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Respiratory syncytial virus (RSV) infection in neonates and young infants often causes life-threatening acute bronchiolitis despite the presence of maternally transferred specific neutralizing antibody (4, 23). Immaturity of the immune system and the fragile anatomy of the infant's bronchioles have been proposed as possible reasons for the severity of RSV disease in young infants (11, 18, 24, 29). However, the pathophysiology of RSV bronchiolitis in infants has not been fully clarified.

Recently, the participation of a wide range of inflammatory or immunoregulatory cytokines, such as interleukin-1 (IL-1), IL-6, IL-8, and tumor necrosis factor alpha in the pathology of RSV lower respiratory tract disease has been suggested in in vivo (13, 21) and in vitro (2, 14, 20, 25) settings. It has been proposed that IL-1 produced in the respiratory tract may act as one of the main inflammatory cytokines during the acute phase of the infection, partly by activating other inflammatory cytokines, but also by activation of inflammatory cells and induction of prostaglandins (1, 19). IL-1 may also promote histamine-induced bronchoconstriction in infants during RSV infection because it is known that injection of IL-1 into mice induces formation of histidine decarboxylase, the enzyme that forms histamine, in various tissues, including lung tissue (5).

In human and mouse monocytes, IL-1-converting enzyme (ICE) has been reported as the cysteine protease required for final IL-1 processing and secretion (28). Recently, a possible role of ICE in apoptosis has also been proposed (16). Furthermore, interferon regulatory factor 1 (IRF-1) upregulates ICE in response to mitogen or gamma interferon (26, 27), and IRF-1 is also known to be activated by viral infection in mouse cells (12). However, the precise kinetics and role of IRF-1 and ICE in IL-1 production in virus-infected human cells have not been investigated. In this study, we investigated the temporal relationship of the expression of IRF-1, the ICE gene, and IL-1 production in RSV-infected neonatal monocytes.

Heparinized (50 µg/ml) cord blood samples obtained from seven healthy neonates were sedimented over Ficoll-Hypaque (Pharmacia, Uppsala, Sweden). Mononuclear cells were collected and washed twice with RPMI 1640 medium. The cell concentration was adjusted to 1.5 × 10⁷/ml in RPMI 1640 medium supplemented with 20% human AB serum. A 300-µl sample was applied to each well of 24-well semimicro plates (Nunc, Roskilde, Denmark). The plates were precoated with human AB serum and incubated for 1 h at 37°C and then incubated for 2 h in 5% CO₂ in air. The plates were washed twice with RPMI 1640 medium to remove nonadherent cells. Over 95% of adherent cells were positive for nonspecific esterase.

RSV strain Long (the prototype RSV group A strain) grown in HEp-2 cells was used for infection. The stock virus titer was 10⁷ PFU/ml. Uninfected HEp-2 cell culture fluid was processed similarly for use in a mock infection.

Adherent monocytes were washed twice in RPMI 1640 medium and counted. Monocytes were inoculated with stock virus at a multiplicity of infection (MOI) of about 2 for 1 h at 37°C. The same volumes and inoculation times were used for noninfected control cells. Cells were then washed with RPMI medium twice and incubated in 0.7 ml of RPMI 1640 medium with 20% fetal calf serum. Culture medium supernatants were collected 2 and 20 h after infection, centrifuged at 400 × g for 5 min, and preserved. To recover the intracellular cytokines at 2 and 20 h after infection, 0.7 ml of distilled water was added to the cells in each well. Ten minutes later, the cell lysates were centrifuged at 1,000 × g for 5 min and the supernatants were collected.

To examine the expression of mRNA for IRF-1, ICE, and IL-1, monocytes were harvested 0 h (preinfection) and 2 h after RSV exposure; monocytes were washed with PBS and treated with 0.8 ml of RNAzol B (Biotecx Laboratories, Houston, Tex.) for RNA extraction.

The IL-1 in the culture supernatants and cell lysates was measured with a quantitative enzyme-linked immunosorbent assay (Quantikine; R and D systems, Minneapolis, Minn.) by following the manufacturer's instructions. All samples were diluted twice before assay. The detection limit of the test is 7.8 pg/ml (manufacturer's information).

Total cellular RNA was isolated from monocytes with or without RSV infection by using RNAzol B and tested for IRF-1, ICE, or IL-1 mRNA by specific reverse transcriptase (RT)-PCR as described previously (14). As an internal control, the activity of -actin mRNA was also determined. Fifty nanograms of the total RNA was used for RT-PCR. For cDNA synthesis, 40 µl of an RNA solution (50 ng) and a random hexamer at 150 pmol/3 µl (Takara, Kyoto, Japan) were heated at 70°C for 10 min and cooled rapidly. After addition of 17 µl of 5× first-strand buffer (250 mM Tris-HCl, 375 mM KCl, 15 mM MgCl₂), 9 µl of 0.1 mM dithiothreitol (GIBCO BRL, Gaithersburg, Md.), 17 µl of 2.5 mM deoxynucleoside triphosphates (Takara), and 200 U of Maloney murine leukemia virus RT (GIBCO BRL), the mixture was stored at 37°C for 1 h. Sequences of the PCR primer pairs and specific probes for Southern blot analysis are shown in Table 1 and were described previously (12, 17, 22). PCR primers and specific probes for ICE were chosen from the nucleotide sequence for ICE p10 (28). The PCR mixture contained 50 ng of cDNA, 10 µl of PCR buffer (500 mM KCl, 10 mM Tris, 1% Triton X-100), 8 µl of 2.5 mM deoxynucleoside triphosophates (Takara), 6 µl of 25 mM MgCl₂, 100 pM 5' and 3' primers, and distilled water for a total volume of 100 µl. After denaturing at 94°C for 10 min and cooling to 65°C, the mixture was seeded with 2.5 µl of thermostable Taq polymerase (Promega, Madison, Wis.). Twenty-five cycles of amplification for -actin, 28 for IRF-1, 29 for ICE, and 24 for IL-1 were carried out with a DNA thermal cycler (Perkin Elmer, Norwalk, Conn.). Each cycle consisted of warming at 95°C for 35 s, 55°C for 2 min, and 72°C for 2 min. Finally, the preparations were incubated at 72°C for 15 min. Ten-microliter samples of the RT-PCR products were analyzed by electrophoresis on a 2% agarose gel, and the amplified products were visualized by UV fluorescence after staining with ethidium bromide. The UV fluorescence signals of specific PCR products in agarose gels were quantified by using a FluorImager SI (Molecular Dynamics, Sunnyvale, Calif.). The specificity of each PCR product was confirmed by determining its predicted size on agarose gels and also by Southern blot analysis. The PCR products were transferred to a nylon membrane and probed with digoxigenin 3'-end-labeled internal oligonucleotide probes (Boehringer GmbH, Mannheim, Germany).

As a positive control for mRNA for IL-1, total RNA from normal adult peripheral blood mononuclear cells which were incubated with concanavalin A at 10 µg/ml for 3 h was used. Adult monocytes exposed to RSV were used as a source of positive controls for IRF-1 and ICE mRNAs.

To quantify relative levels of mRNA, a standard curve was obtained by titration (1/5 dilutions) of first strands obtained from 250 ng of RNA from the positive controls described above (Fig. 1). Relative differences in mRNA expression in the test samples were obtained from the standard curves run for the same number of cycles as the unknown samples. The intensity of fluorescence of DNA amplified from first strands obtained from 250 ng of RNA was defined arbitrarily as 1,000 mRNA equivalents.

View larger version (21K): [in this window] [in a new window]   FIG. 1.   Ethidium bromide-stained gels and standard curves of -actin (A), IRF-1 (B), ICE (C), and IL-1 (D) mRNAs generated by RT-PCR using control RNA. A 250-ng sample of total RNA was reverse transcribed, and fivefold dilutions of the first strand were amplified by PCR. The intensity of fluorescence of the DNA amplified from the first strand obtained from 250 ng of total RNA was defined arbitrarily as 1,000 mRNA equivalents.

The level of -actin in each unknown sample was determined from the actin standard curve, and the levels generally varied less than 30% when the first strand was obtained from the same amount of total RNA (data not shown).

Statistical comparison of the mean levels of IL-1 protein or mRNA equivalents between RSV-infected and mock-infected cells was made by using the Student t test.

Twenty-four hours after exposure to RSV, no cytopathic effect was observed and approximately 30% of RSV-exposed monocytes were weakly positive for viral antigen as determined by immunofluorescent-antibody staining with anti-human RSV strain Long rabbit antibodies (DAKOPATTS, Copenhagen, Denmark) (data not shown). No difference in viability was observed between RSV-exposed and noninfected cells at 24 h after treatment (data not shown).

The expression of mRNA for IRF-1, ICE, and IL-1 was determined in monocyte samples at 0 h (preinfection) and 2 h after RSV exposure. Detectable mRNA levels for these three were observed in all adherent monocytes prior to treatment; however, an apparent increase in the expression of these mRNAs was observed in RSV-exposed monocytes compared to noninfected-cell controls 2 h after treatment. Figure 2 shows the ethidium bromide-stained RT-PCR products and Southern blot analysis of two representative cases. The differences of mean mRNA equivalents for these transcripts between the RSV-treated and mock-infected cells were statistically significant (IRF-1, P < 0.01; ICE, P < 0.05; IL-1, P < 0.01) (Table 2).

View larger version (73K): [in this window] [in a new window]   FIG. 2.   -Actin, IRF-1, ICE, and IL-1 mRNA expression determined by RT-PCR (upper) and Southern blot (lower) analyses of two cord blood monocyte samples exposed to RSV. Lane P, positive control. mRNA expression was assessed before treatment (pre) and 2 h after RSV exposure (2+) or mock infection (2).

The production of IL-1 was examined 2 and 20 h after treatment in culture fluids and cell lysate (Fig. 3). Two hours after RSV exposure, significantly higher levels of intracellular IL-1 protein were observed in infected than in mock-infected cells (mean, 4,020 ± 426 [standard error] and 2,315 ± 836 pg/ml, respectively [P < 0.05]). On the other hand, after 20 h of RSV exposure, moderately higher quantities of soluble IL-1 protein were detected in cell-free supernatants of RSV-exposed cells than in those of mock-infected cells (1,066 ± 258 and 484 ± 227 pg/ml, respectively).

View larger version (17K): [in this window] [in a new window]   FIG. 3.   Immunoactive IL-1 protein concentration in RSV-treated () or mock-infected () monocyte culture supernatants and cell lysates collected at 2 and 20 h after treatment. Mean results ± the standard errors of seven experiments with different donors are shown. Statistical comparison was made by using Student's t test. N.S., not significant.

In this study, neonatal monocytes were exposed to RSV, which is the most important viral pathogen in neonates and young infants. Recent in vitro studies of RSV infection of adult macrophages (25), as well as immunofluorescence analysis of alveolar macrophages of infants with RSV infection (15), suggest that RSV-infected macrophages do produce IL-1 protein. Our study also confirms the prompt production of IL-1 in neonatal monocytes exposed to RSV, with regard to both gene transcription and the immunorelevant IL-1 protein. RSV-exposed neonatal monocytes expressed more IL-1 transcripts and produced more intracellular IL-1 protein than did noninfected control cells as early as 2 h after RSV infection, although there is a possibility that the IL-1 detected in cell lysates is a mixture of the unprocessed IL-1 precursor and intracellular IL-1 because it was reported that the enzyme-linked immunosorbent assay kit used in this study detects but considerably underestimates the IL-1 precursor (9). Subsequently, 20 h after infection, significant IL-1 protein secretion from RSV-exposed cells was observed.

Although the induction of transcription of IL-1 gene occurs in cells by adherence, only limited translational activity is initiated in the absence of a second signal such as lipopolysaccharide (6, 8). In our study, monocytes isolated by adherence also expressed some IL-1 transcript. However, it was obvious that RSV exposure acted promptly, within 2 h, which is apparently the eclipse phase of infection, as a second signal to enhance transcription and to accelerate translation to protein synthesis. This observation suggests that the induction of IL-1 in RSV-treated cells does not depend on viral replication itself in the cells and that adsorption and/or penetration of the virus might trigger induction of IL-1.

Interestingly, monocytes exposed to RSV showed significantly enhanced expression of the IRF-1 and ICE genes the same as the IL-1 gene, at 2 h after exposure. Recently, it has been shown that IRF-1 upregulates ICE gene expression in response to mitogen or gamma interferon (26, 27) and IRF-1 expression itself is activated by viral infection in mouse L929 cells (12). ICE has also been shown to be a prerequisite for final IL-1 processing and secretion (3, 7, 28). Our results include no data on interactions among IRF-1, ICE, and IL-1; however, it might be suggested that RSV infection of neonatal monocytes initially activates IRF-1 to enhance ICE expression and that IL-1 production by RSV-exposed monocytes is enhanced in its later phase by this increased ICE activity.

This is the first report on the effects of viral infection on transcription of the IRF-1, ICE, and IL-1 genes and on the production of IL-1 protein in human cells. Such enhancement might also occur in monocytes and/or macrophages infected with other respiratory viruses. Thus, ICE and IL-1 may be considered as a target for anti-inflammatory drugs in severe viral lower respiratory tract diseases (28).

ICE and other ICE-related proteases have recently been implicated in apoptosis or programmed cell death (7, 16). IRF-1 has also been considered to be correlated with apoptosis through regulation of the ICE gene (26, 27). Miura et al. observed that overexpression of ICE in a rat fibroblast cell line caused apoptosis (16). However, in our study there were no differences in viability between RSV-exposed and mock-infected cells. This observation suggests that the degree of ICE expression able to induce prompt IL-1 excretion from RSV-exposed cells may not be associated with cell apoptosis (10).

We thank M. Akihara and A. Wakamatsu for providing human cord blood samples.