T cells isolated from peripheral venous blood of normal subjects and steroid-naïve stable asthmatic patients expressed STAT6 protein. However there was no increased expression in T cells from asthmatic patients compared to normal subject or in its activation. This is in accordance with a previous study that found similar levels of STAT6 in PBMCs from normal subjects and patients with extrinsic asthma . This suggests that either there is no increase in activated Th2 cells in the blood in the predominantly mild, stable, asthmatic patients studied here or that STAT6 activation is not important for this process. This further suggests that Th2-like gene regulation in man may be controlled, at least in part by the expression of proteins distinct from those predicted by studies in human cell lines or in the mouse. For example, many animal and human in vitro studies suggest that GATA-3 plays an important role in the differentiation of Th2 cells in conjunction with other transcription factors such as nuclear factor of activated T cells (NF-AT)c/B, c-Maf and STAT6 [9, 22].
In both normal subjects and asthmatic patients, alveolar macrophages and bronchial epithelial cells also expressed STAT6 proteins. The predominant sites of STAT6 expression in bronchial biopsies are epithelial cells in all subjects. These results are in contrast to two previous studies using immunohistochemistry in which the authors were unable to find any STAT6 immunoreactivity in nasal  and bronchial epithelial cells  but detected STAT6 localised to the nuclei of some infiltrating cells.
This discrepancy may depend upon differential expression of STAT6 in epithelial cells from upper and lower airways since we found similar results with the two different antibodies by Western blottings. However, we did not use the same STAT6 antibodies (two mouse monoclonals respectively from Transduction Laboratories and Santa Cruz) as Ghaffer and colleagues  and Christodoulopoulos and colleagues  for immunostaining and their sensitivity may differ. However, this is unlikely to account for the different site of expression seen in bronchial airways. Another study using immunohistochemistry with the same antibody as used here has also shown that the bronchial epithelium is the major site of STAT6 expression . In addition, they also reported no difference in the site of expression between asthmatic and normal control subjects .
The presence of STAT6 immunoreactive cells with nuclear localisation of STAT6 in the nasal mucosa of atopic allergic rhinitis may be the result of a selective accumulation of Th2 cells in this disease. This is in accordance with the presence of a minority of T cells with nuclear localisation of STAT6 demonstrated by immunostaining and with the results of a previous study that showed no activation of STAT6 in PBMCs from patients with acute asthma .
Human bronchial epithelial cells and alveolar macrophages express IL-4R complex , respond to IL-4 and IL-13 and produce cytokines such as GM-CSF and eotaxin which contain STAT6-binding sites in the regulatory sequences of their genes [25–30]. In animal models of allergic asthma, blockade of IL-13 markedly inhibits allergen-induced airway hyperresponsiveness, mucus production and eosinophilia . Furthermore, IL-13 delivery to the airway causes all of these effects . Mice lacking STAT6 gene are protected from all pulmonary effects of IL-13 . Reconstitution of STAT6 only in bronchial epithelial cells is sufficient for IL-13-induced airway hyperresponsiveness and mucus production in the absence of inflammation, fibrosis or other lung pathology . These results demonstrate the importance of direct effects of IL-13 and STAT6 on epithelial cells in causing two central features of bronchial asthma .
Interestingly, induction of inducible nitric oxide synthase (iNOS) in both bronchial epithelial cells and alveolar macrophages involves IL-4 and STAT6 [23, 32]. These data suggest that STAT6, which is key components of signals via IL-4 and IL-13, may potentially play an important role in modulating inflammatory gene expression in alveolar macrophages and bronchial epithelial cells. However, our result demonstrated the same level of STAT6 in T-lymphocytes, macrophages and bronchial epithelial cells from all subjects with no difference between normal and asthmatic subjects. There are some potential explanations for these results. Firstly, the process of STAT-6 translocation from cytoplasm into nucleus is dependent of other signals, such as Src kinase and phospholipase C activity [33, 34]. The existence of cytoplasmic STAT6 might denote that other signals such as oxidative stress, which may induce phospholipase C activity, is required for the translocation of STAT6 into nucleus. Secondly, for IL-4/IL-13-mediated pathway activation, there is also a STAT6-independent process such as a acidic mammalian chitinase . In this paper, STAT6 activation was assessed using immunoblotting. Additional data will be required to assess STAT6 activation by quantative ELISA for identifying transcription factor binding to DNA and chromatin immunoprecipitation assay.
In the last decade many clinical studies have examined the effect of blocking Th2 activity in human asthma. Overall, these studies have proved disappointing in that as a group mild-moderate asthmatics, similar to those studied here, did not respond clinically to drugs targeting IL-4, IL-5 and IL-13 [36–38]. This raises the possibility that the underlying concept of asthma being a Th2-driven hyper-eosinophilic disease which has been developed in animal models is not reflected in human disease except in small subsets of patients with selected asthmatic phenotypes [36, 37]. This further highlights the need for more translational research into asthma mechanisms in man if common drivers of disease in human asthma are to be determined.
In summary, T cells from normal subjects and mild-moderate steroid-naïve asthmatic patients in stable phase asthmatics express similar levels of STAT6 protein within each subcellular compartment. Alveolar macrophages and bronchial epithelial cells also express STAT6 proteins with equal expression in normal subjects and asthmatic patients. STAT6 may be important in regulating the expression of Th2-like cytokines in alveolar macrophages and bronchial epithelial cells in addition to T cells in man. Further studies with large number of participants are needed to characterise fully the role of the STAT6 proteins in the regulation of gene expression in these cell types and their potential role in the pathogenesis of asthma, because asthma has various phenotypes. A previous study suggested that treatment with topical glucocorticoids can reduce the number of CD3+/STAT6 immunoreactive cells in nasal mucosa . However at present the ability of glucocorticoids to target STAT6 action in the lower airways is unknown and may determine whether inhibition of STAT6 activity may be a new therapeutic target for anti-asthma drugs.