Numerous studies in vitro have demonstrated that RSV is a potent inducer of chemokine production in infected human respiratory epithelial cells. Indirect evidence suggests that these inflammatory molecules may play a critical role in the pathogenesis of RSV disease in infants. Greater concentrations of MIP-1α and RANTES have been demonstrated in nasopharyngeal secretions and tracheal aspirates of infants with RSV bronchiolitis in comparison to respiratory-disease-free children (36
). Greater quantities of MIP-1α and RANTES were also found in infants intubated because of RSV infection than in control infants that were intubated for nonrespiratory illnesses (17
). We have recently extended these findings, by showing that in comparison to subjects with upper respiratory infections, subjects with bronchiolitis have higher concentrations of MIP-1α and eotaxin in nasopharyngeal secretions, both of which are associated with the development of more severe forms of illness due to RSV (unpublished data). In the present study, we provide novel experimental evidence that chemokines are critically involved in RSV-mediated lung inflammation, an essential pathogenic component of RSV infection. Supporting our conclusion are the following findings. (i) Experimental RSV infection of BALB/c mice results in the inducible expression of lung ELR-containing (MIP-2) and non-ELR-containing (IP-10) CXC chemokines, CC chemokines (RANTES, MIP-1α, MIP-1β, MCP-1, TCA-3), and the C chemokine Ltn. (ii) Virus dose-dependent induction of chemokines is associated with the dose-dependent appearance of lung cellular inflammation. (iii) Genetically altered mice lacking the MIP-1α gene (−/−) have substantial reduction of airway inflammation following RSV infection compared to their control littermates (+/+).
The profile of the inducible lung chemokines shown in this study closely reflects the type of cellular infiltration, dictated by the cell distribution of chemokine receptors, which is characteristic of the RSV-infected mouse model (14
). In this regard, the CCR1- and CCR5-binding chemokines RANTES, MIP-1α, and MIP-1β are potent chemoattractants for monocytes and activated T cells (both CD4+
). MCP-1, which binds to the CCR2 receptor, is a potent chemoattractant and activator of monocytes (31
) and is involved in macrophage activation leading to the release of inflammatory mediators and tissue damage (15
). Neutrophils, which represent a sizeable component of the inflammatory infiltrate in RSV infection, are strongly susceptible to the chemotactic effect of MIP-2, a mouse homologue of IL-8 (9
), and, surprisingly for a member of the CC chemokine family, to TCA-3 (23
). NK cells, whose activity peaks during the first days of primary RSV infection, express a number of chemokine receptors that allow them to respond to several chemokines, including IP-10, lymphotactin, RANTES, MIP-1α, and MCP-1 (reviewed in reference 33
Despite the fact that the CC chemokines eotaxin, RANTES, and MIP-1α are known to be potent chemoattractants for eosinophils, we were unable to demonstrate eosinophils in the lung of RSV-infected BALB/c mice. These data are consistent with several other studies with nonsensitized, nonvaccinated mice infected with RSV (i.e., primary infection) (27
) and suggest that eosinophil-attracting chemokines may be necessary but not sufficient to fully activate the multistep process required for migration of eosinophils into lung tissue. In this regard, the eosinophil growth factors IL-5 and GM-CSF play an essential role in mediating eosinophil infiltration in mouse asthma models (11
), while selective deletion of the eosinophil-specific eotaxin gene has shown partial or no effect on eosinophil recruitment (47
). Although one group has described IL-5-dependent eosinophil lung infiltration in a mouse model of RSV infection (35
), there is no strong experimental evidence that either IL-5 or GM-CSF is induced in the airways of mice following primary RSV infection. On the other hand, the production of GM-CSF by RSV-infected human epithelial cells (25
) may explain why infants with naturally acquired RSV bronchiolitis show evidence of activated eosinophils in their airway mucosa (12
) as well as a positive correlation between the concentrations of eosinophil-specific proteins and those of MIP-1α in respiratory secretions (17
). An interesting finding in this study was the demonstration of a difference in profile of chemokines induced in the lung by replicating versus inactivated purified preparations of RSV. Eotaxin, IP-10, and TCA-3 required viral replication in the lung for their expression, while RANTES, MIP-1β, MIP-1α, MIP-2, and MCP-1 were inducible by inoculation of the mice with inactivated RSV, although in much lower abundance compared to mice infected with intact virus. Since the preparations of RSV used in our protocol were sucrose purified and therefore devoid of a number of contaminating mediators normally present in infected tissue culture preparations (i.e., crude, nonpurified virus) (18
), these results suggest that certain cellular components of the lung are able to respond, either directly to RSV particle binding or indirectly via intermediate mediators released by other cells. While expression of chemokine and cytokine genes in epithelial cells has been extensively shown to require viral replication (reviewed in reference 13
), in other cell types, including macrophages (2
), neutrophils (21
), and eosinophils (R. Garofalo, personal observation), binding of nonreplicating RSV particles or virus-specific surface proteins appears to be able to induce production of certain chemokines and cytokines. These in vitro studies are, however, restricted to single cell types and do not provide insights into the mechanisms that regulate in vivo airborne-pathogen-mediated propagation of inflammatory signals from the airspace to the vascular space. In elegant studies, Kuebler et al. have recently shown that an alveolar stimulus, restricted to the epithelial cells, induced a rapid Ca2+
signaling in endothelial cells of perialveolar capillaries, leading to the expression of leukocyte-endothelium adhesion molecules (22
). It is tempting to speculate that RSV, irrespective of its ability to fully replicate in lung epithelial cells, may be able to rapidly trigger intercompartmental signaling that leads to the expression of certain chemokine genes in other tissue resident cells, such as endothelial cells. Supporting this hypothesis is our observation that the expression of MIP-1α was identified by immunohistochemistry in airway epithelial cells and in the adjacent capillary endothelial cells, a cell type not known to be directly susceptible to RSV infection (Fig. ). The implication in vivo of these observations is not fully understood at the present moment.
Although several chemokines were induced in the lung of RSV-infected mice, our results indicate that the CC chemokine MIP-1α may play a central role in mediating RSV-induced inflammatory process of the airways. Following RSV infection, mice genetically deficient for the MIP-1α gene had significant reduction of total lung cellular inflammation compared to control littermates, without obvious differences in viral replication. Similar results in virus-induced models of lung, myocardium, and cornea inflammation have been previously reported in MIP-1α−/−
mice infected with influenza, pneumonia virus of mice, coxsackievirus, and herpes simplex virus, respectively (6
). In those studies viral clearance was either delayed (6
) or unaffected (42
) compared to that in +/+ animals. Our observation that RSV-infected MIP-1α−/−
mice showed a trend for reduced levels of RANTES, MIP-2, and MCP-1 mRNA, compared to +/+ mice is also in agreement with a previous report (42
): the functional significance of this observation, in the contest of MIP-1α-mediated lung inflammation, is currently unknown.
In conclusion, our studies demonstrate that chemokines, and MIP-1α in particular, are critically involved in the pathogenesis of lung inflammation in mice experimentally infected with RSV. The results presented herein extend previous observations in vitro and support other indirect evidence of the involvement of lung chemokines in the clinical manifestations of naturally acquired RSV infection (bronchiolitis). Additional studies are under way to determine the role of lung chemokines in mediating other pathophysiologic features of RSV infection, such as airway hyperresponsiveness.