CD4+CD252 population, as a majority of cells are negative for any of the transcription factors T-bet, GATA3, RORct and Foxp3 in control animals. However, despite this low baseline expression, the expression of these transcription factors is relatively increased during allergen exposure. Additional T helper subsets are being suggested, and were not identified in the current study, including Th9 [30], Tr1 [31], Th3 [32] and Th22 cells [33], and some of these may indeed contribute to the fraction of CD4+CD25+ and CD4+CD252 that do not express any of the currently studied transcription factors. Clearly, also some of these cells may be involved in the inflammatory process in this model of allergic airway inflammation, and warrant investigation in future studies. All of the effector T cell- and Treg cell- subsets increase their proliferation in lung during allergen exposure (Figure 5), showing by uptake of BrdU. However, the CD4 cells that were CD25+ proliferated to a greater extent than the CD252 cells, indicating a difference in their proliferative ability. Thus, the lung itself is a microenvironment in which all the different studied T-lympho- cytes can proliferate. This is documented not only through BrdU incorporation, but also by cell cycle analysing with 7-AAD, measuring total amount of DNA in cells (Figure 5), indicating that many inflammatory cells are in S-phase or G2/M-phase in the lung during allergic inflammation. Even more, the Th2 cells showed the greatest increase in proliferation after allergen exposure, arguing again that these cells are the dominating effector T cells in allergic inflammation. Several mechanisms for suppression of T effector cells have been proposed. Recent observations have suggested that there could be interaction between different T subsets or with other inflammatory cells, with or without direct cell-to-cell contact [34– 37]. T cells play a role in both endothelium and epithelium. Our data showing that allergen exposure result in an increased accumulation of T-bet+, GATA-3+ and Foxp3+ cells in peribron- chial tissue, argue that these cells may interact in this microenvironment. By contrast, T-bet+ cells accumulate to a significant extent also in alveolar lung tissue, and to a lesser extent in perivascular tissue. Further, GATA-3+ and Foxp3+ cells are accumulated primarily in perivascular tissue. These differences in distribution of T cells argue for the different homing process of these cells to the different microenvironment, which is a unique contribution of the current study to the literature. The lung tissue represents a highly complicated and sophisti- cated network of many different cells that can react differently to a variety of stimuli, resulting in unique local inflammatory milieus of cytokines, chemokines and mediators in different microenviron- ments. The inflammation itself can of course affect different subsets of Th-cells that accumulate in different tissue. For example, it has been shown that NO production from human airway epithelium can affect the Th1/Th2 balance [38]. Another possible mechanism can be that the different T-helper cells that have accumulated in the lungs further drive the inflammation by further release of cytokines. Lordan et al. have shown that addition of Th2 cytokines in an already established allergic environment have the potential to sustain further airway inflammation [39]. To investigate the role of cytokines present in the airways, cytokines were first quantified in whole lung, showing that allergen exposure significantly increased levels of pro-inflammatory cyto- kines such as IL-6 and TNF-a and IL-17, showing the presence of a general inflammation after allergen exposure. These cytokines may be produced by a variety of cells as structural cells i.e. epithelial or fibroblasts or immune cells such as mast cells or eosinophils. However, it is likely that T cells represent a major source of many of the measured cytokines, and that Th1 and Th2 cells produce their corresponding cytokines during proliferation [40]. Lung Expansion of Th1, Th2, Th17 and Tregs We decided to take these findings further, to more exactly determine the local inflammatory milieu in the specific tissue microenvironment were respective T effector cells were found. Thus, laser microdissection and pressure catapulting technology and real-time RT-PCR analysis was combined to document the presence of expression of a broad range of inflammatory genes, mainly cytokines and chemokines, in respective tissue. Our data confirmed the hypothesis that there are differences local inflamma- tory milieus in the lung tissue compartments during allergic airway inflammation. Thus, from the 28 genes that had a fold changed $3 times after allergen exposure only 14, were common in all examined tissues, whereas some were dominating in respective environment. Importantly, even among these genes, differences in levels of expression were documented. For example, eotaxin-2 (Ccl24), which represents a major chemoattractant for eosinophils, showing a big variation in its local expression, can possibly explaining the results of high accumulation of eosinophils in perivascular tissue [10]. This chemokine may also have cytokine properties, as we have shown that it can play a role in eosinophilopoiesis locally in the lung [41]. Another example is the expression of osteopontin (Spp1), which recently has been proposed to enhance Th2 responses and is related to human asthma severity [42]. Chemokines plays a major role in T cell trafficking in allergic and asthmatic inflammation [43]. Therefore it was logical to hypothesise that local chemokine milieu can affect T effector cells accumulation. Indeed we have described extensive chemokine networks expressed in all investigated tissues, but with uneven expression in the different compartments. The pattern of their expression can at least partly explain our immunohistochemistry results. Thus, Th1 reported chemokines such as Ccl17 and Ccl2 [43,44] were primarily expressed in alveolar tissues, where T-bet cells primarily are located. Ccl8 was quite recently reported by Islam et al. [45] as a potent chemoattractant for GATA-3, IL-5+ Th2 cells in skin allergy, which is in line with our finding of expression of this chemokine in primarily the perivascular milieu, where GATA-3 cells were accumulated. Further, the chemokines Ccl19 may explain the microenvironmental localisation of Treg, while the chemokines Ccl20 may not explain Th17 cells distribution [43,46,47]. By Ingenuity Pathways Analysis, we have documented extensive interactions between the chemokines and cytokines, suggesting that a small difference in expression of one may affect the action of the other, and can thus influence the whole inflammatory network in the microenvironment. This becomes clearer when some chemokines such as Ccl4 can influence the traffic of more than one of the studied T effector cells. The interaction between the different chemokines is complicated, since we observed a down-regulation of Pf4, which is related to Th1 responses [48], even though other Th1 chemokines were increased in the same environment. In summary, our current study shows that T-bet, GATA-3, Foxp3 and RORct in both CD25+ and CD252 CD4+ T cells all expand in lung during allergic airway inflammation, and also proliferate within the lung. In this mouse model of allergic airway inflammation, Th2 cells are the most dominant effector T-cell. Within the lungs, these effector T cells are differently distributed, with Th1 primarily being present in alveolar lung tissue, Th2 and Treg cells primarily being observed in perivascular lung tissue, and the numbers of Th17 cells being few but with a dominance in peribronchial lung tissue. The inflammatory milieu in those tissue compartment showed different profiles, both for mediators associated to allergic inflammation, such as chemokines, explain- ing at least partly our results. Overall, this study shows that Th2 cells are prominent in allergic inflammation, but also verify concomitant the activity of Th1, Th2, Th17 and Tregs. PLoS ONE | www.plosone.org 15 May 2011 | Volume 6 | Issue 5 | e19889