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Originally published online as doi:10.1189/jlb.0606412 on October 4, 2006

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(Journal of Leukocyte Biology. 2006;80:1364-1374.)
© 2006 by Society for Leukocyte Biology

Human rhinovirus induces robust IP-10 release by monocytic cells, which is independent of viral replication but linked to type I interferon receptor ligation and STAT1 activation

Nichole L. Korpi-Steiner*, Mary Ellen Bates*, Wai-Ming Lee{dagger}, David J. Hall{ddagger} and Paul J. Bertics*,1

Departments of
* Biomolecular Chemistry and
{dagger} Pediatrics, University Wisconsin-Madison, Madison, Wisconsin, USA; and
{ddagger} Department of Chemistry, Lawrence University, Appleton, Wisconsin, USA

1 Correspondence: Department of Biomolecular Chemistry, University of Wisconsin-Madison, 1300 University Avenue, Madison, WI 53706-0450, USA. E-mail: pbertics{at}wisc.edu


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ABSTRACT
 
Human rhinovirus (HRV)-induced respiratory infections are associated with elevated levels of IFN-{gamma}-inducible protein 10 (IP-10), which is an enhancer of T lymphocyte chemotaxis and correlates with symptom severity and T lymphocyte number. Increased IP-10 expression is exhibited by airway epithelial cells following ex vivo HRV challenge and requires intracellular viral replication; however, there are conflicting reports regarding the necessity of type I IFN receptor ligation for IP-10 expression. Furthermore, the involvement of resident airway immune cells, predominantly bronchoalveolar macrophages, in contributing to HRV-stimulated IP-10 elaboration remains unclear. In this regard, our findings demonstrate that ex vivo exposure of human peripheral blood monocytes and bronchoalveolar macrophages (monocytic cells) to native or replication-defective HRV serotype 16 (HRV16) resulted in similarly robust levels of IP-10 release, which occurred in a time- and dose-dependent manner. Furthermore, HRV16 induced a significant increase in type I IFN (IFN-{alpha}) release and STAT1 phosphorylation in monocytes. Neutralization of the type I IFN receptor and inhibition of JAK or p38 kinase activity strongly attenuated HRV16-stimulated STAT1 phosphorylation and IP-10 release. Thus, this work supports a model, wherein HRV16-induced IP-10 release by monocytic cells is modulated via autocrine/paracrine action of type I IFNs and subsequent JAK/STAT pathway activity. Our findings demonstrating robust activation of monocytic cells in response to native and/or replication-defective HRV16 challenge represent the first evidence indicating a mechanistic disparity in the activation of macrophages when compared with epithelial cells and suggest that macrophages likely contribute to cytokine elaboration following HRV challenge in vivo.

Key Words: monocyte/macrophage • signal transduction • inflammation


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INTRODUCTION
 
Human rhinovirus (HRV) is a primary cause of the common cold and is the virus most frequently detected in children and adults exhibiting exacerbations of asthma [1 ]. Despite the frequency of HRV-associated respiratory disease, its pathogenesis and the intracellular/immunologic mechanisms that link HRV illness to common cold symptoms and asthma exacerbations are understood incompletely [2 ].

Several reports suggest that the pathology induced by HRV respiratory infections and allergic airway inflammation in asthma is a consequence of host defense and inflammatory responses including cytokine elaboration and the recruitment of select immune cells [3 4 5 6 ]. Airway inflammation in asthma is typified by the presence of Th2 lymphocytes and elaboration of cytokines and chemokines associated with T lymphocyte recruitment and activation [3 , 5 ]. However, the inflammatory response in asthma is multifaceted, involving many cell types and inflammatory mediators, including cytokines characteristic of Th1 inflammation [3 ]. In this regard, a Th1-associated chemokine, IFN-{gamma}-inducible protein-10 (IP-10; CXCL10), a CXCR3 ligand that selectively recruits activated Th1 lymphocytes and NK cells, has been implicated recently in modulating airway inflammation in asthma [7 , 8 ]. It has been proposed that IP-10 contributes to airway inflammation and airway hyper-responsiveness in a murine model of asthma [4 ]. Consistent with these findings, it has been reported that human bronchial epithelium and submucosa from asthmatic individuals have significantly greater detectable levels of IP-10 mRNA as compared with normal, control individuals [6 ]. Moreover, submucosal macrophages and neutrophils are the prominent sources of IP-10 mRNA and express significantly higher levels of IP-10 mRNA in asthmatics as compared with control individuals. Furthermore, segmental bronchoprovocation of allergic-asthmatic individuals with allergen results in increased IP-10 levels and T lymphocyte numbers in bronchoalveolar lavage (BAL) fluid, and there is a direct correlation between airway T lymphocyte numbers and IP-10 levels [9 ]. Similarly, individuals challenged with HRV16 intranasally have significantly greater levels of IP-10 protein in nasal lavage fluid, which correlates positively with viral titer and T lymphocyte number in nasal secretions as well as respiratory symptom severity [7 ]. Taken together, it is clear that IP-10 is associated with airway inflammation and lymphocytic infiltration exhibited with asthma and rhinovirus infection; however, the functional role of IP-10 in asthma and the cellular source and intracellular regulation of IP-10 production in response to inflammatory stimuli, specifically HRV, are not well-defined.

Monocytic cells (monocytes and macrophages) are the predominant immune cells present in the lumen of the lower airway, and activation of these cells represents one of the first steps in natural immunity toward virus infection [10 , 11 ]. The ability of HRV to infect upper and lower airway epithelium with similar efficacy thereby suggests that monocytic cells likely have direct interaction with HRV in vivo [12 ]. In addition, given the close association of augmented inflammation of the lower airways and worsening of asthma, HRV interaction with lower airway macrophages is likely relevant to asthma exacerbations [12 13 14 ]. Previous studies from our laboratory and others demonstrate that monocytic cells and epithelial cells express the major group HRV attachment molecule ICAM-1 and respond to in vitro HRV challenge with the release of a numerous cytokines [10 , 15 16 17 ]. Although recent reports have demonstrated that HRV replication is required for the induction of IP-10 expression by primary epithelial cells, it remains unclear if HRV is capable of promoting IP-10 expression in monocytic cells, which do not sustain a productive infection with HRV [7 ].

Expression of the IP-10 gene is regulated by several putative transcription factors including IFN-stimulated response element, STAT, C/EBP-β, NF-{kappa}B, and AP-1 [7 ]. Two recent studies using epithelial cells examined the involvement of intermediate production of type I IFNs, well-known activators of STAT factors, in modulating HRV-induced IP-10 expression; however, conflicting results linking type I IFN receptor ligation to HRV-stimulated IP-10 expression have been reported [7 , 18 ]. Therefore, a better understanding of the intracellular signaling molecules modulating HRV-induced IP-10 expression in airway cells and the role of monocytic cells in IP-10 elaboration in response to HRV challenge is needed. Accordingly, this study addressed the hypotheses that HRV can induce IP-10 expression by monocytes/macrophages, that intracellular HRV16 replication is not required for IP-10 protein expression in these cells, and that HRV16-stimulated IP-10 release is modulated by type I IFN receptor/JAK/STAT pathway activity. Our findings demonstrate for the first time that exposure of primary monocytic cells to native or replication-defective HRV results in robust IP-10 release that is similar to the level of IP-10 release previously reported by HRV-stimulated epithelial cells [7 ]. Furthermore, this work supports a model, wherein HRV16-induced IP-10 release by monocytic cells is modulated via autocrine/paracrine action of type I IFNs and subsequent JAK/STAT pathway activity.


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MATERIALS AND METHODS
 
Reagents
RosetteSep human monocyte enrichment cocktail was purchased from StemCell Technologies Inc. (Vancouver, BC, Canada). Antibodies were purchased from the following sources: anti-ERK1/2 and anti-STAT1 (pY701; Upstate Biotechnology, Lake Placid, NY), anti-CD54 (anti-ICAM-1; Ancell, Bayport, MN), anti-CD118 (anti-IFN receptor type I; Endogen/Pierce, Rockford, IL), anti-Grb2 (Santa Cruz Biotechnology, CA), and anti-CD14 (Becton Dickinson, San Jose, CA). Isotype controls IgG1{kappa} and IgG2a{kappa} were obtained from Sigma Chemical Co. (St. Louis, MO). The compound WIN52035 was obtained from Sterling Drug Inc. (Rensselaer, NY). JAK inhibitor I (JAK-I), SB203580, and SB202474 were purchased from Calbiochem (La Jolla, CA). Recombinant human (rh)IFN-{gamma} was purchased from PeproTech Inc. (Rocky Hill, NJ), and rhIFN-{alpha}2b (Intron A) was obtained from Schering (Kenilworth, NJ). Anisomycin was purchased from Sigma Chemical Co.

Cell culture
Human alveolar macrophages were obtained from atopic donors undergoing BAL at the University of Wisconsin Hospital and Clinics (Madison). Human PBMC were obtained from atopic or nonatopic donors at the University of Wisconsin Hospital Clinics, and peripheral blood monocytes were isolated from the PBMC before plating. The Human Subjects Committee of the University of Wisconsin Hospital approved the experimental protocols. Isolated human BAL macrophages and human peripheral blood monocytes were resuspended in RPMI-1640 medium, supplemented with 10% FBS (Mediatech, Herndon, VA), 2 mM sodium pyruvate, 2 mM L-glutamine, and 100 U/ml penicillin/streptomycin (complete medium), and plated in 24-well tissue-culture plates (Corning Inc./Costar, Acton, MA) at a density of 1 x 106 cells/mL (0.5 mL/well) or 12-well tissue-culture plates at a density of 2 x 106 cells/mL (1 mL/well). Following a 2-h incubation, cells were rinsed two times with HBSS (Mediatech) containing 2% heat-inactivated bovine calf serum (HBSS-BCS), and media were replaced with RPMI-1640 medium supplemented with 1% FBS for immunoblotting experiments or complete medium for experiments examining cytokine elaboration. Cells were incubated overnight at 37°C in a humidified atmosphere with 5% CO2.

Isolation of alveolar macrophages
Bronchoscopy and BAL were conducted as described [19 ]. Briefly, in unchallenged, atopic individuals, two bronchopulmonary segments were identified with a fiberoptic bronchoscope, and lavage was performed with 160 mL 0.9% saline in each bronchopulmonary segment. The BAL fluid recovered from the bronchopulmonary segments was combined and filtered through mesh, and the BAL cells were isolated by centrifugation (400 g for 10 min at 4°C). The BAL cells were washed twice with HBSS supplemented with 2% newborn calf serum (400 g for 10 min at 4°C), and the resulting cell population was evaluated by differential morphological analysis (Diff-Quick Scientific Products, McGaw Park, IN). Morphological examination indicated that typical BAL cell populations were primarily comprised of macrophages (87%), and the remaining cells were primarily lymphocytes (7%), neutrophils (1%), and epithelial cells (2%).

Purification of human monocytes
Heparinized whole blood was obtained from volunteer donors at the University of Wisconsin Hospital and enriched for PBMC via centrifugation (700 g) through a Percoll monolayer (1.090 g/mL Percoll) for 20 min at room temperature (RT). A suspension of erythrocytes and leukocytes (RBC) was also obtained below the Percoll monolayer following centrifugation. Platelets were reduced by subjecting PBMC to three consecutive washes (350 g, 250 g, 150 g for 12 min at RT) in HBSS-BCS. The RBC suspension (0.5 mL) and 45 mL HBSS-BCS were added to 108 PBMC, followed by centrifugation at 250 g for 12 min at RT. The cell pellet was resuspended in 1 mL HBSS-BCS and incubated with 150 µL RosetteSep human monocyte enrichment cocktail for 20 min at RT. The cells were enriched for monocytes by centrifugation at RT over lymphocyte separation medium (Mediatech) for 20 min at 1400 g and washed by centrifuging at RT with HBSS-BCS for 10 min at 350 g. The monocyte pellet was resuspended in complete medium and plated in tissue-culture plates as denoted above. The resulting cell population consisting of adherent and nonadherent monocytes was 85–90% CD14+ (n=4) as determined by flow cytometry.

Production and purification of HRV16, neutral-red (NR)-inactivated HRV16, and 35S-labeled HRV16
Human RV16 was grown in HeLa cells as described previously; in addition, NR-containing HRV16 and 35S-labeled HRV16 were grown in HeLa cells, whereupon the tissue-culture medium was supplemented with 10 µg/mL NR dye at 30 min postinfection and/or 35S-methionine (1 mCi per 4x108 cells; Amersham, Piscataway, NJ) at 3.25 h postinfection, respectively [20 21 22 ]. Infected HeLa cells were harvested 8 h postinfection, and virions were purified from the infected cell lysate by centrifugation through a sucrose cushion and subsequently sedimented through a sucrose-step gradient to remove contaminants as described previously [20 , 21 ]. The yield of the HRV16 particles was measured optically, wherein an OD260 unit correlates with 0.133 µg (9.4x1012 virions) virion protein [21 ]. To inactivate NR-containing HRV16, the viral RNA genome was fragmented by exposure to a 15-W white fluorescent light located 3 inches above the purified viral suspension for 15 min. The multiplicity of infection (MOI), which refers to the number of infectious HRV particles per cell (typically one infectious HRV particle per 300 total particles), was determined by measuring the infectivity of the virions in HeLa cell monolayers as described elsewhere [21 ]. The NR-containing virions exhibited over 99% reduction in infectivity following 15 min of white fluorescent light illumination.

HRV16 treatment with WIN52035 or low pH conditions
Sucrose-purified HRV16 was treated with WIN52035 (20 µg/mL) in RPMI 1640 at RT with agitation for 1 h and added to cells at a final concentration of 2 µg/mL WIN52035. Sucrose-purified HRV16 was treated with low pH (pH<4) in RPMI 1640 at RT with agitation for 30 min with subsequent pH neutralization (pH=7–7.5) for 2 min prior to stimulation of cells. No viral infectivity in HeLa cells could be detected following WIN52035 or low pH treatment of HRV16.

HRV16 and NR-inactivated HRV16 attachment assay
Human peripheral blood monocytes were resuspended in Dulbecco’s PBS (DPBS; Cambrex Bio Science, Walkersville, MD) and aliquoted into four 0.1% BSA-coated Eppendorf tubes at a cell density of 1 x 107 monocytes/250 µL. Appropriate cell suspensions were treated with anti-ICAM-1 antibody (30 µg/mL) or isotype control (IgG1{kappa}; 30 µg/mL) for 30 min at 4°C. Following incubation, the monocytes were sedimented by centrifugation at 500 g for 5 min at 4°C, and the supernatants were discarded. The monocytes were resuspended with 250 µL DPBS, supplemented with 0.1% BSA and 35S-labeled HRV16 (1.6x108 virions/µL) or 35S-labeled, NR-inactivated HRV16 (1.6x108 virions/µL), and incubated for 1 h at RT with agitation to allow virus attachment. Following incubation, the monocytes were sedimented by centrifugation at 500 g for 5 min at 4°C. The cells were next subjected to three consecutive washes (500 g for 5 min at 4°C) using DPBS supplemented with 0.1% BSA. Cells were lysed in DPBS containing 0.2% SDS and 0.05% NaOH. Input 35S-labeled virus and cell lysates (in triplicate) were measured for 35S cpm using a liquid scintillation analyzer (Packard, Meriden, CT). The number of cell-associated virions was determined by normalizing the total cpm/sample to specific activity (cpm/virion). The number of virions/cell was quantified by normalizing the number of cell-associated virions to the number of cells used {virions/cell=[(total cpm/sample)/(cpm/virion)]/1 x 107 monocytes}.

Immunoblotting
Peripheral monocytes and BAL macrophages were lysed with SDS sample buffer (75 µL SDS sample buffer per 2x106 cells), and protein concentrations for each sample were quantified using bicinchoninic acid protein assay (Endogen/Pierce). Equivalent amounts of protein for each sample were resolved on SDS-PAGE gels and transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA). The membranes were blocked in 5% milk in TBST (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.05% Tween 20), and immunoblotting was performed according to the manufacturer’s protocols. The immunoreactive bands were visualized using SuperSignal West Pico chemiluminescent substrate (Endogen/Pierce) and Epichemi II darkroom (UVP, Upland, CA) equipped with a 12-bit cooled charged-coupled device camera.

Detection of IP-10/IFN-{alpha} protein release by monocytic cells
Peripheral monocytes and BAL macrophages were plated in 24-well plates as described above. Following overnight incubation, cells were pretreated with or without neutralizing antibodies as described in figure legends and subsequently stimulated with control vehicle, HRV16, IFN-{alpha}, or IFN-{gamma} for 24 h at 37°C. Following incubation, cell supernatants were harvested and frozen at –80°C until analysis. The protein levels of IP-10 and/or IFN-{alpha} were measured using commercial sandwich ELISA kits according to the manufacturer’s protocols (R& D Systems, Minneapolis, MN, and Endogen/Pierce, respectively).

Cell viability assay
Isolated human peripheral blood monocytes were resuspended in complete medium and plated in 96-well tissue-culture plates at a density of 1 x 106 cells/mL (0.1 mL/well). Cells were pretreated with blocking/isotype antibodies and stimulated with control vehicle or treated/untreated virus for 24 h at 37°C. Following incubation, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) and the electron-coupling reagent phenazine methosulfate were added to monocytes according to the manufacturer’s protocol (Promega, Madison, WI), and the mixtures were incubated for 1 h at 37°C in a humidified atmosphere with 5% CO2. During this incubation period, MTS is converted to aqueous, soluble formazan by endogenous dehydrogenase enzymes; thus, the quantity of formazan product, as measured by the amount of absorbance at 490 nm using an ELx800 ELISA microplate reader (Bio-Tek Instruments Inc., Winooski, VT), is directly proportional to the number of metabolically active/viable cells.


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RESULTS
 
HRV16 induces IP-10 protein release by monocytic cells in a time- and dose-dependent manner
Preliminary analysis of gene expression by human peripheral blood monocytes stimulated with HRV16 (MOI=10) or control vehicle for 4 h was conducted using oligonucleotide mircroarrays (Affymetrix HuGeneFL GeneChip). We observed that HRV16 stimulated an increase in several chemokine transcripts, and the most robust responses (>30-fold) were seen for IP-10, MCP-1, MCP-2, and IFN-inducible T cell-{alpha} chemoattractant, whereas smaller responses (three- to 16-fold) were noted for IL-8, MCP-3, and MIP-1 (data not shown). These data suggest that monocytic cells are active participants in promoting host chemokine elaboration following an in vivo HRV challenge, and given the importance of IP-10 to the recruitment of lymphocytes and NK cells, we tested the concept that monocytes/macrophages respond to HRV16 challenge with the production/release of IP-10 protein. In this regard, Figure 1 reveals that primary BAL macrophage exposure to HRV16 does result in robust IP-10 protein release (Fig. 1A ; HRV16=26.7±4.3 ng/mL vs. control vehicle=0.32±0.22 ng/mL; P=0.003). Human peripheral blood monocytes were also examined for IP-10 release following HRV16 (MOI=10) challenge at various time intervals, which likely correspond to HRV16 exposure in vivo (Fig. 1B) . Increased IP-10 release from monocytes was detected 8 h post-HRV16 infection. Monocyte exposure to HRV16 for 24 h resulted in robust IP-10 release (9.95±1.8 ng/mL; control IP-10 release ≤0.014±0.007 ng/mL), which remained throughout 72 h. Peak levels of IP-10 release (14.3±2.0 ng/mL) by monocytes were detected 48 h post-HRV16 challenge, indicating a time-dependent release of IP-10 by human monocytes (P<0.0001). Similarly, Spurrell et al. [7 ] saw peak levels of IP-10 release by human airway epithelial cells at 48 h post-HRV16 infection. It is noteworthy that the levels of IP-10 release by monocytes (14.3±2.0 ng/mL) and macrophages (26.7±4.3 ng/mL) following HRV16 inoculation are similar to that exhibited by HRV16-stimulated epithelial cells (15.6±5.1 ng/mL [7 ]). Although the monocytes used here were obtained from 10 different individuals, including five allergic-asthmatic and five nonatopic patients, no significant difference in HRV16-induced IP-10 release was detected between the allergic-asthmatic and nonatopic donors (P=0.29).


Figure 1
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  		Figure 1. IP-10 secretion from HRV16-inoculated monocytic cells. Human BAL macrophages or peripheral blood monocytes were stimulated with control vehicle or HRV16 (MOI=10) for the times indicated. (A) The induction of IP-10 protein release from BAL macrophages following HRV16 challenge for 24 h (P=0.003 by paired t-test). Data represent mean ± SEM from three independent experiments. (B) A time course of IP-10 protein release from monocytes following exposure to HRV16 (P<0.0001 by repeated-measure ANOVA analysis). Data represent mean ± SEM from 10 independent experiments. {phi}, Control levels of IP-10 release by monocytes were at or below the minimum level of detection (12.5 pg/mL) of the IP-10 ELISA assay.

To assess if HRV16-induced IP-10 release by monocytic cells is dose-dependent, human monocytic cells were exposed to varying doses of HRV16 (MOI=0, 0.1, 1, 10, 100) for 24 h (Fig. 2 ). Exposure of BAL macrophages and monocytes to HRV16 at a MOI of 0.01 had little to no effect on IP-10 protein release. However, a dose-dependent release of IP-10 was detected following treatment with HRV16 at a MOI of 0.1–100 (BAL macrophages, P=0.004; monocytes, P=0.0002). It is interesting that similar levels of IP-10 release by monocytic cells were detected following HRV16 challenge in a MOI range of 1–10, and lower levels of IP-10 release were seen when the cells were challenged with HRV16 at a MOI of 100 or MOI less than 1. These data demonstrate robust levels of IP-10 release by monocytic cells upon exposure to predicted physiological concentrations of HRV16 (MOI≤10).


Figure 2
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  		Figure 2. A dose-response of HRV16-induced IP-10 release by human monocytic cells. Human BAL macrophages (A) or peripheral blood monocytes (B) were stimulated with control vehicle or various concentrations of HRV16 (MOI=0.01, 0.1, 1, 10, 100) for 24 h (BAL macrophages, P=0.004; monocytes, P=0.0002, by repeated-measure ANOVA analysis). Data represent mean ± SEM from three independent experiments in BAL macrophages and monocytes.

Disrupting HRV16-ICAM-1 interaction attenuates HRV16-stimulated IP-10 release by monocytic cells
The major group HRV (including HRV16) receptor is ICAM-1 [23 ]. To confirm that HRV16-stimulated IP-10 release by monocytic cells is HRV-specific, we used three methods to abrogate HRV16-ICAM-1 interaction: pretreatment of monocytic cells with an anti-ICAM-1 mAb, which has been shown previously to inhibit HRV16 attachment to human monocytes [10 ]; pretreatment of HRV16 with WIN52035, which is a compound that induces conformational changes in the region of the HRV capsid that binds to ICAM-1 [22 , 24 ]; and neutralization of HRV16 via pretreatment at low pH (pH<5.0), which facilitates viral uncoating [25 ]. As shown in Figure 3 , neutralization of cell surface-expressed ICAM-1 on monocytes, using anti-ICAM-1, abolished the ability of HRV16 to induce IP-10 release, whereas the isotype control, IgG1{kappa}, had little effect. Furthermore, inhibiting the ability of HRV16 to bind ICAM-1, via pretreatment of HRV16 with WIN52035 or low pH, also attenuated HRV16-stimulated IP-10 release by monocytes. The decrease in IP-10 release by monocytic cells upon impeding HRV16-ICAM-1 interaction was not a result of cytotoxic effects of the inhibitory methods, as mean cell viability for all treatments was ≥98% relevant to control treatment (n=3; MTS assay as described in Materials and Methods). These data demonstrate that HRV16-induced IP-10 release by monocytic cells requires ICAM-1 for viral attachment and/or signal transduction.


Figure 3
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  		Figure 3. The effect of blocking HRV16-ICAM-1 interaction on HRV16-stimulated IP-10 release by monocytic cells. Neutralization of HRV16 using WIN52035 or low pH was conducted as described in Materials and Methods. Human monocytes were pretreated with anti-ICAM-1 or isotype control (IgG1{kappa}; 10 µg/mL) for 30 min prior to agonist stimulation. Following neutralizing pretreatments, cells were stimulated with the indicated control vehicle or HRV16 (MOI=10) for 24 h. Data represent mean ± SEM from three independent experiments.

Human monocytes release IP-10 following exposure to native and NR-inactivated HRV16
Previous studies by us and others [10 , 17 ] have suggested that monocytic cells do not support a productive infection with HRV16. In contrast, airway epithelial cells are the primary site of HRV16 replication and require viral replication for IP-10 induction [7 , 18 ]. To assess if HRV16 replication is required for IP-10 production by human monocytes, we used NR-containing HRV16, which when exposed to white fluorescent light, displays severely reduced HRV16 infectivity. However, HRV16 inactivation by this method did not affect virus attachment to cells, as human 35S-RV16- and 35S-NR-inactivated HRV16 incubated with human monocytes for 1 h showed no significant difference in the number of particles associated with the cells (Fig. 4A ). Moreover, the attenuation of HRV16 association with monocytes in the presence of anti-ICAM-1 ensures that HRV16 attachment is ICAM-1-dependent. It is interesting that NR-inactivated HRV16 retained the ability to stimulate IP-10 release by human monocytes following a 24-h incubation at a level similar to that exhibited by native HRV16 (Fig. 4B) . These data reveal that unlike HRV16 replication requirements in epithelial cells, HRV16 replication is not required for monocytic cell activation and subsequent cytokine elaboration.


Figure 4
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  		Figure 4. Native and NR-inactivated HRV16 binding to human monocytes and stimulation of IP-10 production. (A) Effect of NR inactivation of HRV16 on viral association with human monocytes, which were pretreated ± control vehicle, anti-ICAM-1 antibody (30 µg/mL), or isotype control (IgG1{kappa}; 30 µg/mL) and then incubated for 1 h with 4000 particles/cell 35S-HRV16- or 35S-NR-inactivated HRV16. Cell-associated radioactivity was measured after sedimenting and washing the cells. Results are expressed as the number of virions per cell (Materials and Methods) and represent mean ± SEM from three separate experiments (HRV16 vs. NR-inactivated HRV16, P=0.25 by paired t-test; *, HRV16 vs. HRV16+anti-ICAM-1 antibody, P<0.05 by paired t-test). (B) IP-10 secretion by human monocytes following native or NR-inactivated HRV16 challenge. Human peripheral blood monocytes were stimulated with control vehicle, HRV16 (MOI=10), or NR-inactivated HRV16 (MOI=10) for 24 h (P=0.11 by paired t-test). Data represent mean ± SEM from seven independent experiments.

JAK/STAT and p38 kinase activity modulate HRV16-induced IP-10 release by human monocytes
Putative transcriptional activators of IP-10 gene expression include the STAT factors [7 ]. Accordingly, the present study examined the STAT isoform 1 (STAT1) phosphorylation state following human monocyte exposure to HRV16 (MOI=10) for various time periods. Immunoblot analysis indicated that HRV16 stimulated STAT1 phosphorylation in monocytes in a time-dependent manner (Fig. 5A ). The phosphorylation of STAT1 in monocytes was not detectable until 2 h post-HRV16 inoculation, whereupon the phosphorylation persisted, although to a lesser degree, throughout 8 h. In addition, monocytes were stimulated with IFN-{gamma}, a well-known activator of STAT1, as a positive control, whereas treatment with anisomycin was used as a negative control for STAT1 phosphorylation. As STAT1 is phosphorylated in monocytes inoculated with HRV16 and is a putative, transcriptional regulator of IP-10, these data suggest that STAT1 activity participates in mediating HRV16-induced IP-10 release.


Figure 5
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  		Figure 5. JAK/STAT1 pathway and p38 kinase involvement in IP-10 production by human monocytes following HRV16 challenge. (A) Time course of HRV16-stimulated STAT1 phosphorylation in human monocytes, which were treated with control vehicle, HRV16 (MOI=10), anisomycin (10 µM), or IFN-{gamma} (100 ng/mL) for the indicated times. Equivalent amounts of protein (50 µg) for each sample were subjected to SDS-PAGE, and immunoblotting was conducted using antiphospho-STAT1 as described in Materials and Methods. A representative immunoblot displaying STAT1 phosphorylation in HRV16-stimulated monocytes is shown (n=3). (B) Effect of JAK-I on HRV16-stimulated IP-10 secretion by human monocytes, which were pretreated with or without control vehicle or increasing concentrations of JAK-I (0.003–1.0 µM) for 2 h. Following pretreatment, the cells were incubated with control vehicle or HRV16 (MOI=10) for 24 h Data represent the mean ± SEM from three independent experiments (P=0.0001 by repeated-measure ANOVA analysis). (C) Effect of p38 kinase inhibition on HRV16-induced IP-10 production by human monocytes, which were pretreated with control vehicle or the active/inactive forms of the p38 kinase inhibitor (active=10 µM SB203580; inactive=10 µM SB202474) for 30 min. Following pretreatment, the cells were stimulated with control vehicle or HRV16 (MOI=10) for 24 h. Data represent mean ± SEM from three independent experiments (*, HRV16 vs. HRV16+SB203580, P<0.05 by t-test).

Activation of STAT molecules occurs upon phosphorylation of select tyrosine residues by members of the JAK family (JAK1, -2, and -3 and Tyk 2) of tyrosine kinases [26 ]. To determine whether JAK/STAT pathway activity modulates IP-10 production by monocytes following HRV16 challenge, the effect of JAK-I, which blocks JAK family member (inhibits JAK1, -2, and -3 and Tyk2) activity on HRV16-induced IP-10 release by monocytes, was evaluated. The JAK inhibitor significantly attenuated HRV16-stimulated IP-10 protein in a dose-dependent manner (P=0.0001; Fig. 5B ) via a noncytotoxic mechanism (mean viability=92%; data not shown; n=3). These data suggest that HRV16-induced IP-10 release by monocytic cells is mediated via JAK/STAT pathway activity.

We reported previously that HRV16 stimulates p38 kinase activity, which mediates, at least in part, MCP-1 protein release by primary monocytes and macrophages [17 ]. In the present study, we examined further if p38 kinase activity modulates HRV16-induced IP-10 release by monocytes. Pretreatment of monocytes with a selective p38 kinase inhibitor (10 µM SB203580) significantly attenuated HRV16-induced IP-10 release (P=0.02), whereas pretreatment with the inactive analog of the inhibitor (10 µM SB202474) caused no reduction (Fig. 5C) .

Intermediate production of a soluble factor mediates HRV16-stimulated STAT1 phosphorylation in human monocytes
Our previous studies have demonstrated that intracellular signaling events in human monocytic cells are initiated within 15 min of HRV16 stimulation [17 ]. The delayed kinetics of HRV16-stimulated STAT1 phosphorylation in monocytes suggests that this process may be secondary to the release/action of an autocrine/paracrine factor (Fig. 5A) . To test this idea, monocytes were exposed to control vehicle or HRV16 (MOI=10) for 2 h, followed by the transfer of the tissue-culture medium to untreated monocytes for 30 min, and the STAT1 phosphorylation status was then evaluated via immunoblotting. As shown in Figure 6A , HRV16 induced STAT1 phosphorylation in monocytes after a 2-h incubation (lane 2). It is interesting that the transfer of tissue-culture medium from monocytes exposed to HRV16 for 2 h to untreated monocytes resulted in STAT1 phosphorylation within 30 min (Fig. 6A , lane 6). The presence of HRV16 in the transfer medium was not responsible for STAT1 phosphorylation at 30 min, as HRV16 stimulation alone did not induce STAT1 phosphorylation within 30 min (Fig. 5A , lane 3; Fig. 6A , lane 8). These findings demonstrate that HRV16-induced STAT1 phosphorylation requires, at least in part, the action of an autocrine/paracrine factor. The ability of IFN-{alpha}/IFN-{gamma} to stimulate STAT1 phosphorylation in monocytes within 30 min further supports the idea of an autocrine/paracrine factor mediating HRV16-induced STAT1 phosphorylation (Figs. 5A and 6A , lanes 9 and 10; see Fig. 7A ).


Figure 6
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  		Figure 6. STAT1 phosphorylation and IFN-{alpha} secretion by human monocytes following HRV16 challenge. (A) The effect of conditioned medium transfer on STAT1 phosphorylation in monocytes, which were treated with control vehicle, HRV16 (MOI=10), IFN-{alpha} (100 ng/mL), or IFN-{gamma} (100 ng/mL) for the times indicated. In addition, tissue-culture supernatants were transferred from cells that were stimulated with control vehicle or HRV16 (MOI=10) for 2 h (lanes 3 and 5, respectively) to monocytes with no prior treatment (lanes 4 and 6, respectively) and incubated at 37°C for 30 min. Equivalent amounts of protein for each sample (50 µg/sample) were subjected to SDS-PAGE, and immunoblot analysis was conducted using antiphospho-STAT1 as described in Materials and Methods. A representative immunoblot displaying STAT1 phosphorylation in monocytes is shown (n=3). (B) IFN-{alpha} secretion by human monocytes following HRV16 challenge. Human peripheral blood monocytes were treated with control vehicle or HRV16 (MOI=10) for 24 h (P=0.03 by paired t-test). Data represent mean ± SEM from five independent experiments.


Figure 7
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  		Figure 7. STAT1 phosphorylation and IP-10 secretion from IFN-{alpha}/IFN-{gamma}/HRV16-stimulated monocytes. (A) Human peripheral blood monocytes were stimulated with HRV16 (MOI=10) for 2 h or various concentrations of IFN-{alpha} (0.1–100 ng/mL) or IFN-{gamma} (0.1–100 ng/mL) for 30 min. Equivalent amounts of protein (40 µg) for each sample were subjected to SDS-PAGE, and immunoblotting was conducted using antiphospho-STAT1 as described in Materials and Methods. Immunoblot analysis using anti-ERK1/2 (Total ERK1/2) was also conducted to confirm equal protein loading. A representative immunoblot displaying STAT1 phosphorylation in agonist-stimulated monocytes is shown (n=3). (B) Human monocytes were incubated with HRV16 (MOI=10), various concentrations of IFN-{alpha} (1, 10, 100 ng/mL), or IFN-{gamma} (1, 10, 100 ng/mL) or a combination of HRV16 (MOI=10) and IFN-{alpha}/IFN-{gamma} (10 ng/mL) for 24 h (P=0.0008 for IFN-{alpha} dose response; P=0.0004 for IFN-{gamma} dose response; P=0.50 for HRV16+IFN-{alpha} vs. HRV16 alone; P=0.006 for HRV16+IFN-{gamma} vs. HRV16 alone; all statistics done by repeated-measure ANOVA analysis). Data represent mean ± SEM from five independent experiments.

Increase in IFN-{alpha} secretion by human monocytes following HRV16 challenge
Type I IFNs are produced by monocytic cells in response to various pathogens and are established activators of STAT1 [27 ]. Hence, autocrine/paracrine action of type I IFNs may mediate HRV16-induced STAT1 phosphorylation. This study tested if monocyte exposure to HRV16 stimulates IFN-{alpha} protein release. Human monocytes obtained from five different donors were challenged with control vehicle or HRV16 at a MOI of 10 for 24 h. As shown in Figure 6B , HRV16 induced a significant increase in IFN-{alpha} release by monocytes (P=0.03). The detected amount of IFN-{alpha} released by monocytes following HRV16 is likely a conservative measurement of total IFN-{alpha} release, as several IFN-{alpha} isoforms exist, which are not detected by this ELISA assay and are likely expressed by monocytic cells upon exposure to HRV16. These data suggest that HRV16-stimulated IFN-{alpha} release acts in an autocrine/paracrine manner to mediate STAT1 phosphorylation and IP-10 production.

IFN-{alpha} and IFN-{gamma} stimulate STAT1 phosphorylation and IP-10 release by human monocytes
Our findings demonstrate that HRV16 stimulates IFN-{alpha} release by human monocytes, which may mediate HRV16-induced STAT1 phosphorylation and IP-10 induction. Accordingly, monocytes were exposed to various concentrations of IFN-{alpha} or IFN-{gamma}, spanning the measured quantity of IFN-{alpha} released by monocytes exposed to HRV16 ex vivo for 30 min or HRV16 at a MOI of 10 for 2 h, and STAT1 phosphorylation in monocytes was examined. Immunoblot analysis demonstrated that IFN-{alpha} and IFN-{gamma} up-regulated STAT1 phosphorylation in monocytes in a dose-dependent manner (Fig. 7A ). Moreover, IFN-{alpha} induced STAT1 phosphorylation to a similar level as that exhibited upon HRV16 stimulation. These findings support the concept that HRV16-stimulated STAT1 phosphorylation in monocytes likely occurs via autocrine/paracrine action of IFNs. In addition, IFN-{alpha} and IFN-{gamma} induced IP-10 release by human monocytes in a dose-dependent manner (P=0.0008 for IFN-{alpha}, and P=0.0004 for IFN-{gamma}; Fig. 7B ). Simultaneous stimulation of monocytes with HRV16 and IFN-{gamma} resulted in increased IP-10 release by monocytes, which was significantly greater than the level of IP-10 release exhibited by HRV16 or IFN-{gamma} stimulation alone (P=0.006). The observation that HRV16 and IFN-{gamma} together stimulate IP-10 release by monocytes in an additive manner suggests that these stimuli induce IP-10 production via independent mechanisms. It is interesting that simultaneous stimulation of monocytes with HRV16 and IFN-{alpha} resulted in IP-10 secretion at levels similar to that induced by HRV16 stimulation alone, thereby suggesting that these stimuli induce IP-10 release via similar mechanisms. Taken together, these data support the idea that autocrine/paracrine action of type I IFN not only mediates HRV16-induced STAT1 phosphorylation in monocytic cells but IP-10 secretion as well.

Neutralization of the type I IFN receptor attenuates HRV16-stimulated STAT1 phosphorylation and IP-10 release by human monocytes
As HRV16-induced STAT1 phosphorylation and IP-10 release by monocytic cells are likely linked to autocrine/paracrine action of type I IFNs, this study evaluated the effect of neutralizing the type I IFN receptor on these processes. Independent stimulation of monocytes with HRV16, IFN-{alpha}, or IFN-{gamma} resulted in STAT1 phosphorylation (Fig. 8A ). As expected of a receptor-mediated response, blocking ligand interaction with the type I IFN receptor using a neutralizing mAb (anti-IFN receptor type I) significantly suppressed IFN-{alpha}-induced STAT1 phosphorylation but had no effect on IFN-{gamma}-stimulated STAT1 phosphorylation in human monocytes. It is noteworthy that neutralization of the type I IFN receptor also attenuated STAT1 phosphorylation in monocytes challenged with HRV16. These findings support the concept that HRV16-induced STAT1 phosphorylation is mediated, at least in part, by autocrine/paracrine action of type I IFNs.


Figure 8
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  		Figure 8. Effect of IFN receptor type I neutralization on HRV16-induced STAT1 phosphorylation and IP-10 release by human peripheral blood monocytes. (A) Human monocytes were pretreated with or without control vehicle or anti-IFN receptor type I neutralizing antibody (anti-IFNRI; 5 µg/mL) for 30 min. Following pretreatment, the cells were stimulated with control vehicle (C; 2 h), HRV16 (MOI=10; 2 h), IFN-{alpha} (1 ng/mL; 30 min), or IFN-{gamma} (1 ng/mL; 30 min). Equivalent amounts of protein for each sample (50 µg/sample) were subjected to SDS-PAGE, and immunoblot analysis was conducted using antiphospho-STAT1 as described in Materials and Methods. Immunoblot analysis using anti-ERK1/2 (Total ERK1/2) was also conducted to confirm equal protein loading. A representative immunoblot displaying STAT1 phosphorylation in agonist-stimulated monocytes is shown (n=3). (B) Monocytes obtained from six different donors were pretreated with or without control vehicle or anti-IFN receptor type I as denoted above. Following pretreatments, cells were stimulated with control vehicle, HRV16 (MOI=10), IFN-{alpha} (10 ng/mL), or IFN-{gamma} (10 ng/mL) for 24 h (P=0.0005 for HRV16+anti-IFN receptor type I vs. HRV16 alone; P=0.0009 for IFN-{alpha}+anti-IFN receptor type I vs. IFN-{alpha} alone; P=0.72 for IFN-{gamma}+anti-IFN receptor type I vs. IFN-{gamma} alone; all statistics done by repeated-measure ANOVA analysis). Data represent mean ± SEM.

The effect of type I IFN receptor neutralization on HRV16-, IFN-{alpha}-, or IFN-{gamma}-induced IP-10 release by monocytes is consistent with the observed effects on STAT1 phosphorylation (Fig. 8A) . Monocytes exposure to HRV16 (MOI=10), IFN-{alpha} (10 ng/mL), or IFN-{gamma} (10 ng/ml) for 24 h resulted in similar levels of IP-10 secretion (Fig. 8B) . However, neutralizing the type I IFN receptor with anti-IFN receptor type I significantly attenuated type I IFN-{alpha}-induced IP-10 release by monocytes (P=0.0009) but had little to no effect on type II IFN-{gamma}-induced IP-10 release (P=0.72; Fig. 8B ). Furthermore, the presence of anti-IFN receptor type I significantly attenuated IP-10 release by monocytes challenged with HRV16 (P=0.0005). Taken together, these data support the concept that HRV16 stimulates type I IFN release by monocytic cells, which subsequently mediates HRV16-induced STAT1 phosphorylation and IP-10 release.


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DISCUSSION
 
Although the intracellular and immunologic mechanisms that mediate HRV-induced pathobiology of asthma remain unclear, it is evident that symptomatic infections are associated with elevated levels of chemokines in airway and nasal secretions as well as inflammatory cell recruitment into lung tissue, airway, and nasal passages [7 , 28 29 30 31 32 33 ]. For example, individuals challenged with HRV16 have significantly greater levels of IP-10 protein in nasal lavage fluid that positively correlates with viral titer and lymphocyte number in nasal secretions as well as respiratory symptom severity [7 ]. However, there is relatively little information about the cellular source and intracellular regulation of IP-10 protein production in response to HRV exposure. In this study, we demonstrate for the first time that monocytic cells, which are the predominant immune cells in the airway lumen, produce robust levels of IP-10 protein in response to HRV16 challenge, which is independent of viral replication. Furthermore, our data indicate that monocytic cell exposure to HRV16 results in STAT1 phosphorylation and that p38 kinase and JAK/STAT pathway activity are important in modulating HRV16-stimulated IP-10 production in monocytes. Our findings further indicate that monocytic cell exposure to HRV16 results in significantly elevated release of IFN-{alpha}. Moreover, HRV16-induced STAT1 phosphorylation and robust release of IP-10 by monocytic cells are linked to type I IFN receptor ligation; therefore, these processes are likely modulated by autocrine/paracrine action of type I IFNs.

Human monocytes and macrophages do not support a productive HRV16 infection [10 , 17 ], yet our data demonstrate that monocytic cells do produce high levels of IP-10 in response to native and/or replication-defective HRV16 challenge. In contrast, HRV16-stimulated IP-10 expression in epithelial cells requires infection with virus capable of replication [7 , 18 ]. A potential mechanism that may explain how HRV16 induces IP-10 release by monocytic cells without viral replication, which is required in epithelial cells, is that HRV16-ICAM-1 interaction at the cellular surface may be sufficient to initiate signal transduction and elicit IP-10 protein expression by monocytic cells. In this regard, previous studies have revealed that ICAM-1 cross-linking initiates rapid cytoplasmic signaling and subsequent phosphorylation and activation of stress-activated protein kinases [34 , 35 ]. Furthermore, this idea is supported by our data demonstrating that blockade of HRV16-ICAM-1 interaction attenuates HRV16-induced IP-10 secretion by monocytic cells. Thus, HRV16 binding to ICAM-1 may initiate signal transduction via ICAM-1, or HRV16-ICAM-1 interaction may be important for HRV16 internalization and subsequent intracellular signaling. Of note, a previous report stated that a HRV16 particle has the capacity to bind ~60 soluble ICAM-1 molecules (one ICAM-1 molecule per icosahedral face of the capsid [25 ]), which suggests that ICAM-1 oligomerization may be required for optimal HRV16-induced signal transduction via ICAM-1 or virus internalization. Our findings indicate that IP-10 release by monocytic cells is maximal following HRV16 stimulation with a MOI range of 1–10 and that lower levels of secreted IP-10 are detected upon stimulation with HRV16 at a MOI of 100. Thus, HRV16 at a MOI of 100 may saturate cell-surface ICAM-1 molecules, thereby preventing ICAM-1 oligomerization and resulting in lower levels of IP-10 secretion.

An alternative explanation for the disparity in monocytes and epithelial cell responsiveness to nonreplicative HRV16 is that HRV16 replication may not be required for IP-10 expression by airway epithelial cells similar to that exhibited by monocytic cells. In this regard, the two published reports suggesting that HRV16 replication is required for IP-10 expression by epithelial cells used UV-inactivated HRV16 to model replication-defective HRV16 [7 , 18 ]. However, subjecting virus to UV irradiation may cross-link viral proteins in addition to introducing defects into the viral genome, thereby repressing the ability of the virus to interact with ICAM-1 or other potential surface recognition molecules. In support of this concern, our unpublished data indicate that UV-inactivated HRV16 exhibited over 80% reduction in binding to HeLa cells as compared with native HRV16 (n=3). In the present study, NR-inactivated HRV16 was replication-defective but maintained virus-cell association and retained the ability to induce IP-10 expression by monocytic cells.

Not only is the mechanism of signal transduction initiation by HRV16 unclear, but little is known about the intracellular regulation of HRV16-induced chemokine expression as well. The IP-10 promoter region contains consensus sites for activated STAT protein, NF-{kappa}B, as well as AP-1 [7 ]. The transcription factor AP-1 is a well-known protein substrate of p38 kinase, and our previous studies demonstrated that HRV16 induces p38 kinase activity in primary monocytic cells [17 ]; therefore, we proposed that p38 kinase activity likely modulates HRV16-induced IP-10 production. This idea was supported by our present study, which indicates that inhibition of p38 kinase activity significantly attenuates IP-10 production by monocytes following HRV16 challenge. Furthermore, our study is the first to report that HRV16 consistently stimulates STAT1 phosphorylation in primary monocytic cells isolated from 12 human donors and that JAK/STAT1 activity mediates, at least in part, IP-10 protein release by monocytes following HRV16 challenge. It is interesting that HRV16-induced STAT1 phosphorylation in monocytic cells did not occur until 2 h postinfection. In contrast, IFN-{alpha} and IFN-{gamma}, well-known activators of STAT1, were able to elicit STAT1 phosphorylation in human monocytes with rapid kinetics (15 min, n=3, data not shown). Moreover, the transfer of tissue-culture medium from monocytes exposed to HRV16 for 2 h to untreated monocytes resulted in STAT1 phosphorylation within 30 min. Taken together, these findings suggest that STAT1 phosphorylation in monocytic cells following HRV16 challenge is likely mediated by the intervening production of soluble factors. Accordingly, a potential mechanism of HRV16-stimulated STAT1 phosphorylation and IP-10 protein production by monocytic cells involves the autocrine/paracrine action of secreted IFNs.

IFNs are essential regulators of various innate immune responses and initiate early host defense mechanisms against viral infections [36 ]. IFNs are commonly classified into two groups. Type I IFNs can be induced by virus infection and are comprised of several IFN-{alpha} isoforms, IFN-β and IFN-{omega}, which are produced by a wide variety of cells. Type II IFN, IFN-{gamma}, is induced primarily by mitogenic or antigenic stimuli and is produced by select cells of the immune system including NK cells, CD4+ Th lymphocytes, and CD8+ cytotoxic suppressor cells [27 ]. Accordingly, this study evaluated if HRV16 has the capacity to induce type I IFN (IFN-{alpha}) production by monocytic cells as well as examined the involvement of IFNs in mediating STAT1 phosphorylation and IP-10 release by monocytic cells. There exist at least 14 identified human isoforms of IFN-{alpha}. We examined IFN-{alpha} release using an ELISA assay, which detected some but not all isoforms, and observed that primary monocytes exposure to HRV16 for 24 h causes a significant increase in IFN-{alpha} release. We also showed that ex vivo stimulation with physiologically relevant concentrations of IFN-{alpha} induced STAT1 phosphorylation and IP-10 release by monocytic cells. In addition, as our results demonstrate that simultaneous stimulation of monocytic cells with HRV16 and IFN-{alpha} results in IP-10 release at similar levels to that released upon HRV16 challenge alone, we suggest that these stimuli use the same intracellular mechanism to regulate IP-10 expression or that HRV16 and IFN-{alpha} together induce maximal IP-10 induction, thereby nullifying an additive effect. However, the latter idea is less likely given the additive effect of IP-10 release by monocytic cells exhibited upon simultaneous exposure to HRV16 and IFN-{gamma}.

To test the concept that HRV16-stimulated STAT1 phosphorylation and IP-10 release by monocytic cells are linked to the autocrine/paracrine action of type I IFNs, we assessed HRV16-induced STAT1 phosphorylation and IP-10 production levels by monocytic cells pretreated with a neutralizing type I IFN receptor antibody. Two recent reports used the same technique to test if HRV16-induced IP-10 expression by epithelial cells was secondary to type I IFN receptor ligation and found conflicting conclusions [7 , 18 ]. Investigations by Chen et al. [18 ] demonstrated that HRV16-stimulated STAT1 phosphorylation and IP-10 mRNA induction in differentiated tracheobronchial epithelial cells isolated from two donors were partially suppressed by the type I IFN receptor neutralizing antibody. Conversely, Spurrell et al. [7 ] observed that neutralizing the type I IFN receptor did not suppress HRV16-induced IP-10 mRNA induction in adenoidal epithelial cells isolated from three human donors but did attenuate IFN-β-stimulated IP-10 mRNA induction. The conflicting results could be a result of differential regulation in epithelial cells derived from different tissues or that Chen et al. [18 ] used a high concentration of neutralizing IFN receptor antibody (20 µg/mL) compared with 5 µg/mL in the study by Spurrell et al. [7 ]. Our findings demonstrate that blocking ligand interaction with the type I IFN receptor, using 5 µg/mL neutralizing antibody, attenuated HRV16- and IFN-{alpha}-stimulated STAT1 phosphorylation and IP-10 release by monocytic cells but did not suppress IFN-{gamma}-induced STAT1 phosphorylation or IP-10 production. These data are the first to demonstrate clearly that HRV16-stimulated STAT1 phosphorylation and IP-10 expression at the protein level in primary monocytic cells are linked to type I IFN receptor ligation.

In summary, although symptomatic HRV infections are associated with elevated levels of chemokines in the airway and nasal secretions, the intracellular regulation and production of cytokines and chemokines by airway immune cells in response to HRV challenge are largely unknown. Our findings indicate that primary monocytic cells become activated and release robust levels of IP-10 protein following HRV16 inoculation, which is independent of viral replication. Phosphorylation of STAT1 in monocytes also occurs following HRV16 challenge, and inhibition of the JAK/STAT pathway activity suppresses HRV16-stimulated IP-10 production. Type I IFN-{alpha} is also produced following monocytic cell exposure to HRV16; moreover, HRV16-induced STAT1 phosphorylation and production of IP-10 are attenuated by neutralization of the type I IFN receptor. Thus, the present work supports a model wherein HRV16-induced IP-10 release by human monocytic cells is modulated via autocrine/paracrine action of type I IFNs and subsequent type I IFN receptor/JAK/STAT pathway activity. Our findings demonstrating robust activation of human monocytic cells in response to replicative and/or nonreplicative HRV16 challenge further support the idea that there is a mechanistic disparity in the activation of airway macrophages compared with epithelial cells and further suggest that macrophages are likely active participants in promoting cytokine elaboration following HRV challenge in vivo.


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ACKNOWLEDGEMENTS
 
This work was supported by the National Institutes of Health Grants HL56396, AI50500, MolRR03186, and T32-HD041921-01. We are grateful to several individuals affiliated with the University of Wisconsin-Madison including Drs. J. Gern and A. Mosser for preparation of virus stocks, Dr. K. Meyer and M. Jackson for subject screening and assisting with bronchoscopies, Dr. N. Jarjour and R. Degraw for preparation of BAL macrophages and providing the IP-10 ELISA kit, Dr. J. Sedgwick and A. Brooks for preparation of PBMC, as well as M. Evans for statistical analysis assistance.

Received June 23, 2006; revised July 23, 2006; accepted August 25, 2006.


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REFERENCES
 
    1
  1. Pattemore, P. K., Johnston, S. L., Bardin, P. G. (1992) Viruses as precipitants of asthma symptoms. I. Epidemiology Clin. Exp. Allergy 22,325-336[CrossRef][Medline]
    2. 2
  2. Gern, J. E., Busse, W. W. (1999) Association of rhinovirus infections with asthma Clin. Microbiol. Rev. 12,9-18[Abstract/Free Full Text]
    3. 3
  3. Busse, W. W., Lemanske, R. F., Jr (2001) Asthma N. Engl. J. Med. 344,350-362[Free Full Text]
    4. 4
  4. Medoff, B. D., Sauty, A., Tager, A. M., Maclean, J. A., Smith, R. N., Mathew, A., Dufour, J. H., Luster, A. D. (2002) IFN-{gamma}-inducible protein 10 (CXCL10) contributes to airway hyperreactivity and airway inflammation in a mouse model of asthma J. Immunol. 168,5278-5286[Abstract/Free Full Text]
    5. 5
  5. Romagnani, S. (2000) The role of lymphocytes in allergic disease J. Allergy Clin. Immunol. 105,399-408[CrossRef][Medline]
    6. 6
  6. Ying, S., O’Connor, B., Ratoff, J., Meng, Q., Mallett, K., Cousins, D., Robinson, D., Zhang, G., Zhao, J., Lee, T. H., Corrigan, C. (2005) Thymic stromal lymphopoietin expression is increased in asthmatic airways and correlates with expression of Th2-attracting chemokines and disease severity J. Immunol. 174,8183-8190[Abstract/Free Full Text]
    7. 7
  7. Spurrell, J. C., Wiehler, S., Zaheer, R. S., Sanders, S. P., Proud, D. (2005) Human airway epithelial cells produce IP-10 (CXCL10) in vitro and in vivo upon rhinovirus infection Am. J. Physiol. Lung Cell. Mol. Physiol. 289,L85-L95[Abstract/Free Full Text]
    8. 8
  8. Taub, D. D., Lloyd, A. R., Conlon, K., Wang, J. M., Ortaldo, J. R., Harada, A., Matsushima, K., Kelvin, D. J., Oppenheim, J. J. (1993) Recombinant human interferon-inducible protein 10 is a chemoattractant for human monocytes and T lymphocytes and promotes T cell adhesion to endothelial cells J. Exp. Med. 177,1809-1814[Abstract/Free Full Text]
    9. 9
  9. Liu, L., Jarjour, N. N., Busse, W. W., Kelly, E. A. (2004) Enhanced generation of helper T type 1 and 2 chemokines in allergen-induced asthma Am. J. Respir. Crit. Care Med. 169,1118-1124[Abstract/Free Full Text]
    10. 10
  10. Gern, J. E., Dick, E. C., Lee, W. M., Murray, S., Meyer, K., Handzel, Z. T., Busse, W. W. (1996) Rhinovirus enters but does not replicate inside monocytes and airway macrophages J. Immunol. 156,621-627[Abstract]
    11. 11
  11. Gern, J. E., Vrtis, R., Kelly, E. A., Dick, E. C., Busse, W. W. (1996) Rhinovirus produces nonspecific activation of lymphocytes through a monocyte-dependent mechanism J. Immunol. 157,1605-1612[Abstract]
    12. 12
  12. Mosser, A. G., Brockman-Schneider, R., Amineva, S., Burchell, L., Sedgwick, J. B., Busse, W. W., Gern, J. E. (2002) Similar frequency of rhinovirus-infectible cells in upper and lower airway epithelium J. Infect. Dis. 185,734-743[CrossRef][Medline]
    13. 13
  13. Halperin, S. A., Eggleston, P. A., Hendley, J. O., Suratt, P. M., Groschel, D. H., Gwaltney, J. M., Jr (1983) Pathogenesis of lower respiratory tract symptoms in experimental rhinovirus infection Am. Rev. Respir. Dis. 128,806-810[Medline]
    14. 14
  14. Gern, J. E., Galagan, D. M., Jarjour, N. N., Dick, E. C., Busse, W. W. (1997) Detection of rhinovirus RNA in lower airway cells during experimentally induced infection Am. J. Respir. Crit. Care Med. 155,1159-1161[Abstract]
    15. 15
  15. Griego, S. D., Weston, C. B., Adams, J. L., Tal-Singer, R., Dillon, S. B. (2000) Role of p38 mitogen-activated protein kinase in rhinovirus-induced cytokine production by bronchial epithelial cells J. Immunol. 165,5211-5220[Abstract/Free Full Text]
    16. 16
  16. Terajima, M., Yamaya, M., Sekizawa, K., Okinaga, S., Suzuki, T., Yamada, N., Nakayama, K., Ohrui, T., Oshima, T., Numazaki, Y., Sasaki, H. (1997) Rhinovirus infection of primary cultures of human tracheal epithelium: role of ICAM-1 and IL-1β Am. J. Physiol. 273,L749-L759[Medline]
    17. 17
  17. Hall, D. J., Bates, M. E., Guar, L., Cronan, M., Korpi, N., Bertics, P. J. (2005) The role of p38 MAPK in rhinovirus-induced monocyte chemoattractant protein-1 production by monocytic-lineage cells J. Immunol. 174,8056-8063[Abstract/Free Full Text]
    18. 18
  18. Chen, Y., Hamati, E., Lee, P. K., Lee, W. M., Wachi, S., Schnurr, D., Yagi, S., Dolganov, G., Boushey, H., Avila, P., Wu, R. (2006) Rhinovirus induces airway epithelial gene expression through double-stranded RNA and IFN-dependent pathways Am. J. Respir. Cell Mol. Biol. 34,192-203[Abstract/Free Full Text]
    19. 19
  19. Liu, L. Y., Jarjour, N. N., Busse, W. W., Kelly, E. A. (2003) Chemokine receptor expression on human eosinophils from peripheral blood and bronchoalveolar lavage fluid after segmental antigen challenge J. Allergy Clin. Immunol. 112,556-562[CrossRef][Medline]
    20. 20
  20. Wilson, J. N., Cooper, P. D. (1965) The effect of light on poliovirus grown in neutral red Virology 26,1-9[CrossRef][Medline]
    21. 21
  21. Lee, W. M., Wang, W. (2003) Human rhinovirus type 16: mutant V1210A requires capsid-binding drug for assembly of pentamers to form virions during morphogenesis J. Virol. 77,6235-6244[Abstract/Free Full Text]
    22. 22
  22. Wang, W., Lee, W. M., Mosser, A. G., Rueckert, R. R. (1998) WIN 52035-dependent human rhinovirus 16: assembly deficiency caused by mutations near the canyon surface J. Virol. 72,1210-1218[Abstract/Free Full Text]
    23. 23
  23. Greve, J. M., Davis, G., Meyer, A. M., Forte, C. P., Yost, S. C., Marlor, C. W., Kamarck, M. E., McClelland, A. (1989) The major human rhinovirus receptor is ICAM-1 Cell 56,839-847[CrossRef][Medline]
    24. 24
  24. Shepard, D. A., Heinz, B. A., Rueckert, R. R. (1993) WIN 52035-2 inhibits both attachment and eclipse of human rhinovirus 14 J. Virol. 67,2245-2254[Abstract/Free Full Text]
    25. 25
  25. Hoover-Litty, H., Greve, J. M. (1993) Formation of rhinovirus-soluble ICAM-1 complexes and conformational changes in the virion J. Virol. 67,390-397[Abstract/Free Full Text]
    26. 26
  26. Schindler, C., Darnell, J. E., Jr (1995) Transcriptional responses to polypeptide ligands: the JAK-STAT pathway Annu. Rev. Biochem. 64,621-651[Medline]
    27. 27
  27. Schroder, K., Hertzog, P. J., Ravasi, T., Hume, D. A. (2004) Interferon-{gamma}: an overview of signals, mechanisms and functions J. Leukoc. Biol. 75,163-189[Abstract/Free Full Text]
    28. 28
  28. Gern, J. E., Martin, M. S., Anklam, K. A., Shen, K., Roberg, K. A., Carlson-Dakes, K. T., Adler, K., Gilbertson-White, S., Hamilton, R., Shult, P. A., Kirk, C. J., Da Silva, D. F., Sund, S. A., Kosorok, M. R., Lemanske, R. F., Jr (2002) Relationships among specific viral pathogens, virus-induced interleukin-8, and respiratory symptoms in infancy Pediatr. Allergy Immunol. 13,386-393[CrossRef][Medline]
    29. 29
  29. Jarjour, N. N., Gern, J. E., Kelly, E. A., Swenson, C. A., Dick, C. R., Busse, W. W. (2000) The effect of an experimental rhinovirus 16 infection on bronchial lavage neutrophils J. Allergy Clin. Immunol. 105,1169-1177[CrossRef][Medline]
    30. 30
  30. Johnston, S. L. (1995) Natural and experimental rhinovirus infections of the lower respiratory tract Am. J. Respir. Crit. Care Med. 152,S46-S52[Medline]
    31. 31
  31. Levandowski, R. A., Weaver, C. W., Jackson, G. G. (1988) Nasal-secretion leukocyte populations determined by flow cytometry during acute rhinovirus infection J. Med. Virol. 25,423-432[Medline]
    32. 32
  32. Turner, R. B., Weingand, K. W., Yeh, C. H., Leedy, D. W. (1998) Association between interleukin-8 concentration in nasal secretions and severity of symptoms of experimental rhinovirus colds Clin. Infect. Dis. 26,840-846[Medline]
    33. 33
  33. Zalman, L. S., Brothers, M. A., Dragovich, P. S., Zhou, R., Prins, T. J., Worland, S. T., Patick, A. K. (2000) Inhibition of human rhinovirus-induced cytokine production by AG7088, a human rhinovirus 3C protease inhibitor Antimicrob. Agents Chemother. 44,1236-1241[Abstract/Free Full Text]
    34. 34
  34. Lawson, C., Ainsworth, M. E., McCormack, A. M., Yacoub, M., Rose, M. L. (2001) Effects of cross-linking ICAM-1 on the surface of human vascular smooth muscle cells: induction of VCAM-1 but no proliferation Cardiovasc. Res. 50,547-555[Abstract/Free Full Text]
    35. 35
  35. Wang, Q., Doerschuk, C. M. (2001) The p38 mitogen-activated protein kinase mediates cytoskeletal remodeling in pulmonary microvascular endothelial cells upon intracellular adhesion molecule-1 ligation J. Immunol. 166,6877-6884[Abstract/Free Full Text]
    36. 36
  36. Samuel, C. E. (2001) Antiviral actions of interferons Clin. Microbiol. Rev. 14,778-809[Abstract/Free Full Text]



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