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Division of Infectious Diseases, University of Colorado Health Sciences Center, Denver, CO 80262
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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. Whereas
IL-1ß down-regulated IL-1RI surface expression
(p < 0.05), there was no change in the
surface expression of IL-1RAcP. Moreover, murine IL-10 increased
surface expression of IL-1RI, but did not affect expression of IL-1RAcP
or the proliferation of D10S cells. Steady state levels of mRNA for
IL-1RAcP and IL-1RI in D10S cells showed a similar pattern to that of
surface expression of the respective receptors. We conclude that 1)
blocking IL-1RAcP inhibits IL-1 signaling in D10S cells, 2) domains-II
and III may be involved in complex formation with IL-1RI, 3) IL-1RAcP
is not regulated as is IL-1RI in the same cells, and 4) IL-1
responsiveness is dependent on the expression of IL-1RI, not IL-1RAcP. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-1ß with different affinities (6, 7). Whereas IL-1RI is
necessary for IL-1 signal transduction (8), the IL-1RII appears to act
as a decoy receptor (9, 10). A number of homologues of IL-1R have been
reported. One of these, T1/ST2, possesses considerable sequence
similarity to the extracellular, ligand-binding portion of both type I
and type II IL-1R (11, 12). Although the native T1/ST2 receptor does
not bind IL-1 (13), the T1/ST2 cytoplasmic domain, when fused to the
murine IL-1R extracellular and transmembrane regions and transfected
into COS cells, can activate nuclear factor-
B DNA binding in
response to exogenous IL-1 (14).
Until recently, the IL-1RI was thought to be the sole receptor that
signaled the cell. However, most cytokines signal cells following the
formation of homo- or heterodimers with two or more chains required for
optimal biologic activity (15). Affinity cross-linking studies of IL-1
to cells expressing natural IL-1R reveal higher molecular mass IL-1R
complexes (>200 kDa) than expected (16, 17). These higher molecular
mass complexes may be indicative of a multisubunit IL-1R complex. A
cell surface protein in close association with IL-1RI has been cloned
and termed as the IL-1R accessory protein
(IL-1RAcP)4 (18). This
IL-1RAcP is a 570-amino-acid glycoprotein with a molecular mass of 66
kDa and is a member of the Ig superfamily. It bears limited homology
throughout the protein to both type I and type II IL-1R. Using
degenerate oligonucleotides of conserved sequences in the IL-1R family,
another IL-1R family member, IL-1R-related protein (IL-1Rrp), has been
described (19). IL-1Rrp has significant sequence homologies to IL-1RI,
IL-1RAcP, and T1/ST2, but does not bind IL-1, IL-1ß, or IL-1Ra.
However, when fused to the extracellular domain of IL-1RI and
transfected into COS cells, IL-1 activates this chimeric receptor, and
thus the IL-1Rrp cytoplasmic portion was capable of eliciting
responses, comparable with those induced via the IL-1RI (19).
Cell lines unresponsive to IL-1 express IL-1RI, but do not express the
IL-1RAcP (20). However, transfection with IL-1RAcP into IL-1
nonresponders restores IL-1 responsiveness (21). These results add
further evidence that IL-1RAcP forms a complex following the binding of
IL-1 with IL-1RI and plays a pivotal role in IL-1 signaling. However,
although soluble IL-1RI or IL-1RII binds IL-1, soluble IL-1RAcP does
not (18). Thus, IL-1RAcP most likely binds to epitopes created on
either IL-1RI or IL-1 itself following the initial binding event of
IL-1 to IL-1RI. In the present study, we identified four regions of
hydrophilic amino acid sequences of the extracellular portion of the
murine IL-1RAcP that could be available for interactions with other
proteins. Anti-IL-1RAcP peptides were prepared and studied for their
ability to affect the biologic response to human and mouse IL-1 and
IL-1ß using proliferation of the Th2 cell line, D10S cells (22). In
addition, these Abs were used to study the surface expression and
regulation of IL-1RAcP on D10S cells treated with IL-1 or IL-10.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The following were purchased: RPMI 1640, penicillin, and streptomycin (Mediatech, Herndon, VA); CNBr-activated Sepharose 4B (Pharmacia Fine Chemicals, Piscataway, NJ); MTS (3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfo-phenyl)-2H-tetrazolium) (Promega Corp., Madison, WI); Con A, phenazine methosulfate (PMS), PBS, SDS, aprotinin, PMSF, BSA, goat anti-rabbit IgG FITC, goat anti-mouse IgG FITC, rabbit IgG, keyhole lympet hemocyanin (KLH), and goat anti-rabbit IgG conjugated to peroxidase (Sigma Chemical Co., St. Louis, MO); Nonidet P-40 (ICN Biomedicals, Aurora, OH); peroxidase-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, PA); enhanced chemoluminescence (ECL) Western blotting detection reagents (Amersham International, Buckinghamshire, U.K.); and FBS and glutamine (Life Technologies, Gaithersburg, MD).
Mouse-conditioned medium was prepared by incubating 2 x
106/ml C57BL6 mouse spleen cells with 5 µg/ml Con A
at 37°C for 48 h in RPMI 1640 supplemented with 10% FBS, 5 µM
2-ME, 25 mM HEPES, and 100 U/ml and 100 µg/ml penicillin and
streptomycin, respectively. The supernatant was centrifuged at 500
x g, mixed with 10 mg/ml -methyl-D-mannoside
(Sigma Chemical Co.), and filtered in 0.22-µm syringe filter
(Millipore, Bedford, MA). rhIL-1ß was kindly provided by Dr. Aldo
Tagliabue (Sclavo, Siena, Italy), and hIL-1 was from Glaxo
(Research Triangle, NC). Rat anti-murine IL-1RI Ab, 35F5, and
murine IL-1 and ß were from Hoffmann-La Roche (Nutley, NJ). Murine
IL-10 was a gift from Schering-Plough Research Institute (Kenilworth,
NJ), and murine soluble IL-1RAcP was from Dr. H. Wesche (Medical School
Hannover, Hannover, Germany). hIL-1Ra was obtained from Dr. Daniel
Tracey (Upjohn, Kalamazoo, MI), and TNF-binding protein (TNFbp) was
provided by Amgen (Boulder, CO).
Cell cultures
EL-4.IL-2 cells and D10S, a subclone of the murine Th cell D10.G4.1 (16, 22), were used in these studies. EL-4.IL-2 was purchased from American Type Culture Collection (Rockville, MD). The EL-4.IL-2 cells were maintained in RPMI 1640 medium containing 10% FBS (same medium described above). D10S cells were maintained in RPMI 1640 medium supplemented with 5% FBS and 10% (v/v) mouse-conditioned medium, as previously described (16, 22). Cell cultures were maintained at 37°C in humidified air with 5% CO2.
Synthetic peptides
Four synthetic peptides, YWTRQDRDLEEPI, VSNNGNYT, WTIDGKKPDDV, and YSSTEDETRTQ, were identified as short hydrophilic regions (amino acids 71–83, 204–211, 282–292, and 304–314, respectively) of the Ig-like domains of the extracellular portion of the murine IL-1RAcP. According to the method for displaying the hydropathic character of a protein (23, 24), the hydropathy value for each of the amino acids in the sequence of the extracellular IL-1RAcP was assigned, successively adding those values, and then the sum was divided by 7. The span of 7 was used, so that a given sum and the average could be plotted above the middle residue of the segment. Peptides were synthesized utilizing diisopropyl carbodiimide/1-hydroxybenzo triazole-activated fluorenylmethyl-oxycarbonyl (F-moc)-protected amino acids (Genzyme-Sygena, Cambridge, MA) on a model 396 Multiple Peptide Synthesizer (Advanced Chemtech, Louisville, KY) at Research Genetics (Huntsville, AL). Peptides were cleaved using Reagent R (25) for 1.5 h and purified on Porous R2/M 16 x 100-mm columns (PerSeptive Biosystems, Framingham, MA) with a gradient of 0 to 80% A to B over 40-column volumes (A = 0.1% trifluoracetic acid (TFA) in distilled water (DW), B = 0.1% TFA in acetonitrile). Confirmation of m.w. was determined by MALDI-TOF mass spectrometry (PerSeptive Biosystems, Framingham, MA). All peptides except peptide282–292 contained greater than 90% of the desired product. The purity of peptide282–292 was 84%.
Antisera
Synthetic peptides were cross-linked with KLH using 0.1% glutaraldehyde, as described (26, 27). Peptide282–292 (WTIDGKKPDDV), having lysines at positions other than that at the amino terminus, was conjugated to KLH by modification of the N-hydroxysuccinimide ester method (28, 29, 30). Briefly, 8 mg of KLH was dissolved in 1.5 ml of coupling buffer (0.2 M sodium bicarbonate buffer, pH 8.8, containing 0.15 M KCl). Then 0.5 ml of dimethylformide containing 5 mg of N-hydroxy-sulfosuccinimide and 5 mg of dicyclohexyl carbodiimide was added and the mixture stirred gently for 1.5 h at 4°C with a magnetic stir bar. The precipitated cyclohexyl urea was removed using centrifugation (1000 x g for 30 min). This cleared supernatant was mixed with 1.5 ml of coupling buffer. Three milliliters of this supernatant were mixed with 5 µmol of peptide282–292 in 2.75 ml of coupling buffer and stirred for 2 h at 4°C. Thereafter, it was dialyzed against 50 mM sodium phosphate, pH 8, filtered with 0.2-µm syringe filter, and stored at -70°C. Antisera were raised in New Zealand-derived rabbits. One milligram of each synthetic peptide was emulsified in CFA (Sigma Chemical Co.) and administered intradermally to several sites along the back. The rabbits were given booster immunizations between 4 and 18 wk later within CFA.
Affinity-purified Abs to synthetic peptides
Each synthetic peptide was immobilized to cyanogen bromide-activated Sepharose 4B (Pharmacia), according to the manufacturer’s protocol. Anti-peptide antisera were first precipitated in 50% saturated ammonium sulfate and then dialyzed against PBS. The IgG-enriched antisera were applied to their respective peptide-immobilized Sepharose columns. After elution of nonspecific proteins from the individual columns with PBS, bound Abs were eluted using 4 M MgCl2 in PBS (pH 7.4) at a flow rate of 20 ml/h at room temperature (RT). Each affinity-purified Ab was then dialyzed against PBS. Protein concentration was determined with bicinchoninic acid (Pierce, Rockford, IL) using rabbit IgG as a standard.
Direct binding of antisera to peptides
Polystyrene microtiter plates (Becton Dickinson, Lincoln Park, NJ) were coated with synthetic peptides at a concentration of 1 µg/100 µl/well in PBS overnight at RT. After three washes with PBS containing 0.05% Tween-20 (PBST), all unoccupied binding sites were blocked for 1 h with PBS containing 5% skim milk and 0.02% azide. Serial dilutions of antisera were added and incubated for 2 h at RT. The plates were washed three times with PBST, and goat anti-rabbit IgG conjugated with peroxidase was added and incubated for 1 h at RT. After three washes with PBST, tetramethylbenzidine substrate (Dako Corp., Carpinteria, CA) was added for 10 min, the enzyme reaction was stopped by adding 0.5 N HCl, and the OD was determined at 450 nm with an ELISA reader.
Identification of murine IL-1RAcP using anti-IL-1RAcP peptide Abs by flow-cytometric analysis
All procedures were conducted at 4°C or as stated otherwise. EL-4 and D10S cells were washed with cold PBS and resuspended with 100 µl of RPMI media containing 1% BSA and 0.02% thimerosal at a final concentration of 5 x 105 cells/tube. The resuspended cells were treated with 6.5 µg of anti-IL-1RAcP peptide Abs for 1 h. After washing with cold PBS, cells were resuspended with 100 µl of PBS and treated with goat anti-rabbit IgG-FITC for 1 h. After washing with cold PBS, cells were resuspended with PBS for flow-cytometric analysis.
Identification of murine IL-1RAcP using anti-IL-1RAcP peptide Abs by Western blot analysis
D10S cells (7 x 107) were washed twice with cold PBS, pelleted, and lysed for 30 min in 500 µl of ice-cold lysis buffer (50 mM Tris/HCl, 100 mM NaCl, 2 mM EDTA, 2 mM EGTA, 1 mM PMSF, 10 µg/ml aprotinin, and 1% Nonidet P-40, pH 7.6). The suspension was gently vortex-mixed for 10 s, and allowed to incubate for 1 h on ice to complete cell lysis. Thereafter, the lysates were centrifuged for 15 min at 14,000 x g at 4°C. Supernatants were collected and the protein content was determined. Samples for Western blot analysis were boiled for 10 min in SDS/PAGE loading buffer and electrophoresed into 12% polyacrylamide gels, as described by Laemmli (31). After electrophoresis, the gels were transferred to polyvinylidene difluoride membrane (Bio-Rad Lab., Hercules, CA) for 1 h at 50 V on a Hoefer SemiDry Transfer Unit (Hoefer Pharmacia Biotech, San Francisco, CA) using a transfer buffer (25 mM Tris/HCl, 192 mM glycine, 15% methanol, and 0.1% SDS, pH 8.3). The membrane was then blocked with 5% fat-free dry milk in PBS. Blots were probed by incubating overnight at 4°C with the anti-IL-1RAcP peptide 4 Ab (5 µg/ml). Normal rabbit IgG was used as a control. After washing three times with PBST, donkey anti-rabbit IgG coupled to horseradish peroxidase was incubated for 1 h. Blots were then washed five times with PBST before visualization. ECL kit (Amersham International) was used for detection.
Expression of IL-1RAcP using RT-PCR
RNA from D10S (2.5 x 106) cells was extracted using Tri-Reagent (Molecular Research Center, Cincinnati, OH), according to instructions. RT-PCR was performed as described (20), with slight modifications using RNA PCR Core Kit (Roche Molecular Systems, Branchburg, NJ). Reverse transcription was conducted in a final volume of 20 µl. For each reaction, 4 µl of 25 mM MgCl2, 1 µl diethyl pyrocarbonate (DEPC) water, 2 µl 10x PCR buffer, 2 µl each of dNTPs (10 mM of dATP, dCTP, dGTP, dTTP each), 1 µl RNA inhibitor (20 U/µl), 1 µl random hexamers (50 µM), 1 µl reverse transcriptase (50 U/µl), and 2 µl sample RNA (0.5 µg/µl) were mixed, overlaid with mineral oil (Sigma Chemical Co.), and placed in a Perkin-Elmer thermocycler (Norwalk, CT) with a program of 30 min at 42°C, 5 min at 90°C, and 5 min at 5°C. As negative control, reverse transcriptase was replaced with DEPC water. The resulting cDNA was amplified for IL-1RI and IL-1RAcP. IL-1RI (product length, 363 bp), primer 1, 5'-CTG GAG ATT GAC GTA TGT ACA GAA TAT CCA AAT-3'; primer 2, 5'-ATC CCC GGC AAT GTG GAG CCG CTG TGG GAA GGT GGC CTG TGT-3'. IL-1RAcP (product length, 677 bp), primer 1, 5'-AAC CAT CGG TCA CTT GGT ATA AGG G-3'; primer 2, 5'-TTC ATC TGT TCC AAA GTG AGC TCG G-3'. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (product length, 452 bp) was used as an internal control, primer 1, 5'-ACC ACA GTC CAT GCC ATC AC-3'; primer 2, 5'-TCC ACC ACC CTG TTG CTG TA-3'. The reactions were performed in a final volume of 50 µl. Two microliters of cDNA solution were added to a mixture of 2 µl (3 µl for IL-1RAcP) 25 mM MgCl2, 5 µl 10x PCR buffer, 36.8 µl (33.8 µl for IL-1RAcP) DEPC water, 0.5 µl (1 µl for IL-1RAcP and GAPDH) each dNTPs, 0.2 µl Taq polymerase (5 U/µl), and 1 µl of each primer 1 and primer 2 (20 µmol/L). The samples were overlaid with mineral oil and placed in the thermocycler with a program of 5 min at 90°C, 5 min annealing at 60°C, 28 cycles with 1 min synthesis at 72°C, 1 min at 90°C, 1 min at 60°C, followed by 7 min at 72°C. The PCR products were analyzed on a 1.5% agarose gel. The RT-PCR for IL-1RI, IL-1RAcP, and GAPDH was optimized for each cytokine separately. The optimal cycle for all three was 28. Under these conditions, the respective PCR product increased in direct ratio for the amount of RNA.
Regulation of surface expression of IL-1RAcP on D10S cells
D10S cells growing in RPMI supplemented with 5% FBS and 10% mouse-conditioned medium were harvested, washed twice with cold PBS/1% FBS, and cultured at the concentration of 5 x 105/ml in RPMI containing 5% FBS for 24 h in the presence or absence of various cytokines. On the day of the experiment, cells were washed with cold PBS, resuspended in 100 µl RPMI/1% BSA/0.02% thimerosal, and treated with anti-IL-1RAcP peptides for 1 h. Detection of bound Abs was performed by flow-cytometric analysis after washing with cold PBS. A FACScan EPICS XL-MCL (Hialeah, FL) equipped with 488-nm argon laser was used. When collecting data, the gate was set to avoid cell debris, damaged cells, and aggregates. A total of at least 5000 cells was analyzed for each sample and data were stored in a histogram mode. The mean fluorescence intensity (MFI) was calculated as follows: MFI = [FI (sample) - FI (negative control)]/[FI (positive control) - FI (negative control)] x 100. For the negative control, cells were stained with secondary Ab only, and for the positive control, cells were not stimulated with cytokines.
Bioassays using MTS/PMS
Bioassays with D10S cells (4.5 x 105/ml) were performed in triplicate in 96-well polystyrene flat-bottom microtiter plates (Becton Dickinson) in volumes of 200 µl/well and incubated for 48 h at 37°C in 5% CO2. D10S cells were assessed for their proliferative response to IL-1 using detection of mitochondrial dehydrogenases by cleavage of a novel tetrazolium compound (3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxy methoxyphenyl)-2H-tetra-zolium, inner salt; MTS). On the day of proliferation assay, MTS/PMS solution was prepared by mixing 25 µl PMS (1.53 mg/ml in PBS) for every 975 µl MTS (1.71 mg/ml in PBS). Fifty microliters of PMS/MTS solution were added to the 96-well culture plate at 37°C for 1 to 3 h. The absorbance of formazan at 490 nm was measured directly from 96-well assay plates without additional processing.
Conjugation of hIL-1ß with FITC
The FITC solution was prepared by dissolving 0.3 mg of the dye in 1 ml of 0.25 M bicarbonate solution with a pH of 8.5. hIL-1ß was conjugated to FITC at 4°C, as described (32). One milliliter of a 0.5 M bicarbonate buffer (pH 8.5) was added to 100 µg of hIL-1ß. The fluorochrome solution was added dropwise to the IL-1 solution under continuous stirring. The mixture was then dialyzed overnight against PBS containing 0.01% thimerosal. The A280/A495 ratio of FITC/IL-1ß was 0.43.
FITC/IL-1ß binding
Studies were carried at 4°C, unless otherwise stated. D10S cells were washed twice with cold RPMI 1640 before resuspension at 5 x 105 cell/ml in binding medium (RPMI 1640 with 1% BSA, 2 mM glutamine). The cells were gently rotated for 1 h in the presence of increasing amounts of FITC/IL-1ß or irrelevant anti-mouse IgG FITC. Then the bound IL-1ß was assessed by FACS. In other studies, D10S were preincubated with nonlabeled IL-1ß, TNFbp, IL-1Ra, or anti-peptide Abs for 1 h, followed by addition of 128 ng of FITC-IL-1ß with an additional rotation for 2 h. Cells were washed with cold PBS/0.1% BSA containing 0.05% azide, and resuspended for flow-cytometric analysis. Nonspecific binding of FITC was determined by adding goat anti-mouse IgG FITC in parallel tubes.
Statistical analysis
ANOVA using Fisher’s least significant difference was used. Data were expressed as the mean ± SEM.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We analyzed the murine IL-1RAcP sequence to find short hydrophilic
regions using a method for displaying the hydropathic character of a
protein (see Materials and Methods). Four peptides of
the Ig-like domains of the extracellular portion (peptide 1, amino
acids 71–83; peptide 2, 204–211; peptide 3, 282–292; and peptide 4,
304–314, respectively) were predicted to be hydrophilic, and hence
were synthesized. The positions of the synthetic peptides relative to
the murine IL-1RAcP as well as the hydropathy of the three Ig-like
domains of the extracellular portion are shown in Figure 1
. The relatedness of these peptides to
comparable regions in IL-1RI and IL-1Rrp is shown in Table I. These synthetic peptides were
conjugated to KLH and used as Ags in preparing antisera that might
recognize stable, hydrophilic regions on the surface of the native
murine IL-1RAcP. Ab titers were determined by ELISA using the synthetic
peptides as Ags. As shown in Table II,
each antiserum recognized the respective peptide and there was little
cross-reactivity with the other synthetic peptides.
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Anti-peptide antisera were purified by affinity chromatography, as
described in Materials and Methods. The binding of
affinity-purified anti-IL-1RAcP Abs to the surface of D10S as well
as the EL-4 subline, EL-4.IL-2, was detected using an anti-rabbit
IgG FITC conjugate. Binding was expressed as MFI. As shown in Figure 2A, compared with the other
Abs, anti-IL-1RAcP peptide 2 Ab bound best to D10S. On the other
hand, anti-IL-1RAcP peptide 1 Ab shows almost same binding as that
of normal rabbit IgG. Similar results using these anti-IL-1RAcP Abs
also bound to EL-4.IL-2 cells with the same specificity as that
observed in D10S cells. Western blot analysis showed that
anti-IL-1RAcP peptide 4 Ab detected intact IL-1RAcP from D10S cell
lysates and soluble IL-1RAcP (Fig. 2B).
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We studied the effects of the different anti-IL-1RAcP Abs on
IL-1-induced proliferation in D10S. D10S cells were incubated with the
different anti-peptide Abs for 1 h at RT and followed by
hIL-1ß for 48 h. As shown in Figure 3, anti-IL-1RAcP Abs inhibited the
hIL-1ß-induced proliferation of D10S cells in a dose-response
fashion. The addition of anti-IL-1RAcP peptide 4 reduced the
biologic activity of hIL-1ß on D10S cells by about 80%, whereas
anti-IL-1RAcP peptide 2 or 3 inhibited by 60%. However,
anti-IL-1RAcP peptide 1, which showed almost no recognition of
IL-1RAcP surface expression (Fig. 2
A), had no
significant effect. hIL-1Ra and an anti-IL-1RI mAb, 35F5, reduced
the IL-1 activity by 73 and 80% in the same assay, respectively (data
not shown).
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Since anti-IL-1RAcP peptide 4 possessed the greatest
IL-1-blocking activity, we compared the effect of this Ab on mouse and
human IL-1 and IL-1ß. D10S cells were treated with
anti-IL-1RAcP peptide 4 Ab for 1 h and stimulated with
different forms of IL-1 for 48 h, and proliferation was assessed,
as described above. As shown in Figure 4,
IL-1ß-induced proliferation was inhibited more effectively than that
of IL-1. In fact, hIL-1ß-induced proliferation appeared more
effectively inhibited by anti-IL-1RAcP peptide 4 than that of mouse
IL-1ß.
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To assess the effect of anti-peptide Abs on IL-1RAcP complex
formation, we examined the direct binding of FITC-labeled hIL-1ß. As
shown in Figure 5A, an
irrelevant anti-murine IgG FITC did not bind to D10S cells, and a
specific dose-dependent binding of FITC/IL-1ß to D10S cells was
observed. D10S cells were next preincubated with anti-peptide
IL-1RAcP Abs or excess hIL-1ß, IL-1Ra, or TNFbp for 1 h at
4°C. Thereafter, hIL-1ß/FITC was added, and the amount of IL-1ß
bound to D10S cells was assessed by flow-cytometric analysis. As shown
in Figure 5B, the binding of IL-1ß/FITC to D10S cells was
specific. The binding of IL-1ß/FITC to D10S cells was blocked by 73
and 88% with hIL-1ß or IL-1Ra, respectively. However, this binding
of IL-1ß/FITC to D10S cells was not blocked by TNFbp. In the presence
of each of the affinity-purified anti-IL-1RAcP Abs, binding of
hIL-1ß to D10S cells was unaffected (Fig. 5B).
These latter results suggest that the epitopes recognized by these Abs
do not affect the IL-1ß binding sites of the IL-1RI and that IL-1ß
does not bind to the extracellular segments of IL-1RAcP. Similar
conclusions were made by Greenfeder et al. (18).
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D10S cells were incubated with hIL-1ß or murine IL-10 for
24 h and stained for FACS analysis. For these studies, we used
affinity-purified anti-IL-1RAcP peptide 4 because peptide 4 shares
little sequence homologies to IL-1RI or IL-1R-related protein (IL-1Rrp)
(see Table I), and this anti-peptide 4 possesses the greatest IL-1
biologic activity (Fig. 3). Unlike a 2-day proliferation assay,
incubation with IL-1 or IL-10 for 24 h did not significantly
affect the number of D10S cells (data not shown). Regardless of the
presence of IL-1 or IL-10, as shown in Figure 6, the level of the surface expression of
murine IL-1RAcP was unchanged when compared with control cells. In
addition, using anti-IL-1RAcP peptide 3, FACS analysis of the
effect of a 24 h-incubation with IL-10 or IL-1 on IL-1RAcP also showed
no change compared with control (data not shown). We also measured the
surface expression of IL-1RI in the same cells using a monoclonal
anti-murine IL-1RI. A concentration of 100 pg/ml of IL-1
down-regulated the surface expression of IL-1RI, confirming previous
studies (33); however, IL-10 increased the surface expression of IL-1RI
by 30% (Fig. 6).
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, RT-PCR revealed that D10S cells have
both IL-1RI (363 bp) and IL-1RAcP (677 bp). As a negative control, in
the absence of reverse transcriptase, no product was detected.
Regulation of the expression of mRNAs for IL-1RAcP and IL-1RI in D10S
cells treated with IL-1 or IL-10 for 24 h was similar to that of
surface IL-1Rs (Fig. 7). However, a high concentration of IL-1 (20
ng/ml) down-regulated the gene expression of IL-1RI. The above results
were similar to those using Northern blot analysis showing that steady
state mRNA levels of IL-1RI decrease in a dose-dependent fashion (33).
On the other hand, the changes of IL-1RAcP mRNA were not significant,
p > 0.1.
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We next measured the proliferation of D10S cells to mouse and
human IL-1 and IL-1ß using mitochondrial dehydrogenases, as
detected by cleavage of a novel tetrazolium compound, MTS. The
advantage of MTS is that the absorbance of the formazan is measured
directly without additional processing. As previously reported (22),
the proliferative responses of D10S cells to any of these forms of IL-1
in the absence of a mitogen were observed consistently in the
subfemtomolar range in over 20 separate assays (data not shown).
Maximal responses were observed at concentration of approximately 500
pg/ml. Human and murine IL-1 showed nearly same dose response of
proliferation in D10S cells (Fig. 8).
Because IL-10 suppresses IL-1-induced cytokine productions in
monocytes, we examined the effect of IL-10 on IL-1-induced
proliferation in D10S. Murine IL-10 had no effect on IL-1-induced
proliferation of D10S cells (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. The anti-IL-1RAcP Abs did not block the binding of
IL-1ß to D10S cells, suggesting that IL-1ß does not bind directly
to the IL-1RAcP. These results are consistent with previous data that
soluble IL-1RAcP is unable to bind IL-1 (18). Our studies thus suggest
that IL-1RAcP is involved in the formation of a heterodimer with IL-1RI
rather than the IL-1 ligand. In most cells, the IL-1RAcP is coexpressed
with the IL-1RI, but certain cell lines lacking expression of IL-1RAcP
are unresponsive to IL-1 (20). Those and our results support the
concept that IL-1RAcP plays an essential role in IL-1 signaling.
Extensive site-directed mutagenesis of IL-1ß and IL-1Ra has been
reported (34, 35). For each of the three members of the IL-1 family
(IL-1, IL-1ß, and IL-1Ra), a primary binding site (site A) exists
(reviewed in 1 ; however, there is a second binding site (site B)
that is missing in IL-1Ra (35). This lack of site B accounts for the
ability of IL-1Ra to bind tightly to the IL-1RI and acts as a receptor
antagonist. Recently, the crystals of IL-1ß and IL-1Ra bound to
soluble IL-1RI have been resolved at 2.5 and 2.7 angstrom, respectively
(36, 37). In those studies, IL-1ß bound to IL-1RI did not undergo a
significant structural change (36). However, when IL-1ß binds to
domains I and II of the IL-1RI, domain III of the receptor flexes to
bind to site B in IL-1ß. Since site B is missing in IL-1Ra, this
flexion in the receptor does not take place. Assuming that flexion of
IL-1RI domain III exposes a new epitope, this would give the IL-1RAcP
an opportunity to make contact with IL-1RI. Hence, the heterodimer of
IL-1RI and IL-1RAcP would presumably dock at domain III of each
receptor chain, the two cytosolic segments would approximate each
other, and signal transduction would be initiated. Since there is no
flexion of domain III in IL-1RI when IL-1Ra binds, no new contact
epitopes are created and there is no approximation of IL-1RAcP to form
the complex. An alternative model could also explain the results.
Aspartic acid 145 has been shown to be critical for biologic activity
of IL-1ß (38). Since this residue does not make contact with domain
III of IL-1RI (37), it has been proposed that IL-1RAcP may bind to this
residue. This possibility, however, awaits structural analysis of
IL-1ß contacts in crystals comprised of IL-1RI, IL-1ß, and
IL-1RAcP.
In the present studies, the inability of anti-IL-1RAcP to block
IL-1ß binding to D10S cells suggests that none of the three domains
of the IL-1RAcP binds to IL-1ß itself, but rather to the complex of
IL-1RI/IL-1ß, perhaps to stabilize the complex. This would facilitate
dimerization of the cytosolic segments of IL-1RI and IL-1RAcP. Since
anti-IL-1RAcP peptide 4 was most effective in blocking the IL-1
bioactivity on D10S cells, we suggest that domain III of IL-1RAcP is
involved in dimerization of the cytosolic segments of IL-1RI and
IL-1RAcP. Several ligand-signaling complexes are stabilized by binding
to receptor molecules that are structurally separate from those
involved in ligand binding (15). Ligand-receptor complexes can also be
stabilized by accessory molecules not directly involved in signaling.
For example, IL-6-induced dimerization of gp130 is dependent on
IL-6R (39). IL-6R appears to stabilize the complex between IL-6
and gp130 rather than to take an active part in intracellular signal
transduction (15). Heparin and heparan sulfate have been found to
potentiate the mitogenic effects of members of the fibroblast growth
factor (FGF) family. Heparin binds acidic FGF in a multivalent manner,
but does not bind the extracellular part of the FGF receptor. Thus,
heparin induces oligomerization of FGF, which, in turn, promotes
oligomerization of receptors (40).
There are several ways to perturb receptor dimerization. An Ab directed
against the fourth Ig domain of the stem cell factor receptor
apparently interferes with signaling by directly blocking dimerization
rather than by blocking ligand binding (41). There are other ways to
perturb assembly of signaling receptor complexes.
Granulocyte-macrophage CSF (GM-CSF) acts by forming a high affinity
signaling receptor complex composed of a GM-CSF-specific -receptor
and a ß-receptor that is shared with IL-3 and IL-5. A mutant form of
GM-CSF with wild-type affinity for the -receptor, but defective in
its ability to form a signaling ß-receptor complex, was found to
antagonize the action of wild-type GM-CSF (42).
In this work, we show that affinity-purified anti-peptide 4 Ab
inhibition of IL-1-induced proliferation of D10S cells was more
effective against IL-1ß than against either human or mouse IL-1
(Fig. 4). This suggests that IL-1ß binding can more easily bring
about dimerization of IL-1RI and IL-1RAcP than IL-1. In fact, that
observation was predicted in the original paper by Greenfeder et al.
(18), which shows that the cross-linking pattern produced with IL-1ß
is more intense than that of IL-1. Interestingly, when we tested
higher concentrations of IL-1ß (1000 pg/ml), these Abs did not block
the proliferation of D10S cells. At a concentration of 5 pg/ml (3
x 10-13 M) of hIL-1ß, only 1.5% of the IL-1R of
D10S cells are occupied, based on a conservative estimate of 11,000
receptors per cell (16). This concentration drives D10S cell
proliferation (22). In addition, this concentration of IL-1ß can
significantly down-regulate surface and gene expression of IL-1RI after
24 h (33). At 8 µg/ml of anti-IL-1RAcP peptide 4, the molar
ratio of anti-peptide to IL-1ß at 5 pg/ml is 1,900, but with
IL-1ß at 1,000 pg/ml, the ratio falls to 9.5. Since D10S cells
proliferate to higher concentrations of IL-1ß in the presence of
anti-IL-1RAcP Abs, the limiting receptor for IL-1 signaling is the
number of available IL-1RAcP molecules. Therefore, under conditions in
which IL-1RAcP is not blocked completely by these Abs, D10S cells will
proliferate to excess IL-1ß.
The affinity-purified anti-IL-1RAcP Abs were highly specific for
their respective synthetic peptides, and bound to D10S and EL-4 cells
(Fig. 2A). These results support the concept that the
hydrophilic regions we selected recognize surface epitopes on the
natural murine IL-1RAcP. In fact, Western blot analysis showed that
anti-peptide 4 recognized both soluble IL-1RAcP and intact IL-1RAcP
(Fig. 2B). Anti-peptide 4, in the Ig-like domain III
(304–314), is of particular importance because this region shares
little sequence homologies to IL-1RI. By using affinity-purified
anti-IL-1RAcP peptide 4, we studied the regulation of IL-1RAcP on
D10S cells. Incubation of IL-1 or IL-10 for 24 h did not affect
the level of the expression of IL-1RAcP (Fig. 6) or the proliferation
of D10S cells when compared with control cells (data not shown). In
these same cells, IL-1 down-regulated the IL-1RI by 25%, confirming
over previous studies (33). Moreover, IL-10 increased the IL-1RI by
30%, but had no effect on IL-1RAcP. The expression of mRNAs for
IL-1RAcP and IL-1RI in D10S cells treated with IL-1 or IL-10 also
showed similar pattern to that of surface IL-1R. It has been reported
that IL-10 significantly up-regulated LPS-elicited IL-1Ra production
(43), and that Th2 cells induce IL-1Ra production upon cell/cell
contact with monocytes (44). Hence, IL-10 may favor the monocyte
production of IL-1Ra by D10S-like cells. However, we did not observe
any significant effects of murine IL-10 on D10S expression of IL-1RAcP
at the surface or RNA level. The overall findings suggest that IL-1RAcP
is not regulated by IL-1 or IL-10 in Th2 cells under the same
conditions that these cytokines regulate IL-1RI. These observations
lead us to conclude that IL-1 signaling requires IL-1RAcP, but surface
expression of IL-1RI regulates the responsiveness to IL-1 in these
cells.
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Acknowledgments |
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Footnotes |
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2 Present address: Korea Research Institute of Bioscience and Biotechnology, P.O. Box 115, Yuseong, Taejon 305-600, Korea.
3 Address correspondence and reprint requests to Dr. Charles A. Dinarello, Division of Infectious Diseases, B168, University of Colorado Health Sciences Center, 4200 East Ninth Ave., Denver, CO 80262.
4 Abbreviations used in this paper: IL-1RAcP, interleukin-1 receptor accessory protein; DEPC, diethyl pyrocarbonate; ECL, enhanced chemoluminescence; FGF, fibroblast growth factor; FI, fluorescence intensity; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GM-CSF, granulocyte-macrophage CSF; h, human; IL-1Ra, interleukin-1 receptor antagonist; IL-1Rrp, interleukin-1 receptor-related protein; KLH, keyhole limpet hemocyanin; MFI, mean fluorescence intensity; MTS, 3-(4, 5-dimethyl thiazol-2-yl)-5-(3-carboxy methyl)-2-(4-sulfophenyl)-2H-tetrazolium; PBST, phosphate-buffered saline containing 0.05% Tween-20; PMS, phenazine methosulfate; RT, room temperature; TNFbp, tumor necrosis factor-binding protein.
Received for publication July 30, 1997. Accepted for publication December 4, 1997.
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, Signal
sequence; 
, extracellular; 
, transmembrane; 
, cytoplasmic
domain; 
, position of sequences chosen for synthetic peptides.
