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Originally published online as doi:10.1189/jlb.1006611 on March 27, 2007

Published online before print March 27, 2007
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(Journal of Leukocyte Biology. 2007;81:1487-1495.)
© 2007 by Society for Leukocyte Biology

RSV-infected airway epithelial cells cause biphasic up-regulation of CCR1 expression on human monocytes

Paul T. Morrison*, Lynette H. Thomas*, Mike Sharland{dagger} and Jon S. Friedland*,1

* Department of Infectious Diseases and Immunity, Imperial College, Hammersmith Campus, London, United Kingdom; and
{dagger} Pediatric Infectious Diseases Unit, St George’s Hospital Medical School, London, United Kingdom

1 Correspondence: Department of Infectious Diseases, Imperial College, Hammersmith Campus, Du Cane Road, London, W12 0NN, UK. E-mail: j.friedland{at}imperial.ac.uk


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ABSTRACT
 
Respiratory syncytial virus (RSV) infection can cause extensive airway inflammation, which is orchestrated by chemokines and their receptors. RSV-infected epithelial cells secrete many cytokines and chemokines, but little is known about regulation of chemokine receptors on target cells. We investigated the effects of conditioned media (CM) from RSV-infected epithelial cells on monocyte CCR1, CCR2, and CCR5 expression. RSV-CM but not control-CM stimulated a biphasic increase in cell-surface CCR1, and levels peaked at 36 h and 96 h poststimulation. Similar CCR1 up-regulation occurred on monocyte-derived macrophages. Cytochlasin D and colchicine blocked both peaks of expression, demonstrating requirement of a functional cytoskeleton. Intracellular staining revealed little internal sequestration of CCR1 protein, and CCR1 up-regulation was inhibited by actinomycin D and cycloheximide, indicating that both waves of RSV-CM-induced surface CCR1 expression were dependent on de novo transcription and protein synthesis. Cytokine-neutralizing experiments showed that the effects of RSV-CM were decreased by blocking TNF-{alpha} (percent inhibition=51±2.3% at 36 h peak and 42±7.7% at 96 h peak) and to a lesser extent, IL-1 (percent inhibition=32±7.2% at 36 h and 23±2.9% at 96 h). In summary, RSV-CM causes a biphasic up-regulation of surface CCR1 on monocytes, which is dependent on an intact cytoskeleton, requires new gene transcription and protein synthesis, and is mediated in part by the proinflammatory cytokines TNF-{alpha} and IL-1.

Key Words: cell networks • chemokine • respiratory epithelium • cytokines • transcription • cytoskeleton


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INTRODUCTION
 
Respiratory syncytial virus (RSV) is the major cause of bronchiolitis and pneumonia in infants and is responsible for serious illness amongst certain adult populations, including the immunosuppressed and the elderly [1 , 2 ]. Further, RSV is a precipitant of asthma attacks, and infection may predispose susceptible children to recurrent wheezing later in life [3 , 4 ]. The primary target of RSV infection is the airway epithelium. Disease pathology is a result of subsequent, excessive inflammation as well as to direct viral, cytopathic effects. Severe infection is characterized by extensive inflammatory cell recruitment into the lower airways initially by neutrophils and at later stages predominately by T lymphocytes and monocytes [5 6 7 ].

The selective recruitment of leukocytes to the infected airway is likely to be initiated by chemokines. A wealth of evidence shows that RSV infection of the airways increases secretion of chemokines, including the neutrophil attractant CXCL8, as well as CCL5 (RANTES), CCL3 (MIP-1{alpha}), and CCL2 (MCP-1), which attract monocytes and lymphocytes [8 9 10 11 12 13 ]. Further, levels of CCL5 and CCL3 correlate with severity of illness [14 15 16 ]. Regulation of the response to infection may be achieved, not only by control of the magnitude and kinetics of chemokine secretion but also potentially by regulation of cell-specific surface chemokine receptor expression. These are members of a family of seven transmembrane-spanning, G protein-coupled molecules and can be subdivided into those for CC or for CXC chemokines [17 , 18 ]. Currently, 10 CCRs have been identified, of which monocytes predominately express three: CCR1, CCR2, and CCR5 [19 , 20 ]. A degree of promiscuity exists, as few receptors bind only one ligand, and many chemokines can bind more than one receptor. For example, the ligands CCL3, CCL4 (MIP-1ß), and CCL5 can activate monocytes via CCR1 or CCR5.

RSV infection of human respiratory epithelial cells in vitro has provided a useful model for extensive characterization of mechanisms controlling chemokine secretion in response to direct infection. Coculture models have shown that RSV-infected epithelial cells secrete mediators, which recruit eosinophils and monocytes, and that interaction of these cells with RSV-infected epithelial cells results in production of additional chemokines [8 ]. We have demonstrated previously that soluble mediators contained in conditioned media (CM) from RSV-infected monocytes are able to amplify chemokine release synergistically from RSV-infected epithelial cells [21 ]. In the present study, we investigate the indirect effects of RSV infection of respiratory epithelial cells on monocytes and have analyzed the expression of CCR1, -2, and -5 on monocytic cells stimulated by CM from respiratory epithelial cells infected with RSV. We report that this RSV-CM causes a biphasic expression of CCR on the surface of monocytes, a response that requires an intact cytoskeleton as well as de novo gene transcription and protein synthesis.


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MATERIALS AND METHODS
 
RSV culture and titration
RSV Strain A2 (kind gift of Peter Oppenshaw, Imperial College, London, UK) was grown in HEp-2 cells, according to methods described previously [22 ]. HEp-2 monolayers were inoculated with 0.1 multiplicity of infection (MOI) RSV and then periodically monitored and harvested when greater than 80% cells were detached. The cell suspension was then spun at 13,000 g, and the virus-containing pellet was resuspended immediately in fresh media, snap-frozen, and stored at –80°C. Viral titer was quanitified according to the microplaque immunoperoxidase method [23 ]. To provide a control, HEp-2 cell supernatants were prepared as above but in the absence of RSV.

Preparation of monocytes and monocyte-derived macrophages (MDM)
Adult human monocytes were isolated from buffy coat residue packs (from the North London Blood Transfusion Centre, Collindale, UK). Briefly, mononuclear cells were separated by density gradient centrifugation over Ficoll-Paque (Amersham-Pharmacia, Bucks, UK), and monocytes were then purified by adhesion onto tissue-culture plastic for 2 h. Nonadherent lymphocytes were removed by thorough washing, and the resultant purified monocytes were used for experiments immediately or allowed to mature into MDM by culturing in RPMI 1640 supplemented with 10% FCS, 2 mM glutamine, and 10 µg/ml ampicillin at 37°C/5% CO2 for 5 days.

Preparation of epithelial cell-derived CM
The Type II alveolar epithelial (A549) cell line [24 ] was cultured in DMEM plus 10% FCS, 2 mM glutamine, and 10 µg/ml ampicillin. Epithelial cell monolayers were infected with RSV (MOI of 0.3). After 48 h, the CM from RSV-infected cells (RSV-CM) was removed, filtered through a 100-kDa filter to remove cells plus RSV, and then aliquoted and stored at –80°C. Control (C)-CM was prepared in exactly the same way but with the addition of irradiated (i.e., inactive) RSV. C-CM or RSV-CM was added to monocytes at dilution ranges of 1:5–1:30 in serum-free RPMI 1640 with antibiotics. After 1 h incubation, FCS was added to make a final culture media containing 10% FCS.

Flow cytometry
Monocytes or MDM were removed from culture plates using dissociation buffer (Invitrogen, Paisley, UK) and transferred to FACS tubes (approximately 1x105 cells/tube) for flow cytometric analysis. After washing with Pharmastain + BSA buffer (Invitrogen), the cells were incubated, according to the manufacturer’s protocols, in the dark on ice for 30 min with one of the following mAb: anti-CD14-PE (Sigma, Poole, UK) or anti-CCR1, anti-CCR1-PE, anti-CCR2-CyCh, or anti-CCR5-FITC (all R&D Systems, UK). Labeled cells were then washed and fixed with 1% paraformaldehyde solution. For all experiments, cells incubated with isoytpe control antibodies (R&D Systems) and were analyzed in parallel as negative controls.

Experimental protocols
The role of the cytoskeleton in regulating CCR surface levels was assessed using colchicine (0.002–2 mM) and cytochalasin (0.01–10 mM; both Sigma). The requirement of new transcription and translation in CCR up-regulation was ascertained using actinomycin D (0.01–10 mg/ml) or cycloheximide (0.002–2 mg/ml; both Sigma), respectively. Concentrations of inhibitors suitable for use were determined according to a protocol described previously [25 ] and were established to have no significant, detrimental effect on cell viability using trypan blue staining and cell fragmentation studies. To examine the effects on the first peak of surface CCR, inhibitors were added concurrently with the CM (1:5 dilution) at 0 h, and cells were assayed for CCR1 at 36 h. To investigate the second peak of CCR1 expression, inhibitors were added into the cultures 48 h following CM treatment (i.e., after the first peak in CCR expression), and cells were assayed after 96 h total culture time.

Cell permeabilization studies
Extracellular CCR1 versus total cell CCR1 content was assessed using permeabilization experiments. Monocytes were incubated with RSV-CM or with C-CM for 36 or 72 h. Cells were then left intact or treated with 5 µg/ml saponin (Sigma) to permeabilize the cells, and then, surface and total CCR levels were assessed by FACS; FACS analysis of the intracellular protein ß-actin in the presence or absence of saponin was used to confirm permeabilization.

Cytokine-neutralizing experiments
To examine the role of specific cytokines within RSV-CM in inducing CCR at each peak of expression, the inhibiting agents anti-TNF-{alpha}, IL-1 receptor antagonist (IL-1-Ra; Peprotech, London, UK), anti-IL-6, and anti-CCL5 (R&D Systems) were used. Cells were treated with RSV-CM (1:5 dilution), and inhibitors were added immediately for the 36-h time-point. To examine the effect on the second (96 h) peak, blockers were added at 48 h post-RSV-CM. We have shown previously that IL-1Ra acts specifically by its ability to inhibit the effect of recombinant (r)IL-1ß (20 ng/ml) but not of human rTNF-{alpha} (10 ng/ml) on epithelial cell IL-8 secretion. Similarly, the specificity of anti-TNF-{alpha} was confirmed by the inhibition of TNF-{alpha} but not IL-1ß-induced IL-8 secretion.


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RESULTS
 
RSV-CM increases CCR surface expression on monocytes
As has been demonstrated previously [19 ], freshly isolated monocytes, which were CD14-positive by FACS analysis, were confirmed to express CCR2 initially but not CCR1 or CCR5 on their surface. CCR2 expression was then lost over the first 12 h in culture (data not shown). Murine models of RSV infection have implicated CCR1 in airway pathology and hyper-reactiveness [26 , 27 ]; however, little is known about how this receptor is altered by the disease. As the initial target for RSV infection is airway epithelium, we investigated indirect effects of RSV infection using CM from RSV-infected epithelial cells. Monocytes were cultured with a 1:5 dilution of RSV-CM or C-CM, and kinetics of CCR expression was assessed by FACS. This revealed a biphasic pattern of RSV-CM-induced surface expression, with peaks of CCR1 at 36 h and again at 96 h and levels returning almost to those of control in between the two peaks (Fig. 1A and 1B ). The effect of RSV-CM was dose-dependent, as demonstrated by experiments, whereby RSV-CM or C-CM was added to monocytes at dilutions from 1:5 to 1:30, and cells were cultured for 36 h prior to FACS analysis. At a 1:5 dilution, RSV-CM caused over an eightfold increase in the expression of CCR1 (Fig. 1C) , whereas even high concentrations of C-CM had little effect (data not shown).


Figure 1
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  		Figure 1. The effect of RSV-CM from epithelial cells on monocyte CCR1 expression. (A) Monocytes were stimulated with C-CM or RSV-CM at a 1:5 dilution and incubated for up to 108 h. Cells were then harvested, and surface CCR1 was analyzed by FACS. Open peaks show C-CM-treated cells, and shaded peaks show RSV-CM-stimulated monocytes. Results were expressed as percent of maximal CCR1 ± SEM (B). (C) Monocytes were incubated with various dilutions of CM for 36 h, and then, CCR1 was measured. Monocytes were matured into macrophages via a 5-day culture (as described in Materials and Methods), and then, the effects of RSV-CM were assessed as in B. Results are the mean ± SEM of at least three independent experiments.

RSV-CM up-regulates CCR1 on MDM
The expression of surface CCR changes according to the differentiation of monocytic cells. Typically, as monocytes mature into macrophage, they acquire a CCR1- and CCR5-positive phenotype and lose surface CCR2 [19 ]. To assess whether differentiation into macrophages alters the response to RSV-CM, isolated monocytes were cultured for 5 days to allow maturation into MDM and then were stimulated with RSV-CM for up to 108 h as before (Fig. 2 ). A similar biphasic pattern of CCR1 expression was observed on MDM, and levels peaked again at 36 and 96 h. Thus, despite a raised basal level of CCR1, MDM display a similar up-regulation of CCR1 to monocytes.


Figure 2
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  		Figure 2. The effect of epithelial cell-derived RSV-CM on MDM CCR1 expression. Monocytes were matured into macrophages via a 5-day culture (as described in Materials and Methods) and then were stimulated with C-CM or RSV-CM at a 1:5 dilution and incubated for up to 108 h. Results were expressed as percent of maximal CCR1 ± SEM, calculated from three independent experiments.

RSV-CM increases CCR2 and CCR5 expression
Along with CCR1, CCR2 and CCR5 are the major CCR, which may be expressed on monocytes. RSV infection in vitro and in vivo results in production of CCL2 [28 , 29 ], which binds solely to CCR2. Further, the RSV-induced chemokines, CCL3 and CCL5 [12 , 14 , 16 ], may also use CCR5 as their receptor. We therefore looked at the effect of RSV-CM on the regulation of CCR2 and CCR5. FACS analysis demonstrated that the biphasic up-regulation of surface expression occurred, not only for CCR1 but also for CCR2 (Fig. 3A ) and for CCR5 (Fig. 3B) , both of which displayed two peaks of expression with a similar pattern of kinetics.


Figure 3
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  		Figure 3. Time course of CCR2 and CCR5 expression on monocytes treated with CM. Monocytes were stimulated with 1:5 dilutions of C-CM or RSV-CM and incubated for the indicated time-points, up to 108 h. Cells were then harvested, and surface levels of (A) CCR2 and (B) CCR5 were analyzed by FACS. Results are the mean ± SEM of at least three independent experiments.

CCR1 expression is dependent on an intact cytoskeleton
The number of receptors on the cell surface depends on the rate of internalization versus the rate of replacement, which in turn, may be regulated by the cell cytoskeleton. Microtubules, along with actin filaments and microfilaments, are the components of the cell skeleton, which determine the internal transport framework. Colchicine binds to the soluble tubulin heterodimer, one of the main constituents of microtubules, thus inhibiting microtubule polymerization. Cytochalasins resemble capping proteins and bind to the barbed end of actin filaments, thus inhibiting the association and dissociation of subunits at that end. Cytochalasin D inhibits CXCR1 and CXCR2 internalization [30 ], suggesting an involvement of actin polymerization in chemokine receptor internalization and recycling. To investigate whether the cell cytoskeleton is necessary for the RSV-induced increase in CCR1 surface levels, cells were treated with the cytoskeletal disruptors at the start of the culture or 48 h post-RSV-CM treatment to dissect out separately any effects on either peak of expression. All doses for these and the other inhibitors used in this study were in accordance with previous work [25 ] and were such that the maximal concentrations of inhibitor left typically greater than 90% viability, as assessed by trypan blue, by the end of the culture period. Cell viability was no different than when cells were incubated in the presence of the diluent DMSO alone. In addition, none of the inhibitors used decreased the proportion of FACS events, falling within a monocyte gate during FACS analysis and confirming that monocytes were not damaged by the conditions. Further preliminary analysis of cell fragmentation by FACS demonstrated that although fragmentation inevitably increased over a total 96-h incubation period, this was not increased above control by any inhibitor. Colchicine and cytochalasin D caused a dose-dependent decrease in monocyte surface CCR1 expression at the 36-h (Fig. 4A and 4B ) and the 96-h (Fig. 4C and 4D) peaks. Thus, an intact cytoskeleton is required for effective transport of CCR1 to the cell membrane and/or for promoting its stability there.


Figure 4
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  		Figure 4. Effect of cytoskeletal inhibitors on CCR1 expression. To examine the requirement of the cytoskeleton for the first peak of CCR1 expression, RSV-CM and (A) colchicine (0.002–2 mM) or (B) cytochalasin (0.01–10 mM) were added to monocytes at Time 0. Cells were then incubated for 36 h prior to analysis of CCR1 by FACS. To investigate the second peak of CCR1, inhibitors were added 48 h following CM treatment, and cells were assayed at 96 h (C, D). Results are the mean ± SEM of at least three independent experiments.

Surface CCR1 is not stored intracellularly
Up-regulation of chemokine receptors may depend on recycling of internalized receptors back to the cell surface and/or on new receptor synthesis. We therefore next investigated intracellular CCR1 expression. Cells were permeabilized with saponin to allow detection of intracellular protein, as confirmed by FACS analysis of the entirely internal, cytoskeletal protein ß-actin (Fig. 5A ). After 36 h and 72 h stimulation with RSV-CM or C-CM, levels of CCR1, in the presence or absence of saponin, were then assessed (Fig. 5B) . Little CCR1 protein was found in unstimulated cells, at the surface or internally, demonstrating that monocytes do not store preformed CCR1. At 36 h, when surface CCR1 expression was maximal, there were only slightly higher levels of total versus external CCR1 (net mean fluorescence intensity for total=8.8±2.8 vs. external only =7.4±4.4, n=3). At 72 h post-treatment, which corresponds to the trough in surface CCR1, as expected, we found low levels of external CCR1; however, total CCR1 was only marginally greater, indicating that recycling of the receptor did not account for the second wave of surface CCR1 expression.


Figure 5
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  		Figure 5. Effect of RSV-CM on intracellular CCR1 storage. (A) Monocytes were left untreated or treated with 5 µg/ml saponin, and then, lysates were analyzed by FACS to detect intracellular ß-actin as a control for permeabilization. (B) Cells were treated with RSV-CM or C-CM for 36 or 72 h and then left intact or permeablized with 5 µg/ml saponin, Extracellular and total CCR1 levels could then be assessed by FACS. Results are the mean ± SEM of at least three independent experiments.

Up-regulation of CCR1 requires transcription and new protein synthesis
As little CCR1 is stored within the cells, basally or subsequent to cell activation, we investigated the requirement for new transcription and translation. Transcription was blocked using actinomycin D, which inhibits DNA-dependent DNA and RNA synthesis specifically. Cycloheximide was used to inhibit protein synthesis via its ability to block the peptidyl synthetase activity of eukaryotic ribosomes. Addition of actinomycin D (Fig. 6A ) or cycloheximide (Fig. 6B) at the start of the culture caused a dose-dependent decrease in the first peak of CCR1 expression at 36 h, and cycloheximide abolished the response to RSV-CM at its highest dose. The second peak of CCR1 expression was similarly blocked when inhibitors were added 48 h after RSV-CM stimulation (Fig. 6C and 6D) . As described above, preliminary data demonstrated that these inhibitors, at the concentrations used, did not alter cell viability or fragmentation significantly during the incubation time. Together, our data therefore indicate that new transcription and de novo protein synthesis are required for RSV-CM-induced CCR1 up-regulation.


Figure 6
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  		Figure 6. Role of transcription and translation in CCR1 expression. Actinomycin D (0.01–10 mg/ml) or cycloheximide (0.002–2 mg/ml) was added to monocyte cultures to block transcription and translation, respectively. To investigate the initial up-regulation of monocyte CCR1 expression, inhibitors were added at the same time as RSV-CM, and then, cells were incubated for 36 h prior to analysis of CCR1 by FACS (A, B). To investigate the second peak of CCR1, inhibitors were added 48 h following CM treatment, and cells were assayed at 96 h (C, D). Results are the mean ± SEM of at least three independent experiments.

Effects of RSV-CM can be blocked by neutralizing cytokines
It is well documented that RSV infection of airway epithelial cells can induce an array of mediators, including TNF-{alpha}, IL-1, IL-6, CCL5, and CXCL8 [13 , 31 , 32 ]. Incubation with purified RSV resulted in production of IL-1{alpha}, IL-1ß, and TNF-{alpha} at mean concentrations of ~120, 20, and 75 pg/ml, respectively [31 ]. RSV-CM therefore represents a cocktail of inflammatory and other agents, many of which are likely to activate monocytes. To dissect out the mediator(s) contributing to the up-regulatory effect of RSV-CM on monocyte CCR, specific blocking agents were used to neutralize the effects of individual cytokines. These experiments showed that blocking TNF-{alpha} reduced the effect of RSV-CM markedly on CCR1 expression, inhibiting expression at the first and second peaks by 51 ± 2.3% and 42 ± 7.7%, respectively. Further, IL-1Ra partially blocked CCR1 expression (percent inhibition=32±7.2 and 23±2.9 for the first and second waves of CCR1 up-regulation, respectively). Blocking IL-1 and TNF-{alpha} simultaneously gave an additive inhibition, and blocking CCL5, a major ligand for CCR1, did not affect CCR1 expression significantly at either point (data not shown). Thus, TNF-{alpha} along with IL-1 are key contributors of CCR1 RSV-CM-induced up-regulation.


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DISCUSSION
 
In this study, we have demonstrated that networks between human epithelial cells and monocytes during RSV infection are important in up-regulating chemokine receptors. RSV-CM but not C-CM caused a prolonged and biphasic up-regulation of CCR1, as well as CCR2 and CCR5, on the surface of monocytes and MDM. Networks acting to increase chemokine secretion are well described. We have shown previously that RSV-CM from monocytes is able to synergize with RSV infection (such as may well occur in infected lung in vivo) to amplify CXCL8 secretion from epithelial cells [21 ]. Similarly, TNF-{alpha} stimulation of nasal epithelial cells in combination with RSV infection induced marked increases in IL-6, CXCL8, and CCL5 when compared with RSV infection alone and TNF-{alpha} stimulation alone [33 ]. In contrast, there have been few previous studies about the role of cytokine networks on expression of cell surface molecules in RSV infection. Jamaluddin et al. [34 ] have demonstrated an important paracrine network in which RSV-CM feeds back onto epithelial cells to induce MHC Class I expression and proteasome activity. Also, vascular endothelial cells exposed to CM from RSV-infected A549 cells express increased surface ICAM-1 and to a lesser extent, VCAM-1 and E-selectin [35 ].

The biphasic nature of the response is interesting in that it may represent an immediate and a delayed response to RSV-CM. This has potential significance pathophysiologically, as although an effective immune response is necessary for viral clearance, it is the excessive and prolonged chemokine secretion that leads to deleterious effects. In particular, CCL3 and CCL5, ligands of CCR1, have been associated with disease severity. The temporal release of chemokines may be key in determining the inflammatory response, and the effectiveness of inhibitors may depend to some extent on the kinetics of the response [36 ]. It may be possible to intervene pharmaceutically to block the actions of IL-1/TNF-{alpha} before the second CCR peak of the response, as this occurs 48–96 h after initial exposure to virus-induced mediators, which is when patients are more likely to present to physicians. Such an approach does not necessarily require knowledge of the cofactors working with these proinflammatory cytokines to up-regulate CCRs, as the aim is only to block synergy. Consistent with this idea, murine models have shown that blocking the actions of CCL5, even 5 days after RSV challenge of animals, was still effective at reducing subsequent airway hyper-reactivity, suggesting that the temporal nature of its actions would make it an effective target even once infection has been established [16 ]. Our work clearly demonstrates CCR up-regulation in response to RSV in vitro and highlights the role that cell networks play in the events following initial infection of the primary target cells. However, further studies are necessary to understand the responses of the chemokine receptor in vivo. Consistent with our findings, a recent study has also found increased expression of CCR1, CCR2, and CCR5 in a mouse model of RSV infection [37 ]. Although in this case, no biphasic response is seen, this is perhaps not surprising, as whole lung mRNA was studied; thus, the primary cell source infected with virus, particularly initially following infection, will be pulmonary epithelial cells.

Our data show that the cytoskeleton is important in the regulation of CCR1 up-regulation, as both peaks of surface CCR1 were inhibited by blockers of microtubule and actin filament function. CCR regulation has been studied mostly in the context of events surrounding receptor activation. After ligation, chemokine receptors may be internalized and then degraded or recycled, leaving the membrane temporarily unresponsive to further stimulation [18 , 38 , 39 ]. Chemokine receptors may also become desensitized via phosphorylation of the C-terminal region, allowing binding of regulatory molecules—arrestins [40 ]. Activation of a Rho family GTPase is critical for CCR1- and CCR5-triggered signaling cascades and induces actin reorganization and the formation of lamellipodia [41 ]. Our data show that the cytoskeleton is required for CCR1 delivery and expression on the monocyte surface after stimulation with RSV-CM. Microtubules were also involved in this process, which is consistent with the fact that cytochalasin D has been shown to block the internalization process of CCR5 [39 ]; our results suggest further that actin filaments are also vital for the presentation of CCR to the cell surface. At this time, we are unable to say whether the receptors we see at the surface also become activated by RSV-CM, which will affect how they are regulated. Work is currently underway to elucidate the functional responsiveness of the up-regulated receptors at different stages in the biphasic response.

There was little sequestered cytoplasmic or endosomal reserve of CCR protein in resting cells; thus, we found no evidence of receptor recycling. Consistent with this, experiments using transcription and translational blockers demonstrated that both phases of CCR expression were preceded by de novo mRNA and protein synthesis. Thus, as yet, undefined events lead to degradation and subsequent resynthesis of CCR after the 36-h peak. Other regulators of CCR expression are also known to act at the level of de novo CCR production, although via different mechanisms. LPS does not inhibit the rate of nuclear transcription of CCR2 but reduces mRNA half-life from 1.5 h to 45 min [42 ], and IL-10 selectively up-regulates the expression of CCR1, -2, and -5 in human monocytes by prolonging their mRNA half-life [43 ].

Cytokine neutralization experiments revealed a key role for TNF-{alpha} in RSV-CM-induced CCR1 up-regulation. This is significant, as a number of studies have demonstrated the presence of TNF-{alpha} in the airways of infants with RSV [44 45 46 ], and TNF-{alpha} has been linked with severity of illness in a murine model [47 ]. We also found a role for IL-1 in the up-regulation of CCR1 and have shown previously that this cytokine may have an important role in amplifying chemokine release from RSV-infected cells [21 ]. IL-1{alpha} has been identified as the predominant endothelial cell-activating factor in CM from RSV-infected epithelial cells [35 ] and is a primary mediator of enhanced CXCL8 production in RSV-infected epithelial cells [48 ]. IL-1{alpha} is also a key RSV-induced mediator involved in autocrine ICAM-1 and IL-6 expression [31 , 49 ]. The effect of these proinflammatory cytokines on CCR1 demonstrate a further key role for TNF-{alpha} and IL-1 in RSV-induced pathology. Although at present, we cannot determine the precise mechanisms behind the biphasic response in CCR1 up-regulation observed, it is possible that this effect relates to a secondary autocrine effect. As monocytes themselves may secrete TNF-{alpha} and IL-1, along with other cytokines and chemokines, when stimulated, it is feasible that the initial stimulus of RSV-CM leads to a second wave of cytokine release. This would involve similar mechanism to that operating when IL-1 stimulates its own gene expression and synthesis in vascular smooth muscle cells, endothelial cells, and monocytes [50 ]. It is alternatively possible that release of other mediators into culture medium or altered cellular responses following prolonged exposure to stimulus may drive a second CCR peak. Such processes may act in synergy with proinflammatory cytokines. Blocking experiments demonstrate the requirement of TNF-{alpha} and IL-1 but reveal nothing as to what other factors interact with these cytokines.

Although blocking TNF-{alpha} and IL-1 had a major effect on CCR increase, this did not abrogate the response completely, suggesting additional mediators, possibly IL-6, may be active at the earlier stage. Such mediators may explain why our data contrast with the finding that IL-1 and TNF-{alpha} may down-regulate CCR2 and to a lesser extent, CCR1 and CCR5 expression on monocytes [42 ]. Other factors within the RSV-CM may well be acting to promote or enhance the IL-1/TNF-{alpha} effect. For example, we have not investigated IL-10, which has a strong up-regulatory effect on monocytes CCR1, CCR2, and CCR5 expression [43 ] and may over-ride the inhibitory effects of LPS, which alone causes strong down-regulation of CCR but in combination with IL-10, results in high expression of CCR1, CCR2, and CCR5 on treated monocytes [51 ].

In summary, we have found that networking between RSV-infected epithelial cells and monocytes leads to a prolonged, biphasic up-regulation of CCR1, which is dependent on an intact cytoskeleton and requires new transcription and translation. The high level of inflammation that occurs in severe RSV disease may be partly a result of overexpression of chemokines and cytokines in conjunction with up-regulation of monocyte chemokine receptors. This will promote inflammatory cell recruitment and activation of the recruited cells at sites of infection. Blockade of CCR1 could help limit excessive cellular influx and be a potential, therapeutic target.


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ACKNOWLEDGEMENTS
 
Asthma UK grant number 01/046 supported this work.

Received October 6, 2006; revised February 7, 2007; accepted February 12, 2007.


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