In the model of virus-induced lung eosinophilia described here, the neutralization of CCL11 reduced eosinophil accumulation to the lung by about 50% and produced a beneficial effect which was seen upon pulmonary histology. Treatment with anti-CCL11 also perturbed the recruitment of CD4+
T cells and reduced the production of the Th2 cytokine IL-5, but not that of IFN-γ, suggesting an important shift in the inflammatory T-helper phenotype. The decrease in lymphocyte accumulation appeared to be the result of reduced trafficking rather than an impaired expansion of effector cells and was accompanied by a shift in the local cytokine milieu towards a more Th1-like environment. These findings are in keeping with studies of the role of eosinophils in allergic disease using neutralizing antibodies (7
) or knockout mice (29
), but to our knowledge we are the first to report a role for CCL11 in virus-induced lung immunopathology. The finding that anti-CCL11 is beneficial in this setting suggests that therapies designed to interfere with CCL11 effects might be useful for such conditions.
Although the observation of a decrease in CD4+
T cells was initially unexpected, a role for CCL11 in the recruitment of CCR3-expressing Th2 cells has been shown by Lloyd et al., who used a model of allergic airway inflammation in which polarized effector Th2 cells were adoptively transferred into naïve mice prior to sensitization with ovalbumin (19
). In this system, treatment with anti-CCL11 specifically inhibited the initial phase of Th2 cell recruitment in the first few days immediately after aerosol challenge, with recruitment via the CCR4/MDC (monocyte-derived chemokine) axis comprising the dominant chemoattractive potential within 1 week of the challenge. The administration of anti-CCL11 inhibited the accumulation of antigen-specific Th2 cells by at least 50% on day 4 after an ovum challenge and significantly reduced both eosinophil infiltration and IL-4 production. This study conclusively demonstrated a role for the CCL11/CCR3 axis in mediating the early recruitment of Th2 cells.
The present data do not rule out secondary effects on CD4+
-T-cell recruitment due to the presence of reduced numbers of infiltrating eosinophils. Such effects include a reduced secretion of eosinophil-derived type 2 cytokines, reduced antigen presentation by endobronchial eosinophils trafficking to the local lymph nodes, and dampening of inflammatory responses elicited by damage to pulmonary epithelium by toxic eosinophil granule proteins. Indeed, some or all of these mechanisms may contribute to the relief from pulmonary CD4+
-T-cell accumulation. A recent investigation confirmed that antigen-loaded eosinophils are able to amplify Th2 responses and, after adoptive transfer into naïve mice, prime de novo Th2-driven allergic lung disease (23
). Nevertheless, these data are also consistent with a role for the CCL11/CCR3 axis in mediating the recruitment of CD4+
T cells to the lungs of infected mice during an RSV challenge. Other groups have been able to selectively inhibit pulmonary eosinophilia (by the neutralization of macrophage inflammatory protein 1α and RANTES, which act predominately via CCR1) without a concomitant reduction in antigen-specific lymphocytes, suggesting that decreased T-cell accumulation is not an obligate consequence of lowered eosinophil recruitment (21
). To the best of our knowledge, this represents the first report of an involvement of CCL11 signaling in the trafficking of CD4+
T cells in a physiological setting. While previous studies have examined the trafficking of strongly committed Th2 cells induced by repeated stimulation under polarizing conditions in vitro (19
), this study suggests a possible role for CCL11 in the migration of T cells generated in vivo during the course of a normal immune response.
Despite considerable efforts, it was not possible to convincingly prove that the differential recruitment of CD4+
T cells in the present study was due to a selective effect upon Th2-type, CCR3-expressing cells. Attempts to confirm the recruitment of such cells to the lungs of rVV-G-primed mice during RSV infection by direct staining with either anti-T1/ST2 or rabbit antibodies specific for murine CCR3 (kindly provided by P. Ponath of LeukoSite, Inc., Cambridge, Mass.) were unsuccessful. In this regard, it may be relevant that responding Th2 cells appear to progressively lose their expression of CCR3 (2
) and consequently may have downregulated this receptor by the time of sampling. Likewise, analyses of IL-4 and IL-5 production by ex vivo-stimulated, G-specific CD4+
T cells did not prove any quantitative differences in Th2 cell accumulation in anti-CCL11-treated mice. Previous studies have suggested that only about 12% of CD4+
T cells in the BAL fluids of rVV-G-primed mice are T1/ST2+
(i.e., Th2 cells) on day 5 of an RSV challenge (39
). The reduction in CD4+
-T-cell accumulation upon neutralization of CCL11 was, on average, closer to 25%, suggesting that the recruitment of both Th2 and non-Th2 subsets is affected by an anti-CCL11 treatment (Fig. ). This is surprising, since CCR3 expression by activated lymphocytes is believed to be restricted to type 2 cells (40
). Treatment with anti-CCL11 did not affect RSV illness or inflammatory cell accumulation in rVV-F- or rVV-β gal-primed mice, in which inflammation was restricted to a Th1 phenotype, confirming that the CCL11/CCR3 axis is not directly involved in Th1 cell recruitment. It is possible that the elimination of non-Th2 cells is a secondary effect due to the decreased eosinophil and Th2 cell recruitment induced by the disruption of the CCL11/CCR3 axis.
Activated eosinophils secrete several granule proteins that can damage the respiratory epithelium (6
). Such damage might exacerbate local inflammation, leading to an augmented recruitment of non-Th2 CD4+
T cells, especially in the context of an ongoing viral infection. The relief of tissue damage upon depletion of CCL11 may therefore result in a dampening of the exuberant immune response, thus explaining the smaller accumulation of CD4+
T cells. Interestingly, a similar reduction in IFN-γ-producing cells upon anti-CCL11 treatment was observed during experimental mycobacterial antigen-elicited granuloma formation (30
). These granulomas, which are normally characterized by the presence of neutrophils and of T cells secreting IFN-γ, but not of eosinophils (i.e., type 1 lesions), were reduced in size by an anti-CCL11 treatment. Furthermore, IFN-γ production by local draining lymph node cells was increased in a dose-dependent manner by exogenous CCL11 protein (30
). The mechanism by which this is achieved is unclear (since Th1 cells do not express CCR3), but it may well contribute to the observed reduction in Th1 cell recruitment in the present study.
Although we could not conclusively demonstrate the effects of anti-CCL11 on the recruitment of Th2 cells, the neutralization of a single chemokine during ongoing complex inflammation with mixed Th1, Th2, and CD8+
-T-cell components is likely to have a limited effect. Furthermore, the neutralization of CCL11 is not equivalent to a complete blockade of CCR3, as several other ligands, including CCL11-2, RANTES, and MCP-3, can also engage this receptor and may compensate to some degree for the absence of its main ligand (41
). Several studies have been unable to detect CCR3 on Th2 cells in vivo (4
), and Th2 cells both develop normally and appear to accumulate efficiently at allergic sites in CCR3−/−
mice, suggesting that adequate compensatory mechanisms for Th2 recruitment exist (22
); these may include the expression of CCR4 (19
) or CCR8 (1
). It remains to be seen whether blocking signaling via these receptors will further impede CD4+
-T-cell accumulation in rVV-G-primed, RSV-challenged mice.
A comparison of anti-CCL11 and anti-T1/ST2 treatments revealed several intriguing similarities, suggesting that overlapping Th2 cell subsets might express both T1/ST2 and CCR3, thereby leading to reductions in eosinophilia, illness, CD4+
-T-cell accumulation, and IL-5 production. The two treatments were not equivalent, however. Importantly, the treatment with anti-T1/ST2 produced a profound suppression not only of CD4+
but also of CD8+
-T-cell recruitment, and it significantly reduced the amount of IFN-γ in BAL fluid (39
). T1/ST2 is expressed predominately on type 2 cytokine-secreting cells, so the treatment was expected to eliminate only the relatively small fraction of Th2 and Tc2 cells. However, treatment with anti-T1/ST2 consistently produced far greater effects upon total T-cell accumulation than might have been predicted. This may again be due to the reduction in pulmonary damage, as suggested for anti-CCL11: the proportionately greater effect of anti-T1/ST2 presumably reflects the relative efficiency of each of these treatments at reducing Th2 cell accumulation.
The observation that IL-5, but not IL-4, was significantly reduced in BAL supernatants from mice that were treated with either anti-CCL11 or anti-T1/ST2 is intriguing. This may reflect the fact that CCR3 and T1/ST2 are expressed on Th2 cells at different stages of their differentiation, and the subsets of CD4+
T cells affected by either treatment might be those which predominately produce IL-5 rather than IL-4. This hypothesis is supported by a report that individual CD4+
T cells in vivo have different levels of commitment to a certain T-helper phenotype and can be distinguished by the expression of one or more type 2 cytokines and/or T1/ST2 (20
). T1/ST2 expression has recently been demonstrated on Tr1 regulatory CD4+
T cells expressing high levels of IL-10 in the respiratory tract (24
). These cells mediate the suppression of Th1 responses in response to Bordetella pertussis
and unrelated pathogens. No role for Tr1-type cells during experimental infection with RSV has been reported to date, although IL-10-producing cells are certainly detected in both primary and secondary disease. Our data describing the global downregulation of both type 1 and type 2 cytokines and of CD4+
T cells by an anti-T1/ST2 treatment suggest that effective Tr1 responses are not induced by G-enhanced RSV disease, a conclusion which is supported by the florid nature of the illness.
Together, our data suggest that selectively targeting the eosinophilic Th2 arm of a mixed type 1 and 2 pulmonary inflammation can have profound effects on global cellular recruitment. This selective targeting can be achieved by the depletion of either CCL11 or T1/ST2+
cells. This compares favorably with the elimination of pulmonary eosinophilia in G-enhanced RSV disease after the administration of IL-12, which skews the local T-helper response in favor of Th1 but can increase weight loss, pulmonary inflammation, and illness (14
). The present data suggest that selectively targeting Th2 responses by treatment with anti-CCL11 will produce beneficial effects on pulmonary eosinophilia without the attendant risks of augmented disease or viral replication. The clinical potential of this study is supported by the fact that one of the chemokine receptors for CCL11, CCR5 (26
), may play a role in the severity of bronchiolitis in humans (11