TNF-α (tumour necrosis factor-α) is known to play a critical role in the pathogenic mechanisms of a number of chronic inflammatory diseases, including RA (rheumatoid arthritis), Crohn's disease and psoriasis. The notion that TNF-α is released in allergic responses from both mast cells and macrophages via IgE-dependent mechanisms, the demonstration that elevated levels of TNF-α are frequently observed in bronchoalveolar fluid of asthmatic subjects undergoing allergen challenge and the results from exposure studies of TNF-α in vivo showing increases in airway responsiveness in both normal and asthmatic subjects emphasize the importance of TNF-α in the initiation of allergic asthmatic airway inflammation and the generation of airway hyper-responsiveness. Drugs targeting TNF-α have been developed to neutralize the deleterious effects of this inflammatory cytokine and have proved to be safe and effective in the treatment of patients with RA, Crohn's disease and psoriasis refractory to conventional treatments. Biological therapies blocking TNF-α are likely to constitute a considerable advance in the management of those difficult cases of asthma that are particularly resistant to typical treatment modalities. In this review article, we intend to address the potential role of TNF-α in asthma and to put forward the idea that drugs that have been developed to neutralize the deleterious effects of TNF-α may also be useful in the management of chronic severe asthma.
- biological therapy
- rheumatoid arthritis
- TNF-α (tumour necrosis factor-α)
Asthma is a complex syndrome with many clinical phenotypes in both adults and children. Its major characteristics include a variable degree of airflow obstruction, BHR (bronchial hyper-responsiveness) and chronic airway inflammation . The current management of asthma focuses on the optimal control of symptoms and the reduction of airway inflammation as a central component of asthma control .
Regrettably, it is estimated that approx. 5–10% of patients with asthma have severe disease that is particularly resistant to typical treatment modalities, including administration of systemic corticosteroids and high-dose bronchodilators [3,4]. This simply reflects the fact that asthma is a heterogeneous disease with heterogeneity in the treatment response. Although this group of patients represents an important subset, our understanding of the pathophysiology of this specific aspect of asthma is deficient. In spite of this lack of information, it is evident that patients with chronic severe asthma suffer from the greatest impairment to their lifestyles and their disease has a substantial burden on the health care system, as their treatment uses a disproportionate amount of health care resources [5–7]. The high direct cost is explained by the treatments themselves and the need for hospitalization, whereas the indirect cost is related to disability induced by the disease, responsible for a loss of work and productivity. Costs could be significantly reduced if disease control was improved. Unfortunately, the drugs used for treating chronic severe asthma appear to have limited effects on disease control and progression. Thus there is a pressing need for the development of new treatment strategies for patients at the more severe end of the disease spectrum who do not respond well to current therapy.
Drugs targeting TNF-α (tumour necrosis factor-α) have gained considerable importance in the therapy of difficult cases of severe inflammatory progression in RA (rheumatoid arthritis), Crohn's disease and psoriasis. Thus it is likely that therapies blocking TNF-α may also represent a considerable advance in the management of those asthma cases who are particularly resistant to typical treatment modalities.
In this review, we intend to address the potential role of drugs in asthma that have been developed to neutralize the deleterious effects of TNF-α.
TNF-α: BIOLOGY, STRUCTURE AND FUNCTION
TNF-α is a non-glycosylated protein of 17 kDa with 157 amino acids and belongs to a family of peptide ligands that activate a corresponding set of structurally related receptors [8,9]. The soluble 17 kDa form of TNF-α is generated by cleaving the 26 kDa trans-membrane precursor by TACE (TNF-α-converting enzyme; also known as ADAM 17). Both soluble and membrane-bound forms of TNF-α are biologically active, although they have different affinities for the two receptors. After separating from the cell membrane, soluble TNF-α aggregates into trimolecular complexes (51 kDa homotrimers) that subsequently bind to the receptors. TACE also cleaves the extracellular domain of its complementary receptor forming sTNFRs (soluble TNF-α receptors) that are free to bind to trimolecular TNF-α rendering it biologically inactive, resulting in diminished cellular signalling of TNF and thus acting as a soluble natural inhibitor of TNF-α bioactivity in vivo.
Biological responses of TNF-α are mediated by specific binding either via a Type I (TNFR1; p55 or CD120a) or a Type II (TNFR2; p75 or CD120b) receptor . These two receptors are expressed on the surface of many cell types, and a recent model of receptor-mediated signalling proposes that TNFR1 is expressed on cells susceptible to the cytotoxic action of TNF-α, whereas TNFR2 is expressed strongly on stimulated B- and T-cells . Binding of TNF-α to its receptors results in activation of intracellular signalling processes that lead to a remarkably diverse set of cellular responses, including differentiation, activation, release of pro-inflammatory mediators and apoptosis, through the recruitment and activation of adaptor proteins (Figure 1) . It is clear that the ratio of TNFR1/TNFR2 dictates the final outcome of the cellular response upon TNF-α stimulation.
TNF-α, a cytokine that plays a role in many inflammatory diseases, is produced by several pro-inflammatory cells (mainly macrophages, but also monocytes, dendritic cells, B-cells, CD4+ cells, neutrophils, mast cells and eosinophils) and structural cells (fibroblasts, epithelial cells and smooth muscle cells) known to be crucial in the pathogenesis of asthma. Large amounts of TNF-α are generated in response to bacteria or parasitic proteins, but all potentially noxious stimuli ranging from physical, chemical to immunological can rapidly induce production and release of TNF-α. Moreover, TNF-α can also be generated as a consequence of stimulation of a wide range of pro-inflammatory cytokines including TNF-α itself. For example, mast cells are known to release and respond to TNF-α, indicating a positive autocrine loop leading to augmentation of mast cell activation .
The biological function of TNF-α includes the modulation of growth differentiation and proliferation of a variety of cell types, but it is also important in the causation of apoptosis . Besides these effects, TNF-α is also a well-known inducer of the inflammatory response and a regulator of immunity. Its inflammatory properties are classically mediated by means of a wide variety of pro-inflammatory cytokines, including IL (interleukin)-1, IL-2, IL-4, IL-6, IL-10, IL-12, IFN-γ (interferon-γ) and TGF-β (transforming growth factor-β), generated mainly through NF-κB (nuclear factor κB) activation .
THE ROLE AND IMPORTANCE OF TNF-α IN ASTHMA
As for the pathogenic mechanisms of RA, there is strong argument for TNF-α playing a critical role in the pathogenesis of chronic inflammatory disorders of the airways such as asthma and COPD (chronic obstructive pulmonary disease). TNF-α is stored in granules and is known to be released during allergic responses from both mast cells and macrophages via IgE-dependent mechanisms . Besides mast cells and macrophages, many other cell types that appear to play a contributory role in the pathogenesis of asthma are also a significant source of TNF-α, including eosinophils , epithelial cells  and neutrophils . In addition, T-cells from asthmatic airways constitutively produce large amounts of TNF-α both at the protein and mRNA levels .
TNF-α mRNA is more frequently expressed in the airways of asthmatic subjects than normal subjects and increased release of this cytokine has been shown from BAL (bronchoalveolar lavage) cells of asthmatic subjects . Also, LPS (lipopolysaccharide) inhalation by mild asthmatic subjects induces TNF-α secretion into BAL fluid and is associated with increased airway reactivity [21,22]. In addition, after allergen challenge, TNF-α is found to be increased in BAL fluid of asthmatic subjects, and their peripheral blood monocytes generate more of this cytokine [23,24].
Once released in the airways, TNF-α acts by inducing a general inflammatory response mainly through the enhanced release of pro-inflammatory/chemotactic mediators and up-regulation of adhesion molecules, such as E-selectin, VCAM-1 (vascular cell adhesion molecule-1) and ICAM-1 (intercellular cell-adhesion molecule-1), thus facilitating the migration of eosinophils and neutrophils [22,25,26]. During this process, they also become primed for mediator secretion. Ultimately, this will lead to chronic inflammation and irreversible airway remodelling, a key feature in bronchial asthma (Figure 2).
Interstitial matrix turnover and tissue remodelling are known to be modulated by the fine interplay between the activity of proteolytic enzymes that degrade the extracellular matrix and the production of extracellular matrix glycoproteins . The proteolytic enzyme MMP-9 (matrix metalloproteinase-9) and the matrix glycoprotein tenascin are abundant in thickened asthmatic subepithelial basement membrane. Their level of expression reflects disease activity in asthma and airway remodelling and it has been demonstrated to be greatly increased in the presence of TNF-α in both airway eosinophils and bronchial fibroblasts via activation of the transcription factor Ets-1 [28,29]. The production of extracellular matrix glycoproteins is mainly derived by the activation of myofibroblasts and fibroblasts in the proximity of the subepithelial basement membrane . TNF-α is known to be implicated in the proliferation and activation of subepithelial myofibroblasts/fibroblasts thus contributing to development of fibrosis below the bronchial basement membrane of the epithelial layer and to tissue remodelling in general [30,31]. Moreover, airway epithelial cells also secrete mucus when stimulated with TNF-α . TNF-α is also likely to have a more integrated role in airway remodelling, since it appears to modulate the EGFR (epidermal growth factor receptor)-dependent stress and repair response that occurs as a result of the inflammatory response that is associated with airway epithelial injury .
BHR AND TNF-α
The role of TNF-α in the pathogenesis of asthma is not just limited to its biological effects in airway inflammation and remodelling; perhaps its most prominent effect is that of induction/potentiation of BHR (Figure 2). BHR, best defined as the abnormal increase in airflow limitation in response to a provoking stimulus, is a major feature of asthma. Although inflammatory mediators and cell infiltration of the airways appear to be implicated in the aetiology of BHR, recent studies have indicated that the ASM (airway smooth muscle) itself has the capacity to modify its contractile susceptibility in the presence of specific pro-inflammatory cytokines [34,35]. This dysfunction in ASM contractility may result either from up-regulation of receptors expressed on the cell surface or from the increased level of receptor–ligand affinity. There are a number of studies indicating that exposure to cytokines can itself induce BHR, and more than one inflammatory mediator has been implicated in this response.
In relation to this, investigators have recently demonstrated that TNF-α regulates the function of ASM by modulating the secretion of other cytokines/chemokines and the level of expression of adhesion molecules . Moreover, TNF-α is also capable of inducing a hypercontractile phenotype by enhancing calcium signalling through different pathways. Considering the nature of these effects, it has been proposed that TNF-α may be implicated in the BHR associated with asthma.
Animal, as well as human, studies appear to indicate that TNF-α has a major role in the pathogenesis of BHR. Earlier work by Kips et al.  has shown that exposure to TNF-α causes BHR and airway inflammation in sensitized rats. In this model, exposure to LPS induced elevated TNF-α concentrations in BAL fluid in association with BHR and pretreatment with anti-TNF-α antibodies before LPS exposure significantly diminished the increase in BHR. Likewise, in tracheal preparations of Dunkin–Hartley guinea-pigs, rhTNF-α (recombinant human TNF-α) increased maximal isotonic contraction to methacholine, which was completely inhibited by co-incubation in the organ bath with a dimeric recombinant TNFR p80 construct . Using a TNFR fusion protein, Renzetti et al.  were the first to provide evidence for the involvement of TNF-α in allergen-induced BHR in sensitized guinea pigs. They also showed that the TNFR fusion protein is as effective as dexamethasone in preventing allergen-induced responses, suggesting that a therapy directed against TNF-α may be useful in the management of asthma. Another argument for the importance of TNF-α in the development of airway inflammation and BHR is that derived from studies in the transgenic mouse model. In TNF-α-deficient mice sensitized to TDI (toluene diisocyanate), non-specific BHR to a methacholine challenge was completely inhibited in association with a marked reduction in airway inflammation . In healthy human volunteers, exogenous TNF-α administered as an aerosol elicits increased airway reactivity in association with a neutrophil influx in BAL fluid . In a follow-up double-blind placebo-controlled randomized crossover study of ten subjects with mild asthma treated with nebulized rhTNF-α, Thomas and Heywood  were able to confirm their earlier observations in healthy volunteers.
TNF PROMOTER POLYMORPHISMS IN ASTHMA
The notion that TNF-α is released in allergic responses via IgE-dependent mechanisms, the demonstration that elevated levels of TNF-α are frequently observed in BAL fluid of asthmatic subjects undergoing allergen challenge and the results from exposure studies of TNF-α showing increases in BHR in both normal and asthmatic subjects suggest that functional variants of the TNF gene could be important in the disease. However, studies on the relevance of TNF promoter polymorphisms in asthma have produced mixed results. Whereas some studies have found evidence for an association between −308A and asthma with the A allele being more frequent in asthma cases [43,44], other investigations failed to find any association [45,46].
Interestingly, a study considering the extensive linkage disequilibrium present on chromosome 6 has found that extended haplotypes account for the association of TNF SNPs (single nucleotide polymorphisms) with asthma . The extended haplotype LTαNco*1/TNF–308*2/HLA-DRB1*02 was observed to have a remarkable association with asthma [OR (odds ratio) 6.68] and this resulted in an even stronger association with BHR (OR 21.9) in a study of over 1000 patients. Another study also found that BHR was associated with −308A, but again through an extended haplotype . As for the genetics of TNF in RA, further studies are needed to demonstrate that TNF promoter polymorphisms in asthma are functional and directly contribute to the pathogenesis of the disease.
TNF-α BLOCKING AGENTS: A PROMISING THERAPEUTIC OPTION IN ASTHMA?
Effective management of the inflamed airways in asthma is based on careful monitoring of the disease and correct use of inhaled corticosteroids . However, when cytokine profiles were investigated in BAL fluid and in the bronchial biopsies of subjects with asthma and nasal polyposis treated by inhaled corticosteroids, it was found that all cytokines under investigation were greatly reduced with the exception of TNF-α [19,50,51]. Thus failure of inhaled corticosteroids to reduce TNF-α to a significant level in asthmatic airways may explain to a certain extent why these anti-inflammatory drugs appear to have limited effects in the more severe forms of asthma.
Considering the critical role of TNF-α in the pathogenesis of asthma and the need for alternative treatments for those asthmatic patients with severe disease who are particularly resistant to conventional therapy, molecules targeted at blocking the effects of TNF-α are likely to constitute a considerable advance in the management of these difficult patients. The currently commercially available TNF-α blockers [infliximab (a chimaeric mouse/human monoclonal anti-TNF-α antibody), etanercept (a soluble fusion protein combining two p75 TNFRs with an Fc fragment of human IgG1), and adalimumab (a fully human monoclonal anti-TNF-α antibody)] (Figure 3) have proved to be remarkably effective and safe in well-conducted clinical trials of patients with RA refractory to conventional therapy . The newer TNF-α targeting immunobiologicals that are being developed are a PEG [poly(ethylene glycol)]-bound p55 TNFR (PEG–TNFR1), PEGylated TNF-α antibody fragments (CDP-870) and TACE inhibitors .
To date there is very limited information on the use of TNF-α blocking agents in asthma. In a retrospective analysis of RA patients receiving infliximab (Remicade), Khalil-Saadeh et al.  were the first to report a significant improvement in FEV1 (forced expiratory volume in 1 s) and exercise tolerance in three patients who were also diagnosed with asthma/COPD. All three patients showed an important clinical improvement whilst on infliximab and discontinued use of their maintenance inhaled corticosteroids. The notion that inhibition of TNF-α may be of prime importance in the control of airway inflammation with improved asthma outcomes was emphasized by subsequent observations by other researchers who added etanercept (Enbrel) in the management plan of ten patients maximally treated for chronic severe asthma . In this small open trial, 25 mg of etanercept administered subcutaneously twice a week for a period of 12 weeks clearly improved lung function and had an important impact on the overall asthma symptoms scores, with all but one patient voluntarily withdrawing completely from the use of their regular bronchodilators by the end of the study. Of particular note is the remarkable 20-fold attenuation in AHR (airway hyper-responsiveness) that occurred after treatment with etanercept. Although this was an open-labelled study, it is difficult to envisage here that a placebo effect could account for such a large change in AHR and it is most unlikely due to the clear improvement in baseline lung function. Taken together, these findings support the notion that TNF-α blocking agents may prove useful in the treatment of chronic severe asthma. In particular, the consideration that no side effects were noted in these studies with asthmatic patients and that none of the patients studied experienced acute attack while on infliximab or etanercept render the use of TNF-α blocking drugs particularly attractive. In relation to the safety of TNF-α blocking agents in asthma, it may be argued that these studies are preliminary and undersized. Although the safety data on TNF-α blocking agents has been mostly gathered from clinical trials of RA patients who failed to respond to conventional therapy, these drugs have proven to be safe and well-tolerated in their respective clinical development programmes .
However, some adverse events have been reported and the long-term safety of all these molecules is not thoroughly recognized [56,57]. Although there is no evidence to suggest a higher infection frequency in patients treated with TNF-α blocking agents , with the increasing usage of these agents the possibility of TB (tuberculosis) reactivation must be kept in mind [57–59]. Therefore a careful screening for latent TB should be made before prescribing TNF-α blocking agents. Recently, demyelinating events were reported in patients treated with a TNF-α antagonist, mainly with etanercept . Therefore it is currently strongly recommended to stop anti-TNF-α treatment when neurological events occur, and these treatments should be strictly avoided in patients with pre-existing demyelinating disease.
Given that TNF-α plays a role in immune surveillance against tumour cells, it is possible that anti-TNF-α agents may be associated with an increased risk of malignancy. However, there is no increase in the rate of malignancies using TNF-α blocking agents compared with that expected in the general population [61,62].
Repeated administration of TNF-α antagonists is required for controlling disease activity. The occurrence of neutralizing antibodies, which may compromise drug efficacy, is a common event particularly with infliximab [56,57]. This may challenge long-term treatment with this agent and validates the need for developing new TNF-α antagonists with lower immunogenicity.
Although chronic severe asthma is a condition characterized by remarkably high direct and indirect costs, the economic cost of TNF-α blocking agents remains a relevant issue [63,64]. Anti-TNF-α treatments are expensive and a comprehensive economic evaluation of the ratio between the total cost and the economic advantages obtained by such treatments will be required.
Emerging evidence from experimental and clinical studies suggests that TNF-α plays a major role in the pathogenesis of human asthma, thus emphasizing the significance of TNF-α as an important therapeutic target in this common medical condition.
A number of drugs targeting and neutralizing the deleterious effects of this cytokine have been developed. Infliximab, etanercept and adalimumab have proved to be effective and safe in trials of patients with RA and it is likely that their safety profile could be mirrored in future studies with asthmatic patients. Moreover, blockade of TNF-α using etanercept has revealed efficacy in a small open study of patients with chronic asthma.
These promising treatment options may constitute considerable progress in the management of difficult cases of asthma, but well-conducted double-blind placebo-controlled studies are needed to establish their therapeutic efficacy in chronic severe asthma and COPD.
The authors' work was supported by an educational grant from the University of Catania.
Abbreviations: AHR, airway hyper-responsiveness; ASM, airway smooth muscle; BAL, bronchoalveolar lavage; BHR, bronchial hyper-responsiveness; COPD, chronic obstructive pulmonary disease; IL, interleukin; LPS, lipopolysaccharide; NF-κB, nuclear factor κB; OR, odds ratio; RA, rheumatoid arthritis; TNF-α, tumour necrosis factor-α; rhTNF-α, recombinant human TNF-α; TACE, TNF-α-converting enzyme; TB, tuberculosis; TNFR, TNF-α receptor; TNFR1 etc., Type I TNFR etc
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