- ing the antiserum against murine IL-lp. In addition, we thank the other members of the Inflammation Project Team at Pfizer for the suggestions and helpful discussions.

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