of July 16, 2015.This information is current as in Primary Human Macrophages

BκMitogen-Activated Protein Kinase or NF-Not Involve Inhibition of p38

Production That DoesαSuppression of TNF-Evidence for a Dual Mechanism for IL-10

Andrews, Dominic Kwaitkowski and Brian M. J. FoxwellWilliams, Cathleen J. Ciesielski, Jamie Campbell, Caroline Agnes Denys, Irina A. Udalova, Clive Smith, Lynn M.

http://www.jimmunol.org/content/168/10/4837doi: 10.4049/jimmunol.168.10.4837

2002; 168:4837-4845; ;J Immunol 

Referenceshttp://www.jimmunol.org/content/168/10/4837.full#ref-list-1

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2002 by The American Association of9650 Rockville Pike, Bethesda, MD 20814-3994.The American Association of Immunologists, Inc.,

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Evidence for a Dual Mechanism for IL-10 Suppression ofTNF-� Production That Does Not Involve Inhibition of p38Mitogen-Activated Protein Kinase or NF-�B in PrimaryHuman Macrophages1

Agnes Denys,2* Irina A. Udalova, † Clive Smith,* Lynn M. Williams,* Cathleen J. Ciesielski,3*Jamie Campbell,* Caroline Andrews,* Dominic Kwaitkowski,† and Brian M. J. Foxwell4*

IL-10 is a potent anti-inflammatory cytokine and inhibitor of TNF- � production. The molecular pathways by which IL-10 inhibitsTNF-� production are obscure, with diverse mechanisms having been published. In this study, a new approach has been takenfor the study of human cells. Adenovirus was used to deliver TNF-� promoter-based luciferase reporter genes to primary humanmonocytic cells. The reporter genes were highly responsive to macrophage activation and appeared to mirror the behavior of theendogenousTNF-� gene. When added, either with or after the stimulus, IL-10 required the 3� untranslated region of theTNF-�gene to inhibit luciferase mRNA and protein expression, indicating a posttranscriptional mechanism. However, if macrophageswere incubated with IL-10 before activation, inhibition of gene expression was also mediated by the 5� promoter, suggesting atranscriptional mechanism. To our knowledge, this is the first time that a dual mechanism for IL-10 function has been demon-strated. Studies to elucidate the mechanisms underlying the inhibition of TNF-� production addressed the effect of IL-10 on theactivation of p38 mitogen-activated protein kinase and NF-�B. However, these studies could demonstrate no requirement for theinhibition of p38 mitogen-activated protein kinase or NF-�B activation as potential mechanisms. Overall, these results may explainthe diversity previously ascribed to the complex mechanisms of IL-10 anti-inflammatory activity. The Journal of Immunology,2002, 168: 4837–4845.

I nflammation is an essential host response to infectious chal-lenge. However, when excessive, the inflammatory responseleads to harmful, or even fatal, consequences as seen in rheu-

matoid arthritis, Crohn’s disease, and septic shock (1). Thus, theinflammatory response must normally be tightly regulated. Amongthe many factors that suppress the inflammatory response, the cy-tokine IL-10 is one of the most important. IL-10 is a potent inhib-itor of monocyte/macrophage activation, blocking the expressionof TNF-� and other proinflammatory mediators. Mice defective inIL-10 expression develop an inflammatory Crohn’s-like diseaseand produce enhanced amounts of TNF-� in response to LPS (2).Furthermore, in murine models, many inflammatory diseases areameliorated by administration of exogenous IL-10 (reviewed byDonnelly et al. in Ref. 3).

Despite considerable scientific and clinical interest, the molec-ular pathways underlying IL-10 inhibition of TNF-� expressionremain obscure. In human PBMC, IL-10 has been reported to sup-press TNF-� gene transcription (4, 5), possibly by inhibiting theactivation of the transcription factor NF-�B (6, 7). However, un-like the inhibition of TNF-� gene transcription (4, 5), the blockadeof NF-�B activation does not appear to require IL-10-induced denovo protein synthesis (6, 7). In contrast, studies performed inmurine macrophages have claimed that IL-10 acts through theposttranscriptional mechanisms by destabilizing TNF-� mRNA (8,9), or, more recently, by inhibiting gene translation via blockingthe activation of p38 mitogen-activated protein kinase (MAPK)5

(10). A role for IL-10-induced de novo protein synthesis has alsobeen described in murine macrophages (9). Furthermore, Riley etal. (11) have demonstrated the absolute requirement of the tran-scription factor STAT-3 in mediating the anti-inflammatory effectsof IL-10. There appears to be no simple explanation for the varietyof mechanisms ascribed to IL-10-mediated inhibition of TNF-�expression except for the different systems used. Thus, it may bepossible that there are major differences between cell systems inhow IL-10 exerts its effect.

For understanding human physiology and disease, a most appro-priate system for the study of IL-10 effects is primary human mono-cyte-macrophages. However, a major drawback in using these cellsfor signaling studies is the inability to transfect them and introducetransgenes. Recently, we have successfully overcome this problem

*Kennedy Institute of Rheumatology Division, Imperial College Faculty of Medicine,Charing Cross Campus, London, United Kingdom; and †University Department ofPaediatrics, John Radcliffe Hospital, Oxford, United Kingdom

Received for publication October 4, 2001. Accepted for publication March 5, 2002.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was funded by the Medical Research Council, the Arthritis ResearchCampaign, and the European Union. A.D. was supported by a European Union Train-ing, Mobility, and Research award.2 Current address: Laboratoire de Chimie Biologique, Unite Mixte de Recherche 8576du Center National de Recherche Scientifique, Universite des Sciences et Technolo-gies de Lille, Villeneuve d’Ascq, France.3 Current address: University of California Stein Clinical Research, La Jolla, CA92093.4 Address correspondence and reprint requests to Dr. Brian M. J. Foxwell, KennedyInstitute of Rheumatology Division, Imperial College Faculty of Medicine, 1 Aspen-lea Road, London W6 8LH, U.K. E-mail address: b.foxwell@ic.ac.uk

5 Abbreviations used in this paper: MAPK, mitogen-activated protein kinase; 3�UTR,3� untranslated region; m.o.i., multiplicity of infection; AdvNF-�B-luc, adeno-NF-�Bluciferase reporter virus; JNK, c-Jun N-terminal kinase; RPA, RNase protectionassay.

The Journal of Immunology

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using adenoviral vectors (12, 13). In this study, we used this approachto study IL-10 regulation of TNF-� at the gene level in primary hu-man macrophages. In particular, adenoviral vectors were constructed,incorporating luciferase reporter genes, under the control of theTNF-� promoter, with or without the 3� untranslated region (3�UTR).Once introduced into primary human macrophages, these reportergenes gave high levels of induction in response to LPS or zymosan.This, to our knowledge, is the first direct examination of TNF-� genefunction in primary human cells. Moreover, this study showed that,depending on the length of exposure of cells to the cytokine, IL-10could inhibit TNF-� expression by either the 5� promoter region or the3�UTR, suggesting that both transcriptional and posttranscriptionalmechanisms could be involved in this single cell type. However, un-like previous studies in other systems, IL-10 was unable to inhibitLPS-induced activation of p38 MAPK and had only a minor effect onNF-�B-induced transcription.

Materials and MethodsCells

Human mononuclear cells were isolated from single donor plateletphoresisresidues obtained from the North London Blood Transfusion Center (Lon-don, U.K.) by Ficoll-Hypaque centrifugation preceding monocyte separa-tion in a Beckman JE6 elutriator (Beckman, High Wycombe, U.K.). Mono-cyte purity was routinely �85% when assessed by flow cytometry (14).The elutriated human monocytes were cultured at 1 � 106/ml in RPMI1640 (BioWhittaker, Verviers, Belgium) with 25 mM HEPES and 2 mML-glutamine supplemented with 10% (v/v) heat-inactivated FCS and 10U/ml penicillin/streptavidin. To optimize infection, macrophages were de-rived from the monocytes by culturing the cells with M-CSF at 100 ng/ml(Genetics Institute, Boston, MA) for 48 h (13).

Plasmids

Human TNF-� promoter (�1173 bp) with 3�UTR of the human TNF-�gene (pGL3-TNF-�-3�UTR), or without the 3�UTR (pGL3-TNF-�) (15),were subcloned into the pAdTrack vector (16) to generate pAdTrack-p5�3�UTR and pAdTrack-p5�. KpnI/SalI fragments containing the humanTNF-� promoter, the luciferase reporter gene, and the SV40 late poly(A)signal were derived from pGL3-TNF-� inserted into KpnI/SalI sites of theAdTrack vector. pAdTrack-p5�3�UTR was obtained by substituting a XbaI/BamHI fragment containing the SV40 late poly(A) signal in the pGL3-TNF-� plasmid for �1 kbp of 3�UTR amplified by PCR with correspond-ing primers: 3�UTR-F (XbaI), aattctagaGGAGGACGAACATCCAAC;and 3�UTR-R(BamHI), aatGgATcCCCAGAGTTGGAAATTC. The KpnI/SalI fragments were subsequently cloned into the pAdTrack vector.

Adenoviral vectors and their propagation

The pAdEasy-1 adenoviral plasmid was provided by Prof. B. Vogelstein(Howard Hughes Medical Institute, Baltimore, MD). Recombinant viruseswere generated by homologous recombination in BJ5183 Escherichia colitransformed by heat-shock with 1 �g of each of the linearized PmeIpAdTrack constructs and 100 ng of pAdEasy-1. Kanamycin-resistant pos-itive recombinant clones were selected and confirmed by restriction en-zyme digestion. Viral DNA was transfected into HEK 293 cells. Viruseswere purified by ultracentrifugation through two cesium chloride gradients,as described in He et al. (16). Plaque assays were performed by HEK 293cells, exposing the cells to each virus for 1 h in serum-free DMEM (LifeTechnologies, Paisley, U.K.) and, subsequently, overlaying the cells withan agarose mixture (1.5% agarose, 2� DMEM with 4% FCS; v/v, 1/1) andincubated for 10–14 days to determine viral titer (16). The I�B� encodingvirus was kindly provided by Dr. R. de Martin (University of Vienna,Vienna, Austria) and adeno-NF-�B luciferase reporter virus (AdvNF-�B-luc), previously described (17), was kindly provided by Dr. P. McCray(University of Iowa, Iowa City, IA).

Infection and IL-10 treatment

Human macrophages were plated at a density of 2 � 105 cells/well in96-well plates and exposed to virus at the optimal multiplicity of infection(m.o.i.; 40:1 for Advp5� and Advp5�3�UTR; 100:1 for Ad0 AdvI�B�;200:1 for AdvNK-�B-luc) for 1 h in serum-free medium, followed bywashing and reculturing in growth medium with 2% (v/v) FCS for 24 h.Infected cells were then stimulated with 10 ng/ml LPS (Salmonella typhi-

murium; Sigma, Poole, U.K.) or 30 �g/ml zymosan (Sigma) for 4 h, unlessotherwise stated, in the presence or absence of IL-10.

Measurement of human TNF-� production

TNF-� levels were measured in cell supernatants by sandwich ELISA aspreviously reported (18).

Luciferase assay

After stimulation, cells were washed once in PBS and lysed with 100 �l ofchloramphenicol acetyltransferase lysis buffer (0.65% (v/v) Nonidet P-40,10 mM Tris-HCL (pH 8), 0.1 mM EDTA (pH 8), 150 mM NaCl). Celllysate (50 �l) was transferred to a luminometer cuvette strip and luciferaseassay buffer (220 �l) was added. Luciferase activity was measured with aluminometer (Labsystems, Chicago, IL) by dispensing 30 �l luciferin (1.5mM; Sigma) per assay point. Cell lysates were assayed for protein con-centration by Bradford assay and luciferase activity was adjustedaccordingly.

RNase protection assay

After M-CSF treatment, cells were plated at 2 � 106/well in a 12-well plateand infected, as described above. In Advp5�- and Advp5�3�UTR-infectedcells, luciferase and GAPDH mRNAs were detected by RNase protectionassay (RPA) by using luciferase and GAPDH riboprobes, respectively. Inparallel, TNF-� and GAPDH mRNAs were detected in Adv0-infectedcells. Riboprobe vectors were constructed as follows. A 352-bp HincII-XbaI luciferase fragment was cloned from pGL3c (Promega, Madison, WI)into pBluescript KS-digested EcoRV and XbaI. A 268-bp TNF-� genefragment was amplified by PCR from human genomic DNA and subclonedinto the SpeI site of pBluescript KS� (kindly provided by Dr. A. Clark,Kennedy Institute, London, U.K.). Riboprobe template constructs were lin-earized by appropriate restriction enzyme and purified by phenol-chloro-form extraction and ethanol precipitation. Luciferase and GAPDH ribo-probes were synthesized using T7 RNA polymerase and TNF-� riboprobeby using T3 RNA polymerase (Boehringer Mannheim, Indianapolis, IN) inthe presence of 50 �Ci of [�-32P]UTP (800 mCi/mmol; Amersham Phar-macia Biotech, Little Chalfont, U.K.). The final concentration of unlabeledUTP in the in vitro transcription reactions was 12 �M, except in the caseof luciferase, where it was 2.4 �M. RPAs were conducted using the DirectProtect kit (Ambion, Austin, TX). Under the hybridization conditionsDNA-RNA heteroduplexes are not detected. Protected RNA fragmentswere resolved by electrophoresis on denaturing 6% polyacrylamide gels,quantified by phosphor imaging (Fuji FLA-2000; Raytek Scientific, Shef-field, U.K.) and visualized by autoradiography. Each experiment was per-formed twice and serial dilutions of lysates were used to check that quan-titations were within the linear range of the assay.

Immunoprecipitation and in vitro kinase assays

MAPKs were immunoprecipitated from cleared cell lysates, as describedpreviously (19). In vitro kinase assays for p38 MAPK were performedusing either His6-MAPKAPK-2 or GST-ATF-2 as a substrate, c-Jun N-terminal kinase (JNK) assays were performed using GST-ATF-2 as a sub-strate, and p42 MAPK assays were performed using myelin basic protein(Sigma) as a substrate. Immunoprecipitates were incubated with 30 �lkinase assay buffer (25 mM Tris (pH 7.5), 25 mM MgCl2, 25 mM �-glyc-erophosphate) containing 20 �M ATP and 0.5 �Ci [�-32P]ATP (Amer-sham Pharmacia Biotech) with 50 �g/ml appropriate substrate protein for25 min at room temperature. Reactions were terminated by the addition ofgel sample buffer and boiling for 5 min. All substrates were separated bySDS-PAGE. Gels were dried and phosphorylated substrates were visual-ized using a Fuji FLA-2000 phosphor imager and by autoradiography at�70°C.

NF-�B EMSAs

Following stimulation, cells were scraped into ice-cold PBS and lysed inhypotonic lysis buffer (0.125% Nonidet P-40, 5 mM HEPES (pH 7.9), 10mM KCl, 1.5 mM MgCl2), and nuclei were harvested by centrifugation(13,000 � g for 30 s). Nuclear protein extracts were prepared by incubatingthe nuclei in hypertonic extraction buffer (5 mM HEPES (pH 7.9), 25%glycerol, 500 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA) for 2 h withconstant agitation. NF-�B DNA binding activities were determined by in-cubating 1–3 �g of each extract with [�-32P]ATP-labeled double-strandedNF-�B consensus oligonucleotide (Promega), followed by resolution on a5% (w/v) nondenaturing polyacrylamide gel. Gels were dried on to filter

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paper and retarded DNA; protein complexes were visualized using Hyper-film MP (Amersham Pharmacia Biotech).

ResultsLPS and zymosan potently activate TNF-� reporter genesdelivered by adenoviral infection into primary humanmacrophages and demonstrate the involvement of the 3�UTR inmRNA stability

The 5� promoter and 5� promoter-3�UTR constructs, described pre-viously (15), were incorporated into recombinant adenoviruses (re-porter viruses, Advp5� and Advp5�-3�UTR, respectively), as pre-viously described by He et al. (16). Primary human macrophageswere infected with these adenoviral constructs at a m.o.i. of 40:1,as previous studies had indicated that this concentration resulted inthe successful infection of �90% of cells (Ref. 13 and data notshown). LPS activation of the reporter gene resulted in a potentstimulation of both the 5� and 5�3�UTR constructs, respectively(Fig. 1). The reporter gene also responded equally well to the yeastproduct, zymosan (Fig. 1), an alternative stimulus of TNF-� pro-duction with a similar potency to LPS (20). A consistent finding inall experiments was the lower absolute response of constructs con-taining the 3�UTR. These data support the view, obtained fromprevious studies in murine macrophage cell lines, that the 3�UTRis generally suppressive to TNF-� expression (21).

Kinetic studies were also performed to compare the behavior ofthe reporter genes with the endogenous gene. In response to LPS,TNF-� production reached a maximum 4 h after stimulation, andthereafter decreased slowly with a t1/2 of �18.5 h (Fig. 2A). Thekinetics of luciferase activity from Advp5�- or Advp5�3�UTR-in-fected LPS-stimulated human macrophages followed a similar pro-file to TNF-�, with maximum expression at 4 h and apparent t1/2

estimated to be 23.5 and 12 h, respectively (Fig. 2, B and C). Theshorter half-life of luciferase, when under the additional control ofthe 3�UTR, might be expected from the overall destabilizing effect

of this element on TNF-� mRNA (21, 22) and explain why theabsolute signals produced in the presence of the 3�UTR are con-sistently lower (Fig. 1). To confirm this, studies on luciferasemRNA stability were performed and showed that the presence ofthe 3�UTR does indeed increase the rate of decay of luciferasemRNA (Fig. 2D). In the presence of the 3�UTR the mRNA had at1/2 of 56 min; however, in the absence of this element there waslittle decay (�15%) within the same timeframe. Similar studies onTNF-� mRNA indicate a t1/2 of �30 min, similar to luciferasemRNA from the 3�UTR constructs (Fig. 2D). One might haveexpected that the half-life of the endogenous TNF-� protein wouldhave been comparable to the 5�3�UTR rather than the 5� construct.However, these data do not take into account potential differencesin the biological half-lives of the TNF-� and luciferase proteins,and one can only assume that the half-life of the endogenousTNF-� would be longer in the absence of the 3�UTR in humans,as shown previously in mouse cells with deletions of the 3�UTR onthe AU-rich region of the 3�UTR (23).

In primary human macrophages, IL-10 requires the 3�UTR tosuppress TNF-� production but has no effect on p38 MAPKactivation

Adenovirus delivery of TNF-�-based reporter gene constructs toprimary human macrophages was then used to investigate whatrole the 5� and 3� regions may play in the IL-10 inhibition ofTNF-� expression. Adeno-reporter virus-infected cells were si-multaneously treated with LPS and various concentrations ofIL-10 for 4 h, after which time TNF-� production and luciferaseactivities were assayed (Fig. 3). IL-10 inhibited TNF-� expressionto a maximum of 80% at 10 ng/ml (Fig. 3). However, the responsesof the two reporter constructs were quite distinct. The 5� constructwas only weakly inhibited by IL-10 (�10%), whereas the5�3�UTR construct showed a dose response profile similar to theendogenous TNF-�, although the maximum inhibition attained

FIGURE 1. Activity of TNF-�-based reporter constructs when in-fected into human macrophages. Cellswere infected with adenoviral con-structs at a m.o.i. of 40:1. FollowingLPS (10 ng/ml) or zymosan (30 �g/ml) stimulation (4 h), cells were har-vested and assayed for luciferase ac-tivity. The data are mean values (�)from duplicates and are representativeof at least four experiments using cellsof different donors.

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was less (60% at 10 ng/ml). The IC50 for IL-10 on TNF-� proteinexpression was 0.2–0.3 ng/ml, compared with 2–3 ng/ml for thereporter gene. However, if the concentration of half-maximal in-hibition is calculated, then the activity of IL-10 is similar for theendogenous gene (�0.1 ng/ml) and the 5�3�UTR construct (0.2–0.3 ng/ml). This suggests that aspects of the inhibitory activity ofIL-10 on the reporter gene and the endogenous gene are similar.The kinetics of IL-10 inhibition of TNF-� expression and lucif-erase expression were also compared using the optimal concentra-tion of 10 ng/ml (Fig. 4). Over the 2- to 24-h period poststimula-tion, the expression of TNF-� and the 5�3�UTR construct gavevery similar profiles with very little activation detected in the pres-ence of IL-10 (Fig. 4, A and B). The effect of IL-10 on the 5�construct was again much weaker, with no significant inhibition of

luciferase activity over the time course (Fig. 4C). These data sug-gest that the major inhibitory effect of IL-10 is mediated via the3�UTR and that there appears to be little effect on the transcriptionof the gene when IL-10 is administered at the same time as LPS.To gain a further insight into the mechanism of IL-10 activity,luciferase mRNA levels were analyzed by RPA. As shown in Fig.5, simultaneous addition of IL-10 caused a marked reduction ofluciferase mRNA from the 5�3�UTR construct, whereas there wasonly a marginal effect on mRNA from the 5� construct. These dataindicate that IL-10 causes a reduction in mRNA levels via the3�UTR. Data obtained with TNF-� mRNA showed similar resultsto the 5�3�UTR construct (Fig. 5).

As the 3�UTR is associated with posttranscriptional control ofTNF-� expression, the data so far suggest that IL-10 mediates its

FIGURE 2. Comparison of the kinetics of LPS-induced TNF-� production and luciferase expression in human macrophages. Cells were uninfected (A),infected with Advp5� (B), or infected with Advp5�3�UTR (C), and untreated or activated with 10 ng/ml LPS. At the indicated times, cell lysates or culturesupernatants were harvested to assay either luciferase or TNF-� production, respectively. In the absence of LPS, TNF-� production was undetectable. D,Cells were infected with Advp5� or Advp5�3�UTR and activated with LPS for 4 h. Actinomycin D was added to stop any further mRNA synthesis and thecells were incubated for a further 0, 15, 30, 45, or 60 min, after which time they were harvested for RPA analysis of luciferase, TNF-�, or GAPDH mRNAs.The results are normalized to 100% at the 0-min point. Each experiment was repeated at least two (D) or three (A–C) times with cells from different donors.

FIGURE 3. IL-10 inhibition of reportergene constructs requires the 3�UTR. Cellswere infected with Advp5� (A) orAdvp5�3�UTR (B) and activated simulta-neously with LPS (10 ng/ml) and variousconcentrations of IL-10. Luciferase activi-ties and TNF-� production were assayed af-ter 4 h of LPS stimulation. The results areexpressed as percentages of luciferase acti-vation and TNF-� production induced byLPS alone. Data are mean values � SEMfrom four separate experiments conductedwith cells from different donors.

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activity at this level. If this is so, IL-10 should still be able toinhibit TNF-� production, at least for a period, if added after LPS.Reporter virus-infected macrophages were stimulated with LPS,IL-10 was added for periods of up to 2 h postactivation, and thecells were harvested at 4 h for assay. As expected, IL-10 had littleeffect on the activity of the 5� construct, regardless of when it wasadded (Fig. 6). The inhibitory effect of IL-10 on TNF-� and theexpression of the 5�3�UTR construct was maintained, even if IL-10was added 1 h after LPS activation, but was greatly reduced if thecytokine was added 2 h postactivation (Fig. 6). These data suggestthat IL-10 does not inhibit the early events that are involved inTNF-� production (e.g., transcription) and instead targets laterevents (e.g., posttranscriptional). These data support the hypothe-sis that posttranscriptional control mediated via the 3�UTR is thetarget of IL-10.

As p38 MAPK has been implicated in the posttranscriptionalcontrol of TNF-� expression (24, 25) and has very recently beenshown to be inhibited by IL-10 in murine macrophages (10), weFIGURE 4. Kinetics of IL-10 inhibitory activity on LPS-induced

TNF-� production and luciferase expression. Human macrophages wereuninfected (A), infected with Advp5�3�UTR (B), or infected with Advp5�(C) (at a m.o.i. of 40:1) and then incubated with LPS in the presence orabsence of IL-10 (10 ng/ml). The experiment was harvested at indicatedtimes and TNF-� and luciferase were assayed. Data are mean values (�)from duplicates and are representative of three separate experiments con-ducted with cells from different donors.

FIGURE 5. Effect of IL-10 on LPS-induced TNF-� and luciferasemRNA levels. Human macrophages infected with Advp3�, Advp5�3�UTR,or Adv0 were treated with IL-10 (10 ng/ml) simultaneously or 24 h beforeLPS. Four hours after LPS activation, cells were harvested and RPAs wereperformed to quantify luciferase or TNF-� mRNA. Upper panel, TNF-�and luciferase mRNA (RPA); lower panel, luciferase or TNF-�:GAPDHratios. These are plotted as percentages of the maximum value in presenceof LPS alone. All quantitations of RNA signal were performed by a phos-phor imager. Data are representative of experiments using cells from twodifferent donors.

FIGURE 6. IL-10 can inhibit TNF-� expression, even when added post-LPS stimulation. Human macrophages, infected with either Advp5� orAdvp 5�3�UTR (m.o.i., 40:1), were incubated for 4 h in the presence ofLPS (10 ng/ml). IL-10 (10 ng/ml) was added at the given times after LPS.The results are expressed as percentages of luciferase activation andTNF-� production induced by LPS alone. Data are mean values (�) fromduplicates and are representative of three separate experiments conductedwith different donors.

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investigated whether this kinase was a target for IL-10 in humanmacrophages. As shown in Fig. 7, IL-10 was unable to inhibitLPS-induced p38 MAPK activation. As the other related p42/44extracellular signal-regulated kinase, MAPKs, and p54 JNK arealso involved in regulating TNF-� expression, the effect ofIL-10 on these kinases was also studied. However, like p38MAPK, there was no inhibitory effect on the activation of thesekinases (Fig. 7).

Preincubation of human macrophages with IL-10 before LPSstimulation reveals a second mechanism of inhibiting TNF-�production through the 5� promoter

As time of exposure to IL-10 could obviously have a bearing on itsfunction, this study was extended to investigate the effect of addingIL-10 before LPS for periods of up to 24 h (Fig. 8). Preincubatingmacrophages with IL-10 for 12 h produced a modest increase inthe inhibition of endogenous TNF-� production or luciferase ac-tivity from the 5�3�UTR reporter gene, when compared with the

effect of simultaneous addition of LPS and IL-10 (Fig. 8). Furtherperiods of preincubation, up to 24 h, did not elicit any major ad-ditional effect. However, pre-exposure of cells to IL-10 had a pro-found effect on the expression of the 5� reporter. Preincubation ofmacrophages with IL-10 for 12 h before LPS stimulus resulted in50% inhibition of the 5� construct that increased to nearly 70%when the preincubation period was extended to 24 h (Fig. 8). Thiswas compared with the 80% inhibition of endogenous TNF-� pro-duction. The effect of preincubating macrophages for 24 h withdifferent concentrations of IL-10 was also examined. As shown inFig. 9, LPS-induced TNF-� protein expression was inhibited to amaximum of 90% (10 ng/ml IL-10) with an IC50 of �0.1 ng/ml,regardless of which reporter construct had been infected into themacrophages. IL-10 also inhibited the expression of the 5�3�UTRreporter to an identical degree to the endogenous gene (Fig. 9B).However, in contrast to data in Fig. 3A, preincubation for 24 h withIL-10 now produced a dose-dependent inhibition of the 5� con-struct that showed a maximum inhibition of 60% at 10 ng/ml (Fig.9A). The IC50 for IL-10 on the 5� construct was 5 ng/ml, but thisreduced to 0.5 ng/ml if the half-maximal inhibition was again cal-culated. These data suggest that, in addition to posttranscriptionalregulation of the TNF-� gene, IL-10 can also inhibit transcriptionof the TNF-� gene if cells are exposed to this inhibitory factor fora sufficient period. Indeed, studies on mRNA levels showed thatpreincubation for 24 h with IL-10 resulted in a decrease in lucif-erase mRNA, regardless of the presence of the 3�UTR (Fig. 5).However, we were unable to perform nuclear run-on experimentsto confirm an effect on transcription, as we cannot obtain sufficientcells from a single donor to perform fully controlled experiments.

FIGURE 7. IL-10 does not inhibit the LPS-induced activation of p38,p54/JNK, or p42/44/extracellular signal-regulated kinase kinases in humanmacrophages. Cells were treated with IL-10 (10 ng/ml) for the given times,followed by activation for 15 min with LPS (10 ng/ml). Cell lysates weregenerated and immunokinase assays were performed, as described in Ma-terials and Methods. Data are representative of at least three experimentsusing cells from different donors.

FIGURE 8. Effect of prolonged exposure to IL-10 before LPS stimula-tion on luciferase expression. Human macrophages infected with eitherAdvp5� or Advp5�3�UTR were treated with IL-10 (10 ng/ml) for the giventimes before LPS stimulation. Four hours after LPS activation, cells wereharvested and luciferase activities and TNF-� expression were assayed.Data are mean values � SEM from duplicates and are representative ofthree separate experiments using cells from different donors.

FIGURE 9. Prolonged exposure to IL-10 before LPS stimulation results in theinhibition of luciferase expression in the absence of 3�UTR. Infected cells (A,Advp5�; B, Advp5�3�UTR) were pretreated for 24 h with various concentrations ofIL-10 before LPS exposure. Cells were harvested after 4 h of LPS stimulation andluciferase activities and TNF-� production were assayed. Data are expressed aspercentages of luciferase activation and TNF-� production induced by LPS in theabsence of IL-10. Data are mean values � SEM from four experiments using cellswith different donors.

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IL-10 inhibition of LPS-induced TNF-� production isindependent of NF-�B

An inhibitory mechanism that involves the 5� promoter region ofthe TNF-� gene suggest that IL-10 may be interfering with thefunction of a transcription factor. As NF-�B has been implicated

previously as a target for IL-10, the effect of the cytokine on theactivation of this transcription factor was investigated. As shownin Fig. 10A, IL-10 had no effect on the activation of NF-�B byLPS, as measured by EMSA, regardless of the length of the ex-posure to the cytokine. As would be expected from this result, wealso observed no effect of IL-10 on I�B� degradation (data notshown). Next, using an adenovirus encoding an NF-�B-driven lu-ciferase reporter gene previously described by Sanlioglu et al. (17),the effect of IL-10 on the transactivating activity of the transcrip-tion factor was also examined. As shown in Fig. 10B, preincuba-tion with IL-10 for 24 h had no effect on the NF-�B transcriptionalactivity, although there was a slight inhibition of the NF-�B re-porter gene (�20%) when IL-10 was added simultaneously withLPS. As expected, the coinfection of macrophages with the I�B�-encoding adenovirus (AdvI�B�) inhibited gene expression by�90%, whereas a control virus had no effect. We extended thisstudy further by addressing whether a role for NF-�B in TNF-�expression was essential for IL-10 inhibition. We have previouslyshown using AdvI�B� that NF-�B is not a requirement for zymo-san-induced TNF-� production (20). In this study, similar datawere obtained with the reporter genes that showed that coinfectionof the cells with the reporter viruses and AdvI�B� resulted in an80–90% inhibition of the response of both constructs to LPS. Incontrast, we observed no significant inhibition in response to zy-mosan (Fig. 10C). A control virus Ad0 had no effect on responsesto either stimulus. However, IL-10 showed the same inhibitoryprofile to zymosan-induced reporter gene activity as seen abovewith LPS, namely inhibition via the 3�UTR, when cytokine andstimulus were added simultaneously, and an effect on the 5� regiononly when cells were preincubated with IL-10 (Fig. 10D). IL-10inhibited zymosan-induced production of TNF-� protein to levelssimilar to those obtained with the reporter genes, except, of course,in the case of the t0 time point and the 5� construct (Fig. 10D).These data support the conclusion that IL-10-induced inhibition ofTNF-� expression is NF-�B independent.

DiscussionThe published literature on the mechanisms of the anti-inflam-matory action of IL-10 is somewhat contradictory. The proposedmechanisms, although incompletely characterized, appear to bediverse and depend on the nature of the stimulus, the gene ofinterest, and the cell system investigated. In the present study, agenetic approach was taken by investigating which regions of theTNF-� gene mediate the suppressive effect of IL-10. A particularaspect to this study was the harnessing of our previously successfulexperience of using adenovirus to deliver transgenes into primaryhuman monocytic cells (12, 13, 20, 26). Using this approach, wewere able to deliver, for the first time to our knowledge, TNF-�gene-based reporter constructs into primary human macrophagesand investigate TNF-� gene regulation in a system highly relevantto human pathology. Unlike previous studies, the data showed thatIL-10 apparently uses two independent mechanisms for inhibitingTNF-� expression, by targeting either the 5� promoter or the3�UTR.

The description of a potential posttranscriptional mechanism viathe 3�UTR for the IL-10-mediated inhibition of TNF-� productionin human cells is novel, as previous studies in human cells haveimplicated a transcriptional target (4, 5, 7, 27). However, none ofthese studies used the approach of analyzing gene function. Thesedata are in agreement with studies in murine macrophages, wherea posttranscriptional mechanism has been proposed (9, 28). Pre-vious studies on the 3�UTR have shown that this region has anoverall suppressive effect (21) on TNF-� expression (which this

FIGURE 10. No evidence for an involvement of NF-�B in IL-10 inhi-bition of TNF-�. A, Cells were pretreated with IL-10 for 24 h (t �24)or treated simultaneously with IL-10 (t 0) at the time of addition ofmedium alone (Unstim) or LPS (10 ng/ml) for 30 min; cells were harvestedand nuclear extracts were prepared for EMSA. B, Cells were infected withAdvNF-�B-luc and some cells were also coinfected with AdvI�B� or Ad0,as shown. Cells were also preincubated with IL-10 for 24 h (t 24) ortreated simultaneously with IL-10 (t 0) at the time of addition of LPS (10ng/ml). After 4 h, cells were harvested and luciferase activity was assayed.Data are presented as a percentage of activation of cells with LPS alone andare mean values � SEM from three experiments using cells from differentdonors. C, Cells were infected with Advp5� or Advp5�3�UTR, with eitherAd0 or AdvI�B�. After 24 h cells were activated with LPS for 4 h, afterwhich time cells were harvested and luciferase activity and TNF-� pro-duction were assayed. Data are represented as the percentage of activationof cells that were infected only with the reporter viruses. Assays wereperformed in triplicate; data are means � SD. D, Infected cells (Advp5� orAdvp5�3�UTR) were treated with IL-10 (10 ng/ml), either simultaneously(t 0) or 24 h prior (t �24) to stimulation with zymosan (30 �g/ml) for4 h. Cells were then harvested and assayed for luciferase activity or TNF-�production. Assays were performed in triplicate; data are means � SD.Each study is representative of experiments on cells obtained from at leasttwo different donors.

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study has now confirmed in human macrophages). However, al-though we were able to show that IL-10 was able to decreasemRNA levels via the 3�UTR, it was not possible to confirm pre-vious findings in murine macrophages (9) that this effect was me-diated by destabilizing TNF-� mRNA (data not shown). A poten-tial target for IL-10 in this context would be p38 MAPK, as thiskinase is involved in the posttranscriptional control of TNF-� ex-pression (24). However, we were unable to show any effect ofIL-10 on the activation of p38 MAPK, the related p54/JNK, orp42/44 MAPK. These results agree with previous findings thatIL-10 did not inhibit LPS-induced phosphorylation of p38 MAPKin human monocytes (3). A recent study has suggested that cyto-kines and growth factors could affect mRNA splicing (29). Theluciferase reporter gene used in this work is, of course, devoid ofintrons; however, the apparent similarity of the responses of theendogenous TNF-� and reporter genes to IL-10 indicate that in-trons and mRNA splicing are not targets for IL-10 activity. There-fore, the mechanism by which IL-10 may inhibit TNF-� expres-sion posttranscriptionally is still unclear. A potential target is theAU-rich ARE regions found in the 3�UTRs of TNF-� and manyother cytokine genes. These regions have been implicated in theregulation of mRNA stability and turnover (30), as well as inmRNA translation (31–34). The recent study in murine macro-phages by Kontoyiannis et al. (10) has identified this region as atarget for IL-10-mediated suppression of TNF-� gene translationby a mechanism involving inhibition of p38 MAPK. However,IL-10 inhibition of TNF-� in human macrophages appears to begrossly different, as there is no inhibition of p38 MAPK. Also, asTNF-� mRNA levels are inhibited, this precludes any major rolefor an effect on TNF-� gene translation. The full understanding ofthe mechanism for the posttranscriptional regulation of TNF-� byIL-10 in human cells will obviously require a more extensive studyand may require the discovery of as yet unknown pathways in-volved in the general posttranscriptional control of this gene.

The second inhibitory mechanism of IL-10 in primary humanmacrophages required the 5� promoter region, suggesting an inhi-bition of gene transcription. However, the amount of materialavailable from individual donors prevented any meaningful run-onexperiments being performed to confirm this. In contrast to theposttranscriptional mechanisms, this inhibitory mechanism re-quired a prolonged pre-exposure to IL-10. The inference fromthese data would be that there is a requirement for gradual changesin cell physiology, e.g., protein synthesis, to mediate this effect ofIL-10. A requirement for IL-10-directed protein synthesis for theinhibition of LPS-induced TNF-� gene transcription is supportedby previous studies in human cells, showing that the protein syn-thesis inhibitor cycloheximide inhibits IL-10 function (4, 5). How-ever, so far our own studies with cycloheximide have proved in-conclusive, possibly because the general inhibition of proteinsynthesis leads to toxicity. It has been reported in human PBMCthat IL-10 can suppress the activation of the key proinflammatorytranscription factor, NF-�B (7), possibly by inhibiting I�B� kinaseactivity (6) or NF-�B DNA binding (6). Previously, we have alsoobserved IL-10-mediated inhibition of LPS-induced NF-�B activ-ity in the murine cell lines RAW 264.7 and the pre-B cell line70Z/3. However, the concentrations required to achieve this effectwere high (an IC50 of �100 ng/ml), far in excess of that requiredto inhibit TNF-� expression (an IC50 of �0.3 ng/ml) (28). How-ever, our studies in human macrophages have failed to show anyeffect of IL-10 (10 ng/ml) on NF-�B activation, as judged byEMSA. Additional studies using an adeno-NF-�B reporter virusshowed that preincubation with IL-10 had no effect on transcrip-tional function. The only effect of IL-10 on NF-�B function weobserved was a 20% decrease in LPS-induced NF-�B reporter

gene activity when IL-10 was added simultaneously with LPS.This does not appear to account for a mechanism that operates viathe 3�UTR or the potent suppression of IL-10 on TNF-� expres-sion. Moreover, our studies with zymosan show that a NF-�B-dependent mechanism of gene induction is not an essential require-ment for the IL-10 inhibition of TNF-� expression. The studiesshowing inhibition of NF-�B reported that IL-10 inhibited activa-tion of the factor with either minimal (5 min) or no pre-exposureof the cells to the cytokine before stimulus. This would not fit withthe observations in this study and the studies of others (4, 5) show-ing the requirement for prolonged exposure to IL-10 (4, 5), whichindicated de novo protein synthesis was required for IL-10 inhi-bition of TNF-� gene transcription. In summary, our data usingprimary human macrophages do not agree with previous studiesthat IL-10 has a profound effect on NF-�B activation and that thisis a mechanism by which IL-10 inhibits TNF-� expression. Wecan only conclude that, to some extent, the differences in theseresults may be due to our use of primary cells rather than cell linesand that changes in signaling mechanisms can occur between suchsystems. Such a conclusion is supported by recent studies on NF-�B-inducing kinase. This kinase was proposed to play an essentialrole in NF-�B activation by many stimuli, including LPS, TNF-�,and IL-1, in cell lines (35). However, this kinase has subsequentlybeen shown to play no such role in response to these stimuli inprimary human or murine cells (36–38). Our data indicate thatanother transcriptional mechanism is the target of IL-10 and thatthe mechanism involved is more likely to be indirect, probablyrequiring the expression of IL-10-induced proteins. The identifi-cation of the transcriptional target for IL-10 may be dependent ona greater understanding of the general mechanisms controllingTNF-� gene transcription.

The previous studies in human cells focusing on transcriptionalregulation have overlooked posttranscriptional mechanisms oper-ating via the 3�UTR. The advantage of this study is that, by cor-relating the effect of IL-10 with different regions of the TNF-�promoter, it was possible to identify and delineate both mecha-nisms and show, for the first time, that both mechanisms couldcoexist. Also, by using the adenoviral system to undertake thisstudy in primary cells, any potential problems with performingstudies in transformed cell lines could be avoided. This report pro-vides some key insights as to why there has been much disagree-ment in the field of IL-10 function, by showing that multiple mech-anisms can coexist and that the nature of the system studied has amajor impact on the result.

AcknowledgmentsWe thank Drs. A. Clark, J. Dean, and U. Sarma, L. Bradley, and Profs. M.Feldmann, J. Saklatvala, R. D. Schreiber, and H. Nagase for their helpfulreading of the manuscript, S. Evans for typing the manuscript, Dr. R. deMartin for the AdvI�B� virus, and Dr. Paul McCray for theAdvNF-�B-luc.

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