Proc. Nati. Acad. Sci. USAVol. 85, pp. 1204-1208, February 1988Immunology

Glucocorticoids selectively inhibit the transcription of theinterleukin 1,f gene and decrease the stability ofinterleukin 1,8 mRNA

(U-937 promonocytic cell line/post-transcriptional regulation)

SIMON W. LEE*, ANN-PING Tsout, HARDY CHANt, JAN THOMAS*, KAREN PETRIEt, ELSIE M. EUGUI*,AND ANTHONY C. ALLISON*f*Department of Immunology, Institute of Biological Sciences, and tDepartment of Molecular Biology, Institute of Bio-organic Chemistry, Syntex Research,3401 Hiliview Avenue, Palo Alto, CA 94303

Communicated by Dale Kaiser, November 2, 1987 (receivedfor review July 23, 1987)

ABSTRACT Transcription of the interleukin 1pf (IL-1,)gene was studied by mRNA hybridization with a cDNA probein the human promonocytic cell line U-937. Phorbol ester andlipopolysaccharide increased the steady-state level of IL-1imRNA. Glucocorticoids markedly decreased IL-1(3 mRNAlevels by two mechanisms. Transcription of the IL-1 gene wasinhibited, as shown by in vitro transcription assays with nucleiisolated from glucocorticoid-treated cells. Moreover, kineticanalyses and pulse-labeling of mRNAs showed that glucocor-ticoids selectively decrease the stability of IL-113 mRNA,without affecting the stability of 3-actin and FOS mRNAs.Inhibition of the formation and effects IL-1 is a mechanism bywhich glucocorticoids can exert antiinflammatory and immu-nosuppressive effects.

Although glucocorticoids are among the most potent andwidely used antiinflammatory agents, their mode of action ispoorly understood. The most popular theory has been thatglucocorticoids induce the formation of a group of proteins,collectively termed lipocortins, that inhibit phospholipase A2activity, thereby decreasing the production of proinflamma-tory prostaglandins and 5-lipoxygenase products (1, 2). Ahuman lipocortin of Mr 40,000 has been cloned and ex-pressed (3). However, proteins of the lipocortin family (alsoknown as calpactins), which are membrane-associated cyto-skeletal proteins serving as substrates for tyrosine kinasephosphorylation (4), are present in rather high concentra-tions in many cell types; it is not clear that they areglucocorticoid-inducible or that they are phospholipase in-hibitors rather than phospholipid-binding proteins (5). Al-though there may be other proteins with properties moreclosely matching those originally postulated for lipocortin,alternative or supplementary mechanisms by which gluco-corticoids might exert antiinflammatory effects deserve con-sideration.One such mechanism is inhibiting the formation of varie-

ties of interleukin 1 (IL-1), which are mediators of inflam-mation and tissue degradation. Proinflammatory effects ofboth forms of IL-1 [interleukin la (IL-la) and 1,B (IL-1,B)]include activation of phospholipase A2 and formation ofprostaglandin E2 in several cell types (6, 7), action onendothelial cells to increase the formation of prostacyclin (8)and to augment attachment of leukocytes (9), and action onbasophils to increase the release of histamine and othermediators of inflammation (10). Catabolic effects of IL-1include action on synovial cells to increase the release ofcollagenase (11), action on chondrocytes to release a neutralmetalloproteinase that degrades cartilage proteoglycan (12,

13), and action on bone to produce demineralization (14).These processes could contribute to joint erosion in rheu-matoid arthritis, loss of alveolar bone in peridontal disease,and other tissue destructive effects associated with chronicinflammation.

Glucocorticoids have been reported to inhibit the releaseof IL-1 by mouse peritoneal macrophages stimulated withlipopolysaccharide (LPS) (15) and rat peritoneal macro-phages stimulated with carrageenan (16). These in vitroeffects are paralleled in vivo: the sera of mice injected withLPS contain IL-1, the level of which is decreased byglucocorticoid treatment (17). Because inhibition of IL-1formation by glucocorticoids has important implications forimmunopharmacology, we have analyzed the phenomenonat the molecular level. Evidence is presented that, in cells ofthe human promonocytic cell line U-937, exposure to gluco-corticoids inhibits the transcription of the IL-1,8 gene anddecreases the stability of IL-1f3 mRNA.

MATERIALS AND METHODSCells and cDNA. U-937 cells, a human promonocytic cell

line (18), were grown in RPMI 1640 medium (GIBCO)supplemented with 7% (vol/vol) heat-inactivated fetal calfserum, penicillin at 50 units/ml, streptomycin at 50 pug/ml,and 2 mM glutamine at 370C in 5% C02/95% air and weresubcultured at 3 x 105 cells per ml every 3-4 days. Cells (3x 106) were washed and induced with LPS (20-50 pg/ml)(Escherichia coli serotype 0111:B4, 'Sigma) and 0.1 AuMphorbol 12-myristate 13-acetate (PMA, Sigma).A human IL-183 cDNA clone was obtained from Immunex

Corporation (Seattle, WA). The 13-actin cDNA was a giftfrom B. Endlich (University of California, San Francisco).c-FOS and MYC DNA clones were purchased from theAmerican Type Culture Collection.RNA Isolation and Hybridization. Cells were induced and

treated as described in the figure legends. RNA for pytoplas-mic dot blots was prepared as described by White andBancroft (19). After lysing U-937 cells in a solution of 140mM NaCl, 10 mM Tris-HCl (pH 8.4), 1.5 mM MgCl2,spermidine at 0.5 mg/ml, heparin at 80 units/ml, and 0.12%Nonidet P-40, total cytoplasmic RNA was isolated byphenol/chloroform, 1:1 (vol/vol), extraction. RNA samples(10 lig) were electrophoresed in a 1% agarose/2.2 M form-aldehyde gel and transferred onto GeneScreenPlus transfermembranes (New England Nuclear). The prehybridizationand hybridization ofRNA blots were done as described (20).

Abbreviations: IL-1, -la, and -1,B, interleukin 1, la, and 1,B; PMA,phorbol 12-myristate 13-acetate; LPS, lipopolysaccharide; Dex,dexamethasone.tTo whom reprint requests should be addressed.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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DNA probes were labeled with 32P by nick-translation to aspecific activity of at least 108 cpm/,ug. Blots were washedfor two 1-hr periods in 2 x SSC/0.2% NaDodSO4 at 420Cand for one 1-hr period in 0.2 x SSC/0.2% NaDodSO4 at650C and exposed at - 70'C to Kodak XAR films withintensifying screens (1 x SSC = 0.15 M NaCl/0.015 Msodium citrate, pH 7.0). The extent of hybridization oncytoplasmic RNA dot blots was quantitated by scanningwith an optical densitometer (Hoefer, San Francisco)equipped with a HP3390 integrator (Hewlett-Packard, Avon-dale, PA).

Cell-Free Nuclear Transcription Assay. Isolation of nucleiand nuclear transcription assays were carried out as de-scribed by Greenberg and Ziff (21). Briefly, nuclei fromU-937 cells [with or without PMA/LPS and dexamethasone(Dex) treatment] were isolated. Nuclei (107 nuclei) wereresuspended in 100 Al of the solution containing 50 mMTris-HCl (pH 8.3), 40% (vol/vol) glycerol, 5 mM MgCl2, and0.1 mM EDTA and mixed with 100 ,ul of reaction buffer [10mM Tris-HCl, pH 8.0/5 mM MgCl2/300 mM KCl/1.0 mMATP/0.5 mM GTP/0.5 mM CTP/5 mM dithiothreitol/200,Ci of [a-32P]UTP (3000 Ci/mmol, 1 Ci = 37 GBq; NewEngland Nuclear). After incubation at 26°C for 45 min,labeled nuclear RNA was isolated by the method of Mc-Knight and Palmiter (22) and hybridized to 10 Mg of linear-ized plasmid DNA immobilized on nitrocellulose filters.Each filter strip was hybridized with 32P-labeled RNA at 3.7x 106 cpm/ml. Hybridization and washing conditions wereaccording to Greenberg and Ziff (21).

Pulse-Labeling, Chase, and Quantitation of Labeled RNA.Following treatment with 0.1 ,uM PMA and LPS at 20 ,g/mlfor 3 hr, 106 U-937 cells per ml were pulse-labeled with[5,6-3H]-uridine at 17 ,uCi/ml (47 Ci/mmol; New EnglandNuclear) for 21 hr. Subsequently, the cultures were chasedfor 8 hr in medium with 5 mM uridine and 2.5 mM cytidine.PMA and LPS were present throughout the chase period.Half of the cultures were then treated with 10 ,M Dex. Totalcytoplasmic RNA (25 jig per sample) at selected intervalsafter Dex treatment was extracted and hybridized to 5 ,g ofIL-1p cDNA immobilized on nitrocellulose filters (23).Blank filters and filters spotted with ,-actin and pBR322plasmid were used as controls. Filters were prehybridized asdescribed (24) and hybridized in the prescribed solution butwith 1 x Denhardt's solution (0.02% polyvinylpyrrolidone/0.02% Ficoll/0.02% bovine serum albumin) in siliconizedvials for 65 hr at 42°C. After washing to remove unhybrid-ized RNA, bound [3H]RNAs were quantitated by scintilla-tion counting for 10 min in 10 ml of Aquasol (New EnglandNuclear).

RESULTSInduction of IL-1f3 mRNA. Cytoplasmic RNAs isolated

from the noninduced and LPS-stimulated U-937 cells reactedonly slightly with the nick-translated IL-1,8 cDNA probe oncytoplasmic RNA dot blots, showing that the basal level ofIL-1f3 transcription in U-937 cells is low. However, follow-ing exposure to PMA, IL-1,B mRNA transcripts were de-tected in U-937 cells. A much higher level of IL-lP mRNAwas detected when cells were incubated with both PMA andLPS (PMA/LPS) (Fig. 1A). The kinetics of IL-1,8 inductionby PMA/LPS are shown in Fig. 1B. Substantial amounts ofIL-1l8 transcripts were produced 4 hr after induction, and amaximal steady-state level was attained by 12 hr. Thisinduction was specific and not a result of generalized in-crease of mRNAs, since hybridization of the blots with theP-actin probe showed no significant change in the f3actinmRNA level throughout the induction period (data notshown).

A

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FIG. 1. Steady-state mRNA levels of IL-1,8 in U-937 cells. (A)Cells were incubated with LPS at 50 Ag/ml, 0.1 ,uM PMA, or bothfor 7 hr at 37°C. Noninduced cells were included as a control. RNAwas analyzed at dilutions of 1:27, 1:9, and 1:3 (Top to Bottom,respectively). Dots at the 1:3 dilution contained RNA from 106 cells.(B) Time course of IL-1P induction by PMA and LPS. Cells wereinduced as described above, and at selected time intervals RNA wasextracted and analyzed. Autoradiograms were scanned, and densi-ties in arbitrary area units were plotted against induction time.

Inhibition of IL-1,B mRNA Induction. In the presence ofDex (100 nM) or hydrocortisone (10 ,M), PMA/LPS treat-ment of U-937 cells failed to induce IL-1,6 mRNA synthesis(Fig. 2A). Dex also exerted an effect on the steady-state levelof IL-1,3 transcripts after induction had already occurred.Fig. 2B shows that when 10 AM Dex was added to cellsalready induced by a 17-hr exposure to PMA/LPS, IL-1,BmRNA levels remained constant for at least 60 min, thendeclined rapidly and retained =20% of initial level after 5 hr.The rate of mRNA decrease was similar when Dex wasadded to cells at various times after maximal IL-1j3 induc-tion. RNA gel blot analysis of RNA isolated fromPMA/LPS-stimulated and Dex-treated cells confirms thisobservation (Fig. 2C). The specific mRNA affected corre-sponded to the reported molecular size of IL-1P mRNA andwas only present in the induced U-937 cells. During thisperiod, the level of ,-actin mRNA remained essentiallyunchanged.

Nuclear Run-On Transcription Assay. The experimentsjust described show that Dex prevents induction byPMA/LPS of high levels of IL-1,8 mRNA. To ascertainwhether Dex, acting through its receptor, has a direct effecton the rate of transcription, nascent RNAs were elongated in

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FIG. 2. Effect of steroids on IL-1P induction. (A) Cells wereinduced for 20 hr with PMA and LPS in the presence of Dex orhydrocortisone (HG). Noninduced cells and cells induced with PMAand LPS in the absence of drugs were included as controls (data notshown). (B) Reduction of IL-1,3 mRNA accumulation by Dex. Cellswere induced for 17 hr, at which time Dex (10 tuM) was added. Atvarious time intervals after treatment, RNA was extracted andanalyzed. Threefold dilutions ofRNAs were spotted and analyzed inA and B as described in Fig. 1. (C) RNA gel blot analysis of IL-1,8and 13-actin mRNA levels. Total RNA (10 jig per lane) was used.Lanes: 1, noninduced cells; 2, induced cells; 3, induced cells treatedwith Dex for 5 hr.

vitro in the presence of [a-32P]UTP by using nuclei isolatedfrom Dex-treated (3 hr) and induced cells. The radioactivelylabeled transcripts were isolated and hybridized to DNAsimmobilized on nitrocellulose filters. Findings presented inFig. 3 show that [a-32P]UTP incorporation into IL-1,8 mRNAwas reduced by at least 80% in the presence of Dex. Nosignificant change was detected in the level of FOS, MYC,or /3-actin nuclear RNAs, and no hybridization to the vectorplasmid was detected (data not shown). Thus Dex has aselective inhibitory effect on the transcription of the IL-1,pgene.

Effects of Actinomycin D and Cycloheximide on IL-1fmRNA Stability. Our finding that addition of Dex to cells inwhich a high level of IL-1,3 mRNA is already present leads,after a lag period of -1 hr, to a rapid decline in the mRNAlevel suggests that Dex may also decrease the stability ofthat mRNA. To analyze this question directly, steady-statelevels of IL-1,3 mRNA were examined in the presence ofactinomycin D and cycloheximide, which inhibit transcrip-tion and translation, respectively. Induced IL-1,8 mRNAwas found to fall steadily for at least 5 hr after actinomycinD addition (Fig. 4A). The slow decay of IL-1,8 mRNA is

I 2 3

FIG. 3. Analysis of IL-1,B transcription in noninduced andinduced cells treated with Dex. Hybridization of nitrocellulose-immobilized IL-1,B cDNA with nascent-labeled nuclear RNA fromnoninduced (dot 1), PMA/LPS-induced (17 hr) (dot 2), andPMA/LPS-induced (17 hr) and Dex-treated (3 hr) (dot 3) U-937cells.

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FIG. 4. Dex-induced IL-1,8 mRNA degradation in U-937 cells.(A) Analysis of the steady-state level of IL-1/3 mRNA by cytoplas-mic RNA dot blot hybridization. Dex (10 uM), actinomycin D (5,ug/ml), and cycloheximide (10 ptg/ml) were added to cells inducedwith LPS (20 ,ug/ml) and PMA (0.1 tLM) as described in Fig. 2B. o,Induced cells; v, induced cells treated with Dex; *, induced cellstreated with actinomycin D; A, induced cells treated with actinomy-cin D and Dex; *, induced cells treated with cycloheximide; o,induced cells treated with cycloheximide and Dex. Intensities ofdots on autoradiogram were quantitated with a scanning densitom-eter, and densities were plotted as percent of initial level before drugtreatment. (B) Analysis of pulse-labeled IL-1,3 mRNA. Cells werepulse-labeled with [H3]uridine for 21 hr, placed in the chase mediumfor 8 hr to deplete the [3H]UTP pool, and washed, and then Dex (10,tM) was added. PMA and LPS were present throughout theexperiment. At selected time intervals after Dex treatment, mRNAswere analyzed. To correct for small variations in the amounts ofinput RNA, the data were normalized with respect to 3-actin mRNAlevels as determined by cytoplasmic RNA dot blot analysis of thelabeled RNA. Specific activities of radiolabeled IL-1,BmRNA elutedfrom filters were plotted (cpm/,tg of RNA). Each point representsthe mean ± SEM from triplicate determinations of the sample. Priorto hybridization, specific activities of total cytoplasmic RNA pre-pared from control and Dex-treated cells at various time intervalsduring the chase were similar (1.55 ± 0.11 x 105 cpm/pg). 0,Induced cells; v, Induced cells treated with Dex (10 uM). Thefractional degradative rate (k) was the slope of the linear regressionline on the logarithmic-transformed data.

unusual for a cytokine mRNA and may be due to thepresence of PMA, which has been reported to stabilizemRNAs of other cytokines, such as granulocyte/macro-phage colony-stimulating factor (25). Dex treatment, on theother hand, led to a more rapid decay of the IL-1p mRNA(i.e., <50% of the initial level after 3 hr, Fig. 4A). Theaddition of both Dex and actinomycin D to induced cells didnot lead to a significant increase in IL-1,8mRNA degradationabove that of the actinomycin D-treated cells. Hence, acti-nomycin D opposes the effect of Dex, indicating that Dex-induced IL-1/3 mRNA degradation requires de novo mRNAsynthesis. In accordance with this interpretation, addition ofcycloheximide with Dex to induced cells also abolished theeffect of Dex, and the IL-1,8 mRNA remained elevated

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throughout the treatment period. Cycloheximide alone in-creased the level of IL-lP transcripts in PMA/LPS-stimulated U-937 cells by =1.5-fold 5 hr after treatment.The experiments just described reveal a significant de-

crease in the stability of IL-113 mRNA after Dex treatmentand allow estimates of fractional degradative rates by usingaccepted calculations (26) of IL-lP mRNA to be made undervarious treatment conditions. The fractional degradative rateof IL-1,1 mRNA in the presence of actinomycin D or Dexwas 0.076 hr-1 or 0.350 hr-1, respectively. During thisperiod, the stability of f3actin mRNA remained unchangedin the presence of Dex (data not shown).An independent measurement of mRNA stability was

carried out by analyzing the kinetics of decay of radioac-tively pulse-labeled mRNA. From the observations in Fig.4B estimates were made of fractional degradative rates ofIL-1,8 mRNA following Dex treatment by the two methods.They showed significant and comparable rate decreases(0.350 hr-1 from steady-state mRNA levels and 0.290 hr-from pulse-labeling).These experiments provide conclusive evidence that glu-

cocorticoids induce degradation of IL-1i mRNA. To exam-ine whether glucocorticoid treatment would accelerate deg-radation of all mRNAs containing an A+ U-rich consensussequence in the 3'-untranslated region, FOS mRNA was alsostudied. In the system described above, RNAs were hybrid-ized with a FOS cDNA. FOS mRNA was found in U-937cells not exposed to PMA and LPS, and the level was notaffected by these agents. A comparable decline in FOSmRNA level was observed under control and various treat-ment conditions (data not shown). Thus it appears that theeffect of Dex was specific for the IL-1,8 mRNA and that anA+ U-rich sequence is not the common recognition targetrequired for Dex-induced IL-1l mRNA degradation.

DISCUSSIONThe main finding reported is that Dex selectively inhibitstranscription of the IL-lP gene and decreases the stability ofIL-lP mRNA. Transcription of the IL-la gene is alsoinhibited by Dex (data not shown). We have made similarobservations with several glucocorticoids in U-937 cells andhuman peripheral blood monocytes in the absence of PMA(data not shown), so these findings appear to be generaleffects of glucocorticoids on human cells of the monocytelineage. The effects are abolished in the presence of theglucocorticoid receptor antagonist RU 486 (data not shown).

Alteration of transcription by glucocorticoids is believedto result from the specific binding of the glucocorticoid-re-ceptor complex to "enhancer" elements of target genes (27,28). Such binding increases transcription by RNA polymer-ase II of many genes. In contrast, glucocorticoids decreasetranscription of the genes for proopiomelacortin in pituitarytumor cells (29), a1-fetoprotein in developing rat liver cells(30), type 1 procollagen in chicken skin fibroblasts (31), andcollagenase in rabbit synovial cells (24). During the shut-down of transcription by glucocorticoid hormones, there isno demonstrably accelerated breakdown of nascent tran-scripts (24, 30, 31).From the nuclear transcription and other experiments now

reported, it is clear that the IL-1,3 gene can be added to thelist of genes the transcription of which is suppressed byglucocorticoids. Moreover, in U-937 cells we have found 1hr after exposure to Dex a marked decrease in the stability ofIL-lP mRNA. This effect of Dex evidently requires de novomRNA and protein synthesis: in the presence of actinomycinD or cycloheximide, Dex has no effect on the stability ofIL-1/3 mRNA. A glucocorticoid-inducible protein might in-fluence the stability of a specific mRNA in several ways asdiscussed below in the context of histone mRNA.

In contrast, Dex does not influence transcription of f3-actin or FOS genes or the stability of the correspondingmRNAs in U-937 cells. The mRNA for FOS shares with IL-1mRNA an A + U-rich sequence in the 3'-untranslated region,which has been postulated as the recognition signal for aprocessing pathway specifically degrading mRNAs for cer-tain cytokines and protooncogenes (25). Since Dex treat-ment markedly decreases the stability of IL-1,p mRNA butnot FOS mRNA, the presence of an A + U-rich sequence inthe 3'-untranslated region as such cannot explain the in-creased rate of degradation of the former.The 5'-untranslated regions of mRNAs can also influence

their stability and rate of translation. Histone mRNA main-tains high steady-state levels during the S phase of the cellcycle and is selectively destabilized thereafter. Fusion geneconstructs provide evidence that the first 20 nucleotides ofthe histone mRNA leader are sufficient for coupling mRNAstability to DNA synthesis (32). Destabilization of histonemRNA requires synthesis of protein, which the authorssuggest might be a nuclease or could direct a nuclease tomRNAs containing the target sequence (32). The 5'-untranslated leader sequence of IL-1,8 mRNA affects therate of translation, as shown by insertion of a plant viralleader sequence in this position (33). The plant viral RNA isunstructured at the 5' terminus, so the secondary structureof IL-1,i mRNA affects its translation efficiency and mayalso influence its intracellular fate and stability. The effect ofglucocorticoids on the stability of IL-1f3 mRNA is unusualalthough other examples of regulation of mRNA stability areknown. While glucocorticoids control the transcription ofseveral genes in many cell types, as mentioned above, theyhave no demonstrable effect on the stability of the mRNAsof proopiomelacortin (29), type 1 procollagen (31), collagen-ase (24), or interleukin 3 (34).The molecular biological studies reported in this paper

support the generalization that IL-1 and glucocorticoidshave antagonistic effects, the former proinflammatory andthe latter antiinflammatory. Glucocorticoids not only sup-press the transcription of IL-1 genes; they reinforce theseeffects by degrading IL-1 mRNA already formed, therebyrapidly inhibiting IL-1 synthesis, and by antagonizing theeffects of IL-1 on target cells. Examples of the latter are theinduction by IL-1 of collagenase (11) and plasminogen acti-vator (35) in synovial cells and decrease in the levels of thecorresponding mRNAs in these cells by glucocorticoids (24,36). IL-1 increases formation of prostaglandin E2 in severalcell types (6, 7) and prostacyclin in endothelial cells (8),whereas glucocorticoids inhibit formation of prostaglandinE2 in several cell types (37) and prostacyclin in endothelialcells (38).

Inhibition of the formation and effects of IL-1 is relevantnot only to the antiinflammatory effects of glucocorticoidsbut also to their immunosuppressive effects. Glucocorticoidscan inhibit the formation of interleukins 2 and 3, granulo-cyte/macrophage colony-stimulating factor, and other lym-phokines by helper T lymphocytes (34, 39). Glucocorticoidscan also inhibit class II major histocompatibility complexexpression on macrophages (15). Cytotoxic clones of Tlymphocytes (40) and B lymphocytes (41) are relativelyresistant to glucocorticoids. Some helper effects of T lym-phocytes are inhibited by glucocorticoids (42), and IL-1overcomes this inhibition (43). IL-1 also reverses inhibitoryeffects of glucocorticoids on granulopoiesis (44). Henceglucocorticoid-mediated inhibition of IL-1 formation, ana-lyzed at the molecular level in this paper, could havewidespread effects on immune responses and hematopoiesisas well as on inflammatory reactions.

We thank those listed in the section on materials who suppliedreagents and Gordon Ringold for critical review of the manuscript.

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1208 Immunology: Lee et al.

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