In this study, we have delineated the role of CCR4, CCR8 and CXCR3 in antigen-specific Th2 cell trafficking. We have shown that while CCR4 deficiency decreases antigen-specific Th2 cell trafficking into the allergic lung, CXCR3 deficiency has no impact and CCR8 deficiency leads to an accumulation of these cells. The roles of CCR4 and CCR8 were specific for antigen-specific Th2 cells as antigen-specific Th1 cell trafficking was not affected by CCR4 or CCR8 deficiency.
There are conflicting reports in the literature on the effect of CCR4 deficiency on allergic airway inflammation. CCR4−/−
mice sensitized with OVA and challenged 14 days later with five daily intranasal OVA doses developed airway inflammation, airway hyperresponsiveness and serum IgE levels comparable to wild type mice 22
. Moreover, the use of CCR4 blocking antibody did not inhibit allergic airway inflammation in a guinea pig model 23
. However, airway hyperresponsiveness and eosinophilia were diminished in CCR4−/−
mice in a chronic Aspergillus model 18
, and in another report using CCL17 blocking antibody in OVA sensitized mice prior to OVA challenges 19
. There is also discrepancy on the effect of CCR8 deficiency on allergic airway inflammation. For example, three studies used an active immunization model with OVA and alum but only one found a difference between wild type and CCR8−/−
mice 17, 20, 21
. In addition, there was no difference in BAL eosinophilia after adoptive transfer of CD4+
cells isolated from OVA-immunized wild type and CCR8−/−
, while CCR8 deficiency led to a decrease in allergic airway inflammation in a mast cell dependent model 12
. In all of these reports, the CCR4 or CCR8 axis was interrupted in all cell types, including Tregs. The outcome of each study most likely differed based on the animal model used and the balance of the effect of CCR4 or CCR8 deficiency on multiple cell types. Our study is the first to isolate the role of CCR4 and CCR8 in the trafficking of antigen-specific Th2 cells from all other cell types by using competitive and separate adoptive transfer models.
Among the chemokine receptors studied, CCR4 was the only receptor required for the efficient recruitment of antigen-specific Th2 cells into the lung and the airways. We show that approximately 60% of antigen-specific Th2 cells express CCR4 prior to their adoptive transfer and after trafficking into the airways. This level of CCR4 expression is consistent with the 50% trafficking defect in CCR4−/−
antigen-specific Th2 cells. It is also similar to what has been described in asthmatic subjects 9, 10
. We also demonstrate that approximately 60% of IL-4 producing antigen-specific Th2 cells express CCR4. This explains the dramatic decline in BAL Th2-type cytokines in CCR4−/−
OTII Th2 cell recipient mice compared with OTII Th2 cell recipients. In addition, CCR4−/−
OTII Th2 cell recipients had a 50% reduction in airway eosinophilia and a modest decrease in overall lung parenchymal inflammation and airway mucus production but no change in airway hyperresponsiveness. This indicates that the residual Th2 cells and Th2-type cytokines were sufficient to preserve some markers of allergic lung and airway inflammation. In this study, we focused on CCR4, CCR8 and CXCR3, and the relative contribution of other chemoattractant receptors to the residual Th2 cell trafficking remains to be determined.
We show that while CXCR3 had no impact on the trafficking of antigen-specific Th2 cells, CCR8−/−
OTII Th2 cells accumulated in the lung, BAL and TLN. This increased accumulation was not due to decreased apoptosis or increased rate of proliferation. However, while the rate of proliferation was the same between OTII and CCR8−/−
OTII Th2 cells, there were more CCR8−/−
OTII Th2 cells proliferating in the TLN. This resulted in more CCR8−/−
OTII Th2 cells being available to leave the TLN and accumulate in the lung over time. The reason for the increased trafficking of CCR8−/−
OTII Th2 cells into the TLN is not clear. However, we speculate that in the absence of CCR8 signaling, CCR8−/−
OTII Th2 cells are not responsive to constitutive CCR8 ligands expressed in organs, such as the skin 36
, and are thus more available for recruitment and subsequent proliferation in the TLN. These data demonstrate that the functional role of any given chemokine receptor and its ligands cannot be predicted from in vitro
studies or studies of ligand and receptor expression and highlight the complexity of the chemokine system in vivo
In summary, we have isolated the role of CCR4, CCR8 and CXCR3 on antigen-specific Th2 cells from the role of these receptors on all other cell types using an adoptive transfer model of allergic pulmonary inflammation. In so doing we have determined that CCR4 is required for the efficient entry of antigen-specific Th2 cells into the allergic lung and the airways.