Rhinovirus Replication in Human Macrophages Induces NF-�B-DependentTumor Necrosis Factor Alpha Production
Vasile Laza-Stanca,1 Luminita A. Stanciu,1 Simon D. Message,1 Michael R. Edwards,1James E. Gern,2 and Sebastian L. Johnston1*
Department of Respiratory Medicine, National Heart and Lung Institute and Wright Fleming Institute ofInfection and Immunity, Imperial College London, London, United Kingdom,1 and Department of
Pediatrics, University of Wisconsin—Madison, Madison, Wisconsin2
Received 24 January 2006/Accepted 12 May 2006
Rhinoviruses (RV) are the major cause of acute exacerbations of asthma and chronic obstructive pulmonarydisease (COPD). Rhinoviruses have been shown to activate macrophages, but rhinovirus replication inmacrophages has not been reported. Tumor necrosis factor alpha (TNF-�) is implicated in the pathogenesisof acute exacerbations, but its cellular source and mechanisms of induction by virus infection are unclear. Wehypothesized that rhinovirus replication in human macrophages causes activation and nuclear translocationof NF-�B, leading to TNF-� production. Using macrophages derived from the human monocytic cell lineTHP-1 and from primary human monocytes, we demonstrated that rhinovirus replication was productive inTHP-1 macrophages, leading to release of infectious virus into supernatants, but was limited in monocyte-derived macrophages, likely due to type I interferon production, which was robust in monocyte-derived butdeficient in THP-1-derived macrophages. Similar to bronchial epithelial cells, only small numbers of cellssupported complete virus replication. We demonstrated RV-induced activation of NF-�B and colocalization ofp65/NF-�B nuclear translocation with virus replication in both macrophage types. The infection inducedTNF-� release in a time- and dose-dependent, RV serotype- and receptor-independent manner and was largely(THP-1 derived) or completely (monocyte derived) dependent upon virus replication. Finally, we establishedthe requirement for NF-�B but not p38 mitogen-activated protein kinase in induction of TNF-�. These datasuggest RV infection of macrophages may be an important source of proinflammatory cytokines implicated inthe pathogenesis of exacerbations of asthma and COPD. They also confirm inhibition of NF-�B as a promisingtarget for development of new therapeutic intervention strategies.
Acute exacerbations of asthma and chronic obstructive pul-monary disease (COPD) are the major causes of morbidity andmortality in both diseases. Rhinoviruses (RVs) are the mostcommon trigger of acute exacerbations (19, 30, 42); however,the mechanisms by which RVs provoke exacerbations are notwell understood. The airway epithelium is thought to be thesite of RV replication, and many studies have observed RVinduction of proinflammatory cytokines, chemokines, and ad-hesion molecules in epithelial cells (28, 47, 52, 59). During RVinfection, the number of epithelial cells infected with virus islow both in vitro and in vivo (8, 39); nonetheless, it iscurrently believed that inflammatory cytokine productionfrom RV-infected epithelial cells is an important mechanismcontributing to the pathogenesis of exacerbations of asthmaand COPD (37).
Tumor necrosis factor alpha (TNF-�) is a potent inflamma-tory cytokine implicated in the pathogenesis of asthma andCOPD (25, 31, 60). TNF-� has multiple biologic effects rele-vant to the pathogenesis of exacerbations of airway disease,including the enhanced release of other proinflammatory/che-motactic mediators, up-regulation of adhesion molecules, en-hanced migration of eosinophils and neutrophils, and induc-
tion of hypercontractile airway smooth muscle (4, 36, 60).TNF-� is detected in increased amounts in bronchoalveolarlavage during experimental RV infection in asthma and isalso increased in acute exacerbations of COPD (1, 9). Thesedata implicate TNF-� in the pathogenesis of acute exacer-bations of both diseases; however, TNF-� cannot be de-tected in significant quantities in the supernatants of RV-infected epithelial cells, suggesting that an alternative cellularsource may exist (59).
Lung macrophages (M�) are an important source forTNF-�, are the most numerous cells in the airway lumen, andare quickly recruited during inflammatory processes of thelung (18). Following interaction with various bacterial and viralpathogens, they become activated and secrete a wide range ofantiviral, proinflammatory, and/or immunomodulatory cyto-kines (62). Their position in the airway and their high levels ofexpression of RV receptors ICAM-1 and low-density lipopro-tein (LDL) receptor suggest M� may be a target for RVinfection and TNF-� production (17, 34). Relatively few stud-ies have investigated interactions between RVs and cells of M�or monocytic origin. Induction of interleukin-8 (IL-8), IL-10,IL-12, TNF-�, and monocyte chemoattractant protein-1(MCP-1) and alteration of surface expression of CD14, CD80,and CD69 in peripheral blood mononuclear cells, monocytes,or M� have all been reported after exposure to RV (14, 16, 21,29, 44, 45, 56). However, the mechanisms responsible for thismonocyte/M� activation are unclear. Production of IL-10,TNF-�, and MCP-1 was reported to be replication indepen-
* Corresponding author. Mailing address: Department of Respira-tory Medicine, National Heart and Lung Institute and Wright FlemingInstitute of Infection and Immunity, Imperial College London, NorfolkPlace, London W2 1PG, United Kingdom. Phone: 44 20 7594 3764. Fax:44 20 7262 8913. E-mail: [email protected].
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dent, while IL-8 secretion and CD69 up-regulation appeared inpart replication dependent, as UV-inactivated RV had a sig-nificantly reduced effect compared to live virus (14, 16, 21, 29,44). When evidence for RV replication in M� was specificallyinvestigated, attachment and entry of virus was detected, butno further evidence of replication was detected, as RV titers insupernatants decreased over time and RV RNA synthesis wasnot observed (14).
In addition, the molecular mechanisms regulating RV induc-tion of inflammatory cytokines in M� are poorly understood,though RV induction of MCP-1 in monocytes and M� is re-ported dependent on the p38 mitogen-activated protein kinase(MAPK) MAPK/AP-1 pathway (21). Studies of RV infectionof respiratory epithelial cells indicate a central role for NF-�Bin up-regulation of several proinflammatory molecules (32, 46,47, 67). We therefore hypothesized that rhinoviruses can rep-licate in human M� and that replication causes activation andnuclear translocation of NF-�B, leading to TNF-� production.To address this hypothesis, we studied RV replication in THP-1-derived M� and primary monocyte-derived M� (MDM),both well-established models of human M� (6, 7, 63). We alsoinvestigated the roles of virus replication and NF-�B in TNF-�production.
MATERIALS AND METHODS
Cell lines and viruses. HeLa and THP-1 cell lines (European Collection ofCell Cultures) were cultured in E-MEM (Invitrogen) and RPMI 1640 (Invitro-gen), respectively, with 10% fetal calf serum (FCS; Invitrogen). RV serotypes 16,9 (major group; receptor, ICAM-1), and 1B (minor group; receptor, LDL re-ceptor) were grown in HeLa cells and prepared as previously described (47).Virus stocks or supernatants of RV-infected M� were titrated on HeLa cells toascertain their 50% tissue culture infective dose (TCID50)/ml (47). The identitiesof all RVs were confirmed by neutralization using serotype-specific antibodies(ATCC). UV inactivation and generation of filtered virus were performed aspreviously described (47). Human respiratory syncytial virus (RSV) strain A2 wasa gift from P. J. Openshaw.
Generation of THP-1-derived M� and MDM. For all experiments, THP-1 cellswere used after differentiation to M�. To induce differentiation THP-1, 0.75 �106/ml in RPMI 1640 with 5% FCS were treated with phorbol myristate acetate(PMA; Sigma) at 200 nM for 24 h and placed for a further 24 h in 5% FCSwithout PMA, to allow the cells to rest.
M� were generated from peripheral blood monocytes using a previously de-scribed protocol that was slightly modified (49). Briefly, mononuclear cells wereseparated from total blood of healthy donors by density gradient centrifugationusing Ficoll-Paque (Sigma). Monocytes, isolated by positive selection using anti-CD14 magnetic beads (magnetic-activated cell sorter), were differentiated to M�by culture for 7 days in macrophage serum-free medium (Invitrogen) supple-mented with 10 ng/ml granulocyte-macrophage colony-stimulating factor (Bio-source) (49). The study was approved by the St. Mary’s NHS Trust ethicscommittee, and informed consent was obtained from all volunteers.
Infection of M�. THP-1-derived M� or MDM generated as described abovein 12-well plates were exposed to RV or RSV at a multiplicity of infection (MOI)of 1 with continuous shaking. After 1 h, unattached virus was removed, cells werewashed extensively, and 1 ml of RPMI 1640 containing 5% FCS for THP-1 ormacrophage serum-free medium for MDM was added to each well. This wasconsidered time point zero (0 h). Supernatants and RNA or protein lysates (forWestern blot analysis) were harvested at different time points and stored at�80°C for further use.
RNA extraction, reverse transcription, and TaqMan real-time PCR for viralRNA quantification. Whole-cell RNA was extracted using TRIzol according tothe manufacturer’s instructions (Invitrogen). Two �g of total RNA was reversetranscribed into cDNA using Omniscript reverse transcriptase and componentsas directed by the manufacturer (QIAGEN). RV cDNA was measured by Taq-man PCR (ABI) and normalized using 18S rRNA. Primers and probe sequencesalong with the protocol used for Taqman real-time PCR have been publishedelsewhere (23, 24). Data were analyzed using version 1.0 ABI Prism 7000 SDSsoftware (ABI) and converted to copy numbers using a standard curve for a
plasmid of known concentration containing the amplified region of the RVgenome.
Western blotting for RV 3C protease expression. THP-1-derived M� werelysed directly into sodium dodecyl sulfate (SDS) sample buffer (Invitrogen).After electrophoresis in a 12% SDS-polyacrylamide gel electrophoresis (PAGE)gel (Invitrogen), proteins were transferred to a polyvinylidene difluoride mem-brane (Amersham). All these steps were performed under reducing conditions.After blocking, membranes were incubated with a 1/5,000 dilution of rabbitanti-3C RV protease antibody (provided by Svetlana Amineva, University ofWisconsin—Madison) (3) followed by horseradish peroxidase-conjugated swineanti-rabbit antibody (Serotec). Bands were visualized by chemiluminescence withthe ECL Western blotting detection reagent (Amersham). After scanning, den-sitometry analysis of the band corresponding to the 3C protease was performed.
Infectious center assay to determine numbers of infected cells. To asses thenumber of virus-releasing cells after RV infection, we employed an infectiouscenter assay using modifications of a published protocol (39). THP-1-derived M�or MDM were infected with RV16 as described above and incubated at 37°C.After 1 h, cells were trypsinized and plated over monolayers of HeLa cells indifferent dilutions. After a further 1-h incubation, to allow the cells to settle,medium was carefully removed and RMPI 1640 containing 5% FCS and 0.3%Indubiose (Invitrogen) was added. Plates were incubated at 37°C for 4 days, andat the end of incubation cells were fixed by addition of 10% formaldehyde andstained with crystal violet, 0.1%. Virus released from infected M� infects onlythe HeLa cells on which infected M� have settled, thus creating a plaque. Thenumber of virus-releasing M� was assessed by enumeration of plaques.
Transient transfection and NF-�B luciferase reporter assay to assess NF-�Bactivation. Before RV16 infection, THP-1-derived M� were transiently trans-fected with a construct containing the luciferase gene under the control of aminimal promoter containing four NF-�B binding sites (BD Clontech) and aconstitutive �-galactosidase-expressing construct (Invitrogen) using JetPEI(Polyplus) according to the manufacturer’s protocol. After RV infection, cellswere incubated for 48 h to allow expression of the reporter gene. Preparation ofcell lysates and luciferase assays were performed as recommended by the man-ufacturer (Promega) using an Autolumat LB953 (Berthold Systems Inc.). Allluciferase measurements were normalized to �-galactosidase activity measuredusing the �-galactosidase enzyme assay system (Promega).
Immunofluorescent staining for confocal microscopy colocalization of virusinfection and NF-�B translocation. THP-1-derived M� or MDM seeded ineight-well microscope slides were infected as described above. At different timepoints, cells were fixed in 4% paraformaldehyde and permeabilized with TritonX-100, 0.2%. After overnight blocking with phosphate-buffered saline containing10% FCS and 1% bovine serum albumin, cells were incubated with rabbitanti-3C RV protease serum, 1/500, with or without monoclonal mouse antibodyanti-p65 (1/200; Santa Cruz) for 1 h in blocking buffer followed by goat anti-mouse AlexaFluor 546, 1/200, and goat anti-rabbit AlexaFluor 488, 1/200 (Mo-lecular Probes) as secondary antibodies in blocking buffer for 45 min. The slideswere coverslipped in 4,6-diamidino-2-phenylindole (DAPI)-containing mount-ing medium and examined using an LSM 510 confocal microscope (Zeiss). Toevaluate the percentage of virus-infected cells, the number of RV 3C-positivecells in 200 nucleated cells was counted and expressed as a percentage of totalcells.
TNF-�, IFN-�, and IFN-� enzyme-linked immunosorbent assay (ELISA).Levels of TNF-� and type I IFNs in supernatants of THP-1-derived M� or MDMwere measured using paired antibodies and standards commercially available forTNF-� (Biosource) or commercially available kits for IFN-� and IFN-� (Bio-source) following the manufacturer’s recommendations. The sensitivity of theassay was 10 pg/ml for TNF-�, 5 IU/ml for IFN-�, and 15 pg/ml for IFN-�.
p38 MAPK and NF-�B inhibition. To evaluate the role of NF-�B or p38MAPK in TNF-� secretion, we carried out inhibition experiments with chemicalinhibitors of these pathways. AS602868 (a gift from Ian Adcock), an inhibitor ofIKK� (12), or CAPE (Calbiochem), an inhibitor of p65 translocation (41), wasused to pretreat the THP-1-derived M� or MDM for 1 h before infection, atconcentrations ranging from 0.01 to 5 �M and 25 to 1.25 �g/ml, respectively (33,41). The same concentration of drug was added to the medium after infection.Similar experiments were carried out with SB203580 (Calbiochem), an inhibitorof p38 MAPK, at a concentration of 10 �M (20).
Statistical analysis. The results were analyzed using GraphPad Prism version4.00 for Windows (GraphPad Software, California). Results of at least threeseparate experiments were expressed as means standard errors of the means(SEM) and analyzed using analysis of variance (ANOVA) for multiple com-parisons, followed where appropriate by paired Student’s t tests for pairedcomparisons.
VOL. 80, 2006 RV REPLICATION INDUCES TNF-� PRODUCTION IN MACROPHAGES 8249
RESULTS
Rhinovirus replicates efficiently in THP-1-derived M�. Toinvestigate whether RV16 can replicate in human M�, we firstused THP-1-derived M�. After differentiation cells were ex-posed to RV16, and supernatants, total cell RNA, or intracel-lular proteins were harvested at different time points. Intracel-lular levels of RV RNA were determined using real-time PCR.Relatively high levels of RV16 RNA were detected at timezero (4.53 0.486 log10 copies/�g total RNA), followed by asmall reduction at 1 h and an eclipse phase at 2 h, when adecrease of almost 1 log10 was observed compared with 0 h(Fig. 1A). Following the eclipse, viral RNA production in-creased rapidly, reaching a peak of greater than 6 log10 copiesat 8 h (a 2.45-log10 increase compared with 2 h [P � 0.01] anda 1.53-log10 increase compared with 0 h [P � 0.05]). Thereaf-ter, viral RNA levels decreased gradually but were still signif-icantly elevated at 24 h and were greater than 5 log10 copies atboth 48 and 72 h (Fig. 1A).
Full productive replication was then investigated by assess-ment of virus release into supernatants of cells. Very low titersof RV16 were detected in supernatants at early time points, 0to 4 h, but they remained unchanged during this period. RV16titers then increased progressively and were significantly ele-vated compared to 0 h at 8, 24, 48, and 72 h, peaking at 24 h
(2.56 log10 TCID50/ml increase compared with 0 h; P � 0.001),followed by a gradual decline thereafter (Fig. 1B).
Synthesis of new RV proteins was also investigated byWestern blot analysis of cell lysates using a polyclonal rabbitserum against RV 3C protease, which is a nonstructuralprotein expressed only during viral replication (3, 54). RV3C protease was undetectable at time zero and thereaftercould be detected in increasing amounts (Fig. 1C). Whenanalyzed by densitometry, the 3C protease expression wassignificantly elevated between 8 and 48 h (P � 0.01), peak-ing at 24 h (P � 0.001) (data not shown). In addition 3ABCprotease, a precursor of 3C protease, was also clearly de-tected at both 8 and 24 h (Fig. 1C).
Despite being acknowledged as the primary site of RV rep-lication in the lower airway, only a small minority of bronchialepithelial cells are infected in vitro and similar frequencies areobserved in vivo (5, 8, 39). Therefore, having demonstratedproductive replication in THP-1-derived M�, we next wishedto determine the frequencies of virus-infected cells, to permitcomparison with epithelial cells. For this, an infectious centerassay was used to determine the number of RV16-releasingcells, using methods adapted from those previously used todetermine frequencies of infected epithelial cells (39). WhenTHP-1 M� were exposed to RV16, 5.66 0.63% of cells
FIG. 1. RV replication in THP-1-derived M�. (A) THP-1-derived M� were infected for 1 h with RV16 (input MOI of 1 TCID50/cell), and RNAwas extracted from cell lysates at 0, 1, 2, 4, 8, 24, 48, and 72 h postinfection. RV RNA expression was quantified by using TaqMan, and data arepresented as the number of copies per �g of total RNA. The results are expressed as means SEM (n � 4). Statistical significance between theeclipse (2 h) and other time points is indicated: �, P � 0.05; ��, P � 0.01; statistical significance between 0 h and other time points is indicatedby # for P � 0.05. (B) Cells were infected as for panel A, supernatants were harvested, and the amount of infectious virus released into thesupernatants was assessed by virus titration. The results are expressed as means SEM (n � 4). ��, P � 0.01; ���, P � 0.001. (C) Cells infectedas for panel A were lysed and analyzed for the presence of RV 3C protease by Western blotting. A representative image of four differentexperiments is shown. (D) THP-1-derived M� were infected with RV16 (MOI of 1) for 1 h, and the number of virus-releasing cells was assessedin an infectious center assay. The numbers of infected THP-1 cells overlaid in duplicate on HeLa cells are indicated at the bottom (see Materialsand Methods for more details). Numbers of plaques counted (each representing an infectious center derived from a rhinovirus-releasing THP-1cell) at each concentration of overlaid THP-1 cells are indicated in the boxes with arrows. The experiment is representative of four, in which 5.66 0.63% of cells were associated with plaque formation.
8250 LAZA-STANCA ET AL. J. VIROL.
(n � 4) released sufficient infective virus to lead to plaqueformation on HeLa cells (Fig. 1D).
We next investigated frequencies of cells in which replica-tion was demonstrable by immunofluorescent staining forRV16 3C protease, with the polyclonal rabbit serum, usingconfocal microscopy to enumerate infected and noninfectedcells. RV 3C protease was detected as early as 2 h after RV16infection of THP-1-derived M� and persisted until 48 h (datanot shown). The highest frequency of positive cells for RV163C protease (9 1.4%; n � 4) was detected at 6 h afterinfection (see Fig. 3B, below). No staining was detected whencells were treated with medium or UV RV16 (data not shown).
Consistent with these infection frequencies and with datarecently reported in primary bronchial epithelial cells fromnormal volunteers (65), no cytopathic effects were observedin RV16-infected THP-1 M� when inspected by invertedmicroscopy.
Limited rhinovirus replication can be detected in MDM. Ithas been reported that human RVs are unable to replicate inM� (14). However, UV inactivation experiments have sug-gested replication is required for up-regulation of severalgenes following RV infection in monocytes or M� (16, 21, 29,44, 45), and we have shown efficient replication in THP-1-derived M�. Therefore, we wished to investigate to whatextent rhinovirus replication can take place in primary M�.For this we used MDM, a model with close resemblance toalveolar M� (2).
MDM were exposed to RV16, and supernatants or total cellRNA was harvested at different time points. RV16 was de-tected at low titers at 0 h and, after a small increase between 2and 8 h, virus titers continuously decreased thereafter; how-ever, virus was still detectable at 72 h (Fig. 2B) (P 0.05). Wenext investigated intracellular levels of RV RNA using real-time PCR. High levels of viral RNA were detected at 0 h (6.767 0.098 log10 copies/�g total RNA), and these levels had de-creased by 1 log by 24 h but then remained constant at around6 logs at 48 and 72 h (Fig. 2A) (P 0.05).
Having observed limited virus release and high levels of viralRNA persisting to at least 72 h, we next investigated if synthe-sis of new viral proteins could be demonstrated by immuno-fluorescent staining for RV16 3C protease, using confocal mi-croscopy. RV 3C protease was clearly detectable at 4 h(frequency of positive cells for RV16 3C protease, 19 1.7%;n � 3) after RV16 infection of MDM and persisted until 24 h.No staining was detected when rabbit polyclonal immunoglob-ulin G (data not shown) was used and when cells were treatedwith medium (Fig. 2C).
To assess frequencies of virus-releasing cells in MDM, wenext used the infectious center assay in a manner similar to thatwith THP-1 M�. Only 0.1 0.01% of cells (n � 4) releasedsufficient infective virus to lead to plaque formation on HeLacells (data not shown).
These data indicate that RV can replicate well in THP-1-derived M�, but replication is clearly limited in MDM. Toattempt to explain these differences, we next investigated re-lease of type I IFNs by THP-1-derived M� and MDM inresponse to RV infection. Both IFN-� (P � 0.05) and IFN-�(P � 0.01) were significantly increased at 24 h in the superna-tants of RV16-infected MDM, while no IFN-� and only trace
amounts of IFN-� were detected in RV16-infected THP-1-derived M� supernatants (Fig. 2D and E).
NF-�B is activated after RV infection of both THP-1-derivedand monocyte-derived M�. As NF-�B activation is implicatedin activation of bronchial epithelial cells by RVs (32, 47, 67)and having demonstrated full RV replication in THP-1-derivedM� and limited replication in MDM, we next examined ifNF-�B was activated following infection of M�. We first as-sessed whether an NF-�B-dependent reporter gene was acti-vated by RV infection. When THP-1-derived M� were tran-siently transfected with an NF-�B–Luc minimal promoter/reporter and analyzed for luciferase expression at 48 h afterinfection, there was a 2.12-fold increase in RV16-infected cellscompared with medium-treated cells (Fig. 3A), (P � 0.05),confirming RV induction of NF-�B activation.
We next sought evidence of NF-�B activation by assessingNF-�B nuclear translocation by immunostaining for p65/NF-�B and analysis by confocal microscopy in infected andnoninfected cells. Clear evidence of NF-�B nuclear transloca-tion was observed in RV16-infected THP-1-derived M� at 3, 6(Fig. 3B), and 24 h, while no nuclear staining was observedwith medium-treated cells or UV-inactivated RV16-treatedcells (data not shown). To determine whether NF-�B translo-cation occurred only in virus-infected cells or in noninfectedcells (as a consequence of paracrine stimulation by cytokinesreleased from infected cells), THP-1-derived M� werecostained for RV 3C protease and NF-�B and analyzed usingconfocal microscopy. p65/NF-�B translocation was clearly co-localized with RV 3C protease staining (Fig. 3B), indicatingthat NF-�B activation occurred only in virus-infected cells.
Finally, to determine whether RV infection resulted inNF-�B activation in primary human MDM, similar experi-ments were carried out with MDM. Both NF-�B activation andRV 3C protease expression were observed in MDM at 2, 4, 8,and 24 h, with peak expression for NF-�B at 4 h (Fig. 3B). Aswith THP-1-derived M�, RV 3C protease and p65 transloca-tion were almost exclusively colocalized, indicating that NF-�Bactivation occurred only in RV-infected MDM (Fig. 3B).
TNF-� is released from RV-infected M� in a time- anddose-dependent manner. We next assessed TNF-� productionby M� after RV16 infection. THP-1-derived M� and MDMreleased TNF-� after RV16 infection in a time-dependentmanner. In the case of THP-1-derived M�, there was a steadyincrease of TNF-� in the supernatant starting at 4 h, reachingstatistical significance at 24 h (P � 0.05) and continuing there-after until 72 h (P � 0.01) (Fig. 4A). For MDM, significantlyelevated concentrations of TNF-� in supernatants were ob-served from 8 to 48 h; however, the peak was reached earlier,at 24 h (P � 0.01) (Fig. 4B). TNF-� was also secreted in adose-responsive manner after RV16 infection. The time pointwith the greatest levels of cytokine in the supernatant waschosen to carry out these experiments. Infection of eitherTHP-1-derived M� or MDM with increasing concentrations ofRV16 resulted in release of increasing amounts of TNF-� intothe supernatants (Fig. 4C and D).
TNF-� secretion is serotype and receptor independent andis largely dependent on viral replication. To investigatewhether RV-induced TNF-� secretion is serotype or receptorrestricted, we investigated two other serotypes in addition toRV16: RV9, another major group serotype, and RV1B, a mi-
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nor group serotype. Because all three virus stocks were used asunpurified preparations, we also wished to confirm that theobserved induction was virus specific and not the result ofother soluble factors present in the inoculum used for infec-tion. For this, the inoculum was molecular weight filtered usinga 30-kDa filter to remove all virus particles but not smallmolecules, such as cytokines (47). We also wished to determinewhether the production of TNF-� is virus replication depen-dent and therefore we used UV-inactivated RV. As for the
dose-response studies, these experiments were carried out attime points of maximum production for each cell type. RV9and RV1B were also capable of inducing the release of similaramounts of TNF-� from THP-1-derived M� and MDM (Fig.5A and B), indicating that induction was not serotype or re-ceptor restricted. When filtered RV16 inoculum was used, theproduction of TNF-� was abolished, confirming that inductionwas virus specific (Fig. 5A and B). When comparing UV-inactivated RV16 with live RV16, the levels of TNF-� mea-
FIG. 2. RV replication in MDM. (A) MDM were infected for 1 h with RV16 (MOI of 1), and RNA was extracted from cell lysates at 0, 2, 4,8, 24, 48, and 72 h postinfection. RV RNA expression was quantified by using TaqMan, and data are presented as the number of copies per �gof total RNA. The results are expressed as means SEM (n � 3). Significance is at a P value of 0.05 by ANOVA. (B) Cells were infected asfor panel A, supernatants were harvested, and the amount of infectious virus released into the supernatants was assessed by virus titration. Theresults are expressed as means SEM (n � 3). Significance is at a P value of 0.05 by ANOVA. (C) MDM, seeded in chambered slides, wereinfected with RV16 (MOI of 1) for 1 h. At 4, 8, and 24 h postinfection cells were fixed, permeabilized, and stained using rabbit anti-RV 3C proteaseserum and an appropriate secondary antibody (green). The slides were coverslipped using DAPI-containing mounting medium and analyzed usingconfocal microscopy. A representative image of three independent experiments is presented. Magnification, �800. Green represents, RV 3Cstaining and overlay with DAPI staining for nuclei (blue). (D and E) THP-1-derived M� or MDM were infected for 1 h with RV16 (MOI of 1),supernatants were harvested at 24 h, and the amount of IFN-� (D) or IFN-� (E) released into supernatants was assessed by ELISA. The resultsare expressed as means SEM (n � 3). �, P � 0.05; ��, P � 0.01.
8252 LAZA-STANCA ET AL. J. VIROL.
sured in supernatants were reduced by 83% in the case ofTHP-1-derived M� and completely suppressed in the case ofMDM (Fig. 5A and B), confirming that induction was largelyor completely replication dependent, respectively. Similar re-sults were obtained with filtered and UV-inactivated RV9 andRV1B (data not shown).
Inhibition of NF-�B, but not p38 MAPK, inhibits TNF-�production from M� in response to RV16 infection. The tran-scription factor NF-�B is required for inflammatory gene up-regulation after RV infection of epithelial cells (32, 47, 67) andis also important for induction of TNF-� in other systems (11,53). Having demonstrated activation of NF-�B after RV infec-
FIG. 3. NF-�B activation after RV infection of M�. (A) THP-1-derived M� transiently transfected with NF-�B–Luc minimal promoter wereinfected with RV16 (MOI of 1) or medium treated and analyzed for luciferase expression at 48 h. The results were normalized to �-galactosidaseand are expressed as the relative fold induction over the medium control. The results are expressed as means SEM (n � 4). �, P � 0.05 comparedwith medium. (B) THP-1-derived M� or MDM in eight-well chambered slides were infected with RV16 (MOI of 1). At 6 h postinfection (forTHP-1-derived M�) or 4 h postinfection (for MDM), cells were fixed, permeabilized, and stained with RV 3C protease rabbit antiserum (green)and mouse anti-p65 monoclonal antibody (red), followed by an appropriate secondary antibody. A representative image of four independentexperiments for THP-1-derived M� and three for MDM is presented. Magnification, �800. Green, RV 3C staining; red, p65 staining; blue, overlaywith DAPI staining for nuclei. Cells with rhinovirus infection are indicated by green arrows, and cells with NF-�B nuclear translocation are shownby the red arrows. In the overlay, most cells have dual staining, indicating that p65 nuclear translocation occurred principally in virus-infected cells.
FIG. 4. TNF-� is released from RV-infected M� in a time- and dose-dependent manner. (A and B) THP-1-derived M� (A) or MDM (B) wereinfected for 1 h with RV16 (MOI of 1), supernatants were harvested at 4, 8, 24, 48, and 72 h, and the amount of TNF-� released into thesupernatants was assessed by ELISA. (C) THP-1-derived M� were infected for 1 h with RV16 at MOIs of 0.01, 0.1, and 1. Supernatants wereharvested 72 h postinfection, and concentrations of TNF-� were assessed by ELISA. (D) MDM were infected for 1 h with RV16 at MOIs of 0.2,0.5, 1, and 5. Supernatants were harvested 24 h postinfection, and concentrations of TNF-� were assessed by ELISA. The results are expressedas means SEM (n � 4). Significance (compared to medium control): �, P � 0.05; ��, P � 0.01.
VOL. 80, 2006 RV REPLICATION INDUCES TNF-� PRODUCTION IN MACROPHAGES 8253
tion of M�, we wished to asses its involvement in TNF-�production. For this we used two inhibitors, AS602868 (a spe-cific inhibitor of the upstream kinase IKK�) and CAPE (aspecific inhibitor of the p65 subunit of the NF-�B/Rel com-plex). AS602868 inhibited RV16-induced TNF-� production inTHP-1-derived M� in a dose-responsive manner, achievingstatistically significant inhibition at concentrations of 0.5 and 1�M (P � 0.05) and complete inhibition at 5 �M (P � 0.001)(Fig. 6A). CAPE also induced a dose-dependent inhibition of
RV-induced TNF-� secretion in THP-1-derived M� but wasless effective than AS602868. Significant inhibition was ob-served at 5 and 10 �g/ml (P � 0.05), and 60.26% inhibition wasreached with a 25-�g/ml concentration (P � 0.001) (Fig. 6B),with higher doses having a toxic effect on cells. To confirm theabove findings in primary MDM, we tested the effect ofAS602868 on TNF-� release from RV-infected MDM. Weinvestigated 5 �M, because this concentration proved to havethe maximal effect without any cell toxicity in THP-1-derived
FIG. 5. RV-induced TNF-� secretion from M� is serotype and receptor independent and is largely dependent on viral replication. THP-1-derived M� (A) or MDM (B) were infected for 1 hour with major group (receptor, ICAM-1) viruses RV16 and RV9 and the minor group(receptor, LDL receptor) virus RV1B (all at an MOI of 1), medium alone (m), UV-inactivated RV16 (MOI of 1; UV), or the same volume of RV16inoculum from which virus had been removed by molecular weight filtration (filtered). Supernatants were harvested at 72 h for THP-1-derived M�or at 24 h for MDM, and the amount of TNF-� released was quantified by ELISA. The results are expressed as means SEM (n � 3). Significance(compared to medium control): �, P � 0.05; ��, P � 0.01; ���, P � 0.001.
FIG. 6. Requirement for NF-�B in TNF-� production from RV-infected M�. THP-1-derived M� (A, B, and D) or MDM (C) were pretreatedfor 1 h with inhibitors at the doses indicated, before infection with RV16 or with RSV (for p38 MAPK inhibition experiments only) at an MOIof 1. The same concentration of drug was added to the medium after infection. Supernatants were harvested at 72 h for THP-1-derived M� orat 24 h for MDM, and the amount of TNF-� released was quantified by ELISA. (A) AS602868, an inhibitor of IKK�, in concentrations between0.01 and 5 �M; (B) CAPE, an inhibitor of p65 translocation, in concentrations between 1.25 and 25 �g/ml; (C) AS602868 at a concentration of5 �M; (D) SB203580, an inhibitor of p38 MAPK, at a concentration of 10 �M. The results are expressed as means SEM (n � 6 [A, B, and C]or n � 4 [D]). Significance (compared to medium control): �, P � 0.05; ��, P � 0.01; ���, P � 0.001.
8254 LAZA-STANCA ET AL. J. VIROL.
M�. Again, an effective inhibition was observed, with TNF-�production after RV infection of MDM being reduced by 95%(P � 0.01) (Fig. 6C).
Another molecule shown to be important for cytokine pro-duction by RV-infected epithelial cells and M� is p38 MAPK(20, 21). To assess its role in TNF-� secretion, the p38 MAPKinhibitor SB203580 was added to the medium at 10 �M beforeand after RV or RSV infection. Inhibition of p38 MAPK withSB203580 had no effect on TNF-� secretion from RV16-in-fected THP-1-derived M� (RV16, 212.4 9.88; RV16 plusSB203580, 268.5 40.10 ng/ml; P 0.05), while RSV-inducedTNF-� secretion was reduced by 63% (P � 0.05) (Fig. 6D).
DISCUSSION
We have investigated the ability of RV to infect human M�and the molecular mechanisms involved in TNF-� productionfrom RV-infected M�. We have demonstrated that RV canreplicate efficiently in THP-1-derived M� by showing increas-ing intracellular levels of viral RNA and viral replicative pro-tein over time and release of infectious virus into supernatant.As has been observed with epithelial cells, 5% of cells releasedsufficient virus to cause plaques in HeLa cells (Fig. 1). Also, weshowed evidence of replication in MDM by showing persis-tence of virus release and high levels of intracellular viral RNAup to 72 h and by demonstrating synthesis of new viral proteins.However, only 0.1% of cells released sufficient virus to causeplaques in HeLa cells, and virus titers released into superna-tants did not increase significantly over time, indicating thatreplication was limited (Fig. 2). For both THP-1-derived M�and MDM, we also demonstrated activation of NF-�B follow-ing RV infection and colocalization of p65/NF-�B nucleartranslocation with viral replication. Virus infection was accom-panied by release of TNF-� in a time- and dose-dependent,serotype- and receptor-independent manner, largely or com-pletely dependent upon viral replication. Finally, we estab-lished the requirement for NF-�B, but not p38 MAPK, in RVinduction of TNF-�.
M� are a heterogeneous population. The differentiationprocess, which is under the control of local environments, willrender M� with significant differences from one body site toanother (26, 34, 64). Therefore, significant differences will bepresent not only between monocytes and M� but also betweenM� from different anatomical locations (2, 22, 34, 49). Al-though using THP-1-derived M� and MDM instead of primaryairway M� could be considered a limitation of this study,primary alveolar M� are difficult to obtain in sufficient numberand purity for extensive experiments, and it is not known howrepresentative they would be of M� found in the trachea andbronchial tree, where rhinovirus infections are likely to occur.For these studies, we therefore chose two models extensivelyused as replacements for alveolar M�. The first used THP-1cells, a monocytic cell line which can be differentiated to amacrophage phenotype by PMA treatment (6, 7, 58, 63). Thesecond used M� derived from peripheral blood monocytes bytreatment with granulocyte-macrophage colony-stimulatingfactor. The M� obtained by this protocol have a close resem-blance to alveolar M� (2).
The first important finding of this study is that RV canreplicate in M�. This confirms the preliminary results of a
previous study which showed a low-grade release of rhinovirusfrom nondifferentiated THP-1 cells (29). The pattern of infec-tion in THP-1-derived M� is similar with that found in respi-ratory epithelial cells, where only a small percentage of thecells release sufficient virus to produce plaques in an infectiouscenter assay (39). Persistence of viral RNA over 72 h, presenceof RV 3C staining in 19% of cells, virus release by 0.1% ofinfected cells, and replication-dependent TNF-� release inMDM suggest RV can replicate, to a limited degree, in thissystem as well. The discrepancy between the number of RV 3Cprotease-positive cells and the number of virus-releasing cellssuggests that virus replication is abortive in the majority ofMDM, with only a minority of cells supporting a completecycle of virus replication. Also, the release of type I IFNs in thesupernatants of MDM is indirect evidence of viral replication,as these cytokines are only induced during viral replication(27). Type I IFN production could also explain the differencesseen in viral replication between THP-1-derived M� andMDM, as type I IFN production was almost absent in THP-1-derived M�. We have recently described profoundly deficientIFN-� production in response to rhinovirus infection of pri-mary bronchial epithelial cells from asthmatic subjects (65). Itis interesting that if a similar deficiency were observed in M�from asthmatic subjects, then THP-1-derived M� could be agood model for M� from asthmatic subjects. Even if this is notthe case, we have provided evidence of limited rhinovirus rep-lication in MDM sufficient to induce NF-�B translocation andTNF-� production. These data suggest rhinovirus infection ofM� in vivo may be an important source of proinflammatorymediators in the context of exacerbations of respiratory dis-ease. The outcome of RV replication in primary macrophagesis similar to the results obtained for RSV and influenza virus.Both viruses can infect airway M�, but the release of virions isabsent or very limited (10, 51).
The differences seen in permissiveness to RV replicationand in type I IFN production between THP-1 M� and MDMwere not found when NF-�B translocation or TNF-� produc-tion was studied; indeed, TNF-� production occurred at levelsapproximately 100-fold higher in THP-1-derived M� com-pared with MDM. These differences imply that the signalingpathways leading to NF-�B activation and TNF-� productionare not affected in THP-1-derived M�, while specific pathwaysinvolved in type I IFN production and antiviral responses aredeficient in THP-1-derived M� compared with MDM.
NF-�B is important for cytokine production from RV-in-fected epithelial cells (32, 47, 67). During viral infections,NF-�B activation can be the result of several different events,including recognition of double-stranded or single-strandedRNA, viral enzymes, stress induced by viral entry and/or rep-lication (38). In the case of RV infection of epithelial cells,oxidative stress has been shown to induce early activation (48).Recognition of double-stranded RNA by protein kinase R hasbeen proposed as another possible mechanism leading to cy-tokine production, but there is no clear evidence at this timefor involvement protein kinase R in cytokine induction afterRV infection (15). Also, RV 3C protease was reported to beimportant in NF-�B activation and cytokine production fromRV-infected epithelial cells (13, 66). In monocytes/M�, tran-sient degradation of I�B was shown to take place and sug-gested to be important for MCP-1 production (21). In this
VOL. 80, 2006 RV REPLICATION INDUCES TNF-� PRODUCTION IN MACROPHAGES 8255
study we showed, using a minimal promoter, that NF-�B acti-vation occurs during RV infection of THP-1-derived M�. Be-cause only a minority of cells proved to support RV replica-tion, we assessed whether NF-�B activation occurred in virus-infected cells or in neighboring cells through paracrine effectsof an unknown mediator. RV 3C protease is a nonstructuralprotein produced either transiently by primary translation ofinput viral RNA or, to a greater degree and at later timepoints, by translation from viral RNA produced as a result ofreplication. Its expression at the levels and time points ob-served therefore constitutes evidence of replication. When RV3C protease was detected in THP-1-derived M� or MDM, itcolocalized with nuclear translocation of p65 (Fig. 3). This isthus the first study to show a direct colocalization betweenRV replication and p65 translocation, indicating that NF-�Bactivation at the time points studied occurs principally invirus-infected and not neighboring cells through paracrinemechanisms.
One of the most important and well-studied NF-�B-depen-dent genes is TNF-�, which has a very-well-established role ininflammation (11, 53). In the airway there are two major po-tential sources: mast cells as a source of preformed TNF-� andM� as a source of newly formed TNF-� (60). Respiratoryviruses are known to induce TNF-� release from M� (40, 43).Because only very low amounts, if any, are released from ep-ithelial cells after RV infection in vitro, it is logical to investi-gate whether M� could be a source during rhinovirus infectionof the lower airways (59). Here we show that infection of M�leads to sustained TNF-� production. This has been suggestedin a previous study; however, in that study TNF-� productionwas reported not to be associated with replication (14). UsingUV-inactivated RVs we showed that that TNF-� secretion isalmost completely dependent on viral replication. These find-ings are in agreement with previous studies which showedreplication to be important for efficient IL-8 production fromRV-infected monocytes (29).
Finally, we showed a clear requirement for NF-�B activationfor TNF-� to be released from RV-infected M� using twospecific chemical inhibitors of NF-�B. NF-�B has been re-ported to be required for induction of several inflammatorymediators induced in response to RV infection of epithelialcells, suggesting inhibition of NF-�B might have therapeuticpotential in treatment of virus-induced exacerbations ofasthma and COPD (32, 46, 47, 55, 67). The present data show-ing that NF-�B is also required for RV induction of TNF-� inM� reinforce the evidence that inhibition of NF-�B could be arewarding approach. At the highest specific doses used, IKK�inhibition was the most efficient approach, inhibiting TNF-�release by 95% in both model systems used (Fig. 6A and C),while inhibition of p65 translocation only partially blockedrelease (by �60%) (Fig. 6B). This may be because the IKK�inhibitor is either more specific or more potent that CAPE. Analternative explanation is that IKK� inhibition would be ex-pected to block activation of all forms of NF-�B, while p65inhibition would block only those species of NF-�B that in-cluded this protein subunit. These data suggest p50 ho-modimers or other Rel family proteins excluding p65 maycontribute to RV induction of TNF-� and that therapeuticapproaches based on NF-�B inhibition may be most successful
if they target upstream activation events that would inhibit allfamily members, rather than just p65.
Previous studies have implicated p38 MAPK in cytokine/chemokine production from RV-infected epithelial cells andmonocyte/M� (20, 21). We therefore also investigated its rolein TNF-� production in response to RV infection of M�. p38MAPK inhibition had no effect of TNF-� production, suggest-ing that although this pathway has been reported to be acti-vated during RV infection of M� and involved in induction ofMCP-1 (21), it does not appear to be required for induction ofTNF-�. However, the same concentration of p38 MAPK in-hibitor efficiently inhibited RSV-induced TNF-� production.Further studies will be required to increase our understandingof signaling pathways involved in induction of inflammatorycytokine production in response to RV infection of M�.
TNF-� has strong proinflammatory activities, and its inhibi-tion has therapeutic effects in a number of chronic inflamma-tory diseases (35, 50, 57, 61). Its role in allergic inflammationhas been recognized, and recent studies implicate it in severestable asthma and COPD (25, 31). There is less informationavailable relating to acute exacerbations, though preliminarystudies suggest it may be important (1, 9). Further studies arerequired to investigate its role in virus-induced asthma andCOPD exacerbations to help determine whether pharmacolog-ical inhibition or use of blocking antibodies/soluble receptorcould be considered as a possible therapeutic intervention inasthma and COPD exacerbations. The present data indicatingthat TNF-� release by M� during RV infection is stronglyinduced to high-nanogram levels suggest it is likely to play animportant role in the exacerbation process and, therefore, in-hibition is likely to be rewarding.
In conclusion, we have demonstrated that RV replicationoccurs in M�, is accompanied by NF-�B activation in virus-infected cells, and strongly induces TNF-� secretion. We alsoshowed that TNF-� secretion is mediated by NF-�B but notp38 MAPK. These studies suggest inhibition of both TNF-�and NF-�B may be useful in treatment of exacerbations ofasthma and COPD and indicate that further study on the roleof RV-induced macrophage activation in the pathogenesis ofthese conditions is required.
ACKNOWLEDGMENTS
This work was supported by an Asthma UK project grant awarded toS.L.J. and L.A.S. (grant number 02/027) and by British Lung Founda-tion/Severin Wunderman Family Foundation Lung Research Pro-gramme grant number P00/2.
REFERENCES
1. Aaron, S. D., J. B. Angel, M. Lunau, K. Wright, C. Fex, N. Le Saux, and R. E.Dales. 2001. Granulocyte inflammatory markers and airway infection duringacute exacerbation of chronic obstructive pulmonary disease. Am. J. Respir.Crit. Care Med. 163:349–355.
2. Akagawa, K. S. 2002. Functional heterogeneity of colony-stimulating factor-induced human monocyte-derived macrophages. Int. J. Hematol. 76:27–34.
3. Amineva, S. P., A. G. Aminev, A. C. Palmenberg, and J. E. Gern. 2004.Rhinovirus 3C protease precursors 3CD and 3CD localize to the nuclei ofinfected cells. J. Gen. Virol. 85:2969–2979.
4. Amrani, Y., H. Chen, and R. Panettieri. 2000. Activation of tumor necrosisfactor receptor 1 in airway smooth muscle: a potential pathway that modu-lates bronchial hyper-responsiveness in asthma? Respir. Res. 1:49–53.
5. Arruda, E., T. R. Boyle, B. Winther, D. C. Pevear, J. M. Gwaltney, Jr., andF. G. Hayden. 1995. Localization of human rhinovirus replication in theupper respiratory tract by in situ hybridization. J. Infect. Dis. 171:1329–1333.
6. Asseffa, A. 1993. Phorbol myristate acetate-differentiated THP-1 cells displayincreased levels of MHC class I and class II messenger RNA and interferongamma-inducible tumoricidal activity. Oncol. Res. 5:11–18.
7. Auwerx, J. 1991. The human leukemia-cell line, THP-1—a multifacetedmodel for the study of monocyte-macrophage differentiation. Experientia47:22–31.
8. Bardin, P., S. Johnston, G. Sanderson, B. Robinson, M. Pickett, D. Fraenkel,and S. Holgate. 1994. Detection of rhinovirus infection of the nasal mucosaby oligonucleotide in situ hybridization. Am. J. Respir. Cell Mol. Biol.10:207–213.
9. Calhoun, W. J., E. C. Dick, L. B. Schwartz, and W. W. Busse. 1994. Acommon cold virus, rhinovirus 16, potentiates airway inflammation aftersegmental antigen bronchoprovocation in allergic subjects. J. Clin. Investig.94:2200–2208.
10. Cirino, N. M., J. R. Panuska, A. Villani, H. Taraf, N. A. Rebert, R. Merolla,P. Tsivitse, and I. A. Gilbert. 1993. Restricted replication of respiratorysyncytial virus in human alveolar macrophages. J. Gen. Virol. 74:1527–1537.
11. Drouet, C., A. Shakhov, and C. Jongeneel. 1991. Enhancers and transcriptionfactors controlling the inducibility of the tumor necrosis factor-alpha pro-moter in primary macrophages. J. Immunol. 147:1694–1700.
12. Frelin, C., V. Imbert, E. Griessinger, A. Loubat, M. Dreano, and J. F.Peyron. 2003. AS602868, a pharmacological inhibitor of IKK2, reveals theapoptotic potential of TNF-alpha in Jurkat leukemic cells. Oncogene 22:8187–8194.
13. Funkhouser, A. W., J. A. Kang, A. Tan, J. Li, L. Zhou, M. K. Abe, J. Solway,and M. B. Hershenson. 2004. Rhinovirus 16 3C protease induces interleu-kin-8 and granulocyte-macrophage colony-stimulating factor expression inhuman bronchial epithelial cells. Pediatr. Res. 55:13–18.
14. Gern, J. E., E. C. Dick, W. M. Lee, S. Murray, K. Meyer, Z. T. Handzel, andW. W. Busse. 1996. Rhinovirus enters but does not replicate inside mono-cytes and airway macrophages. J. Immunol. 156:621–627.
15. Gern, J. E., D. A. French, K. A. Grindle, R. A. Brockman-Schneider, S.-I.Konno, and W. W. Busse. 2003. Double-stranded RNA induces the synthesisof specific chemokines by bronchial epithelial cells. Am. J. Respir. Cell Mol.Biol. 28:731–737.
16. Gern, J. E., R. Vrtis, E. A. Kelly, E. C. Dick, and W. W. Busse. 1996.Rhinovirus produces nonspecific activation of lymphocytes through a mono-cyte-dependent mechanism. J. Immunol. 157:1605–1612.
17. Gliemann, J. 1998. Receptors of the low density lipoprotein (LDL) receptorfamily in man. Multiple functions of the large family members via interactionwith complex ligands. Biol. Chem. 379:951–964.
18. Gordon, S. B., and R. C. Read. 2002. Macrophage defences against respira-tory tract infections: the immunology of childhood respiratory infections. Br.Med. Bull. 61:45–61.
19. Greenberg, S. B., M. Allen, J. Wilson, and R. L. Atmar. 2000. Respiratoryviral infections in adults with and without chronic obstructive pulmonarydisease. Am. J. Respir. Crit. Care Med. 162:167–173.
20. Griego, S. D., C. B. Weston, J. L. Adams, R. Tal-Singer, and S. B. Dillon.2000. Role of p38 mitogen-activated protein kinase in rhinovirus-inducedcytokine production by bronchial epithelial cells. J. Immunol. 165:5211–5220.
21. Hall, D. J., M. E. Bates, L. Guar, M. Cronan, N. Korpi, and P. J. Bertics.2005. The role of p38 MAPK in rhinovirus-induced monocyte chemoattrac-tant protein-1 production by monocytic-lineage cells. J. Immunol. 174:8056–8063.
22. Hart, P., C. Bonder, J. Balogh, H. Dickensheets, R. Donnelly, and J. Finlay-Jones. 1999. Differential responses of human monocytes and macrophages toIL-4 and IL-13. J. Leukoc. Biol. 66:575–578.
23. Hayden, F. G., R. B. Turner, J. M. Gwaltney, K. Chi-Burris, M. Gersten, P.Hsyu, A. K. Patick, G. J. Smith III, and L. S. Zalman. 2003. Phase II,randomized, double-blind, placebo-controlled studies of ruprintrivir nasalspray 2-percent suspension for prevention and treatment of experimentallyinduced rhinovirus colds in healthy volunteers. Antimicrob. Agents Che-mother. 47:3907–3916.
24. Hewson, C. A., M. R. Edbrooke, and S. L. Johnston. 2004. PMA induces theMUC5AC respiratory mucin in human bronchial epithelial cells, via PKC,EGF/TGF-alpha, Ras/Raf, MEK, ERK and Sp1-dependent mechanisms. J.Mol. Biol. 344:683–695.
25. Howarth, P. H., K. S. Babu, H. S. Arshad, L. C. Lau, M. G. Buckley, W.McConnell, P. Beckett, M. A. Ali, A. Chauhan, S. J. Wilson, A. Reynolds,D. E. Davies, and S. T. Holgate. 2005. Tumour necrosis factor (TNF-�) as anovel therapeutic target in symptomatic corticosteroid-dependent asthma.Thorax 60:1012–1018.
26. Hume, D. A., I. L. Ross, S. R. Himes, R. T. Sasmono, C. A. Wells, and T.Ravasi. 2002. The mononuclear phagocyte system revisited. J. Leukoc. Biol.72:621–627.
27. Iwamura, T., M. Yoneyama, K. Yamaguchi, W. Suhara, W. Mori, K. Shiota,Y. Okabe, H. Namiki, and T. Fujita. 2001. Induction of IRF-3/-7 kinase andNF-�B in response to double-stranded RNA and virus infection: commonand unique pathways. Genes Cells 6:375–388.
28. Johnston, S. L., A. Papi, P. J. Bates, J. G. Mastronarde, M. M. Monick, andG. W. Hunninghake. 1998. Low grade rhinovirus infection induces aprolonged release of IL-8 in pulmonary epithelium. J. Immunol. 160:6172–6181.
29. Johnston, S. L., A. Papi, M. M. Monick, and G. W. Hunninghake. 1997.Rhinoviruses induce interleukin-8 mRNA and protein production in humanmonocytes. J. Infect. Dis. 175:323–329.
30. Johnston, S. L., P. K. Pattemore, G. Sanderson, S. Smith, F. Lampe, L.Josephs, P. Symington, S. O’Toole, S. H. Myint, D. A. Tyrrell, et al. 1995.Community study of role of viral infections in exacerbations of asthma in9–11 year old children. BMJ 310:1225–1229.
31. Keatings, V. M., P. D. Collins, D. M. Scott, and P. J. Barnes. 1996. Differ-ences in interleukin-8 and tumor necrosis factor-alpha in induced sputumfrom patients with chronic obstructive pulmonary disease or asthma. Am. J.Respir. Crit. Care Med. 153:530–534.
32. Kim, J., S. P. Sanders, E. S. Siekierski, V. Casolaro, and D. Proud. 2000.Role of NF-kappa B in cytokine production induced from human airwayepithelial cells by rhinovirus infection. J. Immunol. 165:3384–3392.
33. Koch, A., M. Giembycz, K. Ito, S. Lim, E. Jazrawi, P. J. Barnes, I. Adcock,E. Erdmann, and K. F. Chung. 2004. Mitogen-activated protein kinasemodulation of nuclear factor-�B-induced granulocyte macrophage-colony-stimulating factor release from human alveolar macrophages. Am. J. Respir.Cell Mol. Biol. 30:342–349.
34. Laskin, D. L., B. Weinberger, and J. D. Laskin. 2001. Functional heteroge-neity in liver and lung macrophages. J. Leukoc. Biol. 70:163–170.
35. Maini, R. N., and P. C. Taylor. 2000. Anti-cytokine therapy for rheumatoidarthritis. Annu. Rev. Med. 51:207–229.
36. Mantovani, A., S. Sozzani, M. Locati, P. Allavena, and A. Sica. 2002. Mac-rophage polarization: tumor-associated macrophages as a paradigm for po-larized M2 mononuclear phagocytes. Trends Immunol. 23:549–555.
37. Message, S. D., and S. L. Johnston. 2004. Host defense function of theairway epithelium in health and disease: clinical background. J. Leukoc. Biol.75:5–17.
38. Mogensen, T. H., and S. R. Paludan. 2001. Molecular pathways in virus-induced cytokine production. Microbiol. Mol. Biol. Rev. 65:131–150.
39. Mosser, A. G., R. Brockman-Schneider, S. Amineva, L. Burchell, J. B. Sedgwick,W. W. Busse, and J. E. Gern. 2002. Similar frequency of rhinovirus-infectiblecells in upper and lower airway epithelium. J. Infect. Dis. 185:734–743.
40. Nain, M., F. Hinder, J. Gong, A. Schmidt, A. Bender, H. Sprenger, and D.Gemsa. 1990. Tumor necrosis factor-alpha production of influenza A virus-infected macrophages and potentiating effect of lipopolysaccharides. J. Im-munol. 145:1921–1928.
41. Natarajan, K., S. Singh, T. R. Burke, Jr., D. Grunberger, and B. B. Aggarwal.1996. Caffeic acid phenethyl ester is a potent and specific inhibitor of acti-vation of nuclear transcription factor NF-kappa B. Proc. Natl. Acad. Sci.USA 93:9090–9095.
42. Nicholson, K. G., J. Kent, and D. C. Ireland. 1993. Respiratory viruses andexacerbations of asthma in adults. BMJ 307:982–986.
43. Panuska, J. R., R. Merolla, N. A. Rebert, S. P. Hoffmann, P. Tsivitse, N. M.Cirino, R. H. Silverman, and J. A. Rankin. 1995. Respiratory syncytial virusinduces interleukin-10 by human alveolar macrophages. Suppression of earlycytokine production and implications for incomplete immunity. J. Clin. In-vestig. 96:2445–2453.
44. Papadopoulos, N. G., L. A. Stanciu, A. Papi, S. T. Holgate, and S. L.Johnston. 2002. A defective type 1 response to rhinovirus in atopic asthma.Thorax 57:328–332.
45. Papadopoulos, N. G., L. A. Stanciu, A. Papi, S. T. Holgate, and S. L.Johnston. 2002. Rhinovirus-induced alterations on peripheral blood mono-nuclear cell phenotype and costimulatory molecule expression in normal andatopic asthmatic subjects. Clin. Exp. Allergy 32:537–542.
46. Papi, A., and S. L. Johnston. 1999. Respiratory epithelial cell expression ofvascular cell adhesion molecule-1 and its up-regulation by rhinovirus infec-tion via NF-�B and GATA transcription factors. J. Biol. Chem. 274:30041–30051.
47. Papi, A., and S. L. Johnston. 1999. Rhinovirus infection induces expressionof its own receptor intercellular adhesion molecule 1 (ICAM-1) via increasedNF-�B-mediated transcription. J. Biol. Chem. 274:9707–9720.
48. Papi, A., N. G. Papadopoulos, L. A. Stanciu, C. M. Bellettato, S. Pinamonti,K. Degitz, S. T. Holgate, and S. L. Johnston. 2002. Reducing agents inhibitrhinovirus-induced up-regulation of the rhinovirus receptor intercellular ad-hesion molecule-1 (ICAM-1) in respiratory epithelial cells. FASEB J. 16:1934–1936.
49. Pirhonen, J., T. Sareneva, M. Kurimoto, I. Julkunen, and S. Matikainen.1999. Virus infection activates IL-1� and IL-18 production in human mac-rophages by a caspase-1-dependent pathway. J. Immunol. 162:7322–7329.
50. Roberts, S. D., D. S. Wilkes, R. A. Burgett, and K. S. Knox. 2003. Refractorysarcoidosis responding to infliximab. Chest 124:2028–2031.
51. Rodgers, B. C., and C. A. Mims. 1982. Influenza virus replication in humanalveolar macrophages. J. Med. Virol. 9:177–184.
52. Schroth, M. K., E. Grimm, P. Frindt, D. M. Galagan, S. I. Konno, R. Love,and J. E. Gern. 1999. Rhinovirus replication causes RANTES production inprimary bronchial epithelial cells. Am. J. Respir. Cell Mol. Biol. 20:1220–1228.
53. Shakhov, A., M. Collart, P. Vassalli, S. Nedospasov, and C. Jongeneel. 1990.Kappa B-type enhancers are involved in lipopolysaccharide-mediated tran-
VOL. 80, 2006 RV REPLICATION INDUCES TNF-� PRODUCTION IN MACROPHAGES 8257
scriptional activation of the tumor necrosis factor alpha gene in primarymacrophages. J. Exp. Med. 171:35–47.
54. Shin-Ru Shih, S.-J. C., G. H. Hakimelahi, H.-J. Liu, C.-T. Tseng, and K.-S.Shia. 2004. Selective human enterovirus and rhinovirus inhibitors: an over-view of capsid-binding and protease-inhibiting molecules. Med. Res. Rev.24:449–474.
55. Spurrell, J. C., S. Wiehler, R. S. Zaheer, S. P. Sanders, and D. Proud. 2005.Human airway epithelial cells produce IP-10 (CXCL10) in vitro and in vivoupon rhinovirus infection. Am. J. Physiol. Lung Cell Mol. Physiol. 289:L85–L95.
56. Stockl, J., H. Vetr, O. Majdic, G. Zlabinger, E. Kuechler, and W. Knapp.1999. Human major group rhinoviruses downmodulate the accessory func-tion of monocytes by inducing IL-10. J. Clin. Investig. 104:957–965.
57. Stokkers, P. C. F., and D. W. Hommes. 2004. New cytokine therapeutics forinflammatory bowel disease. Cytokine 28:167–173.
58. Takashiba, S., T. E. Van Dyke, S. Amar, Y. Murayama, A. W. Soskolne, andL. Shapira. 1999. Differentiation of monocytes to macrophages primes cellsfor lipopolysaccharide stimulation via accumulation of cytoplasmic nuclearfactor kappa B. Infect. Immun. 67:5573–5578.
59. Terajima, M., M. Yamaya, K. Sekizawa, S. Okinaga, T. Suzuki, N. Yamada,K. Nakayama, T. Ohrui, T. Oshima, Y. Numazaki, and H. Sasaki. 1997.Rhinovirus infection of primary cultures of human tracheal epithelium: roleof ICAM-1 and IL-1�. Am. J. Physiol. 273:L749–L759.
60. Thomas, P. S. 2001. Tumour necrosis factor alpha: the role of this multi-functional cytokine in asthma. Immunol. Cell Biol. 79:132–140.
61. Tobin, A. M., and B. Kirby. 2005. TNF alpha inhibitors in the treatment ofpsoriasis and psoriatic arthritis. BioDrugs 19:47–57.
62. Tracey, K. J., and A. Cerami. 1993. Tumor necrosis factor, other cytokinesand disease. Annu. Rev. Cell Biol. 9:317–343.
63. Tsuchiya, S., Y. Kobayashi, Y. Goto, H. Okumura, S. Nakae, T. Konno, andK. Tada. 1982. Induction of maturation in cultured human monocytic leukemiacells by a phorbol diester. Cancer Res. 42:1530–1536.
64. van Furth, R. 1992. Mononuclear phagocytes, p. 3–12. Kluwer AcademicPublishers, Dordrecht, The Netherlands.
65. Wark, P. A., S. L. Johnston, F. Bucchieri, R. Powell, S. Puddicombe, V.Laza-Stanca, S. T. Holgate, and D. E. Davies. 2005. Asthmatic bronchialepithelial cells have a deficient innate immune response to infection withrhinovirus. J. Exp. Med. 201:937–947.
66. Zalman, L. S., M. A. Brothers, P. S. Dragovich, R. Zhou, T. J. Prins, S. T.Worland, and A. K. Patick. 2000. Inhibition of human rhinovirus-inducedcytokine production by AG7088, a human rhinovirus 3C protease inhibitor.Antimicrob. Agents Chemother. 44:1236–1241.
67. Zhu, Z., W. Tang, A. Ray, Y. Wu, O. Einarsson, M. L. Landry, J. Gwaltney,Jr., and J. A. Elias. 1996. Rhinovirus stimulation of interleukin-6 in vivo andin vitro. Evidence for nuclear factor kappa B-dependent transcriptionalactivation. J. Clin. Investig. 97:421–430.
