The present authors describe for the first time that IFN-γ can exert a simultaneous differential effect on both forms of the rhinovirus receptor, ICAM-1, in target infected cells. Specifically, the membranous form of ICAM-1 is down-regulated in IFN-γ-biased epithelial cells exposed to the major group rhinovirus, HRV-14, whilst sICAM-1 release is enhanced.
The current experiments demonstrate that mICAM-1 expression is induced by both major and minor group rhinoviruses. Since both group viruses utilise distinct cellular receptors [21
], the observed induction in mICAM-1 expression appears to be not receptor restricted. However, when NHBE cells are first pre-conditioned with IFN-γ and subsequently infected, seminal different response patterns are observed between major and minor group rhinoviruses. Expression of mICAM-1 is down-regulated within 24 h in IFN-γ-biased HRV-14-infected cells, whilst mICAM-1 levels remain elevated in equivalent mediator conditioned cells infected with the minor group serotype. It follows that the effects of IFN-γ and major group rhinovirus together are likely to be receptor mediated. These data support previous findings by Sethi et al
(1997) who demonstrated a reduction in mICAM-1 expression in response to IFN-γ and HRV-14 in separate cell lines. Whilst these observations require to be confirmed in vivo
, there are potential implications for the capability of IFN-γ to modulate selectively HRV-14 cell receptor expression, particularly as mICAM-1 is critical in both inflammatory cell mobilisation and virus-host cell binding/internalisation. A down-regulation in mICAM-1 expression on the epithelial cell surface may limit rhinovirus infection by decreasing the number of receptors available for virus binding; whilst possibly also reducing the magnitude of subsequent effector response at the target site.
The current study next explored possible molecular mechanisms by which mICAM-1 down-regulation may occur in IFN-γ-biased cells exposed to HRV-14. As IFN-γ-mediated effects were observed to be sensitive, albeit not wholly, to pharmacological inhibition with cycloheximide and actinomycin D, the authors postulated that modulation of ICAM-1 expression could at least be facilitated at the transcriptional and translational level. There is abundant evidence that STAT-1 signalling is a key regulatory pathway; although not necessarily mediating all effects of IFN-γ [18
], it made sense to first determine whether STAT-1 regulation is involved in mICAM-1 modulation during HRV-14 infection. The present data show that IFN-γ induces phosphorylation of STAT-1; its effects on membranous ICAM-1 levels are inhibited in a dose-dependent fashion by AG490 in the absence of infection. These data suggest that the selective transcriptional actions of IFN-γ are likely to be mediated by a conventional JAK/STAT signal transduction pathway, whether wholly or partly in uninfected cells. Interestingly the present authors did not observe AG490 inhibition on mICAM-1 protein levels in HRV-infected cells. This may be due to the fact that this signalling pathway is suppressed by HRV in infected cells. Moreover, the magnitude of STAT-1 phosphorylation was lower after 30 mins, though not after 15 mins, treatment in HRV-infected cells compared with uninfected equivalents. The authors are aware of several reports on virus interference with IFN-γ signalling. Respiratory viruses such as Sendai, Cytomegalovirus, Adenovirus, Vaccinia, simian virus 5, have been shown to reduce STAT phosphorylation [22
]. It is possible that human rhinoviruses may behave similarly. In addition, HRV proteins may interfere directly with host JAK/STAT signalling mechanism preventing interferon-induced transcriptional responses. Several viruses have been demonstrated to employ this mechanism [27
]. It follows that STAT-1 phosphorylation may be inhibited by HRV without affecting other molecules such as JAK involved in the signalling cascade. However, whilst HRV-14 manipulation of interferon-dependent immunity is possible, further exploration of the interaction between HRV-14/IFN-γ/JAK/STAT signalling is required to explain the observed lack of AG490 inhibition on mICAM-1 levels in HRV-infected cells; equally other signalling molecules may be operative.
The simultaneous effects of IFN-γ and rhinovirus infection were also explored on the soluble form of ICAM receptor. IFNγ induces a small but significant increase at 8–96 h in sICAM-1 release from uninfected cells. In the presence of major group rhinovirus, sICAM-1 release from IFN-γ-biased cells is augmented in a time-dependent manner. In contrast, infection with the minor group rhinovirus had no significant effect on sICAM-1 release. The concurrent enhanced sICAM-1 release and mICAM-1 downregulation during HRV-14 infection could play a pivotal function in limiting HRV-14 infection within a biased IFN-γ milieu. The present authors postulate that this differential ICAM-1 isoform processing may create an anti-viral influence by reducing the density of the HRV docking target on the cell surface, whilst the released soluble ICAM-1 molecules serve as a decoy to prevent further HRV binding to epithelial cells and infection by binding to virus particles.
Whilst HRV replication was not measured directly, the present data demonstrate that changes in relative expression levels of membrane-bound and secreted ICAM-1 forms are followed by reduction in analysed HRV-14 titres. Viral levels were reduced in IFN-γ treated HRV-14 infected cells at 8 h compared to untreated HRV-14 infected cells. It is possible that the observed reduction in viral titres at this time point reflects a direct effect of IFN- γ on viral replication, although this is just hypothesis at the moment. In addition, at 120 and 144 h post infection, epithelial cell infectivity appeared to be reduced in IFN-γ treated HRV-14 infected cells compared to untreated cells infected with HRV-14 and both IFN-γ treated and untreated HRV-1b infected cells. These observations are consistent with previous studies which also demonstrated reduced viral titres in IFN-γ treated and HRV-14 infected H292 cells [14
]. Since these observations appear to be specific to the major group rhinovirus that utilise ICAM-1 as its cellular receptor, the decrease in viral titres are likely to be a consequence of IFN-γ on differential modulation of the ICAM-1 variants. Whilst it is possible that the observed later effects on viral titres may be secondary to de novo
IFN-γ production inhibiting viral replication, in our previous studies we did not observe increasing IFN-γ levels over the experimental period. The relevance of defense mechanisms driven by IFN-γ-induced processing of the HRV-14 receptor for host epithelial cell infectivity remains to be clarified in future studies.
The current investigation conducted preliminary experiments to explain possible source/s for the enhanced soluble isoforms. The underlying mechanisms responsible for IFN-γ induced modulation of sICAM-1 levels during HRV infection include alternative splicing of the ICAM-1 gene and proteolytic cleavage of the membranous form. Alternative splice donor site selection results in a reading frameshift of the ICAM-1 gene with elimination of the transmembrane and cytoplasmic domains [19
]. However, the present authors concluded that gene splicing is not the predominant mechanism driving sICAM-1 release as mRNA transcript expression is not induced by IFN-γ and HRV-14, suggesting other mechanisms are operative. However, Actinomycin D dramatically reduced sICAM-1 release suggesting that gene transcription is involved in the regulation of ICAM-1 receptors in bronchial epithelial cells, albeit not transcription of the actual ICAM-1 gene; possibly the transcription of a gene regulating proteolysis such as MMPs or inhibitors of these proteases. Indeed, separate experiments showed proteolytic cleavage of mICAM-1 as potentially pivotal to sICAM-1 release. Certainly, IFN-γ and HRV-14 together appear to induce an increase in sICAM-1 release, whilst decreasing mICAM-1 expression on the epithelial cell surface. Protease inhibitors with a broad spectrum of activity had no effect on mICAM-1 expression in IFN-γ-stimulated uninfected NHBE cells. However, there was near-complete abrogation of sICAM-1 release from both IFN-γ uninfected and HRV-infected cells incubated with same protease inhibitors. It is thus plausible that during HRV infection, IFN-γ promotes the enzymatic cleavage of mICAM-1 resulting in a downregulation of cell surface mICAM-1, with simultaneous enhanced release of sICAM-1 into the local extracellular environment. Furthermore, the involvement and regulation of selective proteolytic enzymes such as MMPs and caspases in the cleavage of mICAM-1 should also be considered and addressed in future studies, especially in the context of human airway HRV infection. Indeed, keratinocyte and astrocyte models have demonstrated that the sICAM-1 release mechanism is sensitive to metalloproteinase (MMP) inhibitors [9
] suggesting a critical role for MMPs in the production of sICAM-1. Interestingly, in addition to establishing the role of proteases in the production of sICAM-1, the authors demonstrated the JAK/STAT signalling pathway as a potential mechanism in the regulation of ICAM-1 receptors. The cellular response to virus infection is complex and is likely to involve multiple genes, therefore as the JAK/STAT pathway is a generic signalling mechanism, it may be implicated in the activation of accessory genes, such as proteases/protease inhibitors involved in the regulation of ICAM-1 expression as opposed to the ICAM-1 gene itself.
In conclusion, this current study highlights an interesting modulation of ICAM-1 receptor isoforms by the Th-1 mediator, IFN-γ in the presence of HRV-14 infection. Whilst some findings are preliminary, the present in vitro
cell model observations are in line with previous data from human experimental rhinovirus studies. Winther et al
, 2002 [31
], demonstrated that nasal epithelial cell mICAM-1 expression is upregulated within 24 h of HRV inoculation, decreasing rapidly to baseline by day 9; with simultaneous increase in sICAM-1 release into nasal lavage, albeit that significance was only noted on day 3. Separate studies by Hildebrandt et al
] also detected enhanced soluble receptor levels during the acute phase of naturally acquired upper respiratory tract infections. These observations need to be pooled together to design future experiments to determine the relevance of differential ICAM-1 processing on natural rhinovirus infection; this could unravel key molecular targets for anti-HRV pharmacological engineering.