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Eosinophils
Increased numbers of eosinophils in asthmatic airways is an almost constant finding. The eosinophils are also activated and their state of activation and numbers correlate with airwayshyperresponsiveness. Eosinophil-derived proteins such as major basic protein, in the sputum correlate with disease activity and the levels decrease after appropriate treatment. Eosinophil cationic pro-tein (ECP), which is located in the eosinophil granule matrix, is also raised in serum after allergen-provoked asthma and during the pollen season in a topic individuals. Eosinophil precursors increase during the late asthmatic reaction and their numbers fluctuate in relation to season exposure in atopic subjects. This suggests that exposure to allergen stimulates eosinophil production by the bone marrow. Macrophages and monocytes Immuno histochemistry of bronchial biopsy specimens shows that the submucosa had a significantly increased macrophage population in asthmatic patients. The population had phenotypic characteristics of peripheral blood monocytes, suggesting that they had migrated recently into the lung. HLA Class II antigen was expressed on the infiltrating cells of the airway mucosa to a greater extent in asthmatic subjects than in normal individuals. The major activity secreted by peripheral blood monocytes and alveolar macrophages which augment eosinophil survival and increase their capacity to produce pro inflammatory mediators is granulo cyte-macrophage colony stimulating factor (GM-CSF).
Lymphocytes
There is a tendency for increased numbers of T cells in asthmatic airway biopsies, with an increase in the numbers of cells expressing receptors for inter-leukin-2 receptor (CD25), reflecting lymphocyte activation.The numbers of CD25+cells correlated with airways hyper responsiveness. Hamid et al. using the technique of in situ hybridization to examine expression of interleukin (IL)-5 in bronchial biopsies, demonstrated increased IL-5 mRNA in the bronchial mucosa of six out of 10 asthmatic subjects and in none of the nine controls. Although the number of patients studied was small, there was a trend for the IL-5+ asthmatic subjects to have more severe asthma than those in whom no significant IL-5 was observed. Biopsies positive for IL-5 mRNA also had a greater number of CD25+ cells, a greater number of eosinophils and a greater number of EG2+ eosinophils (which stains for the secreted form of an eosinophil granule protein). These data are consistent with the suggestion that IL-5, secreted by activated T lymphocytes, contributes to the recruitment and activation of eosinophils in the bronchial mucosa in asthma. The murine equivalent of TH2-type cells which produce IL-4 and IL-5 (as compared to THl-type cells which generate IL-2 and gamma-interferon) have also been observed in the bronchial mucosa and bronchoalveolar lavage fluid from mild atopic asthmatics. By immuno magnetic enrichment, the IL-4+and IL-5+ cells were entirely in the CD2+ population. The numbers of activated T cells and their products correlate with the severity of disease. In chronic to severe cortico steroid-dependent asthma, activated T cells appear in peripheral blood and their numbers decrease after steroid treatment. In contrast, there is a group of steroid-resistant individuals who have chronically act-ivated CD4+/CD25+ cells which proliferate in vitro, even in the presence of dexamethasone. A recent placebo-controlled, double-blind trial of cyclospor in Ainchronic steroid-dependent asthma showed efficacy with a substantial increase in lung-function. Taken together, these studies suggest that T cell mediated responses, independent of IgE production, are important regulatory pathways of the pathogenesis of airways inflammation.
Cytokines
Now that asthma is seen as a chronic inflammatory disease understanding the role of cytokines in orchestrating the chronic eosinophilic inflam-mation is an important research goal. It is now clear from a number of different studies that asthma, even in its mildest form, is characterized by local infiltration of inflammatory cells in which mononuclear cells and eosinophils are particularly prominent. The mechanism for the selective eosinophilic accumulation is unknown. Although mast cell mediators may contribute to eosinophil recruitment, there is increasing evidence that cytokines may play an important role in the recruitment and activation of these effector cells. For example, IL-3, is a mast cell growth factor and stimulates proliferation of eosinophils from bone marrow stem cells. IL-3, IL-5 and GM-CSF promote maturation and differentiation of eosinophils. They also prolong eosinophil survival in culture and, presumably, persistence of eosinophils from tissues. This may be achieved by inhibition of programmed cell death (apoptosis) in eosino-phils. T cell-derived lymphokines, IL-4 and gamma-interferon are involved in the regulation of IgE production. The T lymphocyte is not the only cell which produces cytokines. There is now increasing data to indicate the eosinophils, endothelial cells, fibroblasts' and epithelialcells alpha' can also secrete cytokines. Of particular note is the finding that eosinophils themselves can express genes for cytokines and release active molecules, such as IL-1, GM-CSF, transforming growth factor alpha(TGFM)andTGFPI3
Adhesion molecules
Adhesion molecules on inflammatory cells and endothelial cells play a critical role ininflammatory cell recruitment. Thus cytokines may lead to selective eosinophil accumulation via enhanced recruitment through the induction of selected adhesion molecules on endothelial cells. IL-5 selectively enhances the in vitro adhesion of eosinophils to endothelium. Of the three major cytokine-induced endothelial adherence molecules, intercellular adhesion molecule-I (ICAM-1), endo-thelium-leukocyte adhesion molecule-I (ELAM-1) and vascular cell adhesion molecule-I (VCAM-1) that have been characterized and identified which may be important in leukocyte adhesion, VCAM-l appears to have selectivity for the eosinophil. Specific anti-VCAM-l antibody-inhibited eosinophil, but not neutrophil adherence. A counter ligand for VCAM-1 is VLA-4, one of the beta-1 integrin adhesion molecules also referred to as CD49/CD29. Neutrophils do not show evidence of VLA-4, whereas eosinophils and basophils both expressed VLA-4. IL-4 selectively induces VCAM-1 expression and VLA-4 has been shown to increase adhesiveness in endothelial cells for eosinophils and basophils, but not neutrophils. The VLA-4 molecule can also function as an alternative receptor for fibronectin and eosino-phils also express VLA-6 which binds laminin. Thus, the ability of eosinophils to remain localized within the extra vascular inflammatory site may be increased in tissues expressing higher levels of laminin and/or fibronectin. The role of adhesion molecules in asthma was strongly supported by evidence from Wegner and colleagues who identified the relationship be-tween eosinophilic infiltration and airways respon-siveness in the primate model of asthma. The administration of anti-ICAM-1 monoclonal anti-body in vivo reduced eosinophil infiltration in airways hyper responsiveness following antigen inhalation challenge. The type of response in the airways was dependent upon the model employed. When a subset of intrinsically hyper eosinophilic animals were given a single inhaled allergen challenge, there was both an immediate and a delayed asthmatic response. The latter was associated with a neutrophil influx into the airways and an associated airways hyper responsiveness which was inhibited by anti-ELAM-l but not anti-ICAM-l.
The beta agonist debate
Inhaled beta2-adrenoceptor agonists are byfar the most effective bronchodilators currently available and their use has increased dramatically in recent years throughout the world.
Mechanism of action
The molecular mechanism of their bronchodilator action has recently been elucidated; although several effects of beta-agonists have been described, the most important mechanism of action involves activation of a large conductance calcium-activated potassium channel (known as the maxi-K channel), which is blocked by the scorpion venom toxin charybdotoxin. Charybdotoxin blocks the bronchodilator action of beta-agonists in both animal and human airways, and there is recent evidence that the beta2-receptor in airway smooth muscle cells is directly coupled via a stimulatory G-protein (Gs) to the maxi-K channel, without the involvement of the second messenger cyclic AMP.
There has been considerable debate about the non-broncho-dilator/anti-inflammatory effects of beta-agonists. Beta-Agonists are effective in inhibiting micro vascular leakage and plasma exudation in the airways, and inhibit mast cell mediator release. In this respect they could be considered as anti-inflammatory agents, but they do not seem to have effects on the chronic inflammatory process and therefore do not have steroid-like actions. This is shown most directly by the lack of effect of treatment with a regular inhaled beta-agonist on the inflammatory cell profile in bronchial biopsies taken from asthmatic patients, whereas inhaled steroids are very effective in suppressing the inflammatory response. Similarly the new long-acting inhaled beta-agonist salmeterol has no effect on the profile or activation of inflammatory cells in biopsies of asthmatic patients. Clinical evidence that this is so is provided by a study which compared regular inhaled beta-agonist with regular inhaled steroids over a 2 year period in over 100 newly diagnosed patients. There was a marked difference between the two groups with more frequent symptoms and use of rescue broncho-dilators, greater diurnal variability and greater airway responsiveness in the group treated with beta-agonists alone.
Questions about safety Although beta-agonists are highly effective as bronchodilators, questions have recently been raised about their long term safety in asthma when used on a regular basis. Epidemiological studies in New Zealand suggested that there may be a link between the use of a particular beta-agonist fenoterol and the risk of asthma death. The original study was criticized because of the matching of controls but a further study which took this into account still showed an association. An epidemiological study in the Province of Saskatchewan in Canada examined the link between death and near death from asthma and the use of anti-asthma medication obtained from computerized records over a 7 year period. There was a significant association between asthma death and near death and the regular use of inhaled beta-agonists, with a very steep dose-response relationship. The risk associated with fenoterol was significantly greater than the risk attached to salbutamol, but fenoterol is approximately twice the strength of salbutamol compared puff for puff. Correction for the bronchodilator dose give inhaled fenoterol and salbutamol approximately equal risk. The most likely explanation for this association between high doses of beta-agonists and asthma death would be that the high use of a beta-agonist inhaler is an indication of severe asthma, giving a higher risk of death. While this is certainly a factor, adjustment for all possible markers of severity fails to alter the association between beta-agonist use and risk of death and near death. Furthermore there is no such association between mortality and the use of inhaled steroids, which would also be used in patients who may have moderately severe asthma. This suggests (but cannot prove) that there may be a causal link between beta-agonist inhaler usage, especially in high doses, and the risk of death or near death from asthma. Studies have also indicated that beta-agonist may be contributing to an increase in asthma morbidity. A widely quoted cross-over study carried out in New Zealand found that regular use of inhaled fenoterol( four times daily) was associated with worse control of asthma (judged by combined symptom scores, peak flow measurements and airway responsiveness) compared with the period when patients took fenoterol on an 'on demand' basis.
Two studies from Holland indicate that this may not be confined to fenoterol, since regular doses of inhaled salbutamol increase (albeit to a small extent) airway responsiveness, whereas when salbutamol was taken as required to did not. Over a 2 year period regular, but not on demand salbutamol, is associated with an increased annual decline in spirometric values, although a similar accelerated fall in lung function is also seen with ipratropium bromide, so the phenomenon may apply to all bronchodilators. These studies have raised concerns, although the Committee of Safety on Medicines has looked at all currently available evidence and is of the opinion there is no cause for concern. It is obviously important that further carefully controlled studies are carried out, particularly since long-acting inhaled beta2-agonists have now been introduced (see later). A Task Force has recently been established by the National Asthma Campaign which includes a Working Party on Therapy which will examine some of these issues.
Mechanism of adverse effects
Since beta-agonists have been implicated invasthma mortality and morbidity, several possible mechanisms have been proposed. The link between beta-agonists and sudden death from asthma has been attributed to cardiac arrythmias induced by beta-agonists, particularly in the presence of hypo-kalaemia and hypoxia, yet there is little direct evidence for this. In a recent series of 'near deaths' from asthma there was no evidence for any cardiac arrythmias. In a recent study it was found that an inhaled beta-agonist protects against induced broncho-constriction up to a point, but at high doses protection is lost and the airway function falls even more precipitously.This may indicate that inhaled, Beta-agonists provide effective protection against day-to-day constrictor stimuli, but that large constrictor stimuli (e.g. massive allergen exposure or upper respiratory tract viral infection) maylead to a sudden, and possibly catastrophic, fall in lung function. The effect of beta-agonists on asthma morbidity may relate to the fact that beta-agonists control asthma symptoms without controlling the underlying chronic inflammation so that patients may fail to take regular inhaled anti-inflammatory treatments. Beta-agonists may paradoxically increase airway hyper responsiveness in asthmatic patients, and there may be a rebound increase in responsiveness when beta-agonists are stopped. Another hypothesis is that beta-agonists inhibit the release of mediators such as heparin from mast cells, since heparin may have anti-inflammatory properties, although the amount of heparin which is released from mast cells is likely to be very low. Other theories suggest that increased viscous mucus secretion may be a factor, whereas others suggest that the bronchodilator response to beta-agonists allows more allergen to be inhaled into the lower airways where a chronic inflammatory response is initiated. In studies in guinea pigs the non-bronchodilating (+) enantiomer of beta-agonists may increase airway responsiveness, suggesting that the normal racemic (±) beta-agonists such as salbutamol, terbutaline and fenoterol may be deleterious, although whether these considerations are clinically relevant remains to be determined. The clinical implication of these studies is that short-acting inhaled beta-agonists should only be used on demand for symptom control and should never be used on a regular basis. In patients who are taking high doses of inhaled beta-agonists anecdotal reports suggest that there may be an improvement in asthma if beta-agonist inhaler use is restricted.
Inhaled steroids
Efficacy
There is little doubt that inhaled steroids are currently the most effective therapy available for asthma. In several countries, guidelines to asthma therapy have been drawnup and recently international guidelines have been published. All of these guidelines stress the introduction of inhaled anti-inflammatory treatment at an early stage and in adult steroids are the preferred therapy. Several studies have now demonstrated convincingly that inhaled steroids taken over a long period will resolve or substantially suppress the inflammatory changes in the airways associated with asthma. Steroids are very effective in controlling the inflammation in asthma, butwhen steroids are withdrawn asthma symptoms, airways hyper responsiveness (and presumably inflam-mation) return. The molecular mechanisms of action of steroids in asthma are still not certain, but are likely to involve an effect at the gene transcription level and particularly on the transcription of critical cytokines such as IL-3, IL-5 and IL-6. Cortico steroids inhibit the survival of eosinophils in response to GM-CSF and IL-5. An additional direct interaction between the occupied gluco corticoid receptor and the transcription factor activator protein-I which may be switched on in inflammation has also been described. In addition to their inhibitory actions on inflammatory cell infiltration and activation steroids may have a direct inhibitory effect on airway micro vascular leakage 'and on airway mucus secretion. This broad spectrum of effects of cortico steroids on inflammatory cells may explain the efficacy of this form of medication in chronic asthma.
Steroid-resistant asthma
Corticosteroid resistance in some patients with asthma has been recognized for many years, but the mechanisms are far from clear. The importance of this phenomenon is that it may provide important clues to understanding the mechanism of action of cortico steroids in asthma. One action of steroids is to increase the synthesis of lipocortin-1 which has some anti-inflammatory effects. Two studies have demonstrated that antibodies to lipocortin-1 are not elevated in steroid-resistant asthmatic. There is evidence that T-lymphocytes show reduced responsiveness to steroids in vitro which is correlated with reduced in vivo responsiveness. This does not appear to be due to a defect in steroid binding to the gluco corticoid receptor, but a defect in binding of the activated steroid receptor to steroid responsive elements on target genes has not been excluded. The defect in steroid responsiveness is also seen in monocytes from these patients. The reduced responsiveness is not confined to inflammatory cells since there is evidence for reduced skin blanching response to steroids in such patients.
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