Adenosine Provocation: A New Test for Allergic Type Airway Inflammation

Stephen T. Holgate, M.D.

School of Medicine, Southampton General Hospital, Southampton, United Kingdom

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The observation that the airways contract too much and too easily when stimulatedbronchial hyperresponsiveness (BHR) is fundamental to our understanding of asthma pathophysiology, and yet the underlying mechanism(s) of BHR remain elusive. Although methacholine and histamine have become gold standards as bronchial provocants to quantify BHR, a further level of complexity has been revealed by using a wide range of different "indirect" stimuli, including adenosine 5'-monophosphate (AMP), that cause bronchoconstriction through the secondary release of mediators. The findings by de Meer and colleagues (1) in this issue of the Journal (pp. 327-331) that the airways' response to inhaled AMP more closely reflects allergic inflammatory processes in the lower airways when compared with methacholine, adds to previous work that compared these forms of challenge and reported that AMP response is better at predicting sputum eosinophilia (2) or corticosteroid sensitivity (3) in chronic airways disease. Although a retrospective study by Fowler and colleagues (4) came to a rather different conclusion, namely that methacholine was a more sensitive index of airway responsiveness, this was based on a different spectrum of subjects who were selected from an asthmatic population with more severe disease, and in whom airway wall remodelling in addition to airway inflammation would be playing an important role. As the authors of this study point out (4), the concurrent use of inhaled steroids by a significant proportion of the subjects may have had a major effect in reducing airway responses to AMP (3).

Overall, the Groningen studies add to the view that this stimulus operates via adenosine A_2B receptor to activate mast cells in inflamed airways (5). Accumulated evidence for this mechanism is now considerable, and includes the inhibitory effects of H₁-histamine, cysteinyl LT₁ antagonists and mast cell-stabilizing drugs such as cromolyn sodium, the detection of mast cell mediators following local upper and lower airway challenge with AMP in rhinitic and asthmatic subjects, and the clear enhancement of IgE-dependent mast cell mediator release that has been demonstrated in vitro (reviewed in 6). A unique feature of de Meer's study is the observation that the dose-response slopes for AMP, when compared to that for methacholine, increased to a greater extent along the asymptomatic to symptomatic rhinoconjunctivitis-asthma axis. The data presented in the paper to support this conclusion indicate that the majority of this differential for the two forms of bronchial provocation lies in the markedly reduced AMP responsiveness of the normal controls and an indication of a threshold effect, whereas for methacholine there appeared to be a smoother transition between the values of the dose-response slopes. In this regard it would have been informative to know if atopy per se, i.e., without any symptoms, is sufficient to create a differential between the airways response to AMP and methacholine.

Thus, although it seems clear that responsiveness of the airways to AMP in asthma and allied disorders more closely reflects Th-2 mediated inflammation than does responsiveness to methacholine, what now needs to be established is how adenosine A_2B receptor function becomes upregulated in allergic airway disease. Human mast cells express both A_2A and A_2B receptors, both of which are linked to the GS-activation of adenylate cyclase. However, only the latter appears to activate Ca⁺⁺-dependent signalling, possibly via Gq-activation of the phosphatidylinositol pathway (5, 7). In vitro studies on enzymically dispersed human lung mast cells and blood basophils show that adenosine is able to selectively enhance IgE-dependent activation for mediator release (8), whereas for mast cells obtained by bronchoalveolar lavage, adenosine alone may serve as a secretogogue (6), as well as to enhance the response to other secretogogues (9). This might suggest that airway mast cells in an inflammatory microenvironment are "primed" for mediator secretion that can be revealed by adenosine exposure. In contrast, skin mast cells even in atopic subjects are nonresponsive to adenosine (10). Taken together, these findings suggest that adenosine responsiveness differs between mast cell subpopulations and that the microenvironment in which the mast cell is situated is a key component for conferring a response to this stimulus. This view is further strengthened by finding a greater response to AMP in atopic subjects who smoke (1). From a mechanistic standpoint, it would be of interest to determine whether specific Th-2 cytokines, e.g., interleukin (IL)-4, IL-9, and IL-13, which are known to affect mast cells in other ways, are responsible for regulating A_2B receptor function.

The clear relationship found between methacholine responsiveness and baseline airway calibre supports a causal relationship based on geometric considerations and explains in part why, in Fowler's study (4), methacholine challenge had greater sensitivity for detecting severe asthmatic patients with low baseline FEV₁ values. Although not revealed in de Meer's study, others have shown a correlation between indices of methacholine and AMP responsiveness, but the relationship is weak, further supporting the idea that adenosine causes bronchoconstriction by indirect mechanisms.

The recent discovery that adenosine deaminase-deficient mice exhibit a lung phenotype with features of asthma, including BHR, enhanced mucus secretion, airway eosinophilia, increased IgE synthesis, and elevated bronchoalveolar levels of IL-5 that could be reversed by exogenous administration of adenosine deaminase (11), raises the interesting possibility that adenosine generated in asthmatic airways could itself contribute causally to asthma pathogenesis. Transcript array technology has been used to examine which genes in the lung become activated by adenosine that accumulates in adenosine deaminase-deficient mice. This has provided additional insights into mechanisms linking adenosine to an asthma phenotype by revealing marked overexpression of the monocyte chemotactic protein-3 gene in the airways paralleled by enhanced protein secretion (12). Monocyte chemotactic protein-3 is a chemokine with powerful eosinophil chemotactic properties. In the same study, the presence of greatly enhanced airway expression of molecules involved in tissue remodelling, including vascular endothelial growth factor, osteopontin, and fibronectin, as well as the cathepsin family of neutral proteases, provides a further important link between adenosine and asthma. The presence of elevated levels of adenosine in asthmatic airways, together with its proinflammatory effects in human and experimental models of asthma, might have therapeutic implications. The benefit observed in asthma with relatively weak but selective A_2B antagonist, enprofylline, gives reason for some optimism (5).

As intimated by the findings of the Groningen group, airway provocation with AMP may provide a discriminatory test for asthma over and above the use of more direct stimuli such as methacholine and histamine (13). de Meer and colleagues point out that AMP responsiveness is enhanced in cigarette smokers and in some patients with COPD. Patients with late onset nonallergic (intrinsic) asthma also respond positively to AMP challenge. Under all of these conditions, AMP-induced bronchoconstriction reflects increased inflammation of a specific type, dominated by activated mast cells and eosinophils (i.e., Th-2 like). After detailed appraisal of a range of indirect forms of bronchial challenge, a European Respiratory Society Workshop Report (14) has included AMP challenge as being one worthy of serious further consideration as a more discriminant test for specific forms of airway inflammation linked to the asthmatic phenotype. However, only with its wider use in a range of airway disorders, both in cross sectional and longitudinal studies, will it be possible to determine whether these experimental findings can be usefully applied in the clinic.

	References

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