Th2 lymphocytes play a crucial role in allergic asthma, and strategies aimed at interfering with the function of these cells or individual cytokines (anti–IL-5, soluble IL-4 receptor) have been proposed as novel forms of therapy (32
). Recently, the immunomodulator FTY720 has been shown to be effective as an oral drug for the treatment of experimental asthma, induced by adoptive transfer of effector Th2 cells (22
). In addition, numerous other Th1-mediated experimental disease models of graft rejection, type 1 diabetes mellitus, autoimmunity, and arthritis have been successfully treated using FTY720, and this drug is currently employed in phase III clinical trials for prevention of allograft rejection (18
). In all of these models, FTY720 has been administered systemically where its major mechanism of action is the inhibition of lymphocyte egress from the LN medulla, resulting in peripheral lymphopenia (17
). S1PR agonists cause sequestration of lymphocytes in secondary lymphoid tissues by acting as functional antagonists that downregulate S1PR1s on lymphocytes or by preventing entry of medullary lymphocytes into the efferent lymphatics for recirculation (17
). Although FTY720 does not impair the activation, expansion, and memory function of lymphocytes (33
), the occurrence of peripheral lymphopenia and, as a consequence, the reduced recruitment of effector lymphocytes to sites of inflammation might constitute a form of immunodeficiency, with its inherent risk of developing opportunistic infections, which is undesirable for the treatment of non–life threatening diseases, such as allergic asthma. To avoid this systemic immunodeficiency, we tested to determine whether local i.t. treatment with FTY720 was similarly able to suppress the cardinal features of asthma. Local FTY720 treatment given prior to each allergen challenge in sensitized mice was able to inhibit the salient pathologies of asthma, including eosinophilic inflammation, goblet cell hyperplasia, Th2 cytokine production, and most importantly, bronchial hyperreactivity to inhaled methacholine, even when the treatment was delayed until after the 3 first aerosol challenges. FTY720 most likely acted on S1PRs, as the antiinflammatory effect was mimicked by S1P; however, the precise receptor subtype involved is unclear. Our preliminary data using a selective S1P1R agonist, SEW2871, suggest that agonism of S1P1R alone is sufficient to inhibit allergic airway inflammation (unpublished observations). In striking contrast to oral FTY720 treatment, with treatment via inhalation we observed no peripheral blood lymphopenia or sequestration of T and B lymphocytes in the secondary lymphoid organs. In addition, lung administration did not lead to measurable plasma FTY720 levels. There was, however, a reduction in allergen-induced BAL fluid lymphocytosis and a reduction in the number of T and B lymphocytes in the lung-draining mediastinal LNs, most likely due to the reduction in airway inflammation in FTY720-treated mice, and a concomitant reduced influx of lung-derived CCR7+
effector Th2 lymphocytes to LNs. Migration of effector lymphocytes to the lung-draining LNs does occur in the lungs of allergen-challenged mice and is CCR7 dependent (34
DCs are crucial in both the initiation and maintenance phase of allergic asthma and therefore constitute ideal targets for the development of novel antiasthmatic drugs (2
). As a proof of concept, selective depletion of DCs during allergen challenge abolishes all the cardinal features of asthma, including bronchial hyperreactivity (30
). DCs are crucial because they can locally activate Th2 effector cells in the airway wall by providing chemotactic cues for Th2 cells (CCL17 and CCL22) and by delivering MHC and costimulatory signals (4
). Additionally, at times of allergen challenge, DCs migrate from the site of allergic inflammation to mediastinal LNs to stimulate new rounds of division in recirculating central memory cells or from naive T cells, thus feeding the inflammatory response with new waves of effector cells (2
). As FTY720 and S1P suppressed inflammation without altering lymphocyte distribution, we studied the effect of FTY720 and S1P on lung DCs and found that freshly isolated lung DCs express all S1PR subtypes, consistent with prior reports on cultured DCs (23
). One of the striking observations we made after local FTY720 treatment was a reduction in the number of mDCs in mediastinal LNs of allergen-challenged mice. Although, again, a reduction in lung inflammation might be responsible for reduced input of immigrating lung-derived DCs in LNs, we also observed a reduction in DC migration to LNs in naive mice when FTY720 was administered together with fluorescently labeled OVA, concomitant with an accumulation of DCs in the lung compartment. The same effect of systemically administered FTY720 and S1P agonists was seen on DC migration from the skin following FITC skin painting (26
We and others have shown that S1P agonists and FTY720 alter the regulation of actin-dependent cytoskeletal processes in DCs by downstream signaling of different S1PRs through small Rho family GTPases, such as Rho and Rac/Cdc42 (23
). It was shown that a correct balance and possibly cyclic activity of Rho and Rac/Cdc42 are essential for DC migration to occur and that this process might be inhibited by FTY720 (38
). However, this explanation seemed less likely as we performed in vitro chemotactic assays. DC migration from the periphery of the lung to the mediastinal nodes is CCR7 dependent (39
). It has been shown by Lan et al. that FTY720 treatment of DCs impairs CCR7-dependent chemotactic migration to CCL19 across endothelial cells in vitro (26
) whereas Czeloth et al. showed that migration to the other CCR7 ligand, CCL21, across Transwell inserts was unaffected (38
). In our experiments, we found that FTY720 treatment of DCs did not affect migration toward CCR7 ligands in vitro whereas, clearly, responsiveness to the chemotactic signal S1P was reduced. We therefore favor the idea that FTY720 specifically suppresses migration of DCs by downregulation of the S1PR1 and not by interference with the actin cytoskeleton.
Inhalation of low-endotoxin OVA is a normally tolerogenic event in which pDCs inhibit the potential of mDCs to prime for effector Th2 cells (3
). By depleting pDCs, this tolerogenic response is turned into robust Th2 priming by mDCs. In this model, FTY720 completely abolished the development of Th2 effector cells, and consequently, asthma did not develop upon repeated OVA challenge. There may be several reasons for the lack of Th2 priming. First, S1P agonists have been shown to alter the function and stability of bronchial epithelial tight junctions (40
). These tightened junctions could be a barrier to the uptake of inhaled OVA, thus leading to a lack of Th2 priming. To exclude this possibility, we also incubated mDCs with FTY720 or S1P prior to adoptive transfer to naive mice. This pretreatment of DCs significantly reduced their potential to induce Th2 priming, and consequently, asthmatic inflammation did not develop. Therefore, the effects of FTY720 are most consistent with an autonomous defect on DCs.
Second, FTY720 inhibited the migration of lung-derived DCs to LNs, thus inhibiting proper T cell priming. Priming of recirculating naive T cells or reactivation of central memory T cells in LNs depends on input of DCs from the periphery of the lung in a process that is CCR7 dependent (39
). However, inhibition by FTY720 of DC-mediated FITC-OVA transport was not absolute, and the DCs that escape FTY720 inhibition might still induce T cell activation. This indeed was the case when we followed primary T cell activation in a cohort of adoptively transferred DO11.10 T cells in vivo (data not shown). Even in FTY720-treated mice, OVA inhalation still led to T cell divisions in mediastinal nodes, although the strength of the T cell response was reduced. We therefore favor the third possibility that FTY720- or S1P-treated DCs are also defective in priming naive and memory T cells due to an intrinsic defect in T cell stimulatory capacity. Consistent with this, we cultured OVA-pulsed BMDCs with OVA-specific T cells from naive mice and observed a strong and dose-dependent reduction in proliferation and production of both Th1 and Th2 effector cytokines when DCs were preincubated with FTY720. In addition, FTY720 treatment of DCs inhibited their potential to stimulate primed effector Th2 cells generated under polarized Th2 conditions in vitro. Similar effects of FTY720 and S1P were noticed on the immunostimulatory capacity of human monocyte–derived DCs matured using LPS although in the human setting, Th1 cytokines were preferentially inhibited while Th2 priming was essentially weak but unaffected (23
). Our results showed that local FTY720 treatment inhibits IL-12 production in lung DCs, a finding similar to that in human data.
FTY720 might modify the potential of DCs to stimulate T cells in several ways. The induction and maintenance of effector cytokine production in T cells in the lungs depends upon an adequate antigen-processing function and maturation of lung DCs (41
). Surprisingly, neither FTY720 nor S1P altered the expression of CD40, CD80, CD83, CD86, programmed death ligand-1 (PDL-1), PDL-2, and MHCII in immature or mature BMDCs (data not shown). Moreover, local FTY720 treatment did not alter the maturation of lung and LN DCs. These data are in accordance with studies in which S1P agonism did not affect maturation of murine and human DCs (23
). FTY720 treatment of DCs could also interfere with their potential to form stable and long-lasting interactions with T cells in the LNs or lung parenchyma. The small Rho family GTPases (such as Rac1 and Rac2) are modified by FTY720, and expression of these factors in DCs has been shown to be essential for the occurrence of DC–T cell interaction (31
). In accordance, FTY720 treatment of DCs indeed abolished the formation of long-lasting DC–T cell interactions typical of an immunological synapse that is critical for induction of cellular immunity (31
). Whether local treatment with FTY720 would also reduce the formation of an immunological synapse between recirculating T cells and lung DCs in the airways or lung parenchyma remains to be shown.
In conclusion, the present study demonstrates for what we believe is the first time that the local administration of FTY720 inhibits Th2-mediated cardinal features of asthma by altering the function of lung DCs without causing any systemic lymphopenia. These findings could pave the way for a feasibility study in which this compound or a more selective S1PR agonist is administered via aerosol to patients with asthma. This paper validates the concept that targeting airway DC function is a powerful method to treat asthma.