Restoration of cytokine imbalance by immunotherapy

It is now over 10 years ago that two populations of T helper cells TH1 and TH2 were recognized in mice [ 1, 2] and led to the recognition of a similar, but not identical, heterogeneity among human CD4+ T lymphocytes [ 3–6]. T helper cells can be broadly subdivided into these two phenotypes based on their patterns of cytokine production, with TH1-type cells producing predominantly IFNγ and IL-2 (activators of macrophages) and TH2 type cell producing IL-4 (promotes IgE switching by B cells) and IL-5 (favours specific eosinophil differentiation, activation and survival) among other cytokines ( Fig. 1). Inappropriate TH2-type responses to allergens may account substantially for both immediate IgE-mediated responses and chronic eosinophilic allergic inflammation. The production of IgE antibody is dependent upon T lymphocyte regulation. Interaction between T and B lymphocytes and regulatory cytokines influence the initiation and maintenance of IgE responses. IL-4 has been shown in mice and subsequently in humans, to be required in all circumstances for normal IgE responses [ 7]. A balance is provided by IFNγ that antagonises the production of IgE antibody [ 8] ( Fig. 2). and TH2 cells can be distinguished by their cytokine profile and result in different immunological response. cells) inhibits certain dendritic cell functions, including their ability to provoke TH1 responses. Analysis of mRNA expression by T cells at sites of allergic inflammation has revealed elevated numbers of IL-4 and IL-5 expressing T cells [ 9–13]. Using in situ hybridization and a panel of riboprobes against cytokine mRNA it has been demonstrated that increased expression of TH2-type cytokines (IL-3, IL-4, IL-5 and GM-CSF) occurs in allergic airway disease [ 9]. Furthermore, allergen-induced late phase cutaneous responses are accompanied by recruitment of CD4+ T lymphocytes and activated eosinophils [ 10, 11] and an increase in cytokine mRNA expression for TH2-type cytokines [ 9]. Allergen-specific T cells from non-atopic individuals are characterized by a TH1-type cytokine production. The secretion of IFNγ but not IL-4 or IL-5 by these TH1-like clones has been observed [ 14]. It is tempting to speculate that treatment for allergic diseases would induce a shift from a TH2-type to a TH1-type T cell phenotype. The application of this hypothesis in specific immunotherapy (IT) has been widely investigated. However, although the clinical benefit of IT has been well established the mechanisms involved are still unclear. The general consensus seems to be the modulation of signalling pathways following engagement of immunoglobulin receptors on mast cells/basophils by incremental doses of allergen, and alterations in T-cell cytokine production from TH2 to TH1. A number of studies have indeed provided evidence for this so called ‘switch mechanism’ of IT [ 15–29]. There have been several reports about the influence of grass pollen IT on cytokine mRNA expression in the nasal mucosa and the skin [ 15–18]. The initial hypothesis was that IT might act by modulating CD4+ and/or CD8+ T lymphocyte infiltration downregulating TH2-type cytokine transcription [ 15]. In situ hybridization in inflammatory cells supported an ongoing T cell activation, with an increase in IL-2 mRNA in half of the actively treated patients and a correlation between IL-2 mRNA expression and CD25+ cell numbers. There was also increased expression of IFNγ in the actively treated subjects but no change in IL-4 or IL-5 [ 15–17]. The results support the view that IT may possibly be working through induction of T cell tolerance. The role of IL-4, IL-1, IL-2, IL-6, GM-CSF and TNFα in nasal biopsies and/or nasal secretions of patients with allergic rhinitis after treatment with corticosteroids or IT was investigated by Fireman et al. [ 19]. The improvement of symptoms induced by corticosteroid therapy was associated with decrease in IL-4 expression and in the case of IT with increased expression of IL-2 and IFNγ. Suppressed T-cell proliferative responses to mite antigen in house dust mite allergic patients after rush IT for Der p 1 were recently reported by Lack et al. [ 20]. There was a marked increase in IFNγ production, particularly by CD4+ T cells in all patients. The correlation between the increases in IFNγ production and the changes in cutaneous reactivity was highly significant. Overall, successful IT inhibited early and late phase responses and was accompanied by local decrease in eosinophils and activated T lymphocytes. Moreover, following IT and allergen challenge T cells at the target tissue and the skin revealed TH1 characteristics and no TH2 cytokine secretion pattern ( Fig. 3). . Cytokine expression on nasal and/or skin tissue of allergic patients after immunotherapy and allergen challenge. Constant increase of IFNγ and other TH1 type cytokines upon findings of nasal and/or skin tissue after immunotherapy and allergen challenge. TH2 type cytokine secretion pattern was unchanged. Many studies involving allergen stimulation of peripheral blood T cells have also been performed on patients undergoing IT ( Fig. 4). The majority of allergen specific T cell clones had a TH2-like pattern of cytokine production before IT, in agreement with the studies investigating the target organ or skin biopsies. Reduced production of IL-4 by allergen specific T cells after IT has been widely demonstrated and it was suggested that the switch from TH2 to TH1 is the underlying mechanism of successful IT. However, the findings about IFNγ and IL-2 expression are remarkably variable. The levels of IFNγ secretion after IT were found increased [ 24–26], but also reduced [ 23] or unchanged [ 21, 22, 28] compared with controls or before treatment. In the study of Van Bever et al. in this issue of Clinical and Experimental Allergy [ 28], further to unchanged levels of IFNγ a decreased secretion of IL-2 was observed in patients receiving IT for house dust mite allergy. These findings seem contradictory to the general assumption that IT results in domination of TH1 type cytokines. Trying to give an explanation for this contradiction the authors suggest that upregulation of mRNA expression of IL-2 in the target organs after IT is resulting in depletion of this cytokine in the circulation. Although this seems logical, altered IL-2 production and responsiveness of antigen stimulated peripheral T cells in the study of Hsieh et al. [ 29] conflicts with the above suggestion. seems to decrease after immunotherapy but there are variable findings about IFNγ and IL-2. It seems likely that different experimental protocols employed could be related for these opposite results. For example, when CD4+ were not separated from CD8+ cells (considering that IFNγ and IL-4 can be produced by both subpopulations [ 27]) there was an increase in IFNγ with CD8+ cell counts elevated [ 15, 26] but there was no change in IFNγ after IT when T cells had been depleted in CD8+ cells [ 21]. The interest in the switch mechanism from TH2 to TH1 was recently re-enforced by the observed increases in IL-12 mRNA cells which accompany the inhibition of allergen-induced late skin responses after 4 years of successful grass pollen IT [ 18]. IL-12 is a cytokine produced by macrophages and B cells and may be involved against viruses. It appears to be the principal cytokine responsible for proliferation of TH1-type cells in response to antigen challenge. IL-12 has also been shown to inhibit proliferation of TH2 T cells and inhibit IL-4-dependent B-cell switching from IgG to IgE production see Fig. 2. It was hypothesized that TH1 responses after IT may occur as a consequence of increased production of IL-12. The authors performed intradermal allergen challenge in patients 4 years after IT. In agreement with previous studies it was shown that prolonged IT in patients with hay fever resulted in almost complete inhibition of allergen-induced late cutaneous responses. In situ hybridization demonstrated highly significant increases in IL-12 mRNA+ cells at allergen-challenged sites, which correlated with the number of IFNγ cells. An inverse correlation was noted between IL-12+ cells and IL-4+cells. Taken together these observations suggest that successful grass pollen IT may modify the T-lymphocyte response to subsequent allergen exposure with an additional TH1 response (with local increases in IFNγ), which may be driven by IL-12. The same cytokine was investigated in PBMCs in the study of Van Bever et al. but there was no difference in IL-12 secretion between subjects receiving IT and subjects who never received IT [ 28]. The authors speculated that this was due to the applied technique, being in vitro secretion of IL-12 by PBMC, while it has been assumed that resident macrophages are responsible for the main secretion of IL-12. One can reasonably wonder how any immunological changes relate to the clinical response and how does IT fit into the delicate balance between tolerance and TH1 or TH2 response. Three possible mechanisms account for loss of allergen-specific T-cell responsiveness after IT and response of the remaining T cells towards TH1-type cytokines: activation-induced programmed cell death (apoptosis), induction of specific suppressor cells and induced antigen-specific unresponsiveness (anergy). The above theories about T-cell responsiveness and other mechanisms like the increase of specific IgG4 antibodies (independent of induction of T-cell anergy or immune deviation) have been suggested to explain the hypothesis of ‘TH2 to TH1 switch’ [ 30]. The results of the studies mentioned seem to agree only partially with the ‘TH1-TH2 switch’ hypothesis. Is it the different experimental protocols employed responsible for the conflicting results or is the demonstration of TH1 responses an in vitro artefact of the cloning process? In an editorial with the subversive title ‘TH1/TH2 lymphocytes: doubt some more’ [ 31], the significance of the TH1 responses demonstrated in healthy subjects and after IT is questioned. It is suggested that the true nature of cytokine responses to antigens must be more complex than can be explained by the TH1/TH2 dichotomy. No firm differentiation markers exist that distinguish a TH1 from a TH2 cell, and these subgroups may not represent distinct subpopulations. Although the role of TH2-like cytokines in the pathophysiology of allergic disorders is convincingly demonstrated, the pattern of cytokine response to allergens in non-allergic individuals or after IT is more complex and doubtful. The belief that IL-12 and IFNγ might be the basis for the beneficial effects of IT is challenged based on three observations: the asthmatic lung and allergic nose already contain IFNγ, therapeutic trials with IFNγ did not show clinical benefit and IL-12 and IFNγ do not behave like ‘magic bullets’ which manage to inhibit IL-4 synthesis, IgE production, and TH2 activity without simultaneously activating accessory cell function, inducing adhesion molecule expression, and stimulating neutrophils and eosinophils. Taking this a little further, it is hypothesized that excessive IL-12 production would be expected to induce cellular inflammation, an undesirable effect. IT may promote immune tolerance by subtly regulating the cytokine milieu, rather than by redirecting inflammation from a TH2 to a TH1 pattern of response. Attempts to reduce each cytokine to a limited function do not reflect the pleitropic nature of these proteins and although appealing in their simplicity, ignore the true complexities of immune regulation. Furthermore, it is entirely possible that cells other than T cells may be affected by or may contribute to this change in cytokine gene expression initiated by IT. The detailed mechanisms of IT are indeed difficult to reveal considering that IT was first practised almost a century ago and although the clinical effectiveness is established, the respond of the immune system is still unclear. The shift from TH2 to TH1-type cytokine expression after IT seems to be a useful model but different arms of the immune system may be involved as well. Respond to allergens by an exclusive single T-cell subset does not seem likely. Any possible mechanism relative to IT must be well defined considering the need and extend of this type of treatment. The use of IT in asthma and in paediatric allergic disease, new forms of IT with different allergen forms and by different route are issues that need further research and precise immunological mechanisms must be revealed so the candidates will be the appropriate ones and the success of treatment will be warranted.