Rhinovirus Regulation of IL-1 Receptor Antagonist In Vivo and In Vitro: A Potential Mechanism of Symptom Resolution

Inflammatory responses to infectious agents need to be robust enough to rid the host of the offending agent yet restrained enough to prevent tissue injury. When appropriately regulated, infection-induced inflammation erradicates the invading pathogen and resolves without inducing adverse structural alterations. Rhinovirus (RV)⁴-induced infections are the prototypes of these regulated transient inflammatory responses. Patients with RV infections experience a short incubation period that is followed by symptomatology (fever, chills, rhinorrhea, sneezing and nasal congestion, etc.) and tissue inflammation (1, 2, 3, 4). In the majority of cases, the symptoms and tissue inflammation resolve over the ensuing few days, giving RV-induced upper respiratory tract infections their highly characteristic transient nature. RV infections are the most common acute infectious illnesses in humans (1, 2, 4). In spite of their obvious medical importance, the pathogenesis of RV infections is poorly understood and virtually nothing is known about the cellular and molecular events that are involved in the resolution of RV-induced inflammation and symptomatology.

RV does not induce mucosal cell cytotoxicity comparable to that caused by many other viral agents (1, 4, 5, 6). Instead, it is believed that the manifestations of RV infections are the result of the host inflammatory response to the virus and that this response is mediated, in great extent, by the production of a variety of proinflammatory mediators by RV-infected epithelial cells (7, 8, 9, 10). Support for this concept and insight into the mechanism(s) of this response comes from studies from our laboratories and others that demonstrated the exaggerated accumulation of nasal kinins, IL-1β, IL-6, and IL-8 in the setting of RV-induced upper respiratory tract infections (7, 8, 9, 10) and that RV stimulates epithelial IL-6 and IL-8 elaboration via an NF-κB-dependent transcriptional mechanism(s) (9, 10). A variety of cytokines and soluble receptors are known to inhibit local tissue inflammation (11, 12, 13, 14, 15, 16). Their endogenous production has been implicated in the resolution and titration of in vivo inflammatory responses, and recombinant versions of these moieties have become potential therapeutic agents in inflammatory disorders. Despite the demonstrated importance of these moieties in the resolution and regulation of tissue inflammation, virtually nothing is known about the ability of RV to stimulate the production of these inhibitors or their importance in the termination of RV-induced inflammation and symptomatology.

The IL-1 receptor antagonist (IL-1ra) is an important inhibitor of tissue inflammation and injury (reviewed in 11). Multiple structural variants of IL-1ra have been described that result from the alternative splicing of a single gene (11, 17, 18, 19). These moieties bind to IL-1R without inducing an intracellular response and thereby regulate the effector functions of competing bioactive IL-1 moieties (11). We hypothesized that alterations in IL-1ra elaboration are present at sites of RV infection and that these alterations contribute to the transient nature of RV-induced inflammation and symptomatology. To test this hypothesis, we characterized the levels of IL-1ra in the nasal secretions of normal volunteers before and after experimental RV infection and determined whether RV induced IL-1ra elaboration from normal airway epithelial cells in vitro. The human studies demonstrate that IL-1ra is present in the nasal secretions of normal volunteers, that the levels of nasal IL-1ra increase impressively in patients with symptomatic RV infections, that maximal IL-1ra induction is prolonged compared with the earlier and transient induction of proinflammatory IL-1 moieties, and that RV stimulates the elaboration of intracellular IL-1ra (icIL-1ra) type I in all and secreted IL-1ra (sIL-1ra) in some volunteers. The in vitro studies demonstrate that airway epithelial cells may be an important source of IL-1ra because normal human bronchial epithelial cells (NHBE) contain icIL-1ra type I mRNA and protein and RV increases the intracellular levels and the extracellular release of this IL-1ra moiety via a novel translational and/or posttranslational mechanism(s).

The clinical study protocol that was employed has been previously described (8, 9). In brief, healthy young adults 18 years of age or older with reciprocal serum neutralizing Ab titers of ≤2 to the challenge virus were recruited from the University of Virginia student body. Viral challenges were performed by administering twice in each nostril 0.25-ml volumes of either RV type 39 or RV strain Hanks (not neutralized by antisera to 89 numbered rhinovirus types, acid sensitive, and chloroform-idoxuridine resistant). Both are major serotype viruses that use ICAM-1 for cell entry. A total dose of 800 tissue culture ID₅₀ of RV strain Hanks or 2500 tissue culture ID₅₀ of RV type 39 were employed. Starting on the morning before challenge and at 24 h intervals thereafter, all volunteers were interviewed regarding the presence and severity of the following 10 symptoms: sneezing, nasal discharge, nasal congestion, malaise, headache, chills, feverishness, sore throat, hoarseness, and cough. Symptoms were rated for severity on a scale from 0 to 3. Patients were designated as having a “cold” if they had a total symptom score of ≥5 over the 5 days after challenge plus either nasal discharge for 3 days or the belief of the subject that a cold had occurred. Sham inoculation was conducted in the same manner except that HBSS instead of viral solution was used.

Nasal lavages were performed with 10 ml of isotonic saline once per day on study day 0 (before challenge) to day 5. One portion of the lavage fluid was used for viral culture. The other aliquot was stored at −70°C until its cytokine content was assayed. Viral culture was accomplished by combining lavage fluid with concentrated veal infusion broth and inoculating monolayers of MRC-5 human fetal lung fibroblasts (BioWhittaker, Walkersville, MD). One isolate from each subject was identified as the challenge virus using a standard neutralization test.

Homotypic neutralizing Ab titers were determined by standard tests on blood collected before and 3 wk after inoculation (20). Volunteers were considered infected if they shed virus or had a 4-fold or greater rise in serum Ab titer. This allowed us to compare volunteers that were exposed to virus and did not get infected or ill (not infected/not ill), volunteers that became infected and had “colds” (infected/ill volunteers), and volunteers that were infected but did not manifest “colds” (infected/not ill volunteers).

RV14 was obtained from the American Type Culture Collection (Manassas, VA). Viral stocks were prepared by infection of sensitive cell systems with a low input multiplicity of infection (MOI). When infection was advanced, cell supernatants were harvested, cells were disrupted by freezing and thawing, and debris was pelleted by low-speed centrifugation. Aliquots of clarified supernatants were frozen at −70°C. Viral adsorption was performed at 37°C. Incubations were performed at 33°C for RV. Titers of infectivity of stock viruses were determined by inoculation of serial dilutions into sensitive cell systems and quantification of plaque formation as previously described (21). IL-1β, TNF-α, and endotoxin were not detectable in the stock preparations.

For selected experiments, RV stock was exposed to UV light or further purified using sucrose gradients. The UV exposure was performed as previously described (9). To accomplish the purification, virus stock was concentrated by centrifugation at 150,000 × g at 4°C for 45 min using a Beckman L5-50 centrifuge and a SW50.1 rotor (Beckman Instruments, Palo Alto, CA). The resulting viral pellet was resuspended in NTE buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.4, 1 mM EDTA) and overlaid onto a two-layer sucrose cushion containing 2.4 ml of 60% sucrose in NTE in the bottom layer and 2.4 ml of 30% sucrose in NTE in the top layer. Centrifugation was then repeated for 90 min, the interface containing virus was collected and filter sterilized, viral titers were quantified via plaque assay, and purified virus was aliquoted and stored at −70°C until used.

NHBE and serum-free bronchial epithelial cell growth medium with supplements were purchased from Clonetics (San Diego, CA). These cells were grown and subcultured following the supplier’s instructions, and only cells in passages 2 and 3 were used for these experiments. On the day of infection, the medium was aspirated and cultures were inoculated with virus stock at an MOI of 3.0. After adsorption at 37°C for 90 min, the viral solution was removed, the cells were washed with PBS, bronchial epithelial cell growth medium was introduced, and the cells were incubated at 33°C for the desired period of time. The supernatants were removed at designated time points, clarified by low-speed centrifugation, and stored at −70°C until analyzed. The cell monolayers were then rinsed with PBS and harvested for quantification of levels of icIL-1ra protein or mRNA as described below.

The levels of immunoreactive IL-1α, IL-1β, IL-6, and IL-1ra in the nasal lavage fluids, NHBE supernatants, and NHBE lysates were quantitated using ELISA kits obtained from R&D Systems (Minneapolis, MN) according to the manufacturer’s protocol. These assays can detect as little as 5–15 pg/ml of the noted moieties.

Cell viability was assessed by trypan blue dye exclusion and lactate dehydrogenase (LDH) release assay. The release of intracellular LDH was determined using an LDH assay kit purchased from Sigma (St. Louis, MO), according to the manufacturer’s instructions.

Total cellular RNA was extracted from cell monolayers at desired time points using the acid-guanidinium thiocyanate-phenol-chloroform extraction protocol described by Chomczynski and Sacchi (22). Equal amounts (10 μg) of RNA from each experimental condition were size fractionated by electrophoresis through 1% agarose, 17% formaldehyde gels, transferred to nylon membranes, and hybridized with ³²P-labeled cDNA probes. Clone HTAAA12, which contains a 1.7-kb IL-1ra cDNA insert that hybridizes with all IL-1ra isoforms, was purchased from the American Type Culture Collection. This clone was isolated using restriction enzymes EcoRI and XhoI and labeled to a high sp. act. (10⁹ cpm/μg DNA) using a random primer method. Hybridization was assessed after washing under conditions of increasing stringency and quantitated via autoradiography. The adequacy of gel loading was routinely assessed by ethidium bromide staining and by stripping the membrane and reprobing with a cDNA encoding β-actin.

Western blot analysis was used to determine which IL-1ra moieties were present in the human nasal lavage fluids, NHBE cell supernatants, and NHBE cell lysates. The approach that was employed was modified from Levine et al. (23). The nasal lavages were performed before and at intervals after RV challenge as described above. Uninfected and RV-infected NHBE cells were cultured for 48 h as noted above. The supernatants were then removed, a protease inhibitor mixture (complete protease inhibitor mixture tablets; Boehringer Mannheim, Mannheim, Germany) was added, and the supernatants were concentrated 50-fold by sequential ultrafiltration with 50 kDa and 10 kDa molecular mass cut-off Amicon filters. The cell lysates were generated by resuspending the cell layers in a cell culture lysis reagent (Promega, Madison WI) (25 mM Tris-HCl, pH 7.8, 2 mM DTT, 2 mM 1, 2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid, 10% glycerol, and 1% Triton X-100) supplemented with complete protease inhibitor mixture tablets.

The total protein content of each of the samples was measured using the bicinchoninic acid protein reagent (Pierce, Rockford, IL). Equal amounts of test and control sample protein and positive controls (see below) were fractionated on 15% SDS-PAGE gels under reducing conditions and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA). The membranes were then incubated overnight in blocking buffer (5% w/v) nonfat dry milk in PBS/0.1% Tween) at 4°C. The membranes were then incubated for 2 h at room temperature with primary goat anti-human IL-1ra (R&D Systems), which recognizes sIL-1ra, icIL-1ra type I, and icIL-1ra type II. The membranes were then washed three times in PBS/0.1% Tween and incubated for 2 h at room temperature with mouse anti-goat IgG (Pierce). Immunoreactive IL-1ra was detected using a chemiluminescent procedure (ECL Western blotting detection system; Amersham Life Science, Buckinghamshire, U.K.) according to the manufacturer’s instructions.

Two positive controls were used when needed in these studies (23). The first was recombinant human nonglycosylated mature sIL-1ra (R&D Systems). This is a 153 aa protein that lacks a leader sequence and migrates with a molecular mass of ∼17 kDa. The second was lysates from PMA-differentiated U937 monocyte-like cells incubated for 48 h with recombinant human IL-4 (10 ng/ml) (R&D Systems). As described (24), these samples contain easily detectable glycosylated mature sIL-1ra and icIL-1ra type I, which migrate with molecular masses of ∼25 and 16–18 kDa, respectively.

The total cellular RNA from uninfected and RV-infected NHBE cells was extracted using Trizol Reagent (Life Technologies) according to the manufacturer’s instructions. Reverse transcription and PCR was performed using the Access RT-PCR kit purchased from Promega according to the manufacturer’s instructions. The protocol and RT-PCR primers that were employed were those described by Muzio et al.(18). All primers were synthesized in the Yale Oligonucleotide Synthesis Laboratory. The primer pairs that were used and the size of their reaction products are sIL-1ra: 5′-GAA TGG AAA TCT GCA GAG GCC TCC GC-3′, 5′-GTA CTA CTC GTC CTC CTG G-3′, 539 bp; icIL-1ra type I: 5′-CAG AAG ACC TCC TGT CCT ATG AGG C-3′, 5′-GTA CTA CTC GTC CTC CTG G-3′, 515 bp; icIL-1ra type II: 5′-CTG ACT TGT ATG AAG AAG GAG GTG G-3′, 5′-GTA CTA CTC GTC CTC CTG G-3′, 536 bp; β-actin: 5′-GCG CTC GTC GTC GAC AAC GG-3′, 5′-GAT AGA CAA CGT ACA TGG CTG-3′, 390 bp.

The RT-PCR reaction products were fractionated on 1% agarose gels, and the ethidium bromide stained bands were visualized and photographed under UV light. The DNA was then transferred to a nylon membrane, hybridized using a ³²P-radiolabeled probe, washed, and evaluated via autoradiography. The internal probe that was employed (5′-GCG AGA ACA GAA AGC AGG ACA AGC G-3′) hybridizes with RT-PCR products from all IL-1ra isoforms.

Data that could not be assumed to be normally distributed is expressed as medians and interquartile ranges and analyzed with the Kruskal-Wallis test when comparing three variables and the Mann-Whitney U test when comparing two variables. Normally distributed data is expressed as means ± SEM and is assessed for significance with the Student’s t test or ANOVA as appropriate.

To begin to address the roles of IL-1 family cytokines in normal nasal physiology, lavages were performed on normal volunteers and nasal IL-1α, IL-1β, and IL-1ra were quantitated by ELISA. The levels of IL-1α and IL-1β in these fluids were near or below the limits of detection of our assays (Tables I–III). In contrast, IL-1ra was readily detected. In the 25 patients studied, the levels of IL-1ra ranged from 1,077 to 14,655. Overall, a median of 1737 pg/ml IL-1ra was appreciated. These studies demonstrate that IL-1ra moieties are present in vast excess compared with IL-1α and IL-1β in the normal nose.

In these experiments, normal volunteers were inoculated with RV and their symptoms were monitored and nasal lavage IL-1α, IL-1β, and IL-1ra were quantitated at 24-h intervals. Before RV infection, statistically significant differences in the levels of IL-1ra were not noted in the nasal lavage fluids from the three study populations (Fig. 1 and Table I). As detailed in Fig. 1 and Table I, symptomatic RV infection (infected/ill patients) was associated with an impressive increase in the levels of immunoreactive IL-1ra. This induction could be seen within 24 h, peaked 48 h after RV administration, and remained at near maximal levels for an additional 24 h (Fig. 1 and Table I). Significant increases in IL-1ra were not noted in patients that became infected but not symptomatic (infected/not ill) or in patients that did not become infected (not infected/not ill). RV-induced increases in IL-1α and IL-1β were also appreciated (Figs. 2 and 3 and Tables II and III). However, the magnitude of these inductive responses were small compared with the induction of IL-1ra. In addition, the kinetic of the IL-1 and IL-1ra responses were quite different, with IL-1α and IL-1β peaking transiently 48 h after RV administration and returning to normal 24 h later. Interestingly, RV-induced symptomatology peaked 48 h after virus administration and decreased substantially 72 h after RV exposure (Fig. 4). These studies demonstrate that symptomatic RV infections are characterized by the transient induction of IL-1 cytokines (IL-1α and IL-1β) and prolonged induction of IL-1ra. They also highlight temporal associations between peak symptomatology and the peak levels of IL-1 cytokines and symptom resolution and the prolonged production of IL-1ra.

To determine whether sIL-1ra, icIL-1ra type I, or icIL-1ra type II were produced in uninfected and/or RV-infected nasal tissues, Western blot analysis was employed. The nasal isoforms that were noted are illustrated in Fig. 5. Immunoblot analysis was not always sensitive enough to detect the IL-1ra in the nasal washings from uninfected volunteers. However, when IL-1ra was appreciated it appeared as a single protein with a slightly higher apparent molecular mass than the recombinant human nonglycosylated mature sIL-1ra control protein, which is consistent with the icIL-1ra type I isoform (23). The IL-1ra isoforms seen after RV infection showed some individual to individual variability. In all cases, the enhanced expression of icIL-1ra type I could be appreciated (patients A and B in Fig. 5). In some patients, a larger IL-1ra moiety consistent with glycosylated sIL-1ra could also be appreciated (patient A; Fig. 5). These studies demonstrate that icIL-1ra type I is present in the normal nose. They also demonstrate that symptomatic RV infection is characterized by the enhanced accumulation of icIL-1ra type I and in some volunteers also sIL-1ra.

Because epithelial cells are the major site of RV infection in vivo, studies were undertaken to determine whether respiratory epithelial cells produce IL-1ra before or after RV infection. Under basal conditions, NHBE cells produced only modest levels of IL-1ra (Fig. 6). However, after RV infection IL-1ra elaboration was significantly augmented. This induction was time dependent with an interesting kinetic. Increases in supernatant IL-1ra could not be detected until 24 h after and were more impressive 48 h after RV infection. This contrasted with the production of IL-6 by these cells, which could be appreciated as early as 4 h after RV infection (Fig. 6 and data not shown). This induction also appeared to be virus mediated because it was decreased by 86.4% when the RV stock was exposed to UV light before monolayer incubation (n = 5) (p < 0.05). These studies demonstrate that epithelial cells are an important source of IL-1ra and that RV augments epithelial IL-1ra elaboration with a slower kinetic than that seen with epithelial IL-6.

To further compare the effects of RV in our in vivo and in vitro systems, we characterized the IL-1ra moieties in the supernatants and lysates of NHBE cells before and after RV infection. Western analysis of the supernatants demonstrated IL-1ra with a molecular mass compatible with icIL-1ra type I (Fig. 7). Bands compatible with sIL-1ra and icIL-1ra type II were not noted. Similarly, lysates from NHBE cells, at baseline and after RV infection, contained IL-1ra moieties compatible with icIL-1ra type I but not sIL-1ra or icIL-1ra type II (Fig. 7). In accord with these findings, RT-PCR revealed mRNA for icIL-1ra type I but not sIL-1ra or icIL-1ra type II at baseline and after RV infection (Fig. 8). These studies demonstrate that icIL-1ra type I is the major IL-1 moiety in and elaborated by NHBE cells at baseline and after RV infection. They also raise the possibility that the icIL-1ra type I in the nasal lavage fluids from uninfected and RV-infected volunteers may be epithelial derived.

To further understand the mechanism by which RV stimulates epithelial cell elaboration of icIL-1ra type I, we quantitated the levels of IL-1ra mRNA and IL-1ra protein in NHBE cells and supernatants before and after virus infection. mRNA-encoding IL-1ra was readily detected by Northern analysis in unstimulated NHBE cells (Fig. 9). Interestingly, RV infection did not cause impressive alterations in the levels of this mRNA transcript. A slight (<2-fold) stimulation of IL-1ra was noted 2–4 h after virus inoculation. Overall, however, significant RV-induced effects were not appreciated (Fig. 9). Similarly, significant alterations in the levels of IL-1ra mRNA were unable to be appreciated by RT-PCR 4–48 h after RV infection (Fig. 8 and data not shown). These studies demonstrate that RV stimulates NHBE cell elaboration of IL-1ra, at least in part, via a translational and/or posttranslational mechanism(s). In accord with this observation (Fig. 10), there were impressive levels of immunoreactive icIL-1ra type I in lysates and minimal icIL-1ra type I in supernatants from unstimulated NHBE cells at baseline, and RV infection increased these levels by ∼2- and 12-fold, respectively (Fig. 10). Interestingly, the increase in icIL-1ra type I in NHBE cell supernatants was not associated with LDH release or evidence of cell cytotoxicity as assessed via trypan blue dye exclusion (data not shown). When viewed in combination, these studies demonstrate that NHBE cells contain significant amounts of icIL-1ra type I at baseline and that RV infection increases the production and extracellular transport of this IL-1ra moiety, at least in part, via a noncytotoxic translational and/or posttranslational mechanism.

On average, adults experience 2–4 and children experience 4–10 RV infections per year (25, 26, 27). A cardinal and universally appreciated feature of these infections is their transient nature. It is reasonable to hypothesize that this transience is the result of the ability of the host to rapidly clear the virus and/or the induction, by the virus, of antiinflammatory cellular events. To address the latter possibility, studies were undertaken to determine whether RV stimulated the production of antiinflammatory cytokines in vivo and/or in vitro. The IL-1 cytokine system was chosen for this analysis because of its known complexity (11, 12) and the potential importance of IL-1 in the pathogenesis of RV-induced disorders (8). These studies add in many important ways to our knowledge of the pathogenesis of RV infections. They demonstrate for the first time that the levels of nasal IL-1ra increase impressively in patients with symptomatic RV infections and that this stimulation is the result of RV-induced elaboration of icIL-1ra type I in all and sIL-1ra in some volunteers. These studies also demonstrate that human RV infection is characterized by the transient elaboration of IL-1 (α and β) and prolonged elaboration of IL-1ra with the peak levels of IL-1 correlating temporally with the period of peak symptomatology and the prolonged production of IL-1ra correlating with the onset of symptom resolution. Lastly, they demonstrate that airway epithelial cells may be an important source of IL-1ra by demonstrating that NHBE cells contain mRNA and protein for icIL-1ra type I and that RV increases the intracellular levels and extracellular release of this IL-1ra moiety via a novel mechanism that is, at least partially, independent of the levels of icIL-1ra mRNA.

In contrast to other visceral structures, the nose and respiratory tract are constantly exposed to inhaled Ags and particulates. In spite of this constant stimulation, normal nasal and respiratory structures do not manifest ongoing inflammation. The cellular mechanisms that prevent these inflammatory responses are poorly understood. Our studies demonstrate that the normal nose contains significant amounts of immunoreactive IL-1ra. This suggests that the constitutive elaboration of IL-1ra may be an important mechanism by which normal respiratory structures are kept free of chronic inflammation. The finding that IL-1ra is also produced in a constitutive fashion in the gastrointestinal tract (28, 29) suggests that the constitutive elaboration of IL-1ra may be a protective mechanism that is common to many mucosal surfaces.

A complete understanding of the effect(s) of an alteration in one component of the IL-1 cytokine system must include an understanding of how the intervention effects the other components and how the components interact with each other. Thus, to understand the role of IL-1ra in the pathogenesis of RV infections, we also characterized the effects of RV on nasal IL-1α and IL-1β production. These studies demonstrate that the nasal lavage fluids from infected/ill volunteers contain higher levels of IL-1α and IL-1β than those from infected/not ill individuals. This is in keeping with the present belief that the host inflammatory response is the major cause of RV-induced symptomatology (reviewed in 2). Importantly, these studies also demonstrate, for the first time, that symptomatic RV infections are associated with the transient induction of proinflammatory IL-1 and prolonged induction of antiinflammatory IL-1ra moieties. Our studies also demonstrate a temporal association between the peak levels of IL-1 and peak volunteer symptomatology and between the prolonged induction of IL-1ra and the onset of symptom resolution. IL-1ra inhibits IL-1 effectively only when present at IL-1ra: IL-1 ratios of 100–1,000:1 or greater (11, 30). Our studies are in accord with this observation because high IL-1ra/IL-1 ratios were noted before RV administration and at the onset of symptom resolution (72 h). Appropriately, the IL-1ra:IL-1 ratios were ≥100:1 for all volunteer groups before RV challenge and ≥100:1 72 h after RV inoculation in infected/ill volunteers. In fact, the only time the IL-1ra/IL-1 ratio was <100:1 was 48 h after RV challenge in the infected/ill volunteers, the time point of peak symptomatology. It is important to point out that these studies show correlation and not cause and effect. Causation is difficult to demonstrate because RV causes symptomatic infections only in humans and cannot be effectively studied in animal model systems. However, our results support the contention that the transient elaboration of IL-1 and prolonged induction of IL-1ra in the setting of RV infection can account, at least in part, for the symptoms and the resolution of symptoms, respectively, seen during the course of RV infection. These postulates are in accord with prior studies of Lyme disease in which elevated levels of IL-1ra were shown to correlate with disease recovery (31) and diminished disease-induced symptomatology (32).

IL-1 and IL-1ra can have beneficial and detrimental effects in the setting of infection. This is nicely illustrated in studies with IL-1ra knockout mice that were more susceptible to lethal endotoxemia and less susceptible than controls to listeria monocytogenes and IL-1ra overproducing mice that were protected from the lethal effects of endotoxin but were more susceptible to listeriosis (33). Thus, inhibition of IL-1 appears to protect the organism from overexuberant responses to infection but at the risk of impairing the host’s ability to eliminate infection (33). Our demonstration that the kinetics of elaboration of IL-1 and IL-1ra differ during the course of RV infection may represent an attempt by the host to coordinate the beneficial and detrimental effects of IL-1 in this setting. The early and transient induction of IL-1 would allow the host to mount needed inflammatory and antiviral responses, while the prolonged induction of IL-1ra would allow the host to titrate these responses after the virus has been controlled.

IL-1ra was originally described as an IL-1 inhibitory activity in the urine of patients with fever and the supernatants from monocytes cultured on adherent IgG (11). sIL-1ra was subsequently cloned and demonstrated to be a 177-aa protein with a 25-aa leader sequence. Additional IL-1 moieties have subsequently been appreciated. Two of these moieties (icIL-1ra types I and II) are created by the alternative splicing of a variety of exons and the use of a different promoter. These moieties lack functional leader sequences and are felt to remain in the cytoplasm (11, 18, 19). Subsequent studies have demonstrated that these isoforms are differentially expressed and regulated with mononuclear cells and granulocytes producing predominantly sIL-1ra and keratinocytes and vaginal and respiratory epithelial cells constitutively producing icIL-1ra (11, 19, 23). To begin to address the regulation of IL-1ra, in vivo studies were undertaken to characterize the isoforms of IL-1ra that were induced during the course of experimental RV respiratory infections. These studies demonstrate that, in all symptomatic patients, RV enhanced the accumulation of a nasal moiety compatible with icIL-1ra type I. In some individuals, increases in sIL-1ra were also appreciated. This is the first demonstration of the in vivo dysregulation of icIL-1ra type I in human disease and the first demonstration of individual-individual variation in the isoforms of IL-1ra produced in response to the same stimulus. Our studies also demonstrate that RV infected airway epithelial cells elaborate large quantities of icIL-1ra type I. When viewed in combination, these studies suggest that the icIL-1ra type I and the sIL-1ra found in the nasal secretions of patients with RV infections are derived from epithelial and nonepithelial sources, respectively. The cellular sources of these moieties and the reason why sIL-1ra is elaborated in large quantities by only a subset of RV-infected individuals will require additional study.

Regulation of the effects of IL-1 in the cellular microenvironment would appear to be the major biologic role of extracellular sIL-1ra. icIL-1ra can have similar effects after being released from dead or dying cells or elaborated in a nontoxic fashion after cytokine stimulation (23). However, the fact that multiple isoforms of icIL-1ra have been maintained during evolution suggests that icIL-1ra moieties may have additional functions inside of cells. Support for the idea that icIL-1ra is an intracrine regulator comes from studies demonstrating that intracellular IL-1α may play a role in the growth and differentiation of human endothelial cells (34), that the prohormone of IL-1β inhibits apoptosis by competing for IL-1 converting enzyme (35), that the N-terminal propiece of IL-1α contains a nuclear localization sequence and can act as a transforming oncoprotein (36), and that high levels of icIL-1ra impair IL-1-induced IL-8 and gro elaboration via the destabilization of chemokine mRNA in ovarian cells (37). Interestingly, high levels of constitutive and cytokine-induced icIL-1ra in keratinocytes and transfected fibroblasts have been associated with the down-regulation of ICAM-1 expression (11). ICAM-1 is the cell surface receptor for 85–90% of all RV (the major serotype RVs) (38). This allows for the exciting hypothesis that RV induction of icIL-1ra type I diminishes the surface expression of ICAM-1, thereby diminishing RV infection and RV-induced inflammation. If this hypothesis is correct, IL-1ra can inhibit RV-induced inflammation by acting as a direct IL-1 antagonist and diminishing ICAM-1-mediated RV binding and internalization.

Although RV-stimulated epithelial cell cytokine production plays an important role in the pathogenesis of RV infections, the molecular mechanism(s) that underlies this stimulation is incompletely understood. The information that is available comes from studies from our laboratories that demonstrated that RV stimulates epithelial cell IL-6 and IL-8 production via an NF-κB-dependent transcriptional mechanism (9, 10). Our more recent studies have demonstrated that RV stimulates epithelial RANTES production via a similar NF-κB-dependent transcriptional pathway (Z.Z. and J.A.E., unpublished observations). The present studies demonstrate, for the first time, that RV stimulates epithelial cell IL-1ra elaboration via an impressively different mechanism. NHBE cells were shown to contain large pools of performed icIL-1ra protein and mRNA encoding icIL-1ra type I at baseline. RV infection increased the levels of icIL-1ra and greatly enhanced the transport of icIL-1ra into the pericellular environment. This increase in protein accumulation and enhanced protein transport were not associated with significant increases in IL-1ra mRNA detectable via Northern blot or RT-PCR. Thus, RV stimulates icIL-1ra type I production and elaboration by NHBE cells, at least in part, via a translational and/or posttranslational mechanism. When viewed in conjunction with our studies of IL-6, IL-8, and RANTES, it is clear that RV interacts with epithelial cell cytokine pathways using multiple complex mechanisms.

RV infections are a major public health problem that use impressive quantities of medical resources. In addition to causing the common cold, RV infections are responsible for the majority of flairs of asthma in school age children (39) and precede and predispose to bacterial sinusitis and otitis media and to exacerbations of chronic bronchitis (4, 40). As a result, any therapeutic intervention that diminishes RV-induced symptomatology and inflammation can have a major impact on medical practice. Our studies demonstrate that IL-1ra induction correlates with symptom resolution in experimental RV infection. This raises the intriguing possibility that IL-1ra might be a useful therapeutic for RV-induced pathologies. In prior studies, the utility of IL-1ra in the treatment of infectious diseases has been limited by the need to obtain high IL-1ra/IL-1 ratios to achieve adequate IL-1 antagonism and the appreciation that IL-1 can play an important role in host defense as well as disease pathogenesis (33). However, the local delivery of IL-1ra may allow both of these issues to be overcome because, in theory, high concentrations of nasal IL-1ra can be achieved and the adverse systemic effects of IL-1 antagonism can be minimized via a topical mode of administration.

In summary, these studies demonstrate that RV is a potent stimulator of IL-1ra elaboration in vivo and in vitro. They also highlight the association between IL-1ra elaboration and symptom resolution in volunteers with experimental RV upper respiratory tract infections, the isoform specificity of the in vivo and in vitro inductive responses and the novel mechanism by which RV stimulates epithelial cell IL-1ra elaboration. They suggest that RV stimulated IL-1ra contributes to the resolution of RV-induced inflammation and symptomatology. They also highlight the complexity of the cytokine-based interactions between RV and airway epithelial cells.

We thank the investigators and institutions that provided the reagents that were used, Kathleen Bertier and Millie Phelps for their excellent secretarial assistance, and Dr. Mark Cullen for his assistance with the statistical approaches that were employed.