Results presented here suggest that RSV, an established infectious agent of the lower respiratory tract, may be considered a pathogen of the eye as well. To our knowledge this is the first detailed report of a direct interaction of RSV with the ocular tissue. Interestingly, RSV infection of the corneal epithelium exhibited many features observed in the infection of the lung epithelium: (i) A strong correlation between respiratory infection and the presence of RSV in the eye (Fig. 1); (ii) A productive multiplication of the virus (Fig. 2, 3); (iii) Liberation of a variety of cytokines (Fig. 4, 5); and (iv) Strong activation of transcription factor NF-κB (Fig. 6). It is to be noted that RSV growth was slower in HCE cells compared to lung epithelial cells. Whereas the virus-induced syncytium is observed in about 36 hrs in A549 lung cells , it was not seen until about 48 hrs in HCE cells. The release of progeny virions and the induction of the cytokines also followed a parallel and slower kinetics in HCE cells (compare, for example, Fig. 2 with the kinetics in A549 cells [6, 23]). These results are in agreement with a role of viral macromolecular synthesis in the activation of these signaling pathways in HCE cells. It is to be mentioned here that specific RSV strains infect nonhuman mammals such as the cattle, sheep, pig and goat, and thus, an extension of these findings may have important relevance in agriculture. The implications of the viral regulation of corneal genes are briefly summarized below.
As noted in the Results (Fig. 4), a considerable number of chemokine genes of both CXC and CC families were induced in the RSV-infected HCE cells. The CX3C member, Fractalkine, was not up-regulated, and the C subfamily chemokines (Lymphotactin and SCM-1β) were not tested. The CXC subfamily  is further divided into two groups based on the presence or absence of the Glu-Leu-Arg (ELR) motif immediately preceding the first Cys residue near the amino terminus. The ELR+ CXC chemokines act primarily on neutrophils as chemoattractants and activators, inducing neutrophil degranulation with release of myeloperoxidase and other enzymes. In contrast, chemokines of the ELR- CXC and the CC subfamilies chemoattract and activate T- and B-lymphocytes and natural killer (NK) cells Interestingly, chemokines that exhibited the highest induction (in the hundreds of fold) by RSV (Fig. 4) belonged to the latter two subfamilies and included three ELR- chemokines, namely MIG (Monokine-induced by IFN-γ), I-TAC (IFN-γ-inducible T cell alpha chemoattractant) and IP-10 (IFN-γ-inducible protein of 10 kDa), and two CC chemokines, MIP-1α and MIP-1β. It is also noteworthy that whereas MIG, I-TAC and IP-10 are all IFN-γ inducible , RSV infection did not activate IFN-γ from the HCE cells (Fig. 4). Clearly, these chemokines must be induced via an IFN-γ-independent pathway activated by RSV, which needs to be elucidated further. It should be noted that recent studies have cast doubt on the original assumption that expression of these chemokines absolutely requires IFN-γ. For example, IFN-γ-independent activation of MIG and IP-10 has been observed in primary human umbilical vein endothelial cells (HUVECs) in response to infection by influenza virus . Moreover, studies in IFN-γ knockout mice have revealed that in the absence of IFN-γ, IFN-α and -β are able to induce MIG in response to a viral infection in vivo .
Although many chemokines can be pluripotent and exhibit redundant functions , we notice an interesting pattern in the HCE chemokines induced by RSV (Fig. 4). It appears that the some of most weakly induced ones (I-309, MDC, TARC) are agonists of CCR8 and CCR4, and therefore, likely to recruit Type 2 (Th2) lymphocytes characteristic of allergy. In contrast, the four most highly induced ones (MIG, I-TAC, IP-10 and MIP-1β) were CXCR3 and CCR5 agonists, and therefore, likely to recruit Th1 lymphocytes, characteristic of an inflammatory response . Further studies are needed to unravel how the Th1 and Th2 balance may affect the pathology of the RSV-infected eye.
The significantly higher induction of IL-1β (Fig. 5) deserves special mention. Ocular IL-1, by virtue of its multipotential nature, plays a regulatory and generally pro-inflammatory role in acute inflammation of the eye . First, because the IL-1β receptor (IL-1 RII) is found on monocytes, neutrophils and T cells , the elaborated IL-1β may directly recruit these cells to the infected cornea, thus promoting inflammation. Ocular chemokines have in fact been shown to play essential roles in blinding keratitis caused by herpes simplex and Pseudomonas aeruginosa [8–10]. Second, IL-1 has the potential to activate other cytokines, including chemokines such as IL-8, GROα, and MIP-1α, many of which may in turn recruit and/or activate inflammatory cells such as eosinophils and neutrophils at later times in the infected eye [8, 9, 36–40]. Results in Fig. 4 and 5 in fact suggest that IL-1β is activated through proteolytic processing by ICE relatively early (about 6 hr) in RSV infection of the corneal epithelial cells. Because the transcription of the IL-1β-inducible chemokines, such as IL-8, GRO, and MIP were all induced hours later (beginning around 12 hours) (Fig. 4, 5), it is quite possible that they were induced, at least in part, by IL-1β.
In a recent study , use of DNA microarray lead to the identification of a spectrum of chemokines inducible by RSV infection from two alveolar epithelial cell lines: A549, a type II-like cancer cell line, and SAE, a primary cell line of type I. In A549, the following chemokines mRNAs were induced: CC (I-309, Exodus-1, TARC, RANTES, MCP-1, MDC, and MIP-1α and -β), CXC (GRO-α, -β, and -γ, ENA-78, IL-8, and I-TAC) and CX3C (Fractalkine). Many of these chemokine mRNAs were also induced in SAE cells following RSV infection; however, important exceptions were MDC, TARC and MCP-1, suggesting that RSV-induced cytokine response differs between different types of airway-derived epithelial cells. The most notable differences between these airway cells and the HCE cells were the absence of induction of RANTES, ENA-78 and Fractalkine in the HCE cells (Fig. 4). Such differences may reflect the innate genetic difference between corneal and alveolar epithelial cells. In either case, just as the respiratory epithelium has been postulated to be a primary initiator of pulmonary inflammation , the corneal epithelium may also act as an initiator of ocular inflammation in RSV infection. Our conclusion should not be interpreted to mean that all conjunctivitis is caused by RSV; in chronic vernal keratoconjunctivitis, for example, RT-PCR analysis of conjunctival biopsy samples (n = 15) failed to detect RSV .
As mentioned earlier, RSV infection activates NF-kappa B in lung epithelial cells; however, the status of corneal NF-κB is relatively unknown. To our knowledge, the results presented here constitute the first documentation of NF-κB activation in HCE cells following a respiratory virus infection. Gene knock out studies have shown that the NF-κB kinase (IKK) – and by inference, NF-κB itself – is essential for the development of the mammalian cornea and conjunctiva . A basal expression of NF-κB in the HCE cells was also observed in our studies (Fig. 6). Thus, it is reasonable to conjecture that the increased NF-κB activity observed following RSV-infection might lead to transcriptional induction of cytokines such as IL-8, MIP-1α, MIP-1β and MCP (Fig. 4). We previously showed that non-steroidal anti-inflammatory drugs (NSAID) aspirin and salicylate inhibited NF-κB activation and induction of a number of cytokines from lung epithelial cells . A comparable ability of the NSAIDs to abrogate NF-κB activation in RSV-infected HCE cells (Fig. 6B) raises the interesting possibility that they may also help to resolve RSV-mediated inflammation of the eye. The other avenues of potential intervention would include inhibition of specific immunomodulatory cytokine(s) by a variety of available antagonists including, but not limited to, antibodies . Antiviral-antichemokine combination therapy is in fact a new strategy worth considering as a general therapeutic approach to viral infections [6, 43]. In a recent example , combination of an antiviral (ribavirin) and anti-CCL3 was synergistic and able to prevent mortality in mice infected with the highly lethal pneumonia virus of mice, a natural mouse pathogen that is very similar to RSV and belongs to the same genus, Pneumovirus.
Lastly, it will be interesting to determine whether RSV can travel from the infected outer cells of the eye to the inner layers and eventually to the respiratory tissues. Clearly, the short- and long-term effects of RSV on the ocular epithelium and their potential impact on vision and respiratory disease will open new directions in ocular immunopathology and the mechanism of RSV spread.