The Interleukin-1 Type 2 Receptor Gene Displays Immediate Early Gene Responsiveness in Glucocorticoid-stimulated Human Epidermal Keratinocytes*

Walter J. LukiwDagger , Jorge Martinez, Ricardo Palacios Pelaez, and Nicolas G. BazanDagger §

From the Dagger  Louisiana State University Medical Center, Neuroscience Center and Department of Ophthalmology, New Orleans, Louisiana 70112-2272 and  Research and Development Department, IFIDESA-ARISTEGUI, Industrial Farmaceutica y De Especialidades SA, Maria de Molina 37, Madrid 28006, Spain

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Human epidermal keratinocytes (HEKs) in primary culture (P2-P4) were used to study glucocorticoid (GC)-mediated transcription of the genes encoding the constitutively expressed interleukin-1 type 1 receptor (IL-1R1) and the inducible interleukin-1 type 2 receptor (IL-1R2). Utilizing Northern dot blot analysis and a quantitative reverse transcription-polymerase chain reaction protocol for IL-1R1 and IL-1R2, dexamethasone and, in particular, the budesonide epimer R were shown to effectively and rapidly induce transcription from the IL-IR2 gene when compared with IL-1R1 or beta -actin RNA message levels in the same sample. Southern blot analysis of newly generated IL-1R2 reverse transcription-polymerase chain reaction products using end-labeled IL-1R2 intron probes suggested that GC enhancement of IL-1R2 expression was regulated primarily at the level of de novo transcription. GC-induced IL-1R2 gene transcription displayed features characteristic of a classical immediate early gene response, including a signal transduction function, a relatively low basal abundance, a rapid, transient induction, cycloheximide superinduction, actinomycin D suppression, and a rapid decay of IL-1R2 RNA message. Parallel time course kinetic analysis of IL-1R2 RNA message levels with Western immunoblotting revealed tight coupling of de novo IL-IR2 gene transcription with translation of the IL-1R2 RNA message; a newly synthesized (~46-kDa) IL-1R2 protein was detected in the HEK growth medium as early as 1 h after budesonide epimer R treatment. These data indicate that different GC compounds can variably up-regulate the IL-1R2 response in HEKs through transcription-mediated mechanisms and, for the first time, suggest that a gene encoding a soluble cytokine receptor can respond like an immediate early gene.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES

The immunosuppressive and anti-inflammatory activities of glucocorticoids (GCs)1 are not well understood, due largely to limited knowledge of their complex activities at the level of gene expression. Among the cytokines that mediate the cellular immune and inflammatory response, the interleukin-1 (IL-1) signaling system plays a central role in diverse cell types (1-3). The ~17-kDa IL-1 affects target cells initially through two distinct types of transmembrane receptor complexes: (a) an integral type 1 (~80-kDa) IL-1 receptor (IL-1R1) protein, and (b) an external type 2 (~68-kDa) IL-1 receptor (IL-1R2; Refs. 4-6) protein. IL-1R1 and IL-1R2 proteins, which are both members of the immunoglobulin gene superfamily, each share virtual identity in their extracellular IL-1 ligand binding domain and transmembrane anchor; however, the IL-1R1 has a 213-amino acid cytoplasmic domain essential for signal transduction, whereas the IL-1R2 has a 29-amino acid cytoplasmic terminus incapable of signal transfer (2-6). Signal transduction through the IL-1R1 can be relayed via cytosolic IL-1R-associated kinase cascades, leading to Ikappa B phosphorylation and degradation, followed by NF-kappa B action on target NF-kappa B-regulated gene promoters (7-9). These target genes can include those coding for: (a) specific IL-1-converting enzymes (10), (b) proteases that cleave the soluble extracellular domain of IL-1R2 (11); (c) the de novo expression of IL-1R1, IL-1R2, and related growth factor genes (2, 11, 12); and (d) transcriptional activator proteins such as AP1, NF-IL6, and NF-kappa B, which, in turn, induce transcription from IL-1-sensitive, pro-inflammatory IEGs, such as cyclooxygenase-2 (13, 14). At least two distinct ligand-mediated mechanisms can modulate autocrine, juxtacrine, or paracrine IL-1 stimulation: (a) the cellular production of the ~20-kDa IL-1 receptor antagonist, which competes with the binding of secreted IL-1 to IL-1R1 at the cell surface (15), and (b) the de novo expression of the non-signal-transducing IL-1R2 "decoy" receptor which, through its prominent cysteine-rich extracellular domain, acts as a local membrane-bound scavenger of IL-1 (but not IL-1 receptor antagonist) or serves as a soluble ~46-kDa molecular sink for IL-1 (4, 11) by virtue of an extracellular protease cleavage site.

Here we have studied the effects of the GCs dexamethasone (DEX), the novel nonhalogenated budesonide epimer R (BUDeR), and an equimolar racemic mixture of budesonide S and R (BUDr; Fig. 1) on IL-1R1 and IL-1R2 RNA message and receptor protein induction using human epidermal keratinocytes (HEK) cells at passages P2-P4 in a primary culture test system. HEK cells respond to a wide variety of cytokines and lipid mediators (12, 14, 16) and provide a useful model to study IL-1 biology because they both synthesize and secrete IL-1 and respond to it via intrinsic IL-1R1- and IL-1R2-mediated pathways (17, 18).2 Northern dot blot analysis, quantitative reverse transcription-polymerase chain reaction (RT-PCR), and multiplex RT-PCR using human-specific IL-1R1, IL-1R2, and beta -actin primer sets radiolabeled to high specific activity (>109 dpm/µg) indicated that after only 1 h of HEK cell stimulation, DEX and, in particular, BUDeR elicited a strong induction of the non-signal-transducing IL-IR2 RNA message but not of the IL-1R1 transcript. Southern blotting and RT-PCR unprocessed transcript assay (19, 20) indicated that IL-1R2 RNA increases were primarily the result of de novo IL-1R2 gene transcription. Temporal analysis of IL-1R2 RNA message abundance with newly synthesized IL-1R2 protein indicated that the induction of the IL-1R2 gene expression pathway was a relatively rapid event, fulfilling each of the criteria for the classical cellular IEG response (21). A ~46-kDa IL-1R2 protein was detected by Western immunoblot analysis in HEK whole cell extracts (WCXTs) and in 200-fold concentrated HEK extracellular growth medium as early as 1 h, and increasing to 12+ h after BUDeR treatment. These results suggest that rapidly induced IL1-R2 gene transcription, tight coupling to IL-1R2 message translation, and secretion of the IL1-R2 protein can contribute to the anti-inflammatory potential of GC compounds in HEKs by scavenging and antagonizing extracellular cytokine IL-1 activity. Enhanced local up-regulation of the IL-1R2 signaling pathway by nonhalogenated 16alpha ,17alpha -substituted GCs such as BUDeR may make these compounds particularly useful anti-inflammatory agents in epidermal keratinocytes that play key structural and physiological roles in primary immune defense and the inflammatory response in vivo.


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Fig. 1.   Comparative structures of DEX and the non-9alpha -fluorinated, 16alpha ,17alpha -acetyl-substituted stereoisomeric glucocorticosteroids BUDeR and budesonide epimer S. BUDr is an equimolar racemic mixture of BUDeR and budesonide epimer S compounds. Me, methyl; Pr, propyl.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Inducers, Antagonists, and Other Reagents-- DEX (9alpha -fluoro16alpha -methyl-11beta ,17alpha ,21-trihydroxy-1,4-pregnadiene-3,20-dione; Sigma D-1756), BUDeR (16alpha ,17alpha -butylidenebis(oxy)-11,21-di-hydroxypregna-1,4-diene-3,20-dione), and BUDr (an equimolar racemic mixture of budesonide S and R; Fig. 1) were solubilized as 5 µg/µl solutions in 99.9% dimethyl sulfoxide (ACS reagent grade; Sigma D8779). Actinomycin D (Sigma A9415 dissolved in dimethyl sulfoxide) and cycloheximide (Sigma C0934 dissolved in ACS grade ethanol) were used at 1 and 10 µg/ml, respectively. 20 ml of keratinocyte growth medium (KGM; Clonetics) were made (100 nM with respect to each GC) to treat HEK cells in T-75 (Corning 25110-75; 75 cm2) tissue culture flasks with these compounds. The ligands DEX, BUDeR, and BUDr were solubilized into the KGM by 5 min of vigorous vortexing. The KGM in which the cells were incubated (37 °C in 5% CO2) was decanted and replaced with ligand-containing KGM and then warmed to 37 °C over a time course of 0-24 h. All other reagents, enzymes, and media were of the highest grades commercially available and were used without further purification.

HEK Cells in Culture-- HEKs respond productively to a wide variety of cytokines, lipid mediators, and GCs. Cryopreserved normal HEK cells (obtained from pooled donors (Clonetics CC-2504; HEK-Neo pooled) and received as frozen primary cultures) were grown to 80% confluence (~2-3 × 106 HEK cells/T-75 flask) in 20 ml of KGM supplemented with a serum containing 7.5 mg/ml bovine pituitary extract, 0.1 µg/ml human epidermal growth factor, 5 mg/ml insulin, 50 µg/ml gentamycin/amphotericin, and 0.5 mg/ml hydrocortisone in T-75 flasks at 37 °C in 5% CO2 according to the manufacturer's specifications (Clonetics). HEK cells responded well to ligands between P2 and P4, after which there was a graded decline in both the growth rate and responsiveness to ligand induction. The HEK cell cultures used here, which were obtained from pooled neonatal or adult donors, revealed little difference in their individual responses to either DEX, BUDr, or BUDeR stimulation, as measured by the induction of the IL-1R2 RNA message (Figs. 3-6). For maximal ligand induction, cells were deprived for 24 h to 0.5% of normal serum levels before the addition of GC test compounds. 100 nM (final concentration) DEX, BUDeR, or BUDr were added to HEKs, and cells were harvested at 0, 1, 3, 6, 12, and 24 h. For each treatment condition, HEKs were cultured in duplicate T-75 flasks for the parallel isolation of either total RNA for Northern blot and RT-PCR analysis or WCXTs for Western immunoblot detection.

Harvesting of HEK and Preparation of WCXTs-- All extraction procedures were performed at 4 °C on wet ice. After each incubation period with GC test compounds, the KGM was decanted and replaced with 20 ml of Dulbecco's phosphate-buffered saline (Life Technologies, Inc.) containing 1 mM phenylmethylsulfonyl fluoride (Sigma P-7626), 0.05 µg/µl aprotinin (Sigma A-6279), and 0.025 µg/µl leupeptin (Sigma L-2884). HEKs were scraped into a suspension of phosphate-buffered saline containing these enzyme inhibitors and pelleted at 4 °C by centrifugation at 1400 × Gav for 10 min. HEK cellular pellets were gently resuspended in 200 µl of a hypotonic buffer consisting of 20 mM HEPES (pKa = 7.55 at 20 °C), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, and 1% (v/v) aprotinin (which was made 0.5% (v/v) with SDS (Sigma L-4509) just before use). HEK WCXTs were thoroughly resuspended using a 200 µl Pipetman with a cut-off tip followed by repeated pipetting on wet ice for 5 min. HEK WCXT protein concentrations were determined using a dotMETRIC protein microassay (Chemicon, Temecula, CA; dot sensitivity, 0.3 ng of protein) using bovine serum albumin (bovine serum albumin radioimmunoassay grade; Sigma A-7888) as a standard.

Isolation of Total Keratinocyte RNA-- After each GC induction time point, the KGM was completely replaced with 7.5 ml of TRIzol reagent (Refs. 22 and 23; Life Technologies, Inc. catalog number 15596-026). Samples were then incubated for 5 min, with gentle shaking at 25 °C to dissociate nucleoprotein complexes. After the addition of 1.5 ml of reagent grade chloroform, suspensions were transferred to sterile, diethylpyrocarbonate (Sigma D5758)-treated 15-ml conical tubes, vigorously shaken for 5 min, incubated at 25 °C for 15 min, and centrifuged at 5000 × Gav for 15 min at 4 °C. The upper aqueous phase, containing total extracted RNA, was transferred into sterile RNase-free 15-ml conical tubes, and 4 ml of 100% (v/v) ACS reagent grade iyl alcohol were added. RNA was precipitated overnight at -80 °C and then centrifuged at 9000 × Gav for 10 min at 0 °C. Supernatants were vacuum aspirated, and visible RNA pellets were washed twice with 10 ml of 80% ACS reagent grade ethanol (v/v) and pelleted by centrifugation at 9000 × Gav for 10 min at 4 °C. Supernatants were aspirated again, and the total RNA pellet was dried under a vacuum for ~10 min over a bed of anhydrous calcium sulfate (Drierite; W. A. Hammond). The partially dessicated RNA pellet was then resuspended in RNase-free water to ~1 µg/µl by incubating for 15 min at 60 °C in an Eppendorf 5436 thermomixer. Total RNA concentrations were determined spectrophotometrically at A260, and samples had A260:A280 ratios of >= 2.1.

Northern Dot Blot Analysis-- Northern dot blots containing up to 10 µg of alkaline-denatured total HEK RNA were prepared using 9 × 12-cm Zeta-Probe GT Nylon membranes and a Bio-Dot SF Microfiltration apparatus (Bio-Rad 170-6543). Membranes were hybridized using ExpressHyb hybridization solution (CLONTECH), and RT-PCR generated IL-1R1, IL-1R2, or beta -actin cDNA probes (Table I) end-radiolabeled by using [gamma -32P]dATP (~3000 Ci/mmol) or by direct incorporation using [alpha -32P]dCTP (~3000 Ci/mmol; Amersham redivue). Membranes were washed twice for 30 min at 61 °C in 1 mM EDTA, 40 mM NaHPO4, pH 7.2, and 5% SDS according to the manufacturer's protocols.

                              
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Table I

RT-PCR and Multiplex RT-PCR Analysis-- Typically, 1.0 µg of the total HEK RNA was reverse transcribed into cDNA using 200 units of SuperScript II reverse transcriptase (Life Technologies, Inc.) in the presence of 5 µM random hexamers, 500 µM each deoxynucleotide triphosphate, 20 units of RNasin (Promega N2115), 50 mM Tris-HCl, 10 mM dithiothreitol, 75 mM KCl, and 3 mM MgCl2 in a 20-µl volume at 42 °C for 90 min. This generated a "mini" cDNA library from total RNA isolated from control and GC-treated HEK cells. After cDNA synthesis, samples were treated with RNase H for 90 min at 37 °C to remove residual template RNA. PCR used ~10% (v/v) of the first strand (reverse transcriptase) reaction as a DNA template in the presence of 200 µM deoxynucleotide triphosphates, 2 mM MgCl2, and 2 units of AmpliTaq polymerase (Perkin Elmer Corp.) in a final volume of 50 µl. This reaction included either separate (standard RT-PCR) or combinations of primer sets (multiplex RT-PCR) of 100 pM of each forward and reverse human-specific primer for IL-1R1, IL-1R2, or beta -actin (Stratagene catalog number 302010; Table I). Either unlabeled (for "cold" multiplex RT-PCR) or [gamma -32P]dATP (>5000 Ci/mmol; Amersham redivue AA0018) 5'-end-labeled primers (for "hot" multiplex RT-PCR) were used in these reactions. [gamma -32P]dATP isotopes were typically utilized >4 days before their quality control reference date. Quantitative IL-1R1 and IL-1R2 RT-PCR amplification (Fig. 2) was performed for 31 cycles after 5 min of denaturation at 94 °C, 1 min of annealing at 60 °C, 1 min of primer extension at 72 °C, and 7 min of final extension at 72 °C after the 31st cycle using a GeneAmp 9700 PCR System (Perkin Elmer Corp.). Typically, 15 µl of each cold multiplex PCR reaction were analyzed on a 1× TBE (89 mM Tris-borate and 1 mM EDTA, pH 8.3) 1.5% agarose gel in the presence of 0.5 µg/ml ethanol-recrystallized ethidium bromide (Life Technologies, Inc.; 5585 UA). PCR products of the hot multiplex PCR were separated on 5% or 10% (acrylamide:bisacrylamide, 30:1) Mini Protean II ready gels (Bio-Rad 161-1101 and 161-1109) in 0.5× TBE buffer. Gels were dried onto Whatman No. 1 filter paper (Bio-Rad gel dryer model 583) for 90 min at 80 °C and quantitated via signal transfer onto PhosphorImager storage screens.

Southern Analysis of Unspliced IL-1R2 Transcript Levels-- Southern blotting and RT-PCR unprocessed transcript assay were performed as described previously (19, 20) with the following modifications. Up to 5 µg of 0, 1, 3, and 6 h BUDeR-induced total HEK RNA was fed directly into a One-Step RT-PCR Amplification System (Life Technologies, Inc., Superscript 10928-026) using either IL-1R2- or beta -actin-specific primers. 35 µl of this integrated RT-PCR reaction were resolved by electrophoresis through 1× TBE, 1.5% agarose gels, followed by Southern transfer onto Zeta-Probe GT Nylon membranes (Bio-Rad). Antisense oligonucleotides derived from human IL-1R2 exon 7, IL-1R2 intron 7 and intron 8, or the beta -actin coding sequence (Table I) were 5'-end-labeled using [gamma -32P]dATP (>5000 Ci/mmol; Amersham redivue AA0018) and T4 polynucleotide kinase (Promega M4101), hybridized at 55 °C to 60 °C using ExpressHyb hybridization solution, and were washed according to the manufacturer's instructions (CLONTECH).

Western Analysis and Immunoblotting-- 30 µg of control or GC-treated HEK WCXTs or HEK KGM concentrated 200-fold using Dulbecco's phosphate-buffered saline and Centricon-10 concentrators (Amicon, Beverly, MA) were analyzed under reducing conditions on 10% acrylamide Tris-glycine SDS gels (Bio-Rad Ready-Gels 161-0907); proteins were transferred onto Hybond-P (Amersham RPN2020F) polyvinylidene difluoride transfer membranes using a mini-Trans-Blot electrophoretic transfer cell (Bio-Rad 170-3930). Membranes were blocked and probed with an anti-human IL-1R2 rat monoclonal primary antibody (Cdw121b) that exhibited no cross-reactivity with human IL-1R1 (Genzyme, Cambridge, MA). Bound primary antibodies were detected with an anti-rat IgG peroxidase-linked secondary antibody (Amersham NA932) and developed with an ECL Plus Western blotting analysis system, according to the manufacturer's instructions (Amersham RPN2132).

Data Analysis and Quantitation-- For cold multiplex-PCR, agarose gels were stained in 0.5 µg/ml ethidium bromide, and images were either photographed using Polaroid instant photography or digitized using the Bio-Rad Gel Doc 1000 UV Gel Documentation System. For hot multiplex-PCR, dried polyacrylamide gels were exposed to phosphorimager storage screens, and signals were analyzed via a raster scanning laser on a GS-250 molecular imager (Bio-Rad). To quantitate Western analysis signals Coomassie Blue-stained gels were placed on a UV/white light conversion screen (Bio-Rad 1707538) and photometrically digitized using the GS-250 molecular imager. Relative intensities of the IL-1R1 and IL-1R2 RNA signals were quantitated against the beta -actin RNA signal in the same sample using phosphorimager analysis and the data acquisition packages provided with each instrument. All p values were derived from protected t tests or least square means from a two-way factorial analysis of variance (ANOVA).

IL-1R Promoter DNA and IL-1R2 RNA Sequence Analysis-- PCR primers were designed using Hitachi Oligo DNA sequence analysis software (Version 5.0). Human IL-1R1 and IL-1R2 gene promoter 5' untranslated region and IL-1R2 mRNA sequence data were obtained through GenBankTM accession numbers L09701, U14177, U14178 and X59770, respectively. RNA sequence analysis was performed using both Kodak/IBI/Pustell (a modified Version 2.04) and Omiga 1.1.3 (Oxford Molecular) RNA subsequence analysis packages.

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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Utilizing human-specific IL-1R1 and IL-1R2 cold or hot primer sets (Table I) and purified IL-1R1 and IL-1R2 cDNA templates, it was determined that at 31 cycles of PCR amplification, the masses of the IL-1R1 and IL-1R2 input cDNAs were linear functions of both the IL-1R1 and the IL-1R2 primary PCR products (Fig. 2). Similarly, quantitative beta -actin PCR analysis using human-specific beta -actin primers at a 60 °C annealing temperature has been described previously (23). To ascertain whether the forward and reverse primer sets specific for human IL-1R1 and IL-1R2 were compatible in the same PCR reaction conditions at a common 60 °C annealing temperature, pilot experiments were performed using mixed IL-1R1 and IL-1R2 primer sets (Table I) after 3 h of either DEX or BUDeR treatment of HEK cells. Results of a cold multiplex PCR experiment using only these two primer sets are shown in Fig. 3A. The gel reveals that only two bands in the GC-treated HEKs were generated at 340 and 574 bp, corresponding to the expected size of the IL-1R1 and IL-1R2 primary PCR products from published DNA sequences (GenBankTM accession numbers L09701 and X59770, respectively). Control HEK samples (no DEX, BUDeR, or BUDr) yielded a strong IL-1R1 (304-bp) signal but only a weak 574-bp band, corresponding to basal levels of the IL-1R2 RNA message under control (uninduced) conditions. In agreement with previous observations (24), this finding demonstrates that human-specific IL-1R1 and IL-1R2 RNA messages could be both reliably and simultaneously quantitated in single 0.5-ml PCR reaction tubes. Notably, simultaneous 3-h incubation of combinations of DEX + BUDr or DEX + BUDeR in serum-deprived HEKs showed no additive function in either IL-1R1 or IL-1R2 RNA message induction (Fig. 3A).


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Fig. 2.   RT-PCR quantitation of IL-1R1 and IL-1R2 RNA expression in HEK cells. At 31 cycles, the relationship of input cDNA to IL-1R1 and IL-1R2 primary PCR product was in the linear range of amplification.


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Fig. 3.   Quantitation of the IL-1R1 and IL-1R2 RNA signal. A, cold (ethidium bromide-stained) RT-PCR utilizing mixed primer sets for IL-1R1 and IL-1R2 in the same sample; B, hot (radiolabeled) RT-PCR utilizing mixed end-radiolabeled primer sets for IL-1R1, IL-1R2, and beta -actin in the same sample. HEK cells were treated with DEX, BUDeR, and/or BUDr for 3 h. M1, 246-bp marker; M2, 200, 400, and 800-bp low DNA mass ladder marker (Life Technologies, Inc.). B, beta -actin shows anomalous band spreading during multiplex RT-PCR. Low intensity IL-1R2 RNA signals are detectable in serum-deprived untreated HEK cells using either technique.

Hot multiplex-PCR at 31 cycles may provide a more precise quantitation of the levels of specific RNA messages within complex populations of cDNA (as derived from the reverse transcription of total RNA) because: (a) the fluorescence of ethidium bromide-stained cold gels is nonlinear with respect to varying DNA concentrations, making RT-PCR products difficult to quantify, and (b) only 5'-end radiolabeled primers labeled to high specific activity are incorporated into the primary PCR product in direct proportion to the amount of existing template in the original cDNA sample. Omission of the reverse transcriptase mix yielded no PCR signals; similarly, RNase-free DNase treatment of the HEK RNA extracts yielded only the expected primary PCR bands at 340 and 574 bp. The induction of the IL-1R1, IL-1R2, and beta -actin RNA messages with BUDeR, DEX, and BUDr in HEK cells using this technique is shown in Fig. 3B. Data on the abundance IL-1R1 and IL-1R2 RNA messages were normalized against the RNA signal in the same sample for beta -actin, a moderate-to-highly abundant DNA transcript (23, 25). IL-1R1 and IL-1R2 RNA message levels in control ranged from less than ~1% to ~4% of signal for beta -actin RNA (Fig. 4). The strongest GC induction at 3 h of the IL-1R2 RNA over the IL-1R2 RNA control signal was by BUDeR (9.2-fold; p < 0.02), followed by DEX (6.6-fold, p > 0.04) and BUDr (5.2-fold, p > 0.06). For each of the GCs studied, the IL-1R1 RNA signal level was slightly depressed at the 1 and 3 h time points when compared with control levels at zero time (Figs. 3 and 4). Using either Northern dot blot analysis (Fig. 4A) or the hot quantitative RT-PCR at 31 cycles for both IL-1R1 and IL-1R2 (Fig. 4B), time points of 0, 1, 3, 6, 12, and 24 h established a time course over which DEX and BUDeR induced transcription from the IL-1R2 gene in HEK cells. De novo IL-1R2 gene transcription was almost completely abolished by pretreating HEKs with the DNA transcriptional inhibitor actinomycin D (Fig. 4, ACTD) at 1 µg/ml in KGM. Cycloheximide (Fig. 4, CHX; 10 µg/ml) superinduced IL-1R2 but not IL-1R1 RNA message abundance (Fig. 4, A-C).


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Fig. 4.   Time course of induction of human IL-1R2 genes with DEX and BUDeR as monitored by Northern dot blot analysis (A) and by quantitative RT-PCR using end radiolabeled primers (B). Using either technique, IL-1R2 was found to be maximally induced at 3 h. In these experiments, BUDeR was a consistently stronger inducer of IL-1R2 gene transcription than DEX (C). Actinomycin D (ACTD) strongly suppressed DEX- or BUDeR-mediated induction of the IL-1R2 transcript. Cycloheximide (CHX) consistently superinduced IL-1R2 but not IL-1R1 RNA message. Exposure time for IL-1R1 and IL-1R2 or beta -actin dot blots was 26 and 8 h, respectively; exposure time for IL-1R1, IL-1R2, and beta -actin RT-PCR signals was 1 h. Significance over IL-1R1 signal at zero time, dashed line; *, p < 0.04; **, p < 0.01; ***, p < 0.001 (ANOVA).

PCR primers that lie in exons 4 and 8 of the IL-1R2 gene and from the beta -actin coding region (Table I) were then used for the direct amplification of 0, 1, 3, and 6 h BUDeR-induced HEK RNA using a single-tube integrated RT-PCR. Samples were then subjected to agarose gel electrophoresis, Southern blotting, and probing with an end-labeled beta -actin oligonucleotide probe to demonstrate equivalent RNA message levels and RT-PCR efficiencies, an end-labeled oligonucleotide from exon 7 to measure processed IL-1R2 RNA levels, and end-labeled oligonucleotides from introns 7 and 8, (separating exons 7 and 8 and exons 8 and 9, respectively) of the IL-1R2 gene to measure the abundance of unspliced IL-1R2 transcripts. To demonstrate the quantitative nature of this assay for IL-1R2-amplified sequences, mixtures of total RNA from control and GC-treated HEKs were subjected to the same RT-PCR and Southern blotting conditions and IL-1R2 exon 7 hybridization protocols and were analyzed along with the other samples. Quantitation of IL-1R2 signal demonstrates the linearity of this assay over the range of IL-1R2 induction observed (Fig. 5, right panel). No unspliced IL-1R2 transcript and little processed IL-1R2 RNA were detectable at 0 h; however both unprocessed (intron 7-probed) and processed (exon 7-probed) IL-1R2 transcripts are detectable in HEK RNA at 1 and 3 h after GC induction. Similar results were obtained using an IL-1R2 intron 8 probe (data not shown). This suggests that GCs are inducing IL-1R2 RNA message, at least in part, by increasing productive transcription from the IL-1R2 gene.


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Fig. 5.   BUDeR induces unprocessed IL-1R2 DNA transcripts and processed IL-1R2 RNA message in HEK cells. HEK cells were induced with BUDeR for 0, 1, 3, and 6 h; total RNA was then isolated and subjected to RT-PCR using primers for both IL-1R2 and beta -actin (Table I). Primary PCR products were then separated by electrophoresis, Southern blotted, and hybridized to labeled oligonucleotides derived from the beta -actin coding region, intron 7 of the IL-1R2 gene, or exon 7 of the IL-1R2 gene. Membranes were phosphorimaged, and exposure times were adjusted for comparison of each probe. Lanes A-E contain total RT-PCR products from HEK cells treated for 3 h with BUDeR and contain 100%, 75%, 50%, 25%, and 0% of the total HEK RNA. These samples were subjected to electrophoresis, Southern transferred, and hybridized with a labeled IL-1R2 exon 7 probe. The linearity of this assay using the standard curve generated by samples A-E is shown in the bottom panel.

The parallel induction of the ~68-kDa IL-1R2 protein by DEX and BUDeR is shown in Fig. 6. De novo IL-1R2 protein synthesis was almost completely blocked with cycloheximide when HEK cells were pretreated at 10 µg/ml in KGM. The rapid appearance of both a ~68-kDa (cell-associated) and a ~46-kDa (soluble) IL-1R2 protein in HEK WCXTs, suggesting rapid IL-1R2 extracellular cleavage, was closely associated with increasing IL-1R2 RNA message output at 3 h. This suggested a tightly regulated transcription-to-translation mechanism operating in the GC-triggered IL-1R2 signaling pathway. Induction coupling of the IL-1R2 RNA message to translation into IL-1R2 protein is shown at the 3 h time point in Fig. 7. Newly synthesized ~46-kDa IL-1R2 protein was rapidly shed into the HEK pericellular medium and was abundantly detected in both HEK WCXTs (Fig. 8A) and in 200-fold concentrated HEK KGM as early as 1 h after GC induction (Fig. 8B).


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Fig. 6.   Induction of the human ~68-kDa (membrane-associated) IL-1R2 protein with DEX and BUDeR in HEK cell WCXTs, as detected by Western immunoblotting using a human IL-1R2-specific monoclonal primary antibody, was significant at 3 h (p < 0.02). A ~46-kDa IL-1R2 (shed) protein was also associated with HEK WCXTs at this time point. Cycloheximide strongly blocked both DEX- and BUDeR-mediated induction of both membrane-associated and soluble IL-1R2 protein. Significance over IL-1R2 signal at zero time, dashed line; *, p < 0.05; **, p < 0.02, ***, p < 0.001 (ANOVA).


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Fig. 7.   Tight coupling of the induction of the IL-1R2 RNA message (top panel) with IL-1R2 protein (bottom panel) in control, DEX-, BUDeR-, and BUDr-treated HEK cells relative to the IL-1R1 control (dashed line) assayed at 3 h after GC induction is typical of an IEG response. *, p < 0.04; **, p < 0.01; ***, p > 0.05 (ANOVA).


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Fig. 8.   Fate of newly synthesized IL-1R2 protein. A, Coomassie Blue-stained 10% TGSDS gel profile of HEK WCXTs 0-24 h after BUDeR induction. B, Western blot of HEK KGM concentrated 200-fold and probed with an anti-human IL-1R2 rat monoclonal primary antibody (Genzyme). M, molecular weight standards; numbers to the left of the gel are the molecular weights of markers in A. Arrows at ~46 kDa indicate the approximate migration of HEK-associated and shed IL-1R2 protein.

Lastly, the 1286-nucleotide IL-1R2 RNA message contains several structural features resembling IEG transcription products, including enrichment in adenine-uridine-rich elements, RNA instability elements associated with rapidly degraded RNA messages (Refs. 26 and 27; Fig. 9). Primary cultures of HEK cells at P2-P4 may provide one preferred test system for evaluating GC-mediated IL-1, IL-1R1, and IL-1R2 gene signaling pathways because these epidermal keratinocytes were found to be at least 10-fold more responsive to both DEX and BUDeR stimulation than HeLa ATCC CCL-2, a transformed, IL-1-responsive human epithelioid cell line. No GC-mediated IL-1R2 genetic response was noted in WI38 (ATCC CCL-75) cells, a diploid human lung fibroblast cell line, when treated under identical assay conditions (Fig. 10).


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Fig. 9.   Schematic diagram showing the positions (arrows) of 17 adenine-uridine-rich RNA message instability elements relative to the polyadenylation signal in the 1286-nucleotide IL-1R2 RNA message (GenBankTM accession number X59770).


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Fig. 10.   Comparison of 3-h DEX- and BUDeR-mediated induction of the IL-1R2 RNA message in HEK neonatal and adult primary cell lines and in HeLa and WI-38 cells, relative to their respective control levels (dashed line). HeLa and WI-38 cell lines, transformed cells of IL-1-responsive epidermal and fibroblast origin, respectively, showed statistically insignificant IL-1R2 induction (p > 0.45); primary cultures of either neonatal or adult HEKs consistently displayed significant IL-1R2 induction by the GCs DEX or BUDeR. Significance over IL-1R2 signal at zero time, dashed line; *, p < 0.04; **, p < 0.01 (ANOVA).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

DEX, BUDeR, GC Receptor Binding, and IL-1R Gene Expression-- Despite a nonsymmetrical 16alpha ,17alpha -acetyl substitution and a lack of a 9alpha -fluoro atom in its steroid nucleus, BUDeR has been shown to have an 11-fold higher binding affinity for the GC receptor when compared with DEX (28). BUDeR also shows greater potency than DEX in modulating NF-kB-DNA binding (14) and in stimulating transcription from synthetic target genes regulated by GC-responsive elements in their promoters (29). Stereospecificity of the BUDeR 16alpha ,17alpha -acetyl substitution at the GC receptor may also be critical in GC-receptor complex activation because BUDeR was found to induce IL-1R2 gene transcription 1.8-fold greater (p < 0.02) than an equivalent concentration of BUDr, a 1:1 racemic mixture of the budesonide R and S epimers (Fig. 1). Notably, no synergism was noted when combinations of the GCs (DEX + BUDr or DEX + BUDeR) were used in treating HEK cells (Fig. 3), suggesting the recruitment of single DEX-, BUDr-, or BUDeR-mediated signaling pathways during IL-1R2 gene induction via productive GC-GC receptor interaction (14, 30-36). BUDeR was found to be the most efficient in rapidly up-regulating IL-1R2 RNA message and IL-1R2 protein abundance; a small effect was also noted on the depression of IL-1R1 RNA and protein levels (Figs. 4 and 7). The high ratio of topical to systemic activity of BUDeR when compared with DEX (28, 29), indicates that BUDeR may be pharmacologically preferred when inhibiting IL-1 biology and IL-1R expression in the treatment of inflammatory conditions associated with epidermal keratinocytes and related cell types.

GCs and the Induction of IL-1R2 Gene Transcription-- GC compounds can inhibit the activation of genes coding for cytokine signaling such as IL-1 and cell surface receptors required in the inflammatory response by limiting the availability of transcription factors to access their target promoters (31-33). A dramatic inhibition of cis-acting transcription factor AP1-like-, gamma -interferon activation sequence-, and especially NF-kappa B-DNA binding by the GCs DEX and BUDeR, temporally correlating to a reduction in cyclooxygenase-2 message synthesis, has recently been demonstrated in HEK cell lines (14). Specifically, AP1 and NF-kappa B are required for the activation of many cytokine and cytokine receptor genes (1, 8, 31). GCs, via the GC-receptor complex, can directly interact with transcription factor AP1 through AP1's N-terminal domain to moderate gene activation (32, 33); similarly, GCs like DEX can inhibit NF-kappa B activation by induction of the Ikappa B-alpha gene and Ikappa B-alpha inhibitory protein, which ultimately sequesters the NF-kappa B regulatory element as an inactive cytoplasmic complex (34, 35). However, GCs appear not to be just broad spectrum pro-inflammatory-gene repressors, but rather utilize pleiotropic strategies to potentiate anti-inflammatory responses. These processes include the transcription-mediated up-regulation of several cell surface and soluble ligand receptors, such as the beta 2-adrenergic receptor (36) and the induction of the IL-1R2 in human B and T lymphocytes (4), mononuclear phagocytes (6, 37), polymorphonuclear cells, IL-1R2-transfected fibroblasts (38) and in epidermal cell lines (16-18).

Our results suggest that in HEKs at P2-P4, BUDr, DEX, and especially BUDeR trigger an up-regulation of basal IL-1R2 gene expression, because low levels of both processed IL-1R2 RNA and IL-1R2 protein were detected in control HEK total RNA and WCXTs at zero time (Ref. 39; Figs. 3-7). No significant induction of IL-1R1 RNA message was noted. The fact that the human IL-1R1 and IL-1R2 genes are encoded by multiple, tandemly linked DNA elements located in an IL-1R coding-rich region on chromosome 2q12-13 is noteworthy; however, each IL-1R promoter appears to be under individual transcriptional control (4, 40). The IL-1R1 and IL-1R2 gene promoters are encoded by three or two different 5' exons, respectively, at this locus (40, 41), therefore depending on the selection of variably spliced IL-1R gene 5' regulatory regions, AP1- and/or NF-kB-DNA binding may be alternately utilized to promote transcription from a specific IL-1R gene isotype. Notably, each IL-1R2 promoter exon but not every IL-1R1 promoter exon contains a GC-responsive element consensus sequence (40). These latter features may allow greater flexibility in the IL-1R response to different concentrations or combinations of extracellular signaling ligands.

The GC-induced IL-1R2 Gene Behaves Like an IEG-- Newly generated IL-1R2 RNA message contains features characteristic of the transcription products of IEGs (21). These include expression in diverse cell types, a signal transduction function, relatively low basal abundance, rapid transient induction after treatment with GCs, cytokines, and mitogens such as phorbol 12-myristate 13-acetate (4, 24, 38), cycloheximide superinduction, actinomycin D repression, and rapid decay of the IL-1R2 RNA message. Moreover, RNA sequence analysis of the 1286-nucleotide IL-1R2 RNA message (GenBankTM accession number X59770) reveals 17 adenine-uridine-rich RNA instability elements typical of cytokine, lymphokine, and proto-oncogene RNA messages that are transiently expressed and rapidly degraded (21, 26, 27; Fig. 9). Northern analysis, RT-PCR assay, unprocessed DNA transcript assay, the rapid disappearance of IL-1R2 RNA signal after a 3-h GC stimulation of HEKs with either DEX or BUDeR, and actinomycin D suppression of this GC-induction suggest that the de novo GC-mediated IL-1R2 expression pathway is regulated primarily at the level of IL-1R2 gene transcription, although post-transcriptional IL-1R2 RNA message stabilization may provide auxiliary controls in other cell types (11, 16). Parallel time course kinetic analysis of newly synthesized RNA levels with Western immunoblotting also revealed a tight coupling of de novo IL-IR2 gene transcription with translation of the IL-1R2 RNA message; a newly synthesized (~46-kDa) IL-1R2 protein was detected in the HEK pericellular environment as early as 1 h after BUDeR induction.

In conclusion, the established function of IL-1R2 protein is to act as a molecular trap to capture extracellular IL-1 and thereby compromise the IL-1 signaling system (6, 11, 42, 43) as a strong negative extracellular regulator of IL-1 action. Our evidence of the rapid coupling of IL-1R2 gene transcription to IL-1R2 RNA translation into protein, followed by the shedding of the ~46-kDa IL-1R2 into the extracellular space, suggests that GC-triggered HEKs can deal rapidly with local IL-1 levels via a classical IEG response. When compared with DEX, nonfluorinated GCs bearing asymmetric 16alpha ,17alpha -acetyl substitutions such as BUDeR may elicit a stronger induction of the IL-1R2 cytokine scavenger system. Such initiator elements of the IL-1R2 signal transduction pathway may therefore present future targets for pharmacologic design in light of altered IL-1R2 gene expression in pathological conditions of the gastrointestinal tract (44), in focal cerebral ischemia (3, 45), in neuroinflammation (46), and in neurodegenerative disorders of the brain (47, 48).

    ACKNOWLEDGEMENTS

We thank Dr. Hilary Thompson (Department of Ophthalmology, Louisiana State University Medical Center) for statistical analysis of the IL-1R1 and IL-1R2 RNA and protein signal data, Joelle Finley and Josephine Roussell (Louisiana State University Medical Center Cell Culture Facility) for the maintenance of human epidermal keratinocytes, and Dr. Harvey Herschman (Molecular Biology Institute, University of California at Los Angeles) for helpful suggestions and critical evaluation of the manuscript.

    FOOTNOTES

* This work was supported by a National Institutes of Health EY02377 core grant and by a gift from the EENT Foundation. Part of this research was presented in abstract form at the 6th International Conference on Platelet-activating Factor and Related Lipid Mediators held September 21-24, 1998 in New Orleans, Louisiana.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed: Louisiana State University Medical Center, Neuroscience Center and Dept. of Ophthalmology, 2020 Gravier St., Suite D, New Orleans, LA 70112-2272. Tel.: 504-599-0831; Fax: 504-568-5801; E-mail: nbazan{at}lsumc.edu.

2 N. G. Bazan and W. J. Lukiw, unpublished observations.

    ABBREVIATIONS

The abbreviations used are: GC, glucocorticoid; HEK, human epidermal keratinocyte; RT-PCR, reverse transcription-polymerase chain reaction; DEX, dexamethasone; BUDeR, budesonide epimer R; IEG, immediate early gene; IL, interleukin; IL-1R1, IL-1 type 1 receptor; IL-1R2, IL-1 type 2 receptor; NF-kappa B, nuclear factor kappa B; AP1, activator protein 1; BUDr, an equimolar racemic mixture of budesonide S and R; WCXT, whole cell extract; KGM, keratinocyte growth medium; ANOVA, analysis of variance; bp, base pair(s).

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
  1. Barnes, P. J., and Adcock, I. (1993) Trends Pharmacol. Sci. 14, 436-441[Medline] [Order article via Infotrieve]
  2. Dinarello, C. A. (1998) Int. Rev. Immunol. 16, 457-499[Medline] [Order article via Infotrieve]
  3. Sharma, B. K., and Kumar, K. (1998) Metab. Brain Dis. 13, 1-8[Medline] [Order article via Infotrieve]
  4. McMahan, C. J., Slack, J. L., Mosley, B., Cosman, D., Lupton, S. D., Brunton, L. L., Grubin, C. E., Wignall, J. M., Jenkins, N. A., Brannan, C. I., Copeland, N. G., Huebner, K., Croce, C. M., Cannizzarro, L. A., Benjamin, D., Dower, D. K., Spriggs, M. K., and Sims, J. E. (1991) EMBO J. 10, 2821-2832[Abstract]
  5. Grant, A. J., Roessler, E., Ju, G., Tsudo, M., Sugamura, K., and Waldmann, T. A. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 2165-2169[Abstract]
  6. Colotta, F., Re, F., Muzio, M., Bertini, R., Polentarutti, N., Sironi, M., Giri, J. G., Dower, D. K., Sims, J. E., and Mantovani, A. (1993) Science 261, 472-475[Medline] [Order article via Infotrieve]
  7. Stylianou, E., O'Neill, L. A., Rawlinson, L., Edbrooke, M. R., Woo, P., and Sklatvala, J. (1992) J. Biol. Chem. 267, 15836-15841[Abstract/Free Full Text]
  8. Baeurle, P. A., and Baltimore, D. (1996) Cell 87, 13-20[Medline] [Order article via Infotrieve]
  9. Muzio, M., Ni, J., Feng, P., and Dixit, V. M. (1997) Science 278, 1612-1615[Abstract/Free Full Text]
  10. William, R., Watson, G., Rotstein, O. D., Parodo, J., Bitar, R., and Marshall, J. C. (1998) J. Immunol. 161, 957-962[Abstract/Free Full Text]
  11. Re, F., Sironi, M., Muzio, M., Matteucci, C., Introna, M., Orlando, S., Penton-Rol, G., Dower, S. K., Sims, J. E., Colotta, F., and Mantovani, A. (1996) J. Exp. Med. 183, 1841-1850[Abstract]
  12. Chedid, M., Rubin, J. S., Csaky, K. G., and Aaronson, S. A. (1994) J. Biol. Chem. 269, 10753-10757[Abstract/Free Full Text]
  13. Ristmaki, A., Narko, K., and Hla, T. (1996) Biochem. J. 318, 325-331[Medline] [Order article via Infotrieve]
  14. Lukiw, W. J., Pelaez, R. P., Martinez, J., and Bazan, N. G. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 3914-3919[Abstract/Free Full Text]
  15. Hammerberg, C., Arend, W. P., Fisher, G. J., Chan, L. S., Berger, A. E., Haskill, J. S., Voorhees, J. J., and Cooper, K. D. (1992) J. Clin. Invest. 90, 571-583[Medline] [Order article via Infotrieve]
  16. Groves, R. W., Giri, J., Sims, J., Dower, S. K., and Kupper, T. S. (1995) J. Immunol. 154, 4065-4072[Abstract/Free Full Text]
  17. Rothwell, N. J., and Hopkins, S. J. (1995) Trends Neurosci. 18, 130-136[CrossRef][Medline] [Order article via Infotrieve]
  18. Rauschmayr, T., Groves, R. W., and Kupper, T. S. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 5814-5819[Abstract/Free Full Text]
  19. Gilbert, R. S., Reddy, S. T., Kujubu, D. A., Xie, W., Luner, S., and Herschman, H. R. (1994) J. Cell. Physiol. 159, 67-75[Medline] [Order article via Infotrieve]
  20. Reddy, S. T., Gilbert, R. S., Xie, W., Luner, S., and Herschman, H. R. (1994) J. Leukocyte Biol. 55, 192-200[Abstract]
  21. Morgan, J. I., and Curran, T. (1995) Trends Neurosci. 18, 66-67[CrossRef][Medline] [Order article via Infotrieve]
  22. Chomczynski, P. (1993) BioTechniques 15, 532-536[Medline] [Order article via Infotrieve]
  23. Lukiw, W. J., Rogaev, E. I., and Bazan, N. G. (1996) Alz. Res. 2, 221-228
  24. Groves, R. W., Sherman, L., Mizutani, H., Dower, S. K., and Kupper, T. S. (1994) Am. J. Pathol. 145, 1048-1056[Abstract]
  25. Lukiw, W. J., and Bazan, N. G. (1997) J. Neurosci. Res. 50, 937-945[CrossRef][Medline] [Order article via Infotrieve]
  26. Shaw, G., and Kamen, R. (1986) Cell 46, 659-667[Medline] [Order article via Infotrieve]
  27. Chen, C.-Y. A., and Shyu, A.-B. (1995) Trends Biochem. Sci. 20, 465-470[CrossRef][Medline] [Order article via Infotrieve]
  28. Dahlberg, E., Thalen, A., Brattsand, R., Gustafsson, J. A., Johansson, U., Roempke, K., and Saartok, K. (1984) Mol. Pharmacol. 1, 70-78
  29. Smith, C. L., and Kreutner, W. (1998) Arzneim. Forsch. 48, 956-960[Medline] [Order article via Infotrieve]
  30. Tsai, M. J., and O'Malley, B. W. (1994) Annu. Rev. Biochem. 63, 451-486[CrossRef][Medline] [Order article via Infotrieve]
  31. Goppelt-Struebe, M. (1997) Biochem. Pharmacol. 53, 1389-1395[CrossRef][Medline] [Order article via Infotrieve]
  32. Kerppola, T. K., Luk, D., and Curran, T. (1993) Mol. Cell. Biol. 13, 3782-3791[Abstract]
  33. Smith, M., Burke, Z., and Carter, D. (1996) Mol. Cell. Endocrinol. 122, 151-158[CrossRef][Medline] [Order article via Infotrieve]
  34. Auphan, N., DiDonato, J. A., Rosette, C., Helmberg, A., and Karin, M. (1995) Science 270, 286-290[Abstract]
  35. Scheinman, R. I., Cogswell, P. C., Lofquist, A. K., and Baldwin, A. S., Jr. (1995) Science 270, 283-286[Abstract]
  36. Mak, J. C., Nishikawa, M., and Barnes, P. J. (1995) Am. J. Physiol. 268, L41-L46[Abstract/Free Full Text]
  37. Brown, E. A., Dare, H. A., Marsh, C. B., and Wewers, M. D. (1996) Cytokine 8, 828-836[Medline] [Order article via Infotrieve]
  38. Orlando, S., Matteucci, C., Fadlon, E. J., Buurman, W. A., Bardella, M. T., Colotta, F., Introna, M., and Mantovani, A. (1997) J. Immunol. 158, 3861-3868[Abstract]
  39. Struhl, K. (1997) Genes Funct. 1, 5-9[Medline] [Order article via Infotrieve]
  40. Sims, J. E., Painter, S. L., and Gow, I. R. (1995) Cytokine 7, 483-490[CrossRef][Medline] [Order article via Infotrieve]
  41. Ye, K., Vannier, E., Clark, B. D., Sims, J. E., and Dinarello, C. A. (1996) Cytokine 8, 421-429[CrossRef][Medline] [Order article via Infotrieve]
  42. Burger, D., Chicheportiche, R., Giri, J. G., and Dayer, J. M. (1995) J. Clin. Invest. 96, 38-41[Medline] [Order article via Infotrieve]
  43. Re, F., Muzio, M., De Rossi, M., Polentarutti, N., Giri, J. G., Mantovani, A., and Colotta, F. (1994) J. Exp. Med. 179, 739-743[Abstract]
  44. Fink, G. W., and Norman, J. G. (1997) Cytokine 9, 1023-1027[CrossRef][Medline] [Order article via Infotrieve]
  45. Wang, X., Barone, F. C., Aiyar, N. V., and Feuerstein, G. Z. (1997) Stroke 28, 155-162[Abstract/Free Full Text]
  46. Gabellec, M. M., Griffais, R., Fillion, G., and Haour, F. (1996) J. Neuroimmunol. 66, 65-70[CrossRef][Medline] [Order article via Infotrieve]
  47. Schneider, H., Pitossi, F., Balschun, D., Wagner, A., del Rey, A., and Besedovsky, H. O. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 7778-7783[Abstract/Free Full Text]
  48. Nishiyori, A., Minami, M., Takami, S., and Satoh, M. (1997) Brain Res. Mol. Brain Res. 50, 237-245[CrossRef][Medline] [Order article via Infotrieve]


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