Anti-IgE has recently been approved for the treatment of severe asthma (18
), but issues surrounding protein therapies, including tolerization and immunogenicity, remain. Therefore, small compounds for inhibiting IgE–Fc
RI signaling are still being actively investigated. In this study, we demonstrated the potency of an oral Syk inhibitor, R406, in inhibiting antigen-induced mast cell activation, degranulation, and cytokine production, as well as in preventing airway inflammation and suppressing allergen-induced AHR. These data add to the growing body of literature of the importance of Syk in the development of allergic airway inflammation (8
). In a dose-dependent manner, R406 prevented development of AHR induced by 10 consecutive d of OVA exposure. Using this protocol, we previously showed that the development of AHR required IgE, mast cell activation, and expression of Fc
). After aggregation of Fc
RI by multivalent allergen, signaling through the receptor is initiated. The pathways may be distinct for degranulation and cytokine production (15
), but upstream of both, Syk activation appears to be essential (20
). Evidence for the role of Syk and the activity of the inhibitor R406 was confirmed at several levels. First, using BMMCs activated through Fc
RI, the inhibitor suppressed mast cell degranulation and cytokine production. Incubation of the inhibitor together with sensitized IgE–BMMCs for 1 h before cross-linking with OVA led to a dose-dependent inhibition of [3
H]-serotonin release, a marker of degranulation. Under similar conditions, inhibition of Syk decreased the production of IL-2, IL-6, IL-13, and TNF-α in a dose-dependent manner. For reasons that are not clear at present, in vitro
BMMC-derived cytokine production appeared to be more sensitive to the inhibitor than degranulation.
The assays are certainly different as are the pathways activated through Syk that appear to be necessary for degranulation as opposed to cytokine production. In the former, a role for mitogen-activated protein/ERK1 kinase (MEK1)–ERK1/2 has been defined (15
). Inhibition of ERK1/2 activation by MEK1 inhibitors interferes with the degranulation response (15
). Cytokine transcriptional regulation appears dependent on the activation of MEK kinase 2 (MEKK2)–MEK5–ERK5 with perhaps some amplification through an MEKK2–c-Jun N-terminal kinase (JNK) pathway. ERK1/2 does not appear to be involved in this pathway, nor does phosphorylation of p38. These MAPK pathways were examined after mast cell activation through Fc
RI. In a dose-dependent manner, the Syk inhibitor decreased the phosphorylation of ERK1/2, JNK1/JNK2, and ERK5. p38 phosphorylation was unaffected. These data confirm and extend the results in Syk-deficient mice where mast cell degranulation and cytokine production were significantly reduced (19
Syk is expressed in mast cells (24
), macrophages (26
), B cells (28
), neutrophils (30
), and dendritic cells (31
); all of these cells may be activated in allergen-induced airway inflammation. The one cell important in the development of airway inflammation and AHR that does not use Syk is the T lymphocyte. Thus, in addition to targeting mast cells, and because of its widespread distribution, Syk is considered to be a good target for the treatment of allergic diseases without strong immunosuppression. This possibility was tested in a mast cell–dependent mouse model of allergen-induced AHR and airway inflammation. In most rodent models of allergen-induced AHR, the animals are sensitized systemically by injection of the allergen together with adjuvant, such as alum, and then challenged via the airways (32
). In these protocols using systemic sensitization with adjuvant, marked inflammatory changes develop in the lung, high serum levels of allergen-specific immunoglobulins are induced, and increased airway reactivity to inhaled spasmogen develop as well as goblet cell hyperplasia. These lung-based allergic responses develop normally in mice that are mast cell deficient (36
) or antibody deficient (34
), excluding a critical role for IgE and mast cells in their development in this approach.
In the present study, mice were exposed to a less potent exposure protocol using aerosolized allergen alone in the absence of adjuvant. After 10-d allergen exposure, the mice developed altered airway responsiveness, detected as heightened responsiveness of tracheal smooth muscle rings to EFS. This response has been associated with muscarinic acetylcholine receptor-2 dysfunction and increased acetylcholine release (37
). This increase in responsiveness after allergen exposure was prevented by R406. This protocol also results in only modest airway inflammation and less goblet cell hyperplasia than is seen after systemic sensitization. Nevertheless, eosinophil accumulation in the lung after the 10-d OVA exposure protocol could be detected using MBP staining of lung sections. Administration of the Syk inhibitor twice daily was effective in preventing this eosinophil accumulation in the lung. Furthermore, we could detect PAS-positive cells identifying goblet cell metaplasia after 10-d allergen exposure, and the Syk inhibitor also prevented the appearance of PAS-positive cells in the airway epithelium. These results effectively demonstrate that the Syk inhibitor inhibited not only AHR but also lung inflammation, pulmonary eosinophilia, and goblet cell hyperplasia.
Virtually identical results were obtained when mice were passively sensitized in vivo
with OVA-specific IgE and exposed to only 2 d of aerosolized allergen challenge. In this IgE–mast cell–Fc
RI–dependent model (12
), R406 also prevented the development of the heightened response to EFS, confirming the effectiveness of targeting Syk in these responses. Whether the effects of inhibiting Syk are being manifested in cells other than mast cells in these responses cannot be ruled out at this time because of the expression of Syk in many cell types that may be involved in the development of AHR (B cells, dendritic cells, eosinophils). The passive sensitization model certainly bypasses the need for endogenous IgE production by B lymphocytes, a cell whose function is highly dependent on Syk activation (20
Mast cells can release many mediators during the degranulation response as well as cytokines after transcription and translation and enhance airway reactivity and inflammation (38
). Among the cytokines released from mast cells, IL-13 has been shown to play a major role in eosinophil recruitment and mucin hyperproduction and goblet cell metaplasia (41
). In the models used in this study, 10-d OVA exposure or passive sensitization, we have shown that, although mast cells are essential, mast cell–derived IL-13 may not be essential (12
). Indeed, recent data suggest that it is perhaps mast cell–derived LTB4 that may be essential to the IL-13 release from other cells (43
). LTB4, interacting with its specific receptor BLT1 on a subset of IL-13–secreting CD8+
T cells, may be responsible for the recruitment to the lung and activation of this subset in the lungs and the development of AHR and airway inflammation (44
). Thus, R406, in preventing the mast cell degranulation response and the release of LTB4, could be preventing the accumulation of CD8+
T effector cells in the lung and hence the failure to develop AHR and airway inflammation. Studies are currently underway examining this potentially important action of R406.
In summary, Syk activation is an important convergence point in the pathways activated through Fc
RI, regulating both degranulation and cytokine production. Targeting of Syk effectively prevented the development of AHR and airway inflammation in model approaches that are dependent on IgE, mast cells, and Fc
RI. In this way, Syk inhibition may represent an important therapeutic avenue for the treatment of allergic asthma.