© 2006 American Thoracic Society DOI: 10.1165/rcmb.f318
Chemokine ComplexityThe Case for CCL5Departments of Medicine and Cell Biology, Washington University School of Medicine, St. Louis, Missouri William of Ockham (of Ockham's Razor fame) and Albert Einstein both urged us to keep it simple, but biology, perhaps in comparison to philosophy or physics, is often a more complicated matrix. An example of the complex nature of nature is the case of chemokine action in mediating inflammatory disease. Thus, chemokines were associated initially with the development of inflammatory and allergic disease primarily through their role as chemoattractant cytokines. In turn, increased lung levels of the chemokine CCL5 in patients with asthma and in mouse models of asthma (1, 2) were presumed to cause further recruitment of inflammatory cells, especially eosinophils, into airway tissue and consequent worsening of airway disease. However, this view of CCL5, and chemokines in general, turns out to be quite naïve, as underscored by an additional article in the current issue of the AJRCMB (3).
In this new article, Koya and colleagues (pp 147–154) show that CCL5 selectively dampens the long-term effect of allergen challenge in mice (3). In this model, prolonged allergen exposure (out to 48 d after initial sensitization) led to airway hyperreactivity and mucous cell metaplasia, and administration of CCL5 decreased these traits, whereas treatment with anti-CCL5 antibody significantly increased them. Administration of CCL5 also caused an increase while anti-CCL5 antibody caused a decrease in BAL fluid levels of IFN- Non–T Cell Explanations: CCL5 and the Macrophage Consistent with the present report by Koya and colleagues, the history of CCL5 is closely linked to its actions in regulating T cell performance (3). Indeed, the initial naming of CCL5 (i.e., Regulated And Normal T cell Expressed and Secreted or RANTES) was based on T cell behavior. Is it possible, however, that CCL5 has a broader activity on other immune cell types that better explains its effects on the allergic response? In support of this prospect, we now recognize that several (non-T) cell types express CCL5-responsive receptors, including macrophages and dendritic cells (via CCR1 and CCR5) as well as eosinophils (via CCR3). In fact, inspection of the present data after allergen challenge indicates that the only significant change in lung immune cell levels takes place in macrophages, and in linkage with functional data, this change in macrophage levels also occurred selectively in the setting of the long-term allergen challenge. But, whether these concomitant changes in lung macrophage levels were driving the change in airway behavior was not pursued. This omission is not unusual. The whole subject of CCL5 signaling to macrophages was barely even considered until recently. Our research group can take some credit for this redirection to the macrophage based on studies of CCL5 function during common respiratory viral infections. For these types of infections, the site of initial viral replication is often the airway epithelium. This pattern is especially typical of the most common cause of serious respiratory infection in childhood (i.e., respiratory syncytial virus [RSV]) as well as other common paramyxoviruses. For these viruses (and others), the initial immune cell response is relatively nonspecific and is conducted by cells of the innate immune system. Natural killer cells and neutrophils are rapidly attracted to the infected tissue and activated at the site of viral replication. However, this response is not likely enough to fully clear the infection. Thus, while this innate immune response is developing, there is a simultaneous development of an adaptive immune response. This response is initiated by maturation and migration of dendritic cells to the draining lymph nodes. Once in the nodal tissue, the dendritic cells instruct rare virus-specific CD4 and CD8 T cells to proliferate and activate. Ultimately, the CD8 T cells will migrate back to the lung and directly kill the infected host cells. The virus-specific CD4 T cells will in turn direct B cells to make neutralizing antibody that will eventually lead to resolution of the viral infection. In addition to these well-characterized events, the period of time following the early recruitment of neutrophils and the subsequent appearance of the adaptive immune response is dominated by the recruitment of macrophages into the airway tissue. Macrophages represent the primary phagocytic cells of the immune system and are therefore critical for the removal of cellular corpses and debris. In addition to these housekeeping duties, macrophages are also capable of presenting antigen to lymphocytes; however, the relative importance of this aspect of their biology in a viral infection is unknown. Macrophages, like airway epithelial cells, may also be productively infected by paramyxoviruses and thereby secrete inflammatory cytokines that help to further activate lymphocytes to clear the virus. The precise contribution of macrophages to antiviral defense was incompletely defined. Nonetheless, it seemed reasonable that macrophage as well as epithelial cell behavior might significantly influence the outcome from viral infection. Our initial approach to these issues aimed at defining the pattern of gene expression in response to RSV infection in primary cultures of human airway epithelial cells. The results revealed that a prominent (perhaps the most prominent) aspect of the epithelial immune response consisted of the production and release of CCL5 (1, 4). This high level of induction was also found for other common respiratory viruses, including rhinovirus and influenza virus. In the case of RSV, viral induction of CCL5 gene expression depended on both transcriptional and post-transcriptional events. The synergy inherent in this combined biochemical mechanism may be responsible for the pronounced induction of CCL5 compared to all other immune-response genes. In any case, the prominence of the induction of CCL5 gene expression suggested a special role for this chemokine in antiviral defense. To test the role of CCL5 in the antiviral response, we developed a Ccl5–/– mouse and examined its response to respiratory viral pathogens (5). For initial experiments, we used mouse parainfluenza virus type I (Sendai virus; SeV) to model respiratory infection, since we found that mice are relatively resistant to infection with RSV. The experimental conditions for SeV infection allow for high-level viral replication and a pattern of illness in wild-type mice that is similar to human paramyxoviral infection (6, 7). In later experiments, we also used a mouse-adapted strain of influenza virus. For both viruses, we found that CCL5 was required for host survival. The same phenotype developed in mice that were deficient in CCR5. This finding fit with coordinated induction of CCR5 in concert with CCL5 expression during viral infection. Other receptors for CCL5 (i.e., CCR1 and CCR3) did not exhibit similar induction under these conditions. Thus, CCL5–CCR5 interaction appeared necessary for the antiviral defense system and specially tailored for activation during viral infection. Since CCL5 is a potent chemotaxin, we initially reasoned that the viral susceptibility of Ccl5–/– and Ccr5–/– mice was due to decreased recruitment and/or activation of immune cells (especially macrophages and effector T cells) at the site of infection (8). However, lymphocyte infiltration into the airways was no different in Ccl5–/– and Ccr5–/– mice compared to wild-type control mice. Moreover, T cell activation levels also appeared unchanged as assessed by flow cytometry of immune cells isolated from spleen, lung, and BAL fluid of Ccl5–/– and control mice. In contrast, loss of CCL5–CCR5 interaction was associated with an increased level of macrophages in the airway tissue. Serial tissue sections indicated that this macrophage population was persistently infected with virus and was undergoing apoptosis at increased levels in Ccl5–/– and Ccr5–/– mice compared to wild-type control mice.
We recognized that the observed phenotype for Ccl5 deficiency was distinct from those found in other experimental models for chemokine blockade. In those models, chemokine deficiency is associated with a decrease (not an increase) in immune cells at the site of infection (9–11). In fact, our results might also have been compatible with defective macrophage traffic due to loss of chemotactic signal, resulting in higher levels of macrophage infection and death rates. However, the distinct phenotype in Ccl5–/– mice was better explained when we found that the CCL5–CCR5 interaction was necessary to prevent virus-induced apoptosis in isolated macrophages, where chemotaxis was no longer a variable. Under these conditions, endogenous CCL5 or exogenous restoration of physiologic levels of CCL5 were each protective against virus-induced apoptosis and so fully reversed the Ccl5–/– defect. In addition, the absence or blockade of CCR5 caused increased virus-inducible apoptosis at levels equivalent to those observed in Ccl5–/– macrophages. Thus, in Ccl5–/– and Ccr5–/– mice, accumulation of apoptotic macrophages in tissue could be explained by premature cell death before reaching the airspace. In addition, similar to mouse macrophages, we found that CCR5 blockade caused increased apoptosis in human macrophages infected with SeV as well as viruses that are commonly pathogenic in humans (e.g., RSV and influenza virus). In each case, physiologic levels of CCL5 activate CCR5 and initiate dual signals to G The Case for the Dendritic Cell These studies established a distinct role for CCL5–CCR5 signaling in the innate immune response to viral infection, but we still questioned whether chemokines in general, and CCL5 in particular, might somehow influence the adaptive immune response to viral infection. As noted above, previous work made it likely that chemokine influence is directed at traffic and activation of immune cells. For example, Ccl3–/– mice exhibit decreased inflammation and delayed clearance of virus during infection with influenza virus or pneumonia virus of mice (PVM) (9, 12). However, we found little evidence of a change in the effector arm of the immune response, at least by 12 d after viral inoculation, in Ccl5–/– mice. By this time, Ccl5 deficiency had no influence on the levels of virus-specific T cells recruited to the lung. We therefore questioned whether earlier events that are important for the initiation of the adaptive immune response might also be influenced by chemokine action. In support of this possibility, others have reported that CCR5 (as well as CCR1) appears to regulate the homeostatic recruitment of lung dendritic cells. For example, treatment with a CCL5 antagonist (met-CCL5) decreases the number of dendritic cells in the rat lung (13). In addition, others recently showed that interferon-producing cells (also known as plasmacytoid dendritic cells) enter the lymph node via high endothelial venules using a CCR5-dependent mechanism during infection with Mycobacterium tuberculosis (14). Whether this response is also dependent upon CCL5 signaling is not yet known. Nonetheless, if plasmacytoid dendritic cells can condition the subsequent antigen-specific T cell response, this trafficking defect represents an additional way in which CCL5–CCR5 interaction may influence the adaptive immune response. Perhaps similar to the findings with tuberculosis models, we have recently found that CCL5–CCR5 interaction may regulate the recruitment of differentiating and maturing dendritic cells from the lung to the draining lymph nodes. Thus, both Ccl5–/– and Ccr5–/– mice appear to have over 3-fold fewer dendritic cells migrating to the draining lymph nodes compared to wild-type control mice at early times (1 d) after inoculation with virus (15). This defect is accompanied by a partial failure to up-regulate CCR7 expression that may at least partially explain the decrease in DC traffic to the lymph node. As noted above, we did not find much difference in the effector arm of the adaptive immune response by 12 d after inoculation, but it is still possible that earlier events might be influenced by CCL5. Whether a significant degree of delay in the development of the adaptive immune response contributes to immune compromise during viral infection still needs to be defined. For example, a significant delay in the development of the adaptive response could contribute to immune compromise during a secondary viral infection. The findings also suggest that the afferent arm of the adaptive immune response may be influenced more strongly by CCL5–CCR5 signals, whereas previous work has focused on the effector arm of the response. A Broader Role for CCL5 in Allergy
We earlier raised the possibility that CCL5 influences the allergic response through non–T cell mechanisms. Based what we have learned about CCL5, is it possible that CCL5 influences the chronic allergic response via actions on macrophages and dendritic cells? For example, could prolonged allergen exposure lead to CCL5-dependent recruitment and survival of macrophages? The observations by Koya and coworkers suggest that this is the case (3). Could these macrophages then contribute to down-regulating the allergic response? This issue was not examined in the present work, but certainly macrophages could be a major source of IFN- It is also possible that CCL5 influences the allergic response via actions on dendritic cells. For example, is the compromise in dendritic cell traffic found in Ccl5–/– and Ccr5–/– mice after viral infection also found after allergen challenge? As noted above, CCR5 is necessary for plasmacytoid dendritic cell traffic to draining lymph nodes after exposure to a microbial stimulus (14). Furthermore, plasmacytoid dendritic cells are a major source of type I IFNs. Thus, regulating the migration of this cell population to the draining lymph node may also influence the development of Th1 cells.
Finally, the role of CCR5 signaling may also extend to other immune cell types. For example, naïve CD8 T cells appear to use CCR5 to move to sites of CD4 T cell–dendritic cell interaction in the draining lymph nodes (16). Presumably, this interaction allows for the generation of memory CD8 T cells. In addition, CCR5 signals can facilitate activation-induced apoptosis of NKT cells as well as skew NKT cell production from IL-4 towards IFN-
Footnotes This research is supported by grants from the National Institutes of Health (NIAID and NHLBI), Martin Schaeffer Fund, and Alan A. and Edith L. Wolff Charitable Trust. Conflict of Interest Statement: Neither author has a financial relationship with a commercial entity that has an interest in the subject of this paper. References
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