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1032 C. Miossec et al. Eur. J. Immunol. 1996.26: 1032-1042Christine Miossec,Mane-Claude D ecoen,Laurence Durand,Florence Fassyand Anita Diu- HercendRoussel Uclaf,Romainville, France

Use of monoclonal antibodies to studyinterleukin -1P-converting enzyme expression:only precursor forms are detected ininterleukin-lg-secreting cellsWe have generated a series of monoclonal antibodies (mAb) using recombinantinterleukin (1L)-lp-converting enzyme (ICE) p20 and p10 subunits as immuno-gens. The mAb have been selected for further study based on their reactivitywith ICE in transfected COS cells and their lack of cross-reactivity with TX, theclosest ICE homolog known to date. Two anti-p20 and one anti-pl0 mAb havebeen used to study ICE expression by Western blotting and immunodetection. InICE-transfected COS cells, the mAb recognize the p45 ICE precursor and thematuration products (p20 or p10 subunits) for which they are specific. In mono-cytes and cell lines expressing ICE, only precursor forms are detected and intra-cellular immunostaining followed by confocal microscopy shows that they arelocated in the cytoplasm. Quantification experiments show that THPl cellsexpress approximately 67000 molecules of ICE precursor per cell, with an esti-mated precursor to mature ratio of at least 100. In these cells as well as in mono-cytes, lipopolysaccharide stimulation did not change the pattern of ICE expres-sion, although efficient secretion of mature IL-lP was measured. However, uponcell disruption, precursor maturation was observed. Our results, therefore, showthat ICE is present in cells as a large pool of intracytoplasmic precursor, and thatvery limited amounts of mature ICE protein are present, but nevertheless suffi-cient to allow efficient IL-1p cleavage. Altogether, these observations suggestthat post-translational maturation of the precursor protein could represent a spe-cific step in the regulation of ICE enzymatic activity.

1 IntroductionProinflammatory cytokines are produced by monocytesand other cell types in response to many inflammatory sti-muli. Among these cytokines, interleukin-1P (IL-1p) has alarge array of biological activities and plays a pivotal rolein inflammation by acting on different cell types and induc-ing the production of others proinflammatory cytokinessuch as tumor necrosis factor-a (TNF-a) [ l , 21. IL-1p issynthesized as an inactive cytoplasmic 31-kDa precursor(proIL-lp) and extracellular release of its mature form of17 kDa requires proteolytic cleavage between Asp 116 andAla 117. This cleavage is performed by an intracellular cys-teine protease called IL-lp-converting enzyme (ICE)which specifically cleaves at Asp-X bonds [3, 41.ICE was initially purified and characterized from thehuman monocytic cell line THPl [3, 41. Active ICE wasshown to be a heterodimeric enzyme containing a 20-kDasubunit (p20) paired with a 10-kDa subunit (p lo), both ofwhich are derived from a 45-kDa precursor (p45) by prote-

[I 153391Received January 22, 1996; accepted February 13, 1996.Correspondence: Christine Miossec, Roussel Uclaf, 102-111route de Noisy, F-93235 Romainville Cedex, France (Fax: +33-1-49 91 52 57)Abbreviations: ICE: Interleukin-lp-converting nzyme GAM:Goat anti-mouseKey words: Interleukin-lp / Interleukin-1p-converting enzyme /Monoclonal antibody / Prccursor maturation / Subunit

olytic cleavage of at least three Asp-N bonds. Recent crys-tallographic studies of ICE have shown that both subunitsare involved in the formation of the enzyme active site. Inaddition, the two subunits associate with each other toform a (p2O/p10), tetramer in the crystal [5 , 61. Althoughmature ICE subunits are derived by cleavage at Asp-Xbonds, which suggests that automaturation could occur,the mechanism of conversion of the proenzyme into theactive heterodimeric ICE remains unclear. Initial studieshave shown that active ICE is able to maturate the p45precursor in vitro and tha t purified p45 or p30 (lacking the119 N-terminal amino-acids) can autoprocess in vitro to theactive form [3, 71. Furthermore, it has been demonstratedthat ICE polypeptides exist as oligomers in transfectedcells and that association of ICE precursor polypeptides inquaternary structure is required for autoprocessing [8].The structural model proposes that each p20/p10 heterodi-mer in the tetramer is derived from two different p45 pre-cursor proteins; the two proenzyme molecules associateand process to form a mature protein in which each cata-lytic domain includes a p10 from one proenzyme and a p20from the other [5, 6, 81. Alternatively, given the distancebetween the C terminus of p20 and the N terminus of p10within each catalytic domain, it has been postulated thatdimerization could occur after proteolytic activation [S].However, under physiological conditions, ICE maturationcould be ensured by an unknown protease or by anothermember of the ICE family. Indeed, ICE was the firstidentified member of a new cysteine protease family, withsimilarities with the product of the nematode cell deathgene CED-3 [9], that now includes five mammalian homo-logs: CPP32 / Apopain / Yama [lo-121; Nedd-2 / ICH-1[13, 141; TX / ICErel-I1 / ICH-2 [15-171; ICErel-111 / TY

0014-2980/96/0505-1032$10.00 + .25/0 0 VCH Verlagsgesellschaft mbH, D-69451 Weinheim, 1996


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Eur. J . Immunol . 1996.26: 1032-1042 Cellular expression of interleukin-lp-converting enzyme 1033[16, 181; and Mch-2 [ls)]. Comparison of their primarysequences reveals conservation of the residues implicatedin the active site for catalysis, stabilization of the inter-mediary substrate or contacts with the substrate Asp resi-due, as determined by the crystal structure of ICE [5, 61.This suggests that all proteins of the family are cysteineproteases with specificity for Asp-X bonds [20]; in addi-tion, all members are likely to mature from a precursorprotein into two subunits. Therefore, one cannot excludeinteractivation of the different family members by a cas-cade of proteolytic events. However, despite their similar-ity with ICE, ICE homologs have been found unable tocleave the IL-lp precursor efficiently, and their specificsubstrates are still mostly unknown.To further study ICE precursor and subunits expression incells, we have generated and characterized a series ofmonoclonal antibodies (mAb) against human ICE. Twoanti-p20 and one anti-pl0 mAb have been studied. Theyhave been selected for recognition of ICE in transfectedcells, and for their lack of cross-reactivity with TX, the clo-sest ICE homolog known to date. Expression of the differ-ent polypeptides corresponding to ICE was studied by Wes-tern blotting and immunodetection in human PBMC andcell lines. In addition, one of the anti-p20/ICE was used inintracellular staining experiments. Our results show thatICE precursor is the predominant form expressed in cellsand is located in the cytoplasm. Neither the p20 or p10 ICEsubunits nor ICE activity can be detected in cell lysates,even during active IL-1p secretion by the cells. Quantifica-tion experiments show that THPl cells express approxi-mately 67000 molecules of ICE precursor per cell, with anestimated precursor to mature ratio of at least 100. Ourresults, therefore, show that very limited amounts ofmature ICE protein are present in cells but are neverthelesssufficient to allow efficient IL-1p maturation.

2 Materials and methods2.1 Obtention of recombinant ICE proteinsThe human p20 (coding for amino acid 120-297) and p10(coding for amino acid 317-404 plus the following tag atthe N terminus: MDYKDADDDR) ICE cDNA wereexpressed in E. coli under the control of the Tac promotor.p20 protein was purified from inclusion bodies by gel filtra-tion in denaturing conditions (7 M urea). p10 protein waspurified from periplasmic fluid by anion exchange chroma-tography. Purified p20 and p10 were generously given byDr. T. Fox (Vertex Pharmaceuticals, Cambridge, MA).The human p30 ICE cDNA coding for amino acid 120-404was expressed in E. coli under the control of the heat-inducible pL promotor. p30 preparations were purified frominclusion bodies in denaturing conditions, then allowed torefold and autoprocess to generate active enzyme.

tions of the same amounts of protein emulsified in IFA at2-week intervals. Two weeks later, 23 pg protein wereinjected intravenously in PBS, followed by splenectomy 3days later. Somatic hybridization of spleen cells with NS-1myeloma cell line was carried out according to classicalprocedures. Hybridoma supernatants were tested byELISA with p20 or p10 as the coated proteins, andselected mAb were produced as ascites fluids i n mice, andconcentrated through ammonium sulfate precipitation.Three of them were characterized further, the anti-p20CAL and LO1 mAb (IgG2b and IgGl isotype, respec-tively), and the anti-pl0 ALP mAb (IgG2b isotype).2.3 In vitro culture of cell linesThe following human myeloid leukemia cell lines wereused: the erythroleukemia cell line K562, the very earlymyeloblast KG1, the promyelocyte HL60 and the myelo-monocytes U937 and THP1. The other cells used were Tcell lines (Jurkat and CCRF-CEM), B cell lines (Raji, HS-SULTAN and RPMI 8226) and nonhematopoietic celllines (WISH amnion cell line and A431 epidermoid carci-noma cell line). COS-7 cells were used for transientexpression of various cDNA.THPl and COS-7 cell lines were kindly provided by VertexPharmaceuticals. All other cell lines used were from theAmerican Type Culture Collection (Rockville, MD). Theywere all tested for the presence of mycoplasmas and foundto be negative. All cell lines were maintained in RPMI1640 medium supplemented with 10% (v/v) heat-inactivated fetal calf serum (FCS), 2 mM L-glutamine, 1mM sodium pyruvate, 10 mM Hepes, 100 IU/ml penicillinand 100 pg/ml streptomycin, except COS-7 cells that weremaintained in supplemented Dulbeccos modified Eaglesmedium (DMEM); 20 WM2-ME was added in TH Pl cellculture medium.2.4 Transient tran sfection of COS-7 cellsThe SV40-based mammalian expression vector pcDL-SRa296 was used for the transient expression of the cDNAin COS cells using the DEAE-dextran method; the pcDL-ICE45 plasmid, containing the complete human ICE codingsequence, and the pcDL-TX plasmid, containing the com-plete human TX coding sequence [151, were kindly providedby D. Livingston (Vertex Pharmaceuticals) and C. Faucheu(Roussel Uclaf, Romainville, France), respectively. COS-7cells were seeded the day prior to transfection in DMEMsupplemented with 10YOFCS at a density of 1.5-2~10~ells/100 mm petri dish. For transfection, cells were treated 4 hwith 5 pg plasmid DNA in 8 ml culture medium containing1% FCS, 400 pg/ml DEAE-dextran and 100 pM chloro-quine. Medium was then removed and replaced for 1min byPBS plus 10% DMSO. After washing, cells were incubatedin complete DMEM medium for 20-30 h.

2.2 Production of the anti-ICE m onoclonal antibodies 2.5 LPS activation of PBMC and T H P l cellsSix-week-old Biozzi mice were immunized with recombi-nant p20 or p10 ICE proteins. The immunization scheduleconsisted of an intraperitoneal injection of 23 pg proteinemulsified in CFA followed by three intraperitoneal injec-

Human PBMC were isolated through Ficoll-Paque densitygradient centrifugation of leukopheresis residues fromblood donors. They were stored frozen and thawed beforethe activation assay. Briefly, PBMC were seeded at 2 . 5 ~ 1 0 ~


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1034 C. Miossecet al. Eur. J . Immunol. 1996.26: 1032-1042ceIIs/mI in supplemented RPMI medium, and LPS ( E . ColiOlll:B4, Sigma, Saint Quentin Fallavier, France, cat. no.L3012) was added at the final concentration of 1 pg/ml.After various periods of incubation, supernatants werecollected and tested by ELISA for their content in IL-1p(R&D Systems, Abingdon, GB, cat. no, 201-LB); cell pel-lets were washed in PBS and resuspended in lysis buffer.THPl cell line was stimulated in the same way, except thatcells were seeded at 2x10' cells/ml and that LPS ( E . Coli055:B5, Sigma Chimie, cat. no . L2880) was used at 5 pg/ml.

2.6 Preparation of cell extracts for electrophoresisCells were washed twice in cold PBS and the cell pellet wasresuspended in ice-cold hypotonic lysis buffer containing20 mM Tris base pH 7.2, 1% Triton X-100, 1 mM EDTA,10pghl trypsin inhibitor, 2 pg/ml aprotinin, leupeptin andpepstatin, and 1 mM N-ethyl maleimide. All steps wereperformed at 4C to prevent autocleavage of ICE proteins.Following a 30-min incubation, the homogenates wereclarified by a 10-min centrifugation at 13000 rpm; thesupernatants were collected, diluted in sample buffer con-taining 2-ME, boiled at 95C for 10 rnin and stored at-80C until electrophoresis. For the autoprocessing assays,lysates were prepared with the same procedure, exceptedthat N-ethyl maleimide was omitted in the lysis buffer; celllysates were incubated for various times at 22C beforedenaturation and electrophoresis.

2.7 Western blotting and immunod etectionLysates from approximately 1.5~10' ells were run on 12%or 16% precast polyacrylamide gels (Novex, San Diego,CA) containing 0.1% SDS, along with plO/ICE or p20/ICE recombinant proteins as positive controls, and pre-stained RainbowTM protein molecular weight markers(Amersham France, Les Ulis, France). Proteins were elec-trotransferred onto polyvinylidene difluoride membranes(Immobilon, Millipore Corporation, Bedford, MA). Theblots were blocked for 1 h at room temperature with 5%skim milk in PBS and probed with an anti-ICE mousemAb (diluted in 5% skim milk and 0.2% Tween 20 inPBS; dilution 1/500000 for CAL and LO1 mAb and1/100000 for ALP mAb) for 1 h at room temperature.After washing, the membrane was further incubated for1 h at room temperature with a horseradish peroxidase-conjugated goat anti-mouse (GAM) IgG in the same dilu-tion buffer (GAM-IgG1 to reveal LO1 mAb and GAM-IgG2b to reveal CAL and ALP mAb). Antibodies boundto the blots were visualized using the enhanced chemilumi-nescence detection system (ECLTM;Amersham France)followed by autoradiography (Hyperfilm ECL, AmershamFrance). To quantify intensity of the bands, films werescanned with a Biocom 2.11 densitometer and integratedareas of signals were calculated using the Lecphor soft-ware.

2.8 ProIL-1 p cleavage assayProIL-1p labeling was performed by simultaneous in vitrotranscription and translation using the TNTTM-coupled

reticulocyte lysate systems (Promega, Madison, WI).Briefly, 1 pg circular plasmid pBS-SK+ containing thecomplete coding sequence of the human IL-1p precursor(kindly given by S . Roman-Roman, Roussel Uclaf) wasincubated with 25 p1 rabbit reticulocyte lysate according tothe manufacturer's protocol, in the presence of 'I7 RNApolymerase and 40 pCi ["S]methionine (AmershamFrance); the reaction was incubated at 30C for 1 h.The labeled proIL-lp was directly used to evaluate ICEactivity in cell lysates. Lysates were prepared as describedfor Western blotting procedures, except that the cysteineproteases inhibitor N-ethyl maleimide was omitted. Celllysate (12.5 pl) was diluted 1:1 with 10 mMTris buffer con-taining 1 mM DTT and 0.1 '% Chaps, then incubated for 1h at 37C with 1 pl [35S]proIL-lp.Recombinant active ICE(autoprocessed and refolded p30 protein) was used inparallel as a control of cleavage activity (3.3 pg ICE wasincubated in the same conditions with 1 p1 proIL-lp). Theproducts of cleavage were resolved on 16% precast polyac-rylamide gel (Novex) in denaturing conditions and visual-ized by autoradiography.2.9 Immunofluorescence, cytofluorimetry and confocalmicroscopyThe cell surface immunofluorescence assays were per-formed according to conventional procedures. Briefly, liv-ing THPl cells from exponentially growing cultures wereincubated for 30 rnin at 4C with predetermined saturatingconcentration of the first antibody (LO1 anti-p20 ICEmAb or irrelevant negative control mAb diluted to1/20000 in PBS), and then with fluorescein-conjugatedGAM Ig serum in a second step (Coulter, Hialeah, FL).CD14 labeling was performed as a direct immunofluores-cence assay by a 30-min incubation at 4C with FITC-conjugated anti-CD14 mAb (Immunotech, Marseille-Luminy, France); negative control was an FITC-conjugated irrelevant murine IgGl mAb (Southern Bio-technology, Birmingham, AL). Samples were run o n aCoulter Epics ELITE flow cytometer (Coulter).For intracellular staining, exponentially growing TH Pl orK562 cells were washed twice in PBS and were fixed in3.7% paraformaldehyde and 0.03 M sucrose for 15 rnin atroom temperature. After quenching for 10 rnin in 50 mMNH4Cl in PBS, the cells were washed once in PBS contain-ing 1mg/ml bovine serum albumin, and permeabilized for15 rnin at 37C in 0.05% saponin in the buffer used forwashing. Cells were then incubated for 1 h at room tem-perature with the first antibodies (LO1 anti-p20/ICE anti-body diluted to 1/20000 in the permeabilizing buffer, orirrelevant IgGl murine mAb). After two washes in thesame buffer, the presence of antibodies was revealed byincubating the cells for 1h at room temperature in permea-bilizing buffer containing labeled second antibody (WOO,FITC-conjugated GAM IgG 1, Southern Biotechnology).After three washes in permeabilizing buffer and one washin PBS, the cells were separated in two samples for cyto-fluorimetric analysis and for microscopic study. Samplesfor cytofluorimetry were analyzed immediately after label-ing o n ELITE apparatus. Samples for microscopy weremounted in 50 mg/ml Dabco (Sigma France), 100 mg/mlmowiol 4.88 (Calbiochem, La Jolla, CA), 25% glycerol,


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Eur. J. Immunol. 1996.26: 1032-1042 Cellular expression of interleu kin- ID-converting enzymc 1035

. .D10 3-S lM D119N120 D297-52911 D316-A317Figure 1. Schematic drawing of human ICE precursor primarystructure . Four Asp-X cleavage sites generatin g previously identi-fied products are indicated. Dashed areas represent the p20 andp10 polypeptides which compose the active heterodimeric ICEenzyme. Arrow s shows various poten tial products of cleavage.

100 mM Tris-HCI pH 8.5. Cells were examined under aconfocal microscope (Leica, Wetzlar, Germany); opticalsections were recorded with a xl00 lens and a pinhole aper-ture such that the thickness of the sections was about 0.5pm. Photographs were taken on Kodak Ektakrome 100ASA.

3 Results3.1 Generation and characterizationof mAb against ICEMice were immunized with p20 or p10 recombinant ICEproteins (Fig. 1)produced in E.coZi, and their splenic cellswere fused with the NS-1 myeloma cell line to generatehybridomas. Supernatants of the growing hybridomaswere screened by ELISA on the recombinant immunizingproteins. This first-line screening identified a series ofreactive clones, from which eight directed against p20/ICEand five directed against plO/ICE were selected for furthercharacterization. The specificity of these mAb for the ICEprotein was tested by Western blot analysis using lysates oftransfected COS cells. The entire cDNA coding sequenceof ICE was transiently expressed in COS cells and controlcultures were performed with the empty vector or aftermock transfection. Furthermore, to detect potential cross-reactivities of the anti-ICE mAb with ICE-related pro-teins, COS cells were also transfected with the codingsequence of the newly identified TX protein, which wasshown to display 59% and 64% amino acid identity withICE in the p20 and p10 regions, respectively [15]. The cellswere lysed 24 h after transfection and proteins were sub-

jected to SDS-PAGE separation, electroblotting and stain-ing with the various anti-p20/ICE and anti-plO/ICE mAb.At that time, about half of these reagents could beexcluded as they reacted with major or minor bands pre-sent in control lysates, i.e. untransfected cells and COScells transfected with vector alone. Of the remaining mAb,two anti-p20/ICE (CAL and LO1 mAb) and one anti-pl0/ICE (ALP mAb) were further characterized, and Fig. 2shows the results obtained with these antibodies. Recom-binant p20 or p10 were used as positive controls and couldbe detected with the corresponding mAb. No protein wasstained in either lysates from mock or empty vector trans-fection (Fig. 2, lanes 1 and 4, respectively), or in lysates ofcells transfected with TX cDNA (Fig. 2, lane 3). TXexpression was verified with a specific anti-peptide antise-rum (data not shown).In ICE-transfected cell lysates, the three mAb CAL, LO1and AL P stained several bands and showed partially simi-lar patterns (Fig. 2, lane 2). A major protein correspondingto the precursor ICE p45 protein was found around 45 kDawith the three mAb (see Fig. 1). At least three or fourother bands were detected between 45 and 30 kDa withthe three mAb. They probably correspond to intermediateforms of processing between p45 and p30 ICE proteins. Inaddition, the anti-p20 CAL and LO1 mAb specificallyreacted with a doublet at about 20 kDa. This doublet mostlikely corresponds to the two forms of the p20 IC E sub-unit, p22 (amino acids 120-297) and p20 (amino acids103-297) previously described [3], the p20 species beingpredominant. Finally, the anti-plO/ICE ALP mAb specifi-cally stained two bands at about 10 kDa that were not seenwith the anti-p20 mAb. These two bands likely correspondto p10 (amino acids 317404) and p10 linked with the 19-amino acid peptide spanning between the p10 and p20 ICEsubunits (amino acids 298404, see Fig. 1). Note thatrecombinant p10 migrates between the two ICE bandsobtained in transfected COS lysates due to the 9-aminoacid tag peptide linked to it.Altogether, the pattern obtained with transfected COScells indicates that the selected anti-ICE mAb are highlyspecific for ICE, do not react with the closely related TXprotein and can detect the p45 ICE precursor protein aswell as processed products including p20 and p10 subunitsin immunoblotting procedures.

CAL mAb LO1mAb ALP mAbFigure 2. mmuno blot analysis of anti-IC E mA b reactivity w ith lysates from p45 ICE-tran sfected CO S cells. C OS cells were transfectedwith different plasmids: pcDL-ICE45 for expression of p45 ICE cDNA (lane 2), pcDL-TX fo r expression of p43 TX cDNA ( lane 3),pcDL-SRa 296 vector alon e (lane 4); mock transfections were performed as controls ( lane 1 ) . Cells were lysed 24 h after transfectionand lysates were subjected to 16% SDS-PAG E, blotted and probed with the anti-ICE mA b, CA L anti-p20, LO1 anti-p20 or AL P anti-p10 as indicated. Recomb inant p20 ICE or recom binan t p10 IC E (10 ngflane) were m igrated as controls. Arrow s indicate position of th ep45, p20 and p10 ICE proteins. Num bers on the left indicate molecular size of standards in kDa .


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3.2 Expression of ICE proteins in transfected COS cellsThe time course of appearance of the various forms of theICE protein was investigated in transfected COS cells.Cells were lysed at different time points after transfection,and immunodetection was carried out with either CALanti-p20/ICE mAb or AL P anti-plO/ICE mAb. The resultspresented in Fig. 3 show that no protein is detectable 2 hafter the beginning of the transfection. ICE proteinexpression appears 14 h after transfection and increasesuntil 26 h. However, when transfection experiment waspursued beyond 26-30 h, a high level of cell mortality wasobserved as previously described [151, and ICE expressioncould not be further studied. At 14 h, only p45 is detect-able with traces of p20 (Fig. 3A). At 20 h, both p45 and~ 2 0 1 ~ 2 2r p45 and plO/p12 are seen with the respectivemAb and their expression increases at 26 h (Fig. 3A, B).

Figure 3. Kinetic analysis of ICE proteinsexpression in transfected COS cells. COS cellswere transfected with pcDL-ICE45 for expres-sion of p45 ICE cDNA (as indica ted ICEc D N A + ) or pcDL-S Ra296 vector alone (asindicated ICE cDNA-). Cells were lysed at dif-ferent time points following transfection andlysates were subjected to 16% SDS-PAGE,blotted and probed with CAL anti-p20 ICEmAb (A) or ALP a n t i-p l0 IC E mAb (B ) . C on-trol recombinant p20 or p10 IC E polypeptides(10 ngllane) are shown. Numbers on the leftindicate molecular size of standards in kD a.ICE expression was also tested with CAL anti-p20/ICEmAb in a series of cell lines including hematopoietic (Jur-kat and CCRF-CEM T cells; Raji, HS-Sultan and RPMI8226 B cell lines) and non-hematopoietic cells (the WISHamnion cell line and the A431 epidermoid carcinoma)(Fig. 4B). The two T cell lines and two of the B cell lines(Raji and HS-Sultan) were found negative for ICE expres-sion. RPMI 8226, WISH and A431 were clearly positive.In all cell types expressing ICE, the same pattern ofexpression was found, with a predominant signal at 45 kDacomposed of two close bands and corresponding to the p45A

These results show that ICE p45 is expressed first andremains the predominant form expressed in transfectedCOS cells. It is slowly converted to p20 and p10 subunitsthat appear later and accumulate in the cells. However,although significant amounts of p20 and p10 are detectedby 20 and 26 h, the intensity of the 45-kDa band does notdiminish, suggesting that synthesis of the p45 protein is acontinuous process in the transfected cells.B

3.3 Expression of ICE proteins in PBMC and cell linesICE expression in PBMC and cell lines of the myelo-monocytic lineage was analyzed with CAL anti-p20 andALP anti-pl0 mAb (Fig. 4A). PBMC and the cell linestested, including THP1, KG1, HL60 and U937, stainedpositively with the mAb, with the exception of K562,which did not show any band. The pattern of expression ofthe ICE proteins was identical with the two mAb and wasvery similar for these different cells: a major signal wasdetected at 45 kDa, which was composed of two bands.For HL60 and THP1, a second signal was generally seen atabout 35-40 kDa, also composed of two close bands.When films where overexposed, a series of minor bandsappeared between 30 and 45 kDa. However, the doubletsp20lp22 and plO/p12 could not be visualized under ourconditions of immunodetection. The LO1 anti-p20 ICEmAb gave similar results (data not shown).

Figure 4 . Expression of ICE proteins in human cell l ines andPB MC . (A) Expression of ICE was analyzed in PBMC andmyelo-monocy tic cell lines by immunob lotting with C A L anti-p20mAb and ALP ant i-p l0 mAb. Lane l= TH Pl , lane 2=U937, lane3=H L60 , lane 4=PBM C, lane 5=K G1, lane 6=K562. Cell lysateswere subjected to 12 % SDS -PAGE, blotted and probed with theindicated mA b. Reco mbinant p20 or p10 IC E polypeptides (10 ng/lane) are shown as controls. (B) Expression of ICE was analyzedby imm unoblotting in various cell l ines: CCRF -CEM (lane l ) ,WISH (lane 2) , Jurka t ( lane 3) , T HP l ( lane 4), A431 (lane 5),K562 (lane 6), RPMI 8226 (lane 7), HS-SULTAN (lane 8). Celllysates were subjected to 16% SDS-PAGE, blotted and probedwith CA L anti-p20 mAb . Reco mbinant p20 ICE polypeptide (10@lane) is shown as a control. Num bers on the left indicate mole-cular size of standards in kD a.


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Eur. J. Immunol. 1996.26: 1032-1042 Cellular expression of interleukin-lp-converting nzyme 1037ICE precursor. Intriguingly, this 45-kDa doublet does notalways migrate at exactly th e same apparent molecularmass; in KG 1 lysate it is slightly higher than in THPl orPBMC lysates, whereas in A431 lysate it is slightly lower.The CAL anti-p20 mAb did not react with lysates fromJurkat cells, thus showing that it does not recognize CPP32and ICH-1 proteins that are expressed in these cell lines([lo] and data not shown).These results show that the three selected anti-ICE mAbspecifically recognize natural human ICE in PBMC andcell lines, and that ICE is constitutively expressed inPBMC and in a number of hematopoietic and non-hematopoietic cell lines. Moreover, we show that the p45ICE precursor is the predominant species in all cell typesexpressing ICE. In contrast to ICE-transfected cells, nop20 or p10 ICE subunits could be detected under our con-ditions in lysates of cells naturally expressing ICE.

3.4 Quantification of ICE in T H P l cellsIn light of these results, it was of interest to quantitate theamount of ICE precursor in the cells and to evaluate thelimit of detection of ICE proteins with our immunostain-ing procedure. Recombinant p20 was used as a standard

A1 2 3 4 5 6 7 0 9

46 -30 -21 -14 -6.5 -

CAL rnAb

B- 4 I

0 500 1000 1500 2000 2500Signal (arbitrary units)Figure 5 . Quantification of ICE precursor in THPl cell lysates.(A) Serial dilutions of THPl cell lysate (corresponding to 10' cells,0.5~10' ells, 0.25~10'cells and 0.12~10'cells; lanes 1,2, 3, and 4,respectively) or recombinant p20 polypeptide (2.5, 1.25, 0.8, 0.5and 0.25 ng; lanes 5, 6 , 7 , 8 and 9, respectively) were subjected to16% SDS-PAGE, blotting and immunodetection with CAL anti-p20 mAb. Numbers on the left indicate molecular size of stan-dards in kDa. (B) Film from autoradiography was scanned andintegrated signals obtained with recombinant p20 were plottedagainst its concentration. The standard curve obtained was used todeduce the amounts of ICE proteins in THPl lysates (Table 1).

and twofold serial dilutions (from 2.5 to 0.18 ng) weremigrated in parallel with various amounts of THPl celllysates (corresponding t o 106-0.125x106 ells). The blot wasprobed with CAL anti-p20/ICE mAb (Fig. 5A) and thefilms obtained were scanned with a densitometer. Signalintensities were plotted versus the amount of recombinantp20 and the resulting curve (Fig. 5B) was used to deducethe amount of p45, assuming that under these denaturingconditions the CAL epitope is recognized in the same wayin recombinant p20 and natural p45. A 2.25 correction fac-tor corresponding to the p45/p20 molecular weight ratiowas introduced to calculate the quantities of p45/ICE inTHPl cell lysates. As shown inTable 1, the quantity of p45/lo6cells was deduced from each signal and was averaged.In two separate experiments we obtained 4.2 and 5.4 ngICE precursor/106 THPl cells, which represents about67000 molecules of ICE p45kell.These ICE p45 quantification assays were carried o u t onfilms with short exposure (15-30 s) to generate signals pro-portional to the emitted chemiluminescence and avoidsaturation of the film. Under these conditions, the p45 pre-cursor was the only detectable band in THP1. The sameexperiments were carried out with overexposed films toincrease the sensitivity of the test, and a different calibra-tion curve was established using lower amounts of recom-binant ICE p20, from 500 to 10 pg. Using this procedure,a p20 signal was still undetectable in THPl lysates corres-ponding to lo6cells, while a faint but readily visible bandwas found with 20 pg recombinant protein (data notshown). This indicates that the p20 protein is expressed inthese cells in quantities below the detection limits of theassay, i . e . below 20 pg/106 cells. This corresponds to 600molecules of ICE p20/THP1 cell. Therefore, each THPlcell contains around 67000 p45 ICE molecules and lessthan 600 p20 molecules, and the ratio of precursor versusmature ICE is at least 100.

Table 1. Quantification of ICE precursor in THPl cell lysateExperiment 1 Experiment 2

TH Pl lysatea) Signalb) p45" (ng) Signalb) p45" (ng)(arbitrary (arbitraryunits) units)10' cells 1373 3.62 1904 4.70

0.5 x 10'cells 648 2.09 907 2.630.25 x 10'cells 303 1.33 283 1.530.125 X 10'cells 107 0.65 71 N D ~ )Mean for lo6 cells') 4.2 5.4a) Serial dilutions of THPl cell lysates were run on 16% SDS-polyacrylamide gels and blotted; immunodetection was per-formed with the CAL anti-p20 ICE mAb.b) Films obtained from autoradiography were scanned to calcu-late integrated areas of signals.c) Quantity of p45 ICE was deduced from a standard curve estab-lished with recombinant p20 (Fig. 5).d) Not determined (value out of the standard curve).e) Mean quantity of p45 ICE in 10' THPl cells (corrected andaveraged data obtained with the various lysate samples, i . e .from lo6 cells to 0.125 x lo6cells).


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1038 C. Miossec e t al.A

E u r . J. Immunol . 1996.26: 1032-1042B

lo without LPS I

l ime in hours

3.5 Expression of ICE pr oteins and activity of ICEduring LPS stimulationTo further investigate these results, ICE expression wasfollowed in PBMC during activation with LPS, whichinduces secretion of mature IL-1p in these cells. Cells werecultured for 6 or 24 h in the absence or in the presence ofLPS. Culture supernatants were tested for release ofmature IL-16 and cell lysates were analyzed for ICEexpression. As shown in Fig. 6A, when PBMC were sti-mulated with LPS, the 45-kDa band was clearly seen at alltime points and did not change in intensity as compared tocontrol over the 24-h period of incubation. In parallel, in

1 2 3 4 5 6

46 -30 -21 -14 -7 kDa+ 6.5 -

Figure 7. ICE activity in PBM C lysates. [35S]proIL -lpwas gener-ated using an in v i m ranscription/translationmethod. It was thenincubated with recombinant active ICE (lane 1) or lysates fromunstimulated PBMC (lane 3), LPS-stimulated PBMC (lane 4),uns t imula ted THPl ( lane 5) or LPS-stimulated THPl (lane 6);control reaction was performed with [3SS]proIL -lpn buffer (lane2). Samples were resolved by 1 6% S DS-PA GE and cleavage prod-ucts were visualized by autoradiography. In this experiment, at 6h, the LPS-stimulated PBM C secreted 17.8 ng/ml IL-1p. Numberson the left indicate molecular size of standards in kDa . The arrowshows the position of mature IL-1p.

Figure 6 . Expression of ICE proteins and secre-tion of IL-1-p by LPS-activated human PBMC.PBMC were cultured in the presence (+ ) or inthe absence (-) of LPS (1 pg/ml) and were har-vested after 6 or 24 h of culture. ( A ) Cell lysateswere subjected to 16% SDS-PAGE, blottedand probed with CAL anti-p20 ICE mAb orALP ant i -p l0 ICE mA b. Recombinant p20 orp10 ICE polypeptides (10 ngAane) are shown ascontrols. Numbers on the left indicate molecu-lar size of standards in kDa. (B) Supernatantfrom unstimulated PBMC (dashed areas) orLPS-stimulated PBMC (filled areas) weretested for the presence of mature IL-1p byELIS A after various periods of culture (0, 6 or24 h).

the same cultures, substantial amounts of IL-lP were mea-sured in the supernatants of LPS-activated cells, i . e . 6 ng/ml and 8 ng/ml at 6 and 24 h of culture, respectively (Fig.6B). In no instance did we detect any p20 or p10 in thelysates of stimulated cells (Fig. 6A, lanes 3 and 5 ) evenwith longer exposures of the film. Note that at time zerotwo additional bands were seen around 35-40 kDa (Fig.6A, lanes 1). This was not seen in all the experiments andwas probably due to cell damage and partial ICE proteoly-sis during thawing of the PBMC (see below). These signalswere not seen upon further culture of the cells (see Fig.6A, lanes 2 and 4, respectively, for 6- and 24-h culture withn o LPS).These results show that the pattern of expression of ICE inPBMC is not influenced by LPS stimulation of the cells.Therefore, IL-1 p secretion can occur in cells where pro-cessed ICE species are undetectable under our Westernblot conditions. Similar results were obtained with THPlcells: upon stimulation with LPS, secretion of IL-lP wasobserved in the supernatants, whereas no ICE subunitcould be detected in the cell lysates (data not shown).However, in these experiments, IL-1P release by PBMC orTH Pl cell line was indeed dependent o n ICE activity sinceit could be inhibited by specific ICE inhibitors such asYVAD-CHO at 10 pM as previously described ([5] anddata not shown).To confirm these results, PBMC and THPl lysates weretested for their content in ICE activity, as measured by theability to cleave in vitro translated [35S]methionine abeledproIL-1P. A purified preparation of active ICE was used asa control in the same experiment and could perform the[35S]IL-1p recursor cleavage to completion within 60 min(Fig. 7, lane 1). When [35S]proIL-1Pwas exposed to unsti-mulated PBMC or THPl cell lysates, no 17-kDa IL-1 wasgenerated (Fig. 7, lanes 3 and 5) indicating that the enzy-matic activity present in these cells was below the detec-tion limit of the test. As shown in Fig. 7, lanes 4 and 6,lysates from LPS-stimulated PBMC or THPl were alsounable to cleave the labeled 31-kDa IL-lP substrate pro-


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Eur . J. Immu nol. 1996.26: 1032-1042 Cellular expression of interleukin-lp-convertingnzyme 1039Time 0 l h 4h 24hLP S - + - + - + - +--

1 2 3 4 5 6 7 8 ~ 2 0

46 -30 -21 -14 -6.5 -

CAL mAbFigure 8. Auto-processing of ICE proteins in TH Pl lysates . TH Plcells were incubated in the presence (+) or in the absence (-) ofLPS and then harvested and lysed after 24 h of culture . Cell lysa-tes were either immediately denatured with sample buffer andheat ( time=0), or incubated a t 22C for 1, 4 r 24 h before dena-turation. Samples were then subjected to 1 6% SD S-PAGE, blot-ted and probed with CA L anti-p20 ICE mAb. Recombinant p20ICE polypeptide (10 @lane) is shown as a control. Numbers onthe left indicate molecular size of standards in kD a.

tein. Therefore, ICE activity in stimulated cells was alsoundetectable in our test. Together, these results show thatLPS stimulation of the cells does not lead to detectablemature ICE expression in the cells nor to measurableproIL-10-converting activity in cell lysates, even when thecells are actively secreting mature IL-10.

A B

. I I I . I . . . . . .Pnl. LOO

3.6 ICE autoprocessingSince processed ICE proteins could not b e detected inresting or in LPS-stimulated PBMC or THPl cells, wedetermined whether ICE precursor maturation couldoccur in vitro. THPl cell lysates were, therefore, incubatedat 22C before being subjected to electrophoresis, and ICEproteins were probed with the anti-ICE mAb by immuno-detection. As shown in Fig. 8, when cell lysates fromTHPlcells, stimulated or not with LPS, were incubated at 22"C,the p20/p22 subunits were detected with the CAL anti-p20mAb after 1 h of incubation. When the incubation waspursued, the amount of subunits detected increased,whereas the intensity of the p45 band diminished, indicat-ing that the p45 ICE precursor was being transformed intothe ICE subunits. After 24 h of incubation, the p45 bandhad almost completely disappeared, whereas p20/p22forms were clearly detected with CAL mAb. However, theintensity of the bands obtained for the subunits after 24 hof incubation was lower than that of the initial p45 band,suggesting that complete degradation of the p45 proteinwas also occurring during this maturation process. LPSstimulation of the cells prior to lysis did not seem to influ-ence this maturation process in vitro.When immunodetec-tion was performed with the ALP anti-pl0 mAb, ~10112species became detectable in parallel with the disappear-ance of p45 (data not shown).These results therefore indicate that, upon cell lysis andincubation, the ICE precursor is maturated and trans-formed into p20 and p10 subunits, although some degrada-tion is also likely to occur. This suggests that a regulatedprocess in living cells holds ICE in a precursor form andthat this process can be disrupted by cell lysis.

C

1 ~ .~.

. I I I. 1 I...PnT* Loo Figure 9. Cellular localization of ICE proteins.Cytofluorimetric analysis of ICE expressionwas performed after cell surface staining of., , I. I,, I,,. T H Pl cells (A ) and a fter intracellular stainingof permeabilized T H Pl cells (B) or permeabil-ized K562 cells (C). The x-axis shows FITC-fluorescence intensity on a logarithmic scaleand y-axis represents the relative frequency ofcells. Confocal microscopic examination wascarried out with fixed and permeabilized TH Plcell line stained with LO1 anti-p20 ICE mAband FITC-conjugated second antibodies, asdescribed in Sect. 2.9. Fluorescence intensitieswere converted into colors, with a scale goingfrom black (negative) to red and yellow withincreasing intensities. Four optical sectionsacross the sam e cell are shown. B ar= 5 pM . (D)

NEGATIVE

1*1n Lm4!izzIl


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1040 C . Miossecet al . Eur. J. Immunol. 1996.26: 1032-10423.7 Cellular localization of IC E proteinsmAb are powerful tools to allow specific detection of agiven protein in tissues and cell lines. We therefore testedour three anti-ICE mAb for their ability to recognizenative ICE by indirect immunofluorescence staining ofpermeabilized cells. Initial experiments showed that theLO1 anti-p20 mAb gave a good fluorescence signal andthis mAb was selected for further studies.Immunofluorescence staining of cell lines was performedwith LO1 mAb to analyze ICE expression either on thesurface of intact cells or inside the cells by cytofluorimetry.As shown in Fig. 9A, no staining was observed when non-permeabilized TH Pl cells were stained with anti-p20 LO1mAb, indicating that no cell surface expression of ICE wasdetectable in these cells, whereas CD14 was clearlydetected using a specific anti-CD14 mAb. When THPlcells were fixed with paraformaldehyde and permeabilizedwith saponin, specific staining was obtained with LO1mAb (Fig. 9B). K562 cells were used as a negative control(Fig. 9C) since they were found to be negative for ICEexpression in immunoblotting with all three anti-ICE mAb(see Fig. 4 for results with CAL and ALP). Reverse tran-scription of K562 mRNA and PCR amplification withICE-specific oligomers showed an absence of ICE messen-gers in these cells (data not shown).The subcellular localization of ICE was then analyzed byfluorescence microscopy. THPl cells were permeabilizedand stained with LO1 mAb and fluorescent second antibo-dies, and examined by confocal microscopy. Fig. 9D showsan example of the results obtained in four optical sectionsof the same cell. Staining for the ICE protein was clearlycytoplasmic and the fluorescence signal showed an evendistribution over the cytosol. No staining was observed inthe nucleus and no particular subcellular structureappeared to be stained.These results show that the LO1 anti-p20 mAb can recog-nize ICE inside the cells in its native form and that com-plete denaturation of the protein as performed in SDS-polyacrylamide gels is not necessary fo r its binding. More-over, since the p45 form is the major species expressed incells, these results indicate that the ICE precursor isevenly located in the cytoplasm of the THPl cells and isnot detectable on the outer cell membrane nor in thenucleus of the cells.

4 DiscussionUsing recombinant ICE p20 and p10 subunits as immuno-gens, we have generated a series of mAb against the intra-cellular enzyme ICE. Three mAb have been selected forfurther study: two anti-p20/1CE mAb (CAL and LOI) andone anti-plO/ICE mAb (ALP). Using transfected cells, w eshow that these antibodies are highly specific for ICE anddo not recognize the TX protein, which represents themost closely related ICE homolog known to date [15]. Inaddition, the antibodies can recognize both the precursorand the ICE subunits as seen in transfected cells. Reactiv-ity of these mAb with PBMC and cell lines shows that theycan be used to detect natural ICE in denatured form as in

immunoblots. One of these reagents (LO1 anti-p20 mAb)can also bind ICE in its native form and allows cytofluori-metry analysis following permeabilization of the cells withsaponin. We therefore describe for the first time a series ofreagents that allow a highly specific recognition of humanICE and we have used these tools to examine expressionof this enzyme in PBMC and in cell lines.The ICE enzyme is composed of two subunits which arederived from a precursor protein of 45 kDa. Maturation ofthe precursor requires proteolytic cleavage at threeaspartic acid sites (Asp 119, Asp 297 and Asp 316, see Fig.1) to generate two subunits that combine to create theactive enzyme [ S , 61. Therefore, we expected to find atleast two forms of the enzyme in cells expressing ICE: a45-kDa form that would be recognized by both the anti-p20 and the anti-pl0 mAb and two subunits that would berecognized separately by the respective mAb. The p45 sig-nal corresponds to the translation of the full-length ICEmRNA and is detected in lysates of cells naturally expres-sing ICE as well as in ICE-transfected COS cells. It is infact composed of a doublet of very close bands, and themigration of this doublet can show minor differencesdepending on the origin of the lysate. The ICE mRNAcontains two initiator methionine codons with appropriateconsensus Kozak translation initiation sequences [4]. Theresulting proteins should have 404 and 388 amino acidswith a molecular mass difference of 2.2 kDa, which couldexplain the doublet observed at 45 kDa. In normal cells,this could also result from alternative splicing of ICEmRNA. Indeed, alternatively spliced mRNA isoforms ofICE have been recently described and sequenced in TH Plcells [21]. One of these, designated ICEP, lacks the entireexon 3 of the gene and would give rise to a protein with adeletion from Asp 92 to Pro 112, resulting in a 2.8-kDa dif-ference with the full-length protein.The p20 and p10 subunits are clearly expressed in ICE-transfected COS cells and specifically detected by therespective mAb. Additional low molecular weight forms ofICE are also expressed in these cells. The p22 form was ini-tially described in THPl cytoplasmic extracts and wasfound in variable amounts in the purification fractions [3].N-sequencing indicated that p22 corresponds to an alter-native proteolytic site (Asp 103) for generating p20 [22].The p12 band most probably corresponds to p10 plus the19-amino acid linker between p20 and p10, since it is onlyrecognized by anti-pl0 mAb. Such an intermediate form ofcleavage has been observed during maturation of recombi-nant ICE p45 in vitro [7]. Indeed, we can find p20/p22 andplO/p12 species when THPl lysate is incubated in condi-tions allowing ICE maturation. In contrast, none of thesemature subunits (p22, p20, p12 and p10) can be detected incells naturally expressing ICE when lysis is performed inthe presence of an ICE inhibitor. Finally, in both ICE-transfected COS cells and in cell lines, several bands areobserved between 30 and 40 kDa that react with both theanti-p20 and the anti-pl0 mAb. The p30 form probablycontains the p20 and p10 subunits and the linker betweenthem. The other proteins in this region could result fromproteolytic cleavages in the N-terminal propiece of ICE.Indeed, there are seven Asp residues in this precursorregion of ICE that could represent alternative cleavagesites for processing. However, some of these bands couldalso be generated by cleavages in the C terminus of ICE [7]


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Eur. J . Immunol . 1996.26: 1032-1042 Cellular expression of interleukin-lp-converting nzyme 1041that would retain the ALP epitope or, in normal cells, byseveral alternative splicing events of the ICE mRNA [21].Therefore, the product of the ICE gene can be expressedby transfection in many different forms. In cells naturallyexpressing ICE, the high molecular weight forms are theonly species detected and there is no accumulation ofmaturation products of the ICE precursor. Moreover,when PBMC or THPl cells are stimulated with LPS andactively secreting IL-lp, the p45 precursor remains theonly detectable form in the cell lysates. In the same experi-ments, no proIL-10 cleavage activity is found in the celllysates from unstimulated or LPS-stimulated cells. There-fore, IL-l(J maturation can take place in cells where pro-cessed ICE subunits are present in very small quantities.One possible explanation is that a very small amount ofmature ICE efficiently cleaves the proIL-lp moleculesexpressed in the cells as a result of LPS stimulation, andthat this accounts for the mature IL-1p secretion observed.From our data, we have estimated the amount of p20 sub-unit in THPl to be below 600 molecules per cell. Underour conditions of stimulation, lo6 THPl cells secreteapproximately 0.5 ng mature IL-lP/h, which representsaround 5 molecules/s and per cell. Considering that thecatalytic constant (kcat)of ICE for the synthetic substrateYVAD-para-nitroanilide was estimated to be 1molecule/s[6], the k,,, for its natural substrate 31 kDa IL-1P can beassumed to be at least 1 molecule/s. In this case, only 5molecules of active ICE per THPl cell would be sufficientfor conversion of the secreted amount of IL-1P. Thus, veryfew active ICE molecules per cell can account for theobserved secretion of IL-1p. An alternative hypothesiswould be that the ICE precursor itself may have a low butsufficient proIL-lp cleavage activity. The ICE precursor isindeed capable of catalytic activity in vitro since the puri-fied p45 molecule can autoprocess under certain condi-tions [7,23]. However, its ability to cleave proIL-1p is notknown and we did not detect any proIL -lp cleavage activ-ity in our cell lysates despite a high amount of p45 ICEmolecules present in these lysates. Therefore, if the p45ICE precursor is responsible for proIL-lp cleavage inthese cells, its specific activity is likely to be very low.Together these observations on ICE expression are inaccordance with previous results obtained with a series ofpolyclonal antisera raised against recombinant ICE or ICEpeptides [24]. A recent publication using electron micros-copy studies reports that the active enzyme could be local-ized in the membrane of PBMC, suggesting that it may beinvolved in both processing and secretion of IL-1p [25].However, the high intensity of the signal obtained in thesestudies does not correlate with the amount of active ICEestimated by standard biochemical methods in the presentwork or in previous reports [24], raising the possibility thatthe reagents used (polyclonal antisera anti-ICE and bioti-nylated ICE inhibitor) may cross-react with some ICEhomolog(s).LPS and other stimuli are known to induce IL-1P mRNAexpression in a variety of cell types. Stimulation of humanmonocytes [26,27] or TH Pl cells [28] with LPS results in arapid ( 2 4 h) and substantial (100-fold) rise in IL-1PmRNA levels. This is due to an increased rate of transcrip-tion [28] and also to a higher mRNA stability [29]. In con-trast, ICE mRNA was shown to be constitutivelyexpressed in monocytes and TH Pl cells [3, 41 and studies

on murine peritoneal macrophages have evidenced amoderate up-regulation of ICE mRNA levels (1.5- to 3-fold) following LPS or IFN-y treatment [30]. Our resultsshow that the ICE protein is constitutively expressed inmonocytes and THPl cells, and that its expression is notinfluenced by LPS stimulation. Therefore, the ICE enzy-matic activity is likely to pre-exist in the cells. According tothis hypothesis, an IL-1P-inducing stimulus, such as LPS,would only trigger intracellular expression of largeamounts of the substrate protein ( i . e . proIL-lP) withoutaffecting the enzymatic activity itself. However, one can-not exclude that LPS-activated cells contain an increasedlevel of ICE activity compared to unstimulated cells sincein both cases the activity in the cell lysates was belowdetection. In addition, the conditions of the test for enzy-matic activity are not exactly comparable for resting andactivated cells since the latter contain large amounts ofunlabeled newly synthesized proIL-1P that compete withthe [35S]proIL-1@ubstrate for ICE active site and lowerthe sensitivity of the test.The process of ICE precursor maturation is not yet fullyunderstood. Early experiments in vitro have suggestedthat p45 ICE can automaturate [ 3,4 ]. Indeed, addition ofactive ICE to precursor protein in vitro leads to precursorcleavage and generation of subunits of the right size [3],and recombinant purified ICE precursor can undergoautoprocessing as well as autodegradation in vitro [7]. Inaddition, analysis of the primary sequences of the ICEfamily members strongly suggest that these proteases, likeICE, are expressed as precursor proteins and requirematuration through proteolysis to become fully activeenzymes [20]. They probably all share specificity foraspartic acid and exhibit maturation sites that correspondto cleavage after an aspartic acid residue. It is thereforepossible that they participate in each others activation.Indeed, TX was shown to process a p30 form of ICE [15]and ICE can cleave TX (C. Faucheu, personal communica-tion) and CPP32 [12]. In spite of this, our results show thatin all normal cells examined, the ICE protein is main-tained as a pool of precursor protein. Active ICE, if any, ispresent in very low quantity compared to substantialamounts of precursor protein. Strikingly, when cell integ-rity is disrupted by hypotonic shock and detergent, weobserve a rapid cleavage of p45 into p20lp22 and plO/p12polypeptides in THPl cells. The size of the polypeptidesobtained are similar to the ones observed in ICE-transfected COS cells and are likely to result from sequen-tial proteolysis of the precursor protein. Although we donot fully understand what happens when cells are dis-rupted, our results suggest that processing of the ICE pre-cursor is tightly regulated in normal cells and that cell dis-ruption can overcome this regulation. In fact, the only liv-ing cells in which we observed high amounts of matureICE are transfected cells but they also undergo apoptosisvery soon after transfection and at the time where weobserve maximum expression of the ICE subunits [15].Indeed, overexpression of ICE o r ICE-related proteins hasalso been shown to induce apoptosis in several expressionmodels such as Sf9 insect cells [lo , 171 and mammalian celllines [14, 311. In addition, although transcriptional regula-tion does not seem to be the major mechanism for ICEregulation, a recent report indicates that ICE-encodingmRNA is induced in mammary epithelial cells under con-ditions that promote apoptosis [32]. Therefore, one can


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1042 C. Miossec et al. Eu r. J . Imrnunol. 1996.26: 1032-1042hypothesize that in healthy cells, maintenance of the pro-tein in its precursor inactive form could be a way to pre-serve cell integrity from autodestruction. This mechanism,also used for other intracellular enzymes, provides ameans for quickly releasing enzyme activity since only afew minutes are needed for a proteolytic enzyme to pro-cess a large pool of precursor into a fully active matureprotein. Maturation of the precursor into a fully activeenzyme is likely to represent an essential step, at the post-translational level, to regulate the enzymatic activity ofICE and ICE-related proteases during inflammation pro-cesses as well as in programmed cell dea th.We want to thank Dr. D . Livingston, Dr. 7: Fox and J . Coll fo r thegenerous gifts of the p20 and p10 human recombinant IC E proteinsand cDNA encoding p4 5 ICE in pcDL plasmid for transfectionstudies. We acknowledge C . Faucheu fo r her help in transfectionstudies and RNA analysis. We thank J . - M . Bruneau for helpfuladvice in Western blotting and immuno detection experimen ts. Wewish to acknowledge D r. A . Dautry-Varsat for help and discussionconcerning immunofluorescence microscopy studies, and Ray-mond Hellio for confocal laser scanning analysis. We also thankDrs. W H . Fridman, T. Hercend and R . Westwood for their adviceand support during this work.

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10 Fernandes-A lnemri, T., Litwack, G. and Alnemri, E. S. , J.Biol. Chem. 1994.269: 30761.11 Nicholson, D. W., Ah, A ., Tho rnberry, N. A., Vaillancourt, J .P., Ding, C.K., Gal lant , M., Gareau, Y., Gr iffi n, P. R.,Labelle, M., Lazebnik, Y. A ., M unday, N . A., Raju , S . M .,Smulson, M. E., Yamin, T., Yu, V. L. and Miller, D. K.,Nature 1995. 376: 37.12 Tewari, M., Q uan, L . T., ORourke, K ., Desnoyers, S.,

Ze ng , Z. , Beidler, D.R., Pokier, G.G., Salvesen, G.S. an dDi xit, V. M ., Cell 1995. 81: 801.13 Kumar, S., Kinoshita, M., Noda, M ., Copeland, N. G . an dJenkins, N. A., Genes Dev. 1994. 8: 1613.14 Wang, L . , Miura , M., Bergeron, L. , Zhu , H. and Yuan, J . ,Cell 1994. 78: 739.15 Faucheu, C. , Diu , A. , Chan, A.W. E. , Blanchet, A.-M.,Miossec, C. , HervC, F., Collard-Dutilleul, V., Gu , Y., Aldape,R. , Lippke , J . , Rocher , C . , Su , M . S . 4 , Livingston, D.J.,Hercend, T. and Lalanne , J . -L. , E M B O J . 1995. 14: 1914.16 Munday, N. A ., Vaillancourt, J . P., Ali, A., C asano, F. J. ,Miller, D. K., Molineaux, S. M ., Yamin, T. yu, V. L. andNicholson, D . W., J . Biol . Chem. 1995. 270: 15870.17 Kamens, J ., Paskind, M., Hugunin, M., Talanian, R. V.,Al len , H. , Banach, D. , Bump, N., Hacket t , M., Johnston, C .G. , Li, P., Mankovich, J. A., Terranova, M. and Ghayur, T.,J. Biol. Chem. 1995. 270: 15250.18 Faucheu, C., Blanchet, A.-M., Collard-Dutilleul, V.,Lalanne, J .-L. and Diu-Hercend, A. 1996. Eur. J . Biochem.1996. 236: 207 .19 Fernandes-Alnemri, T., Litwack, G . and Alnemri , E. S. ,Cancer Res. 1995. 55: 2737.20 Thornberry, N. A., Miller, D.K. and Nicholson, D.W., Per-spect. Drug Discov. Design 1994. 2: 3 89.21 Alnemri , E. S., Fernandes-Alnemri, T. and Litwack, G., J .Biol. Chem 1995.270 : 4312.22 Miller, D. K., Ayala, J. M., Egger, L. A., Raju , S. M.,Yamin,T. , Ding, G . J.-F., Gaffney, E. P., Howard, A . D ., Palyha, 0.C . , R o la ndo , A . M ., Salley, J. P., Thornberry, N . A., Weid-ner, J . R ., Williams, J . H., Chapman, K. T. , Jackson, J . , Kos-tura , M. J . , Limjuco, G. , Molineaux, S. M., Mumford , R . A .and Calayca, J . R . , J . Biol. Chem. 1993.268: 18062.23 Howard, A.D., Pa lyha, O.C., Griffin, P.R., Peterson, E.P.,Lenny, A.B., Ding, G.J.-E, Pickup, D.J., Thornberry, N .A.,Schimdt , J .A. and Tocci, M .J . , J . Immunol . 1995.154: 2321.24 Ayala, J . M ., Yamin, T.-T., Egger, L. A ., C hin, J ., Kostura,M. J . and Mil le r, D. K. , J . Immunol . 1994. 153: 2592.25 Singer, I. I., Scott, S., Chin, J . , Bayne , E . K . , Limjuco, G .,Weidner, J ., Miller, D.K., Chapman, K. and Kostura, M. J .,J. Exp. Med. 1995. 182: 1447.26 Matsushima, K. , Taguchi, M., Kovacs, E.J ., Y oung, H. A.and Oppenheim J .J . , J. Immunol. 1986. 136: 2883.27 Vermeulen, M.W., David, J .R. and Remold H.G., J . Immu-nol. 1987. 139: 7.28 Fenton , M.J., C lark, B.D., Collins, K.L., Webb, A. C., Rich,A . and Auron, P.E. , J. Immunol . 1987. 138; 3972.29 Fenton, M.J., Vermeulen, M.W., Clark, B.D., Webb, A.C.,Rich, A. and Auron, P.E., J. Immunol. 1988. 140: 2267.30 Nett, M .A., Cerretti , D.P., Berson, D.R., Seavitt , J . , Gil-bert , D.J . , Jenkins , N.A., Copeland, N. G., Black, R .A . andChapl in , D.D., J . Immunol. 1992. 149: 3254.31 Miura, M. , Zhu , H. , Rote l lo , R . , Hartwieg, E. A. a ndYua n ,J. , Cell 1993. 75: 653.32 Boudreau, N., Sympson, C.J ., Werb, Z. and Bissell, M.J.,Science 1995. 26 7 891.

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