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- Materials and methods
Background: Allergic asthma and rhinitis are described as associated with a Th2 activation. However, recent works indicate that a Th1 activation can also be associated with these diseases, concomitantly to a defect in regulatory T (Treg) cell activation. Occupational asthma (OA) and occupational rhinitis (OR) are peculiar cases of these diseases in which the T-cell activation profile is largely unknown.
Objective: To characterize T-cell activation induced after a specific inhalation test (SIT) in OA and OR.
Material and methods: A total of 21 subjects with OA, 10 subjects with OR, 10 exposed nonallergic (ENA) subjects, and 14 healthy volunteers were included. The SIT with the incriminated substance was performed in patients and ENA subjects. Blood and induced sputum were obtained before and after SIT. T cells were analysed for CD69, CD25, IL-13, and IFN-γ expression by flow cytometry. IL-4 and IFN-γ were assayed by enzyme-linked immunosorbent assay (ELISA) in cell culture supernatants. Treg cells were identified as CD4+CD25+highCD45RO+CD69− T cells in peripheral blood.
Results: Baseline IFN-γ production was decreased in OA and OR compared with controls. The SIT induced an increase in both Th1 and Th2 cells in blood and sputum from OA. In this group, the proportion of peripheral Treg cells decreased after SIT. Similar results were found in the CD8+ population. ELISA assays were concordant with flow cytometry. In OR, an attenuated activation profile was found, with an increase in the proportion of IL-13-producing T cells after SIT. By contrast, in ENA subjects, SIT induced Th2 activation, with an increase in Treg cells and a decrease in Th1 cells.
Conclusions: Our results demonstrate a gradient of T-cell activation from a tolerating profile in ENA subjects to an inflammatory profile in OA, with an intermediate stage in OR.
Occupational factors have been implicated in 9–15% of adult asthma (1), making occupational asthma (OA) the main cause of occupational respiratory diseases. As a result of the large diversity of causative agents, the pathophysiology of OA is still controversial. It is admitted that immunological and nonimmunological reactions can be involved, and among the former, IgE-dependent and IgE-independent immune responses. IgE-dependent OA is mainly because of high molecular weight (>5000 day) molecules, such as vegetable proteins, and thought to be closely related to asthma unrelated to work. Low molecular weight agents such as isocyanates, aldehydes and other chemicals induce a form of asthma in which the implication of IgE is doubtable (2). However, in both cases of immunological OA, the airway inflammation is similar, with the presence of eosinophils, lymphocytes and mast cells (1). Previous studies in mice have demonstrated a Th2 cell activation, with an increase in IL-5 and IL-4 production in experimental OA (3, 4). Other mice studies have found a Th1 activation associated with the Th2 activation previously described, especially in the case of low molecular weight allergen exposure, with IFN-γ production (5, 6), notably by the CD8+γδ T cells (7). This view is reminiscent of what is currently known in non-occupational asthma, in which a Th2 activation predominates, accompanied by a Th1 activation (8). In addition, in non-occupational asthma, some evidences recently indicated that Th2 and Th1 cell activation could be concomitantly associated with a decrease in the regulatory T (Treg) cell frequency and activation (9). However, to date, no data about Treg cells in OA, in response to a specific inhalation test (SIT), have been published.
Occupational asthma is frequently preceded by occupational rhinitis (OR), notably in cases related to high molecular weight agents (1). Many studies clearly established that allergic rhinitis is Th2-mediated (10). In addition, a Th1 activation was also observed in allergic rhinitis during natural allergen exposure (11), and a deficiency in the Treg cell activation was clearly demonstrated in this disease (12). However, no report concerns specifically the immunopathology of OR.
The aim of this study was thus to characterize the human T-cell activation induced by occupational allergens, in OA and OR subjects. We took advantage, for this purpose, of SIT performed to diagnose an OA or rhinitis.
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- Materials and methods
Allergic asthma is characterized by Th2 activation, mainly attributed to CD4+ T cells, which, by producing IL-4 and IL-5, respectively, induce IgE production and bronchial eosinophilia, two main features of asthma. This concept, named the Th2 paradigm in allergy, is to date considered as a dogma in asthma pathophysiology (16).
Despite an intrinsic defect in Th1 cells concordant with this Th2 paradigm, we found that in OA patients, SIT induced a mixed Th2/Th1 response. In addition, CD8+ cells were the main producers of IFN-γ.
In OA, the T-cell activation was previously studied in a murine model of isocyanate-induced asthma, a major form of OA because of a low molecular weight allergen. In this model, the implication of T cells was clearly demonstrated. Indeed, T cells from sensitized animals were sufficient to transfer the disease to naïve mice (17). Conversely in CD4- but also in CD8-deficient mice, isocyanate-induced airways hyper-responsiveness was impaired, together with airways eosinophilia and specific IgE production. A Th2 activation was demonstrated, with an increase in IL-4 and IL-5 production, but also a Th1 activation mainly characterized by IFN-γ increase (6). In humans, hyper-responsive to isocyanates, bronchial biopsy studies have long been established an in situ activation of eosinophils, CD4+ and CD8+ T lymphocytes (18). Therefore, animal and human data are concordant with our findings of a mixed Th1/Th2 activation with implication of the CD8+ T cells in OA.
This profile could be specific of the low molecular weight agent used in these studies, namely isocyanates: OA is due to a large variety of allergens, among which high molecular weight allergens are frequently distinguished from low molecular weight allergens with regard to the induced pathology. Notably, the former are thought to induce a form of asthma similar to nonoccupational allergic asthma, in which atopy is a risk factor, specific IgEs are present in serum, and skin prick tests are relevant to the diagnosis. In OA related to low molecular weight agents, atopy is not a risk factor, specific IgEs are frequently not found, and thus skin prick tests are poorly contributive to the diagnosis (1). However the T-cell activation profile between both forms of substances involved was similar, which suggest that although IgE are not necessarily involved in both cases, some mechanisms are shared.
In addition, more and more publications indicate that in common non-OA, a Th1 activation accompanies the Th2 cytokine production (8). Indeed, whereas in some models of asthma the induction of Th1 cells was protective (19), in other cases, the presence of IFN-γ-producing cells exacerbated the asthmatic phenotype (20). In addition, in humans, a production of IFN-γ was found, correlated to asthma severity (21), symptoms (22), and bronchial hyper-responsiveness (23). It is noteworthy that IFN-γ-producing T cells in asthma were frequently CD8+ cells (23). This is in keeping with results found in ENA subjects. Indeed, whereas in these patients the SIT-induced Th2 activation was not found at the CD8+ level. In addition, a decrease of IFN-γ-producing T cells occurred, suggesting a crucial role for IFN-γ-producing T cells, notably CD8+, in the inception of asthma in exposed subjects.
This raises the question of the role of CD8+ cells in asthma. These cells are considered as a heterogeneous population, including effector cytotoxic T cells and cytokine-producing T cells but also NK cells, NKT cells, and γδ T cells. Furthermore, the bronchial infiltration by the CD8+ T cells was associated with the decline of FEV1 in asthma (24) and asthma mortality (25).
Therefore, the involvement of IFN-γ+ and of CD8+ cells in asthma could be a trait of increased severity. In this view, it is not surprising to find this trait in OA patients, as this form of asthma is considered as more severe than non-OA (1). No relationship was found between asthma severity or specific or nonspecific bronchial hyper-responsiveness and CD8+ or IFN-γ+ T cells in this study, probably because of the limited number of patients explored.
It was also recently pointed out that a deficiency in the Treg cell population could preclude to the mixed Th2/Th1 activation seen in asthma (26). Indeed, Treg cells regroup small populations of T cells, which constitutively produce immunosuppressive cytokines such as IL-10 and/or TGF-β. These cells are thought to be responsible for the immune homeostasis, preventing the organism from any unwarranted inflammation, notably against self-antigens and allergens (27). In this view, a deficiency in Treg cells could be responsible for the increase of auto-immune and allergic diseases observed in the last decades (28). A Treg cell deficiency was previously showed in asthma (8) and allergic rhinitis (12). The decrease of the Treg cells observed in OA patients after SIT, but overall the increase of Treg cells induced in ENA subjects are concordant with this hypothesis. The variation in the proportion of the Treg population was not previously investigated in OA after inhalation challenge. Hoffman and coll (29) assessed the Treg cells in blood after ingestion challenge in patients with OA, because of cereal flour exposure. In these conditions, they observed a decrease of CD4+CD25+ cells after ingestion but only when compared with placebo and not when comparing baseline to post-challenge data. A similar profile of mixed Th1/Th2 activation and a decrease of Treg cells were recently demonstrated in exacerbations of asthma, a condition that can be compared with allergen challenge (30).
T-cell activation has not been previously investigated in OR. These patients are rare; OR is frequently neglected and therefore unexplored. Herein, we demonstrate a T-cell activation profile reminiscent of what is described in non-occupational rhinitis. Indeed, as asthma, rhinitis is largely associated to a Th2 activation at baseline, enhanced under allergen stimulation (7). We found a deficient IFN-γ production in T cells at baseline, which is in keeping with an intrinsic Th2 skewing and a SIT-induced Th2 activation. This profile can be considered as different from the one seen in asthma, as Th1 activation and Treg cell decrease were not found in OR. However, it is more likely that the OR T-cell activation profile is intermediate, with an elevation of IFN-γ production and a decrease of Treg cells that do not reach significance in our study. Indeed, the mean cytokine and T-cell variation was concordant with OA results in a smaller extent.
In conclusion, our results demonstrate a gradient of T-cell activation from a tolerating profile in ENA subjects (decrease of Th1, increase of Th2 and Treg cells after SIT) to an inflammatory profile in OA (increase of both Th1 and Th2, decrease of Treg cells after SIT), with an intermediate stage in OR.