Allergic inflammation and airway smooth muscle function

The Science of the Total Environment 270 Ž2001. 57᎐61

Allergic inflammation and airway smooth muscle function Emanuele CrimiU , Manlio Milanese, Song Pingfang, Vito Brusasco Centro di Fisiopatologia Respiratoria, Dipartimento di Scienze Motorie e Riabilitati¨ e, Uni¨ ersita ` di Geno¨ a, Largo R Benzi 10, 16129 Geno¨ a, Italy Accepted 14 April 2000

Abstract It is widely accepted that airway smooth muscle ŽASM. contraction plays a key role in asthmatic attacks. Whether abnormalities of contractility or autonomic regulation exist in the asthmatic ASM is still debated. Studies based on isometric contraction failed to show differences in the force-generation capability between asthmatic and normal ASM. Recent studies in vitro have shown that sensitized ASM: Ž1. shortens more and more rapidly than normal ASM; and Ž2. develops a myogenic response to stretching. The increased velocity of shortening may compromise in vivo the ability of tidal cycling to reduce airway tone, which would result in an enhanced response to bronchoconstrictor stimuli. The myogenic response may result in a sustained bronchospasm after a deep inhalation, a maneuver that in normal individuals causes bronchodilatation. Although there is no evidence that neural or humoral abnormalities in the autonomic regulation of ASM tone are central to the pathogenesis of bronchial asthma, recent data suggest that ASM receptor dysfunction may develop secondary to airway allergic response. It has been shown that exposure of passively sensitized human bronchi to allergens in vitro causes M 2- and ␤ 2-receptor dysfunction. Impairment of pre-junctional M 2-autoreceptors may result in an enhancement of neurally mediated bronchoconstrictor responses, whereas ␤ 2-receptor dysfunction may reduce the sensitivity to bronchodilator treatment. Airway inflammation, which is a characteristic feature of bronchial asthma, may alter both the contractile properties and the autonomic regulation of ASM. These changes may contribute to the severity of asthma, as they may cause an imbalance between factors favoring and opposing airway narrowing. 䊚 2001 Elsevier Science B.V. All rights reserved. Keywords: Airway responsiveness; Atopy; IgE; Asthma

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Corresponding author. Tel.: q39-0103537689; fax: q39-0103537690. E-mail address: [email protected] ŽE. Crimi.. 0048-9697r01r$ - see front matter 䊚 2001 Elsevier Science B.V. All rights reserved. PII: S 0 0 4 8 - 9 6 9 7 Ž 0 0 . 0 0 7 8 5 - 3

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1. Introduction

2. ASM receptors and signal transduction

Airway hyper-responsiveness ŽAHR. is a main characteristic of bronchial asthma, but the mechanism leading to the development of AHR is largely unknown. Atopy and allergic inflammation are often associated with the development or worsening of AHR, but the causal relationship between the allergic phenomena and AHR is still uncertain ŽBrusasco et al., 1998.. The mechanical effect of AHR is the airway smooth muscle cell, the only cell capable of constricting the airway tissue and reducing the airway lumen. Therefore, in the pathogenesis of bronchial asthma, knowledge of the behavior of the ASM is of paramount importance. In particular, the question to be answered is whether ASM is abnormal in its functions Žsensitivity and contractility .; is a normal responder to abnormal stimuli; or is normal, but it is working in an abnormal environment. The purpose of this short review is to summarize the recent understanding of the behavior of ASM in the presence of a high titer of IgE and after the development of a specific IgE-mediated response.

The response of ASM to bronchoconstrictor and bronchodilator stimuli is by means of transmembrane receptors ŽFig. 1.. These receptors are coupled to different specific G proteins, which transduce and amplify messages from the microenvironment to the effector enzymes ŽBarnes, 1998.. A cascade of enzymatic reactions leading to an increase in cytoplasmic Ca2q causes bronchoconstriction by rapid cross-bridge formation between myosin and actin. This phenomenon occurs when Gq-coupled receptors are stimulated. Myosin light-chain kinase ŽMLCK. plays a central role in promoting cross-bridge formation. In contrast, stimulation of Gs-coupled ␤ 2-receptors leads to relaxations through the activation of adenylyl cyclase, which, in turn, increases the second messenger, cAMP. Adenylyl cyclase is negatively controlled by Gi-coupled muscarinic Žtype 2. receptors. There is a cross-talk between receptors ŽFig. 1.. Protein kinase C ŽPKC. is activated by diacyl glycerol upon Gq-coupled receptor stimulation, causing phosphorylation and dysfunction of Gs

Fig. 1. In vitro model of passive sensitization. Homologous sensitization Žincubation of human bronchus from non-atopic donor with serum from atopic donor. allows the investigation of ASM hyper-reactivity induced by either sensitization or exposure to allergen. If the IgE level is high, ASM dysfunctions result from the process of sensitization by itself, but the presence of the IgEs is not strictly necessary, as IgE deprivation does not prevent it. If the level of specific IgEs is high but the total IgE level is relatively low, ASM dysfunctions occur only after the development of the allergic reaction and are strictly dependent on the presence of IgE in sera, as deprivation of IgEs prevents it.

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and ␤ 2-receptor. Protein kinase A ŽPKA. is activated by the increase of cAMP upon Gs-coupled ß 2-receptor stimulation. PKA reduces the sensitivity of Ca2q stores to Ca2q releasers, and the sensitivity of the contractile proteins to Ca2q. Moreover, prolonged stimulation of a given receptor by the specific agonist leads to homologous desensitization, whereas stimulation of its second messenger leads to the so-called eterologous desensitization. The result is that the sensitivity of ASM to acetylcholine Žthe physiologic constrictor agent. or adrenaline Žthe physiologic relaxant agent. will vary over time, depending on the presence and functional status of the agonist and the antagonist receptors.

3. ASM and atopy The atopic status is often, but not always, associated with AHR ŽCrimi et al., 1998.. The atopic status is characterized by the presence of increased amounts of immunoglobulin E ŽIgE. and by the presence of specific IgE to various allergens. It is well established that the inhalation of a specific allergen causes an inflammatory response in the airway. The mechanisms involved in this response that may affect ASM function are largely unknown. Moreover, it is only recently that IgEs themselves have been recognized as a possible cause of ASM dysfunction and AHR. For ethical problems, most of the knowledge in this field comes from animal models and in vitro studies, the results of which can only be extrapolated to human asthma with caution.

4. Models Two main models have been developed to investigate the relationship between atopy and ASM functions: the in vitro model of passive sensitization of human bronchial tissue; and the model of exposure to allergen of pre-sensitized tissue, either in vivo or in vitro. In all these models the object of the investigation is whether and how the

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behavior of ASM may change in an atopic microenvironment, or soon after the release of allergic mediators ŽFig. 2.. In the passive sensitization model, human bronchial tissue of non-atopic donors is incubated overnight with atopic sera containing high levels of total IgE, generally ) 1000 Urml. Then the sensitivity of ASM to constrictor and relaxing agonists is tested. It is well established that the process of sensitization increases the velocity of shortening ŽMitchell et al., 1994., reduces the response to many relaxing agonist ŽBlack et al., 1989., and in some cases induces a myogenic response ŽAntonissen et al., 1979; Mitchell et al., 1997., that is, an active tone in response to a quick stretch of the muscle tissue Žsee Table 1.. All these changes may contribute to the development of AHR, and in particular to the loss of the ability to dilate airways by deep inhalations. The suggested mechanism leading to these changes are an increased expression of CD23 ŽFc-␧ II. receptor, the low affinity IgE receptor, whose presence has also been recently shown on the ASM membrane ŽHakonarson and Grunstein, 1998., and the associated autologous overproduction of IL-1␤ . The intracytoplasmatic mechanisms may be: an increased activity of MLCK, causing a higher rate of cross bridge formation; an increased activity of the PKC isoform that causes phosphorylation and functional impairment of the Gs-coupled ␤ 2 receptor ŽRossetti et al., 1995.; and an increased expression of the Gi protein, which is coupled with the M 2 receptor and antagonizes adenylyl cyclase ŽHakonarson and Grunstein, 1998.. If the level of total IgEs in the sensitizing serum is near normal but the titer of specific IgE is high, the process of sensitization per se does not cause any change in ASM function. Only after the exposure to the allergen and the development of the allergic response may changes in ASM function be appreciated ŽFig. 2.. We have found ŽSong et al., 1997. that 1 h after incubation of the sensitized tissue with house dust mite, the relaxing response to salbutamol Ž␤ 2-agonist. of carbachol-precontracted tissue is reduced in allergen-challenged tissues, but not in sensitized tis-

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Fig. 2. Cholinergic and adrenergic receptors on the airway smooth muscle membrane. The cholinergic stimulus activates both M 2 and M 3 receptors. The Gi-coupled M 2 -receptors inhibit adenylyl cyclase, whereas Gq-coupled receptors activate phospholipase C ŽPLC.. From this enzyme starts the enzymatic cascade that leads to phasic contraction. PLC causes also an increase in diacylglycerol ŽDAG., which activates protein kinase C ŽPKC.. This phosphorylating enzyme presents various isoforms with different substrates, among which are the Gs protein and the ␤ 2 receptor. PKC is also involved in tonic contraction. Epinephrine activates Gs-coupled ␤ 2 -receptors. It follows an increase in adenylyl cyclase activity and, in turn, in cAMP concentration. Increased concentration of cAMP activates protein kinase A ŽPKA. that has different activities. Among them is the opening of the Ca2q-dependent Kq-channel, which causes membrane hyperpolarization and ASM relaxation. PKA also causes phosphorylation of the inositol triphosphate channel and the decrease in the response to cholinergic stimuli.

Table 1 Effects of passive sensitization with high level of total IgE and of the allergic reaction on ASM functions Sensitization with high level of total IgE

Effect of exposure to allergen after sensitization with specific IgE

­ velocity of shortening ­ myogenic response ­ response to histamine ­ s x contraction to acetylcholine x relaxation to ␤2 agonists x relaxation to VIP, PgE2 , verapamil, Levocromakalin s relaxation to forskolin

x relaxation to ␤2 agonists x protection by pilocarpine ŽM2 agonist. of contraction induced by electric field stimulus s relaxation to Ca2q dependent Kq channel opener s relaxation to theophylline s relaxation to forskolin

Mechanisms ­ activity of myosin light chain kinase ŽMLCK. ­ activity of protein kinase C ­ expression of Gi protein ­ expression of CD23 ŽFc-␧ IIR. on ASM membrane

x activity of ␤2 ᎐Gs protein complex x activity of M2 ᎐GI protein complex on parasympathetic nerve endings ­ activity of protein kinase C ­ autocrine production of IL-1␤ and PgE2

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sues. We have subsequently shown ŽSong et al., 1998. that leukotrienes are the mediators responsible for the ASM dysfunction, since premedication with a potent anti- LTD4 antagonist ŽIralukast . was able to completely prevent ␤ 2-receptor dysfunction, which was not prevented by cetirizine Žan H1 antagonist. and indomethacin Žcyclo-oxygenase inhibitor.. In addition, nedocromil sodium, a mast-cell stabilizer drug, was able to prevent ␤ 2-receptor dysfunction, suggesting that mediators are mainly produced by these cells. Neither adenylyl cyclase nor Ca2q-dependent Kq channel seem to be disfunctioning after exposure to allergen, since neither the relaxing response to forskolin Žpromoters of adenylyl cyclase activity., nor that to a specific Ca2q-dependent Kq channel opener were modified ŽMilanese et al., 1999. The allergic reaction also affects M 2 receptor function. This receptor is located both on the ASM membrane Žsee Fig. 2. and on the parasympathetic nerve endings, where it functions as an autoreceptor by reducing the acetylcholine release upon stimulation by the acetylcholine itself. Thus, a block of the prejunctional M 2-receptor causes an increase in acetylcholine release that, in turn, leads to ASM shortening. In an animal model of in vivo allergic inflammation, it has been shown that the eosinophil major basic protein causes M 2-receptor dysfunction ŽEvans et al., 1997.. We have also shown that in isolated ASM, i.e. in the absence of eosinophils from blood, the allergic reaction may cause M 2-dysfunction ŽSong et al., 1997.. Indeed the protective effect against electric field stimulation of pilocarpine ŽM 2 agonist. is reduced after exposure to allergen. This suggests that the allergic activation of the resident inflammatory cells may also affect M 2 function.

5. Conclusion Data obtained with the model of passive sensitization suggest that atopic status and allergic inflammation may induce ASM dysfunctions, involving both its contractile properties and its autonomic regulation. These dysfunctions may con-

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tribute to the AHR in allergic asthmatic subjects. Furthermore, mechanisms of ASM dysfunction associated with the atopic status seem different from those induced by the allergic reaction. References Antonissen L, Mitchell ARW, Kroeger EA, Kepron W, Tse KS, Stephens NL. Mechanical alterations of airway smooth muscle in a canine asthmatic model. J Appl Physiol 1979;46:681᎐687. Barnes PJ. Pharmacology of airway smooth muscle. Am J Respir Crit Care 1998;158:s123᎐s132. Black JL, Marthan R, Armour CL, Johnson PRA. Sensitization alters contractile responses and calcium influx in human airway smooth muscle. J Allergy Clin Immunol 1989;84:440᎐447. Brusasco V, Crimi E, Pellegrino R. Airway hyperresponsiveness in asthma: not just a matter of airway inflammation. Thorax 1998;53:992᎐998. Crimi E, Spanevello A, Neri M, Ind PW, Rossi GA, Brusasco V. Dissociation between airway inflammation and airway hyperresponsiveness in allergic asthma. Am J Respir Crit Care Med 1998; 157: 4᎐9. Evans CM, Jacoby DB, Gleich GJ, Fryer AD, Costello RW. Antibody to eosinophil major basic protein protects M 2 receptor function in antigen challenged guinea pigs in vivo. J Clin Invest 1997;100:2254᎐2262. Hakonarson H, Grunstein MM. Autologously up-regulated Fc receptor expression and action in airway smooth muscle mediates its altered responsiveness in the atopic asthmatic sensitized state. Proc Natl Acad Sci 1998;95:5257᎐5262. Milanese M, Song P, Crimi E, Rehder K, Brusasco V. Gs-protein dysfunction after allergen challenge in isolated passively sensitized human bronchi ŽPSHB.. Am J Respir Crit Care Med 1999;159Ž2.:A399. Mitchell RW, Ruhlmann E, Magnussen H, Leff AR, Rabe KF. Passive sensitization of human bronchi augments smooth muscle shortening velocity and capacity. Am J Physiol ŽLung Cell Mol Physiol. 1994;242:L218᎐L222. Mitchell RW, Rabe KF, Magnussen H, Leff AR. Passive sensitization of human airways induces myogenic contractile responses in vitro. J Appl Physiol 1997;83:1276᎐1281. Rossetti M, Savineau JP, Huguette C, Marthan R. Role of protein kinase C in non-sensitized and passively sensitized human isolated bronchial smooth muscle. Am J Physiol ŽLung Cell Mol Biol. 1995;268:L966᎐L971. Song P, Milanese M, Crimi E, Rehder K, Brusasco V. Allergen challenge of passively sensitized human bronchi alters M 2 and ␤ 2 receptor function. Am J Respir Crit Care Med 1997;155:1230᎐1234. Song P, Crimi E, Milanese M, Duan J, Rehder K, Brusasco V. Anti-inflammatory agents and allergen-induced ␤ 2-receptor dysfunction in isolated human bronchi. Am J Respir Crit Care Med 1998;158:1809᎐1814.